Official publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 18, Number 1, February 2021.

Thematic issue: What is a new in the world of orthopedic and trauma? Published and copyright by: Medical Assotiation of Zenica-Doboj Canton; Address: Zenica, 72000, Bulevar kralja Tvrtka I 4, Bosnia and Herzegovina; tel./fax: +387 32 444 270; Email: [email protected], [email protected], web site: http//www.ljkzedo.ba For ordering information please contact: Jasenko Žilo, [email protected]; Access to this journal is available free online trough: www.ljkzedo.ba The Journal is indexed by MEDLINE, EMBASE (Exerpta Medica), Scopus, EBSCO; ISSN 1840-0132 DTP by: Graphic and web design studio “B Panel” Zenica, Zmaja od Bosne bb, www.bpanel.ba, e-mail: [email protected], tel. +387 32 441 290, 441 291; Printed by: PLANJAX PRODUKT D.O.O.; Bobare 55, 74260 Tešanj, Bosna i Hercegovina, Tel.: +387 32 667 350, www.planjaxgroup.com, e-mail: [email protected] Medicinski Glasnik Official Publication of the Medical Association of Zenica-Doboj Canton Bosnia and Herzegovina EDITOR-IN-CHIEF Selma Uzunović, Zenica, Bosnia and Herzegovina

DEPUTY EDITOR Besim Prnjavorac, Tešanj, Bosnia and Herzegovina

RESEARCH INTEGRITY EDITOR Larisa Gavran, Zenica, Bosnia and Herzegovina

MANAGING EDITOR Tarik Kapidžić, Zenica, Bosnia and Herzegovina

EDITORS Solmaz Abdolrahimzadeh, Rome, Italy Luiz Ronaldo Alberti, Belo Horizonte, Brazil Mutay Aslan, Antalya, Turkey Adem Balić, Tuzla, Bosnia and Herzegovina Dubraka Bartolek, Zagreb, Croatia Branka Bedenić, Zagreb, Croatia Iva Christova, Sofia, Bulgaria Asja Čelebić, Zagreb, Croatia Josip Čulig, Zagreb, Croatia Filip Čulo, Zagreb, Croatia Jordan Dimanovski, Zagreb, Croatia Branko Dmitrović, Osijek, Croatia Davorin Đanić, Slavonski Brod, Croatia Ines Drenjančević, Osijek, Croatia Harun Drljević, Zenica, Bosnia and Herzegovina Mukaddes Esrefoglu, Istanbul, Turkey Ivan Fistonić, Zagreb, Croatia Roberta Granese, Messina, Italy Simona Gurzu, Tȋrgu Mureş, Romania Diane Medved Harper, Louisville, United State Lejla Ibrahimagić-Šeper, Zenica, Bosnia and Herzegovina Tatjana Ille, Ajman, United Arab Emirates Slobodan M. Janković, Kragujevac, Serbia Vjekoslav Jerolimov, Zagreb, Croatia Ioan Jung, Tȋrgu Mureş, Romania David Kovacevic, New Haven, United States Sven Kurbel, Osijek, Croatia Snježana Pejičić, Banja Luka, Bosnia and Herzegovina Belma Pojskić, Zenica, Bosnia and Herzegovina Asja Prohić, Sarajevo, Bosna Hercegovina Velimir Profozić, Zagreb, Croatia Amira Redžić, Sarajevo, Bosnia and Herzegovina Halima Resic, Sarajevo, Bosnia and Herzegovina Suad Sivić, Zenica, Bosnia and Herzegovina Sonja Smole-Možina, Ljubljana, Slovenia Vladimir Šimunović, Mostar, Bosnia and Herzegovina Ekaterine Tskitishvili, Liege, Belgium Aylin Türel Ermertcan, Manisa, Turkey Adrijana Vince, Zagreb, Croatia Jasmina Vraneš, Zagreb, Croatia

EDITORIAL ASSISTANT Hakija Bečulić, Zenica, Bosnia and Herzegovina

Secretary: Jasenko Žilo Proofreader: Glorija Alić (English) Cover: "A view from a warm home" (Author: Zdena Šarić Pisker) MEDICINSKI GLASNIK Official Publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 18, Number 1, February 2021 Free full-text online at: www.ljkzedo.com.ba, and www.doaj.org (DOAJ, Directory of Open Access Journals) Content Review Balneogynaecology in the 21st century: increasingly recommended primary and complementary treatment 1 of chronic gynaecological Dubravko Habek, Anis Cerovac, Lejla Kamerić, Enida Nevačinović, Adnan Šerak Original The importance of plasma ferritin values in blood donors for the evaluation of body iron store in a five- 7 month period article Elma Ćatović-Baralija, Gorana Ahmetović-Karić, Sabaheta Hasić, Nesina Avdagić, Nermina Babić Clinical use of an analysis of oxidative stress and IL-6 as the promoters of diabetic polyneuropathy 12 Emina Karahmet, Besim Prnjavorac, Tamer Bego, Adaleta Softić, Lejla Begić, Edin Begić, Esma Karahmet, Lejla Prnjavorac, Irfan Prnjavorac Lived experiences of patients with COVID-19 infection: a phenomenology study 18 Ali-asghar Jesmi, Zohreh Mohammadzade-tabrizi, Mostafa Rad, Elyas Hosseinzadeh-younesi, Ali Pourhabib Comparison of analgesic efficacy of acetaminophen monotherapy versus acetaminophen combinations 27 with either pethidine or parecoxib in patients undergoing laparoscopic cholecystectomy: a randomized prospective study Francesk Mulita, Georgios Karpetas, Elias Liolis, Michail Vailas, Levan Tchabashvili, Ioannis Maroulis Left to right shunt congenital heart as a risk factor of recurrent pneumonia in under five-year-old 33 children: a single centre experience in Bandung Indonesia Sri Endah Rahayuningsih, Rahmat Budi Kuswiyanto, Filla Reviyani Suryaningrat, Heda Melinda Nataprawira, Abdurachman Sukadi Characterization and clonal representation of MRSA strains in Tuzla Canton, Bosnia and Herzegovina, 38 from 2009 to 2017 Fatima Numanović, Urška Dermota, Jasmina Smajlović, Sandra Janežič, Nijaz Tihić, Zineta Delibegović, Amela Bećirović, Edina Muratović, Merima Gegić Efficacy and safety of three plant extracts based formulations of vagitories in the treatment of vaginitis: a 47 randomized controlled trial Kemal Durić, Selma Kovčić Hadžiabdić, Mahira Durić, Haris Nikšić, Alija Uzunović, Hurija Džudžević Čančar Prevalence of Cryptosporidium spp. and Blastocystis hominis in faecal samples among diarrheic HIV 55 patients in Medan, Indonesia Hemma Yulfi, Muhammad Fakhrur Rozi, Yunilda Andriyani, Dewi Masyithah Darlan Anti-fibrotic effect of intravenous umbilical cord-derived mesenchymal stem cells (UC-MSCs) injection in 62 experimental rats induced liver fibrosis Taufik Sungkar, Agung Putra, Dharma Lindarto, Rosita Juwita Sembiring Concordance of non-invasive serology-based scoring indices and transient elastography for liver fibrosis 70 and cirrhosis in chronic hepatitis C Emir Trnačević, Nermin Salkić, Alma Trnačević, Anja Divković, Fatima Hukić, Nusret Butković, Amra Serak, Amer Mujkanović An epidemiological study of neuroendocrine tumours at tertiary hospitals in Bosnia and Herzegovina 77 Maja Konrad Čustović, Ermina Iljazović, Azra Sadiković, Zinaida Karasalihović Appropriateness of colonoscopy at a tertiary care centre – are we overdoing gastrointestinal endoscopy? 84 Nerma Čustović, Lejla Džananović, Ismar Rašić, Nadža Zubčević, Lejla Šaranović-Čečo, Jasmina Redžepagić Oxidative stress markers in initial and remission of nephrotic syndrome and serum 90 malondialdehyde level predictor from routine laboratory test Riska Habriel Ruslie, Oke Rina Ramayani, Darmadi Darmadi, Gontar Alamsyah Siregar Refractive errors in children: analysis among preschool and school children in Tuzla city, Bosnia and 96 Herzegovina Amra Nadarević Vodenčarević, Meliha Halilbašić, Anis Međedović, Vahid Jusufović, Adisa Pilavdžić, Aida Drljević, Mufid Burgić Correlation of signal to noise ratio (SNR) value on distortion product otoacoustic emission (DPOAE) and 102 expression of nuclear factor erythroid 2-related factor 2 (NRF2) in cochlear organ of Corti in rat exposed to noise Diana Amellya, Tengku Siti Hajar Haryuna, Wibi Riawan Correlation between numerical and categorical immunohistochemical score of Ki-67 and HER2 with 107 clinicopathological parameters of breast cancer Mirsad Dorić, Suada Kuskunović-Vlahovljak, Edina Lazović Salčin, Svjetlana Radović, Nina Čamdžić, Mirsad Babić, Haris Čampara Posthumous sperm retrieval: a procreative revolution 114 Francesca Negro, Renata Beck, Antonella Cotoia, Maria Cristina Varone Serum total prostate-specific antigen (tPSA): correlation with diagnosis and grading of prostate cancer in 122 core needle biopsy Nina Čamdžić, Suada Kuskunović-Vlahovljak, Mirsad Dorić, Svjetlana Radović, Edina Lazović Salčin, Mirsad Babić Scrotal trauma: interest of preoperative ultrasound in the prediction of the rupture of the tunica albuginea 128 Stefano Manno, Antonio Cicione, Lorenzo Bagalà, Antonio Catricalà, Piero Ronchi, Simona Tiburzi, Carolina Giannace, Lucio Dell’Atti Workload changes during the COVID-19 pandemic and effects on the flow of cancer patients in the 133 Maxillofacial Department Ana Kvolik Pavić, Vedran Zubčić, Slavica Kvolik Brainstem haemorrhage as a rare complication of burr hole craniostomy 138 Rodolfo Corinaldesi, Corrado Filippo Castrioto, Francesca Romana Barbieri, Luciano Mastronardi, Umberto Ripani Application of a personal Santini technique in the resolution of a complex celiac trunk aneurysm - 143 endovascular treatment Gianpaolo Santini, Pasquale Quassone, Luca Tarotto, Francesco Arienzo, Giuseppe Sarti Current status of localized submental fat treatment with sodium deoxicolate (ATX-101) 148 Cristina Ibáñez-Vicente, Miguel Carrato-Gomez, Luigi Meccariello, Umberto Ripani; Michele Bisaccia Does low intensity direct current affect open fracture wound healing? 153 Yoyos Dias Ismiarto, Kemas Abdul Mutholib Luthfi, Mahyudin Mahyudin, Adriel Benedict Intermittent traction therapy in the treatment of chronic low back pain 158 Edina Tanović, Damir Čelik, Āemil Omerović, Vanda Zovko Omeragić, Amila Jaganjac, Hadžan Konjo, Emina Rovčanin, Hana Omerović Efficacy of Zhu’s acupuncture techniques to improve muscle strength of motion limbs in stroke patients 164 Indri Seta Septadina, Erial Bahar The importance of education in patients with metabolic syndrome with regard to their knowledge and 170 attitudes about the disease, and the impact of education on laboratory parameters Azra Bureković, Elvira Āozo, Anida Divanović Effects of semi-refined carrageenan (food additive E407a) on cell membranes of leukocytes assessed in vivo 176 and in vitro Anton Tkachenko, Anatolii Onishchenko, Alexander Roshal, Oksana Nakonechna, Tetyana Chumachenko, Yevgen Posokhov Thematic issue: What is a new in the world of orthopedic and trauma?

Editorial What is a new in the world of orthopedic and trauma? 185 Selma Uzunović Review Biological augmentation strategies in rotator cuff repair 186 Erdi Özdemir, Dogac Karaguven, Egemen Turhan, Gazi Huri Original Arthroscopic labral repair with all-suture anchors: a magnetic resonance imaging retrospective study with 192 a 2.5-year follow-up article Federico Sacchetti, Martina Di Meglio, Nicola Mondanelli, Nicola Bianchi, Vanna Bottai, Federico Cartei, Fabio Cosseddu, Rodolfo Capanna, Stefano Giannotti Distal biceps tendon repair and posterior interosseous nerve injury: clinical results and a systematic review 196 of the literature Silvio Chiossi, Marco Spoliti, Pasquale Sessa, Valerio Arceri, Attilio Basile, Francesca Romana Rossetti, Riccardo Maria Lanzetti A new technique of flexor carpi ulnaris transfer in multilevel surgery for upper extremity deformities in 202 spastic cerebral palsy Georgy Chibirov, Mairbek Pliev, Dmitry Popkov The challenge of the surgical treatment of paediatric distal radius/forearm fracture: K wire vs plate 208 - outcomes assessment Salvatore Di Giacinto, Giuseppe Pica, Alessandro Stasi, Lorenzo Scialpi, Alessandro Tomarchio, Alberto Galeotti, Vlora Podvorica, Annamaria dell’Unto, Luigi Meccariello Modalities of extensor tendon repair related to etiological factors and associated injuries 216 Sanela Salihagić, Zlatan Zvizdić, Dženana Hrustemović, Redžo Čaušević, Ahmad Hemaidi Traumatic bilateral scaphoid fractures 222 Dariush Ghargozloo, Alessandro Tomarchio, Mauro Ballerini, Gianpaolo Chitoni Impact of load on the knee in relation to a treadmill angle 226 Fikret Veljović, Edin Begić, Avdo Voloder, Reuf Karabeg, Amer Iglica, Nedim Begić, Alden Begić, Adisa Chikha Two-stage bone-and-strut technique in the treatment of septic non-unions in the upper limb 232 Luigi Meccariello, Ante Prkić, Vincenzo Campagna, Alberto Serra, Vincenzo Piccinni, Denise Eygendaal, Michele Bisaccia, Giuseppe Pica, Andrea Schiavone, Giuseppe Rollo Radiographic evaluation of the tunnel position in single and double bundle anterior cruciate ligament 239 reconstruction Michele Losco, Francesco Giron, Luca Giannini, Pierlugi Cuomo, Roberto Buzzi, Stefano Giannotti, Nicola Mondanelli Single use instruments for total knee arthroplasty 247 Michele Romeo, Giuseppe Rovere, Leonardo Stramazzo, Francesco Liuzza, Luigi Meccariello, Giulio Maccauro, Lawrence Camarda Medial pivot vs posterior stabilized total knee arthroplasty designs: a gait analysis study 252 Nicola Bianchi, Andrea Facchini, Nicola Mondanelli, Federico Sacchetti, Roberta Ghezzi, Marco Gesi, Rodolfo Capanna, Stefano Giannotti Allogenic platelet concentrates from umbilical cord blood for knee osteoarthritis: preliminary results 260 Vincenzo Caiaffa, Francesco Ippolito, Antonella Abate, Vittorio Nappi, Michele Santodirocco, Domenico Visceglie Intra-operative local plus systemic tranexamic acid significantly decreases post-operative bleeding and the 267 need for allogeneic blood transfusion in total knee arthroplasty Lidia De Falco, Elisa Troiano, Martina Cesari, Pietro Aiuto, Giacomo Peri, Nicolò Nuvoli, Mattia Fortina, Nicola Mondanelli, Stefano Giannotti Radiographic and functional outcome of complex acetabular fractures: implications of open reduction in 273 spinopelvic balance, gait and quality of life Vitaliano F. Muzii, Giuseppe Rollo, Guido Rocca, Rocco Erasmo, Gabriele Falzarano, Francesco Liuzza, Michele Bisaccia, Giuseppe Pica, Raffaele Franzese, Luigi Meccariello Key factors influencing clinical and functional outcomes in extracapsular proximal femur fractures: the 280 role of early weight-bearing - one-year follow-up cohort of 495 patients Enrique Sanchez-Munoz, Beatriz Lozano-Hernanz, Daniel Vicente Velarde-Garrido, Leticia Alarma-Barcia, Victor Trivino Sanchez-Mayoral, Paula Romera-Olivera, Cristina Lopez Palacios Effectiveness of teriparatide combined with the Ilizarov technique in septic tibial non-union 287 Giuseppe Rollo, Francesco Luceri, Gabriele Falzarano, Carlo Salomone, Enrico Maria Bonura, Dmitry Popkov, Mario Ronga, Giuseppe Pica, Michele Bisaccia, Valentina Russi, Predrag Grubor, Raffaele Franzese, Giuseppe M. Peretti, Luigi Meccariello Rare and uncommon diseases of the hip: arthroscopic treatment 293 Christian Carulli, Alberto Schiavo, Alberto Rigon, Wondi De Marchi, Matteo Innocenti, Luigi Meccariello, Massimo Innocenti A new prognostic pelvic injury outcome score Luigi Meccariello, Cristina Razzano, Cristina De Dominicis, 299 Juan Antonio Herrera-Molpeceres, Francesco Liuzza, Rocco Erasmo, Guido Rocca, Michele Bisaccia, Enzo Pagliarulo, Pietro Cirfeda, David Gómez Garrido, Giuseppe Pica, Giuseppe Rollo Navigated percutaneous screw fixation of the pelvis with O-arm 2: two years’ experienceGianluca Ciolli, 309 Daniele Caviglia, Carla Vitiello, Salvatore Lucchesi, Corrado Pinelli, Domenico De Mauro, Amarildo Smakaj, Giuseppe Rovere, Luigi Meccariello, Lawrence Camarda, Giulio Maccauro, Francesco Liuzza Comparing hand strength and quality life of locking plate versus intramedullary k wire for transverse 316 midshaft metacarpal fractures Andrea Pasquino, Alessandro Tomarchio, Enio De Cruto, Jacopo Conteduca, Damiano Longo, Valentina Russi, Giuseppe Pica, Luigi Meccariello, Giuseppe Rollo Minimally invasive sinus tarsi approach in Sanders II-III calcaneal fractures in high-demand patients 322 Paolo Ceccarini, Francesco Manfreda, Rosario Petruccelli, Giuseppe Talesa, Giuseppe Rinonapoli, Auro Caraffa Balance assessment after altering stimulation of the neurosensory system 328 Valentina Li Causi, Alessandro Manelli, Valentina Gianpaola Marini, Mario Cherubino, Luigi Meccariello, Michael Mazzacane, Mario Ronga

Medicinski Glasnik is indexed by MEDLINE, EMBASE (Exerpta Medica), EBSCO and Scopus REVIEW

Balneogynaecology in the 21st century: increasingly recommended primary and complementary treatment of chronic gynaecological diseases

Dubravko Habek1, 2, Anis Cerovac3, 4, Lejla Kamerić5, Enida Nevačinović5, Adnan Šerak5

¹University Department of and , Clinical Hospital „Sveti Duh“ Zagreb, 2Catholic University of Croatia, Zagreb; Croatia, ³Department of Gynaecology and Obstetrics, General Hospital Tešanj, Tešanj, 4Department of Anatomy, School of , University of Tuzla, Tuzla, 5Clinic for Gynaecology and Obstetrics, University Clinical Centre Tuzla, Tuzla; Bosnia and Herzegovina

ABSTRACT

Balneo-gynaecological treatment methods include external bath hydrotherapy, sedentary baths and topical dressings/cataplasm, and internal (intravaginal or intrarectal use of peloids and mine- ral water). Hyperosmolar thermal spas have been very popular in the treatment of infertility due to the improvement of symptoms of chronic pelvic pain, endometriosis, chronic vascular and in- flammatory pelvic diseases. Acute pelvic inflammatory syndrome Corresponding author: is a contraindication for balneo-hydrotherapy while hyperthermal Anis Cerovac hydrotherapy is contraindicated in endometriosis and neurovege- General Hospital Tešanj tative dystonia due to the stimulation of hyperemia, which worsens Braće Pobrić 17, 74260 Tešanj, the clinical picture. Balneo-hydrotherapy is not recommended in Bosnia and Herzegovina metrorrhagia and malignancies. Balneogynaecological treatment Phone: +387 61 051 929; certainly has its own primary but also complementary role in the treatment of chronic gynaecological diseases and is increasingly Fax: +387 32 650 605; recommended today. E-mail: [email protected] Dubravko Habek ORCID ID: https://orcid. Key words: complementary , hydrotherapy, mud therapy org/0000-0003-1304-9279

Original submission: 26 August 2020; Revised submission: 01 September 2020; Accepted: 09 October 2020 doi: 10.17392/1263-21

Med Glas (Zenica) 2021; 18(1):1-6

1 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION that regulates extra and intravascular fluid with increased diuresis, decreased blood pressure and The climatotherapeutic characteristics of indi- decreased oedema (2-11). All these changes of vidual areas have established numerous natural biochemical markers level have positive effects spas based on climate, mineral and medicinal wa- on muscle relaxation, vasodilation and reduction ter sources, and medicinal peloids. Natural spas of painful conditions (9). Balneotherapy proce- are most commonly called baths on land and coast dures in particular reduce inflammatory cytokine (thalassotherapy). There are numerous records in response and adhesion formation, and they are the noticed by Hippocrates, especially used in , rehabilitation Asclepiades, and others, that indicate the (including chronic pain conditions and fibromyal- good effects of naturopathic treatments, which in gia), followed by postoperative recoveries where addition to the healing properties of natural re- they have a proven antiadhesive effect (2-11). sources, included hygienic components too (1-3). Hyperthermal spa therapy causes local vasodila- Gynaecological balneotherapy (balneo-gynaeco- tion and muscle relaxation, while reducing ten- logy) as a method of therapy has been described sion and pain, plus hot submersion, reduces the throughout the history of medicine and civiliza- production of stress hormones. Hydrotherapy in tion, firstly empirically and scientifically evalu- warm alkaline - chlorine mineral water has been ated by ancient , then pre-renaissance shown to reduce the enzymatic activity of cata- period (Trotula Salernitana), following the deve- lase (12) and to modulate serotonin platelet tran- lopment of iatrophysics and iatrochemistry thro- smission in healthy populations (13). Balneothe- ugh the centuries (1). Physicochemical analysis rapy thus uses the healing properties of natural of climatological and balneological conditions, healing factors of mineral waters and mud such alongside with monitoring of the effects of a tre- as temperature, ionization, composition of micro- atment, have scientifically established indications elements and organic substances (13). Today, in for the treatment of numerous diseases, including addition to balneotherapy (spa therapy), other na- gynaecological ones (3,4) (Table 1). turopathic and methods are used This review offers an insight into balneo-gynae- as complementary to the therapeutic, rehabilitati- cological methods in modern gynaecology in the on and relaxation anti-stress programs (massage, 21st century. psychotherapy, dietetics, phytotherapy, heliothe- BIOCHEMICAL AND PATHOPHYSIOLOGICAL EF- rapy, acupuncture) (1,2,5,6,10,11, 14-16). FECTS OF BALNEOTHERAPY BALNEOTHERAPY PROCEDURES The changes of individual biochemical markers, Balneotherapy procedures have a biphasic sti- such as cortisol, an increase of sodium, potassi- mulating effect of hormesis with proven bioche- um and calcium concentrations in blood, have mical and pathophysiological effects, so patients especially been demonstrated in hydrotherapy need to be informed of the mode of action via procedures, in addition to hydrostatic pressure informed consent after setting an indication for

Table 1. Indications for gynaecological balneotherapy Type of balneotherapy Indication Fango, peloid Intravaginal Hydrotherapy Sitz bad Exercises (cataplasmes) mud application Climacterium + + + + + Psychosomatic disorders (premenstrual syndrome, vaginismus, dyspare- + + - + unia) Neurovegetative syndrome (dystonia) Sterilitas, infertilitas (cervical hypersecretion, luteal insufficiency) + + + + + Postoperative care, reconvalescence + + + + Chronic interstitial cystitis, chronic vulvovaginitis with pruritus + + + + Chronic pelvic congestive syndrome (myoma, functional ovarian cysts) + + + Endometriosis + - - - + Chronic pelvic pain syndrome of extragenital genesis (orthopaedic) + + + + Chronic pelvic pain Dysmenorrhoea + + + + Vulvodynia Vulvar craurosis and urogenital atrophia + + + +

2 Habek et al. Balneogynaecology

balneo-gynaecological treatment (anamnesis, di- to childbirth (Wassergeburt) has been used for agnostics, clinical gynaecological examination, years (2,19,25,28,29). Hot sitz baths (40-46 ºC) laboratory biochemical, haematological and in- are relaxing with antispasmodic effect reached flammatory parameters) (10,11, 17-22). Balneo- in 3-10 minutes, neutral sitz baths (33-36 ºC) gynaecological treatment methods are external are soothing and their recommended duration bath hydrotherapy, sedentary baths and topical is between 15 minutes to 2 hours, and cold sitz dressings/cataplasm, and internal (intravaginal baths (12-29 ºC) are tonifying and last from 30 or intrarectal use of peloids and mineral waters). seconds to 8 minutes. The hydromineral characteristics of spring ther- Local hydropathic antiphlogistic procedures are mal waters will dictate the indications for balneo- also Priesnitze wraps and cold moist wraps that gynaecological treatment (10,11). help reduce congestion, tension and pain (espe- cially used in the treatment of thrombophlebitis Local hydropathic antiphlogistic procedures and mastitis) (10,11,18). Radon water has analgesic, haemostatic, sedative and anti-inflammatory effects in the concentrati- Peloid (mud) therapy on of radon in water of 40-200 nCi/L (21). Peloids (mud), peat soil soaked in natural mine- Salt baths with sodium chloride water have an effect ral water, are organic sludges containing large on the secretion of mineralocorticoids and gluco- amounts of sulfides, phytoestrogens, numerous corticoids, and the activity to sympathetic nervous minerals and residues of organic and inorganic system. Salt baths with sodium chloride water have compounds, and are therefore particularly use- a resorptive effect while salty iodine water has an ful in balneogynaecology (30). Mud wrappers effect on congestive venous syndrome (22). and cataplasms are used as well as intravaginal Arsenic water reduces the intensity of the oxida- applications. Basically, fango is volcanic mud tion-reduction processes, and the nitrogen ther- or paraffin sludge. Peloid baths and catapla- mal water has analgesic effects with poor fibri- smas trigger the strongest reaction of the body, nolytic activity. so it is important to recommend them after the inflammation has ended. Alternating vaginal Iodine-bromine water is especially recommen- irrigations are recommended for chronic pelvic ded in gynaecologic patients but precautionary hyperaemia. Peloid therapy (fango, mud) is re- measures should be taken in thyroid disease, whi- commended in chronic pelvic inflammatory di- le the carbon-acid bath is especially used in those sease (PID) and parametritis for 4-6 weeks. It patients (10,11,17,18, 23-28). should be applied only up to a belly height 2-3 Sulfide water reduces the inflammatory response times per week for 20 minutes. while its tempe- by reducing the exudative and infiltration com- rature should range from 40-42 0C (31). Fango- ponents of inflammation, including the formation shaped peloids are particularly recommended of a fibrotic reaction, and a sulfide concentration in menopausal syndrome, infertility treatment, of 100-150 mg/L is required to treat gynaecologi- and sexual disorders such as vaginismus, dyspa- cal disorders. It is recommended for vulvodynia, reunia, apareunia, and frigidity (10,31,33). In- vulvitis and skin diseases because it is keratolytic travaginal pelotherapy uses applications heated and keratoplastic (10,11). up to 45-50 0C which are applied to the vagina Hyperosmolar (salty iodine) or aromatic baths for 4 hours. The vagina has thermoreceptors up are most commonly used, especially in chronic to 50 0C, so by its trophotropic effect on neural inflammatory and/or painful conditions. plexuses and blood vessels it causes a vasodila- Drinking of spring mineral waters completes bal- tory effect in the small pelvis by improving the neological treatment especially for constipation flow in the uterine artery by 50% in the first 24 that is a consequence of chronic pelvic pain and hours of application. Intravaginal pelotherapy premenstrual syndrome (10,11,17,18, 23-28). improves the tone of periurethral tissue and Warm/hot seating baths are not recommended improves vascularization in cases of urogenital in pregnancy and puerperium, whereas in I and atrophy and as a result reduces urinary inconti- II stage of delivery the hydrotherapy approach nence (10,11).

3 Medicinski Glasnik, Volume 18, Number 1, February 2021

Sitz baths Hyperosmolar thermal spas were very popular in the treatment of infertility due to the impro- Sitz baths in bitter salt (Bittersalz) are recommen- vement of symptoms of chronic pelvic pain, en- ded when the patients suffer from dysmenorrhea, dometriosis, chronic vascular and PID, and cu- chronic pelvic pain and vaginismus, and can be rrent sporadic articles are written about this issue used domiciliary at a temperature of 36-40 0C for (19). The increase of Fallopian tubes motility 10-20 minutes (10,33). In the subacute phase of and the reduction of inflammatory infiltrate are inflammatory pelvic disease, after a week of fe- registered after hydro and pelotherapy, whereas brility, warm baths of 35-37 0C (acratotherme) up an improvement in cervical secretion is seen af- to 40 0C can be applied for 15-20 minutes alter- ter intravaginal pelotherapy (10,31,34). Ameri- nately with mud baths 3-4 times a week. Iodine can authors recommended cold neutral sedentary baths and radioactive baths are recommended in baths for five days to menstrual period in women recurrent pregnancy loss. It is recommended to with uterine myomas with menometrorrhagia, have balneo-gynaecological treatment in the spa followed by sulfur water baths with peloids (9), twice a year, while some of the previously men- while postoperative balneotherapy thermal reha- tioned treatment methods can also be enforced at bilitation after the pelvic surgery was especially home (10,11,17,18,27,33). recommended (17,20,21). Contraindications for balneotherapy The treatment with peloids (pelotherapy) by in- travaginal application has been implemented for Acute pelvic inflammatory syndrome is a contra- decades in gynaecology with good results, es- indication for balneo-hydrotherapy, while hyper- pecially in the treatment of infertility and CPP thermal hydrotherapy is contraindicated in endo- (3,10,35). There is research into the healing pro- metriosis and neurovegetative dystonia due to the perties of the Dead Sea peloid in the treatment stimulation of hyperaemia, which worsens the clini- of chronic endometritis and corpus luteum failu- cal picture. Balneo-hydrotherapy is not recommen- re (31). Hyperosmolar healing water of the Dead ded in metrorrhagia and malignancies (10,11). Sea contains high concentrations of cations (Mg, DISCUSSION K, ca) and anions (F, B, CL) with significant anti- bacterial and hyperaemic effects, especially use- Taking into account previous research on balne- ful in healing dermatological diseases, including ogynaecology and its use in clinical gynaecology vitiligo, and lichen sclerosus et atrophicus (29). there is not much current research into it, while the Artymuk et al. have achieved a significant impro- recommendations relate to the treatment of chro- vement in hormonal profile (oestrogen and pro- nic pain conditions, some forms of infertility, and gesterone elevation) with intravaginal pelothe- functional disorders (8,10, 15-18). This has certa- rapy of the Dead Sea peloid gel during 12 days of inly been influenced by biotechnology in human the menstrual cycle in luteal insufficiency, with a reproduction and minimally invasive endoscopic better Doppler sound record of the ovarian artery in the last thirty years (2,5,6). Also, the on the luteal side in subfertile women (31). Atkin hectic lifestyle and the expectations of patients to et al. have demonstrated that continued low-level have a medicine to quickly resolve a gynaecolo- topical heat therapy has proven to be as effecti- gical problem. On the other hand, healing effect ve in the treatment of dysmenorrhea as ibuprofen and success of balneotherapy has remained the (28), while Czech authors have demonstrated the same (2,5,6). Chronic pelvic pain (CPP) of vario- good effects of balneotherapy in paediatric gyna- us aetiology, vaginal effluvium (leukorrhoea) and ecological diseases, including primary dysme- infertility have been the reasons and indications norrhea (35). Rectal microclysis of mineral wa- for the use of antiphlogistic effects of balneologi- ters has been recommended for dysmenorrhea cal treatment for centuries. Previously recurrent and cataplasm to hypochondrium (17-20, 28). pregnancy losses caused by subacute and chronic The CPP is nowadays a disease affecting 6-27% endometritis, myoperimetritis, and mucopurulent of women, and psychosomatic disorders exacer- cervicitis were especially treated with bath tre- bate the clinical picture (27). Study by Min et al. atments. These pregnancy losses are still a pro- suggests that balneotherapy with 10 heated sea- blem in modern obstetrics today (1-10).

4 Habek et al. Balneogynaecology

water baths and 10 mud-pack applications over with high concentrations of radon water at 6.5 five days could be beneficial for patients with kBk / l (22,35). Therefore, it is evident that there CPP in the short term (27). Zambo et al. (33) is a scientific interest in complementary or pri- demonstrated the effects of a 3-week balneo- mary treatment of CPP caused by endometriosis, therapy of alum-containing ferrous thermal wa- chronic inflammatory disease or chronic varicose ter on chronic PID with 20 min baths in 38 0C pelvic syndrome (36). water every other day, 10 sessions in total, and In conclusion, balneological treatment cannot be demonstrated a significant improvement in gyn- replaced by other forms of treatments. In recent aecological findings and psychic status without decades gynaecological treatment has largely influencing hormonal profile and Doppler sound employed pharmacological and surgical tre- parameter. Similar results in the treatment of atments as faster and more successful methods chronic PID have been found by other authors of treating endometriosis, infertility and sterility. too (31). Gerber et al. (34) found a beneficial ef- Nevertheless, balneological treatment certainly fect of mud baths or mud packs, mineral baths, has its own primary but also complementary role electrotherapies, and gynaecological exercises in in the treatment of chronic gynaecological disea- post-PID patients with antibiotic therapy in re- ses and is increasingly recommended today. ducing pain and insignificant motility of the Fal- lopian tubes. Recommendations for chronic PID FUNNDING is to use intravaginal 45 °C mud applications for No specific funding was received for this study. 120 minutes with a pelotherapy 20-minute bath on every second day (10,31). Contemporary ar- TRANSPARENCY DECLARATION ticles have suggested the reduction of size and Conflict of interest: None to declare. pain of endometriotic foci through balneotherapy

REFERENCES 1. Van Tubergen A, Van der Linden S. A brief history of 10. Beer A, Goecke C. Balneotherapie und Physiothera- spa therapy. Ann Reum Dis 2002; 61:273-5. pie. Als primäre und ergänzende Maßnahmen in der 2. Hrgovic I, Hrgovic Z, Habek D, Oreskovic S, Gynäkologie (Balneotherapy and physiotherapy. Hofmann J, Münstedt K. Use of complementary and As primary and supplementary gynecological tre- in departments of obstetrics in atments) [In German] Gynäkologe 2000; 33:18-27. Croatia and a comparison to Germany. Forsch Kom- 11. Kauffels W, Mesrogli M. Praxis der gynäkologis- plementmed 2010; 17:144-6. chen Balneotherapie (Practice of gynecological 3. Sillo – Seidl G. Intravaginal mud therapy. Z Ge- balneotherapy). In: Hepp H, Berg D, Hasbargen U burtshilfe Gynakol 1962; 158:213-8. (Eds) Gynäkologie und Geburtshilfe [In German] 4. Antonelli M. Donelli D. Effects of balneotherapy Berlin, Heidelberg: Springer, 1994. and spa therapy on levels of cortisol as a stress bio- 12. Bender T, Bariska J, Vághy R, Gomez R, Kovacs I. marker: a systematic review. Int J Biometeorol 2018; Effect of balneotherapy on the antioxidant system-a 62:913-24. controlled pilot study. Arch Med Res 2007; 38:86-9. 5. Habek D. History of phytotherapy in gynecology- 13. Marazziti D, Baroni S, Giannaccini G, DellʼOsso short annotation. Acta Med Croat 2020; 74:65-7. MC, Consoli G, Picchetti M, Carlini M, Massimetti 6. Habek D, Akšamija A. Successful acupuncture tre- G, Provenzano S, Galassi A. Thermal balneotherapy atment of uterine myoma. Acta Clin Croat 2014; induces changes of the platelet serotonin transpor- 53:487-9. ter in healthy subjects. Prog Neuropsychopharmacol 7. Galvez I, Torres-Piles S, Ortega-Rincon E. Balne- Biol 2007; 31:1436-39. otherapy, immune system, and stress response: a hor- 14. Matzer F, Nagele E, Lerch N, Vajda C, Fazekas C. metic strategy? Int J Molec Sci 2018; 19:pii:E1687. Combining walking and relaxation for stress reduc- 8. Ablin JN, Häuser W, Buskila D. Spa treatment (bal- tion - a randomized cross-over trial in healthy adults. neotherapy) for fibromyalgia—a qualitative-narrati- Stress 2018; 34:266-277. ve review and a historical perspective. Evid Based 15. Dikova K, Burgudzhieva T, Slaveĭkova O. Physical Complement Alternat Med 2013:1–5. treatment of obstetrical and gynecological diseases. 9. Falagas ME, Zarkadoulia E, Rafailidis PI. The the- Akush Ginekol 1979; 18:134-9. rapeutic effect of balneotherapy: evaluation of the 16. Habek D, Habek JČ, Barbir A. Using acu- evidence from randomised controlled trials. Intern J puncture to treat premenstrual syndrome. Clin Pract 2009; 63:1068–84. Arch Gynecol Obstet 2002; 267:23-6. 17. Baatz H. Balneo-gynecology. Fortschr Med 1979; 97:1873-74.

5 Medicinski Glasnik, Volume 18, Number 1, February 2021

18. Stark MA, Rudell B, Haus G. Observing position 29. Burgudshieva T, Slaveĭkova O. Comparative he- and movements in hydrotherapy: A Pilot Study. J modynamic changes in the organs of the lesser pel- Obstet Gynecol Neonatal Nurs 2008; 37:116-22. vis of women with inflammatory gynecologic disea- 19. Fiaschetti D, Grignaffini A, Cavatorta E, Rastelli ses and sterility following treatment with Baikal peat AV, Gramellini D, Chiavazza F. Thermal therapy in and sulfide mineral waters. Akush Ginekol (Sofiia) gynecology. Acta Biomed Ateneo Parmense 1982; 1980; 19:518-21. 53:399-403. 30. Beer AM, Kovarik R, Münstermann M. Vaginale 20. Fortuna A, Passerini C, Polanco M, Volenski L. Cri- Moortherapie bei chronischer Salpingitis (Vaginal teria for the effect of thalassotherapy in gynecology. peat therapy for chronic salpingitis) [In German] Minerva Ginecol 1980; 32:817-24. Phys Rehab Kur Med 1994; 4:110-2. 21. Ovsienko AB. Effect of radon baths of various con- 31. Artymuk NV, Kira EF, Kondratieva TA. Intravagi- centrations on patients with genital endometriosis. nal gel prepared from Dead Sea peloid for treating Vopr Kurortol Fizioter Lech Fiz Kult 2003; 6:18-21. luteal-phase defect. Int J Gynaecol Obstet 2010; 22. Dionigi R. Salso-iodic waters in venous changes 108:72-3. in gynecology. Quad Clin Ostet Ginecol 1966; 32. Horejsí J, Kotásek A, Jirásek K, Vĕrná M. Gyneco- 21:1279-85. logic spa therapy for children. Present status, indica- 23. Capoduro R. Does balneology still have gynecologic tions and guidelines for recommending spa therapy indications? Rev Fr Gynecol Obstet 1995; 90:236-9. for children at the Pediatric Gynecologic Spa in 24. Kuca K, Grünner L. New guidelines for indications Frantiskovy Láznĕ. Cesk Gynekol 1981; 46:787-91. for balneotherapy-gynecologic diseases. Cesk Gyne- 33. Zambo L, Dekany M, Bender T. The efficacy of kol 1986; 51:373-75. alum-containing ferrous thermal water in the ma- 25. Vuković Bobić M, Habek D. Complementary met- nagement of chronic inflammatory gynaecological hods of delivery. Liječ Vjesn 2006; 128:25-30. disorders – a randomized controlled study. Eur J Ob- 26. Moses SW, David M, Goldhammer E, Tal A, Suke- stet Gynecol Reprod Biol 2008; 140:252–7. nik S. The Dead Sea, a unique natural health resort. 34. Gerber B, Wilken H, Barten G, Zacharias K. Positive Isr Med Assoc J 2006; 8:483-8. effect of balneotherapy on post-PID symptoms. Int J 27. Min KJ, Choi H, Tae BS, Lee MG, Lee SJ, Hong Fertil Menopausal Stud 1993; 38:296-300. KD. Short-term benefits of balneotherapy for pati- 35. Baskakov VP, Lugovaia LP, Gur'ev AV, Sokolova ents with chronic pelvic pain: a pilot study in Korea. SA. Treatment of endometriosis with radon baths J Obstet Gynaecol 2019; 28:1-6. and sex hormones. Akush Ginekol 1982; 10:44-6. 28. Akin MD, Weingand KW, Hengehold DA, Goodale 36. Habek D, Habek JC, Bobić-Vuković M, Vujić B. MB, Hinkle RT, Smith RP. Continuous low-level to- Efficacy of acupuncture for the treatment of primary pical heat in the treatment of dysmenorrhea. Obstet dysmenorrhea. Gynakol Geburtshilfliche Rundsch Gynecol 2001; 97:343-9. 2003; 43:250-3.

6 ORIGINAL ARTICLE

The importance of plasma ferritin values in blood donors for the evaluation of body iron store in a five-month period

Elma Ćatović-Baralija1, Gorana Ahmetović-Karić1, Sabaheta Hasić 2, Nesina Avdagić3, Nermina Babić3

1Department for Blood-Borne Disease Testing, Blood Transfusion Institute of the Federation of Bosnia and Herzegovina, 2Department of Medi- cal , School of Medicine, University of Sarajevo, 3Department of Human Physiology, School of Medicine, University of Sarajevo

ABSTRACT

Aim To present haemoglobin and ferritin parameters in donors to highlight the importance of serum ferritin testing for the purpose of evaluating iron depots in order to make recommendations for preserving a population of blood donors.

Method A prospective study was conducted on 80 blood donors divided in two groups: group I (regular donors, n =40) and group II (irregular donors, n=40). Haemoglobin and ferritin were measured twice every 45 days, before two consecutive blood donations.

Results By measuring haemoglobin and ferritin values before do- Corresponding author: nation in both groups, a decrease of initial ferritin value in Group I Elma Ćatović-Baralija relative to Group II was observed (without statistical significance). A significant decrease was found between repeated measurements Department for Blood-Borne Disease for both parameters in both groups, indicating equal intensity of Testing, Blood Transfusion Institute of the the decline in value regardless of a donor status. Measurement of Federation of Bosnia and Herzegovina ferritin before and after donation revealed statistically significant Čekaluša 86, 71000 Sarajevo, loss of ferritin in all examinees (p=0.011). The decline in haemo- Bosnia and Herzegovina globin after donation, although significant, did not fall below the Phone: +387 33 567 322; reference value for donation in either women or men. Fax +387 33 567 333; Conclusion Results indicate the need for periodic monitoring of E-mail: [email protected] the plasma value of ferritin in voluntary donors who donate blood ORCID ID: https://orcid.org/0000-0002- more than twice a year and the possible oral supplementation with 4938-9382 iron. Key word: anaemia, blood, donors, haemoglobin

Original submission: 18 June 2020; Revised submission: 11 September 2020; Accepted: 22 September 2020 doi: 10.17392/1223-21

Med Glas (Zenica) 2021; 18(1):7-11

7 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION voluntary donors (4,5,7). We want to show that in our population a haemoglobin estimate may not be The development of modern medicine, diagnostic sufficient to assess donor safety prior to phlebo- and therapeutic procedures have imposed increased tomy, so we can point to the need to review donor demands of blood and blood products. The donor acceptance guidelines. selection process aims to determine general health of a donor in order to be convenient to donate a sin- The aim of this study was to examine haemoglo- gle dose of 450 ml of blood not affecting donor he- bin and ferritin concentration of voluntary blood alth as well as providing a safe dose of blood for a donors before donating blood in two consecuti- patient. In addition to the general requirements for ve measurements (45 days apart) to examine the donation that the donor must meet according to the need for the introduction of additional ferritin World Health Organisation (WHO) recommenda- concentration screening. tions (1), screening of voluntary donors routinely EXAMINEES AND METHODS determines the concentration of haemoglobin. The values of haemoglobin required for blood donati- Examinees and study design on for females are >125 g/L and 135 g/L for males with a minimum period of 4 and 3 months pause, A prospective study was conducted on 80 blood respectively, since the last donation (1). A large donors, aged 22-55 years, both genders, at the Blo- number of studies have shown that frequent blood od Transfusion Institute of the Federation of Bo- donations lead to depletion of iron reserves (2). De- snia and Herzegovina in the period from 15 April creased iron reserves in the blood are a well-known to 30 August 2019. Examinees were classified as risk factor in multiple blood donations. Each 1 mL regular donors if they donated blood at least three of blood contains approximately 0.5 mg of iron, do- times in the last year for males and twice for fema- nating a single dose of blood, an average of 242 mg les, and irregular donors did not donate blood at all of iron is lost (3-5). An increase in the frequency of or did not do a donation in the last 3 years. blood donation among the donor population may According to the criteria, two groups were for- result in excessive iron loss and the development of med: group I – 40 regular donors, both genders, iron deficiency with or without symptoms, as well aged 22-55 years, and group II – 40 non-regular as anaemia. Low serum ferritin values are accurate donors. Group I and group II were designated as indicators of the assessment of iron depot status in Ia and Ib / IIa and IIb, depending on whether it an individual (6). Testing of ferritin concentration was the first or repeated measurement. in voluntary donors is not a routine test and it is Inclusion criteria were: voluntary blood donors recommended only when the haemoglobin level between 22-55 years of age, haemoglobin value does not meet the donation limit, according to the of >125 g/L before donation for females and 135 WHO recommendations (1). g/L for males, and a minimum period of 4 and is on the margins of the he- 3 months from the last donation depending on alth care system. Blood as a medicine obtained examinees’ gender. Exclusion criteria were all from voluntary donors is taken for granted, and no criteria for exclusion of voluntary blood donors account is taken of how much effort is required according to WHO (1). to produce it. Voluntary donors are people with a Voluntary donors were informed of the aim of the strong sense of altruism. In today’s society, this study and signed an informative consent form. is not recognized, there are numerous obstacles The study was approved by the Ethics Committee that make it difficult to reach donors and can ne- of the School of Medicine, University of Saraje- gatively affect their attitude for blood donation. vo, as well as the Blood Transfusion Institute of By taking care of the health of voluntary donors, the Federation of Bosnia and Herzegovina. we can influence their positive attitude with the aim of preserving them. Although several studies Methods have shown that repeated blood donation causes Haemoglobin and ferritin were determined in depletion of iron depots and iron deficiency, all regular and irregular blood donors twice at 45- transfusion centres still do only haemoglobin as an day intervals, every time before blood donations. indirect indicator of iron status in the screening of Determination of haemoglobin was performed

8 Ćatović-Baralija et al. Ferritin values at volunteer blood donors

using capillary blood collected by puncturing lue of haemoglobin after donation in the Group the fingertip of one hand (HemoQue Hb 301, Ib was 149.35 g/L and in the Group IIb 149.03 Ängelholm, Sweden). Blood sample (3mL) for g/L (p=0.916). The analysis of the mean value ferritin measurement (Architect i2000sr, Illinois, of two repeated measurements of haemoglobin USA) was collected in a tube with ethylenediami- of the Group I showed a statistically significant netetraacetic acid (EDTA) from the donation bag. difference (p=0.0004), as it was in the Group II Haemoglobin and ferritin values were expressed (p=0.0001) (Table 2). in g/L and ng/mL. A decrease in iron stores was Table 2. Haemoglobin and ferritin values of regular and defined as plasma ferritin values below 20ng/ non-regular donor groups according to the first and second mL; value of 15 ng/mL was the cut off for iron measurement deficiency that leads to the decreased erythropoi- Group Haemoglobin (mean±SD) Ferritin (percentile) Ia 155.55 ±12.19*‡ 32.83 (18.03-49.22)*‡ esis (7). An iron deficiency anaemia was defined Ib 149.35 ± 13.47† 23.36 (11.15-42.80)† as serum haemoglobin and ferritin below 13 g/dL IIa 155.07 ± 12.04‡ 43.1 (20.64-79.41)‡ and 15 mg/ mL, respectively (2). IIb 149.03 ± 13.76 26.63 (12.50-53.66) The Body Mass Index (BMI) was calculated *non-significant difference Ia vs IIa; †non- significant difference IIa vs IIb; ‡significant difference in two repeated measurements; Ia, the according to the formula for the ratio of body first measurement in regular donors; Ib, the second measurement in weight to the square of a person’s height (9,10). regular donors; IIa, the first measurement in non-regular donors; IIb, the second measurement in non-regular donors; Statistical analysis Measurement of ferritin values between the Gro- ups Ia and IIa before the donation did not reveal a The normality of data distribution was tested with statistically significant difference (p=0.21). After the Shapiro-Wilk test. Numerical variables were the donation, there was a decrease in the value presented as the mean or median with 25th to 75th of ferritin, but there was no statistically signifi- percentile depending on the normality of data dis- cant difference between the Groups Ib and IIb tribution. If assumption of data normality was met, (p=0.31). By measuring the value of ferritin in the difference in the mean values of two groups the Group Ia compared to the Group IIa, a lower was tested using the independent t-test, while the mean value of ferritin was observed in the Group difference in repeated-measures was tested by the Ia (p=0.106). By measuring the value of ferritin dependent t-test. Otherwise, Mann Whitney and in the Group I before (41.54 ng/mL) and after the Wilcoxon rank test were used, respectively. (29.28 ng/mL) donation, a statistically significant The frequency of the categorical variables was difference was found (p=0.0001), and the same presented by the absolute number and percentage. result was shown by measuring the mean value The p<0.05 was taken as statistically significant. before (56.67 ng/ml) and after donation (38.35 RESULTS ng/mL) in the Group II (p=0.0001). After dona- tion in both study groups there was a significant The study involved 26 female and 54 male exami- decrease in haemoglobin and ferritin (Table 2). nees, with an equal ratio in both groups. The age All examinees had haemoglobin reference values of the examinees ranged from 22 to 55 years. In before donation (Table 2), while ferritin values in the Group I the average age was 35.33±9.6 years, 25% of examinees from the Group I and 22.5% and in the Group II 33.20±6.22 years (p=0.2). The from the Group II had ferritin <20 ng/mL before the difference in the BMI between groups I and II was donation, and as much as 42.5% and 37.5%, res- not statistically significant (p=0.46) (Table 1). pectively, in the repeated measurement (Table 3). In Table 1. Average age, gender and body mass index (BMI) both groups of examinees after donation there was distribution of regular and non-regular blood donor groups a decrease in haemoglobin (Table 2), but it was still Parameter Group I (n=40) Group II (n=40) Average age (years) 35.33±9.6* 33.20± 6.22 in the reference values appropriate for donation. Gender/male (n, %) 27 (67.5) 27 (67.5) Table 3. Frequency of examinees with plasma ferritin values Gender/female (n, %) 13 (32.5) 13 (32.5) indicating reduced iron depots BMI (kg/m2) 26.68±2.758* 27.85 ± 6.255 No (%) of donors in the group *non-significant; Group I, regular donors; Group II, non-regular Donor’s category Ia Ib IIa IIb donors; Total number 40 (100) 40 (100) The mean value of haemoglobin before donation Ferritin <20 ng/mL 10 (25) 17 (42.5) 9 (22.5) 15 (37.5) Ferritin <15 ng/mL 8 (20) 13 (32.5) 5 (12.5) 10 (25) in the Group Ia was 155.5 g/L, and in the Gro- Ia, first measurement in regular donors; Ib, second measurement in up IIa 155.07 g/L (p=0.861), and the mean va- regular donors; IIa, first measurement in non-regular donors; II b, second measurement in non-regular donors 9 Medicinski Glasnik, Volume 18, Number 1, February 2021

DISCUSSION of the FBIH on 330 voluntary donors showed that the increase in the number of donations reduced In this study, we investigated the changes of hae- the concentration of serum ferritin in the blood of moglobin and ferritin concentrations in blood voluntary donors, although haemoglobin levels donors especially to draw attention to the im- remained acceptable for donations, and negative portance of testing serum ferritin for assessing correlation was observed between the number of iron depots in order to create recommendations donations and the value of serum ferritin (11). to protect the blood donors. Abdullah SM (4) fo- In both groups analysed in our study a decrease und a statistically significant difference in plasma in haemoglobin values after donation was noti- ferritin values between first-time donors and re- ced, but still in the reference values for donation. gular voluntary donors. The results of our study Other studies (4,5, 12-14) showed that iron defi- have shown reduced initial values of ferritin in ciency was present in males who had four or more the Group I compared to the Group II (but wit- blood donations during 1 year. In addition, Tailor hout statistical significance), although we expec- et al. (15) found a significant correlation between ted statistical significance, since multiple donors the frequency of donations, last donation interval are more exposed to ferritin loss. and the serum ferritin measurement. This suggest Baseline haemoglobin and ferritin parameters that ferritin should be included in the assessment were not significantly different depending on a of regular blood donors to secure adequate iron donor status in our study. A significant decrea- reserves in the donor population and a need to se was found between repeated measurements modify the donor acceptance criteria. Reddy et for both parameters in both groups indicating an al. (5) also showed that the increase in the frequ- equal intensity of the value decline regardless of ency of blood donation was accompanied by a the donor status. In contrast, other authors did not significant decrease in serum ferritin, iron-defici- perform a control measurement between two blo- ent erythropoiesis in 11.2% of the regular donors, od donations, but monitored the values of ferri- and accordingly they are at risk of developing tin before the donation in the control group and iron deficiency anaemia. Saracul et al. (16) have regular blood donors who donated with different found that the incidence of iron deficiency was intensities; they found (2,4,5) that ferritin values higher in male donors with three or more dona- in subjects who had not donated so far were in tions per year. The results of many studies were the reference value and/or higher than in regular similar to our findings (11-15) and showed the voluntary blood donors. In our study it was ob- importance of measuring iron stores as an indica- served that reduced iron stores were presented in tor of selection for blood donation. Voluntary do- almost the same number of examinees who dona- nors with low haemoglobin and iron, due to the ted blood for the first time in relation to regular delays in donating blood for a long period, often voluntary donors. express their dissatisfaction and find it difficult to By analysing the mean value of the Group I hae- return as voluntary donors. Transfusion facilities moglobin before and after the donation we found should consider counselling and iron supplemen- a statistically significant difference, as well as tation for males as well, and not just for females, when measuring values before and after donati- in order to keep voluntary donors at the base (17). on in the Group II; also by measuring the value of According to the WHO recommendation (18), ferritin in the Group I before and after donation. serum ferritin concentrate less than <15 ng/mL The decrease in haemoglobin after donation, alt- indicates depleted iron stores. Haematological hough significant, did not fall below the referen- recovery after donating a single unit of whole ce value for donation in either females or males; blood or erythrocyte depends on the total iron with such haemoglobin values voluntary donors supply in the body (5,17). Several studies have would be eligible for at the next donation, regard- revealed rapid recovery of haemoglobin in vo- less of the expressed low ferritin value. However, luntary blood donors with anaemia due to iron regular donation would lead to depletion of ferri- deficiency supplemented with a daily dose of 200 tin stores and consequent anaemia, which would mg of iron (17,19). cause the voluntary donor to be blocked for an The main limitation of the study is the small stu- extended period of time (11). A study conducted dy group. Despite the small number of respon- in 2018 at the Institute for Transfusion Medicine dents, the results of the study indicate a mismatch

10 Ćatović-Baralija et al. Ferritin values at volunteer blood donors

between the haemoglobin and ferritin levels of ded from donation for a longer period due to voluntary blood donors. We believe that this rese- developed anaemia. In addition, ferritin analysis arch will open the question of the need for addi- would identify at-risk individuals who would be tional testing of the concentration of ferritin in candidates for iron supplementation and thus pre- the blood as a useful laboratory parameter for the vent the development of sideropenic anaemia. assessment of the iron depot of blood donors in Bosnia and Herzegovina. In this way, the possi- FUNDING bility would be avoided that donors with satisfac- No specific funding was received for this study. tory haemoglobin levels and ferritin values risky for the development of anaemia would donate TRANSPARENCY DECLARATION blood, which would result in them being exclu- Conflict of interest: None to declare.

REFERENCES 1. World Health Organization. Screening Donated 11. Ahmetović Karić G, Ćatović Baralija E. Influence of Blood for Transfusion Transmissible Infections. Re- whole blood donation on serum ferritin values in vo- commendations. Geneva: WHO, 2010. https://www. luntary blood donors. In: Book of Abstracts of the 6th who.int/bloodsafety/ScreeningDonatedBloodfor- Transfusion Medicine Congress of Serbia, November Transfusion.pdf (20 May 2020) 07-10 2018, p20. 2. Sarakul O, Sommart P, Tolahan S, Wasana P. Iron 12. Akram A, Mahtab M, Hosein TN, Shirin F. Iron stores supplements and iron status in frequent blood donors. in blood donors: A literature mini review. J Bas Res Hematol Transfus Int J 2017; 5:257-60. Med Sci 2017; 4:50-5. 3. Patel UE, White LJ, Bloch ME, Grabowski KM, Ge- 13. Olowoselu OF, Uche E, Oyedeji O, Otokiti OE, hrie AE, Lokhandwala MP, Brunker ARP, Goel, Shaz Ayanshina OA, Akinbami A, Osunkalu V. A compara- HB, Ness MP, Tobian ARA. Association of blood tive study of serum ferritin levels among unfit and fit donation with iron deficiency among adolescent and blood donors. Niger Med J 2019; 60:312-6. adult females in the United States: A nationally repre- 14. Vijatha T, Arun T. M., Santhi S. A study to assess the sentative study. Transfusion 2019; 59:1723–33. iron stores of regular blood donors. Int J Clinic Bio- 4. Abdullah SM. The effect of repeated blood donation chem Res 2016; 3:466-8. on the iron status of male Saudi blood donors. Blood 15. Tailor HJ, Patel PR, Pandya AN, Mangukiya S. Study Transfus 2011; 9:167-71. of various hematological parameters and iron status 5. Reddy KV, Shastry S, Raturi M, Baliga B P. Impact among voluntary blood donors. Int J Med Public He- of regular whole-blood donation on bodyiron stores. alth 2017; 7:61-5. Transfus Med Hemother 2020; 47:75–9. 16. Sarakul O, Sommart P,Tolahan S, Poophapun W. Iron 6. Devi DG, Arumugam P, Hamsavardhini S, Radhiga supplements and iron status in frequent blood donors. S T. A study of serum ferritin levels among voluntary Hematol Transfus Int J 2017; 5:257-60. blood donors. Int J Res Med Sci 2017; 5:5322-9. 17. Mast EA, Szabo A, Stone M, Cable GR , Spencer 7. Armstrong KL. Blood donation and anemia. Can Fam RB, Kiss EJ. The benefits of iron supplementation 2016; 62(9):730–1. following blood donation vary with baseline iron sta- 8. Manuel M, Susana GR, Martin B, José P, Fernan- tus. Am J Hematol 2020; 95:784-91. do G, Giancarlo M. L, Sunil B, Mercé C, Aryeh S, 18. World Health Organization. Serum Ferritin Concen- Michael A. Current misconceptions in diagnosis and trations for the Assessment of Iron Status and Iron management of iron deficiency. Blood Transfus 2017; Deficiency in Populations. Vitamin and Mineral Nu- 15:422–37. trition Information System. Geneva, World Health 9. Park B, Cho H N, Choi E, Seo DH, Kim N S, Park Organization, 2011 (WHO/NMH/NHD/MNM/11.2). E, Kim S, Park YR, Choi K S. Weight control beha- http://www.who.int/vmnis/indicators/serum_ferritin. viors according to body weight status and accuracy pdf (20 June 2020) of weight perceptions among Korean women: a na- 19. Kiss JE, Vassallo RR. How do we manage iron de- tionwide population-based survey. Sci Rep 2019; ficiency after blood donation? Br J Haematol 2018; 9:9127. 5:590-603. 10. Silveira EA, Pagotto V, Barbosa LS, Oliveira C, Pena GDG, Velasquez-Melendez G . Accuracy of BMI and waist circumference cut-off points to predict obesity in older adults. Cien Saude Colet 2020; 25:1073-82.

11 ORIGINAL ARTICLE

Clinical use of an analysis of oxidative stress and IL-6 as the promoters of diabetic polyneuropathy

Emina Karahmet1, Besim Prnjavorac2,3, Tamer Bego4, Adaleta Softić5, Lejla Begić6, Edin Begić7,8, Esma Karahmet4, Lejla Prnjavorac2, Irfan Prnjavorac2

1 Faculty of , University of Tuzla, Tuzla, 2General Hospital, Tešanj, 3Department of Pathophysiology, Faculty of Pharmacy, Sarajevo, 4Department of Clinical Biochemistry, Faculty of Pharmacy, Sarajevo, 5Department of Biochemistry, University of Tuzla, Tuzla, 6Department of Biochemistry, Faculty of Pharmacy, University of Tuzla, Tuzla, 7Department of , General Hospital "Prim.dr. Abdulah Nakaš", Sarajevo, 8Department of , School of Medicine, Sarajevo School of and Technology, Sarajevo; Bosnia and Herzegovina

ABSTRACT

Aim To investigate interleukin 6 (IL-6) values depending on du- ration of diabetes mellitus (DM) and evaluate possible correlation with diabetic polyneuropathy.

Methods The research study included 90 patients with DM divi- ded into three groups (30 patients each) according to the duration of DM: group A – patients who had DM for less than 10 years, group B - duration of DM was 10 to 20 years, and group C - pa- Corresponding author: tients with DM over 20 years. Control group (K) included 30 he- Besim Prnjavorac althy participants. Department of Pathophysiology, Results IL-6 was significantly higher in the healthy control gro- Faculty of Pharmacy up, 180.318 pg/mL±94.18, than in group A, 47.23pg/ml±34.8, Zmaja od Bosne 8, 71000 Sarajevo, group B, 43.31pg/ml±33.17, and group C, 70.39 pg/ml±59.26 Bosnia and Herzegovina (p=0.0001). All groups had significantly different values of IL-6 between each other (p=0.0001). Level of IL-6 was in correlation Phone: +387 33 586 182; with diabetic polyneuropathy in the group A (the youngest partici- Fax: +387 33 666 189; pants) (p=0.0001). In other groups there was no significant corre- E-mail: [email protected]; lation between IL-6 and diabetic polyneuropathy. Emina Karahmet ORCID ID https://orcid. Conclusion The level of IL-6 was in correlation with neuropathy org/0000-0002-0913-5759 among younger patients. A higher level of IL-6 in the control group than in diabetic groups is a sign of stronger inflammatory response among younger and healthy people than in patients with DM.

Original submission: Key words: diabetes, interleukins, inflammation, neuropathy 23 September 2020; Revised submission: 08 December 2020; Accepted: 23 December 2020 doi: 10.17392/1279-21

Med Glas (Zenica) 2021; 18(1):12-17

12 Karahmet et al. IL-6 and diabetic neuropathy

INTRODUCTION the spine and pancreas (2). Tunica media of many blood vessels produces IL-6 too, and it plays pro- Diabetic peripheral neuropathy is a common inflammatory role. Its anti-inflammatory role is complication of diabetes mellitus (DM), which manifested by inhibition of TNF-α and IL-1, and mainly appears in long duration of the disease. by activation of IL-1ra and IL-10 (5). IL-6 is an It is present in 30-50% of patients with DM, but important inflammatory promoter and reactant of statistical results show that it occurs more frequ- the acute phase of the inflammatory reaction (9). ently in DM type 1 (54-59%) than in DM type 2 A very important fact is that in stress, human body (45%) (1). In 13-26 % of patients it occurs in the starts an intense production of the inflammatory form of painful distal neuropathy (1). cytokines, especially IL-6 (10). Considering all The mechanism of pathophysiology is very com- these facts, it is obvious that inflammatory cytoki- plex. There are several accepted explanations: dis- nes can be a good screening method for considera- order of polyol and myoinositol metabolism, for- tion of diabetic neuropathy (11). mation of highly reactive oxygen species (ROS), The aim of this study was to investigate values of reduction of Na/K ATP-enzyme, ischemic endo-ne- IL-6 and check any dependence of IL-6 on DM du- ural microvascular disorder, neurotrophic disorder, ration and its use as a diagnostic marker for diabe- disorder of axonal transmission and non-enzyma- tic polyneuropathy, as well to test active inflamma- tic glycosylation of proteins (2). Commonly used tory processes related to diabetic polyneuropathy. explanation is oxidative stress. (3) High ROS ma- kes changes to the axonal and dendritic surface and PATIENTS AND METHODS changes its main rule (4). Other effects of oxidative stress are the decrease of glutathione and ascorbate Patients and study design levels, increase of lipid peroxidation and protein This retrospective-prospective study included 90 nitrosylation. In patients treated with antioxidant DM patients (type 1 and type 2) age 18-80, who therapy a very good therapeutic effect was shown were divided into three groups (30 patients in each (5). It is important to consider that spinal microglia group) and a control group (K, 30 healthy parti- is highly activated with these neuronal disorders, cipants). All study subjects were processed at the and it starts to produce CD11b and Iba1 signals, Department of , General Hospital and p38 mitogen protein kinase phosphorylation Tešanj, over the period of 18 months (during 2018 is induced. It results with high production of in- and 2019). flammatory cytokines IL-6, interelukin-1 (IL-1), tumour necrosis factor-α (TNF-α) and transfor- The groups were formed according to the dura- ming growth factor-β (TGF-β) (6). These cytokines tion of DM: group A included patients with the can influence the spinal synaptic transmission by duration of DM less than 10 years, group B with increasing excitability of dorsal horn neurons and the duration of DM 10-20 years and group C with partially decreasing inhibitory synaptic transmissi- more than 20 years of DM duration. on (2). At the same time, glucotoxicity and lipo- An inclusion criteria was the diagnosis of DM, toxicity in pancreas induce releasing of free fatty both type 1 and type 2, by definition given by acids (especially palmitic acid, where the process the International Diabetes Federation (IDF) (12) of lipid peroxidation starts), that results with new and the American Diabetes Association (ADA) oxidative stress and damaging of insulin producing (13), according to which a patient is considered β cells of pancreas (2). Inflammatory cytokines diabetic if he/she has a glycaemia above 11.2 have the main role in this process too because of mmol/L at any time (14). Exclusion criteria were: their inflammatory promoting activity. The same participants who withdrew their consent given in cytokines are included in this process, IL-1β, IL- writing, women who became pregnant during the 6, TNF-α, TGF-β but chemokine, IL-8 and NF-κβ examination, participants who experienced any (nuclear factor) (2). IL-6 is double ruled cytokine, changes during the examination period that they pro-inflammatory and anti-inflammatory myokine could not reasonably explain. (8). It is produced by macrophages and T-cells, as Control subjects, without DM diabetes, were re- an immune answer to stimulation (tissue damaging cruited from the systematic preventive examina- for example) (8), previously explained process in tion of healthy employees of the schools.

13 Medicinski Glasnik, Volume 18, Number 1, February 2021

Each participant signed an informed consent to ted descriptively using appropriate measures of participate in the study. Ethical approval was central tendency (arithmetic mean and median). obtained from the Ethical Committee of the Ge- Quantitative variables were compared using neral Hospital Tešanj, Bosnia and Herzegovina. Student’s t-test with correction for unequal va- riance where needed. The relationships between Methods the variables were tested using the parametric Blood samples were taken during a patients’ hos- Pearson correlation. The variance test (ANOVA) pitalization or a regular follow up. was used to analyze the results of IL-6 measure- ments between the groups. All tests were perfor- All measurements were performed in the Biochemi- med with an accuracy level of 95% (p<0.05). cal Laboratory of the General Hospital, Tešanj, with o fresh or frozen serum samples (-60 C). The whole RESULTS blood sample was centrifuged and the serum was prepared and used for IL-6 determination by ELISA The patients and individuals in the control group test (Quantikinine, Becton Dickinson, USA). (n=120) were between 38 and 82 years old, with mean age of 61.61±20.2 years. For all patients an analysis of IL-6 level (referen- ce value 3- 477.30 pg/mL), glycosylated haemo- IL-6 showed significantly lower levels in the gro- globin (HbA1c), C- reactive protein (CRP), uric ups with DM than in the control group. In the gro- acid, erythrocytes sedimentation rate (ESR) and up A, IL-6 mean level was 47.23±6.15 pg/mL, in leucocytes (Le) in the blood was performed at the the group B, mean IL-6 level was 43.31±5.69 pg/ time of admission. mL, in the group C, 70.39±10.64 pg/mL, while in the control group it was 180.32±94.20.08 pg/ Monofilament test for peripheral neuropathy (15) mL (Table 1). was performed by using the single - fiber nylon thread (a filament of nylon). It was placed on the The control group had a significantly diffe- patient’s skin (preferable on the feet). The patient rent level of IL-6 than the groups A, B, and C was asked whether he/she felt the touch of the (p=0.0001). filament or not. A statistically significant difference in IL-6 va- Sonic fork test was used for the examination of lues ​​between all analyzed groups (p=0.0001), as deep sensitivity (Michigan Neuropathy Scree- well as between group A and group K (p=0.0001) ning Instrument, MNSI) (16). The fork was appli- was found. Group B in relation to group K also ed to the patient’s bone (preferable on distal part showed a statistically significant difference of radius) and activated. The patient was asked (p=0.0001), as well as group B in relation to gro- to signal when she no longer felt the vibration. up C (p=0.041). Group C compared to group K It was monitored how long the examinee felt the also showed a statistically significant difference vibration. The test was described as normal, de- in IL-6 values (p=0.0001). creased or absent sensation of vibration (17). The relationship between polyneuropathy and IL-6 with statistical significance was only found Statistical analysis in the group A (p=0.038). Other groups did not All variables were tested for normal distribution show statistically significant ifference in results. using the Kolmogorov-Smirnov test and presen- In group A, an impairment measured by a decre- ase in deep sensitivity showed statistical signifi- Table 1. Followed parameters in four groups Groups* Variable Reference value A B C K Age (years) 59.44±9.38 67.81±7.32 70.04±6.96 47.91±10 DM duration (years) 3.59±2.56 13.91±2.36 24.17±4.52 - HbA1C (%) 4.5-6.0 7.62±1.27 8.7±1.66 9.2±2.17 - CRP (mg/dL) 0-10 18.51±39.03 18.5±37.52 31.22±42.56 - Uric acid (µmol/L) 360-400 353.24±146.36 313.67±88.94 419.19±114.24 150.3 ESR (mm) <20 34.95±24.24 32.75±27.28 44.70±34.20 - Le (106/mm3) 4.0-10.0 8.18±1.86 7.98±3.11 8.93±3.15 - IL-6 (pg/mL) 3- 477.30 47.23±34.8 43.31±33.17 70.39±59.26 180.32 *according to diabetes mellitus duration: A, <10 years; B, 10-20 years; C, > 20 years; K, control group (healthy participants) DM, diabetes mellitus; HbA1c, glycosylated haemoglobin; CRP, C- reactive protein; ESR, erythrocytes sedimentation rate; Le, leucocytes;

14 Karahmet et al. IL-6 and diabetic neuropathy

cant difference with a decrease in peripheral sen- ve evaluation of symptoms included) (15). In our sation (p=0.0001). Total impairment, expressed study no significant correlation between IL-6 level by MNSI score, showed a significant correlation and diabetic polyneuropathy was shown, but chan- with IL-6 (p=0.038). ges of cytokine levels can be seen in a long term follow-up (31). DISCUSSION It is known that reparation of tissues in patients Risk factors for diabetic neuropathy were an im- with long lasting DM is impaired (32); it is ne- portant issue of many studies during the last few cessary to conduct studies with at least 600-800 years, concluding that DM control, duration, participants in order to establish the cause of it. type, therapeutic modality used in the treatment Also, for some conclusion about details of these and comorbidities all affect the development of processes it is necessary to analyze the patients diabetic neuropathy (18-20). Additionally, hyper- with DM type 1 and DM type 2 separately, becau- glycaemia is one of the most important causes of se the therapy can influence the behaviour of in- oxidative stress (21-24). Many studies published flammatory cytokines. This fact could be the main in recent years have shown that IL-6, with other limitation of this and similar studies. We did not inflammatory cytokines, is a part of this inflamma- separate patients with and without statins therapy. tory process. In this study it was shown that IL-6 As the inflammation is one of the main immu- level is much higher in the control group than in nological barriers, it should be considered that the groups with diabetes. It can be considered that an inappropriate immune response can be seen the inflammatory response is more powerful in in serious metabolic disorders, like in diabetes healthy subjects than in patients with DM (25). (33). Infection of the foot is very common in Magrinelli et al. showed that IL-6 and IL-10 were patients with DM, with very strong influence of related with large nerve fibres damaging, but not cytokine synthesis and release (34). The Further with small nerve fibers and polyneuropathy (26). research should consider comorbidities and the- Korkmaz et al. showed that IL-6 and fibrinogen ir therapy for possible influence to immune res- had higher values in infected than in non-infected ponse. It is known that statins, very often used diabetic foot ulcer (27), suggesting that IL-6 and as hypolipemic therapy in patients with DM, can fibrinogen are certain markers of infection (con- influence inflammation as the anti-inflammatory sequently inflammation too) (19,28). Cox et al. substances (35). suggested the idea of treatment of peripheral ne- In conclusion, there are so many factors that influ- uropathy with low-dose pulsatile IL-6, assuming ence damaged nerve tissues with a lot of overlap- that IL-6 will show a potentially anti-inflamma- ping with oxidative stress in diabetes. Interleukins, tory effect (29). However, the anti-inflammatory like IL-6 could be used as a parameter of tissue effect of IL-6 has not been confirmed up to date. In damage and inflammatory response in patients fact, the idea was to imitate natural response of the with DM. It is very important to point out that the human body to physical activity, by excreting IL-6 damage of nerve tissue was in a statistically signi- in low dose and intervals, like a myokine (30). In ficant correlation with IL-6 in group A (younger contrast, our study showed that IL-6 was signifi- diabetics). The higher levels of IL-6 in the con- cantly higher in the control group (K) than in all trol group than in the diabetic groups is a sign of three groups of patients with DM. This interesting a stronger inflammatory answers in younger and finding suggests that younger and healthy people healthy people than in patients with DM. have stronger immunological response than older ones and patients with DM, even if DM induced FUNDING some tissue damage. In this study we also inclu- ded DM patients with amputation. There were No specific funding was received for this study. not many analyses in the recent literature related TRANSPARENCY DECLARATION to IL-6 and the Michigan Neuropathy Score (mo- nofilament test, test with sonic fork and subjecti- Competing interests: None to declare.

15 Medicinski Glasnik, Volume 18, Number 1, February 2021

REFERENCES 1. Russell JW, Zilliox LA. Diabetic neuropathies. Con- 15. Tesfaye S, Boulton AJM, Dyck PJ, Freeman R, Ho- tinuum (Minneap Minn) 2014; 20:1226-40. rowitz M, Kempler P, Lauria G, Malik RA, Spallone 2. Volmer-Thole M, Lobmann R. Neuropathy and dia- V, Vinik A, Bernardi L, Valensi P, Toronto Diabetic betic foot syndrome. Int J Mol Sci 2016; 17:917. Neuropathy Expert Group. Diabetic neuropathies: 3. Marie-Louise Mallet, Marios Hadjivassiliou, Ptole- update on definitions, diagnostic criteria, estimati- maios Georgios arrigiannis, Panagiotis Zis. The role on of severity, and treatments. Diabetes Care 2010; of oxidative stress in peripheral neuropathy. J Mol 33:10:2285-93. Neurosci 2020; 70:1009–17. 16. Herman WH., Pop-Busui R, Barffett BH., Martin 4. Morali G. Salamone P, Casale F, Fuda, G, Cugnasco CL, Cleary PA, Albers JW. Use of the Neuropathy P, Carosi C, Amroso A, Calvo A, Lapiano L, Colci- Screening Instrument as a measure of distal symme- to D, Chio A. NADPH oxidase 2 (NOX2) enzyme trical peripheral neuropathy in Type 1 diabetes: re- activation in patients with Chronic Inflammatory sults from the Diabetes Control and Complications demyelinating Polyneuropathy. Eur J Neurol 2016; Trial/Epidemiology of Diabetes Interventions and 23:958-63. Complications. Diabet Med 2012; 29:937-44. 5. Villegas-Rivera G, Miguel Román-Pintos L, Germán 17. Park JH, Kim DS. The necessity of the simple tests Cardona-Muñoz E, Arias-Carvajal O, Daniel Ro- for diabetic peripheral neuropathy in type 2 Diabetes dríguez-Carrizalez A, Troyo-Sanromán R, Pache- Mellitus patients without neuropathic symptoms in co-Moisés P, Moreno-Ulloa A Miranda-Díaz AM. clinical practice. J Diabetes Metab J 2018; 42:442-6. Effects of zetimibe/simvastatin and rosuvastatin on 18. Kaplan Y, Kurt S, Karaer Ünaldi H, Erkorkmaz Ü. oxidative stress in diabetic neuropathy: A randomi- Risk factors for diabetic polyneuropathy. Noro Psi- zed, double-blind, placebo-controlled clinical trial. kiyatr Ars 2014; 51:11-4. 2015; 10:75-62. 19. Rashad NM, El-Shabrawy RM, Sabry HM, Fathy 6. Klermm C, Bruchangen Ch, Kruchten A, Niemann HA, Said D, Yousef MS. Interleukin-6 and hs-CRP S, Loffler B, Peters G, Ludwig S, Enhardt C.Mi- as early diagnostic biomarkers for obesity-related togen activated protein kinases (MAPKs) regulate peripheral polyneuropathy in non-diabetic patients. IL-6 over-production during concomitant influenza Egypt J Immunol 2018; 25:153-65. virus and staphylococcus aureus infection. Nature 20. Tuttolomondo A, La Placa S, Di Raimondo D, Bellia 2017; 10:424-57. Ch, Caruso A, Bruna Lo Sasso, Guercio G, Diana g, 7. Bastard JP, Jardel C, Delattre J, Hainque B, Bruckert Marcello Ciaccio, Licata G, Pinton A. Adiponectin, E, Oberlin F. Evidence for a link between adipose resistin and IL-6 plasma levels in subjects with dia- tissue interleukin-6 content and serum C-reactive betic foot and possible correlations with clinical va- protein concentrations in obese subjects. Circulation riables and cardiovascular co-morbidity. Cardiovasc 1999; 99:2221-2. Diabetol 2010; 9:50. 8. Febbraio MA, Pedersen BK. Contraction-induced 21. Skyler JS, Bakris GL, Bonifacio E, Darsow T, Ec- myokine production and release: is skeletal muscle kel RH, Groop L, Groop PH, Handelsman Y, Insel an endocrine organ? Ex. Sport Sci 2005; 33:114–9. RA, Mathieu C, McElvaine AT, Palmer JP, Pugliese 9. Kristiansen OP, Mandrup-Poulsen T. Interleukin-6 A, Schatz DA, Sosenko JM, Wilding JP, Ratner RE. and diabetes: the good, the bad, or the indifferent? Differentiation of diabetes by pathophysiology, na- Diabetes 2005; 54:S11424. tural history, and prognosis. Diabetes 2017; 66:241- 10. Louis E, Raue U, Yang Y, Jemiolo B, Trappe S. Time 55. course of proteolytic, cytokine, and myostatin gene 22. Artasensi A, Pedretti A, Vistoli G, Fumagalli L. Type expression after acute exercise in human skeletal 2 diabetes mellitus: a review of multi-target drugs. muscle. J Appl Physiol (1985); 2007; 103:1744-51. 2020; 25:1987. 11. Ziegler D, Bucholz S, Sohr C, Norrouc Z. Oxidative 23. Bornstein SR, Rubino F, Khunti K, Mingrone G, Hop- stress predicts peripheral and cardiac autonomic ner- kins D, Birkenfeld AL, Boehm B, Amiel S, Holt RI, ve dysfunction over 6 years in diabetic patients. Acta Skyler JS, DeVries JH, Renard E, Eckel RH, Zimmet Diabetol 2015; 52:65-72. P, Alberti KG, Vidal J, Geloneze B, Chan JC, Ji L, 12. Cho NH, J.E.Shaw JE, S. Karuranga S, Huang Y, Ludwig B. Practical recommendations for the ma- Da Rocha JV. IDF Diabetes Atlas: global estimates nagement of diabetes in patients with COVID-19. of diabetes prevalence for 2017 and projections for Lancet Diabetes Endocrinol 2020; 8:546-50. 2045. Diab Res and Clin Practice 2018; 138:271-81. 24. Pizzino G, Irrera N, Cucinotta M, Pallio G, Mannino 13. Vacante M, Malaguarnera M, Motta M. Revision F, Arcoraci V, Squadrito F, Altavilla D, Bitto A. Oxi- of the ADA-classification of diabetes mellitus type dative stress: harms and benefits for human health. 2 (DMT2): the importance of maturity onset diabe- Oxid Med Cell Longev 2017; 2017:8416763. tes (MOD), and senile diabetes (DS). Arch Gerontol 25. Akiboye F, Rayman G. Management of Hyperglyce- Res 2018; 53:113-9. mia and diabetes in . Curr Diab 14. American Diabetes Association. Diagnosis and cla- Rep 2017; 17:13. ssification of diabetes mellitus. Diabetes Care 2010; 33:S62-9.

16 Karahmet et al. IL-6 and diabetic neuropathy

26. Magrinelli F, Briani Ch, Romano M, Ruggero S, 32. Lehmann HC, Burke D, Kuwabara S. Chronic in- Toffanin E, Triolo G, Ruggero S, Toffanin E, Triolo flammatory demyelinating polyneuropathy update G, George Chummar P, Marialuigia P, Francesco M, on diagnosis immunopathogenesis and treatment. J Lauriola M, Giampietro Zanette G, Tamburin S. The Neurol Neurosurg Psychiatry 2019; 90:981–7. association between serum cytokines and damage to 33. He Q, Dong M, Pan Q, Wang X, Guo L. Correlati- large and small nerve fibers in diabetic peripheral on between changes in inflammatory cytokines and neuropathy. J Diabetes Res 2015; 2015:547834. the combination with hypertension in patients with 27. Korkmaz P, Koçak H, Onbaşı K, Biçici P, Özmen type 2 Diabetes Mellitus. Minerva Endocrinol 2019; A, Uyar C, Özatağ DM. The Role of serum procal- 44:252-8. citonin, interleukin-6, and fibrinogen levels in diffe- 34. Das AK., Kalra S, Tiwaskar M, Bajaj S, Seshadri K, rential diagnosis of diabetic foot ulcer infection. J Chowdhury S, Sahay R, Indurkar S, Unnikrishnan Diabetes Res 2018; 2018:7104352. AG, Phadke U, Pareek A, Purkait I. Expert Gro- 28. Obradovic S, Begic E, Jankovic S, Romanovic R, up Consensus Opinion: role of anti-inflammatory Djenic N, Dzudovic B, Jovic Z, Malovic D, Subota agents in the management of type 2 diabetes (T2D). V, Stavric M, Ljuca F, Kusljugic Z. Association of J Assoc Physicians India 2019; 67:65-74. PC and AT levels in the early phase of STEMI trea- 35. Dror E, Dalmas E, Meier D, Wueest S, Thevenet ted with pPCI with LV systolic function and 6-month J, Thienel C, Timper K, Nordmann TM, Traub S, MACE. Acta Clin Belg 2020; 1-7. Schulze F, Item F, Vallois D, Pattou F, Kerr-Conte J, 29. Cox AA, Sagot Y, Hedou G, Grek C, Wilkes T, Vinik Lavallard V, Berney T, Thorens B, Konrad D, Böni- AI, Ghatnekar G. Low-dose pulsatile interleukin-6 Schnetzler M, Donath MY. Postrprandial macropha- as a treatment option for diabetic peripheral neuro- ge-derived IL-1 stimulates insulin, and both synergi- pathy. Front Endocrinol (Lausanne) 2017; 8:89. stically promote glucose disposal and inflammation. 30. Pop-Russi R, Ang L, Holms C. Inflammation as the- Nat Immunol 2017; 18:283-92. rapeutic target for diabetic neuropathies. Curr Diab Res 2016; 16:29. 31. Herder C, Kannenberg JM, Huth C.M, Margit Heier M, Püttgen S, Tonrad B, Peters A, Roden M, Meisin- ger C, Ziegler D. Proinflammatory cytokines predict the incidence of progression of distal senso-motor polyneuropathy. KORA F4/FF4 Study. Diab Care 2017; 40:569-76.

17 ORIGINAL ARTICLE

Lived experiences of patients with COVID-19 infection: a phenomenology study

Ali-asghar Jesmi1, Zohreh Mohammadzade-tabrizi2, Mostafa Rad3, Elyas Hosseinzadeh-younesi4, Ali Pourhabib4

1Department of Nursing, 2Department of Paramedic; Sabzevar University of Medical , Sabzevar, 3Department of Nursing, Nurs- ing and Midwifery School, Iranian Research Centre on Healthy Aging, Sabzevar University of Medical Sciences, Sabzevar; 4Department of Nursing, Faculty of Nursing and Midwifery, Golestan University of Medical Sciences, Gorgan; Iran

ABSTRACT

Aim To describe experiences of patients with COVID-19 infec- tion.

Methods This qualitative research was conducted using a pheno- menological approach, and participants were selected via purpo- sive sampling. In total, 14 patients with COVID-19 were selected (nine women and five men) aged 20-60 years. Data were collected via in-depth interviews with open questions and through observa- tion. Data analysis was performed using Colizzi's phenomenolo- gical approach. Corresponding author: Results Three themes and nine categories were extracted; the Mostafa Rad main themes were mental strains, physical manifestations, and Department of Nursing, coping mechanisms. Mental strains entailed concerns, fears and Nursing and Midwifery School, Iranian isolation, on the other hand, physical manifestations comprised Research Centre on Healthy Aging, nervous, respiratory and gastrointestinal system, and systemic dis- Sabzevar University of Medical Sciences orders. Coping mechanisms included religiosity and home reme- dies categories. Sabzevar, Iran Phone: +98 5144018308; Conclusion Mental strains were the most important issues in the Fax: +98 5144018322; patients with COVID-19, which were interwoven with concerning E-mail: [email protected] physical manifestations. Most of the participants used self-medi- cation and spiritual resources to cope with the disease. Therefore, Ali-asghar Jesmi ORCID ID: https://orcid. it is recommended to carry out proper planning by healthcare per- org/0000-0003-2671-9286 sonnel to psychologically and spiritually support these patients, while alleviating physical manifestations of the disease.

Original submission: Key words: infection, coronavirus, qualitative research, nurses 27 July 2020; Revised submission: 04 September 2020; Accepted: 08 October 2020 doi: 10.17392/1247-21

Med Glas (Zenica) 2021; 18(1):18-26

18 Jesmi et al. Experiences of patients with COVID-19

INTRODUCTION fortunately, personal distancing did not suffice, leading to the increased number of new cases of Coronaviruses are a large group of viruses that COVID-19 and showing the presence of a huge cause mild respiratory infections (e.g. common number of the ‘’silent carriers’’ of the coronavi- cold), as well as severe illnesses, such as SARS rus in the community, which forced some regions and MERS (1). Recently, the virus has been to carry out full quarantine (e.g. China and Italy), known as COVID-19, and its outbreak began in or implement preventive care at the national level December 2019 in Wuhan, China (2). COVID-19 (Iran, the UAE, South Korea) in cities, provinces easily spreads in some geographical regions that or even the whole country (11). are infected with the virus. This local spread cau- ses virus infection of the residents in the region, Despite positive consequences, the implementa- including those who are unsure how and when tion of these health policies has been associated they have been infected (3). The World Health with negative psychological consequences at the Organization (WHO) has confirmed COVID-19 community level (12). In fact, mental health of in the regions such as Africa, America, East Me- community members has been threatened due to diterranean, Europe, Southeast Asia, and the We- fear of the disease, fear of mortality, publishing stern Pacific (4). The symptoms of the virus infec- misinformation and rumours, interference with tion vary from mild to extremely severe; the signs daily activities, regulations for the prohibition or of the infection include fever, coughing, and diffi- restriction of travel and commute, decreased so- culty breathing (5). Anxiety is a common sign in cial relations (co-workers, friends, family), finan- patients with chronic respiratory diseases, which cial and occupational problems, and tens of other could considerably affect their quality of life. consequences in this situation (10). In most cases, anxiety may give rise to physical Phenomenological methods allow researchers to issues that overlap with the symptoms of chronic assess internal aspects of the lived experiences of respiratory disease and medication side effects (6). individuals. With a participatory design, these met- In addition, clinical anxiety affects two-thirds of hods provide an opportunity for the participants to the patients with chronic respiratory disease and express their inner feelings narratively (13). Un- may reduce their quality of life and physical per- derstanding the lived experience of patients with formance. However, data are scarce regarding the COVID-19 is of paramount importance to develop experience of anxiety in the patients diagnosed the perception of care. Phenomenological methods with severe respiratory symptoms (7). help improve the health and social consequences Currently, the trend of COVID-19 morbidity and of the disease for the patients (15). Given the close mortality in Iran is distressing (8). Restrictions contact of nurses with patients, one of the main and deprivations due to international sanctions, responsibilities of nurses is to help decrease the the unknown nature of the disease, inefficient use patients’ concerns. As such, it is extremely crucial of the information technology capacity to mana- to recognize the problems and concerns of patients ge the public opinion, and lack of a multi-sectoral and their feeling about the disease. view of health have caused fear and emotional The aim of this study was to evaluate the lived reactions in the society (9). Fard et al. in the study experience of patients with COVID-19 infection. aimed to predict psychological health of patients based on anxiety and social solidarity caused by PATIENTS AND METHODS Corona in 2020, suggesting that the quarantine Patients and study design has had positive and negative psychological and social effects and practical implications for the This qualitative research with descriptive phe- formulation of crisis interventions during the Co- nomenology approach attempted to capture the rona outbreak (10). On 4 February 2020, WHO essence of lived experience of patients with CO- declared the outbreak of COVID-19 as a public VID-19 infection. The data were gathered throu- health emergency, recommending that the con- gh interactive interviews. Eight patients were in- tact of people (especially patients and staff of terviewed face to face and six were interviewed healthcare departments) should be reduced to by phone call. All interviews were simultaneo- control the spread of the disease in the world. Un- usly recorded by tape-recorder and transcribed

19 Medicinski Glasnik, Volume 18, Number 1, February 2021

verbatim during the first 24 hours. Memos and participants who were interviewed by phone in- field notes were used to enrich the interviews. form consents were gathered verbally. The study The purposive and snow ball sampling methods was approved by the Research Deputy and Re- were used to invite participants for interviews. Par- search Ethics Committee of Sabzevar University ticipants were recruited from discharged patients of Medical Sciences (No 99030, Ethics Code: from Sabzevar Vasei Hospital (a governmental IR.MEDSAB.REC.1399.024). COVID central hospital), Iran. Inclusion criteria Methods were confirmed diagnosis of COVID-19 infection by a chest CT scan and PCR test, being 18 years old A semi structured interview with open-ended or older, being able to communicate and willing to questions was used for data collection. The par- share their experiences. A total of 14 participants, ticipants were asked to describe their experience who had experienced severe disease conditions and of involving with COVID-19 infection. Some passed the acute phase of the infection, were inter- of the questions were: Please describe your ex- viewed in this study. Nine of the participants (64%) perience of infection with COVID-19. Can you were females, seven of them (57%) were housewi- describe your feelings when you were informed ves; the age range was 25-53 year. about your COVID test result? Compare your ex- The first participant was a faculty member. He perience from COVID-19 infection with what the was interviewed face to face in his workplace; se- people and audiovisual media describe. Can you ven of the participants were interviewed at Vasei describe the experience of one day dealing with Educational Centre, Sabzevar, Iran in a quiet room this disease? What comes to your mind when you located outside the wards; six participants were in- hear the word COVID-19 infection? Which chal- terviewed by telephone due to disagreement with lenges did you experience with COVID-19 infec- the face-to-face interview (the reason of their di- tion? What were your expectations from those sagreement was fear of transmission of the infecti- around you? What comes to your mind when you on to others or re-infection). Interviews were held think of a problem? The main questions probed between April 2020–July 2020, and lasted for 20- to deepen participants’ experiences: Could you 50 minutes with respect of participants' tolerance. please explain more? What do you mean? etc. All interviews were held by the first author. Data Participants’ nonverbal reactions were recorded saturation was reached by 14 participants (Table 1). by the interviewer and used in the data analysis. Table 1. Characteristics of 14 participants All interviews were recorded by voice recorder and Ordinal number listened by the researcher many times for deep in- Gender Age Job position Marital status of the patient volvement. On the same day, the interviewer trans- M 36 Faculty member Married cribed the tape recording verbatim and reviewed it F 38 HW Married frequently with frequent stops. Questions were ad- F 50 Lab technician Widow M 53 Nurse (infected) Married ded or revised according to emerging information F 43 HW Married during the review process. Members of the team F 32 HW Married then revised the text of the transcript. Interviews F 50 HW Married F 25 HW Married were continued until no new themes emerged. The F 26 Psychologist Single Colaizzi phenomenological approach was used for F 43 HW Married data analysis, including seven steps: 1) read all the F 36 HW Married M 29 Teacher Married patients’ descriptions about COVID-19 Infection, M 42 HW Married 2) extract significant statements, 3) formulate the M 25 Nurse (infected) Single same meaning statements, 4) categorize the classi- F, female; M, male; HW, house wife fied meanings into clusters of themes, 5) integrate All ethical research codes such as the confiden- the findings into an exhaustive description of the tiality of participants' identity, taking permission phenomenon COVID-19 infection, 6) return the for audio recording of the interview and the right descriptions to some patients to assess how they to withdraw from the study, were considered. All compare with their experiences, and 7) correct participants received required information about suggested changes in the final description of the the purpose, method and ethical rights, and eight essence of the phenomenon (11). One note softwa- of them completed an informed consent. For the re was used for the data analysis.

20 Jesmi et al. Experiences of patients with COVID-19

Trustworthiness of this study was based on Lincoln Mental strain and Guba's evaluative criteria (15). Credibility is The mental strains reported by the participants one of the most important criteria for establishi- were divided into three categories of concerns, ng trustworthiness. Results are credible when the fears and isolation. phenomenon under the study was recognized by participants and experts and it reflects their -per Concerns. The majority of the participants re- sonal experience. The authenticity criterion refers ported similar concerns, such as worrying about to the fact that results must be in line with or re- the deterioration of the symptoms, losing the job flect the experiences described and lived by par- position, future of children, persisting complica- ticipants. With the aim of meeting these criteria, tions, and disease disclosure. the principal investigator used bracketing, read the One of the most common concerns among the interviews many times over, went back and forth participants was worrying about the deterioration between the data collection and analysis, reached of the symptoms. The majority of the participants data saturation, used peer reviewing and held de- were concerned about the deterioration of their briefing sessions with other authors regarding the own and their family members’ symptoms, espe- data collected, analysis and interpretation. In order cially shortness of breath. These concerns led to to authenticate the data a member check was used. anxiety and tension and increased sleep disorders In the member check, the interview codes were re- in the participants (e.g. insomnia and nightma- turned to eight participants, and they verified the res). For example, participant number six clai- codes extracted by the researcher. med, “I was constantly worrying about the chan- ging symptoms, especially since my mother had RESULTS the disease at the same time as me. I was more In total, 406 initial codes were extracted. The worried about her and checked up on her frequ- combination of similar meaning and concept ently. I constantly asked her about her breathing codes led to the remaining 230 initial codes. Af- and whether she was experiencing shortness of terwards, the initial codes were classified into breath. I was particularly concerned about her th subcategories based on meaning and conceptual because she has hypertension” (6 participant). similarity. Overall, 31 subcategories were for- Another participant mentioned, “I constantly med, subsequently they made 9 categories, and thought about my breathing becoming worse. I finally three themes included mental strains, would take frequent deep breaths to see whether st physical manifestation, and coping mechanisms I can breathe easily” (1 participant). emerging from the data analysis (Table 2). One of the concerns of the participants (especi- ally the males) was the loss of their job, which Table 2. Themes and sub-themes that emerged from data was more evident in those who were self-em- analysis ployed or had no job security. In this regard, par- Themes Categories Subcategories ticipant number nine expressed, “One of my ma- Worsening of the symptoms Losing the job position jor concerns during the quarantine was the fear Mental strain Concerns Future of children of the lack of improvement and inability to return Persisting complications Disease disclosure to work, which would create economic issues for Fears Fear of death, Fear of dependence my family. This is most concerning because of Isolation Loneliness, Boredom the current situation of the country. My wife was Physical ma- Nervous system Headache, Impaired sense of smell and very comforting though” (9th participant). nifestations disorder taste, Dizziness, Insomnia, Nightmare Respiratory Coughing, Dyspnoea, Shortness of Another concern mentioned by some of the fema- system disorder breath, Chest burning, Throat ache le participants was worrying about the future of Gastrointestinal Loss of appetite, Nausea, and Vomi- disorders ting, Diarrhoea, Constipation. their children. The participants were concerned Systemic dis- Fever, Chills, Weakness , Myalgia, about this issue mostly because of their fear of orders Illness death. For example, participant number eight re- Coping Religious activities Religiosity marked, “I always wondered what would happen mechanisms Religious beliefs Family support to my children if I died. Sometimes I cried and Home remedies Complementary therapies was very worried about them.” (7th participant)

21 Medicinski Glasnik, Volume 18, Number 1, February 2021

One of the major concerns of the participants home even my children avoided me because of was the lack of improvement and fear of persi- fear of transmission. I spend two full weeks in a stent complications for life. In this respect, one room and just my wife fed me. Nobody came to of the participants stated, “I always thought that our home. Even neighbours avoided my wife and I would not get better, especially in the first days, children. It really bothered me” (5th participant). I was so busy thinking that I even had a panic Another issue raised by the participants was their th attack” (6 participant). boredom over house quarantine. They spent the Some of the participants expressed concern about time watching TV, listening to music, studying, the disclosure of the disease to their relatives and saying prays, sleeping or chatting with friends. family members and were reluctant to inform One of the participants stated, “Sometimes I them about the disease. The main reason for this didn’t know what I can do. I was quarantined in a subject was avoiding others and not making close room. I was really boarded. I wished to get rele- relatives such as parents worried. In this regard, ased from that situation, sometimes I prayed for one of the participants remarked, “We did not gaining my health again and leaving the house” inform our neighbours in the building and even (9th participant). the family members in order not to make them anxious” (4th participant). Physical manifestations Fear. The main fear of the majority of the parti- Various forms of physical manifestations were cipants was fear of death and dependence. The expressed by the participants. The virus had participants viewed daily media reports of deaths attacked several body systems of the patients, and shortness of breath as the main causes of es- and the physical manifestations included nervous calating tensions and fears. system, respiratory system, gastrointestinal, and One of the common symptoms of the disease as systemic manifestations. reported by the participants was shortness of bre- Nervous system disorders. Theses manifestati- ath, which caused a sense of impending doom. ons were among the early symptoms of the disea- For instance, one of the participants stated, “…I se in most patients, and the main cause of referral was always afraid that I would die, especially to medical centres was reported to be neurological when I was hospitalized and experiencing severe manifestations such as headache, impaired sen- shortness of breath” (13th participant). se of smell and taste, insomnia, and nightmares. One of the issues mentioned by some participants However, the most common nervous system ma- was fear of disability and dependence on other nifestation was headache, which was reported in family members (spouse and children), which the range of mild to extremely severe pain. In this was due to the weakness and inability of the pati- regard, one of the participants stated, “I had extre- ents to meet their basic needs. In this respect, one me headache, and I had never experienced these of the participants stated, “I was always worried symptoms in my life. I visited a physician prima- that the disease would be permanent. I would not rily due to headache that had become intolerable” st be able to take care of my personal affairs, and (1 participant). One of the manifestations repor- my spouse would be forced to take care of me. ted by the participants during the peak period of I was really sick at the time and very weak” (3rd the disease was the loss of the sense of smell and participant). taste, and intensification of the sense of smell in some cases. Some of the participants considered Isolation. This category comprises of social iso- this disorder to be the cause of their loss of appe- lation and mood declining. tite. For instance, one of them mentioned, “My Most participants experienced feeling of loneli- condition deteriorated after experiencing shortne- ness in the period of the disease. The main re- ss of breath. I lost my sense of taste but had an ason for this subject was quarantining at home intensified sense of smell. For instance, the smell to prevent transmission of the disease to others, of oil would make me nauseous” (3rd participant). avoiding others, even relatives, from the patient In addition, some of the participants experienced and mental pressures. For example, one of the sleep disorders (e.g. insomnia and nightmares) in participants said, “When I was quarantined at the acute period of the disease. Some participants

22 Jesmi et al. Experiences of patients with COVID-19

attributed this to anxiety and mental stress during worries. These categories comprised of religious the illness, while others attributed it to the rise of activities and beliefs. the body temperature. For instance, one of the par- Two main activities reported by the participants ticipants affirmed, “I had nightmares at night, I do included praying and reading the Quran that is not know why. I did not have stress at all before rooted in the Muslim cultural and social context. the symptoms started, but when my symptoms be- In this regard, one of the participants who was came severe, I had nightmares. One of the frequ- pregnant asserted, “I read the Quran, prayed and ent nightmares was that I transmitted the disease to asked God that this infection was not transmitted th my parents” (8 participant). to my son (foetus). It really makes me calm” (11th Respiratory system disorders. Respiratory dis- participant). orders were the most important cause of referral to The participants disclosed their beliefs upon God medical centres and patient hospitalization. In this and appeals to him magnified in the period of ill- regard, the patients mostly complained of issues ness; nearly all participants declared this subject such as coughing, shortness of breath, chest pain, increased their energy and gave them hope for and sore throat. The disorder was mild in some ca- recovery. For example, participant number five ses, and the patients had no complaint of respira- said, “When I was ill I could really hardly bre- tory problems. Nevertheless, the main complaint athe. God helped me and declined my anxiety. of some of the patients was respiratory disorder It was not predictable, I might have died. Thank symptoms. In this respect, one of the subjects God I survived. It’s a miracle.” (1st participant) expressed, “I was choking and could not breathe Home remedies. Most patients used several met- easily. I felt extreme aching in the chest area and hods at home to reduce the symptoms of the di- had difficulty breathing” (9th participant). sease. Most of these remedies were taken to alle- Gastrointestinal disorders. Another common viate the systemic symptoms of the disease such complaint among the participants was gastroin- as fever, chills, and myalgia, as well as to reduce testinal problems, including the loss of appetite, respiratory symptoms such as throat ache and nausea and vomiting, diarrhoea and constipati- shortness of breath. These categories included on. For instance, one of the subjects claimed, “I complementary therapies and family support. wanted to eat but could not, as if something was Consuming supplements such as vitamins, eating blocking the path to my stomach and intestines. soup, herbal tea were common complementary the- My wife would open my mouth and my mother rapies that participants did for strengthening their would pour a spoonful of soup into my mouth. I physical condition. They believed that these thera- felt like I was eating poison” (1st participant). pies were effective for regaining their lost energy. Systemic manifestations. Systemic manifestati- For example, participant number six said, “I lost ons were also common among the participants, my appetite, lost my weight and got truly weak. which affected their daily activities and led to My husband made soup for me; I would eat only their dependence on the family members to four or five spoons. It was really effective. For my meet their basic needs. In this regard, some of throat ache the doctor recommended eucalyptus in- the common systematic manifestations included cense. It was effective too” (6th participant). fever, chills, weakness and lethargy, myalgia, bo- One of the important and effective factors in the redom, and muscles were jelly. “I could not stand recovery of patients was the care of relatives such on my feet and felt extreme pain in my arms and as spouse, parents and children help reducing the legs” (14th participant). symptoms of the disease. These actions include Coping mechanisms both physical and mental care. Participants dis- closed that without family support surviving was The participants applied different mechanisms very hard. One of the participants stated, “Wit- to address the challenges of this infection. These hout my wife's support and care, I could have mechanisms were divided into two categories of hardly survived. She gave me hope when I was religiosity and home remedies. really disappointed. She fed me patiently and Religiosity. Several participants used religio- when I was suffering from fever she applied wet sity as a mechanism to reduce their tensions and sponge frequently to my forehead and feet for

23 Medicinski Glasnik, Volume 18, Number 1, February 2021

declining my body temperature. I owe my reco- rantine. In the current research, the participants very to my wife” (1st participant). expressed their concerns in this regard as well. Since the psychological symptoms are common DISCUSSION in these patients, it is recommended that he- In the present study, most of the participants alth care providers pay special attention to this suffered from psychological strains such as fear, dimension of this new emerging disease (2). It concern, and anxiety about the disease. The fear seems that in some cases this aspect is dominant of death, dependence, deterioration of symptoms, to physical dimension and needs medical inter- and transmission of the disease to family mem- vention (4). The clinical manifestations of the bers were the most common psychological issues disease are not clear, and the disorders related to reported by the participants. In this regard, the the disease vary from mild to severe (23). Several results of several studies conducted on patients physical issues were reported by the participants with COVID-19 in China during the outbreak of in the present study in addition to the psycholo- the infection were indicative of the high preva- gical aspects of the disease. In this regard, some lence of psychological disorders such as anxiety, of the most common physical issues were fever fear, depression, emotional changes, and insomnia and coughing. In the studies by Shi et al., Tian et (16,17). Some of the most important influential al., and Song et al., the prevalence of fever and factors in the psychological health of individuals coughing was reported to be 72.8% and 59.2%, infected with the virus were an unbridled surge 82% and 47.8%, and 96% and 47%, respectively in the spread of the virus, unfavourable status of (24-26). In this systemic manifestation, fever, the patients isolated in the ICU with acute respi- weakness and myalgia were common among our ratory problems, lack of an effective treatment participants; in addition, the most common mani- for the disease, and mortality due to the disease festations of the disease in the presented research (18,19). Home quarantine was another issue that included myalgia, fatigue, pneumonia, and shor- might have caused psychological disorders in the tness of breath. However, the lower prevalence patients. Overall, the studies assessing the psycho- of symptoms such as headache, diarrhoea, and a logical disorders of quarantined individuals have runny nose has also been reported (27), which is denoted several signs of psychological damage consistent with our findings. In our study, in or- such as emotional distress, depression, stress, der to alleviate these symptoms most of the parti- mood fluctuation, irritability, insomnia, decreased cipants took measures such as gargling salt water, attention, post-traumatic stress disorder (PTSD), drinking herbal teas, eating soups, and taking wet anger, and emotional instability (20,21). sponge. Furthermore, the patients used various strategies to cope with the disease and alleviate According to the results of the present study, their concerns, the most important of which was the patients with COVID-19 infection had a low religious activities such as praying. Several stu- psychological capacity and experienced psycho- dies have confirmed the positive effects of reli- logical disorders (e.g. anxiety, fear, depression, gious activities on the improvement of the mental and negative thoughts) due to the current sta- and psychological status of various patients. For tus of the disease in the world. Sleep disorders example, Nadeem et al. referred to religion as an were another issue in these patients, which might alleviator of stress, anxiety, and depression (28). occur in isolated wards or during the quarantine. In another research by Chong et al. religious acti- The results of a study performed in China during vities were reported to decrease internal conflicts the outbreak of the virus in Wuhan indicated an in cancer patients (29). Moreover, Lee et al. re- extreme decrease in the sleep quality of the pati- ported a significant decrease in the anxiety and ents infected with the virus (22). Meanwhile, it depression of patients with seizures through reli- seems that some of the influential factors in the gious activities (30). occurrence of psychological symptoms in the general public could be linked to the concerns In conclusion, the patients with COVID-19 in- about the risk of the disease, future employment fection experienced various challenges such as status, and sources of income for individuals and clinical manifestations and mental strains that families, as well as long periods of home qua- ranged from mild to severe depending on the pa-

24 Jesmi et al. Experiences of patients with COVID-19

tients’ conditions. Furthermore, most of the pati- titude to the vice-chancellor for research of the ents used spiritual strategies to reduce these stra- University for the financial support to this study. ins. They applied home remedies concomitant We would also like to thank all the participants with medical prescriptions in order to alleviate for assisting us in this research project. their clinical symptoms. Since psychological symptoms are common in these patients, it is re- FUNDING commended to seek help from a psychotherapist This study was funded by the Research and Tech- in treating these patients. nology Deputy of Sabzevar University of Medi- cal Sciences. ACKNOWLEDGMENTS This article was extracted from a research project TRANSPARENCY DECLARATION approved by Sabzevar University of Medical Sci- Conflict of interest: None to declare. ences (code: 99030). Hereby, we extend our gra- REFERENCES

1. Cui J, Li F, Shi ZL. Origin and evolution of pathoge- 12. Parmet WE, Sinha MS. Covid-19 - The law and li- nic coronaviruses. Nat Rev Microbiol 2019; 17:181- mits of quarantine. N Engl J Med 2020; 382:e28. 92. 13. Jesmi AA, Jouybari L, Sanagoo A. The lived expe- 2. Tong ZD, Tang A, Li KF, Li P, Wang HL, Yi JP, riences of patients with spinal muscular atrophy: Zhang YL, Yan JB. Potential pre-symptomatic A phenomenological study. J Nurs Midwifery Sci transmission of SARS-CoV-2, Zhejiang province, 2019; 6:57-62. China, 2020. Emerg Infect Dis 2020; 26:1052. 14. Shosha GA. Employment of Colaizzi’s strategy in 3. Jernigan JA, Low DE, Helfand RF. Combining clini- descriptive phenomenology: A reflection of a resear- cal and epidemiologic features for early recognition cher. Eur Sci J 2012; 8:31-43. of SARS. Emerg Infect Dis 2004; 10:327. 15. Cypress BS. Rigor or reliability and validity in qua- 4. Ludwig S, Zarbock A. Coronaviruses and SARS- litative research: Perspectives, strategies, reconcep- CoV-2: a brief review. Anesth Analg 2020; 131:93-6. tualization, and recommendations. Dimens Crit Care 5. Wu Z, McGoogan JM. Characteristics of and impor- Nurs 2017; 36:253-63. tant lessons from the coronavirus disease 2019 (CO- 16. Yang L, Wu D, Hou Y, Wang X, Dai N, Wang G, VID-19) Outbreak in China: summary of a report Qig Yang Q, Wenhui Zhao W, Lou Z, Ji Y, Ruan L. of 72 314 cases from the Chinese Center for Dise- Analysis of psychological state and clinical psycho- ase Control and Prevention. JAMA 2020; Published logical intervention model of patients with CO- online ahead of print. VID-19. medRxiv 2020. 6. Dong XY, Wang L, Tao YX, Suo XL, Li YC, Liu 17. Liu S, Yang L, Zhang C, Xiang YT, Liu Z, Hu S, F, Zhao Y, Zhang Q. Psychometric properties of the Bin Z. Online mental health services in China during Anxiety Inventory for Respiratory Disease in pati- the COVID-19 outbreak. Lancet Psychiatry 2020; ents with COPD in China. Int J Chron Obstruct Pul- 7:e17-18. mon Dis 2016; 12:49-58. 18. Xiang YT, Yang Y, Li W, Zhang L, Zhang Q, Cheung 7. Willgoss TG, Goldbart J, Fatoye F, Yohannes T, Ng CH. Timely mental health care for the 2019 AM. The development and validation of the anxi- novel coronavirus outbreak is urgently needed. Lan- ety inventory for respiratory disease. Chest 2013; cet Psychiatry 2020; 7:228-9. 144:1587-96. 19. Bo H-X, Li W, Yang Y, Wang Y, Zhang Q, Cheung 8. Takian A, Raoofi A, Kazempour-Ardebili S. CO- T, Wu X, Xiang Y-T. Posttraumatic stress symptoms VID-19 battle during the toughest sanctions against and attitude toward crisis mental health services Iran. Lancet 2020; 395:1035-36. among clinically stable patients with COVID-19 in 9. Doshmangir L, Mahbub Ahari A, Qolipour K, Aza- China. Psychol Med 2020; 27:1-2. mi-Aghdash S, Kalankesh L, Doshmangir P, et al . 20. Brooks SK, Webster RK, Smith LE, Woodland L, East Asia's strategies for effective response to CO- Wessely S, Greenberg N, Rubin GJ. The psychologi- VID-19: lessons learned for Iran. Manage Strat He- cal impact of quarantine and how to reduce it: rapid alth Syst 2020; 4:370-3. review of the evidence. Lancet 2020; 395:912-20. 10. Alirezafard S., Safarinia M. Pishbini salamate ravan 21. Rubin GJ, Wessely S. The psychological effects of bar asase ezterab va hambastegie ejtemaee nashi az quarantining a city. BMJ 2020; 368:m313. bimari corona (The prediction of mental health ba- 22. Zhang F, Shang Z, Ma H, Jia Y, Sun L, Guo X, Wu L, sed on the anxiety and the social cohesion that cau- Sun Z, Zhou Y, Yan Wang, Liu N, Liu W. High risk sed by Coronavirus) [in Persian]. Quarterly Social of infection caused posttraumatic stress symptoms in Psychlogical Research 2020; 9:129-41. individuals with poor sleep quality: a study on influ- 11. World Health Organization. Coronavirus dis- ence of coronavirus disease (COVID-19) in China. ease 2019 (‎‎COVID-19)‎‎: situation report, 88. medRxiv. 2020. 2020. ttps://apps.who.int/iris/bitstream/han- 23. Talevi D, Socci V, Carai M, Carnaghi G, Faleri S, dle/10665/331851/nCoVsitrep17Apr2020-eng. Trebbi E, di Bernardo A, Capelli F, Pacitti F. Men- pdf?sequence=1&isAllowed=y (17 April 2020) tal health outcomes of the CoViD-19 pandemic. Riv Psichiatr 2020; 55:137-44.

25 Medicinski Glasnik, Volume 18, Number 1, February 2021

24. Shi H, Han X, Jiang N, Cao Y, Alwalid O, Gu J, Fan 28. Nadeem MA, Ali A, B, Buzdar MA. The association Y, Zheng C. Radiological findings from 81 patients between Muslim religiosity and young adult college with COVID-19 pneumonia in Wuhan, China: a des- students’ depression, anxiety, and stress. J Relig He- criptive study. Lancet Infect Dis 2020; 20:425-34. alth 2017; 56:1170-9. 25. Tian S, Hu N, Lou J, Chen K, Kang X, Xiang Z, Chen 29. Ng GC, Mohamed S, Sulaiman AH, Zainal NZ. H, Wang D, Liu N, Liu D, Gang Chen 7, Zhang Y, Li Anxiety and depression in cancer patients: the asso- D, Li J, Lian H, Niu S, Zhang L, Zhang J . Charac- ciation with religiosity and religious coping. J Relig teristics of COVID-19 infection in Beijing. J Infect. Health 2017; 25: 575-90. 2020; 80:401-6. 30. Lee SA, Ryu HU, Choi EJ, Ko MA, Jeon JY, Han 26. Song F, Shi N, Shan F, Zhang Z, Shen J, Lu H, Ling SH, Lee G-H, Lee MK, Jo K-D . Associations Y, Jiang Y, Shi Y. Emerging 2019 Novel Corona- between religiosity and anxiety, depressive symp- virus (2019-nCoV) pneumonia. 2020; toms, and well-being in Korean adults living with 295:210-17. epilepsy. Epilepsy Behav 2017; 75:246-51. 27. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Li Zhang L, Fan G, Xu J, Gu X, Zhenshun Cheng, Yu T, Xia J, Wei, Wu W, Xie X, Yin W, Li H, Liu M, Xiao Y, Gao H, Guo L, Xie J, Wang G, Jiang R, Gao Z, Jin Q, Wang J, Cao B. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020; 395:497–506.

26 ORIGINAL ARTICLE

Comparison of analgesic efficacy of acetaminophen monotherapy versus acetaminophen combinations with either pethidine or parecoxib in patients undergoing laparoscopic cholecystectomy: a randomized prospective study

Francesk Mulita1, Georgios Karpetas2, Elias Liolis3, Michail Vailas1, Levan Tchabashvili1, Ioannis Maroulis1

1Department of , 2Department of Anaesthesiology, 3Department of Internal Medicine; University General Hospital, Patras, Greece

ABSTRACT

Aim To investigate analgesic effect of three different regimens of combination of analgesics administered to patients undergoing la- paroscopic cholecystectomy.

Methods Patients undergoing laparoscopic cholecystectomy were randomly allocated to one of three groups on admission, depen- ding of a prescribed post-operative analgesic regimen. Patients allocated to the group A received a combination of intravenous (IV) acetaminophen and intramuscular (IM) pethidine, patients in the group B received a combination of IV acetaminophen and IV parecoxib, and the patients of the group C received IV ace- Corresponding author: taminophen monotherapy. Analgesic therapy was administered at regular intervals. Pain was evaluated utilizing the numeric rating Francesk Mulita scale (NRS) at 5 time points: the first assessment was done at 45 Department of Surgery, minutes, the second, third, fourth and fifth at 2, 6, 12, and 24 hours General University Hospital post-administration, respectively. Postoperative pain intensity was Rio 265 04, Patras, Greece measured by NRS within the groups and between the groups at Phone: +30 6982785 142; each time they analysed using one-way repeat measured ANOVA +30 2610 455 541; and Post Hoc Test-Bonferroni Correlation. E-mail: [email protected] Results A total of 316 patients were enrolled. The analgesic re- ORCID ID: https://orcid.org/0000-0001- gimens of groups A and B (combination regimens consisting of 7198-2628 IV acetaminophen and intramuscular pethidine and IV acetami- nophen and IV parecoxib, respectively) were found to be of equi- valent efficacy (p=1.000). In contrast, patients in group C (ace- taminophen monotherapy) had higher NRS scores, compared to both patients in groups A (p<0.01) and B (p<0.01).

Original submission: Conclusion This study confirms the notion of a significant opio- 27 July 2020; id-sparing effect of parecoxib in postoperative Revised submission: after laparoscopic cholecystectomy. 04 September 2020; Key words: analgesia, numerical rating scale, post-operative pain Accepted: 23 September 2020 doi: 10.17392/1245-21

Med Glas (Zenica) 2021; 18(1):27-32

27 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION There are two non-opioid analgesics, parecoxib and acetaminophen, with proven effectiveness One of the most common minimally invasive after different surgical procedures (13). Wide use surgical procedures is laparoscopic cholecystec- of non-opioid analgesics can reduce opioid-indu- tomy. This technique has almost replaced the ced side-effects (14,15). open technique for routine cholecystectomies since early 1990s (1). Currently, this procedure The aim of this prospective interventional cohort is indicated for the treatment of both acute and study was to compare the analgesic efficacy of chronic cholecystitis, symptomatic cholelithia- three analgesic regimens in the setting of lapa- sis, biliary dyskinesia, acalculous cholecystitis, roscopic cholecystectomy: acetaminophen mo- gallstone pancreatitis, and gallbladder tumours notherapy versus acetaminophen combinations or polyps (2). Although, the greatest advantages with either pethidine or parecoxib. Although of laparoscopy is the reduction of postoperative there are many studies in the literature about pain, when compared to open surgery, this still post-operative management after laparoscopic remains a considerable factor affecting periope- cholecystectomy. studies investigating and con- rative course of the patients (3). trasting analgesic effect of combined pethidine/ acetaminophen and parecoxib/acetaminophen Pain leads to increased morbidity and is the pri- have not been published so far. mary reason for prolonged hospitalization after laparoscopic cholecystectomy (4,5). Patients’ PATIENTS AND METHODS most common complains are back and shoulder pain and discomfort at port-site incisions (6). Patients and study design Shoulder and sub-diaphragmatic pain occur in about 12- 60% of patients (7). Peak intensity of This prospective, randomized trial was conduc- pain occurs during the first few postoperative ted at the University Hospital of Patras in Gree- hours and usually declines after 2 or 3 days (8). ce between February 2017 and May 2019, and included 316 patients undergoing elective lapa- Several methods of pain control have been harne- roscopic cholecystectomy. ssed in this setting, such as the administration of intravenous (IV) non-steroidal anti-inflammatory All patients provided a written informed consent. drugs (NSAIDs), intramuscular opioids or intra- Ethical approval was obtained from the Ethics peritoneal local anaesthetics, with questionable Committee of the General University Hospital of outcomes regarding the optimum approach to Patras. (No 838-10/3/2017). pain management (9). The NSAIDs inhibit the Inclusion criteria were age between 35 and 65 ye- enzymes cyclooxygenase (COX) -1 and -2. Only ars, American Society of Anesthesiologists physi- the inhibition of COX-2 is thought to be involved cal status classification I or II (16), and diagnosis in analgesic, anti-inflammatory, and antipyretic of cholelithiasis that was scheduled to be treated effects of NSAIDs (10). by elective laparoscopic cholecystectomy. Pre- Albeit, mechanism of action of acetaminophen operative evaluation for general anaesthesia was remains unclear. In contrast to opioids, acetami- performed. Exclusion criteria were heart failure, nophen has no known endogenous binding sites liver failure, renal dysfunction, diabetes, severe and, unlike NSAIDs, causes only weak inhibition bronchial asthma, neurological or psychiatric dise- of peripheral cyclooxygenase activity, with appa- ase, history of chronic pain or opioid intake, diffi- rent selectivity for COX-2. There is increasing culties in communication due to language barriers evidence of an additional central antinociceptive or intellectual disability, and history of adverse effect (11). Although the mechanism of analge- events after NSAIDs (acetaminophen, parecoxib) sic efficacy of paracetamol remains intangible, it or pethidine administration. The day before sur- may also involve direct and indirect inhibition of gery, the patients gave informed written consents central cyclooxygenases. Furthermore, the acti- to the study. The day prior to surgery patients were vation of the endocannabinoid system and spinal introduced to the numerical rating scale (NRS) for serotonergic pathways also seems to be essential pain documentation (17). in its analgesic action (12) All participants were randomly assigned to each of the three groups before surgery using a com-

28 Mulita et al. Analgesic treatment after cholecystectomy

puter-generated random number generator and one-way repeat measured ANOVA and Post Hoc sequentially numbered opaque sealed envelopes. Test-Bonferroni Correlation. The p<0.05 was The patients in group A were randomized to re- considered significant. Normality of the data was ceive IV acetaminophen 1000 mg every 8 hours tested using Shapiro-Wilk test for normality. and intramuscular pethidine 50 mg every 12 hours, the patients in group B were randomized RESULTS to receive a combination of IV acetaminophen A total of 316 patients, including 152 males and 1000 mg every 8 hours and IV parecoxib 40mg 164 females, were enrolled in the study. The every 12 hours, and the patients in group C were youngest patient was 36, the oldest one was 63 randomized to receive IV acetaminophen 1000 years old. A total of 106 patients received IV mg every 8 hours only. The patients who asked paracetamol and IM pethidine as an analgesic for more postoperative analgesics were excluded therapy, 113 received IV paracetamol and IV from this trial. parecoxib postoperatively, whereas 107 patients All operations were conducted by the same group received IV paracetamol (monotherapy). Thirty of surgeons and anaesthesiologists. General ana- patients from group C asked for more postopera- esthesia consisted of IV fentanyl 0.5-1.5 μg/kg tive analgesics and were excluded from this trial and propofol. All patients received IV acetami- (Table 1, Figure 1). nophen 1000 mg, IV parecoxib 40 mg and intra- muscular pethidine 50 mg during the procedure.

Methods After patient’s extubating in the operating room, surgical information was recorded such as sur- gery time, intra-operative complications, and analgesics used. Following surgery, patients were transferred to the surgical ward. Patients were evaluated at the bedside at 45 minutes, 2 hours, 6 hours, 12 hours and 24 hours after receiving the first analgesic dose from their allocated regimen. Figure 1. Flowchart of 316 patients who underwent laparo- scopic cholecystectomy Patients’ NRS pain ratings were recorded on po- stoperative monitoring charts. The scale ranged All patients were discharged following one day from 0-10: 0 means no pain and 10 corresponds of postoperative hospitalization. No intra-opera- to the maximum possible pain. tive complications were recorded. The mean NRS for patients that were treated with Statistical analysis IV paracetamol and IM pethidine (Group A) was Data were collected and expressed as mean ± 5.18 at 45 minutes (0.75 hours), 3.73 at 2 hours, standard deviation. The analysis of pain scores 2.55 at 6 hours, 1.82 at 12 hours and 0.98 at 24 was expressed as mean and 95% confidence in- hours (Figure 2). The mean NRS for patients that terval. The postoperative pain intensities mea- were treated with IV paracetamol and IV pare- sured by NRS within the groups and between the coxib (Group B) was 5.02 at 45 minutes (0.75 groups at each time interval were analysed using hours), 3.87 at 2 hours, 2.61 at 6 hours,1.89 at 12

Table 1. Number of patients, gender, mean age, hospitalization and duration of surgery according to the patient’s group Group Α Group Β Group C Variable Paracetamol and pethidine Paracetamol and parecoxib Paracetamol (monotherapy) Number of patients (n=286) 106 113 67 Males/Females (137/149) 52/54 54/59 31/36 Mean age (No) (years) 48 (38-60) 51 (41-63) 47 (36-59) Hospitalization (± SD) (days) 1 1 1 Intraoperative complications (No) 0 0 0 Mean operative (± SD) time (minutes) 36.8±9.1 39.4±7.4 38±6.3 Paracetamol 1gr/8h Paracetamol 1gr/8h Dosage Paracetamol 1gr/8h Pethidine 50mg/6h Parecoxib 40mg/12h

29 Medicinski Glasnik, Volume 18, Number 1, February 2021

hours and 1.01 at 24 hours, while the mean NRS using standardized surgical and anaesthetic tech- for patients that were treated with only IV para- niques. Pain was evaluated utilizing the numeric cetamol (Group C) was 5.81 at 45 minutes (0.75 rating scale (NRS). This scale was chosen because hours), 4.89 at 2 hours, 3.63 at 6 hours, 2.90 at 12 comparing to other pain intensity scales it is more hours and 1.84 at 24 hours (Figure 2). preferable by patients, as well as in comparison to other pain scales (such as the Visual Analogue Sca- le, VAS) (18), it is more sensitive in calculating the pain intensity changes that occur (18, 19). The outcomes of this randomized, prospective study suggest that there was no statistically si- gnificant difference in postoperative analgesic treatment among acetaminophen/parecoxib and acetaminophen /pethidine. It is noteworthy to Figure 2. Mean numerical rating scale (NRS) between the pa- mention that both aforementioned combinations tients of group A (paracetamol and pethidine) group B (par- acetamol and parecoxib) and group C (paracetamol -mono- were found to be superior when compared to aceta- therapy) based on time minophen monotherapy in achieving pain control, in patients with laparoscopic cholecystectomy and The NRS scores of the group C (paracetamol mo- should therefore be preferred in this setting. Based notherapy) were significantly higher than those of on the fact that these two pharmacologic regimens the groups A (pethidine + paracetamol, p<0.01) of analgesics appear to be equivalent in efficacy, and B (paracetamol + parecoxib, p<0.01), while the combination of acetaminophen and parecoxib there was no significant difference between the pa- might be preferable over acetaminophen and pet- tients of group A and group B (p=1.00) (Table 2). hidine in order to reduce opioid consumption and Table 2. Mean numerical rating scale (NRS) between the associated adverse events (20,21). three groups of patients based on time of administration Mean NRS according in the group Parecoxib is the first parenteral COX-2 inhibitor Time of administration Group Α Group Β Group C available for clinical use in pain management (hours) Paracetamol Paracetamol Paracetamol (22,23). It is well known from previous clinical and pethidine and parecoxib (monotherapy) trials that its peak serum concentrations occur at 0.75 5.18 5.02 5.81 about 30 minutes after intravenous (IV) admi- at 2 3.73 3.87 4.89 at 6 2.55 2.61 3.63 nistration and 1 hour after intramuscular (IM) at 12 1.82 1.89 2.9 injection. Its first perceptible analgesic effect at 24 0.98 1.01 1.84 occurs within 7-13 minutes, with clinically mea- ningful analgesia demonstrated within 23-39 mi- DISCUSSION nutes and a peak effect within 2 hours following According to the results of our study, the combi- administration of single doses of 40 mg by IV nations of pethidine/paracetamol and parecoxib/ or IM injection (23). The analgesic efficacy of paracetamol showed a comparable analgesic effec- parecoxib sodium 20 and 40 mg, IV or IM, has tiveness and they were better than paracetamol been found to be similar to that of ketorolac 15- monotherapy for the management of postoperati- 30 mg IV and 30-60 mg IM, and IV morphine ve pain after laparoscopic cholecystectomy. One 12 mg (23-25). The advantages of this analgesic of the most important interference in minimizing include its morphine-sparing effects as shown in sedation, impaired pulmonary function and consti- multiple studies (23, 26, 27). Several randomi- pation among operated patients is the reduction in zed controlled trials indicated a reduction in po- doses of opioids by using postoperative non-opioid stoperative opioid consumption after parecoxib analgesics (13). In our study, we have rummaged in different operations such as total hip or knee the effect of paracetamol and its combination with arthroplasty, hysterectomy and laparoscopic cho- parecoxib and pethidine on postoperative pain in a lecystectomy (13). However, studies investiga- randomized, controlled trial. All patients who were ting and contrasting the analgesic effect of com- included in this study were treated with laparos- bined pethidine/acetaminophen and parecoxib/ copic cholecystectomy under general anaesthesia acetaminophen have not been published so far.

30 Mulita et al. Analgesic treatment after cholecystectomy

One limitation of this study that should be consi- cholecystectomy. Both combinations of postope- dered is that we did not record data during mobi- rative analgesics outweigh the paracetamol mo- lization, as pain scores were recorded only at rest. notherapy and should be therefore preferred in The pain rating at rest alone is not very helpful laparoscopic cholecystectomy. Furthermore, our as it is the functional outcome that is of clinical study confirms the notion of a significant opioid- interest. Evaluation of pain during movement sparing effect of parecoxib in postoperative pain might be the initiative for a further study to be management after laparoscopic cholecystectomy. conducted (28). FUNDING In conclusion, the combination of postoperative analgesic treatment with IV paracetamol and IV No specific funding was received for this study. parecoxib IV seems to be equivalent to the com- TRANSPARENCY DECLARATION bination of IV paracetamol and intramuscular pethidine in patients undergoing laparoscopic Conflict of interests: None to declare.

REFERENCES 1. Kapoor T, Wrenn SM, Callas PW, Abu-Jaish W. 10. Munsterhjelm E, Niemi TT, Ylikorkala O, Neuvonen Cost analysis and supply utilization of laparosco- PJ, Rosenberg PH. Influence on platelet aggregation pic cholecystectomy. Minim Invasive Surg 2018; of i.v. parecoxib and acetaminophen in healthy vo- 2018:7838103. lunteers. Br J Anaesth 2006; 97:226–31. 2. Kiriyama S, Takada T, Strasberg SM, Solomkin JS, 11. Slater D, Kunnathil S, McBride J, Koppala R. Phar- Mayumi T, Pitt HA, Gouma DJ, Garden OJ, Büc- macology of nonsteroidal antiinflammatory drugs hler MW, Yokoe M, Kimura Y, Tsuyuguchi T, Itoi T, and opioids. Semin Intervent Radiol 2010; 27:400- Yoshida M, Miura F, Yamashita Y, Okamoto K, Ga- 11. bata T, Hata J, Higuchi R, Windsor JA, Bornman PC, 12. Graham GG, Davies MJ, Day RO, Mohamudally Fan ST, Singh H, de Santibanes E, Gomi H, Kusachi A, Scott KF. The modern pharmacology of para- S, Murata A, Chen XP, Jagannath P, Lee S, Padbury cetamol: therapeutic actions, mechanism of action, R, Chen MF; Tokyo Guidelines Revision Committee. metabolism, toxicity and recent pharmacological New diagnostic criteria and severity assessment of findings. Inflammopharmacology 2013; 21:201–32. acute cholecystitis in revised Tokyo Guidelines. J 13. Gehling M, Arndt C, Eberhart LHJ, Koch T, Kruger Hepatobiliary Pancreat Sci 2012; 19:548–56. T, Wulf H. Postoperative analgesia with parecoxib, 3. Choi GJ, Kang H, Baek CW, Jung YH, Kim DR. acetaminophen, and the combination of both: a ran- Effect of intraperitoneal local anesthetic on pain domized, double-blind, placebo-controlled trial in characteristics after laparoscopic cholecystectomy. patients undergoing thyroid surgery. Br J Anaesth World J Gastroenterol 2015; 21:13386–95. 2010; 104:761–7. 4. Bisgaard T, Kehlet H, Rosenberg J. Pain and conva- 14. Ng A, Parker J, Toogood L, Cotton BR, Smith G. lescence after laparoscopic cholecystectomy. Eur J Does the opioid-sparing effect of rectal diclofenac Surg 2001; 167:84-96. following total abdominal hysterectomy benefit the 5. Bisgaard T, Klarskov B, Rosenberg J, Kehlet H. Fac- patient? Br J Anaesth 2002; 88:714–6. tors determining convalescence after uncomplicated 15. Schüchen RH, Mücke M, Marinova M, Kravchenko laparoscopic cholecystectomy. Arch Surg 2001; D, Häuser W, Radbruch L, Conrad R. Systematic re- 136:917–21. view and meta-analysis on non-opioid analgesics in 6. Morsy KM, Mohamad Abdalla EE. Postoperative palliative medicine. J Cachexia Sarcopenia Muscle pain relief after laparoscopic cholecystectomy: intra- 2018; 9:1235-54. peritoneal lidocaine versus nalbuphine. Ain-Shams J 16. De Cassai A, Boscolo A, Tonetti T, Ban I, Ori C. Anaesthesiol 2014; 7:40-4. Assignment of ASA-physical status relates to ane- 7. Alkhamesi N, Peck D, Lomax D, Darzi A. Intraperi- sthesiologists’ experience: a survey-based national- toneal aerosolization of bupivacaine reduces posto- study. Korean J Anesthesiol 2019; 72:53-9. perative pain in laparoscopic surgery: a randomized 17. Pathak A, Sharma S, Jensen MP. The utility and vali- prospective controlled double-blinded clinical trial. dity of pain intensity rating scales for use in develo- DarziSurg Endosc 2007; 21:602–6. ping countries. Pain Rep 2018; 6:e672. 8. Acharya R, Karan D, Khetan M. Postoperative 18. Shagufta F, Zankhana M, Carlos F, Bertarnd B, Me- analgesia with intraperitoneal ropivacaine with and llar D. A comparison of Numeric Pain Rating Scale without dexmedetomidine after total laparoscopic (NPRS) and the Visual Analog Scale (VAS) in pa- hysterectomy: a randomized, double-blind, con- tients with chronic cancer-associated pain. J Clin trolled trial. Asian J Pharm Clin Res 2016; 9:76-9. Oncol 2017; 35:217 9. Saadati K, Razavi MR, Nazemi Salman D, Izadi 19. Safikhani S, Gries KS, Trudeau JJ, Reasner D, S. Postoperative pain relief after laparoscopic cho- Rüdell K, Coons SJ, Bush EN, Hanlon J, Abraham lecystectomy: intraperitoneal sodium bicarbonate L, Vernon M. Response scale selection in adult pain versus normal saline. Gastroenterol Hepatol Bed measures: results from a literature review. J Patient Bench 2016; 9:189–96. Rep Outcomes 2018; 2:40.

31 Medicinski Glasnik, Volume 18, Number 1, February 2021

20. Nong L, Sun Y, Tian Y, Li H, Li H. Effects of pa- 25. George BB, Zahid HB, Derek JD, Louise T, Richard recoxib on morphine analgesia after gynecology tu- CH. A clinical trial demonstrates the analgesic ac- mor operation: A randomized trial of parecoxib used tivity of intravenous parecoxib sodium compared in postsurgical pain management. J Surg Res 2013; with ketorolac or morphine after gynecologic sur- 183:821–6. gery with laparotomy. Am J Obstet Gynecol 2004; 21. Fu W, Yao J, Li Q, Wang Y, Wu X, Zhou Z, Li WB, 191:1183–91. Yan JA. Efficacy and safety of parecoxib/phloro- 26. Diaz-Borjon E, Torres-Gomez A, Essex MN, Salo- glucinol combination therapy versus parecoxib mon P, Li C, Cheung R, Parsons B. Parecoxib pro- monotherapy for acute renal colic: a randomized, vides analgesic and opioid-sparing effects following double-blind clinical trial. Cell Biochem Biophys major orthopedic surgery: a subset analysis of a ran- 2014; 69:157–61. domized, placebo-controlled clinical trial. Pain Ther 22. Essex MN, Xu H, Parsons B, Xie L, Li C. Pare- 2017; 6:61-72. coxib relieves pain and has an opioid-sparing effect 27. Liu WF, Shu HH, Zhao GD, Peng SL, Xiao JF, following major gastrointestinal surgery. Int J Gen Zhang GR, Liu KX, Huang WQ. Effect of parecoxib Med 2017; 28:319-27. as an adjunct to patient-controlled epidural analgesia 23. Baharuddin KA, Rahman NH, Wahab SF, Halim after abdominal hysterectomy: a multicenter, ran- NA, Ahmad R. Intravenous parecoxib sodium as an domized, placebo-controlled trial. PLoS One 2016; analgesic alternative to morphine in acute trauma 11:e0162589. pain in the emergency department. Int J Emerg Med 28. Shen SJ, Peng PY, Chen HP, Lin JR, Lee MS, Yu 2014; 7:2 HP. Analgesic effects of intra- articular bupivacaine/ 24. Siribumrungwong K, Cheewakidakarn J, Tangtra- intravenous parecoxib combination therapy versus kulwanich B, Nimmaanrat S. Comparing parecoxib intravenous parecoxib monotherapy in patients re- and ketorolac as preemptive analgesia in patients un- ceiving total knee arthroplasty: a randomized, dou- dergoing posterior lumbar spinal fusion: a prospec- ble-blind trial. Biomed Res Int 2015; 2015:450805. tive randomized double-blinded placebo-controlled trial. BMC Musculoskelet Disord 2015; 16:59.

32 ORIGINAL ARTICLE

Left to right shunt congenital heart disease as a risk factor of recurrent pneumonia in under five-year-old children: a single centre experience in Bandung Indonesia Sri Endah Rahayuningsih, Rahmat Budi Kuswiyanto, Filla Reviyani Suryaningrat, Heda Melinda Na- taprawira, Abdurachman Sukadi

Department of Child Health, Faculty of Medicine Universitas Padjadjaran / Hasan Sadikin General Hospital, Indonesia

ABSTRACT

Aim Children with congenital heart diseases are at the greater risk of respiratory tract infection such as pneumonia. Recurrent pneu- monia is one of the most major challenge for paediatric physicians. The aim of this study is to investigate risk factors of congenital heart diseases to recurrent pneumonia children.

Methods This was a retrospective study of under five-year-old children hospitalized in Hasan Sadikin General Hospital Bandung Indonesia from 2015 to 2018. Congenital heart diseases and pne- umonia, as well as recurrent pneumonia, were identified. Conge- nital heart diseases diagnosis with and without pneumonia were Corresponding author: reviewed. Sri Endah Rahayuningsih Results Of 6997 hospitalized children, in 1258 (18.0%) congenital Department of Child Health, heart diseases were found, of which 232 (18.4%) had recurrent Faculty of Medicine, pneumonia. Most of those had left to right (L to R) shunt, 213 niversitas Padjadjaran/ Dr Hasan Sadikin (91.8%). Congenital heart diseases in children aged under 1 year, General Hospital 144 (62%) were more preponderant than in those aged 1–5 years. Bandung Jalan Pasteur no. 38 Bandung More than a half, 119 (51.3%) were males. Left to right shunt was documented as having recurrent pneumonia, of which patent duc- 40161, West Java, Indonesia tus arteriosus and ventricular septal defect were the most common Phone: +62 81 648 709 62; type in congenital heart diseases. Ventricular septal defect had a Fax: +62 22 203 22 16; possibility for recurrent pneumonia by 1.551 times, and malnutri- E-mail: [email protected] tion 2.591 times. ORCID ID: https://orcid.org/0000-0003- Conclusion Ventricular septal defect and malnutrition were identi- 1396-5285 fied as risk factors for recurrent pneumonia. Those patients require multidisciplinary approach to prevent respiratory complications. Original submission: 04 May 2020; Key words: cardiac defect, child, complication, heart murmur, Revised submission: ventricular septal defect 12 June 2020; Accepted: 28 September 2020 doi: 10.17392/1196-21

Med Glas (Zenica) 2021; 18(1):33-37

33 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION dikin Hospital, Bandung, Indonesia is the largest tertiary hospital in West Java, Indonesia, serving The incidence of congenital heart diseases is about more than 38 thousand population every year. 1% in the general population and ranges from 4-50 This hospital is a referral hospital, where most cases per 1000 live births (1). In Indonesia, inciden- of the paediatric heart problems are referred to. ce for live birth is around 4,6 million every year (2). Paediatric cardiac interventions are performed It is estimated that every year many babies were by one paediatric cardiologist. Catheterization is born with congenital heart diseases. The frequency only done 2 days in a week. of CHD is almost the same in every ethnic group and region of the world (3). Left to right (L to R) This study was approved by the Health Research shunt was the most common type of congenital he- Ethics Committee of Hospital Dr Hasan Sadikin art diseases, with ventricular septal defect at least Bandung Indonesia. 20% from all defects, atrial septal defect up to 2% Methods and patent ductus arteriosus approximately 10% (4). The presence of congenital heart diseases caused This study was conducted by identifying conge- a significant defect of structure or function of the nital heart diseases (Q20–Q26 and pneumonia circulation system, which will then also affect the children (J18) (International Statistical Classifi- respiratory system function (5). Direct pulmonary cation of disease ICD-10) (9). Further, recurrent complications of congenital heart diseases are either pneumonia, defined as the repeated pneumonia by structural impact on the airways, abnormal pat- more than one time in a year with clinical im- hophysiological mechanisms leading to increased provement but without documented clearance of lung water and/or significant pulmonary disease (5). chest x ray, was identified. Patients with congenital heart diseases often We reviewed congenital heart disease diagnoses come to hospital with non-cardiac complicati- with and without pneumonia, a type of congenital ons such as infection, especially respiratory tract heart diseases including left to right (L to R) shunt infection like pneumonia (6). Pneumonia as the type (isolated atrial septal defect, ventricular sep- comorbidity in the paediatric patient of congeni- tal defect and patent ductus arteriosus), cyanotic tal heart diseases may extend the length of stay type (tetralogy of fallot and transient great artery, and complicate the treatment for the congenital obstruction type (aorta stenosis) post correcti- heart disease (6). In developing countries infants on surgery/intervention, and epidemiology data with hemodynamically significant congenital he- (gender and age, cerebral palsy, nutritional sta- art diseases at 70% had recurrent pneumonia (7). tus, breastfeeding, low birth weight history and The incidence of left to right shunt with recurrent environmental factors - smoke exposure). pneumonia has not been studied yet. Statistical analysis In developing countries, mortality of the children with congenital heart disease and pneumonia is Both descriptive and analytical analysis were increasing (8). A lack of epidemiological studies conducted. Descriptive analysis was performed from developing countries, including Indonesia, with data expressed as frequency and percenta- makes it difficult to plan even national/local stra- ge for categorical data. For analytical analysis tegies for the prevention and treatment. χ2 test or Fisher exact test was used. Bivariate statistical analysis continued with multivariate The aim of this study was investigate left to right logistic regression was conducted to analyse pro- shunt type of congenital heart disease as a risk minent risk factors that relate simultaneously to factor of the recurrent pneumonia in the children. the outcome. Risk was analysed using odd ratio PATIENTS AND METHODS (OR) with confidence interval 95%. Statistical si- gnificance of p<0.05 was applied. Patients and study design RESULTS This was a retrospective study of under five-ye- Of 6997 hospitalized children in the 2015-2018 ar-old children hospitalized in Hasan Sadikin period, in 1258 (18.0%) congenital heart disea- General Hospital from 2015 to 2018. Hasan Sa- ses were identified. Of those, 232 (18.4%) had

34 Rahayuningsih et al. Left to right shunt as a risk of pneumonia

recurrent pneumonia. Frequently identified con- trition, only 27 patients were with history of low genital heart diseases type was L to R shunt, 1155 birth weight (LBW). Cerebral palsy was found in (91.8%). Only the patients with recurrent pneu- 28 (13.1%) patients. monia with left to right shunt were included in Of seven risk factors analysed for recurrent the analysis, considering only a few patients with pneumonia, two showed statistical significan- cyanotic type, obstructive and post transcatheter ce (p>0.25), namely isolated atrial septal defect closure. Two patients with post patent ductus ar- and LBW; so that for multivariable analysis with teriosus closure were identified as having recu- multiple logistic regression these two variables rrent pneumonia with cerebral palsy and Down were not included in the analysis. Syndrome as an underlying disease (Figure 1). Ventricular septal defect had a possibility for re- current pneumonia by 1.551 times greater than atrial septal defect. Patent ductus arteriosus times was by 0.652 lower than atrial septal defect for the possibility of becoming recurrent pneumonia. As other comorbidities, malnutrition had 2.591 times risk for recurrent pneumonia (Table 2). Table 2. Multivariable analysis of comorbidities in congenital heart diseases left to right shunt in children with recurrent pneumonia (final model)

Variable p ORadj (95% CI) Isolated VSD 0.042 1.551 (1.020 – 3.201) Isolated PDA 0.033 0.652 (0.444 – 0.956) Malnutrition 0.001 2.591 (1.791 – 3.749) Exclusive breastfeeding 0.001 0.280 (0.172 – 0.456) VSD, ventricular septal defect; ASD, atrial septal defect; PDA, patent Figure 1. Congenital heart disease and recurrent pneumonia in ductus arteriosus; ORadj (95% CI), odds ratio adjusted and 95% the period 2015 – 2018 confidence interval;

Congenital heart diseases among children under DISCUSSION 1 year of age were more preponderant than those aged 1-5 years, 144 (62%) and 88 (38%, respec- This study found 18% of congenital heart dise- tively. More than a half, 119 (51.3%) were males. ases from the total number of hospitalized chil- dren under five years of age. So far we have not Ventricular septal defect (VSD) was the most found similar data in any other study. According common type of congenital heart diseases to one single centre study in Vietnam, 10074 con- followed by patent ductus arteriosus (PDA), and genital heart diseases patients were hospitalized both of those significantly become a risk factor during the past 6 years (10). To our knowledge, in recurrent pneumonia patients (p=0.001 and this is the first study to explore congenital heart p=0.001 respectively) (Table 1). diseases with recurrent pneumonia in our setting. Table 1. Risk factors and comorbidities in children with and The previous study in Canada demonstrated that without recurrent pneumonia most child patients hospitalized with recurrent No (%) of patients pneumonia were known to have an underlying ill- Variable Recurrent Without p pneumonia pneumonia ness at the time of pneumonia recurrence, one of Isolated VSD 93 (41.7) 143 (24.8) 0.001 those was congenital heart disease (8). We found Isolated ASD 41 (19.2) 115 (20.0) 0.823 18.4% recurrent pneumonia cases among the chil- Isolated PDA 79 (37.1) 318 (55.2) 0.001 dren under five years of age with congenital heart Exclusive breast feeding 26 (12.2) 146 (25.3) 0.0001 Malnutrition 134 (62.9) 252 (43.8) 0.0001 disease. The most frequently identified congenital Low birth weight 27 (12.7) 82 (14.2) 0.573 heart disease type was left to right shunt (91.8%). Cerebral Palsy 28 (13.1) 108 (18.8) 0.064 Total 213 576 We are still using “recurrent pneumonia” as ter- VSD, ventricular septal defect; ASD, atrial septal defect; PDA, patent minology in this study, but we cannot conduct ductus arteriosus radiologic examination after the patients’ reco- There were only 26 patients who had history of very to confirm the diagnosis due to our limitati- exclusive breastfeeding; 147 patients had malnu- ons such as the cost for patient care and queuing

35 Medicinski Glasnik, Volume 18, Number 1, February 2021

for X-ray examination by inpatients. A previous In our study malnutrition was the only risk factor study in Turkey in three years of observation de- the increase 5.556 times for recurrent pneumo- monstrated that 50 patients with congenital heart nia. Previous studies showed that mortality risk diseases were hospitalized due to pneumonia, increased among severely malnourished children of which acyanotic type was the most common with pneumonia (15). High mortality risk can be (76%) and 68% of patients were under 1 year of linked to immunodeficiency associated with mal- age (1). In our study, there were 62% of children nutrition, high rates of comorbidities, delayed he- under 1 year of age with congenital heart disease alth-seeking behaviour among families of children who had recurrent pneumonia comparing to 38% with malnutrition, and potential delays in diagno- of children aged 1-5 years with the most frequ- sis due to the insensitivity of clinical signs (15). ently identified congenital heart disease type left History of low birth weight in our study was fo- to right shunt (91.8%). In general, our research und in 12 patients. Previous studies showed that has a similar result with the study conducted in low birth weight had been associated with the de- Turkey (1). A previous study showed the highest velopment of severe pneumonia, as well as to be incidence of pneumonia occurred in the youngest a risk factor for increased mortality (14). patients with the incidence decreasing gradually Initial pulmonary hypertension (PH) that occurs with increasing age: the attack rates for pneumo- in the L to R shunt, such as VSD, atrial septal nia are 1/100 in infants (<1 year), 4/100 preschool defect and PDA, will cause high flow PH; howe- age, 2/100 at school age (5-9 years), and 1/100 at ver, if this continues, it will cause damage to the ages 9-15 years (11). intima media of lung tissue and will be replaced To diagnose the recurrent pneumonia, clinical by fibrosis which will cause high PH resistance evaluation should be conducted carefully. The that has a poor prognosis (5). In our study not all evaluation also included history of age of the patients with PH performed right heart catheteri- first chest infection, cough nature, duration and zation to determine PH high flow or PH high re- pattern, premature delivery, growth history, tu- sistance, suggesting that it is not known whether berculosis (TB) history, and parental smoking the recurrence is caused by a high resistance PH

(12). Recurrent pneumonia is still a diagnostic or other lung abnormalities (5). challenge in paediatrics. Early treatment of the The most common causes of recurrent pneumonia child’s underlying condition is crucial in order are L to R shunt of congenital heart diseases, VSD, to stabilize lung disease and thus prevent pro- atrial septal defect and PDA, which cause an over- gressive pulmonary function deterioration (13). flow to the lungs (5). According to our study VSD We also collected other risk factor data in our had a significant influence for higher prevalence study such as smoke exposure, Down syndrome, of recurrent pneumonia. In our research VSD was hypothyroid congenital, TB history and prematu- higher comparing to PDA. Other studies explain re delivery but we excluded all those because of that hospital admission for pneumonia in young incomplete data from medical records. children with haemodynamically significant con- In this study, there were only 32 (out of 213) pa- genital cardiac disease is mainly associated with tients who were known to have been exclusively non-cardiac conditions, which may be genetic, breastfed. A previous study in India stated that respiratory or caused by malnutrition (3). a lack of breastfeeding had been reported to be There are some limitations of the study. The sin- associated with an increased risk of the deve- gle-centre study population may also not be re- lopment of severe pneumonia by 1.5 to 2.6 times presentative of all children with congenital heart (14). It was known that lack of exclusive breas- diseases and recurrent pneumonia. Besides, we tfeeding was identified to be an important deter- did not further divide malnutrition to subcatego- minant associated with the need for a change in ries (wasted, severely wasted or obesity). antibiotics and prolonged hospital stay in severe pneumonia (14). Furthermore, acute malnutriti- In conclusion, ventricular septal defect and mal- on and lack of breastfeeding are considered to nutrition are both risk factors for recurrent pne- be factors associated with increased mortality in umonia. Those patients require multidisciplinary children due to pneumonia (14). approach to prevent respiratory complications.

36 Rahayuningsih et al. Left to right shunt as a risk of pneumonia

ACKNOWLEDGEMENT FUNDING The authors would like to thank Hadyana MD, No specific funding was received for this study. PhD as a statistical analyst for our study, staff of the paediatric respiratory division for managing TRANSPARENCY DECLARATION respiratory illness cases (Sri Sudarwati and Diah Conflict of interest: None to declare. Asri Wulandari) and staff of medical records for their assistance with this study.

REFERENCES 1. Sahan YÖ, Kiliçouglu E, Tutar ZÜ. Evaluation of 9. The ICD-10 Classification of Mental and Behavioral children with congenital heart disease hospitalized Disorder: Clinical Descriptions and Diagnostic Gu- with the diagnosis of lower respiratory tract infection. idelines. Geneva: World Health Organization, 1992. J Pediatr Res 2018; 5:32. 10. Phuc VM, Tin DN, Giang DTC. Challenges in the 2. Badan kependudukan dan keluarga berencana nasio- management of congenital heart disease in Vietnam: nal. Survey demografi dan kesehatan indonesia (Indo- a single center experience. Ann Pediatr Cardiol 2015; nesian demographic and health survey) [in Indonesi- 8:44-6. an] Jakarta: Indonesian Ministry of Health, 2017. 11. Murphy TF, Henderson FW, Clyde Jr WA, Collier 3. Kenny D, Stuart AG. Long-term outcome of the child AM, Denny FW. Pneumonia: an eleven-year study in with congenital heart disease. Paediatr Child Health a pediatric practice. Am J Epidemiol 1981; 113:12– (Oxford) 2009; 19:37–42. 21. 4. Medrano C, Guereta LG, Grueso J, Insa B, Ballestero 12. Yousif TI, Elnazir B. Approach to a child with recu- F, Casaldaliga J, Cuenca V, Escudero F, Calzada LG, rrent pneumonia. Sudan J Paediatr 2015; 15:71. Luis M, Luque M, Mendoza A, Prada F, Rodriguez 13. Montella S, Corcione A, Santamaria F. Recurrent pne- MM, Suarez P, Quero C, Guilera M. Respiratory in- umonia in children: a reasoned diagnostic approach fection in congenital cardiac disease. Hospitalization and a single centre experience. Int J Mol Sci 2017; in young children in Spain during 2004 and 2005: the 18:296. CIVIC Epidemiologic Study. Cardiol Young 2007; 14. MacDonald NE, Hall CB, Suffin SC, Alexson C, 17:360–71. Harris PJ, Manning JA. Respiratory syncytial viral 5. Aditia I, Kothari SS, Feinstain JA. Pulmonary hyper- infection in infants with congenital heart disease. N tention associated with congenital heart disease. Engl J Med 1982; 307:397–400. Pulmonary vascular disease: the global perspective. 15. Chisti MJ, Faruque ASG, Ashraf H, Hossain MI, CHEST 2010; 137(suppl):S52–61. Islam MM, Das SK, Ahmed T. Pneumonia in seve- 6. Daubeney PEF, Rigby ML, Niwa K, Gatzoulis MA, rely malnourished children in developing countries: editors. Pediatric Heart Disease a Practical Guide. 1st nutrition approaches to prevention and ed. UK: Wiley Blackwell 2012; 62–8. early treatment. In: Favilene C, Brown M, editors. 7. Adela S, Elena P, Ina P, Ninel R. The clinical course Public health nutrition: principles and practice in of acute respiratory infections in children with conge- community and global health. Burlington, MA, USA: nital heart disease. Arch Dis Childr 2017; 102 (suppl Jones & Bartlett Learning 2014: 399–407. 2):A1–192. 8. Owayed AF, Campbell DM, Wang EEL. Underlying causes of recurrent pneumonia in children. Arch Pe- diatr Adolesc Med. American Medical Association 2000; 154:190–4.

37 ORIGINAL ARTICLE

Characterization and clonal representation of MRSA strains in Tuzla Canton, Bosnia and Herzegovina, from 2009 to 2017

Fatima Numanović1,2, Urška Dermota3, Jasmina Smajlović1, Sandra Janežič3, Nijaz Tihić1, Zineta Delibegović1, Amela Bećirović1, Edina Muratović4, Merima Gegić1

1Institute of Microbiology, Polyclinic for Laboratory Diagnostics, University Clinical Centre Tuzla, 2School of Medicine, University of Tuzla; Tuzla, Bosnia and Herzegovina, 3National Laboratory for Health, Environment and Food (NLZOH), Centre for Medical Microbiol- ogy, Slovenia, 4Department of Microbiology, Primary Health Care Centre, Tuzla; Bosnia and Herzegovina

ABSTRACT

Aim To characterize methicillin-resistant S. aureus (MRSA) stra- ins phenotypically and genotypically and to determine their clonal affiliation, representation and antibiotic resistance profile.

Methods A total of 62 randomly selected MRSA isolates of diffe- rent clinical samples collected from 2009 to 2017 were phenotypi- cally and genotypically analysed. Phenotypic analyses were per- formed by standard microbiological procedures, and using VITEK 2/AES instrument as well as MALDI-TOF (matrix-assisted laser desorption/ionization) technology. Genotypic characterization included spa, MLST (multilocus sequence typing) and SCCmec typing, and detection of the Panton-Valentine leukocidin (PVL) Corresponding author: and other enterotoxin encoding genes. Fatima Numanović Results The largest number of isolates, 21 (33.87%) belonged Institute of Microbiology, to ST228-MRSA-I, spa type t041, t1003 and t001. Other major Polyclinic for Laboratory Diagnostics, clones were: ST239-MRSA-III, spa type t037 and t030 (27.41%); University Clinical Centre Tuzla ST8-MRSA-IV, spa type t008 and t121 (12.9%); ST247-MRSA-I, Prof. dr. Ibre Pašića bb, 75000 Tuzla, spa type t051 (4.83%). PVL was detected in 10 isolates (SCCmec Bosnia and Herzegovina IV/V). During 2009 and 2010 the most frequent MRSA strain was Phone: +387 35 303 564; South German clone, ST228-MRSA-I (80% and 90%, respective- ly), while in later years it was replaced with Brazilian-Hungari- E-mail: [email protected] an clone ST239-MRSA-III (75% in 2015 and 2016). The South ORCID ID https://orcid.org/0000-0003- German clone, spa type t041 in 90.48% of cases was resistant to 1194-2653 clindamycin, ciprofloxacin, erythromycin, cefoxitin, gentamicin, kanamycin, tobramycin and penicillin, while 70.58% samples of the Brazilian-Hungarian clone spa type t037 were additionally re- sistant to tetracycline and rifampicin. Original submission: Conclusion This research can supplement the existing knowledge 28 August 2020; about the clonal distribution of MRSA in Bosnia and Herzegovina Revised submission: and their sensitivity to antibiotics in order to improve the national 29 October 2020; control of these infections. Accepted: Keywords: antibiotic resistance, multi-locus sequence typing 23 November 2020 (MLST), SCCmec, spa typing doi: 10.17392/1265-21

Med Glas (Zenica) 2021; 18(1):38-46

38 Numanović et al. Molecular characterization of MRSA

INTRODUCTION ging. Antibiogram typing is the main method used in most hospital outbreaks since it is highly availa- Staphylococcus aureus (S. aureus) is the causa- ble and standardized, although with a disadvanta- tive agent of many human infections, including ge in resistance variability (11). The most reliable nosocomial ones (1). Antibiotic resistance incre- typing methods are pulse-field gel electrophoresis ases the complexity in the treatment of S. aureus (PFGE) and multilocus sequence typing (MLST). infections, especially infections caused by methi- PFGE, although the gold standard, is very deman- cillin-resistant S. aureus (MRSA) that developed ding, expensive and difficult to reproduce between methicillin-resistance by the acquisition of mecA laboratories (5). MLST is a preferred technique be- or mecC gene (2). cause data can be exchanged between laboratories. Initially, only hospital-associated MRSA (HA- First, MLST is used to group strains into sequence MRSA) infections were present, but in the 1990s, types (STs), and then closely related STs into clonal community-associated MRSA (CA-MRSA) was complexes (CCs). MLST is used in combination found to disseminate among healthy individu- with PCR analysis of SCCmec element for the de- als in Australia and the United States (3). Unlike termination of MRSA clonal types (12). HA-MRSA, CA-MRSA isolates were non multi- Moreover, spa typing is one of the least laborious drug resistant and genetically different from other and inexpensive MRSA typing methods based on MRSA strains present at that time (4). Generally, the DNA sequencing of the polymorphic X re- CA-MRSA is susceptible to narrow-spectrum gion of staphylococcal protein A (spa) (12). Spa non-beta lactams such as clindamycin, tetracycli- typing enables more precise analysis of MRSA nes and trimethoprim sulfamethoxazole, while strains, allowing the evolution of the molecular resistant to penicillin, oxacillin and erythromycin, epidemiology of MRSA to be examined (13). where HA-MRSA is multiresistant (4,5). The nomenclature of MRSA is currently based Gene(s) responsible for resistance to methicillin on the ST type and the type of SCCmec element. and other beta-lactam antibiotics are situated on The majority of HA-MRSA clones belong to five a mobile genetic element, the staphylococcal ca- phylogenetically distinct CCs: CC5 (ST5-I, -II, ssette chromosome mec (SCCmec). Seven major -IV, -VI; ST228-I), CC8 (ST 247-I, ST239-III, variants of SCCmec (I-VII) have been detected ST8-IV), CC22 (ST22-IV), CC30 (ST36-II) and (6). Generally, HA-MRSA harbours SCCmec CC45 (ST45-II, -IV) (6). During the last two de- type I-III, while CA-MRSA carries SCCmec type cades, several CA-MRSA clones have emerged IV, V or VII (7). In addition, CA-MRSA often worldwide: CC80 (ST80-IV), CC30 (ST30-IV), contains the genes encoding Panton-Valenti- CC8 (ST8-IV/USA300), CC1 (ST1-IV/USA400) ne leukocidin (PVL) toxin associated with skin and CC5 (ST59-IV/USA1000) (8,14). and soft tissue infections, sometimes considered a marker of CA-MRSA infections (6,7). Howe- In recent years, the major epidemiological chan- ver, in recent years, this genetic diversity betwe- ges have occurred to clonal replacement of en HA-MRSA and CA-MRSA has started to MRSA strains, shifts over time have been obser- fade, with no single distinguishing characteristic ved in countries, small regions within one coun- between them. PVL positive, multidrug-resistant try, or single hospitals (15). Therefore, typing of HA-MRSA carrying SCCmec IV/V, as well as a MRSA isolates and the understanding of clonal portion of multiresistant CA-MRSA strains have distribution at the local and international level is been reported (6-8). of great importance for controlling and monito- ring MRSA infections. The global epidemiology of MRSA is heterogeneo- us, where the dominance and presence of certain Since the molecular characterization of MRSA clones differ between geographic regions (9). Nu- is already known in other cantons of Bosnia and merous phenotypic and genotypic techniques are Herzegovina (B&H), we intended to complete used for monitoring and typing of MRSA isolates, these data with the results from our canton. with the notable advantage of the later ones (10). Se- The aim of this study was to phenotypically and lecting the most appropriate typing tool in terms of genotypically characterize MRSA strains in Tu- cost, performance and interpretation can be challen- zla Canton, B&H, and to determine their clonal

39 Medicinski Glasnik, Volume 18, Number 1, February 2021

affiliation, representation and antibiotic resistan- μg), trimethoprim/sulfamethoxazole (1.25/23.75 ce profile, as well as to compare genotypically μg), tetracycline (30 μg), chloramphenicol (30 determined MRSA clones with their antibiotic µg), mupirocin (200 µg) and tobramycin (10 sensitivity/resistance profile. µg). The zone of inhibition was read according to EUCAST Clinical Breakpoint Tables (2017) (3). PATIENTS AND METHODS Vancomycin sensitivity was performed by Vitek 2 antibiogram card 580 (Vitek 2 Compact, bi- Patients and study design oMérieux, France). Each cefoxitin-resistant iso- In this prospective study, 62 methicillin-resistant late was tested for the presence of PBP2A protein Staphylococcus aureus isolates were analysed phe- using an agglutination test (bioMerieux, France), notypically and genotypically, out of a total of 282 and Vitek 2 instrument (antibiogram card 580). MRSA isolates obtained from patients hospitalized Phenotyping confirmation was performed at the at the University Clinical Centre (UCC) Tuzla from National Laboratory for Health, Environment January 2009 to December 2017. The phenotypic and FOOD, Centre for , study was performed at the Institute of Microbio- Republic of Slovenia (NLZOH), using Matrix- logy, UCC Tuzla, while additional phenotypic and assisted laser desorption/ionization) (MALDI- genotypic analysis was conducted at the National TOF) technology (MALDI-TOF MS, Biotyper, Laboratory for Health, Environment and FOOD Bruker Daltonic GmBH, Bremen, Germany). (NLZOH), Centre for Medical Microbiology, Re- Molecular analysis of genes mecA, mecC, and public of Slovenia. Only the first isolate of each PVL was performed by PCR using GenoType patient was taken. The MRSA isolates were obtai- MRSA kit (Hain Lifescience, Germany). Genes ned from patients hospitalized in the Intensive Care encoding staphylococcal enterotoxins (sea, seb, Unit-Surgical Block, Department of Lung and Ge- sec, sed and see), toxic shock syndrome toxin neral Abdominal Surgery, Clinic for Orthopaedics (tst), locus enterotoxin gene cluster (egc) and and Trauma, Clinic for Internal Medicine, Clinic staphylococcal exfoliative toxins (eta, etb, etd) for , Clinic for Children’s Diseases, were detected by multiplex PCR (17). SCCmec Clinic for Skin Diseases and Clinic for Cardiovas- typing was performed using a multiplex PCR cular Surgery. The youngest patient was a neonate method as described previously (12). Amplifica- and the oldest was 86 years old. tion, sequencing and analysis of the polymorphic Ethics clearance and approval of the study were region of protein A (spa typing) were performed granted by the Ethical Committee of the Univer- according to the method described previously, sity Clinical Centre Tuzla. and analysed with Ridom SpaServer (18). STs were assigned based on spa types as published Methods on http://spaserver.ridom.de (8,19,20).

Isolation and identification of S. aureus were Statistical analysis performed by standard microbiological methods (16). Different clinical samples (aspirate, swabs Descriptive statistics, frequency, minimum and of wound, wound drainage, naval, skin, central maximum values and percentages were used. venous catheter, throat, vagina, ear, cannula, RESULTS and conjunctiva) were cultured on blood agar and glucose broth and incubated at 37 °C for The MRSA was mostly isolated from the patients 18-24 hours. Identification was performed by from the Intensive Care Unit-Surgical Block, 26 catalase and coagulase tests. For coagulase-po- (41.23%), and all of them obtained from aspira- sitive S. aureus, antibiotic sensitivity/resistance tes. MRSA was isolated in large numbers from by disk diffusion method was performed (3) to wound swabs in 10 (16.12%) patients and most the following antibiotics (Liofilchem, Italy): of them were hospitalized in the Clinic for Ortho- clindamycin (2 μg), ciprofloxacin (5 μg), eryt- paedics and Trauma, seven (70%). The smallest hromycin (15 μg), fusidic acid (10 μg), cefoxitin number of MRSA was isolated from swabs taken (30 μg), gentamicin (10 μg), kanamycin (30 μg), from throat, vagina, ear, cannula, conjunctiva and linezolid (10 μg), penicillin (units), rifampicin (5 abscess, one of each, from patients hospitalized at other clinics/departments.

40 Numanović et al. Molecular characterization of MRSA 2 4 1 3 4 8 2 7 5 62 26 Total 1 1 MRSA- IV spa type t11509 1 1 clone variant spa type t1003 South German 1 1 clone variant spa type t892 South German 3 1 1 1 spa type t005 and t1895 UK-EMRSA-5 1 1 128 spa type USA 400 USA 1 2 1 and 015 Berlin clone spa type t390 No of isolates 2 1 1 spa type t595 Balkan clone 2 2 clone European European spa type t359 1 3 1 1 Iberian spa type t051 North German / 8 2 2 1 1 1 1 and t121 USA 300 USA spa type t008 1 2 4 5 1 2 2 17 spa type t030 and t037 Hungarian / Bra - zilian 1 1 3 21 16 spa type t041, t1003 and t001 South German

(26) Other Clinics† (4) Other (other)* Total Cardiovascular Surgery (2) (CVC) Children’s Diseases Children’s (naval 2, skin 2) Skin Diseases (1) (skin) Intensive Care Unit, Intensive Care Surgical Block (aspirates) Lung Surgery (8) (wound 3, wound drainage 5) Abdominal General Surgery (2) (wound drainage) Orthopaedics and (7) (wounds) Trauma Internal Medicine (6) (wound 2, skin other* 1) (3) Neurosurgery (wounds) Table 1. Distribution of MRSA clones/types by Department/Clinic Department / Clinic (No of isolates) (sample type) Throat Clinic, Clinic for Pulmonary Diseases; CVC, central venous catheter cannula and conjunctiva; †Eye Clinic; Clinic for Gynaecology Obstetrics; Ear Nose *vagina, ear,

41 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 2. Distribution of MRSA clones by year No (%) of isolates during the year MRSA clone / spa type 2009 2010 2011 2012 2013 2014 2015 2016 2017 Total ST228-MRSA-I t041, t1003 and t001 4 (80) 9 (90) 1 (25) 3 (50) 2 (18.2) 1 (25%) 1 (12.5%) 21 (33.8) ST239-MRSA-III t030 and t037 1 (10) 2 (25) 2 (50) 1 (16.6) 1 (9) 3 (75) 6 (75) 1 (16.6) 17 (27.4) ST8-MRSA-IV t008 and t121 3 (37.5) 4 (36.4) 1 (12.5) 8 (12.9) ST247-MRSA I t051 1 (12.5) 2 (33.3) 3 (4.8) ST22-MRSA-IV t005 and t1895 1 (20) 2 (33.3) 3 (4.8) ST152/377-MRSA-V t595 1 (25) 1 (9) 2 (3.2) ST97-MRSA-IV t359 1 (12.5) 1 (16.6) 2 (3.2 ST45-MRSA-IV (NT) t390 and t015 2 (18.2) 2 (3.2) ST1-MRSA-IV t128 1 (16.6) 1 (1.6) ST111/228-MRSA-I t892 1 (12.5) 1 (1.6) ST1481-MRSA-I t1003 1 (9) 1 (1.6) MRSA-IV t11509 1 (16.6) 1 (1.6) Total 5 10 8 4 6 11 4 8 6 62 NT, non-typeable;

Typing of 62 MRSA isolates carrying mecA gene from 2017. ST228-MRSA-I clone was identified showed that the greatest number of isolates, 21 during 2009 and 2010 in four (out of five; 80%) (33.87%) belonged to ST228-MRSA-I, spa type and in nine (out of 10; 90%) cases, respectively t041, t1003 and t001 (South-German clone); 17 (Table 2). This apparent predominance of the (27.41%) were identified as ST239-MRSA-III, ST228-MRSA-I clone disappeared after 2010, spa type t037 and t030 (Brazilian-Hungarian clo- with a variety of other strains emerging. In 2010, ne), eight (12.9%) ST8-MRSA-IV, spa type t008 ST239-MRSA-III was detected for the first time, and t121 (USA300), three (4.83%) ST247-MR- in one (out of 10) isolate, and in 2015 and 2016 it SA-I, spa type t051 (North German strain/Ibe- was the most frequent clone, in three (out of four; rian clone), and three (4.83%) ST22-MRSA-IV, 75%) cases each year. spa type t005 (UK-EMRSA-15) and t1895. Two In 2011, ST8-MRSA-IV was detected in three (3.22%) isolates of each: ST152/377-MRSA-V, (out of 8; 37.5%) isolates, and again in 2014 in spa type t595 (Balkan clone), ST97-MRSA-IV, four (out of 11; 36.36%) isolates. ST247-MR- spa type t359 and ST45-MRSA-IV/NT (non- SA-I was also identified in 2011 in one (out of typeable), spa type t390 (Berlin clone) and t015 8; 12.5%) isolate, and the second time in 2017 in were found. There was a single (1.6%) isolate of two (out of 6; 33.3%) isolates (Table 2). each: ST1-MRSA-IV, spa type t128 (USA400); Antibiotic susceptibility testing showed that almost ST111/228-MRSA-I, spa type t892 (South-Ger- all 19 (out of 21; 90.48%) ST228-MRSA-I clo- man clone variant); ST1481-MRSA-I, spa type nes were resistant to clindamycin, ciprofloxacin, t1003 (South German clone variant); and MRSA- erythromycin, cefoxitin, gentamicin, kanamycin, IV, spa type t11509 (unclassified ST) (Figure 2). tobramycin, and penicillin (Table 3). Two (out Out of the total of 62 MRSA isolates, 26 (41.93%) of 21; 9.52%) ST228-MRSA-I, spa type t1003 were from the Intensive Care Unit-Surgical Block, and one ST1481-MRSA-I were also resistant to of which 16 (61.53%) belonged to the South-Ger- chloramphenicol and intermediately sensitive to man clone, 10 (16.12%) were from the Department mupirocin. ST239-MRSA-III was mostly resi- of Lung Surgery and General Abdominal Surgery stant to clindamycin, ciprofloxacin, erythromycin, (of which six belonged to the Brazilian-Hungarian cefoxitin, gentamicin, kanamycin, tobramycin, clone). There were seven isolates from the Clinic tetracycline, rifampicin, and penicillin, 12 (out of for Orthopaedics and Trauma that belonged to as 17; 70.58%). However, five isolates had different many as 5 different clones. Out of the total of four antibiotic susceptibility profiles, with three of the- MRSA obtained from patients from the Clinic for se (spa type 030) being resistant to trimethoprim/ Children’s Diseases, two (50%) belonged to the sulfamethoxazole and susceptible to erythromycin USA 300 clone (Table 1). and clindamycin, and two (spa type 037) suscepti- MRSA isolates for genotyping and phenotyping ble to tetracycline. were selected from different years: five (out of 70) from 2009, 10 (out of 57) 2010, eight (out ST8-MRSA-IV, spa type t008, was resistant to eryt- of 41) 2011, four (out of 20) 2012, six (out of hromycin, cefoxitin, kanamycin and penicillin in 22) 2013, 11 (out of 30) 2014, four (out of 21) six (out of 8; 75%) cases each, while two spa type 2015, eight (out of 11) 2016 and six (out of 10) t121 clones were also resistant to ciprofloxacin.

42 Numanović et al. Molecular characterization of MRSA

Table 3. Antibiotic susceptibility profile of genotyped MRSA clones Antibiotic MRSA clone CC CIP ER FA FOX GM K LZD P RA SXT Te† C MUP† NN† ST228-SCCmec I ST111/228 MRSA-I R R R S R R R S R S S S S/R S/R R ST1481-MRSA-I ST239-SCCmec III S/R R S/R S R S/R S/R S/R R S/R S/R S/R S/R S/R S/R ST8-SCCmec IV S S/R R S R S R S R S S S S S S ST247-MRSA R R R S R R R S R R S R S S R ST22-MRSA-IV S S/R§ S S R S S S R S S S S S S ST152/377-SCCmec V S S S S R R R S R S S R S S R ST97-SCCmec IV S S S S R S S S R S S S S S S ST45-MRSA-IV/NT‡ S S S S R S/R S/R S R S S S S S S/R ST1-SCCmec IV R S R S R S R S R S S R S S S MRSA-IV R S R S R S S S R S S S S S S spa type t11509 †susceptibility testing to chloramphenicol, tobramycin and mupirocin was performed only in Slovenia; ‡ST45-MRSA-NT clone (spa type t015) of non-typeable SCCmec type had a more resistant antibiotic profile (S/R = t390/t015);§ spa type t005 clone was susceptible to ciprofloxacin, whilespa type t1895 clones were resistant; CIP, ciprofloxacin (5 µg); E, erythromycin (15 µg); FA, fusidic acid (10 µg); FOX, cefoxitin (10 µg); GM, gentamycin (10 µg); K, kanamycin (30 µg); LZD, linezolid (10 µg); P, penicillin (1 µg); RA, rifampicin (5 µg); SXT, trimethroprim-sulfamethoxazole (5 µg); Te, tetracycline (30 µg); C, chloramphenicol (30 µg); MUP, mupirocin (200 µg); NN, tobramycin (10 µg); S, sensitive; R, resistant;

Out of 62 genotyped isolates, PVL was present In the current study, phenotypic and genotypic in ten, eight ST8-MRSA-IV and two ST152/377- analysis of 62 MRSA isolates showed that the most MRSA-V clones (Table 4). Enterotoxin A was frequent type was ST228-MRSA-I (South-Ger- found in 23 isolates, ten ST239-MRSA-III, nine man clone) (predominantly spa type t041; 18/21, ST228-MRSA-I, three ST247-MRSA-I and one 85.71%). During the 1990s, a new multi-resistant ST1481-MRSA-I clone. Ten isolates were positi- MRSA clone (gentamicin resistant), ST228-MR- ve for enterotoxin gene cluster (egc) G, I, M, N, SA-I, was discovered in Italian hospitals (22). It and O, while three of them were also positive for was also described in Germany among 1997-1998 enterotoxin C. None of the isolates were positive (although rifampicin sensitive). It emerged el- for TSST or exfoliative toxins. sewhere in Europe, and in Hungary it represented 28% of all MRSA isolates between 2001 and 2004 DISCUSSION (11,23). Until 2008, this clone was constantly pre- Although the prevalence of a reported MRSA sent but with some time and geographic variations. decline in Europe, MRSA still remains an impor- In Switzerland, an increasing number of infections tant pathogen, both in hospitals and the commu- caused by the ST228-MRSA-I was recorded from nity due to a high level of resistance to multiple 2008-2010, but the prevalence dropped signifi- classes of antibiotics (3). Typing of MRSA stra- cantly from 2010 to 2014 (24). Our data showed ins is a valuable tool for understanding and con- a predominance of the ST228-MRSA-I clone from trolling their transmission (21). 2009-2010, while its presence dramatically decli- ned after 2010. Two distinct antibiotic profiles of Table 4. Detection of toxins among MRSA strains ST228-MRSA-I clone detected in our study were Number of due to genetic differences in spa type t1003 that Toxin* MRSA strain Total isolates accounted for the resistance and decreased sen- ST-8-MRSA-IV 8 PVL 10 sitivity to chloramphenicol and mupirocin, res- ST-152/377-V 2 pectively. Increasing mupirocin resistance among ST228-MRSA-I 9 ST239-MRSA-III 10 MRSA isolates has been associated with an incre- Enterotoxin A 23 ST247-MRSA-I 3 ased risk of staphylococcal infections and a failure ST1481-MRSA-I 1 to control MRSA transmission especially in a he- ST228-MRSA-I 4 ST45-MRSA-IV 1 althcare setting (25). egc (G, I, M, N, O) ST22-MRSA-IV 3 10 ST1481-MRSA-I 1 In accordance with our data, reports from neighbou- MRSA-IV, spa type t11509 2 ring Slovenia showed a high prevalence of ST228- ST45-MRSA-NT 1 Enterotoxin C 3 MRSA-I (spa type t041, t003 and t001) between ST22-MRSA-IV 2 2006 and 2007 (26). In the Cantonal Hospital Ze- *No toxins were detected in 16 MRSA isolates; PVL, Panton-Valenti- ne leukocidin; egc, enterotoxin gene cluster; NT, non-typeable nica, out of the total of 23 MRSA isolates obtained from neonates and children up to one year of age,

43 Medicinski Glasnik, Volume 18, Number 1, February 2021

87% belonged to spa type t355 (MLST CC152) coming extremely rare in regions it was previously (27). Also, spa type t041 was the most common readily detected. Not surprisingly, only three isola- spa type detected among isolates from the Clinical tes of this clone were detected in our study. Hospital Centre Zagreb, Croatia, while t001 was One of the most surprising moments in dealing the most common isolate in Mostar (B&H) (28). with MRSA infections in recent years has been the The analysis of 248 MRSA isolates from 20 Cro- emergence of CA-MRSA strains (6). Prior to 2001, atian cities in 2004 showed that most isolates be- the most common CA-MRSA was ST1-MRSA- longed to ST111-MRSA-I (ST228-MRSA-I vari- IV (USA400 clone), the first known PVL-positive ant) (29). We have identified one ST111-MRSA-I MRSA (5). Afterwards, a new CA-MRSA clone, isolate in 2011, and a different variant in 2014 ST8-MRSA-IV (USA300), has emerged (37). Sin- (ST1481-MRSA-I). ce CA-MRSA classification cannot be based so- The second most frequent MRSA clone described lely on genotype, without additional information in our study was ST239-MRSA-III spa type t037 none of our clones can be classified with certainty and t030 (17/62, 27.4%). This clone, although pre- as CA-MRSA (4). Our major potential CA-MR- sent among isolates in 2010, became predominant SA isolate was ST8-MRSA-IV (spa type t008 and only later, in 2015 and 2016. ST239-MRSA-III clo- t121), while others belonged to ST152/377-MR- ne was at first described in the late 1970s and early SA-V and ST1-MRSA-IV. In recent years, ST8- 1980s in Australia, Great Britain and South Ameri- MRSA-IV (spa type t008) was the predominant ca as resistant to gentamicin (30). ST239-MRSA-III type of CA-MRSA in the United States (38). A clone at first emerged in Brazil, subsequently spre- large multicentre study from 16 European countri- ad to South American and European countries, and es also showed the prevalence of ST8-MRSA-IV became the most common MRSA clone between clone among CA-MRSA isolates (39). 1994 to 2008 (31). All isolates of this clone were re- ST8-MRSA-IV is a notable PVL-positive MRSA sistant to ciprofloxacin, erythromycin, lincomycin, clone, with the most common spa types t008 and tetracycline, and trimethoprim/sulfamethoxazole. t002 (38,40). All eight of our ST8-MRSA-IV iso- In China, isolates of ST239-MRSA-III clone were lates harboured the PVL gene, while the remai- also resistant to clindamycin and gentamycin (32). ning two PVL-positive isolates were ST152/377- MRSA-V. ST152 clone has often been associated Studies showed that the ST239-MRSA-III clone with Balkan countries and reported to carry the had undergone significant changes; thus, in the PVL gene (41). It cannot be distinguished from pre-2000 period spa type t037 was dominant and clone ST377 using standard MLST primers, hen- was rapidly replaced by spa type t030. Unless con- ce the ST152/377 classification. All other toxins trol measures are taken, due to a faster growth rate tested (enterotoxin A and C, egc) were expressed of spa type 030, other countries or continents may by strains usually defined as HA-MRSA (17). also experience broad dissemination of this mul- ti-antibiotic-resistant MRSA clone (33). Our data Interestingly, ST97-MRSA-IV detected in our stu- showed that, ST239-MRSA-III, spa type t037 clo- dy has usually been isolated from cattle and rarely ne was still the dominant type, while ST239-MR- from humans (8). However, without additional in- SA-III, spa type 030 was detected in only three formation, this clone cannot be classified as either isolates (in 2012 and 2014). As expected, the two community- or livestock-associated (LA-MRSA). spa types differed in antibiotic profiles, with spa Due to economic reasons, only 62/282 of our iso- type t030 exhibiting resistance or decreased sen- lates were randomly characterized genotypically. sitivity to both rifampicin and trimethoprim/sulfa- Although, somewhat ambiguous due to atypical methoxazole. In contrast to our study, no SCCmec and overlapping profiles, phenotypic methods III isolates were detected by PFGE analysis in iso- can still be used for the classification of MRSA lates from Sarajevo, B&H (34). isolates with typical antibiograms (42). ST247-MRSA-I is an ancient strain, the first reco- One of the possible shortcomings of our research vered in the UK (also known as North German and could be the small number of samples in relation Iberian strain; Archaic/Iberian clone). It is respon- to the examined period. We could not avoid this sible for outbreaks in Barcelona in 1989, and more possible shortcoming due to the limited number recently it had been detected in Australia, Croatia, of samples we could process genotypically. Czech Republic and Italy (29,35,36). However, In conclusion, by analysing our isolates over a ST247-MRSA-I seems to be receding and it is be- long period of time, we confirmed that the clo-

44 Numanović et al. Molecular characterization of MRSA

nal distribution of MRSA is very variable. In ACKNOWLEDGEMENTS Bosnia and Herzegovina different clonal types We thank to the staff at the Institute of Microbi- dominate depending on whether MRSA infecti- ology, UCC Tuzla and Institute of Microbiology on is acquired in a hospital or outpatient setting. (B&H) and the National Laboratory for Health, The antibiotic profile of individual clonal types Environment and Food (NLZOH), Centre for is largely uniform and as such is useful in epide- Medical Microbiology, Republic of Slovenia for miological monitoring of MRSA and in smaller their assistance in performing tests. communities. In our study, we also demonstrated that the clonal types of MRSA that are potentially FUNDING associated with outpatient setting are producers of different types of toxins suggesting even grea- No specific funding was received for this study. ter importance of characterizing MRSA through TRANSPARENCY DECLARATION phenotypic and genotypic studies. Competing interests: None to declare.

REFERENCES 9. Seidl K, Leimer N, Palheiros Marques M, Furrer A, Holzmann-Bürgel A, Senn G, Zbinden R, Zinker- 1. Dayan GH, Mohamed N, Scully IL, Cooper D, Begier nagel AS. Clonality and antimicrobial susceptibility E, Eiden J, Jansen KU, Gurtman A, Anderson AS. of methicillin-resistant Staphylococcus aureus at the Staphylococcus aureus: the current state of disease, University Hospital Zurich, Switzerland between pathophysiology and strategies for prevention. Expert 2012 and 2014. Ann Clin Microbiol Antimicrob 2015; Rev Vaccines 2016; 15:1373-92. 14:14. 2. Peacock SJ, Paterson GK. Mechanisms of methicillin 10. Miao J, Chen L, Wang J, Wang W, Chen D, Li L, Li resistance in Staphylococcus aureus. Annu Rev Bio- B, Deng Y, Xu Z. Current methodologies on genoty- chem 2015; 84:577-601. ping for nosocomial pathogen methicillin-resistant 3. European Centre for Disease Prevention and Control Staphylococcus aureus (MRSA). Microb Pathog (ECDC). Surveillance report: Surveillance of anti- 2017; 107:17-28. microbial resistance in Europe 2017. Annual report 11. Mick V, Domínguez MA, Tubau F, Liñares J, Pujol M, of the European Antimicrobial Resistance Surveillan- Martín R. Molecular characterization of resistance to ce Network (EARS-Net). Stockholm: ECDC; 2018. Rifampicin in an emerging hospital-associated met- https://www.ecdc.europa.eu/en/publications-data/ hicillin-resistant Staphylococcus aureus clone ST228, surveillance-antimicrobial-resistance-europe-2017 Spain. BMC Microbiol 2010; 10: 68. (18 August 2020) 12. Tokajian S. Molecular typing of Staphylococcus aure- 4. Parvez MAK, Ferdous RN, Rahman MS, Islam S.J. us: understanding and controlling epidemic spread. J Healthcare-associated (HA) and community-associa- Forensic Res 2012; 3:e104. ted (CA) methicillin resistant Staphylococcus aureus 13. Goudarzi M, Fazeli M, Goudarzi H, Azad M, Seyedja- (MRSA) in Bangladesh - source, diagnosis and tre- vadi SS. Spa typing of Staphylococcus aureus stra- atment. Genet Eng Biotechnol 2018; 16:473-8. ins isolated from clinical specimens of patients with 5. Rebić V, Budimir A, Aljičević M, Bektaš S, Mahmu- nosocomial infections in Tehran, Iran. Jundishapur J tović Vranić S, Rebić D. Typing of methicillin resi- Microbiol 2016; 9:e35685. stant Staphylococcus aureus using DNA fingerprints 14. Dabul AN, Camargo IL. Clonal complexes of by pulse-field gel electrophoresis. Acta Inform Med Staphylococcus aureus: all mixed and together. FEMS 2016; 24:248-52. Microbiology Letters 2014; 351:1 7–8. 6. Liu J, Chen D, Peters BM, Li L, Li B, Xu Z, Shirliff 15. Zarfel G, Luxner J, Folli B, Leitner E, Feierl G, Kittin- ME. Staphylococcal chromosomal cassettes mec ger C, Grisold A. Increase of genetic diversity and (SCCmec): a mobile genetic element in methicillin- clonal replacement of epidemic methicillin-resistant resistant Staphylococcus aureus. Microb Pathog Staphylococcus aureus strains in South-East Austria. 2016; 101:56-67. FEMS Microbiology Letters 2016; 363:14. 7. Dhawan B, Rao C, Udo EE, Gadepalli R, Vishnubha- 16. Murray PR. Manual of Clinical Microbiology. 8th ed. tla S, Kapil A. Dissemination of methicillin-resistant Washington, D. C: American Society for Microbio- Staphylococcus aureus SCCmec type IV and SCC- logy Press, 2003. mec type V epidemic clones in a tertiary hospital: 17. Chiefari AK, Perry MJ, Kelly-Cirino C, Egan CT. De- challenge to infection control. Epidemiol Infect 2015; tection of Staphylococcus aureus enterotoxin produc- 143:343-53. tion genes from patient samples using an automated 8. Monecke S, Coombs G, Shore AC, Coleman DC, Akpa- extraction platform and multiplex real-time PCR. Mol ka P, Borg M, Chow H, Ip M, Jatzwauk L, Jonas D, Cell Probes 2015; 29:461-7. Kadlec K, Kearns A, Laurent F, O’Brien FG, Pearson 18. Harmsen D, Claus H, Whitte W, Rothganger J, Claus J, Ruppelt A, Schwarz S, Scicluna E, Slickers P, Tan H, Turnwald D, Vogel U. Typing of methicillin-resi- HL, Weber S, Ehricht R. A field guide to pandemic, stant Staphylococcus aureus in a university hospital epidemic and sporadic clones of methicillin-resistant setting by using novel software for spa repeat deter- Staphylococcus aureus. PLoS ONE 2011; 6:e17936. mination and database management. J Clin Microbiol 2003; 41:5442-8.

45 Medicinski Glasnik, Volume 18, Number 1, February 2021

19. Budimir A, Deurenberg RH, Bošnjak Z, Stobberingh 30. Pavillard R, Harvey K, Douglas D, Hewstone A, EE, Ćetković H, Kalenić S. A variant of the Southern Andrew J, Collopy B, Asche V, Carson P, Davidson German clone of methicillin-resistant Staphylococcus A, Gilbert G, Spicer J, Tosolini F. Epidemic of hos- aureus is predominant in Croatia. Clin Microbiol In- pital-acquired infection due to methicillin-resistant fect 2010; 16:1077-83. Staphylococcus aureus in major Victorian hospitals. 20. Mediavilla JR, Chen L, Mathema B, Kreiswirth BN. Med J Aust 1982; 29:451-4. Global epidemiology of community-associated met- 31. Aires de Sousa MA, Sanches IS, Ferro ML, Vaz MJ, hicillin resistant Staphylococcus aureus (CA-MR- Saraiva Z, Tendeiro T, Serra J. de Lencastre H. In- SA). Curr Opin Microbiol 2012; 15:588-95. tercontinental spread of a multidrug-resistant methi- 21. Mehndiratta PL, Bhalla P. Typing of methicillin re- cillin-resistant Staphylococcus aureus clone. J Clin sistant Staphylococcus aureus: A technical review. Microbiol 1998; 36:2590-6. Indian J Med Microbiol 2012; 30:16-23. 32. Kong H, Yu F, Zhang W, Li X, Wang H. Molecular 22. Mato R, Campanile F, Stefani S, Crisóstomo MI, San- epidemiology and antibiotic resistance profiles of tagati M, Sanches SI, de Lencastre H. Clonal types methicillin-resistant Staphylococcus aureus strains and multidrug resistance patterns of methicillin-re- in a tertiary hospital in China. Front Microbiol 2017; sistant Staphylococcus aureus (MRSA) recovered 8:838. in Italy during the 1990s. Microb Drug Resist 2004; 33. Shang W, Hu Q, Yuan W, Cheng H, Yang J, Hu Z, 10:106-13. Yuan J, Zhang X, Peng H, Yang Y, Hu X, Li M, hu J, 23. Wisplinghoff H, Ewertz B, Wisplinghoff S, Stefanik Rao X. Comparative fitness and determinants for the D, Plum G, Perdreau-Remington F, Harald S. Mo- characteristic drug resistance of ST239-MRSA-III- lecular evolution of methicillin-resistant Staphylo- t030 and ST239-MRSA-III-t037 strains isolated in coccus aureus in the metropolitan area of Cologne, China. Microb Drug Resist 2016; 22:185-92. Germany, from 1984 to 1998. J Clin Microbiol 2005; 34. Rebić V, Budimir A, Aljičević M, Bektaš S, Mahmu- 43:5445-51. tović Vranić S, Rebić D. Typing of methicillin resi- 24. Senn L, Clerc O, Zanetti G, Basset P, Prod’hom G, stant Staphylococcus aureus using DNA fingerprints Gordon NC, Sheppard AE, Crook DW, James R, by pulse-field gel electrophoresis. Acta Inform Med Thorpe HA, Feil EJ, Blanc DS. The stealthy super- 2016; 24:248-52. bug: the role of asymptomatic enteric carriage in 35. Campanile F, Bongiorno D, Borbone S, Stefani S. maintaining a long-term hospital outbreak of ST228 Hospital-associated methicillin-resistant Staphylo- methicillin-resistant Staphylococcus aureus. MBio coccus aureus (HA-MRSA) in Italy. Ann Clin Micro- 2016; 19:e02039-15. biol Antimicrob 2009; 8:22. 25. Simor AE, Stuart TL, Louie L, Watt C, Ofner-Ago- 36. Melter O, Aires de Sousa M, Urbaskova P, Jakubu V, stini M, Gravel D, Mulvey M, Loeb M, McGeer A, Zemlickova H, de Lencastre H. Update on the major Bryce E, Matlow A. Canadian Nosocomial Infecti- clonal types of methicillin-resistant Staphylococcus on Surveillance Program. Mupirocin-resistant, met- aureus in the Czech Republic. J Clin Microbiol 2003; hicillin-resistant Staphylococcus aureus strains in 41:4998–5005. Canadian hospitals. Antimicrob Agents Chemother 37. Carrel M, Perencevich EN, David MZ. USA300 met- 2007; 51:3880–6. hicillin-resistant Staphylococcus aureus, United Sta- 26. Cvitković Špik V, Grmek Košnik I, Lorenčič Robnik tes, 2000–2013. Emerg Infect Dis 2015; 21:1973-80. S, Žohar Čretnik T, Sarjanović Lj, Kavčič M, Har- 38. Diekema DJ, Richter SS, Heilmann KP, Dohrn CL, lander T, Štrumbelj I, Tomič V, Vajdec Piltaver I, Riahi F, Tendolkar S, McDanel JS, Doern CL. Con- Fišer J, Kolman J, Premru Mueller M. Genetic char- tinued emergence of USA300 methicillin-resistant acterization of methicillin-susceptible and methicil- Staphylococcus aureus in the United States: results lin-resistant Staphylococcus aureus strains isolated from a nationwide surveillance study. Infect Control from bloodstream infections in Slovene hospitals us- Hosp Epidemiol 2014; 35:285-92. ing spa typing. Zdrav Var 2008; 48:78-84. 39. Rolo J, Miragaia M, Turlej-Rogacka A, Empel J, 27. Uzunović S, Bedenić B, Budimir A, Kamberović F, Bouchami O, Faria NA, Tavares A, Hryniewicz W, Ibrahimagić A, Delić-Bikić S,·Sivec S, Meštrović, Fluit AC, Lencastre H. CONCORD Working Group. Varda Brkić D, Rijnders MI, Stobberinghet EE. High genetic diversity among community-associated Emergency (clonal spread) of methicillin-resistant Staphylococcus aureus in Europe: results from a mul- Staphylococcus aureus (MRSA), extended spectrum ticenter study. PLoS One 2012; 7:e34768. (ESBL)--and AmpC beta-lactamase-producing Gram- 40. Carrel M, Perencevich EN, David MZ. USA300 met- negative bacteria infections at Pediatric Department, hicillin-resistant Staphylococcus aureus, United Sta- Bosnia and Herzegovina. Wien Klin Wochenschr tes, 2000–2013. Emerg Infect Dis 2015; 21:1973-80. 2014; 126:747-56. 41. Dermota U, Mueller-Premru M, Švent-Kučina N, 28. Ostojić M, Hukić M. Genotyping and phenotyping Petrovič Ž, Ribič H, Rupnik M, Janežič, Zdovc I, characteristics of Methicillin-resistant Staphylo- Grmek-Košnik I. Survey of community-associated- coccus aureus (MRSA) strain, isolated on three diffe- methicillin-resistant Staphylococcus aureus in Slove- rent geography locations. Bosn J Basic Sci 2015; nia: Identification of community-associated and live- 15:48-56. stock-associated clones. Int J Med Microbiol 2015; 29. Budimir A, Deurenberg RH, Bošnjak Z, Stobberingh 303:505-10. EE, Ćetković H, Kalenić S. A variant of the Southern 42. Bazzi AM, Al-Tawfiq JA, Rabaan AA, Neal D, Ferra- German clone of methicillin-resistant Staphylococcus ro A, Fawarah MM. Antibiotic based phenotype and aureus is predominant in Croatia. Clin Microbiol In- hospital admission profile are the most likely pre- fect 2010; 16:1077-83. dictors of genotyping classification of MRSA. Open Microbiol J 2017; 11:167-78.

46 ORIGINAL ARTICLE

Efficacy and safety of three plant extracts based formulations of vagitories in the treatment of vaginitis: a randomized controlled trial Kemal Durić1, Selma Kovčić Hadžiabdić2, Mahira Durić3, Haris Nikšić1, Alija Uzunović4, Hurija Džudžević Čančar1

1Department of Pharmacognosy, Faculty of Pharmacy, University of Sarajevo, Sarajevo, 2Faculty of Pharmacy, University of Tuzla, Tu- zla, 3Institute for Health Protection of Woman and Maternity of Canton Sarajevo, Sarajevo, 4Agency for Medicinal Products and Medical Devices, Sarajevo; Bosnia and Herzegovina

ABSTRACT

Aim There are more and more herbal preparations that are used for the purpose of treatment and improvement of the clinical ma- nifestation of vaginitis not only by patients themselves, but also by healthcare professionals. Plant species, St. John’s wort, chamomi- le, calendula, yarrow, shepherd’s purse and tea tree oil are all well known for their anti-inflammatory, antimicrobial and wound healing activity. This paper presents the results of a clinical study in which three herbal formulations/vagitories, based on extracts of St. John’s wort, chamomile, calendula, yarrow, shepherd’s purse and tea tree oil, were investigated for their effectiveness on vaginitis. Corresponding author: Kemal Durić Methods This was a randomized controlled clinical study that Faculty of Pharmacy, included 210 women with diagnosed vaginitis. Patients were di- vided into two basic groups, women in reproductive period and University of Sarajevo postmenopausal period. Three subgroups including 30 patients Zmaja od Bosne 8, 71000 Sarajevo, each received one of the three vagitorie formulations for 5 days, Bosnia and Herzegovina after which the effects on subjective and objective symptoms were Phone: + 387 33 586171; monitored. Fax: +387 33 586171; Results Three types of vagitories based on plant extracts had a E-mail: [email protected] positive effect in the treatment of vaginitis. Vagitories based on tea ORCID ID: https://orcid.org/0000-0002- tree oil showed better efficiency compared to vagitories with St. 7613-9178 John’s wort and vagitories based on extracts of five plants. Women in postmenopausal group reported better tolerability of St. John’s Policy of trial registration: ClinicalTrial.gov wort-based and five herbs-based vagitories compared to tea tree - Registration number NCT04558697 oil based vagitories.

Conclusion Investigated vagitories showed a positive effect on Original submission: both objective and subjective symptoms of vagitnis. No serious 26 August 2020; side effects were reported. Revised submission: 28 September 2020; Key words: calendula, chamomile, tea tree oil, St. John’s wort, vaginal inflammation Accepted: 01 November 2020 doi: 10.17392/1261-21

Med Glas (Zenica) 2021; 18(1):47-54

47 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION patients (adult women who already had sexual intercourse) selected among 1205 women pre- Vaginitis is the most common reason for visiting a sented to the Institute for Health Protection of gynaecologist and includes a range of symptoms Woman and Maternity of Canton Sarajevo with related to the lower genital tract and occurs be- symptoms of vaginitis during the period March cause of infection, irritation, or a systemic to June 2019. Exclusion criteria were microbi- disease (1). The most significant symptoms that ologically confirmed bacterial vaginosis, tre- characterize vaginitis are changes in the amount, atment with antibiotic therapy according to offi- colour, odour and pH of vaginal discharge, itching, cial protocols and guidelines, to one annealing, irritation, redness, poor bleeding, pain- of the plant species included in the vaginal test, ful sexual intercourse, painful and frequent urina- diabetes mellitus, pregnancy, lactation, immu- tion (2,3). Disorders in the vaginal environment nodeficiency disorder, severe chronic illness, can allow the proliferation of pathogens that lead previous radiotherapy and chemotherapy, and to inflammation of the vaginal mucosa. Antibiotics, biological therapy. contraceptives, sexual intercourse, stress and hor- mones (e.g. hormone replacement therapy, HRT) After signing up an informed consent for volun- can lead to overgrowth of pathogens (4). Chemi- tary participation in the study, all women com- cal vulvovaginitis can be caused by coloured and pleted a survey form that included questions re- perfumed soap, toilet paper, irrigators, cartridges, garding their reproductive age, number of births, tampons (5). Vulvovaginitis can simply be a res- life habits, and sexual behaviour. ponse to the current period of body imbalance such The study was approved by the Ethics Com- as stress at school, at work, at home, overuse of mittee for scientific research of the University of sugar, alcoholic beverages, increased sexual acti- Tuzla, Bosnia and Herzegovina (No: 03/7-1441- vity (5). In such cases, a simple lifestyle change 2/19; 2019) with appropriate drug applications locally may be The patients were divided into two basic grou- adequate treatments (6). Recurrent vulvovaginitis ps: women in reproductive period (Group 1) and may be part of a broader picture of chronic lifestyle women in postmenopausal period (Group 2). In imbalance, which may be underlying conditions further triage, each group (Group 1 and Group 2) for vaginal flora disorders (6). were divided into a control group that included Previous experience and clinical research suggest 15 patients with a confirmed diagnosis of vagini- that women tend to look over the herbal prepara- tis and treated with vaginal probiotics, and three tions and alternative therapies for the treatment of experimental subgroups treated with one of the vaginal infections and vaginitis (7). For many of three tested types of plant extract based plant- these preparations there is a scientific justification based extract vagitories. The allocation sequence for use, while others are still in the research phase was conducted by a person not involved in the of investigation (8). In general, plant material con- patient recruitment. taining essential oils (9), polyphenols (10), flavo- noids (11), tannins and phenyl carbon acid deriva- Methods tives (12) showed tendency to reduce or eliminate Intervention. Vagitories were prepared at the the factors that stimulate infection or reproduction laboratory Galas Sarajevo. The plant of pathogens, restore normal vaginal environment material, Calenduale flos, Capsellae bursae-pa- and flora, and relieve the symptoms of vaginitis. storis herba, Matricarae flos, Hyperici herba and The aim of this study was to investigate efficacy Millefolii herba, each separately, were soaked in and safety of different types of vagitories prepa- olive oil and macerated for 5 days at room tem- red with extracts of St. John’s wort, shepherd’s perature. Macerate was separated by draining and purse, pot marigold, chamomile, yarrow and tea pressing of and allowed to stand for two days, tree essential oil in the treatment of vaginitis. then filtered. The resulting five extracts were PATIENTS AND METHODS used for a preparation of vagitories. Tea tree oil, chemotype containing at least 30% of terpinen- Patients and study design 4-ol, was also used for preparation of vagitories. This interventional, randomized, controlled, cli- Three types of formulations were prepared for nical trial with four parallel arms included 210 the purpose of the clinical study:

48 Durić et al. Botanicals in the treatment of vaginitis

formulation serial number 1460219 (Vagitories RESULTS A) containing Calendulae extractum oleosum A total of 1205 woman who reported having 5.5% w/w, Bursae pastoris extractum oleosum symptoms related to vaginitis were assessed for 5.5% w/w, Matricariae extractum oleosum 5.5% eligibility criteria, of which 995 were excluded w/w, Hyperici extractum oleosum 5.5% w/w for various reasons (vaginitis associated with and Millefolii extractum oleosum 5.5% w/w as pathogen microorganisms, no objective signs of an active component; formulation serial number vaginitis, or not eligible for inclusion). 0511118 (Vagitories B) containing tea tree oil, A total of 210 woman were eligible for the study, 200 mg per each vagitorie as an active compo- and they randomized in two main groups, each nent: formulation serial number 0650119 (Vagi- of them divided into subgroups assigned to inter- tories C) containing Hyperici extractum oleosum ventions (Figure 1). 32% w/w as an active component. The duration of the therapy was 5 days, asking participants to use one vagitorie before going to bed. At the end of the therapy, the patients came back for a pre-arranged check-up, at which a gyna- ecologist determined the degree of changes (de- crease) in the monitored subjective complaints and objective parameters of vaginal inflammation. Outcomes and data collection. The primary outco- me was measured by a change in objective symp- toms of non-specific vaginitis, assessed by gyna- ecological examination. The clinical symptoms including redness, hyper-secretion, local oedema, pain and annealing were diagnosed at the baseline and 5 days after the beginning of the treatment. The frequency of subjective symptoms of vagi- nal inflammation reported by participants, inclu- ding vaginal secretion, itching and dryness of the Figure 1. Flow chart of study vaginal mucosa were monitored and evaluated according to the given scale as well. No significant difference between the number of women who were in a permanent relationship and According to patients’ responses to the questi- who smoked in relation to vaginal inflammation. ons included in the survey form, a correlation of However, there was a significantly higher num- vaginal inflammation with reproductive age, life ber of premenopausal women who occasionally habits and sexual behaviour was also evaluated. consumed alcohol than postmenopausal ones, 21 A very important secondary outcome was the (18.9%) and eight (8.9%), respectively (p=0.04). occurrence of possible side effects during the the- Most women, 109 (94.5%) reported up to two rapy. Patients were asked to report any side effects partners, while one (0.5%) reported more than as well as the gynaecologist who performed the 10 partners. examination after the end of the treatment period. The majority of the women did not use any con- Statistical analysis traceptives, 150 (75.0%). The most commonly used contraceptive was a condom, reported by 21 Statistical analysis was performed using an in- (10.2%) women. tention-to-treat approach. A χ2 test was used for Regardless of menopausal period, 95 (46.3 %) a comparison of the proportion of the symptoms woman reported that they had bacterial vaginosis and clinical signs between the groups, at the ba- before, while 24 (11.7%) had no previous disease seline and after the treatment. For comparison of and 41 (20.0%) did not know (p<0.004). pre- and post- intervention scores within the gro- Behind bacterial vaginosis, Trichomonas sp. was ups, T-test was used. The p< 0.05 was considered the next most frequently reported cause of the in- statistically significant. fection, in 19 (9.3%) women (p<0.004).

49 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 1. Changes in objective symptoms of inflammation at baseline and at 5 days post intervention follow-up in women in the reproductive period No (%) of women in reproductive period Symptom Vagitories A Vagitories B Vagitories C Vagitories-Probiotic Before (N=25) After (N=25) Before (N=49) After (N=49) Before (N=22) After (N=22) Before (N=17) After (N=17) Redness Yes 18 (72) 4 (22.2) 46 (93.9) 2 (4.3) 20 (90.9) 4 (20) 15 (88.2) 4 (26.7) No 7 (28) 14 (77.8) 3 (6.1) 44 (5.7) 2 (9.1) 16 (80) 2 (11.8) 11 (73.3) p <0.01 <0.001 <0.001 p<0.013 Vaginal secretion Yes 21 (84.0) 11 (52.4) 47 (95.9) 3 (6.4) 21 (95.5) 15 (71.4) 17 (100) 15 (88.2) No 4 (16.0) 10 (47.6) 2 (4.1) 44 (93.6) 1 (4.5) 6 (28.6) 0 2 (11.8) p <0.002 <0.001 <0.01 <0.25 Oedema Yes 11 (44.0) 1 (9.1) 29 (59.2) 0 16 (72.7) 3 (18.8) 9 (52.9) 6 (66.7) No 14 (56.0) 10 (90.9) 20 (40.8) 29 (100) 6 (27.3) 13 (81.2) 8 (47.1) 3 (33.3) p <0.06 <0.001 <0.01 <0.03 Pain Yes 9 (36.0) 1 (11.1) 19 (38.8) 0 10 (45.5) 4 (40.0) 5 (29.4) 4 (80.0) No 16 (64.0) 8 (88.9) 30 (61.2) 19 (100) 12 (54.5) 6 (60.0) 12 (70.6) 1 (20.0) p <0.04 <0.01 <0.02 <0.07 Bacterial vaginosis was reported in 52 (46.4%) vaginal secretion, 167 (81.9%) (p<0,001) and women in the reproductive period and 43 itching, 121 (59.3%) (p<0.04). Considering the (48.3%) woman in the postmenopausal period menopausal period, major subjective symptoms (p<0.004). A significantly higher number of in women in the reproductive period were vagi- women in the reproductive period had tricho- nal secretion, 107 (94.7%) (p<0.001) and itching moniasis (p<0.004) and human papilloma virus 70 (62.0%) (p<0.04), while in the postmenopau- (HPV), 15 (13.4%) and four (4.5%), respecti- sal group, besides vaginal secretion and itching, vely (p<0,004) compared to women in the pos- vaginal dryness was also a significant subjective tmenopausal period (Table 1, Table 2). symptom, 27 (29.7%) (p<0.001). Vagitories B showed the best activity in reduc- Regardless of the menopausal period, the frequ- tion of all objective symptoms in the group of ency of major subjective symptoms at 5 days women in the reproductive period (from 93.6% post intervention follow up were significantly to 100%) (p<0.001). Vagitories A and C showed lower in the groups with vagitorie formulations, better reduction of the mucosal oedema in the compared to probiotic vagitories. Vaginal secre- postmenopausal group, compared to Vagitories B tion, as the most commune symptom in women (100% vs. 66.7%) (p<0.001). in the reproductive period, was reduced in 44 The most frequent subjective symptoms at ba- (93.6%) women in the group with Vagitories B, seline, regardless of menopausal status, was 10 (47.6%) in the group with Vagitories A, six

Table 2. Changes in objective symptoms of inflammation at baseline and at 5 days post intervention follow-up in women in the postmenopausal period N (%) of women in postmenopausal period Symptom Vagitories A Vagitories B Vagitories C Vagitories-Probiotic Before (N=35) After (N=35) Before (N=13) After (N=13) Before (N=29) After (N=29) Before (N=15) After (N=15) Redness Yes 33 (94.3) 7 (21.2) 10 (77.0) 0 23 (79.3) 3 (13.0) 12 (80.0) 6 (50.0) No 2 (5.7) 26 (78.8) 3 (23.0) 10 (100) 6 (20.7) 20 (87.0) 3 (20.0) 6 (50.0) p <0.001 <0.01 <0.001 <0.003 Vaginal secretion Yes 20 (57.1) 11 (55.0) 10 (76.9) 0 29 (100) 15 (51.7) 10 (66.6) 5 (50.0) No 15 (42.9) 9 (45.0) 3 (23.1) 10 (100) 0 14 (48.3) 5 (33.4) 5 (50.0) p <0.01 <0.01 <0.01 <0.5 Oedema Yes 18 (51.4) 0 3 (23.1) 1 (33.4) 12 (41.4) 0 8 (53.3) 6 (75.0) No 17 (58.6) 18 (100) 10 (76.9) 2 (66.6) 17 (58.6) 12 (100) 7 (46.7) 2 (25.0) p <0.01 <0.04 <0.001 <0.001 Pain Yes 19 (54.3) 0 2 (15.4) 0 13 (44.8) 1 (7.7) 8 (53.3) 3 (37.5) No 16 (45.7) 19 (100) 11 (84.6) 2 (100) 16 (55.2) 12 (92.3) 7 (46.7) 5 (62.5) p <0.01 <0.8 <0.01 <0.01

50 Durić et al. Botanicals in the treatment of vaginitis

Table 3. Changes in main subjective symptoms of inflammation at baseline and at 5 days post intervention follow-up in women in the reproductive period N (%) of woman in reproductive period Symptom Vagitories A Vagitories B Vagitories C Vagitories - Probiotic Before (N=25) After (N=25) Before (N=49) After (N=49) Before (N=22) After (N=22) Before (N=17) After (N=17) Vaginal secretion Yes 25 (100) 14 (56.0) 44 (89.8) 3 (6.8) 21 (95.5) 15 (71.4) 17 (100) 15 (88.2) No 0 11 (44.0) 5 (10.2) 41 (93.2) 1 (4.5) 6 (28.6) 0 2 (11.8) p <0.001 <0.001 <0.001 <0.45 Painful/difficult urination* Yes 1 (4.0) 1 (100) 2 (4.1) 1 (50) 0 0 0 0 No 24 (96.0) 0 47 (95.9) 1 (50) 0 0 0 0 Itching Yes 13 (52) 5 (38.5) 35 (71.4) 2 (5.7) 9 (40.9) 3 (33.3) 13 (76,5) 10 (76.9) No 12 (48.0) 8 (61.5) 14 (28.6) 33 (94.3) 13 (59.1) 6 (66.6) 4 (23.5) 3 (23.1) p <0.08 <0.001 <0.03 <0.25 Dryness of vaginal mucosa Yes 0 0 0 0 0 0 0 0 No 0 0 0 0 0 0 0 0 * Due to the small sample it was not possible to determine the p value (28.6%) in the group with Vagitories C, vs. two with probiotics did not show activity in terms of (11.8%) in the group with probiotic vagitories reducing this symptom (0.0%) (p<0.3). (p<0.001) (Table 3). Similar percentage of re- In the group of women in the reproductive period, duction of this symptom was in the group of wo- no change in the frequency of difficult and painful men in the postmenopausal period. Symptom of urination as symptoms of inflammation was found itching was also reduced in the largest number in both subgroups with Vagitorie A and Vagitorie of women especially in the group of women in B, due to the small sample and the frequency of postmenopausal period: eight (88.9%) in the gro- these symptoms. Although the frequency of diffi- up of Vagitories B, 16 (88.9%) Vagitories C, 10 cult painful urination in the postmenopausal wo- (58.8 %) Vagitories A, vs. five (71.4%) women men was reduced in the groups with Vagitorie A, in the group vagitories with probiotic (p<0.06). Vagitorie C and probiotic vagitories, statistically In postmenopausal women, the best activity in it was not significant due to the small sample and reduction of the symptom of vaginal dryness frequency of this symptom (p<0.8). was found in the group with Vagitories C, in 10 Women from the postmenopausal group on the (66.6%) women (p<0.3), while reduction in Vagi- treatment with Vagitories B reported an increase tories A was in seven (50%) and Vagitories B in in irritation lasting for 1-2 days. one (50%) woman (p<0.3) (Table 4). Vagitories

Table 4. Changes in main subjective symptoms of inflammation at baseline and at 5 days post intervention follow-up in women in the postmenopausal period N (%) of woman in postmenopausal period Symptom Vagitories A Vagitories B Vagitories C Vagitories - Probiotic Before (N=35) After (N=35) Before (N=13) After (N=13) Before (N=29) After (N=29) Before (N=15) After (N=15) Vaginal secretion Yes 19 (54.3) 10 (52.6) 11 (84.6) 1 (9.1) 21 (72.4) 6 (20.7) 9 (60) 4 (44.4) No 16 (45.7) 9 (47.4) 2 (15.4) 10 (90.9) 8 (27.6) 23 (79.3) 6 (40) 5 (55.6) p <0.01 <0.01 <0.01 <0.01 Painful/difficult urination* Yes 5 (14.3) 1 (20.0) 0 0 5 (17,2) 1 (20.0) 1 (6,7) 0 No 30 (85.7) 4 (80.0) 0 0 24 (82.8) 4 (80.0) 14 (93.3) 1 (100) Itching Yes 17 (48.6) 7 (41.2) 9 (75.0) 1 (11.1) 18 (62.1) 2 (11.1) 7 (46.7) 2 (28.6) No 18 (51.4) 10 (58.8) 4 (25.0) 8 (88.9) 11 (37.9) 16 (88.9) 8 (53.3) 5 (71.4) p <0.002 <0.01 <0.001 <0.06 Dryness of vaginal mucosa Yes 14 (40.0) 7 (50.0) 2 (15.4) 1 (50.0) 6 (20.7) 2 (33.3) 5 (33.3) 5 (100) No 21 (60.0) 7 (50.0) 11 (84.6) 1 (50.0) 23 (79.3) 4 (66.6) 10 (66.6) 0 p <0.03 <0.07 <0.01 <0.6 * Due to the small sample it was not possible to determine the p value

51 Medicinski Glasnik, Volume 18, Number 1, February 2021

DISCUSSION ined in this formulation. Today, herbal preparations present a very im- The formulation Vagitorie B was prepared based portant part of pharmacotherapy, as a comple- on tea tree oil for which there is a large number mentary but also the therapy of first choice. The of publications that confirm its anti-inflammatory biggest challenge of modern pharmacy and medi- and antimicrobial activity (23-25). Tea tree oil, cine, regarding herbal preparations, is to provide used for preparation of the vagitories in this study evidence of efficacy, evaluation and confirmati- was chemotype terpinen-4-ol (at least 30%). This on of safety of use and development of modern chemotype of essential oil has been labelled in phyto preparations in accordance with pharma- the literature with the best antimicrobial activity ceutical quality standards. Herbal preparations (26, 27). In vitro studies have shown that tea tree investigated in this paper satisfy the principles of oil acts against the causative agent of bacterial rational phytotherapy as their formulations and vaginitis, Trichomonas vaginalis, Gardnerella composition have scientific basis. vaginalis and Streptococcus spp., with a MIC of 0.03 - 0.06% (28). Each formulation of Vagitorie Herbal formulation of Vagitorie A, is prepared B used in this research contained 200 mg of tea from five different plant extracts. Three of them tree oil, corresponding to the single dose used in are obtained from Chamomillae flos, Calendu- clinical studies that confirmed efficacy in previo- lae flos and Hyperici herba, which are marked usly conducted studies (29). The results obtained by the European agency for use in the with Vagitories B showed that this formulation treatment of inflammation of the mucous mem- was most effective compared to other two tested branes of the throat and mouth, skin and mucous vagitories, as well as vagitories with probiotic. membrane irritation in the anal and genital region Similar results were obtained by preliminary cli- and inflammation of skin (sunburn) as well as an nical study conducted by a group of authors from aid in healing minor wounds (13-15). Millefolii Italy, who examined the effectiveness of vagi- herba is recognized as a traditional herbal me- tories based on tea tree oil and probiotics in the dicinal product for the symptomatic treatment of therapy of vaginitis caused by C. albicans (30). minor spasm associated with menstrual periods and a product for the treatment of small super- Vagitorie C contained Hyperici extractum oleo- ficial wounds (16). The fifth extract is Bursa sum 32% w/w as an active component. St. John’s pastoris, traditional herbal medicinal herb used wort contains naphthodiantrones (hypericin and for the reduction of heavy menstrual bleeding of isohypericin), flavonoids (quercetin and hyperosi- women with regular menstrual cycles, after serio- de) and tannins (condensed tannins 6-15%). The us conditions have been excluded by a medical synergistic effect of these ingredients exhibits very doctor (17). Bursa pastoris is also marked to be strong anti-inflammatory and antimicrobial effects an anti-inflammatory agent (18,19). Based on our proven by a large number of studies (31,32). In our findings, no clinical studies have been reported study Vagitories C showed better tolerance and with preparations that contain a combination, but efficacy in women in the postmenopausal group. only single plant extract. A study comparing the Based on the results of the conducted clinical effect of marigold-based vagitoria versus clotri- study, it can be concluded that three types of va- mazole in vaginal candidiasis showed excellent gitories had a positive effect in the treatment of results of marigold extract (20). Clinical trial of vaginitis. All three types of plant extract vagitories the influence of chamomile vaginal gel -confir showed a superior effect in comparison with probi- med reduction of symptoms of itching, vaginal otic vagitories used in the control group. Although dryness, discomfort and painful intercourse in probiotics have a beneficial effect on the regulation the postmenopausal woman (21). Pharmacologi- of the vaginal flora, whose disorder can lead to in- cal investigations confirmed anti-inflammatory, flammation of the vaginal mucosa (33), the results antioxidant and antimicrobial activity of topical indicate that in the acute phase, the tested three application of St. John’s wort as well (22). The plant extract formulations have a better effect on herbal formulation Vagitories A showed a good the vaginal mucosa inflammation. Plants used in effect on both subjective and objective symptoms this study contain saponins, flavonoids and pheno- of vaginal inflammation, confirming the synergi- lic compounds, which exhibit very strong anti-in- stic effect of mixture of five plant extracts, conta- flammatory activity through various mechanisms.

52 Durić et al. Botanicals in the treatment of vaginitis

Those mechanisms include prevention of release Duration of the therapy for all patients included of histamine and productions of prostaglandin (34), in the study was five days. Considering the posi- and reduction of the white blood cells migration to tive effects of the investigated vagitories in the the inflamed tissue (35). Since the symptoms of va- stated period of treatment on one hand, and the ginitis can lead to sexual dysfunctions (36), the use absence of side effects on the other hand, further of tested plant extract vagitories can have a positive studies are needed including a larger number of impact on quality of life. treatment days. The main limitation of this study is the exclusi- ACKNOWLEDGEMENT on of women with vaginosis caused by microor- ganisms, because these women had to be treated This article is a part of research conducted wit- according to official protocols. By reviewing the hin the doctoral dissertation of Selma Kovčić available literature, studies conducted with tea tree Hadžiabdić. oil confirm its very strong antimicrobial activity The research project was approved by the Ethics on microorganisms identified as the main causes Committee (No:03/7-1441-2/19). of vaginal infections, which indicates the possibi- We are thankful to personnel of the Institute for lity and justification of the use of vagitories with Health Protection of Woman and Maternity of tea tree oil in the treatment of vaginal candidiasis Canton Sarajevo for their cooperation. We give (37), trichomoniasis and bacterial vaginosis (38). our deep appreciation to all women who partici- Another limitation of this study is given by the pated in this trial because with their participation uneven representation of respondents by groups. they can help women with vaginal inflammation In any case, most statistical calculations had a p but also offer new therapeutic opportunities for value less than 0.05. future generations. In conclusion, vagitorie formulations investiga- FUNDING ted in this study appear to have a positive effect This work was supported by “JU Apoteke Saraje- on the symptoms of vaginitis. Apart from the vo” in providing sufficient number of vagitories appearance of mild dryness of the vaginal muco- necessary for this investigation. No other funding sa and annealing sensation in patients in the pos- was received for this study. tmenopausal period, who were on therapy with vagitories based on tea tree oil, no other side TRANSPARENCY DECLARATION effects were reported. Conflicts of interest: Conflict of interest: None to declare. REFERENCES

1. Mehta SD. Systematic review of randomized trials of 7. Aviva R. Botanical Medicine for Woman’s Health. 1st treatment of male sexual partners for improved bac- ed. Philadelphia: Churcill Livingstone, 2009. teria vaginosis outcomes in women. Sex Transm Dis 8. González-Burgos E, Gómez-Serranillos MP. Natural 2012; 39:822-30. products for vulvovaginal candidiasis treatment: Evi- 2. Berić B, Popović D. Klinička kolposkopija (Clinical dence from clinical trials. Curr Top Med Chem 2018; colposcopy) [in Croatian]. Medicinska knjiga: Beo- 18:1324-32. grad-Zagreb, 1975. 9. Karaman IM, Bogavac M, Radovanović B, Sudji J, 3. Nyirjesy P. Management of persistent vaginitis. Ob- Tešanović K, Janjušević L. Origanum vulgare essen- stet Gynecol 2014; 124:1135-46. tial oil affects pathogens causing vaginal infections. J

4. Gonçalves B, Ferreira C, Tiago Alves C, Henriques Appl Microbiol 2017; 122:1177-85. M, Azeredo J, Silva S. Vulvovaginal candidiasis: Epi- 10. Wenche Jøraholmen M, Basnet P, Jonine Tostrup M, demiology, microbiology and risk factors. Crit Rev Moueffaq S, Škalko-Basnet N. Localized therapy of Microbiol 2016; 42:905-27. vaginal infections and inflammation: liposomes-in-

5. Beyitler I, Kavukcu S. Clinical presentation, diagno- hydrogel delivery system for polyphenols. Pharmace- sis and treatment of vulvovaginitis in girls: a current utics 2019; 11:53. approach and review of the literature. World J Pediatr 11. Lin Z, Lin Y, Zhang Z, Shen J, Yang C, Jiang M, Hou 2017; 13:101-5. Y. Systematic analysis of bacteriostatic mechanism 6. Babu G, Singaravelu BG, Srikumar R, Reddy SV, of flavonoids using transcriptome and its therape- Kokan A. Comparative study on the vaginal flora and utic effect on vaginitis. Aging (Albany NY) 2020; incidence of asymptomatic vaginosis among healthy 12:6292-305. women and in women with infertility problems of re- productive age. J Clin Diagn Res 2017; 11:DC18-22.

53 Medicinski Glasnik, Volume 18, Number 1, February 2021

12. Duarte de Freitas AL, Kaplum V, Pereira Rossi DC, 25. Golab M, Skwarlo SK. Mechanisms involved in the Buffoni Roque da Silva L, Carvalho Melhem MS, anti-inflammatory action of inhaled tea tree oil in Pelleschi Taborda C, Palazzo de Mello JC, Nakamu- mice. Exp Biol Med 2007; 232:420-6 ra CV, Ishida K. Proanthocyanidin polymeric tannins 26. Cordeiro L, Figueiredo P, Souza H, Sousa A, Andrade- from Stryphnodendron adstringens are effective aga- Júnior F, Medeiros D, Nóbrega J, Silva D, Martins E, inst Candida spp. isolates and for vaginal candidiasis Barbosa-Filho J, Lima E. Terpinen-4-ol as an antibac- treatment. J Ethnopharmacol 2018; 216:184-90. terial and antibiofilm agent against Staphylococcus 13. European Medicines Agency. Committee on Herbal aureus. Int J Mol Sci 2020; 21:4531. Medicinal Products: European Union herbal mono- 27. Sharifi-Rad J, Salehi B, Varoni EM, Sharopov F, Yo- graph on Matricaria recutita L., aetheroleum. July usaf Z, Ayatollahi SA, Kobarfard F, Sharifi-Rad M, 2015 EMA/HMPC/55843/2011. https://www.ema. Afdjei MH, Sharifi-Rad M, Iriti M. Plants of the Me- europa.eu (07 June 2020) laleuca genus as antimicrobial agents: From farm to 14. European Medicines Agency. Committee on Herbal pharmacy. Phytother Res 2017; 31:1475-94. Medicinal Products: European Union herbal mo- 28. Hammer KA, Carson CF, Riley TV. In vitro suscepti- nograph on Calendula officinalis L., flos. 27 March bilities of lactobacilli and organisms associated with 2018 EMA/HMPC/437450/2017. https://www.ema. bacterial vaginosis to Melaleuca alternifolia (tea tree) europa.eu (09 June 2020) oil. Antimicrob Agents Chemother 1999; 43:196. 15. European Medicines Agency. Committee on Herbal 29. Di Vito M, Mattarelli P, Modesto M, Girolamo A, Ba- Medicinal Products: Community herbal monograph llardini M, Tamburro A, Meledandri M, Mondello F. on Hypericum perforatum L., Herba. London, 12 No- In vitro activity of tea tree oil vaginal suppositories vember 2009 Doc. Ref.: EMA/HMPC/101304/2008. against Candida spp. and probiotic vaginal microbio- https://www.ema.europa.eu (15 June 2020) ta. Phytother Res 2015; 29:1628-33. 16. European Medicines Agency. Committee on Herbal 30. Barnes J, Arnason JT, Roufogalis BD. St John’s wort Medicinal Products: Community herbal monograph (Hypericum perforatum L.): botanical, chemical, on Achillea millefolium L., Herba. 12 July 2011 pharmacological and clinical advances. J. Pharm. EMA/HMPC/290284/2009. https://www.ema.euro- Pharmacol 2019; 71:1-3. pa.eu (15 June 2020) 31. Di Vito M, Fracchiolla G, Mattarelli P, Modesto M, 17. European Medicines Agency. Committee on Herbal Tamburro A, Padula F, Agatensi L, Romana FG, Gi- Medicinal Products Community herbal monograph orlamo A, Carbonara GG, Carrieri A, Corbo F, Mon- on Capsella bursa-pastoris (L.) Medikus, Herba. 25 dello F. Probiotic and tea tree oil treatments improve November 2010 EMA/HMPC/262766/2010. https:// therapy of vaginal candidiasis: A Preliminary clinical www.ema.europa.eu (15 June 2020) study. Med J Obstet Gynecol 2016; 4:1090. 18. Min Cha J, Se Suh W, Lee TH, Subedi L, Yeou Kim S, 32. Bölgen N, Demir D, Yalçın MS, Özdemir S. Deve- Ro Lee K. Phenolic glycosides from Capsella bursa- lopment of Hypericum perforatum oil incorporated pastoris (L.) Medik and their anti-inflammatory acti- antimicrobial and antioxidant chitosan cryogel as a vity. Molecules 2017; 22:1023. wound dressing material. Int J Biol Macromol 2020; 19. Peng J, Hu T, Li J, Du J, Zhu K, Cheng B, Li K. 61:1581-1590. Shepherd’s purse polyphenols exert its anti-in- 33. Serignoli Francisconi R, Maquera Huacho PM, Co- flammatory and antioxidative effects associated with radi Tonon C, Alves Ferreira Bordini E, Ferreira suppressing MAPK and NF-κB pathways and heme Correia M, de Cássia Orlandi Sardi J, Palomari Spo- oxygenase-1 activation. Oxid Med Cell Longev 2019; lidorio DM. Antibiofilm efficacy of tea tree oil and 2019:1-14. of its main component terpinen-4-ol against Candida 20. Saffari E, Mohammad ACS, Adibpour M, Mirgha- albicans. Braz Oral Res 2020; 34:e050. fourvand M, Javadzadeh Y. Comparing the effects 34. Homayouni A, Bastani P, Ziyadi S, Mohammad of Calendula officinalis and clotrimazole on vaginal ACS, Ghalibaf M, Mortazavian AM, Mehrabany VE. candidiasis: a randomized controlled trial. Women Effects of probiotics on the recurrence of bacterial va- Health 2017; 57:1145-60. ginosis: a review. J Low Genit Tract Dis 2014; 18:79- 21. Pazyar N, Yaghoobi R, Bagheran Ni, Kazerouni A. A 86. review of applications of tea tree oil in . 35. Cha JM, Suh WS, Lee TH, Subedi L, Kim SY, Lee Int J Dermatol 2013; 52:784-90. KR. Phenolic glycosides from Capsella bursa-pasto- 22. Bosak Z, Iravani M, Moghimipour E, Haghighizadeh ris (L.) Medik and their anti-inflammatory activity. MH, Jelodarian P, Khazdair MR. Evaluation of the Molecules 2017; 22:1023. influence of chamomile vaginal gel on dyspareunia 36. Szakiel A, Ruszkowski D, Janiszowska W. Saponins and sexual satisfaction in postmenopausal women: in Calendula officinalis L. – structure, , A randomized, double-blind, controlled clinical trial. transport and biological activity. Phytochemistry Re- J Phytomed 2020; 10:481-91. views 2005; 4:51–158. 23. Wölfle U, Seelinger G, Schempp CM. Topical appli- 37. Faubion SS, Rullo JE. Sexual Dysfunction in Wo- cation of St. John’s wort (Hypericum perforatum). men: A Practical Approach. Am Fam Physician 2015; Planta Med 2014; 80:109-20 92:281-8. 24. Carson CF, Hammer KA, Riley TV. Melaleuca alter- 38. Van Kessel K, Assefi N, Marrazzo J, Eckert L. nifolia (Tea Tree) oil: a review of antimicrobial and Common complementary and alternative therapies other medicinal properties. Clin Microbiol Rev 2006; for yeast vaginitis and bacterial vaginosis: a systema- 19:50-62. tic review. Obstet Gynecol Surv 2003; 58:351-8.

54 ORIGINAL ARTICLE

Prevalence of Cryptosporidium spp. and Blastocystis hominis in faecal samples among diarrheic HIV patients in Medan, Indonesia

Hemma Yulfi1, Muhammad Fakhrur Rozi2, Yunilda Andriyani1, Dewi Masyithah Darlan1,3

1Department of Parasitology, Faculty of Medicine, Universitas Sumatera Utara, Medan; Indonesia, 2Faculty of Medicine, Universitas Suma- tera Utara, Medan; Indonesia, 3Pusat Unggulan Tissue Engineering, Faculty of Medicine; Universitas Sumatera Utara, Medan, Indonesia

ABSTRACT

Aim To investigate the prevalence of Cryptosporidium sp. and B. hominis among human immunodeficiency virus (HIV) positive pa- tients in two different outpatient clinics, Haji Adam Malik General Hospital and Primary Care Centre of Padang Bulan, Medan, Indo- nesia, between two interval periods.

Method Cryptosporidium spp. microscopic examination, as well as Jones’ medium for B. hominis, were conducted in the Parasi- tology Laboratory, enzyme-linked immunosorbent assay (ELISA) was done in the Multidisciplinary Laboratory, Faculty of Medici- ne, Universitas Sumatera Utara. This was a cross-sectional study, involving 54 diarrheic HIV positive patients (44 males, 10 fema- Corresponding author: les). The data were analysed by Spearmen rank correlation, in- Dewi Masyithah Darlan terrater agreement, and 2 tests. Parasitology Laboratory, Results Infection rate for Cryptosporidium spp. and B. hominis was Faculty of Medicine, 24% (13 patients) and 9% (five patients), respectively. The preva- Universitas Sumatera Utara lence of CD4 cell count below 200 cell/mm3 was relatively high, Jl. Dr. Mansur No. 5, 20155 Medan, 29.6% (16 patients). There was a significant relationship between North Sumatera, Indonesia cryptosporidiosis and CD4 cell count (p=0.01; OR 1.57; 95% CI Phone +62 811 1644 545; 1.25-1.99). Microscopic examination was superior over ELISA, whose diagnostic value for sensitivity and specificity was 46.15% Email: [email protected] and 100.0%, respectively, and Kappa (K) coefficient of 0.56. Hemma Yulfi, ORCID ID: https://orcid. org/0000-0002-6837-1559 Conclusion The prevalence of cryptosporidiosis among HIV pa- tients was still relatively high. CD4 count showed a significant relationship with Cryptosporidium spp. infection, but not with Blastocystic hominis. Microscopic examination is still the most Original submission: reliable technique to diagnose the parasites in faecal samples. 10 September 2020; Keywords: enzyme-linked immunosorbent assay, immunocompro- Revised submission: mised, intestinal parasite, Kinyoun-Gabbett, opportunistic protozoa 12 October 2020; Accepted: 17 November 2020 doi: 10.17392/1271-21

Med Glas (Zenica) 2021; 18(1):55-61

55 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION for Blastocystis sp. colonization (9). There are at least 17 distinct subtypes observed in several host Cryptosporidium spp. has gained significant im- species, humans and animals (10). The diagnosis portance, particularly among the immunocom- of blastocystosis exposes a challenge for clinician, promised populations. In 1976, Cryptosporidium particularly since its appearance in wet mount is became more recognizable for its position cau- confused with Cyclospora spp., yeast, fat globules, sing gastroenteritis, followed by evidence of the helminths’ ova, even sometimes leucocytes (11). It infection associated with immunocompromised is also widely known that the infection commonly condition (1). Since then, Cryptosporidium spp. does not produce symptoms, yet symptomatic plus has emerged as one of the most common inte- any presence of B. hominis from direct smear exa- stinal parasitic species worldwide. Cryptospo- mination in patient stool could develop diagnosis ridium spp. is responsible for several outbreaks and initiate medication (12). leading to higher mortality across the globe, particularly among the HIV population (2). This Since both parasites cause non-specific symp- opportunistic intestinal protozoan has been co-in- toms and serve as opportunistic agents, clinicians fecting with HIV, especially those with low CD4 do not always give enough consideration to these counts (3). Cryptosporidium spp. oocysts tran- protozoans, even though a meta-analysis study smit mainly by the faecal-oral route; complemen- has shown that Cryprosporidium spp. infection tary transmission route was also included, such can lead to prolonged diarrhoea and devastating as direct contact from an infected individual or symptoms (3). This is partly caused by the lack of oocyst contaminated-food or water (4), although evidence on theses parasites, especially in Indo- autoinfection within the same host is also possi- nesia and other South East Asian countries. ble (5). A spectrum of clinical manifestations de- The aim of the study was to investigate Cryp- veloped in accordance with age, immune status, tosporidium spp. and B. hominis infection rates and nutritional status, and the agent’s virulence among diarrheic HIV patients in two different and pathogenicity, whose factors highly depend outpatient clinics, as well as the detection rate of on its special structure, rhoptries and microne- ELISA-based method compared to the microsco- mes (5). The symptoms are mostly self-limited, pic examination using Kinyoun-Gabbett stain for e.g. watery diarrhoea, abdominal cramps, light Cryptosporidium spp. fever, and nausea and vomiting (6). A recent meta-analysis study has proven the association PATIENTS AND METHODS between antiretroviral therapy with the occu- Patients and study design rrence of cryptosporidiosis in HIV patients, thus proper management with chemoprophylaxis is This cross-sectional study was conducted in two recommended to reduce the risk of infection (3). different locations, i.e. HIV outpatient clinic in A stramenopile B hominis is the only organism in Haji Adam Malik General Hospital, and Padang the group that could lead to human infection wit- Bulan Primary Care Centre, both located in the hout any describable pathogenesis. There has been city of Medan, Indonesia. The data were collec- an extensive genetic variation of the organism cau- ted within two interval periods, during June-Au- sing elaborate investigation to understand its life gust 2018 and June-September 2019, through a and reproductive cycle (7). The advancement in consecutive sampling process (all suitable sam- molecular technique has recently unfolded the ge- ples were included in the study). All participants netic diversity of the organism that finally reveals suffered from acute and chronic diarrhoea for se- the detection and classification adequately. There veral hours or days (three or more loose or liqu- are four main morphological forms of Blastocystis id stools per day, based on the WHO criteria for spp. in stools, i.e. vacuolar, granular, amoeboid, diarrhoea) (13). Nevertheless, the study did not and cyst forms which proposed as infective stage note this finding for further results/discussion. (8). The prominent pathogenesis of B. hominis de- Faecal samples were collected from each pati- pends on its proteases activity and host intestinal ent in two separate containers to accommodate microorganisms, all of which depend on organi- unpreserved and formalin-preserved samples for sm subtypes (STs) that can be more supportive further examination.

56 Yulfi et al. Cryptosporidium and Blastocystis in HIV

A total of 54 HIV positive patients had given the- Enzyme-linked immunosorbent assay (ELI- ir consent to enrol in the study and a brief oral SA). The method was prepared to detect Cryp- explanation of the study protocol was conducted tosporidium parvum antigen in faecal samples prior to the execution of the sampling process. using the principle of microplate-based sandwich Additionally, the data of accompanying varia- ELISA (Epitope Diagnostics, Inc., United Sta- bles, such as HIV positive status, age, gender, the tes). The equipment for parasitic detection utilizes latest CD4 cell count, and any presence of diarr- microtiter well, whose wall has been coated with hoea was noted in a short questionnaire. highly purified antibody. It also consists of anti- The Ethical Committee for Medical Research of Cryptosporidium tracer antibody, tracer antibody Faculty of Medicine, Universitas Sumatera Utara diluent, wash concentrate, HRP substrate, stop Indonesia has approved the study protocol (Refe- solution, and Cryptosporidium sp. antigen control rence number: 136/TGL/KEPK FK USU-RSUP (contained purified Cryptosporidium sp. oocysts). HAM/2019). The interpretation was as follows: positive when patient’s sample extinction was greater than the Methods positive cut-off, and negative if patient’s sample extinction was less than the negative cut-off. Parasitological examination. Acid fast staining methods have been applied for the identification Statistical analysis of Cryptosporidium spp (modified Ziehl-Neelsen and Kinyoun-Gabbett methods) (14,15). Kinyo- The data were presented in univariate and biva- un solution consisted of 4 grams of fuchsin al- riate modes, as well as further analysis to determi- kaline, 8 grams of phenol, 5 mL of 95% alcohol, ne superiority between microscopic examination and 100 mL of sterilized water. Gabbett solution and ELISA for Cryptosporidium spp. detection. was made of 1 gram of methylene blue, 20 mL of The test included sensitivity and specificity, as 96% sulphuric acid, 30 mL of absolute alcohol, well as interrater agreement (Kappa, K) and Spe- and 50 mL of sterilized water. The application arman correlation rank test. of KG staining is like ZN staining, in which the RESULTS sample is firstly applied with Kinyoun solution, followed by inundating the sample with Gabbett A total of 54 HIV positive patients (44 males and solution to maintain acid-fast stain nature of the 10 females) had voluntarily enrolled into the stu- oocyst under microscopic examination. An appe- dy and gave their faecal samples the following arance of a 4-6 µL pinkish-red round shape was day. All participants (30 patients) admitted to interpreted as Cyrptosporidium spp. oocysts. the outpatient clinic in Haji Adam Malik Gene- B. hominis was revealed primarily using direct ral Hospital had low level of CD4 cell count and stain using Lugol’s iodine and then positive B. proved their continuation in receiving antiretro- hominis samples that underwent in vitro mul- viral therapy (ART). One patient had infection tiplication using Jones’ culture medium (16). with both Cryptosporidium spp. and B. hominis About 50 mg of unpreserved faecal samples were (with 10-day diarrhoea as the main symptom in inoculated into Jones’ medium and incubated at addition to mild-moderate dehydration). 37 °C; the growth was observed periodically for An exceptionally low level of CD4 cell count 24 hours. The culture would permit the appearan- (below 200 cell/mm3) was evident among 10 ce of reproductive and morphological stages of hospitalized patients compared to six patients B. hominis. The procedure was also conducted to in the primary care centre (Table 1). Thirteen ensure the sample’s positivity, both that the pre- (24.1%) patients were positive for Cryptospori- sence of at least one morphological form of B. dium spp. (eight from the hospital and five from hominis during direct microscopic examination primary care centre) and five (9.3%) patients with was identified, and that was obtained from diarr- blastocystosis (from the hospital and the primary heic patients with negative results. Furthermore, care centre, three and two, respectively). any positive appearance of B. hominis which was Significant relationship between low level of obtained from direct stain examination of cultu- CD4 cell count and Cryptosporidium sp. infec- red samples was declared positive. tion (p=0.01; OR 1.57; 95% CI: 1.25-1.99) was

57 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 1. Baseline characteristic of 54 HIV patients DISCUSSION Characteristic N (%) of patients Age (years) In this study, Cryptosporidium spp. infection < 30 24 (44.4) rate among diarrheic HIV positive patients was ≥30 30 (55.6) 24% with the highest prevalence (84.6%) among Gender Male 44 (81.5) the patients with very low CD4 cell count (<200 Female 10 (18.5) cells/mm3), whereas the remaining positive sam- CD4 cell count (cell/mm3) ples (2 patients) was in 350-499 cells/mm3 group. >500 15 (27.8) 350-499 20 (37.0) Although all patients were admitted with diarr- 200-349 3 (5.6) hoea, most patients did not demonstrate oocysts <200 16 (29.6) in microscopic examination. B. hominis infection Cryptosporidium spp. rate was only 9.25% (five positive faecal sam- Positive 13 (24.1) Negative 41 (75.9) ples). In Indonesia, a recent literature review has Blastocystis hominis shown that Cryptosporidium spp. was found in Positive 5 (9.3) 4-11% among diarrheic children, approximately Negative 49 (90.7) 39% of among HIV/AIDS hospitalized patients with or without diarrhoea, and approximately found, but not in the B. hominis infection, since 18.5% among HIV/AIDS hospitalized patients only five samples were positive with the parasite with tuberculosis (17). Cryptosporidium spp. (p=0.52; OR 0.54; 95% CI: 0.08-3.61). Spearman has emerged as one of the most common ente- correlation rank test demonstrated significant fin- ropathogens causing gastroenteritis in adult pa- dings between Cryptosporidium sp. and CD4 cell tients, as well as one of the neglected tropical count (p=0.000; r=0.6) (Table 2). infectious diseases, which is due to the transfor- Table 2. The infection prevalence and CD4 cell count among mation in the environment, populations and de- 54 HIV positive patients mographics (18). Based on the Global Burden of N (%) of patients with CD4 Disease data in 2010, WHO has estimated more 3 Causative agent cell count (cell/mm ) p OR (95% CI) than 60,000,000 illnesses, 27,000 deaths, and 2.1 Low Normal Total million disability adjusted life years (DALYs) (<500) (≥500) Cryptosporidium spp. lost were caused by Cryptosporidium spp. (19). Positive 13 (100.0) 0 (0.0) 13 (100) 1.57 Epidemiological trends of Blastocystis spp. in- 0.01 Negative 26 (63.4) 15 (36.6) 41 (100) (1.25-1.99) fection demonstrate more frequently in human Blastocystis hominis faecal samples and were found higher than Giar- Positive 3 (60.0) 2 (40.0) 5 (100) 0.54 0.52 Negative 36 (73.5) 13 (26.5) 49 (100) (0.08-3.61) dia lamblia, Entamoeba spp., and Cryptosporidi- um spp. in some regions (20). Studies in Ethiopia The comparison of two diagnostic methods found and Iran found the prevalence of Blastocystis in- that the microscopic examination was superior to fection ranging around 10.6-18.4% in HIV/AIDS ELISA. The greater number of findings (13 sam- patients (21-23), and significantly higher among ples) was obtained using microscopic examina- those without ART (24). Most of those studies tion, while only six samples had positive results confirmed significant association between the in- by using ELISA. Therefore, the diagnostic value fection and diarrhoea (22-24). The rate of infecti- of ELISA could be calculated, i.e. sensitivity on was found much higher in Indonesia, which is 46.15%, specificity 100.0%, positive predictive 73.6% of 318 HIV patients (25). value 100.0%, negative predictive value 85.42% The ability of Cryptosporidium spp. to produce with K=0.56 (Table 3). symptoms fully depends on parasite characteri- stics and host factors, such as the immune com- Table 3. Comparison of diagnostic findings of Cryptosporid- ium sp. between Kinyoun-Gabbett (KG) stain and enzyme- petence and the frequency of exposure to parasite linked immunosorbent assay (ELISA) from infected individuals. All factors might result No (%) of patients in KG stain Weighted Kappa in varying symptoms, ranging from asymptomatic ELISA Positive Negative Total (95% CI) to life-threatening illness (26). One study repor- Positive 6 0 6 (11.1) ted significant association between cryptosporidi- Negative 7 41 48 (88.9) 0.56 (0.29- 0.83) Total 13 (24.1) 41 (75.9) 54 (100.0) osis and each of CD4 count, diarrhoea duration,

58 Yulfi et al. Cryptosporidium and Blastocystis in HIV

abdominal cramps, fever, as well as nausea and microscopic examination. The enzyme assay has vomiting (27). Dehydration can occur among vul- been evaluated for decades with varying degree of nerable population, including children and immu- accuracy, from low sensitivity (40.9%) and higher nocompromised patients, and associated with specificity (78.9%) using animal faecal samples, higher mortality rates (28). There are numerous which was positive for Cryptosporidium spp. reports on extraintestinal cryptosporidiosis, inclu- (38), whilst some other reports found excellent ding in respiratory tract, pancreas, and biliary tract diagnostic value of ELISA-based method, who- (29-32). In normal conditions, Cryptosporidium se specificity and sensitivity were ranging around spp. will develop a membrane in the apical surfa- 92.25-96% and 87.38-100% respectively (39-43). ce of intestinal epithelium that would not provoke The method also showed best performance com- systemic infection (5). Thus, it impairs the secre- pared to other techniques in testing B. hominis, tory and absorptive function of the gut epithelium including direct wet mount, trichrome stain, and recruiting inflammatory cells in localized tissue in vitro culture (44). Notwithstanding its fair/sub- (33). Similarly, the unknown mechanism of B. stantial agreement of Kappa value (0.56), our stu- hominis infection also leads to the confusion with dy was not successful to prove ELISA superiority other gastrointestinal infections. Diarrhoea, abdo- to microscopic examination. There are several as- minal cramps, nausea vomiting, or other non-spe- pects affecting ELISA results, mainly the technical cific symptoms could also accompany the presen- aspects of the equipment and experts who conduct ce of B. hominis in fecal samples (34). the examination relating to the multiple steps of Immune response is problematic for patients with sandwich ELISA (45). The disadvantage of direct impaired cellular immune function, in which the microscopic examination is greater affected by the major reaction will involve T-cell immunity that operator who conducts the procedure, particularly weakens the eradication process of the infection relating to the presence of oocyst or other morpho- (35). It is obvious that the self-limiting nature logical form of organism (46). However, the direct of cryptosporidiosis will vanquish and become microscopic examination is significantly cheaper more disastrous in immunosuppressed populati- and more affordable when conducted properly. on (5). There were no reports of symptoms and In conclusion, Cryptosporidium spp. infection patient’s quality of life in this study, but it confir- should be regarded in managing immunocompro- med the relationship of lower level of CD4 cell mised patients, especially those with low CD4 le- count with higher incidence of cryptosporidiosis. vels. In addition, microscopic examination rema- In contrast, B. hominis infection has no signifi- ins the gold standard for Cryptosporidium spp., cant relationship with CD4 cell count, yet des- since it creates less technical problems relating criptively it was evident that the infection rate of to multistep procedure, particularly for low-cost B. hominis was higher among lower CD4 group. laboratories in developing countries. The pathogenesis of infection remained elusive ACKNOWLEDGEMENT for several decades, but several proposed pat- hogeneses have been reported in many studies, The authors would like to acknowledge that the which is related to cysteine proteases and other present research is supported by the Ministry of proteolytic enzyme secretion (36,37). The pro- Research and Technology, the Higher Education duction of lysates could induce dramatic changes of Republic of Indonesia, as well as the Research in cytoskeleton that finally promotes apoptosis in and Community Service Institution of Universi- epithelial cells, while cysteine protease will be tas Sumatera Utara. encoded by B. hominis leading to IL-8 producti- on from intestine epithelial cells (36). FUNDING Our study also demonstrated low sensitivity of Financial support was provided through the rese- ELISA compared to microscopic examination as arch grant TALENTA 2017 (No: 5338/UN5.1. R/ a golden standard through KG stain. There were PPM/2017). also high false negative samples among cryptos- TRANSPARENCY DECLARATION poridiosis patients, which profoundly presented in Conflict of interest: None to declare.

59 Medicinski Glasnik, Volume 18, Number 1, February 2021

REFERENCE 1. Vanathy K, Parija S, Mandal J, Hamide A, S Kris- 17. Wijayanti T. Kriptosporidiosis di Indonesia (Cryp- hnamurthy. Cryptosporidiosis: a mini review. Trop tosporidiosis in Indonesia) [in Indonesian] Balaba Parasitol 2017; 7:72–80. 2017; 13:73-82. 2. Rossle NF, Latif B. Cryptosporidiosis as threatening 18. Wahdini S, Kurniawan A, Yunihastuti E. Deteksi health problem: a review. Asian Pac J Trop Biomed Koproantigen Cryptosporidium sp pada Pasien HIV/ 2013; 3:916–24. AIDS dengan Diare Kronis (Detection of Cryptos- 3. Ahmadpour E, Safarpour H, Xiao L, Zarean M, Ha- poridium sp. coproantigen in HIV/AIDS patients tam-Nahavandi K, Barac, Stephane AP, Rahimi MT, with chronic diarrhea) [in Indonesian]. eJournal Ke- Rubino S, Mahami-Oskouei M, Spotin A, Nami S, dokt Indones 2016; 4:49–53. and Baghi HB. Cryptosporidiosis in HIV-positive 19. Torgerson PR, Devleesschauwer B, Praet N, Spey- patients and related risk factors: a systematic review broeck N, Willingham AL, Kasuge F, Rokni MB, and meta-analysis. Parasite 2020; 27:27. Zhou XN, Fevre EM, Sripa B, Gargouri N, Furst 4. Bhalchandra S, Cardenas D, Ward HD. Recent bre- T, Budke CM, Carabin H, Kirk MD, ANgulo FJ, akthroughs and ongoing limitations in Cryptospori- Havelaar A, deSilva N. World Health Organization dium research. F1000Res 2018;7:1–9. estimates of the globarl and regional disease burden 5. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. of 11 foodbourne parasitic diseases, 2010: a data Cryptosporidium pathogenicity and virulence. Clin synthesis. PLoS Med 20154; 12:e1001920. Microbiol Rev 2013; 26:115–34. 20. Mackey TK, Liang BA, Cuomo R, Hafen R, Brou- 6. Utami WS, Murhandarwati EH, Artama WT, Ku- wer KC, Lee DE. Emerging and reemerging neglec- snanto H. Cryptosporidium infection increases the ted tropical diseases: a review of key characteristics, risk for chronic diarrhea among people living with risk factors, and the policy and innovation envi- HIV in Southeast Asia: a systematic review and me- ronment. Clin Microbiol Rev 2014; 27:949–79. ta-analysis. Asia Pac J Public Health 2020; 32:8–18. 21. Hailemariam G, Kassu A, Abebe G, Abate E, Damte 7. Khalil S, Mirdha BR, Sinha S, Panda A, Singh Y, D, Mekonnen E, Ota F. Intestinal parasitic infections Joseph A, Singh Y, Joseph A, Deb M. Intestinal pa- in HIV/AIDS and HIV seronegative individuals in rasitosis in relation to anti-retroviral therapy, CD4+ a teaching hospital, Ethiopia. Jpn J Infect Dis 2004; T-cell count and diarrhea in HIV patients. Korean J 57:41-3. Parasitol 2015; 53:705–12. 22. Alemu A, Shiferaw Y, Getnet G, Yalew A, Addis Z. 8. Tan KSW. New insights on classification, identifica- Opportunistic and other intestinal parasites among tion, and clinical relevance of Blastocystis spp. Clin HIV/AIDS patients attending Gambi higher clinic Microbiol Rev 2008; 21:639–65. in Bahir Dar City, North West Ethiopia. Asian Pac J 9. Wawrzyniak I, Poirier P, Texier C, Delbac F, Vis- Trop Med 2011; 4:661-5. cogliosi E, Dionigia M, Texier C, Delbac F, El 23. Zali MR, Mehr AJ, Rezaian M, Meamar AR, Vaziri Alaoui H. Blastocystis, an unrecognized parasite: an S, Mohraz M. Prevalence of intestinal parasitic pat- overview of pathogenesis and diagnosis. Ther Adv hogens among HIV-positive individuals in Iran. Jpn Infect Dis 2013; 1:167–78. J Infect Dis 2004; 57:268-70. 10. Lepczyńska M, Białkowska J, Dzika E, Piskorz- 24. Adamu H, Wegayehu T, Petros B: High prevalence Ogórek K, Korycińska J. Blastocystis: how do spe- of diarrhoegenic intestinal parasite infections among cific diets and human gut microbiota affect its de- non-ART HIV patients in Fitche Hospital, Ethiopia. velopment and pathogenicity? Eur J Clin Microbiol PLoS One 2013; 8:e72634. Infect Dis 2017; 36:1531–40. 25. Kurniawan A, Karyadi T, Dwintasari SW, Sari IP, 11. Zierd CH. Blastocystis hominis – Past and Future. Yunihastuti E, Djauzi S, Smith HV. Intestinal parasi- Clin Microbiol Rev 1991; 4:61-79. tic infections in HIV/AIDS patients presenting with 12. Badparva E, Ezatpour B, Mahmoudvand H, Behza- diarrhoea in Jakarta, Indonesia. Trans R Soc Trop difar M, Behzadifar M, Kheirandish F. Prevalence Med Hyg 2009; 103:892-8. and genotype analysis of Blastocystis hominis in 26. Beyhan YE, Yilmaz H, Cengiz ZT, Ekici A. Clinical Iran: a systematic review and meta-analysis. Arch significance and prevalence of Blastocystis hominis Clin Infect Dis 2017; 12:1–9. in Van, Turkey. Saudi Med J 2015; 36:1118–21. 13. World Health Organization. Diarrhoeal disease. 27. Yang Y, Zhou YB, Xiao PL, Shi Y, Chen Y, Liang https://www.who.int/news-room/fact-sheets/detail/ S, Yihuo WL, Song XX, Jiang QW. Prevalence of diarrhoeal-disease (24 October 2020) and risk factors associated with Cryptosporidium 14. Casemore DP, Armstrong M, Sands RL. Laboratory infection in an underdeveloped rural community of diagnosis of Cryptosporidiosis. J Clin Pathol 1985; Southwest China. Infect Dis Poverty 2017; 6:1–10. 38:1337-41. 28. Pantenburg B, Cabada MM, White AC Jr. Treatment 15. Hok TT. A Simple and Rapid Cold-Staining Met- of cryptosporidiosis. Exp Rev Anti Infect Ther 2009; hod for Acid-fast. Am J Respir Crit Care Med 1962; 7:385-91 85:753-4. 29. Sponseller JK, Griffiths JK, Tzipori S. The evolution 16. Padukone S, Mandal J, Rajkumari N, Bhat BV, Swa- of respiratory Cryptosporidiosis: evidence for tran- minathan RP, Parija SC. Detection of Blastocy- smission by inhalation. Clin Microbiol Rev 2014; stis in clinical stool specimens using three different 27:575–86. methods and morphological examination in Jones' medium. Trop Parasitol 2018; 8:33-40.

60 Yulfi et al. Cryptosporidium and Blastocystis in HIV

30. Reina FTR, Ribeiro CA, de Araujo RS, Matte MH, 40. Marques FR, Cardoso LV, Cavasini CE, De Almeida Castanho REP, Tanaka II, Viggiani AMFS, Martins MC, Bassi NA, Gottardo De Almeida MT, et al. Per- LPA. Intestinal and pulmonary infection by Cryptos- formance of an immunoenzymatic assay for Cryp- poridium parvum in two patients with HIV/AIDS. tosporidium diagnosis of fecal samples. Brazilian J Rev Inst Med Trop Sao Paulo 2016; 58:21. Infect Dis 2005; 9:3–5. 31. Hawkins S, Thomas R, Teasdale C. Acute pancrea- 41. Hawash Y. Evaluation of an immunoassay-based titis: a new finding in cryptosporidium enteritis. Br algorithm for screening and identification of Giar- Med J 1987; 294:483–4. dia and Cryptosporidium antigens in human faecal 32. Naseer M, Dailey FE, Juboori AA, Samiullah S, specimens from Saudi Arabia. J Parasitol Res 2014; Tahan V. Epidemiology, determinants, and manage- 2014. ment of AIDS cholangiopathy: a review. World J Ga- 42. El-Sayed NM, Abdel-Wahab MM. Detection of bla- stroenterol 2018; 24:767-74. stocystis in stool specimens using parasitological 33. Darlan DM, Rozi MF, Andriyani Y, Yulfi H, Sara- methods and commercial antigen detection enzyme- gih RH, Nerdy N. Cryptosporidium Sp. Findings linked immunosorbent assay: a comparative study. and its symptomatology among immunocompromi- Egypt J Med Sci 2011; 32:327–38. sed patients. Open Access Maced J Med Sci 2019; 43. Gawad SSA, Ismail MAM, Imam NFA, Eassa AHA, 7:1567–71. abu-Sarea EY. Detection of Cryprosporidium spp. in 34. Certad G, Viscogliosi E, Chabé M, Cacciò SM. Pat- diarrheic immunocompetent patients in Beni-Suef, hogenic mechanisms of Cryptosporidium and Giar- Egypt: insight into epidemiology and diagnosis. Ko- dia. Trends Parasitol 2017; 33:561–76. rean J Parasitol 2018; 55:113-9. 35. Borad A, Ward H. Human immune responses in 44. El-Sayed NM, Abdel-Wahab M. Detection of Bla- cryptosporidiosis. Future Microbiol 2010; 5:507-19. stocystis in stool specimen using parasitological 36. Roberts T, Stark D, Harkness J, Ellis J. Update on methods and commercial antigen detection enzyme- the pathogenic potential and treatment options for linked immunosorbent assay: a comparative study. Blastocystis sp. Gut Pathog 2014; 6:1–9. Egypt J Med Sci 2011; 32:327-38. 37. Puthia MK, Sio SWS, Lu J, Tan KSW. Blastocystis 45. Nydam D V., Lindergard G, Guard CL, Schaaf SL, ratti induces contact-independent apoptosis, F-actin Wade SE, Mohammed HO. Serological detection of rearrangement, and barrier function disruption in exposure to Cryptosporidium parvum in cattle by IEC-6 Cells. Infect Immun 2006; 74:4114-23 ELISA and its evaluation in relation to coprological 38. Jeremiah S, Parija S. Blastocystis: taxonomy, bio- tests. Parasitol Res 2002; 88:797–803. logy and virulence. Trop Parasitol 2013; 3:17. 46. Kotgire Santosh A. Microbiological stool examinati- 39. Danišová O, Halánová M, Valenčáková A, on: Overview. J Clin Diagnostic Res 2012; 6:503–9. Luptáková L. Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in ani- mals. Brazilian J Microbiol 2018; 49:177–83.

61 ORIGINAL ARTICLE

Anti-fibrotic effect of intravenous umbilical cord-derived mesenchymal stem cells (UC-MSCs) injection in experimental rats induced liver fibrosis Taufik Sungkar1, Agung Putra2-4, Dharma Lindarto5, Rosita Juwita Sembiring6

1Divison of -, Department of Internal Medicine, Faculty of Medicine, Universitas Sumatera Utara, Medan, 2Stem Cell And Cancer Research (SCCR), Faculty of Medicine, 3Department of Postgraduate Biomedical Science, 4Department of ; Faculty of Medicine, Sultan Agung Islamic University, Semarang, 5Division of and Metabolism, Department of Internal Medicine, 6Department of ; Faculty of Medicine, Universitas Sumatera Utara, Medan; Indonesia

ABSTRACT

Aim To investigate the effect of umbilical cord-derived me- senchymal stem cells (UC-MSCs) administration among liver fibrosis experimental rat model via the regulation of angiotensin II type 1 receptor (AT1R) and platelet-derived growth factor-β (PDGF-β) due to their therapeutic potential to replace liver tran- splantation for advanced liver fibrosis. Yet the mechanism of ac- tion has been questionably associated with UC-MSCs fibrosis re- gression properties.

Corresponding author: Methods Sprague-Dawley (SD) rats (n=18) were separated into three groups (control, untreated liver fibrosis, and UC-MSCs tre- Taufik Sungkar ated group). Serum PDGF-β level was determined by enzyme- Division of Gastroenterology-Hepatology, linked immunosorbent assay (ELISA) following 14 days of UC- Department of Internal Medicine, MSCs injection. Meanwhile, AT1R expression was interpreted Haji Adam Malik General Hospital, based on immunoreactive score (IRS) stained using polyclonal Faculty of Medicine, antibody and liver fibrosis stained with hematoxylin & eosin was Universitas Sumatera Utara graded using the METAVIR score. Kampus USU Padang Bulan, Medan Results UC-MSCs were isolated successfully from rat umbilical 20155, Indonesia cord. Liver fibrosis was observed following 14 weeks 4 ofCCl Phone: +62 81 163 9323; injection concurrent with higher serum level of PDGF-β, but the Fax: +62 61 821 6264; UC-MSCs-treated group had lower level (980.08 ±289.41 and 606.42±109.85 for untreated liver fibrosis and UC-MSCs treated E-mail: [email protected] group, respectively; p=0.004). There was also a high expression ORCID ID: http://orcid.org/0000-0002- of AT1R among untreated liver fibrosis group, as well as high- 7736-8677 grade liver fibrosis versus localized fibrosis and low level of AT1R expression among UC-MSCs treated-group (p=0.001). Original submission: Conclusion UC-MSCs administration could ameliorate liver fi- 28 May 2020; brosis by reducing the AT1R expression and PDGF-β serum le- Revised submission: vels, and intervention through this signaling pathway could be 13 August 2020; alternative evidence for the causative of positive outcome. Accepted: 17 August 2020 Key words: immunohistochemistry, liver cirrhosis, mesenchymal stem cell transplantation doi: 10.17392/1211-21

Med Glas (Zenica) 2021; 18(1):62-69

62 Sungkar et al. Anti-fibrotic effect of UC-MSCs

INTRODUCTION Therefore, managing liver fibrosis directed aga- inst AT1R and PDGF-β inhibition could become Liver fibrosis is an abnormal response of hepa- one of the promising approaches. tocyte producing collagenous composition re- sulting from repetitive noxious exposure as eti- The elucidation of liver fibrosis pathogenesis co- ologic agents of chronic liver diseases (CLDs) uld unfold several accessible options to halt or (1). Several causative factors are associated with reverse its progression. Meanwhile, the definitive CLDs ranging from genetic diseases, viral infec- treatment for advanced- liver fibrosis only reli- tions, cholestasis, metabolic disorders, alcohol, es on liver transplantation (LT) that is frequently and other hepatotoxic substance exposures (2). In overshadowed by several drawbacks, such as do- severe form of liver fibrosis, so-called cirrhosis, nor availability, long term use of immunosuppre- it contributes to higher mortality and the leading ssive medication, host rejection, and high cost cause of disability-adjusted life-years (DALYs) (17). Nevertheless, the presence of mesenchymal worldwide (3). There are more than 300 million derived- stem cells (MSCs) could become a re- or 5% of the World population who suffered from placement for LT since it has successfully ame- CLDs, with two million premature deaths predo- liorated liver cirrhosis in several studies but no minantly caused by hepatitis B viral infection findings of any sole mechanism that has been accounting for 257 million cases in 2015 (4). attributed to the positive outcomes (18). The selection of stem cells source must also be con- The pathomechanism of liver fibrosis involves sidered based on various indicators. However, the interaction between immunologic cascade umbilical cord-derived MSCs (UC-MSCs) are by inducing extracellular matrix production, and superior as they offer several advantages, such as harmful stimuli (5). Initially, the accumulation of high content sources for pluripotent stem cells, extracellular matrix (ECM) becomes a hallmark accessibility, and they also highly regenerate to for fibrotic development until it finally impairs hepatocyte (19,20). liver function. This hostile environment activates quiescent hepatic stellate cells (HSCs) to myofi- The aim of this study was to analyze and de- broblasts (MFBs); MFBs will act as the essential monstrate the fibrotic regression function of source for ECM (6-8). In recent findings, plate- UC-MSCs administration by reducing the AT1R let-derived growth factor-beta (PDGF-β) also expression and PDGF-β serum level in experi- plays pivotal roles as a potent mitogen for cultu- mental rats induced liver fibrosis. re-activated HSCs as well as a chemotactic fac- MATERIALS AND METHODS tor (9). Higher levels of PDGF-β also ramp the proliferative response in HSCs among cirrhotic Materials and study design liver patients compared to healthy controls (10). In another theory subset, angiotensin II and its This laboratory experimental study with rando- receptor, angiotensin II type 1 receptor (AT1R) mized post test control group design was conduc- is also involved in the HSCs activation (11). Ac- ted at the Stem Cell and Cancer Research (SCCR) tivated-HSCs can promote AT1R expression or Laboratory and the Animal House Integrated Bi- vice versa through the AT1R signaling pathway omedical Laboratory facility, Faculty of Medici- that activates quiescent HSCs via phosphorylati- ne, Sultan Agung Islamic University, Semarang, on of Janus kinase-2 (12,13). The AT1R activati- Indonesia, during the period April-July 2019. on enhances angiotensin-II effect that perpetually A total of 18 Sprague-Dawley (SD) rats with si- insults the liver by triggering the activation of the milar characteristics, including aged 12-14-week- renin-angiotensin-aldosterone system (RAAS); it old and 200-250 g each, was housed under a con- will promote oxidative stress and up-regulation trolled environment (12h:12h light/dark cycle, of proinflammatory cytokines (14,15). In a pre- temperature 22± 2°C, 55% of humidity with vious study, there was low-grade liver fibrosis in accessible food and water sufficient for 104 days the AT1R deficient rat model with less inflamma- before the study) and umbilical cord-derived me- tory response versus markedly increased fibrosis senchymal stem cells (UC-MSCs) were used. progression in wild type rats after chronic ad- Carbon tetrachloride (CCl4) (Sigma–Aldrich, ministration of carbon tetrachloride (CCl4) (16). USA) was injected into the intraperitoneal site of

63 Medicinski Glasnik, Volume 18, Number 1, February 2021

SD rats to induce liver fibrosis at a dose of 1 mL/kg incubated at 37 °C with 5% humidified CO2 for 14 body weight twice weekly (12 weeks of duration). days following manufacturer’s instructions. Cell The SD rats were then allocated in randomized growth continuation was observed every 24 hours mode into three groups with six rats in each group: under an inverted microscope with replacing the group I (G1) as control group that received normal medium every 2-3 days until it approached 80% saline injection, group II (G2) as untreated liver of confluence. It proceeded into two times-washi- fibrosis group, which was only administered the ng processes using phosphate buffer saline (PBS)

CCl4 injection, and group III (G3) as UC-MSCs followed by trypsinization with 1 mL of 0.25%

treated-group; this group conceived rats with CCl4 trypsin-EDTA (Gibco-BRL, NY, USA) while sha- injection as well as UC-MSCs (1×106 cells per rat) king the tube. Inactivation of Trypsin-EDTA was via the tail vein. Blood sampling and liver tissue done using a new medium then the cells were tran- extraction were conducted on the fourteenth day sferred into a new conical tube for centrifugation after UC-MSCs injection (Figure 1). in 1900 rpm for 10 minutes. Pellet form cells was re-suspended into complete medium and incubated in new flask disks, mentioned as first-passage cul- tures until it sub-cultured in the fourth passage that had been used for the experiment. Flow cytometric analysis. The immunophe- notypes of UC-MSCs were analyzed at the fourth passage using conjugated antibodies, na- mely fluorescein isothiocyanate (FITC) CD90, allophycocyanin (APC) CD73, peridinin chlo-

Figure 1. Flow chart of the experimental study which was rophyll protein complex (PerCP) CD105 and divided into three groups, G1, G2, and G3; G1, control group phycoerythrin (PE) lin monoclonal antibodies. received normal saline injection; G2, untreated liver fibrosis group After this, flow cytometry (BD Bioscience, Fran- (only administered the CCl4 injection); G3, UC-MSCs treated- klin Lakes, NJ, USA) determined the fluorescen- group (rats with CCl4 injection as well as UC-MSCs of 1×106 cells per rat). ce intensity of the cells. Anesthetized-rats were euthanized through cer- In vitro differentiation of UC-MSCs to the oste- vical dislocation procedure at the end of the ogenic lineage. Mesenchymal stem cells must observation based on animal euthanasia guideli- demonstrate their ability to differentiate into me- nes (American Veterinary Medical Association/ sodermal or osteogenic lineage cells. Cells of the 3 AVMA) (21). The study protocol was also decla- fourth passage were used at a density of 5x10 2 red following the guidelines for the care and use cells/cm and cultured in DMEM medium supple- of laboratory animals and approved by the Ethi- mented with 10% FBS, 10 mmol/L β glycerop- -7 cal Commission of Faculty of Medicine, Univer- hosphate, 10 mol/L/ 0.1 µM dexamethasone, sitas Sumatera Utara, Medan, Indonesia. 50µmol/L ascorbate 2-phosphate (all from Sigma- Aldrich, Louis St, MO) to induce osteogenic diffe- Methods rentiation. Cell culture was then incubated at 37 °C and 5% CO , as well as twice a week of me- Isolation, purification, and cultivation of UC- 2 dium replacement. It was ultimately stained with MSCs. The UC-MSCs were developed from the 0.2% alizarin red solution (Sigma-Aldrich, USA); umbilical tissue of single pregnant Sprague-Dawley the appearance of bright red-stain was interpreted (SD) rats (8 x 106 cells). Small pieces of umbilical as osteocytes, which represents calcium depositi- cord were then inundated with Dulbecco’s Modi- on as well as its multipotency properties. fied Eagle’s medium/ DMEM (Sigma-Aldrich, Louis St, MO) in T25 culture flask supplemented Histopathological analysis. The SD rats were with 10% Fetal Bovine Serum (FBS) (GibcoTM anesthetized and sacrificed on days 14 after UC- Invitrogen, NY, USA). Nucleated cells were iso- MSCs injection in advance of liver extraction. lated in a complete culture medium, which was The analysis was aimed at evaluating the extent also supplemented with 100 IU/mL or 1% of pe- of fibrosis among the experimental groups (G1, nicillin/ streptomycin (Sigma-Aldrich, USA) and G2, and G3). Firstly, liver fixation used pot-con-

64 Sungkar et al. Anti-fibrotic effect of UC-MSCs

taining 10% formalin to preserve cells and tissue Platelet-Derived Growth Factor-β (PDGF-β) components. Secondly, paraffin-embedded tissue analysis. Blood was extracted from the perior- was cut into smaller pieces (3-5 µm of thickness) bital venous plexus under general anesthesia using a microtome. Lastly, routine hematoxylin- following 14 days of MSCs administration. Pla- eosin (H&E) staining was applied to tissue sam- telet-derived growth factor-β (PDGF-β) serum ples and observed under microscope at high ma- levels were measured by sandwich enzyme-lin- gnification. Liver fibrosis was graded based on ked immunosorbent assay (ELISA), from Rat the histological scale of the METAVIR score; it PDGF-β ELISA kit (MyBioSource, United Sta- consists of five-point scale ranging from F0 to tes), which perform under the manufacturer’s in- F4 (F0, no fibrosis; F1, localized fibrosis enclose structions as well as the interpretations (formu- portal area; F2, periportal fibrosis or mild portal- lated through the analysis of the standard curve portal septa; F3, bridging fibrosis or septa fibro- and optical density each sample spectrophotome- sis surrounded portal tracts and terminal hepatic trically at wavelength of 450 using software). vein; F4, diffuse nodule or cirrhosis) (22). Statistical analysis Immunohistochemistry. Immunohistochemical staining employed angiotensin II receptor type- A one-way ANOVA determined the mean diffe- 1 (AT1R) polyclonal antibody (MyBioSource, rence of serum PDGF-β level with an LSD com- San Diego, USA). The samples were also pre- parison post hoc test preceded by normality test. served using 10% formalin and embedded in pa- The AT1R expression was determined by Kruskal raffin to accommodate microtome cut. Staining Wallis and post hoc with the Mann Whitney test. A was further applied to samples according to the p<0.05 was considered as statistically significant. manufacturer’s instructions. The interpretation RESULTS of immunohistochemistry was grouped based on immunoreactive score (IRS) from the multiplica- Umbilical cord-derived mesenchymal stem cells tion between given score for positive cell covera- were expressed CD105 (95.9%), CD73 (99.2%), ge (0, no immunoreactive cells; 1, <10%; 2, 10- and CD90 (99.9%), but were negative for Lin 50%; 3, 51-80%; 4, > 80%) and colour intensity (2%) through flow cytometric analysis (Figure (0, no colour; 1, mild; 2, moderate; 3, intense). 2A). The UC-MSCs appeared as long spindle- The IRS score was divided into four categories shaped cells under a microscope (Figure 2B), and (0-1= negative; 2-3= mild; 4-8= moderate; and, it adhered to plastic in cell culture. Additionally, 9-12= strongly positive) (23). UC-MSCs should also demonstrate osteogenic

Figure 2. Characteristics, isolation, and differentiation of UC-MSCs. A-C) Most UC-MSCs expressed positive markers (CD90, CD105, and CD73) with D) negative marker Lin; E) UC-MSC candidates appear as fibroblast-like cell characteristics (magnification 10x, scale bar 200 μm); F) osteogenic differentiation (magnification 40x, scale bar 50 μm); UC-MSCs, umbilical cord-derived mesen- chymal stem cells;

65 Medicinski Glasnik, Volume 18, Number 1, February 2021

Figure 3. Histology of liver sections from rats in various groups. A) no fibrosis (G1); B) untreated liver fibrosis group (G2) was posi- tive with a fibrous expansion of the most portal areas with marked bridging (F3); C) UC-MSCs treated-group (G3) had eliminated the effect of CCl4 injection with mild fibrosis (F1) (magnification 400x); UC-MSCs, umbilical cord-derived mesenchymal stem cells; G1, control group received normal saline injection; G2, untreated liver fibrosis group (only administered the CCl4 injection); G3, UC-MSCs treated-group (rats with CCl4 injection as well as UC-MSCs of 1×106 cells per rat); differentiation capability, which was noticeable fibrosis; specifically, it included isolated fibrosis through the appearance of calcium deposition surrounding the portal area (F1) compared with stained red by Alizarin red dye (Figure 2C). the untreated liver fibrosis group (G2). In other words, UC-MSCs injection was associated with CCl4 successfully induced fibrosis in the experi- mental rat model; it demonstrated significant hi- a lower accumulation of collagenous scar. stological alteration encompassing from septal fi- UC-MSCs injection into fibrosis-induced rat gro- brosis that expanded to portal tracts and terminal up remarkably reduced serum PDGF-β level com- hepatic vein until conspicuous bridging fibrosis pared to control (G1) and untreated liver fibrosis (F3) (Figure 3). It was also notable that the UC- group (G2) with a significant mean difference at MSCs-treated group (G3) had low-grade hepatic day 14 (606.42±109.85 vs. 762.11 ±235.12 vs.

Figure 4. The effect of UC-MSCs injection on serum level of PDGF-ß and AT1R expression. A) Lower serum level of PDGF-ß fol- lowing 14 days of UC-MSCs administration compared to untreated liver fibrosis group (G2) (p=0.003); B) AT1R expression also significantly reduced in UC-MSCs treated groups versus untreated liver fibrosis group (p=0.003); C-E) immunohistochemistry analysis showed that UC-MSCs attenuated AT1R expression, as well as fibrosis up-regulated AT1R abundantly (left to right G1-G3) (magnification 400x); UC-MSCs, umbilical cord-derived mesenchymal stem cells; PDGF-ß, platelet-derived growth factor-beta; AT1R, angiotensin II type 1 receptor; G1, control group received normal saline injection; G2, untreated liver fibrosis group (only administered the CCl4 injection); G3) UC-MSCs treated-group (rats with CCl4 injection as well as UC-MSCs of 1×106 cells per rat);

66 Sungkar et al. Anti-fibrotic effect of UC-MSCs

980.08 ±289.41, respectively) (p=0.004). Post hoc immunomodulatory and paracrine effect, hepa- analysis using LSD also demonstrated significant tocyte proliferation, and restoration (32). The mean differences between each group (Figure 4A). role of MSCs has been studied for their activity Low immunoreactive score (IRS) of AT1R in UC- to restrain myofibroblast differentiation, and they MSCs treated group (G3) compared to G2 (1 (1-2) act in deterring quiescent HSCs transformation vs. 4 (4-6), p=0.001) was also successfully proved to myofibroblast in several stages, thus reducing (Figure 4B). The G2 showed a strong expression fibrosis progression (33,34). The presented study of AT1R, which was represented by the appearance showed considerable reduction of PDGF-β level of dark-brown colour through polyclonal antibody- following UC-MSCs injection intravenously to targeted AT1R staining, but no AT1R expression the experimental rats indicating inhibitory effects was demonstrated in G1. Immunohistochemistry of UC-MSC to prevent catastrophic implications interpretation verified low expression of AT1R of PDGF/PDGFR signaling pathway; on the 14th among UC-MSCs treated group as well as fibrosis day, PDGF-β level decreased in the UC-MSCs- induced up-regulation of AT1R expression (Figure treated group, but there was no significant diffe- 4C). The post hoc analysis of immunoreactive sco- rence between the control group and UC-MSCs res of AT1R expression and PDGF-β level for three treated-group. This phenomenon is relatable to groups is depicted in Figure 4. the fact that MSCs will actively engage in the induction of endogenic stem cells to trigger he- DISCUSSION patocyte regeneration via paracrine effect during the initial phase of administration (35). Repetitious administration of CCl4 establishes liver fibrosis; it initially triggers an acute respon- The renin-angiotensin system (RAS) is also in- se, including interstitial edema, focal or centrilo- volved in the process of fibrogenesis. Several bular necrosis, and inflammatory cell infiltration investigations reported that RAS re-distributed followed by HSCs activation that increases the only in chronically injured livers and activated production of extracellular matrix (ECM), and it HSCs de novo, which then generates angiotensin ultimately transforms normal liver parenchyma II (36). Subsequently, angiotensin II accumulates into well-established liver fibrosis (24-27). In the at the sites of parenchymal injury and binds to angiotensin II type 1 receptor (AT1R) in myofi- presented study, CCl4 induction produced patho- logical changes consisting of high-grade fibrosis broblasts to promote the recruitment of inflamma- among experimental rats. tory cells, secretion of extracellular matrix prote- ins, and inhibition of collagen degradation (37). The major problem relating to chronic liver di- Albeit angiotensinogen is the only component of seases, as well as liver fibrosis, is the continu- the RAS expressed in the healthy rat liver, the ation of collagenous and ECM deposition in expression of angiotensin-converting enzyme the liver tissue (28). The immune arm would (ACE) and AT1R are evident in the fibrotic rat react, subsequently resulting in the activation livers. In humans, there was up-regulation of of quiescent HSCs that directly play pivotal ro- ACE and chymase, a serine protease, in the liver les in the progression of hepatic fibrosis per se. with severe fibrosis, whereas AT1R expression is Several growth factors and cytokines will also re-located to fibrotic areas (38). In the previous overwhelm liver milieu to turn on dormant HSC study, the administration of human adipose-de- (29). Therefore, experimental studies have shown rived MSCs down-regulated AT1R expression in that suppression of HSCs proliferation could be addition to α-SMA, TGFβ1, Col I, and Col III beneficial against liver fibrosis. The PDGF is one reduction in cardiac myofibroblast (39). of the efficient mitogens that could up-regulate supportive protein expression for HSCs proli- Regarding the direct anti-fibrotic effects of MSCs feration and migration. Several reports have in- against HSCs, MSCs can inhibit the proliferation vestigated PDGFRβ up-regulation during HSCs of HSCs as well as its apoptosis inducer (40). In activation and positively correlated with liver fi- our study, prominent pathological abnormalities brogenesis (25,30,31). In general, mesenchymal among the untreated liver fibrosis group occurred stem cells (MSCs) can produce cytokines and in linear with the strong expression of AT1R; in signaling molecules, which finally produce an contrast, UC-MSCs treated group showed weak

67 Medicinski Glasnik, Volume 18, Number 1, February 2021

expression of AT1R. Therefore, the primary suring AT1R expression was still acceptable to outcome relating to the UC-MSCs administrati- determine the linkage between its overexpression on is to down-regulate the AT1R expression that and fibrosis development. subsequently halts fibrosis progression. In conclusion, the attenuation of liver fibrosis occurred following UC-MSCs administration via CCl4 injection in our study was administered biweekly via intraperitoneal for 12 weeks until AT1R down-regulation and PDGF-β serum level it developed liver fibrosis, as confirmed throu- reduction. This supportive mechanism directly gh histopathological examination. Liver fibrosis implicated the disease progression of the experi- was significantly mild in rats which received mental rat model. The study results indicated that UC-MSCs might have a potential as anti-fibrotic UC-MSCs and CCl4 injection, acknowledging the anti-fibrotic effect of UC-MSCs specifically treatment through downregulating PDGF-β and through the reduction of PDGF-β serum level AT1R signaling pathways. and AT1R expression; the amalgamation of the FUNDING processes synergistically decreased or inhibited HSCs activation from proliferating. No specific funding was received for this study. Our study did not escape some limitations, inclu- The study approved by the Ethical Commission ding several markers for HSCs activation that of Faculty of Medicine, Universitas Sumatera were not investigated; the HSCs activity was Utara, Medan, Indonesia (No: 542/TGL/KEPK purely based on the final results of the induction FK USU-RSUP HAM/2019). and UC-MSCs transplantation. In addition, some transcription factors associated with AT1R upre- TRANSPARENCY DECLARATION gulation have not yet been elucidated, but mea- Conflict of interest: None to declare.

REFERENCES 1. Friedman SL. Liver fibrosis–from bench to bedside. 9. Yoshida S, Ikenaga N, Liu SB, Peng ZW, Chung J Hepatol 2003; 38:38-53. J, Sverdlov DY, Miyamoto M, Kim YO, Ogawa 2. Weiskirchen R, Weiskirchen S, Tacke F. Recent ad- S, Arch RH, Schuppan D, Popov Y. Extrahepatic vances in understanding liver fibrosis: bridging ba- platelet-derived growth factor-β, delivered by pla- sic science and individualized treatment concepts. telets, promotes activation of hepatic stellate cells F1000Res 2018; 7:1-17. and biliary fibrosis in mice. Gastroenterol 2014; 3. Asrani SK, Devarbhavi H, Eaton J, Kamath PS. Bur- 147:1378–92. den of liver diseases in the world. J hepatol 2019; 10. Borkham-Kamphorst E, Weiskirchen R. The PDGF 70:151-71. system and its antagonists in liver fibrosis. Cytokine 4. Mokdad AA, Lopez AD, Shahraz S, Lozano R, Mo- Growth F R 2016; 28:53–61. kdad AH, Stanaway J, Murray CJ, Naghavi M. Liver 11. Yoshiji H, Kuriyama S, Yoshii J. Angiotensin-II type cirrhosis mortality in 187 countries between 1980 1 receptor interaction is a major regulator for liver fi- and 2010: a systematic analysis. BMC Med 2014; brosis development in rats. Hepatol 2001; 34:745-50. 12:145. 12. Bataller R, Ginès P, Nicolás JM, Görbig MN, Gar- 5. Liu Y, Wang Z, Kwong SQ, Lui ELH, Friedman cia–Ramallo E, Gasull X, Bosch J, Arroyo V, Rodés SL, Li FR, Lam RWC, Zhang GC, Zhang H, Ye T. J. Angiotensin II induces contraction and prolifera- Inhibition of PDGF, TGF-β, and Abl signaling and tion of human hepatic stellate cells. Gastroenterol reduction of liver fibrosis by the small Bcr- 2000; 118:1149-56. Abl tyrosine kinase antagonist Nilotinib. J Hepatol 13. Bataller R, Sancho-bru P, Ginès P, Lora JM, Al-ga- 2011; 55:612–25. rawi A, Solé M, Colmenero J, Nicolás JM, Jiménez 6. Lei X, Fu W, Kaneyama JK, Omoto T, Miyazaki T, W, Weich N, Gutiérrez-ramos JC. Activated human Li B, Miyazaki A. Hic-5 deficiency attenuates the hepatic stellate cells express the renin-angiotensin activation of hepatic stellate cells and liver fibrosis system and synthesize angiotensin II. Gastroenterol through upregulation of Smad7 in mice. J Hepatol 2003; 125:117-125. 2016; 64:110–17. 14. Shim KY, Eom YW, Kim MY, Kang SH, Baik SK. 7. Hernandez-Gea V, Friedman SL. Pathogenesis of Role of the renin-angiotensin system in hepatic fi- liver fibrosis. Annu Rev Pathol Mech Dis 2011; brosis and portal hypertension. Korean J Intern Med 6:425-56. 2018; 33:453-61. 8. Gressner AM, Weiskirchen R. Modern pathogenetic 15. Zhang Y, Yang X, Wu P, Xu L, Liao G, Yang G. concepts of liver fibrosis suggest stellate cells and Expression of angiotensin II type 1 receptor in rat he- TGF-β as major players and therapeutic targets. J patic stellate cells and its effects on cell growth and Cell Mol Med 2006; 10:76-99. collagen production. Horm Res 2003; 60:105-10.

68 Sungkar et al. Anti-fibrotic effect of UC-MSCs

16. Kanno K, Tazuma S, Chayama K. AT1R-deficient 29. Perepelyuk M, Terajima M, Wang AY, Georges PC, mice show less severe progression of liver fibrosis Janmey PA, Yamauchi M, Wells RG. Hepatic stella-

induced by CCl4. Biochem Biophys Res Commun te cells and portal fibroblasts are the major cellular 2003; 308:177-83. sources of collagens and lysyl oxidases in normal li- 17. Forbes SJ, Gupta S, Dhawan A. Cell therapy for liver ver and early after injury. Am J Physiol Gastrointest disease: from liver transplantation to cell factory. J Liver Physiol 2013; 304:G605-14. Hepatol 2015; 62:157-69. 30. Foo NP, Lin SH, Lee YH, Wu MJ, Wang YJ. 18. Raphael PH, Mahou R, Morel P. Microencapsulated α-Lipoic acid inhibits liver fibrosis through the atte- human mesenchymal stem cells decrease liver fibro- nuation of ROS-triggered signaling in hepatic stella- sis in mice. J Hepatol 2015; 62:634–41. te cells activated by PDGF and TGF-β. Toxicology 19. Yin F, Wang WY, Jiang WH. Human umbilical cord 2011; 282:39–46. mesenchymal stem cells ameliorate liver fibrosis in 31. Ezhilarasan D, Sokal E, Najimi M. Hepatic fibro- vitro and in vivo: from biological characteristics to sis: It is time to go with hepatic stellate cell-specific therapeutic mechanisms. World J Stem Cells 2019; therapeutic targets. Hepatobiliary Pancreat Dis Int 11:548-64. 2018; 17:192–197. 20. Tsuchiya A, Takeuchi S, Watanabe T, Yoshida T, No- 32. Berardis S, Sattwika PD, Najimi M, Sokal EM. Use jiri S, Ogawa M, Terai S. Mesenchymal stem cell of mesenchymal stem cells to treat liver fibrosis: cu- therapies for liver cirrhosis: MSCs as “conducting rrent situation and future prospects. World J Gastro- cells” for improvement of liver fibrosis and regene- entero 2015; 21:742. ration. Inflamm Regen 2019; 39:1-6. 33. Sriramulu S, Banerjee A, Di Liddo R, Jothimani G, 21. Underwood W, Anthony R, Cartner S, Corey D, Gopinath M, Murugesan R, Marotta F, Pathak S. Grandin T, Greenacre CB, Gwaltney-Bran S, Concise review on clinical applications of conditi- McCrackin MA, Meyer R, Miller D. AVMA gu- oned medium derived from human umbilical cord- idelines for the euthanasia of animals: 2013 editi- mesenchymal stem cells (UC-MSCs). Int J Hematol on. Schaumburg, IL: American Veterinary Medical Oncol Stem Cell Res 2018; 12:230. Association, 2013. 34. Zhang LT, Peng XB, Fang XQ, Li JF, Chen H, Mao 22. Bedossa P, Poynard T. An algorithm for the grading XR. Human umbilical cord mesenchymal stem cells of activity in chronic hepatitis C. Hepatol 1996; inhibit proliferation of hepatic stellate cells in vitro. 24:289-93. Int J Mol Med 2018; 41:2545-52. 23. Remmele W, Stegner H. Recommendation for uni- 35. Putra A, Antari A, RetnoKustiyah A, SorayaNurIn- form definition of an immunoreactive score (IRS) tan Y, Anna C, Sadyah N, Wirawan N. Mesenchymal for immunohistochemical estrogen receptor detec- stem cells accelerate liver regeneration in acute li- tion (ER-ICA) in breast cancer tissue. Pathologe ver failure animal model. Biomed Res Ther 2018; 1987;8:138–40. 5:2802-10. 24. Fortea JI, Fernandez-Mena C, Puerto M, Ripoll 36. Munshi MK, Uddin MN, Glaser SS. The role of the C, Almagro J, Banares J, Bellon JM, Banares R, renin–angiotensin system in liver fibrosis. Exp Biol Vaquero J. Comparison of two protocols of carbon Med 2011; 236:557-66. tetrachloride-induced cirrhosis in rats – improving 37. Okamoto K, Tajima H, Nakanuma S, Sakai S, Maki- yield and reproducibility. Sci Rep 2018; 8:9163. no I, Kinoshita J, Hayashi H, Nakamura K, Oyama 25. Chen C, Li X, Wang L. Thymosin β4 alleviates cho- K, Nakagawara H, Fujita H. Angiotensin II enhances lestatic liver fibrosis in mice through downregula- epithelial-to-mesenchymal transition through the in- ting PDGF/PDGFR and TGFβ/Smad pathways. Di- teraction between activated hepatic stellate cells and gest Liver Dis 2020; 52:324–30. the stromal cell-derived factor-1/CXCR4 axis in 26. Lu B, Xu Y, Xu L, Cong X, Yin L, Li H, Peng J. intrahepatic cholangiocarcinoma. Int J Oncol 2012; Mechanism investigation of dioscin against CCl4 in- 41:573-82. duced acute liver damage in mice. Environ Toxicol 38. Moreno M, Bataller R. Cytokines and renin-angio- Phar 2012; 34:127–35. tensin system signaling in hepatic fibrosis. Clin Li- 27. Wang R, Wang J, Song F, Li S, Yuan Y. Tanshi- ver Dis 2008; 12:825–52.

nol ameliorates CCl4 induced liver fibrosis in rats 39. Yong KW, Li Y, Liu F, Gao B, Lu TJ, Abas WA, through the regulation of Nrf2/HO-1 and NF-κB/ Safwani WK, Pingguan-Murphy B, Ma Y, Xu F IκBα signaling pathway. Drug Des Dev Ther 2018; and Huang G. Paracrine effects of adipose-derived 12:1281–92. stem cells on matrix stiffness-induced cardiac myo- 28. Mormone E, George J, Nieto N. Molecular patho- fibroblast differentiation via angiotensin II type 1 genesis of hepatic fibrosis and current therapeutic receptor and Smad7. Sci Rep 2016; 6:33067. approaches. Chem Biol Interact 2011; 193:225-31. 40. Alfaifi M, Eom YW, Newsome PN, Baik SK. Me- senchymal stromal cell therapy for liver diseases. J Hepatol 2018; 68:1272–85.

69 ORIGINAL ARTICLE

Concordance of non-invasive serology-based scoring indices and transient elastography for liver fibrosis and cirrhosis in chronic hepatitis C Emir Trnačević1, Nermin Salkić2, Alma Trnačević3, Anja Divković1, Fatima Hukić1, Nusret Butković1, Amra Serak4, Amer Mujkanović5

1Department for Laboratory Diagnostics, University Clinical Centre Tuzla, 2Department of Gastroenterology and Hepatology, Internal Medicine Hospital, University Clinical Centre Tuzla, 3Infectious Disease Hospital, University Clinical Centre Tuzla, 4Department of , Public Health Centre Tuzla, 5University Clinical Centre Tuzla, Surgery Hospital, Department for ; Tuzla, Bosnia and Herzegovina

ABSTRACT

Aim To assess concordance of eight frequently used serology-ba- sed scoring indices for liver fibrosis and cirrhosis with transient elastography (TE) in chronic hepatitis C (CHC) patients in order to determine serum indices with the highest concordance and clinical usability in clinical practice.

Methods In this prospective study, 63 CHC patients were included and TE results were compared with eight non-invasive indices. The diagnostic performance of these tests was assessed using re- ceiver operating characteristic curves with kappa index calculated Corresponding author: for the concordance analysis. Nermin Salkić Results Median age of 63 patients was 54 years (interquartile ran- Department of Gastroenterology and ge: 42 to 63); 27 (42.9%) were females. According to areas under Hepatology, University Clinical Centre the Receiver Operating Characteristics (AUROC), the best perfor- Tuzla ming serum markers for significant liver fibrosis (METAVIR ≥F2), Prof. Ibre Pašića bb, 75000 Tuzla, advanced liver fibrosis (≥F3) and cirrhosis (F4) determined by Bosnia and Herzegovina TE measurements (≥7.1kPa, ≥9.5kPa and ≥12kPa, respectively) were Fibrotest (AUROC=0.727 for ≥F2) and FIB-4 score (AU- E-mail: [email protected] ROC=0.779 for ≥F3 and AUROC=0.889 for F4). Fibrotest cut-off Phone: +387 35 303 358; at >0.50 was concordant with TE for presence of significant fibro- Fax: +387 35 250 474; sis in 30 (out of 45; 66.7%), FIB-4 cut-off at <1.45 was concordant ORCID: https://orcid.org/0000-0003- for absence of significant fibrosis in 13 (out of 18; 72.2%) and 4727-9267 Goeteborg University Cirrhosis Index (GUCI) cut-off at >1 was concordant for presence of cirrhosis in 16 (out of 22; 72.7%) pati- ents, but not for exclusion of cirrhosis. Original submission: Conclusion Serology-based scoring indices had moderate overall 08 September 2020; concordance with TE. We propose that FIB-4 score, Fibrotest and Revised submission: GUCI be used in routine practice to exclude and diagnose signifi- 15 September 2020; cant fibrosis and diagnose cirrhosis, respectively. Accepted: Key words: diagnosis, hepatic cirrhosis, hepatic fibrosis, non-in- 20 September 2020 vasive markers, vibration controlled transient elastography doi: 10.17392/1269-21

Med Glas (Zenica) 2021; 18(1):70-76

70 Trnačević et al. Concordance of tests for liver fibrosis in CHC

INTRODUCTION another standard in non-invasive assessment of liver fibrosis and cirrhosis (18). Chronic hepatitis C (CHC) is one of the most frequent causes of liver cirrhosis and its compli- In countries with limited resources such as B&H, cations. (1). Despite recent advances in the tre- elastography is not widely available, even more, atment, epidemiological data from Bosnia and there are only four centres that routinely perform Herzegovina (B&H) show that there is up to 1% liver biopsy. The need for cheap and widely ava- prevalence of CHC in general population, and ilable method for assessment of liver fibrosis and the most of the patients are with advanced liver cirrhosis in routine clinical practice and even pri- fibrosis (2,3). mary care is obvious. Although there are numero- us papers that compared serology-based indices The stage of liver fibrosis in CHC patients has a (or serum markers as this group of tests are also huge impact on prognosis, treatment strategy and referred to in literature) and elastography aga- follow-up, with or without treatment (4). Liver inst liver biopsy (16,18–20), studies dealing with biopsy is a traditional method of reference used concordance of serum markers with elastography to assess fibrosis stage in CHC (4); however, it as newly established standard are sparse. is an invasive procedure with serious complicati- ons in up to 0.5% of procedures, biopsy samples The aim of this study was to assess concordance <15mm in length are not reliable and its accuracy of eight of the most frequently used serum mar- is limited by heterogeneity of samples and inter- kers for liver fibrosis and cirrhosis with TE in po- observer and intra-observer variability (4–7). pulation of CHC patients in order to determine serum marker with the highest concordance and Due to inherent limitations of liver biopsy as a dia- clinical usability in routine clinical practice. gnostic procedure, several serology based non-in- vasive methods (indices) for assessments of liver METHODS fibrosis and cirrhosis have been developed with variable clinical accuracy and applicability (8- Patients and study design 15). Fibrotest, aspartate aminotransferase (AST)- to- alanine aminotransferase (ALT) ratio (AAR This prospective study recruited all consecutive score), AST to Platelet Ratio Index (APRI score), adult patients with chronic hepatitis C that were re- FIB-4 test, Goteborg University Cirrhosis Index ferred for transient elastography to the Department (GUCI), Forns score, Lok score, Hui score are of Gastroenterology and Hepatology of University all serological clinical indices combining several Clinical Centre Tuzla from 1 July 2019 to 30 June biochemical and clinical variables into mathema- 2020, as a part of patient’s pre-treatment evaluati- tical formula, and were extensively evaluated and on. Patients were considered for inclusion if they validated for various aetiologies of chronic liver had no treatment during the last 6 months. The disease (8–15). Yet, all these serological indices CHC was confirmed by HCV–RNA polymerase have limitations as blood tests can be influenced chain reaction analysis of serum (Cobas Amplicor by other associated diseases, comorbidities or HCV v2.0, Roche, Switzerland) (21). Cirrhotic even laboratory equipment and/or technique (16). patients were compensated and asymptomatic at the time of the inclusion. Patients with co-existing As a viable and more accurate alternative, a liver diseases other than CHC and post-transplant physical method was developed in form of tran- patients were also excluded. All patients gave con- sient elastography (Fibroscan, Echosens, Paris, sent for the study. France), which is based on liver stiffness mea- surement with comparable or even improved re- The study was conducted in accordance with the liability and clinical accuracy when compared to Helsinki Declaration, and it was approved by the serum indices (17). Despite the fact that transient institutional Ethics Committee of the University elastography (TE) also has its limitation (particu- Clinical Centre of Tuzla. larly in obese patients, those with ascites, or more Methods than 5-fold increase in liver transaminase levels), as well as that proprietary equipment is expen- Serum markers. Demographic and anthropo- sive and not widely available, it has become metric data were recorded at the time of TE and fasting blood samples were also collected at the

71 Medicinski Glasnik, Volume 18, Number 1, February 2021

same time by venepuncture. Standard and identi- Statistical analysis cal batches of tubes for all patients were used. All Descriptive results expressed as the mean (standard parameters used to calculate non-invasive indi- deviation), median (interquartile range), or as the ces were measured at the Department of Labo- number (percentage) of patients. An assessment of ratory Diagnostics, University Clinical Centre of serological tests vs. TE was made with Pearson or Tuzla using serum samples frozen and stored at Spearman correlation where appropriate. The di- -80 °C until assayed. All tests were performed on agnostic performance of serum markers was also the same day in a single laboratory by operators assessed using Area Under the Receiver Operating blinded of clinical and other data about patients. Characteristic (AUROC) analysis, with TE as a Based on measured serum parameters the follow- reference method, albeit imperfect due to indirect ing non-invasive serum tests were calculated: nature of TE itself. The comparison of AUROCs AAR (AST/ALT ratio), Aspartate Aminotrans- was performed according to the method described ferase-to-Platelet Ratio Index (APRI), GUCI, by DeLong (29). The AUROCs were also used to FIB-4, Forns index, Lok score, Hui score and Fi- assess best preforming threshold values of serum brotest, according to the most recently published markers for the prediction of fibrosis and cirrhosis formulas (8–15,22). grades according to TE. Previously published cut- There are two distinct threshold values for FIB-4 off values for all indices of interest also used to described (10): one threshold set at 1.45, which create dichotomous variables in order to calculate has an excellent negative predictive value for si- concordance coefficient kappa (κ). All statistical te- gnificant fibrosis (F2 or greater) and other set at sts were 2-tailed, with type I error of 5% (p<0.05). 3.25, which has an excellent positive predictive value for cirrhosis. For GUCI score a threshold RESULTS of 1.0 was described as predictive for cirrhosis A total of 65 patients with CHC were prospecti- and 0.33 as predictive for absence of significant vely recruited with only two (3.1%) unsuccessful fibrosis (14,23). For Fibrotest, a threshold <0.50 measurements by TE, resulting in 63 patients was described as predictive for absence of signi- included in the study (Table 1). ficant fibrosis, while a threshold of >0.75 was described as predictive for presence of cirrhosis Table 1. Baseline characteristics for 63 patients with chronic hepatitis C (8,24,25). Variable Mean SD Transient elastography. Liver stiffness mea- Gender (females) (No; %) 27 (42.9) surement by transient elastography (Fibroscan, Age (years) 52.30 13.76 Height (cm) 173 13 Echosens, Paris, France) was made in a fasting Weight (kg) 77 13 patient on the right lobe of the liver, through the Body Mass Index (kg/m2) 25.75 4.20 appropriate intercostal space with prone patient Platelets (109/L) 184.00 78.32 and with the right arm in maximal abduction. The Prothrombin time (sec) 14.26 12.83 INR 1.30 1.44 tip of the transducer probe was covered with cou- Total bilirubin (μmol/L) 16.56 10.01 pling gel and placed on the skin in the appropriate ALT (IU/L) 116.62 169.93 intercostal space, usually in medio-axillary line. AST (IU/L) 72.98 96.53 GGT (IU/L) 60.63 52.29 At least 10 valid measurements of liver stiffness Albumin (g/L) 44.57 4.87 were considered as technically appropriate; mea- Cholesterol (mmol/L) 4.53 0.90 surement failure was defined as zero valid shots Haptoglobin 0.78 0.60 Alfa-2 macroglobulin 3.08 1.55 after at least 10 attempts and unreliable measure- Apolipoprotein A1 1.31 0.40 ments were defined as fewer than 10 valid shots Transient elastography (No; %) or an interquartile range of stiffness median value Significant fibrosis (≥F2; ≥7.1 kPa) 45 (71.4) Advanced fibrosis (≥F3; ≥9.5 kPa) 34 (54.0) greater than 30% (26,27). Elastography threshold Cirrhosis (F4; ≥12.0 kPa) 22 (34.9) values corresponding with significant fibrosis INR, international normalized ratio; ALT, alanine aminotransferase; (METAVIR F2 or greater; 7.1 kPA), advanced AST, aspartate aminotransferase; GGT, gamma glutamyl transferase; fibrosis (METAVIR F3 or greater; 9.5 kPA) and In order to test for initial correlation of serum mar- cirrhosis (METAVIR F4; 12.0 kPA) were used, as kers, a correlation analysis was performed with previously described for patients with CHC (28). matrix table (Table 2). There was a statistically si-

72 Trnačević et al. Concordance of tests for liver fibrosis in CHC

gnificant correlation of all serum markers with TE, with strongest coefficient for FIB-4 and Hui score.

Table 2. Correlation matrix of serum markers with transient elastography Serology based index of Transient elastography liver fibrosis/cirrhosis Correlation coefficient p AAR score 0.415 <0.001 APRI score 0.430 <0.001 GUCI score 0.467 <0.001 FIB-4 score 0.740 <0.001 Forns index 0.602 <0.001 Lok score 0.602 <0.001 Hui score 0.758 <0.001 Fibrotest 0.456 <0.001 AAR, AST (aspartate aminotransferase) to ALT (alanine aminotran- sferase) ratio; APRI, AST to platelets ratio index; GUCI, Goteborg University Cirrhosis Index

The diagnostic performance of serum markers was tested against TE with AUROC (Table 3, Figure 1). For F2 or greater fibrosis, Fibrotest showed the highest AUROC, closely followed by FIB-4 test. For both F3 and F4 fibrosis, FIB-4 showed the highest AUROC when compared with fibrosis grades determined with TE measurements.

Table 3. Area under the receiver operating characteristics (AUROC) for serum markers tested against liver fibrosis grades according to transient elastography (TE) measurements

Serum markers for 95% confidence interval prediction of F2 or (CI) of AUROC AUROC p greater according Lower limit Upper limit to TE of CI of CI AAR score 0.623 0.130 0.472 0.774 APRI score 0.657 0.053 0.526 0.788 GUCI score 0.680 0.026 0.552 0.809 FIB-4 score 0.684 0.023 0.552 0.816 Forns index 0.657 0.053 0.506 0.808 LOK score 0.650 0.065 0.513 0.787 HUI score 0.633 0.100 0.491 0.776 Fibrotest 0.727 0.005 0.589 0.866 Serum markers for prediction of F3 or greater according to TE AAR score 0.668 0.022 0.534 0.801 APRI score 0.729 0.002 0.599 0.860 GUCI score 0.759 <0.001 0.633 0.884 FIB-4 score 0.779 <0.001 0.665 0.892 Forns index 0.716 0.003 0.588 0.844 LOK score 0.710 0.004 0.584 0.836 HUI score 0.683 0.013 0.550 0.816 Fibrotest 0.747 0.001 0.624 0.871 Serum markers for prediction of F4 according to TE AAR score 0.702 0.009 0.565 0.839 APRI score 0.877 <0.001 0.777 0.977 GUCI score 0.863 <0.001 0.760 0.965 FIB-4 score 0.899 <0.001 0.808 0.990 Forns index 0.863 <0.001 0.765 0.960 Figure 1. Area under the Receiver Operating Characteristics LOK score 0.782 <0.001 0.667 0.898 (AUROC) for serum markers tested against liver fibrosis grades HUI score 0.840 <0.001 0.728 0.952 according to transient elastography (TE) measurements. A) sig- Fibrotest 0.774 <0.001 0.639 0.909 nificant liver fibrosis (≥F2; ≥7.1 kPa); B) advanced liver fibro- AAR, AST (aspartate aminotransferase) to ALT (alanine aminotran- sis (≥F3; ≥9.5 kPa); C) liver cirrhosis (F4; ≥12 kPa) sferase) ratio; APRI, AST to platelets ratio index; GUCI, Goteborg University Cirrhosis Index

73 Medicinski Glasnik, Volume 18, Number 1, February 2021

Pairwise comparison of AUROCs did not show According to our results, we propose that for a qu- significant differences (p>0.05) between serum ick screening in routine clinical use or primary care markers for F2 or greater and F3 or greater. A settings it is the best to use FIB-4 score for exclu- significant difference was detected for F4 where ding significant liver fibrosis (<1.45) thus postpo- FIB-4 had higher AUROC than Fibrotest, AAR ning referral and to use GUCI score (>1.0) for non- and APRI score (p<0.05). invasive detection of cirrhosis and prompt referral Considering the highest AUROC values for all to hepatologist. Where available, Fibrotest owing three clinically significant fibrosis thresholds FIB- to the highest AUROC (0.727) and concordance 4, GUCI and Fibrotest were selected for further rate with TE (66.7%) seems to be appropriate. analysis, as serum markers with best concordance Non-invasive staging of liver fibrosis and detection according to correlation and AUROC analysis. of cirrhosis is incorporated in current guidelines for When compared with TE values, FIB-4 at cut-off the diagnosis and management of CHC (18). For a point of 1.45 was concordant with TE in 13 (out clinician there are two distinct fibrosis thresholds of 18; 72.2%) patients for exclusion of presen- that are clinically relevant: occurrence of signifi- ce of significant fibrosis with overall κ=0.265 cant fibrosis, defined as F2 or higher according to (p=0.021). At the cut-off point of 3.25, FIB-4 was METAVIR classification which signals the need concordant with TE in 14 (out of 22; 63.6%) pa- for active treatment and occurrence of liver cirrho- tients for the prediction of cirrhosis with overall sis, defined as F4 according to METAVIR which κ=0.661 (p<0.001). signals the need for the treatment and prevention of cirrhosis and its complications (32). The GUCI score at threshold value of 1.0 was concordant with TE in 16 (out of 22; 72.7%) with Serum markers have been heavily evaluated for overall κ value of 0.551 (p<0.001). At the cut- different liver pathologies with variable clinical off value of 0.33 GUCI score was concordant accuracy. As previously reported, all types of with TE in 12 (out of 18; 66.7%) patients for the serum markers of liver fibrosis have AUCs clu- exclusion of presence of significant fibrosis with stering around the value of 0.85, which is a con- κ value of 0.14 (p=0.262). sequence of inherent insufficiency of liver biopsy to perform at the level of a true gold standard Fibrotest at threshold value of <0.50 was concor- (33,34). When discussing the accuracy of any dant with TE in 13 (out of 18; 72.2%) patients marker in a case of discordant results, the cau- with overall κ=0.33 (p=0.005). For the thres- se of discordance can be either failure of fibrosis hold value >0.50, Fibrotest was concordant for marker or failure of biopsy to detect a true stage, diagnosing presence of significant fibrosis with since sensitivity and specificity of liver biopsy TE in 30 (out of 45; 66.7%) patients. At the cut- are 90% and even in perfect conditions, AUROC off point of 0.75, Fibrotest was concordant for for the perfect non-invasive marker would be presence of cirrhosis with TE in 15 (out of 45; 0.90 (35,36). This is important to bear in mind 68.2%) patients with overall κ=0.425 (p=0.001). when comparing serum marker with TE, as both DISCUSSION are derived, developed and compared against im- perfect gold standard (LB), so possible causes for The results of this study have demonstrated that discordance are variable. among eight most frequently used serum mar- kers, FIB-4, GUCI and Fibrotest have shown Transient elastography is one of the most utilized the best overall concordance with TE. Areas non-invasive tools for evaluation of liver fibrosis under the ROC curves for those scores do not in patients with CHC with better diagnostic accu- conflict with previously published results regar- racy as compared to serum tests (37). Still, despite dless of the reference standard used (TE or li- the fact that TE has an excellent overall accuracy, ver biopsy) (16,19,30). Yet, the specific concor- as with all other non-invasive tests, and even liver dance for detecting clinically relevant cut-offs biopsy (37), it is insufficient in differentiating in- is heterogenous and moderate, suggesting the termediate stages of fibrosis (i.e., F2 vs F3) (38). need for utilisation of several serum markers at Therefore, there are several combination algo- different cut-offs for various clinically relevant rithms that advocate combining TE with serum bi- thresholds according to the best detection rate, omarkers, either sequentially or concomitantly in as proposed previously for chronic hepatitis B order to increase diagnostic accuracy of clinically patients (31). relevant thresholds (18,30,39,40). This approach

74 Trnačević et al. Concordance of tests for liver fibrosis in CHC

is very convenient for primary care, as primary may attempt to combine serological markers in a care physicians can make initial screening of pati- combination algorithm, similar to the one we pre- ents with cheap, available and easy serum markers viously developed for chronic hepatitis B (www. before referral to a specialized centre for a more chb-lfc.com) (31) that would use best performing advanced procedure such as TE or even biopsy. threshold points from several serum markers and There are several limitations of this study that incorporate them into a single screening tool sui- need to be addressed. The sample size is at its table for everyday clinical practice. lowest limit for statistical analysis, yet it is suffi- In conclusion, serology based non-invasive indices cient for addressing the aim of this study. It wo- have moderate overall concordance with transient uld be much better to compare the results against elastography, with FIB-4, Goteborg University the liver biopsy, but with the availability of TE Cirrhosis Index (GUCI) and Fibrotest having the and current recommendations that most pati- best performance. For CHC patients and in routine ents with CHC can be evaluated with non-inva- clinical practice, even in primary care, we propose sive means, we used TE as surrogate reference that FIB-4 score, Fibrotest and GUCI be used to standard (18,32). For the purpose of this study, exclude significant fibrosis, diagnose significant concordance of serological tests with TE was the fibrosis and diagnose cirrhosis, respectively. primary aim, we did not attempt to re-evaluate previously established thresholds for TE and FUNDING serum markers, and we believe that results are No specific funding was received for this study. sufficiently reliable for derived conclusions, as is the case with previously published studies (19). TRANSPARENCY DECLARATION Further studies including larger sample sizes Conflicts of interest: None to declare. REFERENCES 1. Pimpin L, Cortez-Pinto H, Negro F, Coubould E, fibrosis in patients with hepatitis C virus infection: a Lazarus JV, Webber L, Sheron N, EASL HEPAHE- prospective study. Lancet 2001; 357:1069–75. ALTH Steering Committee. Burden of liver disease 9. Wai C. A simple noninvasive index can predict both in Europe: epidemiology and analysis of risk fac- significant fibrosis and cirrhosis in patients with chro- tors to identify prevention policies. J Hepatol 2018; nic hepatitis C. Hepatology 2003; 38:518–26. 69:718-35. 10. Vallet-Pichard A, Mallet V, Nalpas B, Verkarre V, 2. Petrovic J, Salkic NN, Ahmetagic S, Stojic V, Mott- Nalpas A, Dhalluin-Venier V, Fontaine H, Pol S. FIB- Divkovic S. Prevalence of chronic hepatitis B and he- 4: An inexpensive and accurate marker of fibrosis in patitis C among first time blood donors in Northeast HCV infection. comparison with liver biopsy and fi- Bosnia and Herzegovina: an estimate of prevalence in brotest. Hepatology 2007; 46:32–6. general population. Hepat Mon 2011; 11:629–33. 11. Forns X. Identification of chronic hepatitis C patients 3. Leblebicioglu H, Arends JE, Ozaras R, Corti G, without hepatic fibrosis by a simple predictive model. Santos L, Boesecke C, Ustianowski A, Duberg A-S, Hepatology 2002; 36:986–92. Ruta S, Salkic NN, Husa P, Lazarevic I, Pineda JA, 12. Lin Z-H, Xin Y-N, Dong Q-J, Wang Q, Jiang X-J, Pshenichnaya NY, Tsertswadze T, Matičič M, Puca Zhan S-H, Sun Y, Xuan S-Y. Performance of the as- E, Abuova G, Gervain J, Bayramli R, Ahmeti S, Ko- partate aminotransferase-to-platelet ratio index for ulentaki M, Kilani B, Vince A, Negro F, Sunbul M, the staging of hepatitis C-related fibrosis: An updated Salmon D, ESGHV (part of ESCMID). Availability of meta-analysis. Hepatology 2011; 53:726–36. hepatitis C diagnostics and therapeutics in European 13. Lok ASF, Ghany MG, Goodman ZD, Wright EC, and Eurasia countries. Antiviral Res 2018; 150:9–14. Everson GT, Sterling RK, Everhart JE, Lindsay KL, 4. Cadranel J-F, Rufat P, Degos F. Practices of liver li- Bonkovsky HL, Di Bisceglie AM, Lee WM, Morgan opsy in France: results of a prospective nationwide TR, Dienstag JL, Morishima C, HALT-C Trial Group. survey. Hepatology 2000; 32:477–81. Predicting cirrhosis in patients with hepatitis C based 5. Regev A, Berho M, Jeffers LJ, Milikowski C, Molina on standard laboratory tests: Results of the HALT-C EG, Pyrsopoulos NT, Feng Z-Z, Reddy KR, Schiff cohort. Hepatology 2005; 42:282–92. ER. Sampling error and intraobserver variation in 14. Islam S, Antonsson L, Westin J, Lagging M. Cirrhosis liver biopsy in patients with chronic HCV infection. in hepatitis C virus-infected patients can be excluded Am J Gastroenterol 2002; 97:2614–8. using an index of standard biochemical serum mar- 6. Bedossa P. Intraobserver and interobserver iariations kers. Scand J Gastroenterol 2005; 40:867–72. in liver biopsy interpretation in patients with chronic 15. Hui AY, Chan HL-Y, Wong VW-S, Liew C-T, Chim hepatitis C. Hepatology 1994; 20:15–20. AM-L, Chan FK-L, Sung JJ-Y. Identification of chro- 7. Rousselet M-C, Michalak S, Dupré F, Croué A, Bedo- nic hepatitis B patients without significant liver fibro- ssa P, Saint-André J-P, Calès P. Sources of variability sis by a simple noninvasive predictive model. Am J in histological scoring of chronic viral hepatitis. He- Gastroenterol 2005; 100:616–23. patology 2005 19; 41:257–64. 16. Zarski J-P, Sturm N, Guechot J, Paris A, Zafrani E-S, 8. Imbert-Bismut F, Ratziu V, Pieroni L, Charlotte F, Be- Asselah T, Boisson R-C, Bosson J-L, Guyader D, nhamou Y, Poynard T. Biochemical markers of liver Renversez J-C, Bronowicki J-P, Gelineau M-C, Tran

75 Medicinski Glasnik, Volume 18, Number 1, February 2021

A, Trocme C, Ledinghen VD, Lasnier E, Poujol-Ro- 27. Lucidarme D, Foucher J, Le Bail B, Vergniol J, Caste- bert A, Ziegler F, Bourliere M, Voitot H, Larrey D, ra L, Duburque C, Forzy G, Filoche B, Couzigou P, de Rosenthal-Allieri MA, Fouchard Hubert I, Bailly F, Lédinghen V. Factors of accuracy of transient elasto- Vaubourdolle M. Comparison of nine blood tests and graphy (fibroscan) for the diagnosis of liver fibrosis in transient elastography for liver fibrosis in chronic he- chronic hepatitis C. Hepatology 2008; 49:1083–9. patitis C: The ANRS HCEP-23 study. J Hepatol 2012; 28. Castéra L, Vergniol J, Foucher J, Le Bail B, Chantelo- 56:55–62. up E, Haaser M, Darriet M, Couzigou P, de Lédinghen 17. Friedrich–Rust M, Ong M, Martens S, Sarrazin C, V. Prospective comparison of transient elastography, Bojunga J, Zeuzem S, Herrmann E. Performance of Fibrotest, APRI, and liver biopsy for the assessment transient elastography for the staging of liver fibrosis: of fibrosis in chronic hepatitis C. Gastroenterology a meta-analysis. Gastroenterology 2008; 134:960- 2005; 128:343–50. 974.e8. 29. DeLong ER, DeLong DM, Clarke-Pearson DL. Com- 18. EASL-ALEH Clinical Practice Guidelines: non-inva- paring the areas under two or more correlated recei- sive tests for evaluation of liver disease severity and ver operating characteristic curves: a nonparametric prognosis. J Hepatol 2015; 63:237–64. approach. Biometrics 1988; 44:837–45. 19. Paranaguá-Vezozzo DC, Andrade A, Mazo DFC, 30. Agbim U, Asrani SK. Non-invasive assessment of li- Nunes V, Guedes AL, Ragazzo TG, Moutinho R, ver fibrosis and prognosis: an update on serum and Nacif LS, Ono SK, Alves VAF, Carrilho FJ. Concor- elastography markers. Expert Rev Gastroenterol He- dance of non-invasive mechanical and serum tests for patol 2019; 13:361–74. liver fibrosis evaluation in chronic hepatitis C. World 31. Salkic NN, Cickusic E, Jovanovic P, Denjagic MB, J Hepatol 2017; 9:436. Iljazovic-Topcic S, Bevanda M, Ahmetagic S. Online 20. Leroy V, Halfon P, Bacq Y, Boursier J, Rousselet combination algorithm for non-invasive assessment MC, Bourlière M, de Muret A, Sturm N, Hunault of chronic hepatitis B related liver fibrosis and cirr- G, Penaranda G, Bréchot M-C, Trocme C, Calès P. hosis in resource-limited settings. Eur J Intern Med Diagnostic accuracy, reproducibility and robustness 2015; 26:628–34. of fibrosis blood tests in chronic hepatitis C: a me- 32. European Association for the Study of the Liver. ta-analysis with individual data. Clin Biochem 2008; EASL Recommendations on treatment of hepatitis C 41:1368–76. 2018. J Hepatol 2018; 69:461–511. 21. Lee SC, Antony A, Lee N, Leibow J, Yang JQ, Sovie- 33. Pinzani M, Vizzutti F, Arena U, Marra F. Technology ro S, Gutekunst K, Rosenstraus M. Improved versi- Insight: noninvasive assessment of liver fibrosis by on 2.0 qualitative and quantitative Amplicor reverse biochemical scores and elastography. Nat Clin Pract transcription-PCR tests for hepatitis C virus RNA: Gastroenterol Hepatol 2008; 5:95–106. calibration to international units, enhanced genoty- 34. Salkic NN, Jovanovic P, Hauser G, Brcic M. Fibro- pe reactivity, and performance characteristics. J Clin Test/Fibrosure for significant liver fibrosis and - cirr Microbiol 2000; 38:4171-79. hosis in chronic hepatitis B: a meta-analysis. Am J 22. Bourliere M, Penaranda G, Renou C, Botta-Fridlund Gastroenterol 2014; 109:796–809. D, Tran A, Portal I, Lecomte L, Castellani P, Ro- 35. Poynard T, Munteanu M, Imbert-Bismut F, Char- senthal-Allieri MA, Gerolami R, Ouzan D, Deydier lotte F, Thabut D, Le Calvez S, Messous D, Thibault R, Degott C, Halfon P. Validation and comparison of V, Benhamou Y, Moussalli J, Ratziu V. Prospective indexes for fibrosis and cirrhosis prediction in chro- analysis of discordant results between biochemical nic hepatitis C patients: proposal for a pragmatic markers and biopsy in patients with chronic hepatitis approach classification without liver biopsies. J Viral C. Clin Chem 2004; 50:1344–55. Hepat 2006; 13:659–70. 36. Mehta SH, Lau B, Afdhal NH, Thomas DL. Excee- 23. Westin J, Ydreborg M, Islam S, Alsiö A, Dhillon AP, ding the limits of liver histology markers. J Hepatol Pawlotsky J-M, Zeuzem S, Schalm SW, Ferrari C, 2009; 50:36–41. Neumann AU, Hellstrand K, Lagging M, DITTO- 37. Bedossa P, Dargere D, Paradis V. Sampling variabi- HCV Study Group. A non-invasive fibrosis score pre- lity of liver fibrosis in chronic hepatitis C. Hepatology dicts treatment outcome in chronic hepatitis C virus 2003; 38:1449–57. infection. Scand J Gastroenterol 2008; 43:73–80. 38. Calès P, Boursier J, Lebigot J, de Ledinghen V, Aubé 24. Poynard T, Morra R, Halfon P, Castera L, Ratziu V, C, Hubert I, Oberti F. Liver fibrosis diagnosis by blo- Imbert-Bismut F, Naveau S, Thabut D, Lebrec D, Zo- od test and elastography in chronic hepatitis C: agree- ulim F, Bourliere M, Cacoub P, Messous D, Munte- ment or combination? Aliment Pharmacol Ther 2017; anu M, de Ledinghen V. Meta-analyses of FibroTest 45:991–1003. diagnostic value in chronic liver disease. BMC Ga- 39. Sebastiani G, Vario A, Guido M, Noventa F, Plebani stroenterology 2007; 7(1). M, Pistis R, Ferrari A, Alberti A. Stepwise combina- 25. Shaheen AAM, Wan AF, Myers RP. FibroTest and Fi- tion algorithms of non-invasive markers to diagnose broScan for the prediction of hepatitis C-related fibro- significant fibrosis in chronic hepatitis C. J Hepatol sis: a systematic review of diagnostic test accuracy. 2006; 44:686–93. Am J Gastroenterol 2007; 102:2589–600. 40. Sebastiani G. Non-invasive assessment of liver fi- 26. Ziol M, Handra-Luca A, Kettaneh A, Christidis C, brosis in chronic liver diseases: Implementation in Mal F, Kazemi F, de Lédinghen V, Marcellin P, Dhu- clinical practice and decisional algorithms. World J meaux D, Trinchet J-C, Beaugrand M. Noninvasi- Gastroenterol 2009; 15:2190. ve assessment of liver fibrosis by measurement of stiffness in patients with chronic hepatitis C. Hepato- logy 2004; 41:48–54.

76 ORIGINAL ARTICLE

An epidemiological study of neuroendocrine tumours at tertiary hospitals in Bosnia and Herzegovina

Maja Konrad Čustović, Ermina Iljazović, Azra Sadiković, Zinaida Karasalihović

Pathology Department, Polyclinic for Laboratory Diagnostics, University Clinical Centre of Tuzla, Bosnia and Herzegovina

ABSTRACT

Aim Neuroendocrine neoplasms (NENs) are a heterogeneous gro- up of tumours with varying clinical expression and behaviour. Be- cause of indolent behaviour of NENs, reviewing and evaluation of epidemiological characteristics is a challenge. The aim of this study was to assess prevalence of NENs at tertiary hospitals consi- dering age, gender, location, and grade.

Methods Electronic files were used for a retrospective assessment of the patients with NENs of the gastroenteropancreatic tract and bronchopulmonary system in tertiary hospitals in Bosnia and Her- zegovina over the past 15 years (2005-2020).

Corresponding author: Results Among 438 patients, 291(66.4%) were males and 147 Maja Konrad Čustović (33.6%) females; the median age was 62 years. The lungs were Pathology Department, the most frequent site, 304 (69.4%), followed by the pancre- as, 22 (5.0%), colon, 14 (3.2%), stomach, 13 (2.9%), appendix, Policlinic for Laboratory Diagnostics, 13 (2.9%), rectum,11 (2.5%), small intestine, eight (1.8%) and University Clinical Centre of Tuzla gallbladder, one (0.2%). Metastases were most frequently found in Prof. dr. Ibre Pašića bb, 75000 Tuzla, the liver, 35 (8%) and lymph nodes, 15 (3.42%). Bosnia and Herzegovina Phone: +387 35 303 566; Conclusion The results were largely consistent with those in lite- rature, including age, gender, location, and the degree of differen- fax: +387 35 303 300; tiation. Most metastases originated from high-grade tumours and E-mail: [email protected] greater impairment of the liver. ORCID ID: https://orcid.org/0000-0001- 9422-6980 Key words: epidemiology, incidence, neuroendocrine tumours, tertiary care centres

Original submission: 15 June 2020; Revised submission: 24 September 2020; Accepted: 06 October 2020 doi: 10.17392/1219-21

Med Glas (Zenica) 2021; 18(1):77-83

77 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION (such as screening and diagnostic endoscopy), physicians’ awareness, and possible environmen- Neuroendocrine neoplasm (NENs) is a hetero- tal factors (such as proton pump inhibitors) are geneous group of tumours with differences in considered responsible for the incidence increase clinical expression and behaviour, ranging from (18,19). relatively indolent to highly aggressive and fatal (1,2). These neoplasms originate from the cells of In recent years, several useful and evidence-ba- the diffuse neuroendocrine system distributed in sed classifications of NENs have been developed, many sites, amongst which most common are the differing between organ systems, and currently gastrointestinal tract and pancreas, followed by causing considerable confusion (20). In 2017, the bronchopulmonary system (3). The NENs se- major changes in the grading and staging systems crete peptides and neuroamines that may cause di- for pancreatic NENs were published: WHO gra- stinct symptoms termed “carcinoid syndromes”, ding systems changed the definition of grade 3 ne- in which case they are marked as “functional tu- uroendocrine tumours (NETs) to neuroendocrine th mours” (4). The behaviour and aggressiveness of carcinomas (NECs); the 8 American Joint Com- this neoplasm depend on a tumour size, secretion mittee on Cancer (AJCC) changed its tumour-no- and histological grade of malignancy (5). des-metastasis (TNM) based staging system for NEN; the treatment strategies changed according Unfortunately, because of their rarity, tumour he- to the new concepts of grade 3 NET/NEC (21). terogeneity and usually nonspecific presentation In many organ systems NENs are graded based of symptoms, 40-50% of NENs patients have an on mitotic count and/or Ki-67 labelling index, advanced disease at the time of the diagnosis (6). as well as the presence of necrosis (20). In the Metastases may occur more frequently to the li- lungs, NENs are currently classified as low-grade ver and are often presented by the time of pri- typical carcinoid (TC), intermediate-grade atypi- mary diagnosis in 45-95% cases, causing shorter cal carcinoid (AC), and the high-grade large cell overall survival (7,8). neuroendocrine carcinoma (LCNEC), and small The diagnosis of a suspected neuroendocrine ne- cell lung carcinoma (SCLC) (10). oplasm requires a confirmation of neuroendocri- The aim of this study was to investigate epidemi- ne differentiation, using a panel of conventional ological features of neuroendocrine tumours of neuroendocrine immunohistochemical markers the gastroenteropancreatic tract and the broncho- such as chromogranin, synaptophysin, and CD56 pulmonary system of patients from hospital cen- (9). Mitotic counts and the Ki-67 protein-based tres in Bosnia and Herzegovina (B&H) (gender, cell proliferation index have become useful to- age, primary site, the existence or absence of me- ols in assessing the malignant potential of NENs tastases). This is the first multicentre study rela- (5,10). Ki-67 proliferation marker had become ted to neuroendocrine neoplasms in Bosnia and an integral part of the World Health Organizati- Herzegovina. on (WHO) classification, as early as in the 2004 edition (11), as opposed to the lung NENs that PATIENTS ANDMETHODS mainly use a number of mitoses. Epidemiological studies based on the US Nati- Patients and study design onal Cancer Institute’s Surveillance, Epidemi- Electronic files were used for a retrospecti- ology and End Results (SEER) cancer registry ve assessment of patients with the diagnosis of (12-14) and data from the National Cancer Regi- neuroendocrine neoplasm of the gastroentero- stry of England for the period from 1971 to 2006, pancreatic tract and bronchopulmonary system, showed an increased global incidence of NENs from the Department of Pathology of the Univer- in recent decades (15). It increases with age with sity Clinical Centre of Tuzla, University Clini- a peak between 50 and 70 years of age. cal Centre of Sarajevo, East Sarajevo Hospital, The etiologic factors behind the increasing in- General Hospital dr. Abdulah Nakaš (Sarajevo), cidence and mortality for NENs are unclear Brčko District Hospital, and Cantonal Hospital (16,17). The improvement in the classification dr. Irfan Ljubijankić (Bihać), over the last 15 ye- system, increased use of diagnostic techniques ars (2005 to 2020).

78 Konrad Čustović et al. Neuroendocrine tumours in B&H

The data of the patients with neuroendocrine tu- The number of patients increased over the years, mours were recorded and, based on pathology re- with 185 in the period 2005-2014, and 251 in the ports, analysed by gender, age, diagnosis, tumour period 2015-2020, resulting in the increase of location, primary site, and presence or absence 35.7%. Incidence showed an ascending trend from of metastases. Patients without pathology reports 2005 to 2016, from 0.03/100,000 to 1.61/100,000 were not included. No patients with mixed ade- per year, respectively, and with a drastic decrease no-neuroendocrine tumours (MANETs) and car- in 2020, of 0.23/100,000 (Table1). cinomas (MANECs) were found. Table 1. Incidence of neuroendocrine neoplasm (NEN) in The study was approved by the Ethics Committee Bosnia and Herzegovina (B&H) in the period October 2005 - February 2020 of the University Clinical Centre of Tuzla. Number of B&H Incidence per Year patients population 100,000 Methods 2005 1 3842527* 0.03 2006 3 3842762* 0.08 Formalin fixed and paraffin embedded tissue 2007 9 3842562* 0.23 blocks from biopsies and surgical materials were 2008 12 3842265* 0.31 available for each patient. Hematoxylin and eo- 2009 19 3842566* 0.49 2010 23 3843126* 0.60 sin stained slides were re-examined to confirm 2011 16 3843000* 0.42 the original diagnosis. Conventional immuno- 2012 22 3839737* 0.57 histochemical markers of neuroendocrine diffe- 2013 42 3531159† 1.19 2014 40 3531159† 1.13 rentiation (synaptophysin, chromogranin, CD56) 2015 50 3531159† 1.42 were utilized to confirm the diagnosis. The Ki-67 2016 57 3531159† 1.61 immunohistochemical staining was performed on 2017 43 3531159† 1.22 each specimen. Each neoplasm from the gastroen- 2018 49 3531159† 1.39 2019 44 3531159† 1.25 teropancreatic tract was classified according to the 2020 8 3531159† 0.23 WHO classification th4 Edition for digestive system Total 438 based on the mitotic count and Ki-67 index (5): G1 *Population estimate – Agency for Statistics of B&H; †Population Census in B&H (2013) –Agency for Statistics of B&H NETs - mitotic count of <2 per 10 high-power fiel- ds (HPF) and/or Ki-67 labelling index of ≤2%, G2 Of 438 patients, 291(66.4%) were males and 147 NETs- mitotic counts of 2 to 20 per 10 HPF and/ (33.6%) were females. The median age was 62 ye- or Ki-67 labelling index of 3% and 20% and NEC ars (range 17-85). The mean age was 61.28 years; had mitotic counts of >20 per 10 HPF and/or Ki67 males were older compared to women, 61.90±1.24 labelling index of >20%. Neuroendocrine tumour and 60.05±1.92 years, respectively (p<0.101). of the lung was classified according to the WHO The most frequent age group was ˃60 years, 258 classification th4 Edition (10). (59.2%),158 (36.2%) were between 40-60 years, and 20 (4.6%) were below 40 years of age. Statistical analysis Among 304 lung NENs, small cell lung carcino- From the descriptive statistical parameters, abso- ma was mostly presented, in 271 (89.1%) pati- lute and percentage frequencies, and arithmetic ents (Table2). means with corresponding standard deviations Table 2. Characteristics of different histological types of lung were calculated. From non-parametric statisti- neuroendocrine neoplasms Number (%) of Mean Male/Female cal methods, the χ²test was applied, while from Histological type patients age(years) ratio parametric statistical methods Student's t-test of SCLC 271 (89.1) 62.2 2.9 independent samples was applied. LCNEC 19 (6.25) 63.9 2.8 AC 8 (2.63) 59.9 1.7 RESULTS TC 6 (1.97) 54.0 0.5 Total 304 (100) 60.0 2.7 A total of 438 patients with neuroendocrine (NEN) SCLC, small cell lung carcinoma; LCNEC, large cell neuroendocrine tumours of the gastroenteropancreatic tract and carcinoma; AC, atypical carcinoid, TC, typical carcinoma bronchopulmonary system was recorded in the In eight (15.4%) patients with small cell carci- period between October 2005 and February 2020: noma, biopsy was obtained from the metastases, 304 (69.4%) were lung, 82 (18.7%) gastroentero- which were most frequently found in the liver, patic (GEP) and 52 (11.9%) metastatic NEN. lymph nodes, and brain.

79 Medicinski Glasnik, Volume 18, Number 1, February 2021

NENs of the gastroenteropancreatic tract were tract and bronchopulmonary system from hospi- found in 82 (18.72%) patients. According the tals in Bosnia and Herzegovina were described. anatomical site, pancreas was most frequent, 22 This is the first multicentre study which included (26.8%), followed by the colon, 14 (17.1%), sto- the distribution of neuroendocrine tumours from a mach, 13(15.8%), cecal appendix, 13 (15.8%), tertiary hospital database over the last 15 years. A rectum, 11(13.4%), small intestine, eight (9.7%) limiting factor of this study was the lack of com- and gallbladder in one (1.2%) patient. Tumours plete data, but considering the fact that in Bosnia originating from the colon and stomach were and Herzegovina there is no register for this type mostly poorly differentiated, 11 (33.3% and 9 of neoplasm, our study is very important. (27.3%), respectively. NET G2 was most frequ- Based on the current medical literature, the ove- ent in the pancreas, 12 (60%), and NET G1 in rall incidence of NENs is rising (3). The SEER cecal appendix, 10 (34.5%) (Table 3). (Surveillance, Epidemiology, and End Results) Table 3. Characteristics of gastroenteropancreatic neuroen- database and national cancer registries in We- docrine neoplasms according to the site and grade stern Europe are probably some of the most pu- Number (%) of patients Characteristic blicly available cancer registries (22). Our study G1 G2 NEC Total Anatomical site showed a significant increase in the number of Pancreas 6 (27.3) 12 (54.5) 4 (18.2) 22 cases (of 35.7 %) for the period between 2015 Colon 3 (21.4) 11 (78.6) 14 and 2020, compared with the period 2005-2014, Stomach 1 (7.7) 3 (23.1) 9 (69.2) 13 Cecal appendix 10 (76.9) 3 (23.1) 13 mainly due to a greater access to complementary Rectum 5 (45.5) 6 (54.5) 11 diagnostic tests, such as imaging technique, en- Small intestine 4 (50) 2 (25) 2 (25) 8 doscopic procedures and histopathology exami- Gallbladder 1(100) 1 Mean age (years) 53.90 53.10 64.42 57.94 nation of these tumours. Accordingly, an increase Male/ Female ratio 1.23 0.82 1.36 1.16 in incidence was also noticed, from 0.03 in 2005 Total 29 20 33 82 to 1.61 in 2016, which is consistent with SEER G1, grade 1; G2, grade 2; NEC, neuroendocrine carcinoma database showing incidence steadily increasing Among 52 metastatic NENs, 44 (84.6%) of the over the past four decades (from 4.2 in 1993- gastroenteropancreatic tract were detected, the 1997 to 5.8 in 2000-2004) (13). most frequently in the liver and lymph nodes. The incidence of NENs increases with age, and it Of 35 (79.5%) cases in the liver, NEC was most peaks between the six and the seventh decade. In frequent, with 21 (60%) cases, followed by 9 the gastrointestinal tract, the median age is less (25.71%) cases of NET G2 and five (14.28%) than 50 years of age, for appendix and pancreatic cases of NET G1. Lymph nodes accounted to 9 NENs, and more than 60 for other organs (23). In (20.5%) cases of metastatic neuroendocrine tu- the bronchopulmonary system contrary to small mour of the gastroenteropancreatic tract (data cell lung carcinoma, the lung carcinoid occurs in not shown). The vast majority of metastatic cases younger and non-smoker patients (10) and can was neuroendocrine carcinoma but some cases be cured with surgical excision. The mean age were classified as NET G1 and NET G2 presen- (61.28 years) in our study was similar to a stu- ted with metastases even when they had a low dy from Norway (61.5 years) (24), but slightly proliferative index. higher compared to studies in France, China, Regarding the age and grades of NENs, NET G1 Germany and Spain (56 years) (25-28). and NET G2 frequently appeared below 40 years There are some minor differences relating to the of age, while SCLC, LCNEC, and NEC of the gender and race, between the countries/conti- gastroenteropancreatic tract were most frequent nents, and in some cases the changes over time after 60 years of age. Metastatic NENs in the li- have been noticed (22). Our study showed a male ver and lymph nodes were found mostly in the predominance, which was consistent with results patients above 60 years of age. of Norway, France, China, Germany and Spain studies (24-28). DISCUSSION Neuroendocrine tumours are commonly found in In this study epidemiological features of neuro- the gastrointestinal tract, pancreas and the lung endocrine tumours of the gastroenteropancreatic but it is not unusual to find neuroendocrine tu-

80 Konrad Čustović et al. Neuroendocrine tumours in B&H

mours in the thyroid, skin, breast and other or- the data in the literature (23,43); G2 pancreatic gans (29-31). Consistent with the Kentucky Can- NENs were most frequently encountered, which cer Registry (KCR) and SEER, between 1995 is consistent with a study from Germany (35). G1 and 2015 (30.6%) (32), as well as a nation-wide and G2 were the most frequent grades among ce- study from the Netherlands (72.7%) (23), the cal appendix tumours (none of which presented most common NEN sites in our study was the with metastases), and NET G1 among tumours lungs (69.4%). The distribution of the types of of the small intestine, which also consistent with lung neuroendocrine tumours was similar to that findings reported in the literature (44). in the literature, with small-cell carcinomas being In conclusion, to our knowledge this is the first the most frequent tumour (33,34). multicentre study of NENs epidemiology in Bo- Regarding the most frequent primary site in the snia and Herzegovina. Compared with the results gastroenteropancreatic tract, our data was consi- of other studies, our results were largely consistent stent with studies from Germany, Italy, Lebanon, with those in the literature, especially regarding Spain and Mexico (35-39), with the pancreas be- the age group, gender, and location. Because of the ing the most frequently affected organ. Although heterogeneous nature of these tumours that vary in the small intestines are currently in some parts of origin, morphology, molecular profile, type and si- Europe the most common primary sites for NENs te-specific prognosis, aggressiveness and response of the gastroenteropancreatic tract (24,25,40), the to therapy, the management of these tumours requ- highest increase in prevalence rate in recent years ires a multidisciplinary approach. Future studies to has been observed for gastric and rectal NENs, clarify etiologic factors are needed. which is consistent with our data. The widespre- ad usage of proton-pump inhibitors has been ACKNOWLEDGMENTS proposed as a possible risk factor for the highest Authors would like to thank Nina Z. Biser for increase of gastric NENs (41). Appendix NEN her helpful assistance in proofreading (language was more frequent among Western countries editing) the manuscript. (Nina Z. Biser; Bachelor (16.7%), which could be explained by accidental of Arts (Cum Laude) in English and Secondary findings on appendectomy performed for acu- Education from DePaul Universities, Chicago IL, te appendicitis (42). Rectal NEN was the most USA). common site in Asian populations (30.6%) (43). Our study showed 15.85% cases of appendix and FUNDING 9.76% of small intestine NEN. No specific funding was received for this study. Prevalence of NENs of the gastroenteropancre- atic tract in our population is consistent with TRANSPARENCY DECLARATION Competing interests: None to declare. REFERENCES 1. Modlin IM, Kidd M, Latich I, Zikusoka MN, Shari- 5. Bosman FT, Carneiro F, Hruban RH, Theise ND. po MD. Current status of gastrointestinal carcinoids. In: WHO Classification of tumors of the digestive Gastroenterology 2005; 128(suppl 6):1717-51. system. 4ͭ ͪEd. Geneva: World Health Organization, 2. Yang Z, Tang LH, Klimstra DS. Effect of tumor he- 2010. terogeneity on the assessment of Ki67 labeling index 6. Pavel M, O’Toole D, Costa F, Capdevila J, Gross D, in well-differentiated neuroendocrine tumors meta- Kianmanesh R, Krenning E, Knigge U, Salazar R, static to the liver: implications for prognostic strati- Pape UF, Öberg K, Vienna Consensus Conference fication. Am J Surg Pathol 2011; 35(suppl 6):853-60. participants. ENETS Consensus Guidelines Update 3. Dasari A, Shen C, Halperin D, Zhao B, Zhou S, Xu for the management of distant metastatic disease of Y, Shih T, Yao JC. Trends in the incidence, prevalen- intestinal, pancreatic, bronchial neuroendocrine ne- ce, and survival outcomes in patients with neuroen- oplasms (NEN) and NEN of unknown primary site. docrine tumors in the United States. JAMA Oncol Neuroendocrinology 2016; 103:172-85. 2017; 3(suppl 10):1335-42. 7. Saeed A, Buell JF, Kandil E. Surgical treatment of 4. Kyriakopoulos G, Mavroeidi V, Chatzellis E, Kalt- liver metastases in patient with neuroendocrine tu- sas GA, Alexandraki KI. Histopathological, immu- mors. Ann Trans Med 2013; 1(suppl 1):6. nochistochemical, genetic and molecular markers of 8. Kandil E, Saeed A, Buell J. Surgical approaches for neuroendocrine neoplasms. Ann Transl Med 2018; liver metastases in carcinoid tumors. Gland Surg 6(suppl 12):252. 2015; 4(suppl 5):442-6.

81 Medicinski Glasnik, Volume 18, Number 1, February 2021

9. Hofland J, Kaltsas G, de HerderWW. Advances in 23. Korse CM, Taal BG, van Velthuysen ML, Visser O. the diagnosis and management of well-differentia- Incidence and survival of neuroendocrine tumours ted neuroendocrine neoplasms. Endocr Rev 2020; in the Netherlands according to histological grade: 41(suppl 2):371-403. experience of two decades of cancer registry. Eur J 10. Travis WD, Brambilla E, Muller-Hermelink HK, Cancer 2013; 49(suppl 8):1975-83. Harris CC. Pathology &Genetics: Tumors of the 24. Sandvik OM, Søreide K, Gudlaugsson E, Kvaløy Lung, Pleura, Thymus and Heart. Lyon: IARC Pre- JT, Søreide JA. Epidemiology and classification ss, 2014. of gastroenteropancreatic neuroendocrine neopla- 11. Kleihues PSL. World Health Organization classifi- sms using current coding criteria. Br J Surg 2016; cation of Tumours. Pathology and Genetics of En- 103:226-32. docrine Organs. Lyon: IARC Press, 2004. 25. Lombard-Bohas C, Mitry E, O’Toole D, Louvet C, 12. Modlin IM, Lye KD, Kidd M. A 5-decade analysis Pillon D, Cadiot G, Borson-Chazot F, Aparicio T, of 13,715 carcinoid tumors. Cancer 2003; 97(suppl Ducreux M, Lecomte T. Thirteen-month registrati- 4):934-59. on of patients with gastroenteropancreatic endocri- 13. Hauso O, Gustafsson BI, Kidd M, Waldum HL, ne tumours in France. Neuroendocrinology 2009; Drozdov I, Chan AK, Modlin IM. Neuroendocrine 89:217-22. tumor epidemiology: contrasting Norway and North 26. Jiao X, Li Y, Wang H, Liu S, Zhang D, ZhouY. Cli- America. Cancer 2008; 113(suppl 10):2655-64. nicopathological features and survival analysis of 14. Lawrence B, Gustafsson BI, Chan A, Svejda B, Kidd gastroenteropancreatic neuroendocrine neoplasms: a M, Modlin IM. The epidemiology of gastroentero- retrospective study in a single center of China. Chin pancreatic neuroendocrine tumors. Endocrinol Me- J Cancer Res 2015; 27(suppl 3):258-66. tab Clin North Am 2011; 40(suppl 1):1-18. 27. BegumN, MaasbergS,PlöckingerU, AnlaufM, Rin- 15. Ellis L, Shale MJ, Coleman MP. Carcinoid tumors keA, PöpperlG, Lehnert H, IzbickiJR, Krausch of the gastrointestinal tract: trend in incidence in M, VashistYK, Raffel A, Bürk CG, Hoffmann J, Go- England since 1971. Am J Gastroenterol 2010; retzki P, Pape UF. Neuroendocrine tumours of the 105(suppl 12):2563-9. GI tract--data from the German NET Registry. Zen- 16. Leoncini E, Carioli G, La Vecchia C, Boccia S, Rin- tralbl Chir 2014; 139(suppl 3):276-83. di G. Risk factors for neuroendocrine neoplasms: 28. GalvánJA, Astudillo A, Vallina A, Fonse- a systematic review and meta-analysis. An Oncol ca PJ, Gómez-Izquierdo L, García-Carbonero 2016; 27:68-81. R, González MV. Epithelial-mesenchymal transition 17. Leoncini E, Boffetta P, Shafir M, Aleksovska K, markers in the differential diagnosis of gastroentero- Boccia S, Rindi G. Increased incidence trend of low- pancreatic neuroendocrine tumors. Am J Clin Pathol grade and high-grade neuroendocrine neoplasms. 2013; 140(suppl 1):61-72. Endocrine 2017; 58:368-79. 29. Yao JC, Hassan M, Phan A, Dagohoy C, Leary C, 18. Hodgson N, Koniaris LG, Livingstone AS, Frances- Mares JE, Abdalla EK, Fleming JB, Vauthey J, Ras- chi D. Gastric carcionoids: a temporal increase with hid A, Evans DB. One hundred years after “carci- proton pump introduction. Surg Endoscop 2005; noid”: Epidemiology of and prognostic factors for 19(suppl 12):1610-2. neuroendocrine tumors in 35,825 cases in the United 19. McCarthy MD. Proton Pump Inhibitor Use, hyper- States. J Clin Oncol 2008; 26:3063-72. gastrinemia, and gastric carcinoids-what is the rela- 30. Modlin IM, Champaneria MC, Chan AK, Kidd M. A tionship? Int J Mol Sci 2020; 21(suppl 2):662. three-decade analysis of 3,911 small intestinal neu- 20. Rindi G, Klimstra DS, Abedi-ArdekaniB,Asa SL, roendocrine tumors: The rapid pace of no progress. Bosman FT, Brambilla E, Busam KJ, de Krijger Am J Gastroenterol 2007; 102(suppl 7):1464-73. RR, Dietel M, El-NaggarAK,Fernandez-Cuesta L, 31. Fraenkel M, Kim MK, Faggiano A, Valk GD. Epi- Klöppel G, McCluggage WG, Moch H, Ohgaki H, demiology of gastroenteropancreatic neuroendocri- Rakha EA, ReedNS,RousBA,SasanoH,Scarpa A, ne tumors. Best Pract Res Clin Gastroenterol 2012; Scoazec JY, Travis WD, TalliniG,Trouillas J, van 26(suppl 6):691-703. Krieken JH, Cree IA. A common classification 32. Chauhan A, Yu Q, RayN,FarooquiZ,Huang framework for neuroendocrine neoplasms: Interna- B, DurbinEB,TuckerT,Evers M, Arnold S, Anthony tional Agency for Research on Cancer (IARC) and LB. Global burden of neuroendocrine tumors and World Health Organization (WHO) expert consen- changing incidence in Kentucky. Oncotarget 2018; sus proposal. Mod Pathol 2018; 31:1770-86. 9(suppl 27):19245-54. 21. Choe J, Kim KW, Kim HJ, Kim DW, Kim KP, Hong 33. Travis WD. Pathology and diagnosis of neuroen- SM, Ryu JS, Tirumani SH, Krajewski K, Ramaiya docrine tumors: lung neuroendocrine. Thorac Surg N. What Is New in the 2017 World Health Organi- Clin 2014; 24:257-66. zation Classification and 8th American Joint Com- 34. Siegel RL, Miller KD, Jemal A. Cancer stati- mittee on Cancer Staging System for pancreatic stics,2018. CA Cancer J Clin 2018; 68:7-30. neuroendocrine neoplasms? Korean J Radiol 2019; 35. Ploeckinger U, Kloeppel G, Wiedenmann B, Loh- 20(suppl 1):5-17. mann R. The German NET-Registry: An audit on 22. Fraenkel M, Kim M, Faggiano A, de Herder WW, the diagnosis and therapy of neuroendocrine tumors. Valk GD. Incidence of gastroenteropancreatic neu- Neuroendocrinology 2009; 90:349-63. roendocrine tumours: a systematic review of the lite- rature. Endoc Relat Cancer 2014; 21:153-63.

82 Konrad Čustović et al. Neuroendocrine tumours in B&H

36. Faggiano A, Ferolla P, Grimaldi F, Campana D, 40. Riihimäki M, Hemminki A, Sundquist K, Sundqu- Manzoni M, Davi M, Bianchi A, Valcavi R, Papi- ist J, Hemminki K. The epidemiology of metasta- ni E, Giuffrida D. Natural history of gastro-ente- ses in neuroendocrine tumors. Int J Cancer 2016; ro-pancreatic and thoracic neuroendocrine tumors. 139:2679-86. Data from a large perspective and retrospective Ita- 41. Jianu CS, Fossmark R, Viset T, Qvigstad G, Sordal lian epidemiological study: the NET management O, Mårvik R, Waldum HL. Gastric carcinoids af- study. J Endocrinol Invest 2012; 35:817. ter long-term use a proton pump inhibitor. Aliment 37. Kourie HR, Ghorra C, Rassy M, Kesserouani C, Pharmacol Ther 2012; 36(suppl 7):644-9. Kattan J. Digestive neuroendocrine tumor distribution 42. Moris D, Tsilimigras DI, VagiosS, Ntanasis-Statho- and characteristics according to the 2010 WHO Cla- poulos I, Karachaliou GS, Papalampros A, Alexan- ssification: a single institution experience in Lebanon. drou A, Blazer DG 3rd, Felekouras E. Neuroen- Asian Pac J Cancer Prev 2016; 17:2679-81. docrine neoplasms of the appendix: a review of the 38. Garcia-Carbonero R, Capdevila J, Crespo-Herre- literature. Anticancer Res 2018; 38(suppl 2):601-11. ro G, Diaz-Perez J, Martínez Del Prado M, Alonso 43. Guo LJ, Wang CH, Tang CW. Epidemiological fea- Orduna V, Sevilla-Garcia I, Villabona-Artero C, tures of gastroenteropancreatic tumors in Chengdu Beguiristain-Gómez A, Llanos-Munoz M. Inci- city with a population of 14 milion based on date dence, patterns of care and prognostic factors for from a single institution. Asian Pac J Clin Oncol outcome of gastroenteropancreaticneuroendocrine 2016; 12(suppl 3):284-8. tumors (GEP-NETs): results from the National Can- 44. TangLH, ShiaJ, SoslowRA, DhallD, WongWD, cer Registry of Spain (RGETNE). Ann Oncol 2010; O'ReillyE, QinJ, PatyP, WeiserMR, GuillemJ, Tem- 21:1794-803. pleL, SobinLH, KlimstraDS.Pathologic classificati- 39. Medrano-Guzman R. Clinical and epidemiologi- on and clinical behavior of the spectrum of goblet cal features in 495 gastroenteropancreatic neuro- cell carcinoid tumors of the appendix. Am J Surg endocrine patients in Mexico. J Clin Oncol 2017; Pathol 2008; 32:1429-43. 35:e15687.

83 ORIGINAL ARTICLE

Appropriateness of colonoscopy at a tertiary care centre – are we overdoing gastrointestinal endoscopy?

Nerma Čustović¹, Lejla Džananović², Ismar Rašić3, Nadža Zubčević¹, Lejla Šaranović-Čečo4, Jasmina Redžepagić5

¹Clinic for Gastroenterohepatology, Clinical Centre, University of Sarajevo, ²Statistics and Epidemiology Department, School of Medi- cine, University of Sarajevo,³ Department of Surgery, General Hospital “Prim.dr. Abdulah Nakaš”; Sarajevo,4 Department for Gastroen- terohepatology, Cantonal Hospital Zenica, 5Clinic for Pathology, Clinical Centre, University of Sarajevo; Bosnia and Herzegovina

ABSTRACT

Aim To evaluate the pattern of indications and a spectrum of colo- nic pathology, and to determine appropriateness of indications for colonoscopy in order to improve patient selection for colonoscopy.

Methods This retrospective study includes 294 patients who were referred to the Gastroenterology Department from a primary care physician in order to approach endoscopic examination. Study data included patients’ anamnestic data (comorbidities, positive family history, performed radiological examinations) an indication for the procedure, and colonoscopy findings.

Results Haematochezia was confirmed in 186 (63.26%), positive Corresponding author: radiologic finding in183 (62.24%) and anaemia in 157 (53.40%) Nerma Čustović patients. Adenoma and colorectal carcinoma were detected in 40 Clinic for Gastroenterohepatology, (13.6%) and 53 (18%) patients, respectively. A significant associa- Clinical Centre, University of Sarajevo tion between haematochezia and colorectal neoplasm was confir- Bolnička 25, 71000 Sarajevo, med (p=0.019), haematochezia and inflammatory bowel disease Bosnia and Herzegovina (p=0.027), and between radiological finding and colorectal neo- plasm (p=0.018). There was no significant association between Phone: +387 33 297 242; anaemia and any of the colonoscopic findings. According to EPA- Fax: +387 33297 822; GE II criteria indications were appropriate in 187 (63.6%), uncer- E-mail: [email protected] tain in 67 (22.8%) and inappropriate in 40 (13.6%) patients. ORCID ID: http://orcid.org/0000-0002- Conclusion This study confirmed a slightly larger number of un- 1424-1628 certain and inappropriate indications for colonoscopy compared to other studies that examined indications for colonoscopy, which can be attributed to a high number of patients with functional bowel disorders. Original submission: 29 July 2020; Keywords: colonic diseases/diagnosis, colorectal neoplasm, Revised submission: polyps, utilization 08 September 2020; Accepted: 15 October 2020 doi: 10.17392/1248-21

Med Glas (Zenica) 2021; 18(1):84-89

84 Čustović et al. Overdoing gastrointestinal endoscopy?

INTRODUCTION The aim of this study was to evaluate the pattern Colonoscopy provides an excellent view of the of indications and the spectrum of colonic patho- mucosa of the entire colon and terminal ileum. logy of patients at a tertiary health care facility in Colonoscopy is safe and effective not only for a Bosnia and Herzegovina in order to evaluate the diagnosis, but also for therapeutic interventions. appropriateness of colonoscopy. In recent years, indications for colonoscopy and PATIENTS AND METHODS its use in gastroenterology have increased mainly due to conscious sedation, safety, and techno- Patients and study design logical developments (1). The main indications are examination after polypectomy or colorectal This is a retrospective study carried out at the cancer resection, haematochezia, iron deficiency Clinic for Gastroenterohepatology, Clinical Cen- anaemia, uncomplicated abdominal pain and blo- tre of the University of Sarajevo in the period Ja- ating, chronic diarrhoea and constipation (2). nuary 2018 to January 2019 including all patients There are many reasons for a patient experiencing reported to the Gastroenterology Department for symptoms, such as chronic constipation, lower the first time (from a primary care physician), in abdominal pain and bloating. The prevalence order to approach endoscopic examination. of constipation in the general population ranges Study data included patients’ demographic and from 2-30 %, with a female to male ratio of 2:1 anamnestic data (comorbidities, positive family (3). Bloating is experienced at least once a month history, radiological examinations performed), in 16 % of healthy individuals and symptoms are an indication for the procedure, and colonoscopy consistent with irritabile bowel syndrom (IBS) in findings. 10-30% of adults in the general population (3). Inclusion criteria were: haematochezia, chronic It is very important to make a distinction between diarrhoea (more than 3 watery stools with or wit- organic and functional disorders. Although dia- hout mucus during the day for at least a month), gnostic colonoscopy may be useful for patients abdominal pain, constipation, hypochromic with functional disorders, its appropriateness anaemia (haemoglobin level <120 g/L), signifi- should be revised (4). Some studies report ove- cant weight loss (>10% during a period of three ruse of endoscopy and questionable indications months), radiologically suspected colon cancer in 30% of performed procedures, while others re- and positive family history of colon cancer. Pa- port that one of ten patients undergo inappropria- tients under surveillance after polypectomy or te colonoscopy (4). The reasons include cancer colorectal cancer resection, and patients with si- phobia, the investigation of accidentally identifi- gmoidoscopy were excluded from the study. ed suspicious carcinoma found on other radiolo- Our study was a retrospective, observational stu- gical imaging methods and overuse of colonos- dy which did not influence the patient care, hence copy in functional bowel disorders (5). no approval from our institutional ethical com- Diagnostic yield in relation to each indication is mittee was required. defined as the ratio between significant findings Methods detected on colonoscopy and the total number of procedures performed for that indication (6). The All patients underwent bowel preparation for presence of any of the following lesions was con- colonoscopy according to the standard protocol: sidered as a significant finding on colonoscopy: a soup at 12 hours, 2 tablets of laxative at 14 hours, pre-malignant or malignant lesion, IBD, polyps, bitter salt at 16 hours (4x67 mL within 1 hour), while haemorrhoids and diverticulosis were not laxative suppository at 18 hours, in the period 19- considered as significant findings (7). Various 22 h drinking 3 L of liquid. Those who had arri- scientific institutions, such as the European Pa- ved for colonoscopy with poor bowel preparation nel of Appropriateness of Gastrointestinal En- were asked to continue with the preparation until doscopy (EPAGE) (8) and the American Society the next day. for Gastrointestinal Endoscopy (ASGE) (9) have Digital rectal examination was performed on all developed different guidelines on the appropria- patients before the colonoscope insertion. Colo- teness of indications for colonoscopy. noscopy was thereafter performed using Olym-

85 Medicinski Glasnik, Volume 18, Number 1, February 2021

pus Exera III Videocolonoscope (CF HQ190L, The most common indication was haematochezia, Tokyo, Japan) with the patient being placed in in 140 (47.6%), followed by chronic diarrhoea in the left lateral position. Supine posture and ab- 93 (31.6%), abdominal pain in 88 (29.9%), consti- dominal pressure were applied where necessary. pation in 71 (24.1%), anaemia in 60 (20.4%), si- The analysis of the number of symptoms and indi- gnificant weight loss in 50 (17.0%), radiologically cations in correlation with the endoscopic finding suspected colon cancer in 37 (12.5%) and positive was performed, in order to assess diagnostic yield family history in 7 (2.3%) patients (Table 1). for each indication. Diagnostic yield is defined as Table 1. Patients’ demographic and anamnestic characteristics the ratio between significant findings detected on Variable No (%) of patients colonoscopy and the total number of procedures Gender performed for that indication. Colorectal neopla- Males 165 (56.1) sm, as well as all lesions that increase the risk of Females 129 (43.9) Haematochezia developing colorectal cancer (polyps and IBD) are Yes 140 (47.6) considered as significant lesions on colonoscopy. No 154 (52.3) Benign lesions are considered as haemorrhoids, Diarrhoea diverticulosis or normal finding. Yes 93 (31.6) No 201 (68.4) Low risk adenoma (LRA) is defined as one or Constipation two adenomas or tubular adenomas <10 mm in Yes 71 (24.2) No 223 (75.9) size. High-risk adenoma (HRA) refers to patients Anaemia with tubular adenoma >10 mm, 3 or more ade- Yes 60 (20.4) nomas, adenoma with villous histology or high- No 234 (79.6) Abdominal pain grade dysplasia. Adenoma detection rate (ADR) Yes 88 (29.9) is a benchmark quality measure for colonoscopy. No 206 (70.1) It is defined as a proportion of patients with at Significant weight loss Yes 50 (17) least one colorectal adenoma detected among all No 244 (83) patients examined by an endoscopist. Radiological finding A comparison of the colonoscopic findings in Negative 257 (87.4) Positive 37 (12.6) order to assess the appropriateness of the indi- Positive family history cations for colonoscopy was performed for pa- Yes 7 (2.4) tients with/without haematochezia, anaemia and No 287 (97.6) positive radiological finding, and EPAGE score was calculated. According to EPAGE criteria (8), According to the EPAGE II criteria indications appropriateness of colonoscopy is classified into were appropriate in 185 (63.6%), uncertain in 67 3 categories: appropriate (≥7), uncertain (4–6) (22.8%) and inappropriate in 40 (13.6%) patients. and inappropriate (≤3). Out of 294 patients, 56 (19.0%) were patients Statistical analysis with normal colonoscopy. For the remaining 238 patients, the abnormal findings were as follows: The statistical analysis included descriptive sta- haemorrhoids (85; 35.7%), colon neoplasms (43; tistics and the calculation of indication rates for 18%), polyps (47; 19.7%) of which 33 (13.8%) distal endoscopy, endoscopic and pathohistologi- were adenomas; 21 (8.8%) patients had haemorr- cal examination, and EPAGE scoring system. Chi hoids and polyps concurrently, inflammatory square tests were performed to evaluate a degree bowel disease (IBD) was found in 34 (14.3%) of significance with 95% confidence interval (CI) and diverticulosis in 29 (12.2%) patients. and significance level at p=0.05. Diagnostic yields were as follows: haematoche- RESULTS zia 186 (63.2%), positive radiologic finding 183 Out of 294 patients, 165 (56.1%) were males (62.2%), diarrhoea 158 (53.7%), anaemia 157 and 129 (43.9%) females (male to female ratio of (53.4%), and obstipation 124 (42.1%). 1.27). The mean age of the patients was 62 years The most common indications for colonoscopy (range of 19–93 and 20-82 years for males and in 56 patients who subsequently had normal co- females, respectively). lonoscopy findings were constipation in 20 (35.7

86 Čustović et al. Overdoing gastrointestinal endoscopy?

%), abdominal pain in 18 (32.1%), and diarrhoea DISCUSSION in 18 (32.1%) patients. Anaemia (as a single or This study revealed a predominance of males associate with other symptoms) was observed in presented for colonoscopy compared to females, 13(23.2%) patients. which was also observed in the study of Cahyono In 140 patients with haematochezia haemorrhoids et al. (10). Austin et al. verified an increasing rate of were verified in 46 (32.9%), colorectal neoplasm rectal cancer among younger adults, while among in 27 (19.3%) (p=0.019), IBD in 23(16.4%) pati- older ones it continues to decrease (11). In the ents (p=0.027) (Table 2). presented study, the most common indication for colonoscopy was haematochezia, similar to obser- Table 2. Spectrum of colonoscopic diagnoses in patients with/without haematochezia, anaemia and positive radiologi- vations in other European studies (8,12). The ove- cal finding rall diagnostic yield of colonoscopy in this study No (%) of patients was 55%, with highest values for haematochezia Haematoche- Positive radiolo- Endoscopic finding Anaemia (63.5%) and positive radiologic findings (62.1%). zia gical finding A lower overall diagnostic yield (48.4%) was also No Yes No Yes No Yes recorded in a few studies, with haematochezia ha- 48 8 43 13 49 7 Normal (31.2) (5.7) (18.4) (21.7) (19.1) (18.9) ving the highest diagnostic yield (11,13). 15 27 30 12 32 10 Colorectal neoplasm The most common abnormalities detected during (9.7) (19.3) (12.8) (20.0) (12.5) (27.0) Inflammatory bowel 12 23 30 5 29 6 colonoscopy were haemorrhoids, polyps and co- disease (7.9) (16.4) (12.8) (8.3) (11.1) (16.3) lon neoplasms. Some studies reported haemorrho- 9 7 13 4 78 1 Diverticulosis ids, colorectal cancer and inflammatory bowel di- (5.8) (5) (5.6) (6.6) (30.4) (2.5) sease as the most common colonoscopy-detected 20 6 21 8 13 4 Polyps (13) (4.3) (9) (13.3) (5.1) (10.8) diagnoses, while others reported polyps, due to an 39 46 70 15 24 7 Haemorrhoids increase in the number of individuals undergoing (25.3) (32.9) (29.9) (25.0) (9.3) (18.9) 1 colonoscopy, the use of a high-resolution colonos- Haemorrhoids and 5 8 9 1 13 (2.5) diverticulosis (3.2) (5.7) (3.8) (2.1) (5.1) cope, as well as lifestyle and diet change (2,13). A Haemorrhoids and 6 15 18 2 19 1 strong increase in adenoma detection rate (ADR) polyps (3.9) (10.7) (7.7) (3) (7.4) (2.5) and neoplasm detection rate (NDR) was observed 154 140 234 60 257 37 Total in few studies (14,15). Our study showed a lower (52.3) (47.6) (79.6) (20.4) (87.4) (12.6) ADR (13.8%) than previous studies, but a higher NDR detection rate (18%). The reason for these In the group of patients with verified polyps the- values may be the lack of screening programs in re were 19 low risk adenomas (LRA), 14 high Bosnia and Herzegovina, as well as delayed repor- risk adenomas (HRA), and 13 non adenomatous ting of patients to primary health care. polyps, with an adenoma detection rate (ADR) of 13.8%. By analysing the symptoms separately, the most common indication for colonoscopy in our pati- Of 60 patients with anaemia, haemorrhoids were ents who subsequently had normal colonoscopy observed in 15 (25.0%) patients, 13 (21.7%) pa- findings were constipation, abdominal pain and tients had normal finding, and colorectal neo- diarrhoea. In a Japanese study (16) 45.6% of pa- plasms were found in 12 (20.0%) patients (Table tients with indication for colonoscopy were dia- 2). No significant association between anaemia gnosed with functional bowel disorders (accor- and any of the colonoscopic findings was found ding to the Rome III diagnostic criteria). Patients (p>0.05). with functional bowel disorders had significantly Patients with positive radiological finding had higher rates of abdominal pain, hard or lumpy colorectal neoplasm in 10 (27.0%), haemorrho- stools, watery stools and bloating compared to ids and normal findings in seven (18.9%) - pati controls (16). A similar prevalence of functio- ents each, and inflammatory bowel disease in nal bowel disorder was reported in other studies six (16.3%) (Table 2). A statistically significant (17,18). Our results verifying a high percent of association was shown only between radiological patients with normal colonoscopy (19%) are in finding and colorectal neoplasm (p=0.018). correlation with some other studies (16-18), ju- stifying a large number of normal colonoscopy

87 Medicinski Glasnik, Volume 18, Number 1, February 2021

findings in patients with presented symptomato- in 22.8%, and inappropriate in 13.6% which is si- logy of functional bowel disorders. milar with a Spanish study, 73.68%, 16.57%, and In our study, there was a significant association 9.74%, respectively (8). Patients with appropria- between haematochezia and colorectal neoplasm. te or uncertain indications based on the EPAGE Several studies showed increasing prevalence of II criteria had more relevant endoscopic findings colorectal cancer in younger patients (18-20), than those with inappropriate indications (13). suggesting that haematochezia in young patients The rate of unnecessary colonoscopy is high, should not be neglected and attributed to hae- especially in patients younger than 50 years of morrhoid nodules, even when they are palpable. age, among whom there is a higher incidence of Among patients with anaemia, there was no si- irritable bowel syndrome (24). Considering the gnificant association between anaemia and any above, it is possible that a larger number of ina- of colonoscopy findings in our study. Some other dequate indications in our study is a consequence studies showed a significant correlation between of a higher percentage of patients with symptoms anaemia and proximal colorectal cancer (cancer of irritable bowel syndrome. of ascending colon and ileocecal region) (21,22). In conclusion, the obtained results can be used in In our study, nearly a quarter of patients with nor- making a local guideline for colonoscopy indi- mal colonoscopy findings had anaemia at the first cations, especially in countries where screening visit, which indicates the necessity to perform programs are not implemented, such as Bosnia many other examinations before the colonoscopy and Herzegovina. Rationalization of the demand in order to find the cause of anaemia. for endoscopy is mandatory to prevent overbur- A statistically significant association was shown dening endoscopy units, decrease waiting lists between radiologically suspected carcinoma for outpatient colonoscopy, improve efficiency in (described as thickening of the intestinal wall, colonoscopy and reduce costs and potential risks narrowing of the intestinal lumen, or suspected arising from inadequate colonoscopy referrals. infiltrative process on CT colonography) and colo- FUNDING rectal neoplasm. Our results are in accordance with those reported in Halligan et al. study comparing No specific funding was received for this study. CT colonography (CTC) and colonoscopy (23). According to the EPAGE II criteria, in our study TRANSPARENCY DECLARATION indications were appropriate in 63.6%, uncertain Conflicts of interest: None to declare.

REFERENCES 1. Gimeno-Garcia AZ, Quintero E. Colonoscopy 6. Jacob K, Dennis P, Charles B, Richard B, James appropriateness: really needed or a waste of time? AB, Mark T. Diagnostic yield of gastrointestinal en- World J Gastrointest Endosc 2015; 7:94-101. doscopy in North West Region Cameroon and trends 2. Akere A, Oke TO, Otegbayo JA. Colonoscopy at in diagnosis over time. Pan Afr Med J 2018; 29:178. a tertiary healthcare facility in Southwest Nigeria: 7. Bohara TP, Laudari U, Thapa A, Rupakheti S, Jo- Spectrum of indications and colonic abnormaliti- shi MR. Appropriateness of indications of upper es. Ann Afr Med 2016;15:109-13. gastrointestinal endoscopy and its association with 3. Soncini M , Stasi C , Satta PU , Milazzo G , Bi- positive finding. JNMA J Nepal Med Assoc 2018; anco M, LeandroG, Montalbano LM , Muscatiello 56:504-9. N , Monica F , Galeazzi F, Bellini M , AIGO. IBS 8. Marzo-Castillejo M, Almeda J, Mascort JJ, Cunille- clinical management in Italy: The AIGO survey. Dig ra O, Saladich R, Nieto R, Pineiro P, Lagostera M, Liver Dis 2019;51:782-9. Cantero Fx, Segarra M, Puente D. Appropriatene- 4. Shaheen NJ, Fennerty MB, Bergman JJ. Less Is ss of colonoscopy requests according to EPAGE- More: A minimalist approach to endoscopy. Gastro- II in the Spanish region of Catalonia. BMC Fam enterology 2018; 154:1993-2003. Pract 2015; 16:154. 5. Samarakoon Y, Gunawardena N, Pathirana A, 9. ASGE Standards of practice committee, Early DS, Hewage S. Appropriateness of colonoscopy accor- Ben-Menachem T, Decker GA, Evans JA, Fanelli ding to EPAGE II in a low resource setting: a cross RD, Fisher DA, Fukami N, Ha Hwang J, Rajeev J, sectional study from Sri Lanka. BMC Gastroenterol Jue TL, Khan KM, Malpas PM, Maple JT, Sharaf 2018; 18:72. RS, Dominitz AS, Cash BD. Appropriate use of GI endoscopy. Gastrointest Endosc 2012;75:1127–31.

88 Čustović et al. Overdoing gastrointestinal endoscopy?

10. Cahyono SB, Bayupurnama P, Ratnasari N, Triwi- 18. Long Y, Huang Z, Deng Y, Chu H, Zheng X, Yang katmani C, Indrarti F, Maduseno S, Nurdjanah S. J, Zhu Y, Fried M, Fox M, Dai N. Prevalence and Evaluating indications and diagnostic yield of colo- risk factors for functional bowel disorders in South noscopy in Sardjito general hospital. J Int Med Acta China: a population based study using the Rome III Int 2014; 4:51-6. criteria. Neurogastroenterol Motil 2017; 29:e12897. 11. Austin H, Henley SJ, King J, Richardson LC, Ehe- 19. Olivo R, Ratnayake S. Colorectal cancer in young man C. Changes in colorectal cancer incidence rates patients: a retrospective cohort study in a single in- in young and older adults in the United States: what stitution. ANZ J Surg 2019; 89:905-7. does it tell us about screening. Cancer Causes Con- 20. Ashktorab H, Vilmenay K, Brim H, Laiyemo AO, trol 2014; 25:191-201. Kibreab A, Nouraie M. Colorectal cancer in young 12. Eskeland SL, Dalen E, Sponheim J, Lind E, Bru- African Americans: is it time to revisit guidelines nborg C, de Lange T. European panel on the appro- and prevention? Dig Dis Sci 2016; 61:3026-30. priateness of gastrointestinal endoscopy II guidelines 21. Atkin W, Wooldrage K, Shah U, Skinner K, Brown help in selecting and prioritizing patients referred to JP, Hamilton W, Kralj-Hans I, Thompson MR, Flas- colonoscopy - a quality control study. Scand J Ga- hman KG, Halligan S, Thomas-Gibson S, Vance M, stroenterol 2014; 49:492-500. Cross AJ. Is whole-colon investigation by colonos- 13. Dakubo JC, Seshie B, Ankrah LN. Utilization and copy, computerized tomography colonography or diagnostic yield of large bowel endoscopy at Kor- barium enema necessary for all patients with colo- le-Bu Teaching Hospital. J Med Biomed Sci 2014; rectal cancer symptoms, and for which patients wo- 3:6-13. uld flexible sigmoidoscopy suffice? A retrospective 14. Brenner H, Altenhofen L, Kretschmann J, Rosch T, cohort study. Health TechnolAssess2017; 21:1-80. Pox C, Stock C, Hoffmeister M. Trends in adenoma 22. Cross AJ, Wooldrage K, Robbins EC, PackK, detection rates during the first 10 years of the Ger- BrownJP, Hamilton W, Thompson MR, Flashman man Screening Colonoscopy Program. Gastroente- KG, Halligan S, Thomas-Gibson S, Vance M, Sa- rology 2015; 149:356-66. unders BP, Atkin W. Whole-colon investigation vs. 15. Karsenti D, Tharsis G, Burtin P, Venezia F, Tordj- flexible sigmoidoscopy for suspected colorectal can- man G, Gillet A, Samama J, Nahon-Uzan K, Cattan cer based on presenting symptoms and signs: a mul- P, Cavicchi M. Adenoma and advanced neoplasia ticentre cohort study.Br J Cancer 2019; 120:154-64. detection rates increase from 45 years of age. World 23. Halligan S, Dadswell E, Wooldrage K, Wardle J, J Gastroenterol 2019; 25:447-56. von Wagner C, Lilford R, Yao GL, Zhu S, Atkin W. 16. Ono M, Kato M, Miyamoto S, Tsuda M, Mizushi- Computed tomographic colonography compared ma T, Ono S, Nakagawa M, Mabe K, Nakagawa with colonoscopy or barium enema for diagnosis of S, Muto S, Shimizu Y, Kudo M, Katsuki S, Megu- colorectal cancer in older symptomatic patients: two ro T, Sakamoto N. Multicenter observational stu- multicentre randomised trials with economic evalu- dy on functional bowel disorders diagnosed using ation (the SIGGAR trials). Health Technol Assess Rome III diagnostic criteria in Japan. J Gastroente- 2015; 19:1-134. rol 2018; 53:916-23. 24. Andújar X, Sainz E, Galí A, Loras C, Aceituno 17. Wang X, Luscombe GM, Boyd C, Kellow J, Abra- M, Espinós JC, Viver JM, Esteve M, Fernández- ham S. Functional gastrointestinal disorders in Bañares F. Inappropriateness rate for colonoscopy eating disorder patients: Altered distribution and indications in an open access unit. Gastroenterol He- predictors using ROME III compared to ROME II patol 2015; 38:313-9. criteria. World J Gastroenterol 2014; 20:16293-9.

89 ORIGINAL ARTICLE

Oxidative stress markers in initial therapy and remission of nephrotic syndrome and serum malondialdehyde level predictor from routine laboratory test

Riska Habriel Ruslie1, Oke Rina Ramayani2, Darmadi Darmadi3, Gontar Alamsyah Siregar3

1Department of Child Health, Faculty of Medicine, Universitas Prima Indonesia, 2Department of Child Health, 3Department of Internal Medicine; Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia

ABSTRACT

Aim To compare oxidative stress state of children with nephro- tic syndrome at the first week of treatment and in remission, and to predict malondialdehyde (MDA) level from routine laboratory tests.

Methods This cross-sectional study involved 80 1-18 years old children with nephrotic syndrome, who were divided into two gro- ups: initial group (40 children in the first week of therapy) and remission group (40 children in remission). Demographic charac- teristics of the patients were taken by a questionnaire. Laboratory tests were measured in the initial group; in the remission group negative or trace proteinuria was measured for three consecuti- Corresponding author: ve days. Serum urea, creatinine, albumin, total cholesterol, MDA, Riska Habriel Ruslie superoxide dismutase, glutathione peroxidase, and urine albumin- Faculty of Medicine, to-creatinine ratio (UACR) were measured and compared between Universitas Prima Indonesia the groups. Albumin, total cholesterol, and UACR were subjected Ayahanda 68A, Medan, Indonesia to predict high serum MDA using a mean of all patients’ MDA level as a cutoff. Phone: +62 618 453 2820; E-mail: [email protected] Results There were higher albumin levels and lower UACR, total ORCID ID: https://orcid.org/0000-0001- cholesterol, and MDA in the remission group compared to the ini- 7779-6535 tial group. Albumin and UACR showed good accuracy, and total cholesterol showed very good accuracy to predict serum MDA le- vel more than 1.35 µmol/L.

Conclusion Children with nephrotic syndrome in the first week of therapy showed a higher oxidative stress state than the children in remission. Serum albumin, serum total cholesterol, and UACR can Original submission: predict serum MDA level with good accuracy. 04 May 2020; Key words: antioxidant, glutathione peroxidase, nephrosis, reacti- Revised submission: ve oxygen species, superoxide dismutase 29 June 2020; Accepted: 23 July 2020 doi: 10.17392/1192-21

Med Glas (Zenica) 2021; 18(1):90-95

90 Ruslie et al. MDA predictor of nephrotic syndrome

INTRODUCTION 18 years old with nephrotic syndrome in the first week of therapy, confirmed by massive proteinu- Nephrotic syndrome is a common paediatric kid- ria (˃40 mg/m2 per hour or urinary protein/crea- ney disease characterized by hypoalbuminemia, tinine ratio (PrU/CrU) in urine >2 mg/mg or in oedema, and hyperlipidemia arising from large urine dipstick ≥ 2+), hypoalbuminemia ≤ 2.5 g/ urinary losses of protein (1,2). Mostly of idio- dL, and oedema; remission (complete) confirmed pathic cause, it is classified based on patients’ by negative or trace proteinuria for three conse- response to corticosteroid medication (3). Hi- cutive days. Chronic kidney disease patients with stological classification of idiopathic nephrotic glomerular rate ≤ 60 mL/minute per syndrome in childhood includes minimal change 1.73 m2 and patients with systemic disease such disease (MCD), focal segmental glomeruloscle- as malignancy, pulmonary tuberculosis, severe rosis (FSGS), and other nephropathies (1). malnutrition, obesity, cardiac diseases, liver dise- Previous research has shown the association ases, systemic lupus erythematosus, and Henoch- between oxidative stress and nephrotic syndrome Schonlein purpura were excluded. (4). Persistent oxidative stress can result in DNA A written informed consent was obtained from damage (5,6). Excessive oxidative stress in glo- all patients’ legal guardians prior to the study en- meruli can cause oedema, foot process fusion, and rollment. The study was approved by the Health epithelial vacuolization thus contributing to the Research Ethical Committee, Faculty of Medici- development of the nephrotic syndrome (7). Pati- ne, Universitas Sumatera Utara. ents with high oxidative stress were correlated to a higher frequency of relapse (4,8) and are more This study involved 40 nephrotic syndrome chil- likely to be steroid-resistant or steroid-dependent dren in the first week of therapy (initial group) (9). Some studies reported the roles of malondi- and 40 nephrotic syndrome children in complete aldehyde (MDA), superoxide dismutase (SOD), remission (remission group). All patients or pa- and glutathione peroxidase (GPX) as oxidative tients’ legal guardians were interviewed using stress markers (5,10,11). Lipid peroxidation pro- a structured questionnaire to note gender, age, duces MDA directly, therefore MDA is believed to nutritional status, and known systemic diseases. be the optimal biomarker of oxidative stress (12). Weight-for-height was used to determine nutriti- Antioxidant enzymes such as SOD and GPX de- onal status based on the WHO paediatric growth grade oxidative stress and its concentration alte- indicators (14). red during the development of oxidative stress in Methods nephrotic syndrome children (13). However, oxi- dative stress biomarkers were expensive and not Six millilitres of blood samples were collected readily available, especially in developing coun- from all patients after 8-10 hours of fasting, and tries. Therefore, other laboratory parameters were they were subjected to measuring serum urea, cre- needed to predict the oxidative stress biomarkers atinine, albumin, total cholesterol, MDA, SOD, in patients with nephrotic syndrome. and GPX level. Serum urea, creatinine, albumin, The aim of this study was to investigate oxidative and total cholesterol were measured using the stress state of children with nephrotic syndrome SMAC autoanalyzer (Technicon, Tarrytown, NY, in the first week of treatment and to compare it USA). Serum MDA level was measured by high with the children in remission, and to predict performance liquid chromatographic (HPLC) MDA level from routine laboratory tests. using Agilent 1200 HPLC system (San Jose, CA, USA) with commercial MDA kits (Immundia- PATIENTS AND METHODS gnostik AG, Bensheim, Germany). Serum SOD and GPX were measured using Advia 1800 in- Patients and study design strument (Siemens Healthcare GmbH, Germany) This study was conducted as a cross-sectional with Ransel Glutathione Peroxidase kit (Randox study at the Paediatric Outpatient and Paediatric Laboratories, London, UK) and Ransod kit (Ran- Ward Departments of the H. Adam Malik Gene- dox Laboratories, London, UK). All urine samples ral Hospital, Medan, Indonesia during the period were collected and subjected to measure albumin January - December 2018. All patients were 1 to and creatinine by urine albumin-to-creatinine ra-

91 Medicinski Glasnik, Volume 18, Number 1, February 2021

tio (UACR) assay kit (MyBioSource, US). Serum Table 1. Demographic characteristics of 80 patients with albumin, UACR, and total cholesterol were cate- nephrotic syndrome gorized with albumin <2.43 mg/dL, UACR ˃1.8 Characteristic Initial group Remission group p Gender (No %) mg/g, and total cholesterol ˃220 mg/dL as the cu- Male 31 (77.5) 27 (67.5) 0.317 toff. Serum MDA was categorized using the mean Female 9 (22.5) 13 (32.5) value of all patients’ serum MDA as the cutoff. Nutritional status (No, %) Underweight 5 (12.5) 4 (10) 0.731 Serum albumin, UACR, and total cholesterol were Normal weight 32 (80) 31 (77.5) subjected to predict high serum MDA. Overweight 3 (7.5) 5 (12.5) Mean age (SD) (years) 4.54 (1.58) 4.18 (1.22) 0.390 Renal biopsy was not performed as most children BMI, body mass index; with steroid-sensitive idiopathic nephrotic syn- drome fitted the standard clinical presentation of There were significantly higher albumin levels minimal change nephrotic syndrome that did not (p<0.001) and lower UACR (p<0.001), total cho- require a routine renal biopsy (15). lesterol (p<0.001), and MDA levels (p<0.001) in the remission group compared to the initial group Statistical analysis (Table 2). Demographic characteristics were analysed for Table 2. Serum biomarker in patients with nephrotic syn- differences between the initial group and the drome in the initial group and the remission group remission group by using the ꭓ2 test for ca- Mean biomarker Initial Remission Mean difference p tegorical data, independent t-test for normally value (SD) group group (CI 95%) distributed numerical data, and Mann-Whit- 24.96 24.91 0.05 Urea (g/dL) 0.986 ney U test if the distribution was not normal. (5.18) (5.3) (-4.61- 6.39) 0.7 0.64 0.06 Creatinine (g/dL) 0.232 Fisher’s exact test was used if ꭓ2 test assump- (0.09) (0.07) (-0.001 - 0.16) tions were not met. Serum urea, creatinine, al- 1.8 3.04 -1.2 Albumin (g/dL) <0.001 bumin, UACR, total cholesterol, MDA, SOD, (0.34) (0.26) (-1.45 - -1.01) 3.36 0.48 2.88 UACR (mg/g) <0.001 and GPX were compared between the initial (0.41) (0.16) (2.65 - 3.24) group and the remission group by using inde- Total cholesterol 279.66 163.81 115.9 <0.001 pendent t-test if data were normally distribu- (mg/dL) (41.32) (10.07) (96.13 - 152.23) ted, otherwise, Mann-Whitney U test was used. Oxidative stress 1.52 1.14 0.38 MDA (µmol/L) <0.001 Pearson’s correlation test was used to determine (0.22) (0.49) (0.28 - 0.56) the correlation between albumin, UACR, total 1155.38 1279.89 124.5 SOD (U/gHb) 0.095 cholesterol with MDA. Overall accuracy was (184.9) (270.37) (-28.1 - 198.9) 30.71 33.56 2.85 GPX (U/gHb) 0.132 evaluated using the area under curve (AUC) of (6.35) (5.29) (-1.02 - 4.8) receiver operating curve (ROC), and specific UACR, urine albumin/creatinine ratio; MDA, malondialdehyde; diagnostic accuracy was evaluated using sensi- SOD, superoxide dismutase; GPX, glutathione peroxidase; CI, confidence interval; tivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), There were no significant differences in urea positive likelihood ratio (PLR), and negative (p=0.986), creatinine (p=0.232), SOD (p=0.095), likelihood ratio (NLR), then diagnostic effecti- and GPX level (p=0.132) between the groups. veness (accuracy) was measured. The mean serum MDA level of all patients 1.35 Differences were considered statistically signifi- µmol/L would be used as a cutoff point. Predic- cant at p<0.05. tive analyses showed good accuracy of albumin and UACR (AUC 0.75 and 0.76, respectively), RESULTS and very good accuracy of total cholesterol Gender, age, and nutritional status (weight-for- (AUC 0.84) (Figure 1). height) were similar among the initial group There was moderate negative correlation and the remission group (Table 1). There were between albumin and serum MDA level (r=- no significant differences in gender (p=0.317), 0.596; p<0.001). Moderate positive correlation age (p=0.390), and nutritional status (p=0.731) was found between UACR and serum MDA le- between the initial and the remission group. vel (r=0.485; p=0.002). A strong positive corre-

92 Ruslie et al. MDA predictor of nephrotic syndrome

cholesterol higher than cutoff score to predict high MDA higher than 1.35 µmol/L were found (Table 3).

Table 3. Specific diagnostic accuracy of variables to predict high serum MDA level in nephrotic syndrome children* Variable Sn Sp PPV NPV Accuracy PLR NLR (cutoff point) (%) (%) (%) (%) (%) Albumin 76 84 83 78 4.75 0.29 80 (at 2,43 g/dL) UACR 84 72 75 82 3 0.22 78 (at 1,8 mg/g) Total cholesterol 72 96 95 77 18 0.29 84 (at 220 mg/dL) *Serum MDA level of 1.35 µmol/L was used as cutoff ; Sn, sensiti- vity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value; PLR, positive likelihood ratio; NLR, negative likelihood ratio

DISCUSSION The association between nephrotic syndrome and oxidative stress state has been reported in previous studies (10–13). Overproduction of re- active oxygen species (ROS) and impairment of antioxidant enzymes can cause oxidative stress state (5,8,13). Excessive oxidative stress resul- ting from an inflammation reaction can cause the injury of glomerular filtration membrane through the destruction of the electrostatic barrier, and injury of endothelial cells and podocytes (16). Lipid peroxidation, a particular reaction of ROS with lipids, produces MDA as its direct byproduct (12,17). This study showed the oxidative stress sta- te in paediatric nephrotic syndrome patients, espe- cially in the initial group with higher serum MDA level compared to the remission group. This is in concordance with a previous study by Reddy et al. (5) that showed higher MDA level in the active group (children with nephrotic syndrome during first episode/relapse) compared to the remission group, and even in the remission group compared to the control group (children without nephrotic syndrome). This result indicated that the changes in oxidative stress persist even after remission. The first line of defence against ROS in vivo is antioxidant enzyme SOD (18). This enzyme Figure 1. Receiver operating curves of A) albumin, B) urine protects the cell from harmful superoxide by re- albumin/creatinine ratio (UACR), C) total cholesterol level to dox reaction to decrease the level of superoxide predict serum MDA level higher than 1.35 µmol/L in nephrotic and mitigate oxidative stress (19). Another an- syndrome children tioxidant enzyme, GPX, is a selenium-depen- lation was found between total cholesterol and dent enzyme that reduces intracellular hydrogen serum MDA level (r=0.674; p<0.001). Good peroxide and lipid peroxides by redox reaction performance diagnostic accuracy of albumin (20). These antioxidant enzyme (SOD and GPX) lower than cutoff score and UACR and total activities were altered during the development

93 Medicinski Glasnik, Volume 18, Number 1, February 2021

of oxidative stress in nephrotic syndrome (4). (23). Therefore, it is concordant to this study that However, this study did not show significant showed good accuracy of UACR, albumin, and differences in antioxidant enzymes (SOD and total cholesterol to predict the oxidative stress GPX) between the initial group and the remissi- state of patients with nephrotic syndrome. on group. These findings are discordant with pre- Some limitations of this study should be noted. vious studies by Reddy et al. (5) and Fydryk et This study did not compare MDA level in the al. (11,21). This might be due to the fast reaction same patient before and after therapy with con- rate and short half-life of SOD (19) and due to trols, and also steroid-sensitive with steroid-resi- selenium dependency of GPX activities (21). stant nephrotic syndrome patients. The correlation between high oxidative stress In conclusion, this study is the first study to predict state with hypoalbuminemia, proteinuria, and the oxidative stress by MDA level from routine la- hypercholesterolemia was found in our study. boratory tests. Children with nephrotic syndrome This is concordant to previous studies which in the first week of therapy have higher oxidative revealed the association between oxidative stre- stress state than those in remission. Oxidative stre- ss with proteinuria (UACR), hypoalbuminemia, ss state of children with nephrotic syndrome can and hypercholesterolemia (22,23). A previous be predicted by measuring routine laboratory te- study by Zhou et al. (22) showed oxidative stre- sts such as albumin, total cholesterol, and UACR. ss-induced podocyte dysfunction via Wnt/b-cate- Serum albumin level less than 2.43 mg/dL, serum nin activation, whereas, hypoalbuminemia decre- total cholesterol more than 220 mg/dL, and UACR ases sphingosine 1 phosphate (S1P) availability more than 1.8 mg/g can predict MDA level more in the endothelium thus inducing oxidative stress than 1.35 µmol/L with good accuracy. and increasing vascular permeability (24,25). Hypercholesterolemia, however, induces oxidati- FUNDING ve stress in endothelial cells and initiates peroxi- No specific funding was received for this study. dation of cell membranes and unsaturated fatty acids by itself thus producing oxidized low-den- CONFLICTS OF INTEREST sity lipoproteins intensifying the oxidative stress Competing interests: None to declare

REFERENCES 1. Downie ML, Gallibois C, Parekh RS, Noone DG 7. Balamurugan R, Bobby Z (e-mail: zacbobby@yahoo. (e-mail:[email protected]). Nephrotic com), Selvaraj N, Nalini P, Koner BC, Sen SK. Incre- syndrome in infants and children: pathophysiology ased protein glycation in non-diabetic pediatric nep- and management. Paediatr Int Child Health 2017; hrotic syndrome: possible role of lipid peroxidation. 37:248–58. Clin Chim Acta 2003; 337:127–32. 2. Noone DG, Iijima K, Parekh R (e-mail: rulan.pa- 8. Fan A, Jiang X (e-mail: jiangxiaoyun2015@126. [email protected]). Idiopathic nephrotic syndrome in com), Mo Y, Tan H, Jiang M, Li J. Plasma levels of children. Lancet 2018; 392:61–74. oxidative stress in children with steroid-sensitive nep- 3. Hodson EM (e-mail: [email protected]. hrotic syndrome and their predictive value for relapse gov.au), Wong SC, Willis NS, Craig JC. Interventions frequency. Pediatr Nephrol 2016; 31:83–8. for idiopathic steroid-resistant nephrotic syndrome in 9. Gopal N (e-mail: [email protected]), Koner children. Cochrane Database Syst Rev 2016; 10. BC, Bhattacharjee A, Bhat V, Murugaiyan SB, Mud- 4. Kamireddy R, Kavuri S, Devi S, Vemula H, Chan- degowda PH. Assay of urinary protein carbonyl con- dana D, Harinarayanan S, James R, Rao A (e-mail: tent can predict the steroid dependence and resistance [email protected]). Oxidative stress in pe- in children with idiopathic nephrotic syndrome. Saudi diatric nephrotic syndrome. Clin Chim Acta 2002; J Kidney Dis Transpl 2017; 28:268. 325:147–50. 10. Arumugam V, Saha A (e-mail: drabhijeetsaha@ya- 5. Reddy P, Sindgikar SP (e-mail: drseema2482@re- hoo.com), Kaur M, Deepthi B, Basak T, Sengupta S, diff.com), Shenoy RD, Shenoy V. Oxidative stress in Bhatt A, Batra VV, Upadhyay AD. Plasma free ho- childhood steroid sensitive nephrotic syndrome and mocysteine levels in children with idiopathic nephro- its correlation with DNA damage. Int J Contemp Pe- tic syndrome. Indian J Nephrol 2019; 29:186–90. diatrics 2016; 3:768–72. 11. Fydryk J (e-mail: [email protected]), Jacobson 6. Darmadi (e-mail: [email protected]), Siregar E, Kurzawska O, Małecka G, Gonet B, Urasiński T, GA, Dairi LB. Association between degree of gastri- Brodkiewicz A, Bukowska H. Antioxidant status of tis and malondialdehyde level of gastritis patients at children with steroid-sensitive nephrotic syndrome. Adam Malik General Hospital Medan. Indones J Ga- Pediatr Nephrol 1998; 12:751–4. stroenterol Hepatol Dig Endosc 2017; 18:80.

94 Ruslie et al. MDA predictor of nephrotic syndrome

12. Mao S, Zhang A, Huang S (e-mail: edjk123456@ 20. Huang J-Q, Zhou J-C, Wu Y-Y, Ren F-Z, Lei XG (e- sina.com). Serum levels of malondialdehyde, vitamin mail: [email protected]). Role of glutathione pe- C and E in idiopathic nephrotic syndrome: a meta- roxidase 1 in glucose and lipid metabolism-related analysis. Ren Fail 2014; 36:994–9. diseases. Free Radic Biol Med 2018; 127:108–15. 13. Al-Eisa A (e-mail: [email protected]), Dhaunsi GS. 21. Fydryk J (e-mail: [email protected]), Olszewska NOX-mediated impairment of PDGF-induced DNA M, Urasiński T, Brodkiewicz A. Serum selenium synthesis in peripheral blood lymphocytes of chil- level and glutathione peroxidase activity in steroid- dren with idiopathic nephrotic syndrome. Pediatr Res sensitive nephrotic syndrome. Pediatr Nephrol 2003; 2017; 82:629–33. 18:1063–5. 14. Dibley MJ (e-mail: [email protected]), 22. Zhou L, Chen X, Lu M, Wu Q, Yuan Q, Hu C, Miao Staehling N, Nieburg P, Trowbridge FL. Interpretati- J, Zhang Y, Li H, Hou FF, Nie J, Liu Y (e-mail: on of Z-score anthropometric indicators derived from [email protected]). Wnt/β-catenin links oxidative stress the international growth reference. Am J Clin Nutr to podocyte injury and proteinuria. Kidney Int 2019; 1987; 46:749–62. 95:830–45. 15. Kliegman RM. Nelson textbook of . 21st 23. Jabarpour M, Rashtchizadeh N (e-mail: rashtchiza- edition. Philadelphia, MO: Elsevier; 2019. [email protected]), Argani H, Ghorbanihaghjo A, 16. Sutariya B, Saraf M (e-mail: madhusudan.saraf@gmail. Ranjbarzadhag M, Sanajou D, Panah F, Alirezaei A. com). α-asarone reduce proteinuria by restoring antioxi- The impact of dyslipidemia and oxidative stress on dant enzymes activities and regulating necrosis factor vasoactive mediators in patients with renal dysfuncti- κB signaling pathway in doxorubicin-induced nephrotic on. Int Urol Nephrol 2019; 51:2235–42. syndrome. Biomed Pharmacother 2018; 98:318–24. 24. Proia RL, Hla T ([email protected]). Emer- 17. Tsikas D (e-mail: tsikas.dimitros@mh-hannover. ging biology of sphingosine-1-phosphate: its role de). Assessment of lipid peroxidation by measuring in pathogenesis and therapy. J Clin Invest 2015; malondialdehyde (MDA) and relatives in biological 125:1379–87. samples: Analytical and biological challenges. Anal 25. Udwan K, Brideau G, Fila M, Edwards A, Vogt B, Biochem 2017; 524:13–30. Doucet A (e-mail: [email protected]). Oxi- 18. Elchuri S, Oberley TD, Qi W, Eisenstein RS, Rober- dative stress and nuclear factor κB (NF-κB) increa- ts LJ, Remmen HV, Epstein CJ, Huang T-T (e-mail: se peritoneal filtration and contribute to ascites for- [email protected]). CuZnSOD deficiency leads mation in nephrotic syndrome. J Biol Chem 2016; to persistent and widespread oxidative damage and 291:11105–13. hepatocarcinogenesis later in life. Oncogene 2005; 24:367–80. 19. Azadmanesh J (e-mail: jahaun.azadmanesh@unmc. edu), Borgstahl GEO. A review of the catalytic mechanism of human manganese superoxide dismu- tase. Antioxidants 2018; 7:25.

95 ORIGINAL ARTICLE

Refractive errors in children: analysis among preschool and school children in Tuzla city, Bosnia and Herzegovina

Amra Nadarević Vodenčarević1, Meliha Halilbašić1, Anis Međedović1, Vahid Jusufović1, Adisa Pilavdžić1, Aida Drljević2, Mufid Burgić3

1Eye Clinic, University Clinical Centre Tuzla, Tuzla, 2Medical Centre’’Plava Poliklinika’’ Tuzla; Bosnia and Herzegovina, 3Al Emadi Hospi- tal, Doha, Qatar

ABSTRACT

Aim To establish the prevalence of refractive errors in preschool and school children between 4 and 15 years of age, living in Tuzla, Bosnia and Herzegovina.

Methods Children from all elementary schools in the city of Tuzla and as well from eight day-care centres were screened for refrac- tive errors in the period 2015-2019. Any child, who failed to pass the screening examination, was referred to an ophthalmologist for complete ophthalmological evaluation. The obtained data were analysed using non-parametric statistics.

Results The highest number of children who were tested after Corresponding author: the screening process was during 2015. A total of 7415 children Amra Nadarević Vodenčarević (3790 males and 3625 females), in the age range of 4-15 were Eye Clinic, University Clinical Centre Tuzla screened. In the total sample of children who were completely eva- Prof I Pašića b.b., 75000 Tuzla, luated (n=145; 290 eyes) the most common refractive error was Bosnia and Herzegovina astigmatism, in 152 (52.4%) eyes. In the preschool children (n=18; Phone: +387 35 303 230; 36 eyes), the most common refractive error was astigmatism, in 19 (52.8%) eyes, followed by hyperopia, in 9 (25%) eyes. In the E-mail: [email protected] school children (n=127) (254 eyes), the most common refractive ORCID ID: https://orcid.org/0000-0002- error was astigmatism, in 133 (52.4%) eyes, followed by myopia, 4588-8571 in 92 (36.2%) eyes. The overall prevalence of refractive errors was 1.95% (145 with refractive error out of 7415 screened).

Conclusion Prevalence of refractive errors is high enough to ju- stify a school eye screening programme.

Key words: astigmatisms, hypermetropia, myopia, visual scree- Original submission: ning 28 February 2020; Revised submission: 21 April 2020; Accepted: 27 July 2020 doi: 10.17392/1153-21

Med Glas (Zenica) 2021; 18(1):96-101

96 Nadarević Vodenčarević et al. Prevalence of refractive errors in Tuzla city

INTRODUCTION nation. In our country there is small amount of in- formation available on the incidence of refractive It is well known that children with different un- errors in our population, especially among pres- corrected refractive errors may experience diffe- chool and school children (14). To our knowled- rent kinds of problems in life, including headache ge, a similar study only with school children was and persistent ocular discomfort, particularly for done in Brčko District (15) and only one similar near work which can impair reading efficiency study that evaluated the frequency of refractive and their performance in school activities. Chil- errors in premature children in retinopathy of dren with uncorrected refractive errors can have prematurity (ROP) screening (16) was done in poor school performance as well (1). There are the Federation of Bosnia and Herzegovina. three types of refractive errors: myopia, hype- ropia and astigmatism. Myopia is a condition in There was also a study in Croatia, comparing the which the eye is long and causes a reduction in difference between screened and unscreened po- visual acuity (VA) that cannot be overcome by pulation (17). accommodation (2,3). In addition, highly myopic The aim of this study was to analyse and evaluate eyes, of −6 dioptres (D) or more, may develop si- refractive errors in paediatric population in the ght-threatening complications (4). Hyperopia, by city of Tuzla, Bosnia and Herzegovina. contrast, is a condition in which the eye is shor- ter (5). Although distance VA may be unaffected, PATIENTS AND METHODS especially in mild hyperopia, it can create visual Patients and study design disturbances which can affect optimum functio- nal performance of school children or cause squ- All pre-school children from 7 day-care cen- int (5,6). Astigmatism is another form of refrac- tres and school children from all 24 elementary tive error which is caused by differences in the schools in Tuzla, who failed vision screening refractive power of the optical system in different and were referred to the Eye Clinic, University axes. This is caused by irregular curvature of the Clinical Centre (UCC), Tuzla for full eye exam, cornea and less commonly the crystalline lens were included in this study. This study took place (7). The importance of astigmatism in children between November 2014 and November 2019 in lies in the fact that it is a correctable cause of vi- Tuzla, Bosnia and Herzegovina. sual impairment in these ages, and it can coexists An approval of the Ethics Committee Board of with spherical error (8). In addition, astigmatism the University Clinic Centre Tuzla was obtained increases the incidence of amblyopia in children, to conduct this study in accordance with the Dec- and even treatment results are affected by the laration of Helsinki. type of astigmatism (9,10). Anisometropia is the Inclusion criteria were age of 4-15 years on the condition in which two eyes have unequal refrac- examination day, failed vision screening, parents tive power; its severe forms can affect binocular or legal guardians signed an informed consent, vision (11). During the growth of the eye, the and no history of systemic diseases. The exclusion process of emmetropization normally occurs. It is criteria were children who had eye injuries or eye known that hyperopia, from +3.0D to +4.0D that diseases of any kind, children who were allergic is present at birth, usually decreases during the to any ingredient in 1% cyclopentolate solution, preschool period to +0.50D, emmetropia or even children who refused to continue the examinations converges to small myopia (12). Minor amount due to eye discomfort during cyclopentolate admi- of anisometropia remains undetected and does nistration (e.g. burning, photophobia, irritation). not cause any significant visual problem. Howe- ver, a difference of ≥1.0D in a child can lead to Methods amblyopia and development of squint (13). Re- fraction of the eye changes through life. The vision screening process and examinations of children were performed by medical teams In Bosnia and Herzegovina most children, in our consisting of medical students (volunteers), re- experience, are usually examined very late by the sidents in , ophthalmologist and ophthalmologist and many of them are forced to ophthalmic nurse and optometrist. live visually impaired life prior to the first exami-

97 Medicinski Glasnik, Volume 18, Number 1, February 2021

Ophthalmologic examinations included external amination, 18 preschool children and 127 school eye examination, visual acuity, biomicroscopic children. The mean age of children was 9.50 ± examination of the anterior segment of the eye, 2.91 years (range of 4 to 15 years). intraocular pressure, dilated fundus and as well In the period 2016-2019 at UCC Tuzla there was as ocular motility examination. The examinati- fewer number of children who failed screening on process began with testing uncorrected visual tests and were examined in our department: in acuity. To measure visual acuity Snellens charts, 2016 30, in 2017 79, in 2018 48, and in 2019 46 tumbling E or Lea charts were used. A cover-un- children. The average visual acuity (VA) of the cover test was then performed to detect if stra- right eye without correction in all patients was bismus was present. The eye movements were 0.54 (SD=+/-0.27). The most frequently repeated tested in 9 cardinal directions. Anterior segment visual acuity of the right eye without correcti- was examined with slit lamp to detect any ocular on was 0.80. The minimum visual acuity of the pathology of anterior segment like corneal patho- right eye without correction was 0.03, while the logy, cataract, congenital anomalies and evidence maximum visual acuity of the right eye without of previous . The children underwent correction was 0.90. The overall average visual a full ocular examination, and any pathology acuity (VA) of the left eye without correction was involving the anterior and posterior ocular se- 0.53 (SD = +/-0.29). The most frequently repea- gments was documented. Detailed history about ted visual acuity of the right eye without correc- present and past ocular problems and treatment, tion was 0.80. The minimum visual acuity of the history of any medical or surgical treatment, and right eye without correction was 0.01, while the family history were taken. maximum without correction was 0.90. Refraction was performed under cycloplegia, During 2015, 2016, 2017, 2018 and 2019 we fo- with cyclopentolate 1% administrated three times und 36 (24.8%), 10 (33.3 %), 25 (31.64 %), 15 at 5 minutes intervals. Retinoscopy was per- (31.25%), and 15 (32.60%), respectively, chil- formed 45 minutes following the first instillation dren with anisometropia of≥1.0D. of drops, followed by dilated fundus exam. Sub- As the higher number of examined children was jective refraction was also performed if the child during 2014-2015 we gave a special focus on collaborated. Myopia was considered when mea- this period. The most common refractive error sured objective refraction was ≥ −0.75 spherical in the 2014-2015 period was astigmatism, in 152 equivalent dioptres in one or both eyes. Hypero- (out of 290; 52.4%) of eyes. In both preschool pia was considered when the measured objec- (n=18; 36 eyes) and school children (n=127; 254 tive refraction was greater than +2.00 spherical eyes), the most common refractive error was equivalent dioptres in one or both eyes provided astigmatism, which was recorded in 19 (52.8%) no eye was myopic. Astigmatism was considered and 133(52.4%) eyes, respectively, followed by to be visually significant if ≥1.00D. Anisometro- hyperopia, 9 (25%) and myopia, 8 (22.2%) eyes pia was defined as unequal refractive power in in preschool children, and in school children two eyes. followed by myopia, 92 (36.2%) and hyperopia, Statistical analysis 29 (11.4%) (Table 1).

Relevant data were presented as frequencies, me- Table 1. Distribution of refractive errors among preschool ans, and standard deviations. and school children during 2015 Children No (%) of eyes RESULTS group Astigmatism Hyperopia Myopia Total Preschool 19 (52.8) 9 (25) 8 (22.2) 36 (100) In the period between November 2014 and No- School 133 (52.4) 29 (11.4) 92 (36.2) 254 (100) vember 2015 a total of 7415 children were ex- Total 152 (52.4) 38 (13.1) 100 (34.5) 290 (100) amined. There were 3790 (51%) male and 3625 (49%) female children. The number of children During 2016, among 30 children (60 eyes) 5 referred to the ophthalmologists for further eval- children were of school age, while 25 were pre- uation and management was 409, of which 145 schoolers. The most common refractive error was children completed full ophthalmological ex- hyperopia, in 25 (41.7%), followed by myopia,

98 Nadarević Vodenčarević et al. Prevalence of refractive errors in Tuzla city

20 (33.3%), and astigmatism, in 15 (25%) chil- for preschool children. In the last few years there dren. During 2017, among 79 children (158 eyes) are strong initiatives in neighbouring countries the most common refractive error was hyperopia, for systemic screening at the age of 4 (17). Our 80 (50.6%), followed by astigmatism, 48 (30.4%) results showed that the prevalence of refractive and myopia, 30 (19%). During 2018, among 48 errors was 1.95%; however, considering that chil- children (96 eyes) the most common refractive dren who previously had corrected visual acuity error was astigmatism, 47 (50%), followed by with appropriate glasses or contact lenses were myopia, 25 (26%), and hyperopia, 23 (24%). not included, we can estimate the prevalence of During 2019, among 46 children (92 eyes) the refractive errors in the Tuzla city is much higher. most common refractive error was astigmatism, It can be estimated that the prevalence in the city 44(47.8%), followed by myopia, 33 (35.9%), and of Tuzla could be expected to be around 9.96%. hyperopia 15 (16.3%) (Table 2). The prevalence of refractive errors varies from one country to another. Several studies reported Table 2. Distribution of refractive errors in the 2015-2019 period the prevalence of refractive errors between 2.9% Total number of No (%) of eyes and 18.5% (20-22). Variations within the country Year children/eyes Astigmatismus Myopia Hypermetropia are also noticeable (22,23). The reason for this 2015 145/290 152 (52.4) 100 (34.5) 38 (13.1) variation is probably because some studies were 2016 30/60 15 (25) 20(33.3) 25 (41.7) 2017 79/158 48 (30.4) 30(19) 80(50.6) conducted in rural and other in urban areas (22, 2018 48/96 47 (50) 25 (26) 23 (24) 23). In the period 2016-2019 fewer children were 2019 46/92 44(47.8) 33 (35.9) 15 (16.3) examined in UCC Tuzla probably because te- As we gave special focus to the 2014-2015 peri- achers had sent children to primary health centres od, we asked parents about medical history of the in our region, and many children were examined children. Of the total number of children (n=145) by private practitioners. 140 were delivered without any complication, In a study from Niš (Serbia) conducted on the while five pregnancies were maintained by hor- sample of 620 children (1240 eyes), hyperme- mone therapy. All children had normal postnatal tropia was the most common refractive error, development. In the family history, we did not 54.11%, followed by astigmatism, 42.91%, while receive information from the parents about ocu- myopia was detected in 2.98% children (12). A lar diseases (glaucoma, cataracts or strabismus) study in Novi Sad (Serbia), where 200 children in immediate family members. From the total (400 eyes) aged 3-18 were examined, showed number (n=145) of children, 118 (81.4%) were prevalence of hyperopic astigmatism (farsighted without refractive error (RE) in the family. In astigmatism) of 40.8%, followed by hyperme- six (4.2%) children, some of the refractive errors tropia, 21.3% (24). In Lithuania, in a study in- were found in siblings, five had astigmatism whi- volving 839 (1678 eyes) children aged 2-6 years, le one sibling had high myopia. In four (2.8%) different grade hyperopia was present in 43.26% children, both parents wore glasses, while in se- and hypermetropic astigmatism in 23.08% (25). ven (11.8%), one parent wore glasses or lenses. Also, the rate of myopia in the United States in patients aged 12-17 years increased significantly DISCUSSION from 24.5% during 1971-1972 to 34.8% during

In this study we analysed refractive errors in scho- the 1999 -2004 periods (26).In Taiwan the preva- ol and preschool children living in Tuzla city. The lence of myopia is 20-30% among 6-7 year olds, results showed that refractive errors were very and as high as 84% in high school students (27). common and undiagnosed. Detection and correc- In Hong Kong, in a study of 4257 children aged tion of refractive anomalies are especially impor- 6–8 years, 25.0% were myopic (28). tant in the paediatric population, because they Screening programs can significantly reduce am- can be the cause of amblyopia and strabismus, blyopia, as showed in our neighbouring countries if detected late (18). Our screening program re- (17). Preschool and school children are not qu- lating to refractive errors was the biggest one in ick in sharing their visual problems and parents Bosnia and Herzegovina after the war, since the- are very often unaware of the children problems. re is still no population-based screening program Refractive errors are common in Bosnian chil-

99 Medicinski Glasnik, Volume 18, Number 1, February 2021

dren and often remained undiagnosed due to low be easily diagnosed and corrected at a relatively standard of living and low level of social-eco- small cost. Conducting preventive programs di- nomic development in some areas, and there is rected to preschool and school children requires not enough attention paid to children's vision and participation of several sectors of the community refraction. We also emphasize that it is necessary involving physicians, educators, family members to implement mandatory examination of all chil- and volunteer personnel. dren at the age of 4 years. ACKNOWLEDGMENT The obstacles in this study came from the fact that many examinations were performed in pri- The authors would like to thank all patients who vate practices with no legal register. Also, a high agreed to participate in this study. Also, we wo- number of children was not further examined uld like to thank the entire team of the Eye Clinic even though it was suggested after the screening. at the University Clinical Centre in Tuzla, Bosnia and Herzegovina, especially employees of the In conclusion, our study presents evidence that a Department for the treatment of amblyopia and national screening program in Bosnia and Her- strabismus. zegovina is needed in order to include a larger number of children and to obtain even more FUNDING accurate data. We strongly believe it is necessary No specific funding was received for this study. to conduct a massive screening program in the whole country to determine the correct inciden- TRANSPARENCY DECELERATION ce and prevalence of refractive errors which can Conflicts of interest: None to declare.

REFERENCES 1. Harrington SC, Stack J, Saunders K, O´Dwyer V. 12. Kostovska V, Stanković-Babić G, Smiljković- Refractive error and visual impairment in Ireland Radovanović K, Cekić S, Vujanović M, Bivolarević school children. Br J Ophthalmol 2019; 103:1112-8. I. Analysis of refractive errors in children aged up 2. Goss D, Grosvenor T. Optometric Clinical Practice to 15 years. Acta Medica Medianae 2013; 52:33–40 Guideline Care of Patient with Myopia. Reference 13. Deng L and Gwiazda JE. Anisometropia in children Guide for Clinicians. St Louis, MO: American Opto- from infancy to 15 years. Invest Ophthalmol Vis Sc metric Association, 2006: 3-10. 2012; 53:3782-8 3. Lagrèze WA, Schaeffel F. Preventing Myopia. Dtsch 14. Vodencarevic AN, Jusufovic V, Halilbasic M, Ali- Arztebl Int. 2017;114:575-80. manovic E, Terzić S, Čabrić E, Drljević A, Burgić 4. Williams K, Hammond C. High myopia and its ri- M. Amblyopia in children: analysis among prescho- sks. Community Eye Health 2019; 32:5-6. ol and school children in the city of Tuzla, Bosnia 5. Verhoeven VJ, Wong KT, Buitendijk GH, Hofman and Herzegovina. Mater Sociomed 2017; 29:164-7. A, Vingerling JR, Klaver CC. Visual consequen- 15. Popovic-Beganovic A, Zvorničanin J, Vrbljanac V, ces of refractive errors in the general population. Zvorničanin E. The prevalence of refractive errors Ophthalmology 2015; 122:101–9. and visual impairment among school children in 6. Bruce A, Fairley L, Chambers B, Wright J, Sheldon Brčko District, Bosnia and Herzegovina. Semin TA, Impact of visual acuity on developing literacy at Ophthalmol 2018; 33:1-11. age 4-5years: a cohort-nested cross-sectional study. 16. Pidro A, Alajbegović-Halimić J, Jovanović N, Pidro BMJ Open 2016; 6:e010434 A. Evaluation of refractive errors in retinopathy of 7. Grosvenor T. How much do we know about astigma- prematurity screening. Med Glas (Zenica) 2019; tism? Clin Exp Optom 2007; 90:3-4. 16:204-8. 8. Sayed, K.M. Analysis of components of total asti- 17. Busic M, Bjelos M, Petrovecki M,Kuzmanovic Ela- gmatism in infants and young children. Int Ophthal- bjer B, Bosnar D, Ramic S, Miletic D, Andrijasevic mol 2017; 37:125–29. L, Kondza Krstonijevic E, Jakovljevic V, Biscan 9. Yap TP, Luu CD, Suttle C, Chia A, Boon MY. Effect Tvrdi A, Predovic J, Kokot A, Biscan F, Kovacevic of stimulus orientation on visual function in children Ljubic M, Motusic Aras R. Zagreb Amblyopia Pres- with refractive amblyopia. Invest Ophthalmol Vis chool Screening Study: near and distance visual acu- Sci 2020; 61:1-9. ity testing increase the diagnostic accuracy of scre- 10. Harvey EM. Development and treatment of astigmati- ening for amblyopia. Croat Med J 2016; 57:29-41. sm-related amblyopia. Optom Vis Sc 2009; 86:634 -9. 18. Al-Tamimi ER, Shakeel A, Yassin SA, Ali SI, Khan 11. Murray SJ, Codina CJ. The role of binocularity in UA. A clinic-based study of refractive errors, stra- anisometropic amblyopia. J Binocul Vis Ocul Motil bismus and amblyopia in pediatric age-group. J Fa- 2019;69:141-52. mily Community Med 2015; 22:158-62.

100 Nadarević Vodenčarević et al. Prevalence of refractive errors in Tuzla city

19. Muma MK, Kimani K, Kariuki-Wanyoike MM, Ila- 24. Bolinovska S. Hipermetropija kod dece predškol- ko DR, Njuguna MW. Prevalence of refractive errors skog i školskog uzrasta. Med Pregl 2007; 60:115-21. among primary school pupils in Kilungu Division 25. Majauskiene O, Aukstikalniene R, Ceponiene R, of Makueni District, Kenya. Med J Zambia 2009; Majauskaite I. The evaluation of visual disorders 36:165-70. in preschool children. Medicina (Kaunas) 2005; 20. Hashemi H, Rezvan F, Yekta AA, Hashemi M, No- 41:240-5. rouzirad R, Khabazkhoob M. The prevalence of asti- 26. Vitale S, Sperduto RD, Ferris FL 3rd. Increased gmatism and its determinants in a rural population prevalence of myopia in the United States between of Iran: the “Nooravaran Salamat” mobile eye clinic 1971-1972 and 1994-2004. Arch Ophthalmol 2009; experience. Middle East Afr J of Ophthalmol 2014; 127:1632-9. 21:175-81. 27. Wu PC, Huang HM, YU HJ, Fang PC, Chen CT. Epi- 21. Maul E, Barroso S, Munoz SR, Sperduto RD, Ellwe- demiology of myopia. J Ophthalmol 2016; 5:386-93. in LB. Refractive error study in children: Results 28. Yam JC, Tang SM, Kam KW, Chen LJ, YU M, Law from La Florida, Chile. Am J Ophthalmol 2000; AK, Yip BH, Wang YM, Cheung CYL, NG DSC, 129:445-54. Young AL, Tham CC, Pang CP. High prevalence of 22. Shrestha GS, Sujakhu D, Joshi P. Refractive error myopia in children and their parents in Hong Kong among school children in Jhapa, Nepal. J Optom Chinese population: the Hong Kong Children Eye 2011; 4:49-55 Study Acta Ophthalmol 2020. Online ahead of print. 23. Pokharel GP, Negrel AD, Munoz SR, Ellwein LB. Refractive error study in children: results from Mec- hi Zone, Nepal. Am J Ophthalmol 2000; 129:436-44.

101 ORIGINAL ARTICLE

Correlation of signal to noise ratio (SNR) value on distortion product otoacoustic emission (DPOAE) and expression of nuclear factor erythroid 2-related factor 2 (NRF2) in cochlear organ of Corti in rat exposed to noise

Diana Amellya1, Tengku Siti Hajar Haryuna1, Wibi Riawan2

1Department of Otolaryngology, Head and Neck Surgery, Faculty of Medicine, Universitas Sumatera Utara, 2Department of Biochemis- try and , Faculty of Medicine, Brawijaya University; Indonesia

ABSTRACT

Aim To investigate the changes in the value of the signal to noise ratio (SNR) and to assess changes in the expression of nuclear factor erythroid 2-related factor 2 (NRF2) in the organ of Corti of rat exposed to noise.

Methods This study used a randomized post test only control gro- up laboratory experimental design with 27 male Wistar strain Rat- tus norvegicus. The study group was divided into 3 groups (n = 9): group I (control), group 2 (2 hours of 100 dB noise exposure) and Corresponding author: group 3 (2 hours of 110 dB noise exposure). Tengku Siti Hajar Haryuna Faculty of Medicine, Results There was no significant difference in the SNR in the gro- Universitas Sumatera Utara up 1 on day 0, 2 and 4 (p>0.05). However, there was a significant difference in the SNR in the group 2 and the group 3 on day 0, Jln. Dr Mansyur no 5, Padang bulan, 2 and 4 (p<0.05). There was a significant difference in the mean Medan city, North Sumatera, levels of NRF2 expression in the cochlear organ of Rattus norve- 20155, Indonesia gicus in all groups (p<0.05). There was no correlation between the Phone: +62 812 606 1694; SNR and the NRF2 expression in group 2 (p> 0.05), but there was Fax: +62 61 821 6264; a correlation between the SNR and the NRF2 expression in the E-mail: [email protected] group 3 (p<0.05). ORCHID ID: https://orcid.org/0000-0002- Conclusion There was found a correlation between the SNR value 9984-6166 on distortion product otoacoustic emission (DPOAE) examination and NRF2 expression in the cochlear organ of Corti of Rattus nor- vegicus exposed to 110 dB noise. Original submission: Key words: animal, experimental, immunohistochemistry 16 October 2020; Revised submission: 24 November 2020; Accepted: 16 December 2020 doi: 10.17392/1292-21

Med Glas (Zenica) 2021; 18(1):102-106

102 Amellya et al. SNR and NRF2 value in rat noised model

INTRODUCTION Hearing loss due to noise that causes damage to the outer hair cells of the cochlea is irreversible It is estimated that there are 1.3 billion people with and cannot be operated or treated (10). Since the hearing loss in the world, and according to WHO, discovery of otoacoustic emissions (OAE) by Da- around 10% who are exposed to noise have the vid Kemp, there have been many investigations potential to get noise-induced hearing loss (NIHL) regarding the relationship between NIHL and the (1). Based on the multi-centre study, there were damage to hair cells outside the cochlea, where several countries that experienced a high preva- OAE is one of the technologies in the field of au- lence of hearing loss due to noise including Nepal diology used to detect damage to hair cells outside (16.6%), Thailand (13.3%), Sri Lanka (9%), Ban- the cochlea due to noise exposure (1,11). Distorti- gladesh (9%), Myanmar (8%), Maldives (6%), on product otoacoustic emission (DPOAE) is a he- India (6%), and followed by Indonesia (4.6%) (2). aring test that is more sensitive than conventional Hearing loss can be caused by strong metabolic audiometry to detect a NIHL (12). activity which is caused by noise stimulation that This study is different from the previous study can cause the hair cells to experience oxidative by Honkura et al. (6). We used normal rat, while stress and will cause cell death until NIHL occurs. Honkura et al. (6) used 2 types of mice (wild mice This will interfere with blood flow to the cochlea and Nrf-/- type mice); in our study we were chec- which is an important factor for hearing function, king the length of time to rest after giving noise vasoconstriction caused by noise can also cause after 2 days, while Honkura et al. (6) after 7 days; NIHL (3). Excessive noise exposure is associated the difference in an amount of giving noise in the with damage to sensory cells in the inner ear, espe- Honkura study was 96 dB, while we used 100 db cially the outer hair cells. Noise with an intensity and 110 dB; while Honkura et al. used auditory of 85 dB or more can cause damage to the hearing brainstem response (ABR), we used DPOAE. receptors, especially the frequency of 3000-6000 Hz and the heaviest of 4000 Hz (4). The aim of this study was to investigate a corre- lation between the signal to noise ratio (SNR) va- Noise-induced hearing loss is one of the most lues in​​ the DPOAE examination of NRF2 expre- common sensorineural hearing disorders (5).The ssion in the cochlear organ of Corti in the rats study shows that the pathogenesis of NIHL is exposed to noise. closely related to cochlear ischemia-reperfusion injury, which is caused by decreased blood flow MATERIAL AND METHODS and free radical production due to excessive noise. Nuclear factor erythroid 2-related factor 2 (NRF2) Material and study design is a transcription activator that plays an important role in defence mechanisms against oxidative stre- In this study a randomized post-test only control ss and also maintaining the reduction of intracellu- group laboratory experimental design was used lar homeostasis (6). Biological function of NRF2 with 27 healthy, adult, male (2-3 months), Rat- is to activate transcription factors from cytoprotec- tus norvegicus Wistar rats, weighing 150-250 tive gene sequences that are able to counteract the grams. The animals were divided into 3 groups harmful effects of oxidative stress (7). (nine animals in each): group 1 (control), group 2 (2 hours 100 dB noise exposure) and group 3 (2 The NRF2 is a regulatory factor that arises from hours 110 dB noise exposure). cellular resistance to oxidants. It also controls ba- sal expression and induction of a ARE-dependent The maintenance of experimental animals was genes series to regulate physiological and patho- carried out at the Animal House Laboratory of logical outcomes from oxidant exposure (8). The the Faculty of Mathematics and Natural Scien- NRF2 is a central mediator of the foremost cel- ces, Universitas Sumatera Utara, the manufacture lular defence system, the transcription factor of of tissue paraffin blocks was carried out at the NRF2 protects against oxidative tissue damage Laboratory of Medical through ARE-mediated transcription activation Faculty, Universitas Sumatera Utara, and cutting of several phase 2 detoxification enzymes and of tissue blocks, hematoxylin eosin staining, antioxidant enzymes (9). immunohistochemical examination techniques carried out in the Biochemistry and Molecular

103 Medicinski Glasnik, Volume 18, Number 1, February 2021

Biology, Faculty of Medicine, Brawijaya Univer- Score) formula was used by multiplying the per- sity, Indonesia. centage of cells stained brown with the intensity The study has received ethical approval from the of the streaks. The distribution of cells dyed brown Ethics Commission of the Faculty of Medicine, was divided into 4 categories: 0 = no cells dyed Universitas Sumatera Utara. brown, 1 = <10%, 2 = 11-50%, 3 = 51-80 % and ≥4 = 81% of cells stained brown; then the inten- Methods sity assessment was carried out by category; 0 - no Noise exposure was given to Rattus norvegicus colour, 1 - weak intensity, 2 - moderate intensity, rats in a voice box measuring 64.5 x 45 x 40 cm and 3 - strong intensity. The calculation results will and made of foam-coated cork. The speaker was show a minimum score of 0 and a maximum score placed on the roof of the box cover, then a hole of 12 (14). After all the results had been calculated, was made at the bottom of the box to measure the a statistical analysis was carried out. intensity of the noise where a tool for measuring Statistical analysis noise intensity was called sound level meter; this measurement was carried out at eight points where Data were analysed using the One Way Anova the noise difference did not exceed 1 dB. The so- test, the Post-Hoc Bonferoni test and Pearson und source was provided by a Compact Disc (CD) correlation test was used to analyse the differen- which contained a sound recording, CD player, ces/correlation in each group,. The statistical test and amplifier that produces noise with a frequency was considered significant if p<0.05. of 1-10 kHz; intensities of 100 dB and 110 dB gi- RESULTS ven for 2 hours and 2 days. DPOAE (Elios Elito Otodia brand, Echodia Ltd, London, UK) exami- The difference in NRF2 expression was found: nation was carried out on all experimental animals decreased in the group 3 compared both to the with anaesthetic with ketamine 50 mg/kg body groups 1 and 2 (Figure 1). weight (13). The probe was adjusted in size and placed in the ear canal. DPOAE was assessed 3 times: the initial assessment before the treatment, 2 days after noise exposure, and then 2 days after the noise rest was over. The examination of NFR2 expression was carri- ed out by exposing the rats first with ether in- Figure 1. An overview of NRF2 expression (at 40x magnifica- halation, then performing necropsy of the rat’s tion). The circle shows the depiction of NRF2 expression in the cochlear organ of corti of Rattus norvegicus, which is marked temporal bone tissue. Tissue samples were taken, in brown. A) group 1 (control); B) group 2 (2 hours of 100 dB fixed with 10% formalin buffer solution and de- noise exposure); C) group 3 (2 hours of 110 dB noise exposure) calcified with EDTA for 4 weeks. Furthermore, There was no significant difference in the SNR laboratory examinations were carried out through value of the DPOAE examination on day 0, 2 and tissue fixation by making tissue paraffin blocks 4 (p>0.05). However, in the group 2 and 3 the- and sliced into​​ 4 µm thick sections, then placed re was a significant difference in the SNR value in a slide to be stained with haematoxylin-eo- from the DPOAE examination on days 0, 2 and sin and immunohistochemical staining, namely 4 (p<0.05). There was a significant difference in NRF2 with primary antibody NRF2 H-300: sc- the mean of NRF2 expression in the cochlear or- 13032 (Santa Cruz, United States). To assess the gan of corti of Rattus norvegicus in all groups NRF2 expression, an Olympus XC 10 microsco- (p<0.05) (Table 1). pe was used using 40x magnification. In the group 2 (110 dB) there was no correlation The expression assessment was carried out by asse- between the SNR value on the DPOAE exami- ssing the distribution and intensity of the immuno- nation and the NRF2 expression (p>0.05), with histochemical streaks. The distribution assessment a positive correlation direction (r=0.040). In the was carried out by counting the percentage of cells group 3 (110 dB) there was a correlation betwe- stained with brown in the entire microscope field en the SNR value on the DPOAE examination of view using 40x magnification. Where to asse- and the NRF2 expression (p<0.05), with a strong ss the NRF2 expression, the IRS (Immunoreactive positive correlation direction (r=0.792) (Table 2).

104 Amellya et al. SNR and NRF2 value in rat noised model

Table 1. Differences in the mean of signal to noise ratio Our research showed no significant difference in (SNR) value on distortion product otoacoustic emission the SNR before rats exposed to noise, in group (DPOAE) examination on the day 0, 2 and 4, and differences in nuclear factor erythroid 2-related factor 2 (NRF2) expres- 1 on day 0, 2 and 4, suggesting that NRF2 does sion in Rattus norvegicus cochlear organ of corti not play a role in the development of cochlear Mean±SD on the day SNR p function, similarly with Honkura’s study (6). Af- Day 0 Day 2 Day 4 ter giving 100 dB and 110 dB noise for 2 hours Group 1 11.36±2.50 10.62±2.43 9.44±2.14 0.245 and 2 days our results showed that the SNR value Group 2 10.63±2.05 4.23±0.96 4.26±1.38 <0.001 Group 3 9.73±3.09 4.11±1.15 4.33±1.21 <0.001 decreased (TTS occurred) and after 2 days of rest NRF Mean±SD Min-Max there was an improvement in the SNR value but Group 1 8.33±3.08 3.00-12.00 Group 2 3.67±1.12 2.00-6.00 <0.001 did not return to the initial value before the tre- Group 3 4.00±2.00 1.00-8.00 atment, which indicated that PTS had occurred.

Table 2. Correlation of signal to noise ratio (SNR) value on We found that the correlation between SNR valu- day 4 on distortion product otoacoustic emission (DPOAE) es on​​ DPOAE examination and NRF2 expression examination and nuclear factor erythroid 2-related factor 2 in the cochlear organ of corti of Rattus norvegi- (NRF2) expression at 100 dB & 110 dB noise cus exposed to 110 dB noise on day 4 could be Group Variable Mean±SD r p due to an increase in the reactive oxygen speci- 2 (100 dB) 0.040 0.918 SNR 3.66±1.11 es (ROS). Kim et al. showed that increased noise NRF2 4.27±1.38 exposure can be harmful, which triggers the for- 3 (110 dB) 0.792 0.011 mation of molecules, such as ROS, and induction SNR 4.00±2.00 NRF2 4.27±1.38 of inflammatory genes in the ear, which results in r, Pearson correlation; damage or death of hair cells. These cells are very delicate and sensitive and do not regenerate once DISCUSSION they are damaged or lost. In addition, there are no The susceptibility of developing NIHL varies known therapeutic treatments to restore damaged greatly, there are some individuals who can to- hearing (3). In our study there was a decrease in lerate high noise levels for a long time, but there SNR value due to noisy administration, which can are some individuals in the same environment also damage the hair cells in the cochlea. with rapid hearing loss, which is related to the Under normal circumstances NRF2 binds to Kelch- duration and intensity of exposure and genetic like ECH-associated protein-1 (Keap1) in the cyto- susceptibility to noise trauma (15). Grondlin plasm where this prevents NRF2 from transloca- et al. also found that individual susceptibility ting to the nucleus and binding to the antioxidant to NIHL depends on two factors, namely envi- response element (ARE). If there is noise exposure ronmental factors that can increase physiological that exceeds the threshold, the bond between NRF2 stress, inflammation and oxidative stress as well and Keap1 will be released and NRF2 translocate as genetic factors (16). to the nucleus and binds to the ARE located in the Honkura et al. compared wild-type and NRF2- gene promoter region that encodes cytoprotective /- mice were exposed to 96dB noise for 2 hours proteins, which are antioxidant enzymes (6,17). continuously, and then ABR were observed 1 The phosphorylation process is an important factor day before, 4 hours and 7 days. On the second in the detachment of the NRF2 bond with Keap-1 day after noise exposure, the ABR threshold and translocating to the nucleus (6,17). before noise exposure was almost comparable We conducted this study because there was no between the two groups of mice; this indica- previous study on the correlation of SNR values tes that NRF2 does not play a role in the deve- in DPOAE examination with NRF2 expression in lopment of cochlear function (6). The ABR in noise-exposed rat’s cochlea. Urono and Motohas- 4 hours after noise did not show any difference hi in 2011 stated that NRF2 is very important for between the two groups of mice, but after 7 days protection from oxidative stress and xenobiotic of rest from noise exposure, there was a perma- detoxification (18), so that cell damage on Cor- nent threshold change which was significantly tiary organs caused by oxidative stress due to no- greater in NRF2-/-β; type ratio was NRF2-/- in ise exposure can be repaired and protected through wild types which were more susceptible to noi- NRF2 by triggering the formation of endogenous se, and will impair the recovery ability of thre- antioxidant enzymes to prevent cell damage which shold shift (TTS) in the absence of NRF2 (6). led to NIHL.

105 Medicinski Glasnik, Volume 18, Number 1, February 2021

In conclusion, our study showed a correlation assistance of the TALENTA research fund with between the SNR value on DPOAE examination the research implementation contract of the Uni- and NRF2 expression in the cochlear organ of versitas Sumatera Utara (No: 2590/UN.5.1R/ Rattus norvegicus exposed to 110 dB noise. The PPM/2018). The author is also grateful to the De- research we conducted was based on the lack partment of Pathology Anatomy, Faculty of Me- of research on the correlation of SNR values on​​ dicine, Universitas Sumatera Utara, Department DPOAE examination with NRF2 expression in of Biochemistry, Faculty of Medicine, University noise-exposed rat cochlea, so it is hoped that this of Brawijaya which helped to provide scientific study can provide new knowledge and future be- equipment. nefits for NIHL patients where with endogenous antioxidants that are still unable to help repair, it FUNDING is expected that exogenous antioxidants are given. No specific funding was received for this study.

ACKNOWLEDGEMENT TRANSPARENCY DECLARATION The author is very grateful to the Research In- Conflicts of interest: None to declare. stitute of the Universitas Sumatera Utara for the

REFERENCES 1. Basner M, Babisch W, Davis A, Brink M, Clark C, 11. Helleman HW, Elsing H, Limpens J, Dreschler WH. Janssen S, Stansfeld S. Auditory and non-auditory Otoacoustic emission versus sudiometry in monito- effects of noise on health. Lancet 2014; 383: 1325–32. ring hearing loss after long term noise exposure- a 2. World Health Organization. Situation Review and systematic review. Scand J Work Environ Health Update on Deafness, Hearing Loss and Intervention 2018; 44:585-600. Programmes. New Delhi: World Health Organizati- 12. Mehrparvar AH, Mirmohammadi SJ, Davari MH, on Regional Office for South East Asia, 2007. Mostaghachi M, Mollasadeghi A, Rahaloo M, 3. Kim MS, Kwak S, Baek H, Li Z, Choe SK, Song K. Hashemi SH. Conventiomal audiometry, extended Protective effect of Yang Mi Ryung extract on noise- high-frequency audiometry, and DPOAE for early induced hearing loss in mice. Evid Based Comple- diagnosis of NIHL. Iran Red Crescent Med J 2014; mentary Altern Med 2017; 2017:9814836. 16: e9628. 4. Sliwinska-Kowalska M, Zaborowski K. WHO Envi- 13. Institute for Laboratory Animal Research Com- ronmental noise guidelines for the european region: mittee for the Update of the Guide for the Care and A systematic review on environmental noise and Use of Laboratory Animals. Guide for the care and permanent hearing loss and tinnitus. Int J Environ use of laboratory animals. Washington. D.C.: Natio- Res Public Health 2017; 14:1139. nal Academy Press, 2014:1-220. 5. Le TN, Straatman LV, Lea J, Westerberg B. Current 14. Czogalla B, Kahaly M, Mayr D, Schmoeckel E, insights in noise-induced hearing loss: a literature Niesler B, Kolben T, Burges A, Mahner S, Jeschke review of the underlying mechanism, pathophysio- U, Trillsch F. Interaction of ER and NRF2 impacts logy, asymmetry, and management options. J Oto- survival in ovarium cancer patients. Int J Mol Sci laryngol Head Neck Surg 2017; 46:41. 2019; 20:112. 6. Honkura Y, Matsuo H, Murakami S, Sakiyama M, 15. Moussavi-Najarkola SH, Khavanin A, Mirzaei R, Sa- Mizutari K, Shiotani A, Yamamoto M, Morita I, Shi- lehnia M, Muhammadnejad A, Akbari M. Temporary nomiya N, Kawase T, Katori Y, Motohashi H. NRF2 and permanent level shifts in distortion product otoa- is a key target for prevention of noise-induced hea- coustic emission following noise exposure in an ani- ring loss by reducing oxidative damage of cochlea. mal model. Int Occup Environ Med 2012; 3:145-52. Sci Rep 2016; 6:19329. 16. Grondin Y, Bortoni ME, Sepulveda R, Ghelfi E, Bar- 7. Li C, Cheng L, Wu H, He P, Zhang Y, Yang Y, Chen tos A, Cotanche D, Clifford RE, Rogers RA. Genetic J, Chen M. Activation of the KEAP1-NRF2-ARE polymorphisms associated with hearing threshold signalling pathway reduces oxidative stress in hep2 shift in subjects during first encounter with occupa- cells. Mol Med Rep 2018; 18:2541-50. tional impulse noise. PlosOne 2015; 10:e0130827. 8. Ma Q. Role of NRF2 in oxidative stress and toxicity. 17. Hybertson BM, Gao B, Bose SK, McCord JM. Oxi- Annu Rev Pharmacol Toxicol 2013; 53:401-26. dative stress in health and disease: the therapeutic 9. Hosokawa K, Hosokawa S, Ishiyama G, Ishiyama potential of Nrf2 activation. Mol Aspects Med 2011; A, Lopez IA. Immunohistochemichal localization 32:234-46. of NRF2 in the human cochlea. Brain Res 2018; 18. Urono A, Motohashi H. The Keap1-Nrf2 system 1700:1-8. as an in vivo sensor for electrophiles. Nitric Oxide 10. World Health Organization. Hearing loss due to re- 2011; 25:153-60. creational exposure to loud sounds: a review. https:// apps.who.int/iris/handle/10665/154589 (01 Decem- ber 2020).

106 ORIGINAL ARTICLE

Correlation between numerical and categorical immunohistochemical score of Ki-67 and HER2 with clinicopathological parameters of breast cancer

Mirsad Dorić, Suada Kuskunović-Vlahovljak, Edina Lazović Salčin, Svjetlana Radović, Nina Čamdžić, Mirsad Babić, Haris Čampara

Department of Pathology, School of Medicine, University of Sarajevo, Bosnia and Herzegovina

ABSTRACT

Aim To evaluate the relationship between numerical and catego- rical immunohistochemical score of Ki-67 and human epidermal growth factor of receptor 2 (HER2) with clinicopathological para- meters of breast cancer (BC).

Methods The study included 311 patients with invasive BC di- agnosed at the Department of Pathology, School of Medicine in Sarajevo, Bosnia and Herzegovina, during the period 2015-2019. The expression level of Ki-67 and HER2 was detected by immu- nohistochemical analysis.

Results The expression of Ki-67, as a numerical variable corre- Corresponding author: lated significantly with tumour grade (p=0.025), progesterone re- Mirsad Dorić ceptor (PR) (p=0.034) and categorical score of HER2 (p=0.028). When Ki-67 was categorized into high (>14%) and low (≤14%) le- Department of Pathology, vel groups, a statistically significant association was found betwe- School of Medicine, en Ki-67 level groups and HER2 status (categorical and nume- University of Sarajevo rical) (p=0.001 and p=0.043, respectively), as well as significant Čekaluša 90, 71000 Sarajevo, negative linear correlation with PR (p=0.037). The expression of Bosnia and Herzegovina HER2, as a numerical variable, showed a statistically significant Phone: +387 33 666 964; correlation with tumour grade (p=0.038), PR (p=0.025) and cate- gorical Ki-67 (p=0.043). Categorical score of HER2 correlated si- E-mail: [email protected] gnificantly with age (p=0.025), histologic type (p=0.039), tumour ORCID ID: https://orcid.org/0000-0002- grade (p=0.016), estrogen receptor (ER), (p=0.002) progesterone 8595-2064 receptor (PR) (p=0.0001), and categorical and numerical value of Ki-67 (p=0.0001 and p=0.0001, respectively).

Conclusion The results demonstrated that the categorical immu- nohistochemical score of HER2 provided a greater association Original submission: with clinicopathological parameters than numerical score of BC. 14 May 2020; Furthermore, a slightly better correlation with clinicopathological Revised submission: parameters was shown by the numerical value than by the catego- 16 June 2020; rical score of Ki-67 by applying a cut-off value of 14%. Accepted: Key words: breast neoplasms, carcinoma, prognosis, proliferative 23 July 2020 activity doi: 10.17392/1203-21

Med Glas (Zenica) 2021; 18(1):107-113

107 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION PATIENTS AND METHODS Breast cancer (BC) is the most common can- Patients and study design cer and the leading cause of death in women worldwide (1). The expression of biomarkers in A retrospective analysis was conducted using BC is important to identify prognosis. Several cla- data of 311 patients with invasive BC diagnosed ssic BC markers, such as estrogen receptor (ER), at the Department of Pathology, School of Medi- progesterone receptor (PR), Ki-67 proliferative in- cine Sarajevo, Bosnia and Herzegovina, during dex, and human epidermal growth factor of recep- the period from 2015 to 2019. All patients with tor 2 (HER2) are relevant for therapeutic strategy BC underwent partial or total mastectomy with and prognosis (2-4). In addition to these factors, axillary lymph node dissection. No neoadjuvant the assessment of tumour proliferation pattern is chemotherapy or radiotherapy was administe- important for a treatment decision (2-4). red before surgical treatment. Tissues specimens Uncontrolled proliferation of tumour cells is a di- were fixed in 10% buffered formalin, paraffin stinct feature of malignancy and can be assessed embedded, processed, and stained with hema- by various methods (5). Dowsett et al. found that toxylin and eosin. the most commonly used measurement is immu- The study analysed patient's age, histologic tu- nohistochemical evaluation of Ki-67 antigen (6). mour type according to the WHO (17); tumour Although Ki-67 is the most commonly used mar- size (using the TNM staging system) T1 (includ- ker to evaluate proliferative index in BC, clearly ing T1a, T1b, and T1c) ≤2 cm, T2 ˃2 ≤ 5 cm, T3 defined cut-off values for high Ki-67 index have ˃5 cm T4 (including T4a, T4b, T4c, and T4d) tu- not been defined yet (7). mour of any size growing into the chest wall or HER2 is an important regulator of the cell cycle, skin (inflammatory breast cancer) (18); tumour including cell proliferation, cell survival, and grade (using Nottingham histological score, apoptosis (8,9). The amplification or overexpre- Elston and Ellis histologic grading criteria (19): ssion of HER2 serves as a prognostic factor and grade I (well differentiated), grade II (moderately also has a therapeutic significance (10,11). differentiated), and grade III (poorly differenti- ated); lymph node (LN) metastasis (negative, The identification of the optimal method or met- positive); and lymphatic vessel invasion (LVI) hodologies for the assessment of proliferative ac- (absent, present). tivity in BC has been the subject of several pre- vious studies (12-13). Despite numerous studies Methods in this field, the relationship between the Ki-67 and other clinicopathological prognostic factors Immunohistochemical analysis. 4-µm-thick remains uncertain (14,15). The association of sections of formalin-fixed, paraffin-embedded HER2 status and Ki-67 is still controversial, as tissue were mounted on coated slides. The immu- some researchers have found a positive associati- nostained slides were examined for nuclear stai- on with Ki-67, but others have not (14,16). ning in the case of estrogen receptor (ER), proge- sterone receptor (PR), and Ki-67, and membrane In routine clinical practice, the results of immu- staining in the case of HER2. nohistochemical analysis of Ki-67 and HER2 are presented as numerical and categorical values. The primary antibody against the ER was perfor- Numerical score is used in determination of ca- med in humidity chamber in EDTA buffer (pH 9) tegorical score, which primarily has predictive for 40 min (clone 1D5, Dako Cytomation, Glo- significance but also is important in prognosis strup, Denmark, dilution 1:30). The protocols for identification. staining PR and Ki-67 included a microwave an- tigen retrieval step, 3 times for 5 minutes: anti-PR The aim of this study was to evaluate the relati- (clone PgR, Dako Cytomation, Glostrup, Den- onship between numerical and categorical immu- mark; dilution 1:30), anti-Ki-67 (clone MIB-1, nohistochemical score of Ki-67 and HER2 with Dako Cytomation, Glostrup, Denmark; dilution clinicopathological characteristics of BC in order 1:10). Antigen retrieval for HER2 using Hercep- to determine which immunohistochemical score Test was performed following the manufacturer's provides better prognostic significance. protocol (Dako Cytomation).

108 Dorić et al. Immunohistochemical score of Ki-67 and HER2

Immunohistochemical evaluation. For hormo- bles between the mean score of Ki-67 and HER2 ne receptors, the proportion of positive staining as a numerical variable with clinicopathologic tumour cells (expressed in percentage) and the factors. For all statistical analyses, a p≤0.05 was average intensity of staining were evaluated ba- considered significant. sed on Allred score method (20). Tumours were considered positive for ER and PR when at le- RESULTS ast 1% of the tumour cells showed unequivocal The present study was conducted on 311 breast nuclear staining according the American Society cancer patients with the mean age of 60.65 of Clinical and the College of Ameri- ±11.25 years and age range of 32 to 89 years. can Pathologists (ASCO/CAP) guidelines (21). Breast cancer was most common in postmeno- Interpretation of HER2 staining and scoring. pausal women, 256 (82.3%). The majority of the HER2 was scored according to the pattern of tumours ranged between 2 and 5 cm in size (pT2), membranous staining and percentage of stained 167 (53.7%). Invasive ductal carcinoma was seen malignant cells. HER2 staining was scored from 0 in 299 (73.6%), lobular carcinoma in 34 (10.9%) to 3+ (Hercep Test score) (according to the manu- and other types in 48 (15.4%) patients (Table 1). facturer) as follows: 0 - no staining or faint incom- Table 1. Clinicopathological characteristics of 311 patients plete staining in <10% of cells; 1- faint incomplete with breast cancer staining in >10% of cells; 2 - weak to moderate Variables Value complete staining in >10% of cells; 3 - strong Mean age (years) (±SD) 60.65 ±11.25 Menopausal status (No, %) complete staining in >10% of cells. In categorical Premenopausal 55 (17.7) scoring only score 3 was considered positive; if Postmenopausal 256 (82.3) Tumour size (AJCC) (No, %) IHC is 0 or 1+, the tumour was considered HER2 pT1 116 (37.3) pT2 167 (53.7) negative; samples with HER2 score of 2+ was pT3 16 (5.1) confirmed as HER2-negative or HER2-positive pT4 12 (3.9) Histological type (No, %) using chromogenic in situ hybridization (CISH). Ductal (NOS) 229 (73.6) Interpretation of Ki-67 staining and scoring. Lobular 34 (10.9) Other 48 (15.4) As Ki-67 is a nuclear protein, only nuclear stai- Nottingham grade (No, %) ning (plus mitotic figures stained with Ki-67) was G1 49 (15.8) G2 213 (68.5) incorporated into the Ki-67 score. The fraction of G3 49 (15.8) ER status (No, %) proliferating cells was based on a count of at least Negative 75 (24.1) 500 tumour cells. The Ki-67 proliferative index Positive 236 (75.9) PR status (No, %) for each of the cases provided Ki-67 results using Negative 91 (29.3) numerical and categorical scores. Numerical score Positive 220 (70.7) HER-2 score (No, %) in the range 0-100 corresponded to the percenta- 0 173 (55.6) ge of positive tumour cells (the Ki-67 values were 1 + 71 (22.8) 2 + 34 (10.9) expressed as the percentage of positive cells in 3 + 33 (10.6) each case). In categorical score, cases with ≥ 14% HER-2 status (No, %) Negative 257 (82.6) positive nuclei were classified as positive (high) Positive 54 (17.4) Ki-67 expression, and those with < 14% were cla- Ki-67 (categorical) (No, %) Low (< 14%) 155 (49.8) ssified as negative (low) Ki-67 expression. High (≥ 14%) 156 (50.2) Mean Ki-67 (numerical) (±SD) 24.12±26.82 Statistical analysis LN status (No, %) Negative 147 (47.3) Positive 164 (52.7) Patients and clinicopathological characteri- LVI (No, %) stics were evaluated using descriptive statistics. Absent 143 (46.0) Present 168 (54.0) The correlation between Ki-67 and HER2 as a AJCC, American Joint Committee on Cancer; NOS, not otherwise categorical variable with other clinicopatholo- specified; ER, estrogen receptor, PR, progesterone receptor; HER, gic parameters were evaluated using Pearson’s human epidermal growth factor; LN, lymph node status; LVI, lymp- hovascular invasion; χ² test and Spearman rank correlation test. Kru- skal-Wallis or Mann-Whitney U test were used The numerical score of Ki-67 ranged from 1 to to evaluate the difference in the continuous varia- 95% (mean 24.12±26.82%). The expression of

109 Medicinski Glasnik, Volume 18, Number 1, February 2021

Ki-67 as a numerical variable, showed a statisti- Categorical score of HER2 showed a statisti- cally significant correlation with tumour grade cally significant correlation with histologic (p=0.025), PR (p=0.034), categorical score of type (p=0.039), tumour grade (p=0.016), ER HER2 (p=0.0001). There were no correlations (p=0.002), PR (p=0.0001), age (p=0.025), as well between age, menopausal status, tumour size, LN as with categorical and numerical value of Ki-67 status, LVI, ER, and categorical HER2 (p>0.05). (p=0.0001 and p=0.0001, respectively). No sta- However, 155 (out of 311; 49.8%) patients were tistical differences in menopausal status, tumour in low, and 156 (50.2%) were in high Ki-67 expre- size, LN status and LVI were observed (Table 3). ssion group. High expression of Ki-67 was more frequent in the patients with high grade tumours, DISCUSSION but without statistical significance (p =0.069), and To the best of our knowledge, this is the first stu- showed correlation with HER2 status: categorical dy to compare the relationship between numeri- and numerical (p=0.001 and p=0.043, respecti- cal and categorical immunohistochemical score vely). Also, there was a significant negative linear of Ki-67 and HER2 with clinicopathological cha- correlation with PR (p=0.041) (Table 2). racteristics of breast cancer patients. However, Table 2. Correlation of categorical and numerical scores of the association of Ki-67 index with prognostic Ki-67 with clinicopathological characteristics of 311 patients parameters of BC has been extensively studied with breast cancer (7,22,23). The correlation of Ki-67 with clinico- Categorical Ki-67 Numerical Ki-67 Variable Correlation Correlation pathologic factors varied, although the purpose p p Coefficient Coefficient of these studies was the assessment of prognosis Age -0.017 0.768 -0.051 0.337 and predictive value determination (4,24,25). Menopausal status 0.044 0.443 0.002 0.957 pT (tumour size) 0.001 0.981 -0.025 0.659 The results of this study showed a correlation Histological type -0.083 0.145 -0.100 0.078 between the expression of Ki-67 (as numerical Tumour grade 0.103 0.069 0.127 <0.025 variable) and tumour grade, PR, and numerical LVI -0.029 0.607 -0.055 0.333 LN status -0.081 0.156 -0.077 0.174 score of HER2. However, no correlation was ob- ER -0.081 0.155 -0.066 0.248 served between Ki-67 index and age, menopausal PR -0.118 <0.037 -0.120 <0.034 status, tumour size, histologic type, ER, LN sta- HER2 categorical 0.219 <0.0001 0.240 <0.0001 HER2 numerical 0.115 <0.043 0.101 0.076 tus, LVI and categorical HER2. LVI, lymphovascular invasion; LN, lymph node status; ER, estrogen re- Recent studies have shown that absolute (nu- ceptor; PR, progesterone receptor; HER, human epidermal growth factor; merical) high expression of Ki-67 is associated The expression of HER2, as a numerical varia- with higher tumour size, higher LN status, higher ble, showed statistically significant correlation tumour grade, ER/PR negativity, HER2 and LVI with the tumour grade (p=0.038), PR (p=0.025) positivity (26,27). Our findings were not consi- and categorical Ki-67 (p =0.043). No association stent with the results of previous studies. These was found between numerical score of HER2 and discrepancies may be related to the patients and other clinicopathological parameters (Table 3). tumour heterogeneity. The mean Ki-67 score in Table 3. Correlation of categorical and numerical scores of the presented study was 24.31%, in contrast with human epidermal growth factor of receptor 2 (HER2) with clin- results of Sun et al., with 31.22% (26). icopathological parameters of 311 patients with breast cancer Categorical HER2 Numerical HER2 When Ki-67 was categorized into high (>14%) Variable Correlation Correlation and low (≤14%) level groups, a statistically si- p p Coefficient Coefficient gnificant association was revealed between Ki-67 Age -0.127- <0.025 -0.085 0.133 expression and HER2 status (numerical and cate- Menopausal status -0.010 0.860 -0.011 0.848 pT (tumour size) 0.022 0.697 0.061 0.283 gorical), and significant negative linear correlation Histological type -0.117 <0.039 -0.095 0.093 with PR. No significant correlation was observed Tumour grade 0.136 <0.016 0.118 <0.038 with the rest of the clinicopathologic parameters. LVI 0.014 0.804 0.035 0.544 LN 0.009 0.876 0.045 0.427 A number of previous studies have investigated ER -0.178 <0.002 -0.087 0.124 the correlation between Ki-67 and other clinico- PR -0.246 <0.0001 -0.127 <0.025 Ki-67 categorical 0.219 <0.0001 0.115 <0.043 pathological parameters, using Ki-67 as a catego- Ki-67 numerical 0.240 <0.0001 0.101 0.076 rical variable (23, 27-29), however, the findings HER, human epidermal growth factor; LVI, lymphovascular invasion; were controversial. The earliest study conducted LN, lymph node status; ER, estrogen receptor; PR, progesterone receptor;

110 Dorić et al. Immunohistochemical score of Ki-67 and HER2

in the United Kingdom, demonstrated a signifi- gnificance of HER2 overexpression (positivity). cant association between the Ki-67 index and the In all previous analyses, the prognostic value of histological grade, size and type of the tumours HER2 was determined using a categorical result. (30). A study that included a cohort of Pakistani In the presented study, HER2 overexpression was patients revealed a significant association betwe- statistically significant with respect to age, histo- en Ki-67 expression and tumour grade, PR, HER2 logical type, tumour grade, ER, PR, categorical and lymph node status (23). Alco et al. reported and numerical values ​​of Ki-67. No statistical the results of the largest study from Turkey in differences in menopausal status, tumour size, 2015 and revealed that the Ki-67 index correlated LN status and LVI were observed. positively with an increasing tumour size (28). In Numerous earlier researches have enrolled cases our study, an association was found between Ki- with HER-2 overexpression and reported their 67 level (numerical and categorical) and tumour correlation with a high tumour grade, absence of staging, but without statistical significance. This ER or PR expression and high Ki-67 (31, 37-40). correlation was demonstrated in many previous The coincidence of HER2 overexpression with studies (14, 27-27, 31). Consistent with the ob- Ki-67 high expression, PR and ER negativity in- servations of other studies (32-34) our observa- dicates that there may be some regulatory relati- tion that Ki-67 positivity leads breast carcinoma onship between HER2 and these genes in signal in progression to higher histological grade, impli- transduction pathways (39). Many studies have es that Ki-67 high expression promotes tumour reported that age is an independent factor for poor growth in breast cancer patients (34). prognosis in BC (41-43), which is consistent with Several methods for assessing HER2 status are the results of our study, but there are also studies currently available, and each method has its that did not confirm these findings (44). Moreo- proponents. Immunohistochemistry is the most ver, various studies have a lack of relationship frequently used, convenient and cost-effec- between histologic type and HER2 status, which tive initial test for HER2 protein expression. is inconsistent with our results (44,45). The results of immunohistochemistry are gene- In conclusion, the results of this study demon- rally divided into four scale scores (range 0 to strate that the categorical immunohistochemical 3+), depending on the percentage of positive score of HER2 provided a greater association tumour cells and staining intensity (numerical with clinicopathological characteristics than nu- score), then categorized into positive and nega- merical score of BC. This can be explained by tive (categorical score). HER-2 status is crucial equivocal HER2 +, which in the categorization is in the guidance of treatment decisions for the unequivocal and defined as positive or negative, use of trastuzumab and is becoming a standard by retesting using in situ hybridization methods. recommendation in the pretreatment work-up of Also, there were small differences found between patients with invasive breast cancer (35). We did the correlation of numerical and categorical valu- not find any data which investigate the correla- es of​​ Ki-67 with clinicopathological parameters. tion between numerical score of HER2 and cli- A better association was shown by using the nu- nicopathological parameters in literature except merical value of Ki-67 than by using categorical our results. We found that HER2 overexpression score applying cut-off value of 14%. This indica- correlated negatively with PR expression, while tes a necessity of new researches that would more correlating positively with the tumour grade and precisely determine the cut-off value for Ki-67. categorical Ki-67 positivity. No association was found between numerical score of HER2 and FUNDING other clinicopathological parameters. No specific funding was received for this study The overexpression or amplification of HER2 is an indicator of likelihood of response to an- TRANSPARENCY DECLARATION ti-HER2 therapies (36). This is the predictive si- Competing interests: None to declare.

111 Medicinski Glasnik, Volume 18, Number 1, February 2021

REFERENCES 1. Siegel R, Jemal A. Cancer Facts and Figures. Atlan- 13. Curigliano G, Burstein HJ, Winer EP, Gnant M, Du- ta: American Cancer Society, 2013. bsky P, Loibl S, Colleoni M, Regan MM, Piccart- 2. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Gebhart M, Senn HJ, Thürlimann B, André F, Ba- Jeffrey SS, Rees CA, Pollack JR, Ross DT, John- selga J, Bergh J, Bonnefoi H, Brucker SY, Cardoso sen H, Akslen LA, Fluge O, Pergamenschikov A, F, Carey L, Ciruelos E, Cuzick J, Denkert C, Di Leo Williams C, Zhu SX, Lønning PE, Børresen-Dale A, Ejlertsen B, Francis P, Galimberti V, Garber J, AL, Brown PO, Botstein D. Molecular portraits of Gulluoglu B, Goodwin P, Harbeck N, Hayes DF, human breast tumours. Nature 2000; 406:747-52. Huang CS, Huober J, Khaled H, Jassem J, Jiang Z, 3. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler Karlsson P, Morrow M, Orecchia R, Osborne KC, S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Pagani O, Partridge AH, Pritchard K, Ro J, Rutgers Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown EJT, Sedlmayer F, Semiglazov V, Shao Z, Smith I, PO, Botstein D, Lønning PE, Børresen-Dale AL. Toi M, Tutt A, Viale G, Watanabe T, Whelan TJ, Gene expression patterns of breast carcinomas dis- Xu B. De-escalating and escalating treatments for tinguish tumor subclasses with clinical implications. early-stage breast cancer: the St. Gallen Internati- Proc Natl Acad Sci USA 2001; 98:10869–74. onal Expert Consensus Conference on the Primary 4. Cuzick J, Dowsett M, Pineda S, Wale C, Salter J, Therapy of Early Breast Cancer 2017. Ann Oncol Quinn E, Zabaglo L, Mallon E, Green AR, Ellis IO, 2017; 28:1700-12. Howell A, Buzdar AU, Forbes JF. Prognostic value 14. Wiesner FG, Magener A, Fasching PA, Wesse J, of a combined estrogen receptor, progesterone re- Bani MR, Rauh C, Jud S, Schrauder M, Loehberg ceptor, Ki-67, and human epidermal growth factor CR, Beckmann MW, Hartmann A, Lux MP. Ki-67 as receptor 2 immunohistochemical score and compa- a prognostic molecular marker in routine clinical use rison with the Genomic Health recurrence score in in breast cancer patients. Breast 2009; 18:135–41. early breast cancer. J Clin Oncol 2011; 29:4273–8. 15. Tanei T, Shimomura A, Shimazu K, Nakayama T, 5. Soliman NA, Yussif SM. Ki-67 as a prognostic mar- Kim SJ, Iwamoto T, Tamaki Y, Noguchi S. Progno- ker according to breast cancer molecular subtype. stic significance of Ki-67 index after neoadjuvant Cancer Biol Med 2016; 13:496-504. chemotherapy in breast cancer. Eur J Surg On- 6. Dowsett M, Nielsen TO, A'Hern R, Bartlett J, Co- col 2011; 37:155–61. ombes RC, Cuzick J, Ellis M, Henry NL, Hugh 16. Bottini A, Berruti A, Bersiga A, Brizzi MP, Bruzzi P, JC, Lively T, McShane L, Paik S, Penault-Llorca Aguggini S, Brunelli A, Bolsi G, Allevi G, Generali F, Prudkin L, Regan M, Salter J, Sotiriou C, Smith D, Betri E, Bertoli G, Alquati P, Dogliotti L. Rela- IE, Viale G, Zujewski JA, Hayes DF; International tionship between tumour shrinkage and reduction Ki-67 in Breast Cancer Working Group. Assessment in Ki67 expression after primary chemotherapy in of Ki67 in breast cancer: recommendations from the human breast cancer. Br J Cancer 2001; 85:1106–12. International Ki67 in Breast Cancer working group. 17. Sinn HP, Kreipe H. A brief overview of the WHO J Natl Cancer Inst 2011; 103:1656-64. classification of breast tumors, 4th edition, focusing 7. Hashmi AA, Hashmi KA, Irfan M, Khan SM, Edhi on issues and updates from the 3rd Edition. Breast MM, Ali JP, Hashmi SK, Asif H, Faridi N, Khan A. Care (Basel) 2013; 8:149-54. Ki67 index in intrinsic breast cancer subtypes and its 18. Amin M B, Edge S, Greene F, Byrd DR, Brookland association with prognostic parameters. BMC Res RK, Washington MK, Gershenwald JE, Compton Notes 2019; 12:605. CC, Hess KR, Sullivan DC, Jessup JM, Brierley JD, 8. Ménard S, Tagliabue E, Campiglio M, Pupa SM. Gaspar LE, Schilsky RL, Balch CM, Winchester DP, Role of HER2 gene overexpression in breast carci- Asare EA, Madera M, Gress DM, Meyer LR. AJCC noma. J Cell Physiol 2000; 182:150–62. Cancer Staging Manual. 8th ed. Chicago: Springer 9. Harari D, Yarden Y. Molecular mechanisms un- International Publishing AG, 2018. derlying ErbB2/HER2 action in breast cancer. On- 19. Elston CW, Ellis IO. Pathological prognostic factors cogene 2000; 19:6102–14. in breast cancer. I. The value of histological grade in 10. Lebeau A, Deimling D, Kaltz C, Sendelhofert A, breast cancer: experience from a large study with long- Iff A, Luthardt B, Untch M, Löhrs U. Her-2/neu term follow-up. Histopathology 1991; 19:403-10. analysis in archival tissue samples of human bre- 20. Remmele W, Stegner HE. Recommendation for uni- ast cancer: comparison of immunohistochemistry form definition of an immunoreactive score (IRS) and fluorescence in situ hybridization. J Clin Oncol for immunohistochemical estrogen receptor detec- 2001; 19:354–63. tion (ER-ICA) in breast cancer tissue. Pathologe 11. Varga Z, Noske A. Impact of modified 2013 ASCO/ 1987; 8:138–40. CAP guidelines on HER2 testing in breast Cancer. 21. Deyarmin B, Kane JL, Valente AL, van Laar R, One Year Experience PLoS One 2015; 10:0140652. Gallagher C, Shriver CD, Ellsworth RE. Effect of 12. Coates A. S, Winer E. P, Goldhirsch A, Gelber R. ASCO/CAP guidelines for determining ER status on D, Gnant M, Piccart-Gebhart M, Thürlimann B, molecular subtype. Ann Surg Oncol 2013: 20:87-93. Senn H-J and Panel Members. Tailoring therapies– improving the management of early breast cancer: St Gallen International Expert Consensus on the Primary Terapy of Early Breast Cancer. Ann Oncol 2015; 26:1533–46.

112 Dorić et al. Immunohistochemical score of Ki-67 and HER2

22. Luporsi E, André F, Spyratos F, Martin PM, Jacque- 33. Shapochka DO, Zaletok SP, Gnidyuk MI. Relati- mier J, Penault-Llorca F, Tubiana-Mathieu N, Sigal- onship between NF-kappaB, ER, PR, Her2/neu, Zafrani B, Arnould L, Gompel A, Egele C, Poulet Ki67, p53 expression in human breast cancer. Exp B, Clough KB, Crouet H, Fourquet A, Lefranc JP, Oncol 2012; 34:358-63. Mathelin C, Rouyer N, Serin D, Spielmann M, Hau- 34. Nishimura R, Osako T, Okumura Y, Hayashi M, gh M, Chenard MP, Brain E, de Cremoux P, Bellocq Toyozumi Y, Arima N. Ki-67 as a prognostic marker JP. Ki-67: level of evidence and methodological con- according to breast cancer subtype and a predictor of siderations for its role in the clinical management of recurrence time in primary breast cancer. Exp Thera- breast cancer: analytical and critical review. Breast peutic Med 2010; 1:747-54. Cancer Res Treat 2012; 132:895-915. 35. Mendoza G, Portillo A, Olmos-Soto J. Accurate bre- 23. Haroon S, Hashmi AA, Khurshid A, Kanpurwala ast cancer diagnosis through real-time PCR her-2 MA, Mujtuba S, Malik B, Faridi N. Ki67 index in gene quantification using immunohistochemically- breast cancer: correlation with other prognostic mar- identified biopsies. Oncol Lett 2013; 5:295-8. kers and potential in pakistani patients. Asian Pac J 36. Wolff AC, Hammond ME, Schwartz JN, Hagerty Cancer Prev 2013; 14:4353–58. KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons 24. Denkert C, Loibl S, Müller BM, et al. Ki67 levels as PL, Hanna WM, Langer A, McShane LM, Paik S, predictive and prognostic parameters in pretherape- Pegram MD, Perez EA, Press MF, Rhodes A, Stur- utic breast cancer core biopsies: a translational in- geon C, Taube SE, Tubbs R, Vance GH, van de Vij- vestigation in the neoadjuvant GeparTrio trial. Ann ver M, Wheeler TM, Hayes DF; American Society of Oncol 2013; 24:2786–93. Clinical Oncology; College of American Pathologi- 25. Aman NA, Doukoure B, Koffi KD, Koui BS, Traore sts. American Society of Clinical Oncology/College ZC, Kouyate M, Toure I, Effi AB. Immunohistoche- of American Pathologists guideline recommendati- mical evaluation of Ki-67 and comparison with cli- ons for human epidermal growth factor receptor 2 te- nicopathologic factors in breast carcinomas. Asian sting in breast cancer. J Clin Oncol 2007; 25:118-45. Pac J Cancer Prev 2019; 20:73-9. 37. Shokouh TZ, Ezatollah A, Barand P. Interrelati- 26. Sun J, Chen C, Wei W, Zheng H, Yuan J, Tu YI, Yao onship Between Ki67, HER2/neu, p53, ER, and PR F, Wang L, Yao X, Li J, Li Y, Sun S. Associations and Status and Their Associations with Tumor Grade indications of Ki67 expression with clinicopatholo- and Lymph Node Involvement in Breast Carcinoma gical parameters and molecular subtypes in invasive Subtypes. Medicine (Baltimore) 2015; 94:1359–64. breast cancer: A population-based study. Oncol Lett 38. Liu Z, Zhang C, Zhuo P, He K, Wang X, Yu Q, Huo 2015; 10:1741-48. Z, Wang F, Yu Z. Characteristic of ER+/PR- and 27. Kanyılmaz G, Yavuz BB, Aktan M, Karaağaç M, Ki67 value with breast cancer. Int J Clin Exp Med Uyar M, Fındık S. Prognostic importance of Ki-67 in 2017; 10:3533-9. breast cancer and its relationship with other progno- 39. Ding L, Zhang Z, Xu Y, Zhang Y. Comparative stu- stic factors. Eur J Breast Health 2019; 15:256-61. dy of Her-2, p53, Ki-67 expression and clinicopat- 28. Alco G, Bozdogan A, Selamoglu D, Pilanci KN, Tu- hological characteristics of breast cancer in a cohort zlali S, Ordu C, Igdem S, Okkan S, Dincer M, Demir of northern China female patients. Bioengineered G, Ozmen V. Clinical and histopathological factors 2017; 8:383-92. associated with Ki-67 expression in breast cancer 40. Soliman NA, Yussif SM. Ki-67 as a prognostic mar- patients. Oncol Lett 2015; 9:1046-54. ker according to breast cancer molecular subtype. 29. Kilickap S, Kaya Y, Yucel B, Tuncer E, Babacan Cancer Biol Med 2016; 13:496-504. Akgul N, Elagoz S. Higher Ki67 expression associa- 41. AlZaman AS, Mughal SA, AlZaman YS, AlZaman tes with unfavorable prognostic factors and shorter ES. Correlation between hormone receptor status survival in breast cancer. Asian Pac J Cancer Prev and age, and its prognostic implications in bre- 2014; 15:1381-85. ast cancer patients in Bahrain. Saudi Med J 2016; 30. Pinder SE, Wencyk P, Sibbering DM, Bell JA, El- 37:37-42. ston CW, Nicholson R, Robertson JF, Blamey RW, 42. Lobbezoo DJ, van Kampen RJ, Voogd AC, Dercksen Ellis IO. Assessment of the new proliferation mar- MW, van den Berkmortel F, Smilde TJ, van de Wouw ker MIB1 in breast carcinoma using image analysis: AJ, Peters FP, van Riel JM, Peters NA, de Boer M, associations with other prognostic factors and survi- Borm GF, Tjan-Heijnen VC. Prognosis of metasta- val. Br J Cancer 1995; 71:146-9. tic breast cancer subtypes: the hormone receptor/ 31. Inwald EC, Klinkhammer-Schalke M, Hofstädter HER2-positive subtype is associated with the most F, Zeman F, Koller M, Gerstenhauer M, Ortmann favorable outcome. Breast Cancer Res Treat 2013; O. Ki-67 is a prognostic parameter in breast cancer 141:507-14. patients: results of large population-based cohort 43. Gabriel CA, Domchek SM. Breast cancer in young of a cancer registry. Breast Cancer Res Treat 2013; women. Breast Cancer Res 2010; 12:212. 139:539-52. 44. Liu X, Zheng Y, Qiao C, Qv F, Wang J, Ding B, Sun 32. Zhou SJ, Guo H. Ki-67 expression and significance Y, Wang Y. Expression of SATB1 and HER2 in bre- of different molecular subtypes of breast invasive ast cancer and the correlations with clinicopatholo- ductal carcinoma. Zhonghua Yi Xue Za Zhi 2013; gic characteristics. Diagn Pathol 2015; 10:50. 93:2895-7. 45. Aman NA, Doukoure B, Koffi KD, Koui BS, Traore ZC, Kouyate M, Effi AB. HER2 overexpression and correlation with other significant clinicopathologic parameters in Ivorian breast cancer women. BMC Clin Pathol 2019; 17:19:1.

113 ORIGINAL ARTICLE

Posthumous sperm retrieval: a procreative revolution

Francesca Negro1, Renata Beck2, Antonella Cotoia2, Maria Cristina Varone1

1Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Rome, 2Anesthesia and Intensive Care Unit, Department of Medical and Surgical Sciences, University of Foggia, Policlinico "AUO Riuniti", Foggia; Italy

ABSTRACT

Aim Postmortem sperm retrieval with consequent artificial inse- mination has become a technically possible option for future use in assisted reproductive technology (ART). The authors have set out to discuss the social and ethical significance of posthumous sperm retrieval, and the laws currently in force in Italy, the United States and elsewhere.

Methods International literature from 1997 to 2020 has been reviewed from Pubmed database, Google Scholar and Scopus, drawn upon American, Italian and international sources (an ethi- cally acceptable solution can only be achieved through an over- haul of the laws currently in effect). One of the most contentious issues was about donor consent. In Italy, a donor's will to retrieve Corresponding author: his sperm in the event of premature disappearance can be proven Renata Beck according to the Law 219/2017, through advance health care di- Anaesthesia and Intensive Care Unit, rectives. Department of Medical and Surgical Sci- Results A substantial increase, both in requests and protocols, was ences, University of Foggia, documented in the United States. In Italy, over the last two years, Policlinico "AUO Riuniti" three rulings were issued concerning posthumous insemination. Viale Pinto 1, 71100 Foggia, Italy However, no official standardized protocols, guidelines or targeted Phone: +39 0881 732 387; legislation exist at the national level to regulate medical activity in E-mail: [email protected] that realm, whereas established laws often set implicit limitations. Francesca Negro ORCID ID: https://orcid. Conclusion Current legal frameworks appear to be inadequate, org/0000-0003-2966-1112 because in most cases they were conceived under conditions that have radically changed. The need for newly-updated regulatory frameworks to promptly bridge that gap is increasingly clear, if current social needs related to reproductive rights are to be met in Original submission: the foreseeable future. 21 August 2020; Key words: ART, ethics, gamete supply, posthumous reproducti- Revised submission: on, posthumous sperm use 01 October 2020; Accepted: 29 October 2020 doi: 10.17392/1256-21

Med Glas (Zenica) 2021; 18(1):114-121

114 Negro et al. Posthumous sperm retrieval

INTRODUCTION partner; however, in order to proceed it is nece- ssary to obtain a permission by a Court, which will The innovations in the field of Medically Assisted evaluate the requests on a case-by-case basis (10). Procreation (MAP) and the success that modern in- semination techniques have had in terms of treating In the United States, although there is no go- couple sterility meant that the posthumous sperm vernment regulation on PSR, several scientific so- retrieval and the ensuing production of embryos cieties have weighed in on the subject. The Ameri- have become reality. The first instance of posthu- can Society for states: "A mous sperm retrieval (PSR) occurred in 1980, when spouse’s request that sperm or ova be obtained ter- a 30-year-old man was declared brain dead after a minally or soon after death without the prior con- car accident and his family asked for his sperm to sent or known wishes of the deceased spouse need be preserved (1). Between 1980 and 1995, Kerr et not be honored". (11). Instead, the 2006 Universal al. (2) reported a total of 82 such requests in 40 fa- Anatomic Gift Act allows the recovery of organs cilities in the United States. None of these facilities and tissues after death upon consent of the closest had established protocols or guidelines in place for relative, unless there is evidence that the deceased PSR, while the first case of a child born following would not have consented (12). posthumous conception took place in 1999 (2). In Italy PSR is prohibited, under articles 5 and From 1997 to 2002, Hurtwitz et al. (3), noticed a 12, 2nd subsection of Law 40/2004, unless the 60% increase in PSR requests in the United States, application of the reproductive technique has with the number of approved requests also growing already led to the formation of embryos and the by 68%, in lockstep with the improvement of assi- implantation is allowed in order to uphold "the sted reproductive technologies (ART) (3). rights of all the subjects involved, including the The methods mainly used for PSR of the sperm conceived", as stated in Art.1 of the Law n.40, samples include the direct recovery from the epi- according to the rules laid out in Article 6 of the didymis: through percutaneous epididymal sperm same legislation (13). aspiration (PESA) or microsurgical epididymal Many countries do not have standardized proto- sperm aspiration (MESA). Other methods entail cols yet, and the existing ones differ significantly the collection of testicular tissue and/or the remo- from each other, particularly concerning evidence val of the testicles. The sperm should be recovered requirements of prior consent, waiting periods be- within 24-36 hours of death to ensure gamete via- fore being allowed to use the sperm sample, retrie- bility. Following recovery, the sperm is generally val methods, preservation-related logistic aspects, cryopreserved in a sperm bank until its use (4). and financial costs of the procedure (14,15). There are numerous ethical implications associated The aim of this study was to map out and expo- with PSR, including concerns about informed con- und upon the legal, ethical and social implicati- sent, the rights of the deceased, the interests of the ons which PSR entails, through an analysis of se- requesting party, the best interests of the minor (5), lected relevant cases and judicial and legislative as well as the underlying delicate position of both approaches. Given the complexities and challen- doctors and fertility clinics (6,7). Such multifaceted ges that arise when competing interests and rights challenges have led many countries to enact strict are at stake, it is worth exploring all aspects re- regulations on PSR. France, Canada, Germany, lative to PSR and the various approaches taken Sweden and some states in Australia have banned over the years by scientific societies, regulatory PSR altogether, regardless of consent (8). In the bodies and courts. This article was in fact meant United Kingdom, the deceased is required to have as a starting point for a broader discussion of the signed valid informed consent documentation in practical role, ethical significance, social value of order for his sperm to be legally retrieved. Moreo- PSR, and of the laws currently in force in Italy, ver, according to the Human Fertilization and Em- the United States and other countries. bryology Act of 1990, it is illegal in the UK to store MATERIALS AND METHODS sperm without a signed consent from the donor (9). In Israel, a twofold protocol has been enacted, Materials and study design according to which sperm can be recovered by a A systematic review of studies published on sci- living man or after his death at the request of his entific databases from 1997 to 2020 has been

115 Medicinski Glasnik, Volume 18, Number 1, February 2021

carried out in order to investigate and shed light sulting in litigation at times, given the conflicting on the current state of ART techniques, and to interests of the actors involved: intended parents, compare them to the current laws and guidelines donors, children and health care professionals and governing MAP, including PSR, in different co- facilities; the bone of contention is not only legal untries. The study was conducted over the years in nature, but social and anthropological as well, 2019-2020 at the University of Rome “Sapien- since it has to do with how societies progress and za”, Department of Anatomical, Histological, Fo- evolve, particularly with respect to family structu- rensic and Orthopaedic Sciences, in a collaborati- re; such major changes often outpace legislators, on with the Department of Medical and Surgical thus giving rise to a vacuum which may result in Sciences, University of Foggia. individual rights being put in jeopardy.

Methods RESULTS Pubmed, Google Scholar, Scopus, as well as legal The research analysis conducted has laid bare a databases (Lexis, Justia, Kleagle) were reviewed lack of uniformity and homogeneity in the way by applying various combinations of terms in the PSR is approached and regulated; that in turn re- following three categories. Search terms 1: "po- verberates on the rights and prospects of those sthumous", ‘’post-mortem", "deceased", "death", involved, who often have to rely on lengthy court "end of life"; search terms 2: "sperm", "gametes", proceedings and trials to have their rights upheld, "reproduction"; search terms 3: "policy", "proto- given the lack of clean-cut legislative framewor- col", "guidelines". The searches have produced ks in the countries herein analysed. In addition, 5640 results on Google Scholar, 166 publications such imbalances and discrepancies in the way the on Pubmed and 121 on Scopus. Only 64 sources issue is regulated negatively impact those who were ultimately deemed fit for the article’s main lack the financial means to travel abroad to co- purpose, i.e. those with direct correlation with ART untries with more permissive regulations in place and PSR techniques within the broader context of (so-called “procreative tourism”), adding to the policy-making, regulations and court rulings. sense of social inequality. From the standpoints of legislative and regulatory Some works, similar to protocol proposals, which initiatives targeted to PSR, Italian scientific pro- aim to standardize PSR procedures were found duction appears to be lagging behind compared (16). Most protocols call for the acquisition of to the overall international scenario: very few re- informed consent (17), in some cases directly in ports and recommendations have been issued by writing and signed by the donor (18), in other ca- Italian medical societies and bioethics committees ses as a verbal memory documented before the and scientific institutions. Hence, the main frame donor's death and defined as a verbal conversa- of reference is still the national legislation which tion with a doctor or another figure who must governs access to medically-assisted procreati- not be the beneficiary of the subject's provisions on procedures, whose effects have been herein (19). Some protocols admit the possibility of an highlighted through an analyses of relevant court implicit consent or the designation of a surrogate decisions. Besides, in order to better figure out decision maker, which could be a spouse/partner the relationship between Italy and PSR practices, or the closest relative (20). we have fine-tuned Google searches by using the The first case of international resonance was from following two categories: term 1 - "ruling", "Tri- 1997: a widow requested that sperm be retrie- bunal", "Supreme Court"; term 2 - "posthumous ved from the gonads of her late husband, brain fertilization", "end of life". The research produced dead because of a fulminant meningitis (21); her various commentaries on legal cases and topics request was initially granted (the sperm sample such as posthumous fertilization, but no result on was collected and stored). However, she was for- PSR. Relevant sources were sifted through and ced to file an application to the British Court of taken into account in terms of their relevance in Appeal in order to subsequently use this sample the broader analysis of PSR, within the realm of for conception. An oversight commission was medically-assisted procreation; MAP procedures, summoned, which did not prohibit the recovery particularly those which entail heterologous ferti- and storage of gametes, but nonetheless stated in lization, have often sparked controversy, even re- its decision: "The posthumous use of gametes is a

116 Negro et al. Posthumous sperm retrieval

practice which we feel should be actively discou- sted) party be associated with a "surrogate decision raged" (21). At the end, however, she obtained the maker", tasked with representing the deceased. The legal right to export the retrieved sperm to Belgi- Italian legal system, with the Englaro ruling, has um, where an artificial fertilization procedure was laid out for the first time a set of requirements ai- carried out. She managed to give birth to a viable med at defining the profile of the surrogate decision male child, who was the first baby born as a result maker, in order to identify the forms of assessment of PSR. In 1999, a woman became the first Ameri- best suited and/or respectful of the dying subject’s can to obtain sperm from her deceased husband for autonomy: the Court in fact believes that the substi- the purpose of fertilization. The sperm sample was tute judgment mechanism, which is consolidated in collected 30 hours after the death of her husband the US legal tradition, can be instrumental in uphol- and kept for 15 months. At the end of this peri- ding the self-determination of the incapable, clearly od of mourning, she underwent intracytoplasmic straying from the theory of best interest. Therefore, sperm injection (ICSI) and managed to give birth current legislation requires doctors to follow the to a baby girl (22). These two cases of considera- patient's wishes and, where necessary, binds them ble legal relevance were followed by others which to file an application to the Court in order to ascer- concerned the posthumous acquisition of sperm tain the patient's alleged ante-mortem wishes, if the material, which resulted in pregnancies (23,24). patient has not left advance directives, in order to From the women’s perspective, the issue of PSR relieve the medical staff of any responsibility (34). only concerns the use of those recovered during As for the children thus conceived, by definition, a life (25,26). On the other hand, for men, ethi- deceased sperm donor cannot be a social presence cal issues arise both from the recovery and the in the life of his child (5). Therefore, pursuing PSR post-mortem use of sperm, which translate into can be considered a choice mainly in favour of the the tortuous moral and legislative pathways, ne- requesting party and which, consequently, may cessary either with or without written informed not take into account all the needs for the care of consent by the deceased. the resulting child, for whom the possibility of ha- Recently, PSR has been the subject of a report by ving a second "social parent" is forgone (35, 36). the Ethics Committee of the American Society for Moreover, the right of donor-conceived children Reproductive Medicine. The document stresses that to know their biological donor parent cannot be the posthumous use of gametes for fertility tech- exercised for obvious reasons (37). niques is legally feasible only after a written docu- There are so few cases of PSR in the world that mentation is produced, reflecting the will of the it is not possible to establish with a reasonable deceased. In the absence of written documentation, degree of certainty what consequences could only requests made by the surviving spouse/partner affect the child. These methods could therefore should be considered, especially if they were filed be integrated with psychological follow-up pro- when both spouses were still alive (27,28). The ar- grams after childbirth, in order to best serve their gument for allowing the PSR is based on the rea- interests (38,39). sonably inferred concept of consent: that is, acting Given the lack of international protocols and/or on behalf of the deceased in a way that is logically guidelines defining the areas, procedures and ti- consistent with how he would have acted if he had mes to be implemented in such cases, doctors or been able to choose (17,29,30). From such perspec- fertility centres take on a highly controversial role tive, Italian law 91/99 on “tacit agreement” could indeed. In fact, PSR is not a part of the essential be applied (31). In light of a statement issued by the procedures of the care process; on the other hand, Italian Ministry of Health on 20 August 2019, in the doctor who decides to carry out this proce- the absence of an explicit refusal in life, organs and dure becomes morally responsible for upholding other tissues can be used in transplants after death the rights of the deceased in terms of procreation. (32). It is worth bearing in mind, however, that PSR Hence, doctors should not be bound to meet such use may also be motivated by personal interests, requests (40, 41). In addition, a sentient new life e.g. financial ones related to future inheritance (33). could result from the doctor's decision to proceed Since reproductive decisions are highly personal with PSR. According to the ASRM, "a pre-embryo in nature, it is essential that the requesting (intere- deserves greater respect than that of another hu-

117 Medicinski Glasnik, Volume 18, Number 1, February 2021

man tissue because of its potential for life, but less DISCUSSION respect than that accorded actual persons" (42). In recent years, demand for PSR procedures has The safety of PSR is another aspect of fundamen- risen, along with the development of ART met- tal importance. The sperm sample collected po- hods. The legislative statutes that regulate such sthumously, as well as that collected from a living practices are relatively few. In the US states of donor, should undergo some screening protocols North Dakota and Virginia, the provisions of the to minimize the risk of disease transmission or Uniform Status of Children of Assisted Concep- infections, and to ensure the good health of the tion Act of 1988 are partially adopted, by which mother and foetus. Although PSR should be carri- a person may not be legally viewed as a parent if ed out within a short period following death (from his sperm or oocyte has been used to conceive a a urological point of view, sperm can be collected child after his death; the same reasoning appli- within 36 hours after death) (43), the actual use of es to cryopreserved embryos prior implantation. sperm for procreation should be delayed to allow The same view is shared by the Uniform Parenta- for a mourning period. Most centres with existing ge Act of 2000 (46). protocols suggest a period of time from 6 months The American Bar Association Model Act has to 1 year to complete appropriate medical scree- provided a further limitation to the concept of ning procedures and psychological vetting (16). posthumous legal parent, establishing that if ga- These reasons, combined with the unstable emo- metes (either female or male) are used for repro- tional circumstances stemming from the death of ductive purposes after 3 years from the death of a spouse, would recommend a 6-month waiting the donor, the donor cannot be legally recognized period for the use of the cryopreserved sample in as parent (47). Furthermore, according to a 1985 assisted procreation procedures. decree by the New York State Task Force on Life Another aspect that has stood out from the lite- and the Law, a child does not have any inheritan- rature herein examined, and which could encou- ce rights unless the deceased has expressly left rage the use of cryopreserved posthumous sperm specific provisions in his will (48,49). taken from one's partner, has to do with the po- In the United States, therefore, the absence of ssible medico-legal litigation related to the degree protocols and/or regulations recognized at the of reliability of fertility clinics, which the woman national level means that jurisprudence guides would have to contact for ART practices where it the legal framework in case of PSR requests, as is not allowed to use her own partner's sperm (44). demonstrated by a judgment from the Massachu- There have been cases that have cast doubt on the setts Supreme Court issued in January 2002, the role of clinics and sperm banks. A case dates back first ruling in the United States on PSR, in which to 2015, when a Canadian couple of two women the Social Security Commissioner established that approached an American sperm bank (Xytex) and children born using posthumous gametes are to all selected a sperm donor. Seven years after the birth intents and purposes legal heirs (36,50), meaning of the child, the couple received the name of the that a child born through PSR methods, in order to donor, which allowed them to identify him as a be considered in all respects a legal heir, must be schizophrenic who had dropped out of university genetically related to the deceased and the decea- and had just been accused of burglary. In March sed must have consented to the conception of the 2015, the Canadian couple filed a lawsuit against child prior to his death and committed himself to the sperm-supplying bank, which was however di- supporting it. The ruling also noted that the child smissed by a judge from Fulton County, Georgia born through PSR has the same legal succession (45). Another egregiously important aspect that rights as the heirs already born. This ruling can be may be inferred from the Canadian affair is the considered by any measure the milestone to better growing difficulty in guaranteeing sperm donor define the inheritance rights for children concei- anonymity; that is partly due to the amount of in- ved through posthumous sperm retrieval. formation which can be gathered through the new methods of DNA identification (7), as well as the In Italy, in order to access medically assisted pro- possible issues arising from the use of sperm from creation, both partners in a couple must be alive. an unknown subject. Doubts have arisen as to whether art. 5 of the l. 40/2004 refers only to the time of the request for

118 Negro et al. Posthumous sperm retrieval

access to the reproductive technique or if, on the related to reproductive issues are to be met in the contrary, it should be construed as valid for the foreseeable future (62). entire duration of the procedure and up to the mo- Still, there are no official laws regulating the use ment of conception. of posthumous reproductive technologies at the in- At any rate, post-mortem procreation is made po- ternational level; some countries, apparently more ssible by cryopreservation, which can affect both responsive to this need, have enacted formal sets gametes and embryos already formed in a test of regulations (63,64). Nonetheless, the growing tube. Numerous ethical questions arise regarding number of requests for these procedures around embryo selection, genetic repairs of human ge- the world has brought to the forefront the issue of nome, such as the recent human embryo-editing reconciling PSR practices with the legal and ethi- experiments (51,52), all of which could signifi- cal landscape of the reference country (65). cantly affect the development and evolution of hu- In Italy, the situation is remarkably different from manity itself (53-57), while posing new, extremely the international one. The inertia of the Italian legi- challenging ethical and legal quandaries (58). slative and judicial system rests upon a cultural, so- Italian Law 40/2004 allows for the cryopreser- cial and religious subtext that strongly affects and vation of gametes, both male and female (Article influences bioethical thought and policy-making 2), while it directly bans it for embryos, except initiatives. Such an ecosystem makes the Italian for those cases where it is necessary, according to legislative system obsolete, unable to keep up with the specialists, “for serious and documented for- the rate of scientific progress in the ART field, as ce majeure stemming from the woman’s health demonstrated by the inherent contradictions of the condition, which was not foreseeable at the time current legal regulations regarding PMA (66) and of the treatment" (Art. 14) (13). In this scenario, by the most recent judgments of the Italian Supre- PSR for procreation purposes may run counter to me Court (67), which has recently ruled on the con- the right to self-determination and reproductive troversial topic of "posthumous" medically assisted freedom protected by Italian law. The fundamen- procreation. Furthermore, as widely highlighted, it tal issue of PSR aimed at ensuring the production is of utmost importance to be able to prove patient of an embryo to be implanted into the womb of a consent in life to posthumous procreation. widowed woman. Of course, there are many con- Therefore, in accordance with the 2017 Law 219, sequent ethical, logistical and legal implications; it is necessary to foster information and raise awa- yet, it is important to take into consideration the reness, particularly among new generations of ci- issue as a logical achievement, within the fra- tizens, of ART-related issues, and what these entail mework of a trend already established at both the in terms of opportunities for couples, in order to international and Italian level (59,60). overcome ethical controversies and expedite the We can conclude that heterologous fertilization bureaucratic procedures in case of PSR request. techniques have radically changed the traditional The presence of consent granted during life sho- notion of family (61). The conventional family uld be the first step towards a greater degree of structure can in fact no longer be considered as uniformity and harmonization of the legal rules ai- the only one capable of providing children with med at regulating and governing MAP practices in balance, a favourable environment in which Italy. By virtue of the inalienable right of surviving to grow, as validated abroad in various court spouses to exercise their reproductive freedom and judgments that recognized as families same-sex fulfil the wish of the deceased spouse to become unions with children born to homosexual pa- a father, PSR should be effectively regulated by rents through ART. Our research has laid bare a striking a balance between law and ethics with the widespread and substantial inadequacy of legal involvement of all stakeholders, no matter how frameworks and statutes, which in most cases challenging and controversial that may be. are conceived and modelled according to social and scientific conditions that have rapidly and FUNDING radically changed. The need for regulatory fra- No specific funding was received for this study meworks to promptly bridge that gap is increa- TRANSPARENCY DECLARATION singly clear and urgent, if the new social needs Conflict of interest: None to declare.

119 Medicinski Glasnik, Volume 18, Number 1, February 2021

REFERENCES 1. Rothman CM. A method for obtaining viable sperm 20. Collins R. Posthumous reproduction and the pre- in the postmortem state. Fertil Steril 1980; 34:512. sumption against consent in cases of death caused 2. Kerr SM, Caplan A, Polin G, Smugar S, O’Neill K, by sudden trauma. J Med Philos 2005; 30:431-42. Urowitz S. Postmortem sperm procurement. J Urol 21. Shenfield F. Consent and intent in assisted reproduc- 1997; 157:2154-58. tion. Law Med 2000; 3:317-25. 3. Hurwitz JM, MacDonald JA, Lifshitz LV, Batzer FR, 22. Planchon S. Comment the application of the dead Caplan A. Posthumous sperm procurement: an upda- man’s statutes in family law. Journal of American te. Fertil Steril 2002; 78(suppl 1):S2-42. Academy of Matrimonial Lawyers 2001; 13:561-77. 4. Jequier AM, Zhang M. Practical problems in the 23. Check M, Summers-Chase D, Check JH, Choe J, posthumous retrieval of sperm. Hum Reprod 2014; Nazari A. Sperm extracted and cryopreserved from 29:2615-9. testes several hours after death results in pregnancy 5. Freeman T. Gamete donation, information sharing following frozen embryo transfer: case report. Arch and the best interests of the child: an overview of the Androl 1999; 43:235-7. psychosocial evidence. Monash Bioeth Rev 2015; 24. Belker AM, Swanson ML, Cook CL, Carrillo AJ, 33:45-63. Yoffe SC. Live birth after sperm retrieval from a 6. Robertson JA, Kempers RD, Cohen J, Haney AF, moribund man. Fertil Steril 2001; 76:841-3. Younger JB. Posthumous Reproduction. Fertility 25. Zaami S, Busardò FP. Elective egg freezing: can you and Reproduction Medicine. New York: Elsevier really turn back the clock? Eur Rev Med Pharmacol Science, 1998. Sci 2015; 19:3537-8. 7. Negro F, Varone MC, Del Rio A. Advances in 26. Wang A, Kumsa FA, Kaan I, Li Z, Sullivan E, Farqu- medically-assisted procreation technologies: can har CM. Effectiveness of social egg freezing: proto- malpractice claims and "reproductive damage" be col for systematic review and meta-analyses. BMJ identified. Clin Ter 2020; 171:e225-8. Open 2019; 9:e030700. 8. Jones S, Gillett G. Posthumous reproduction: con- 27. Zaami S, Stark M, Malvasi A, Marinelli E. Eggs sent and its limitations. J Law Med Ethics 2008; Retrieval. Adverse Events, Complications, and 16:279-87. Malpractice: A Medicolegal Perspective. In: Malva- 9. Bahadur G. Death and conception. Hum Reprod si A, Baldini D, Eds. Pick Up and Oocyte Manage- 2002; 17:2769-75. ment. Switzerland, Cham: Springer, 2020: 347-59. 10. Landau R. Posthumous sperm retrieval for the pur- 28. Ethics Committee of the American Society for Re- pose of later insemination or IVF in Israel: an et- productive Medicine. Posthumous retrieval and use hical and psychosocial critique. Hum Reprod 2004; of gametes or embryos: an Ethics Committee opini- 19:1952-6. on. Fertil Steril 2018; 110:45-9. 11. Ethics Committee of the American Society for Re- 29. Orr RD, Siegler M. Is posthumous semen retrieval et- productive Medicine. Posthumous reproduction. hically permissible? J Med Ethics 2002; 28:299-302. Fertil Steril 2004; 82(suppl 1):S260-2. 30. Panagiotopoulou N, Karavolos S. "Let Me Keep My 12. Spielman B. Pushing the dead into the next repro- Dead Husband's Sperm": Ethical Issues in Posthu- ductive frontier: post mortem gamete retrieval under mous Reproduction. J Clin Ethics 2015; 26:143-51. the uniform anatomical gift act. J Law Med Ethics 31. Law n. 91, enacted on 1st April 1999. Disposizioni in 2009; 37:331-43. materia di prelievi e di trapianti di organi e di tessuti. 13. Law 40/2004, enacted by the Italian Parliament http://www.parlamento.it/parlam/leggi/99091l.htm on 19th February, 2004. www.camera.it/parlam/ (09 September, 2020) leggi/04040l.htm (08 August 2016) 32. Italian Ministry of Health statement n. 110, issued 14. Finnerty JJ, Karns LB, Thomas TS, West RW, Pin- on 20th August, 2019. http://www.salute.gov.it/por- kerton JV. Gamete retrieval in terminal conditions: tale/news/p3_2_4_1_1.jsp?lingua=italiano&menu is it practical? What are the consequences? Curr Wo- =salastampa&p=comunicatistampa&id=5253 (09 mens Health Rep 2002; 2:174-8. September, 2020) 15. Tash JA, Applegarth LD, Kerr SM, Fins JJ, Ro- 33. Napoletano S, Del Rio A. Reproductive medicine senwaks Z, Schlegel PN. Postmortem sperm retrie- between advancement and ethics. Clin Ter 2018; val: the effect of instituting guidelines. J Urol 2003; 169:e108-9. 170:1922-5. 34. Supreme Civil Court – 1st Section. Ruling 16. Bahm SM, Karkazis K, Magnus D. A content n.21748/2007. Caso Englaro- Interruzione dei tratta- analysis of posthumous sperm procurement proto- menti e incapacità Sentenza di Cassazione. https:// cols with considerations for developing an institutio- www.aduc.it/generale/files/allegati/cassazione_en- nal policy. Fertil Steril 2013; 100:839-43. glaro.pdf (09 September, 2020) 17. Hostiuc S, Curca CG. Informed consent in posthu- 35. Kroon F. Presuming consent in the ethics of posthu- mous sperm procurement. Arch Gynecol Obstet mous sperm procurement and conception. Reprod 2010; 282:433-8. Biomed Soc Online 2016; 1:123-30. 18. Goulding EA, Lim BH. Life after death: posthumous 36. Tremellen K, Savulescu J. Posthumous conception sperm procurement. Whose right to decide? BJOG by presumed consent. A pragmatic position for a rare 2015;122:394. but ethically challenging dilemma. Reprod Biomed 19. Batzer FR, Hurwitz JM, Caplan A. Postmortem pa- Soc Online 2016; 3:26-9. renthood and the need for a protocol with posthumo- 37. Zaami S. Assisted heterologous fertilization and the us sperm procurement. Fertil Steril 2003; 79:1263-9. right of donor conceived children to know their bio- logical origins. Clin Ter 2018; 169:e39-43.

120 Negro et al. Posthumous sperm retrieval

38. Montanari VG, Marinelli E, di Luca NM, Zaami S. 53. Baldini D, Beck R, Negro F, De Viti D. Assisted Gamete donation: are children entitled to know their reproductive technologies and metabolic syndrome genetic origins? A comparison of opposing views. complications: medico-legal reappraisal. Clin Ter The Italian State of Affairs. Eur J Health Law 2018; 2019; 170:e364-7. 25:322–37. 54. Asplund K. Use of in vitro fertilization-ethical 39. Tremellen K, Savulescu J. A discussion supporting issues. Ups J Med Sci 2020; 125:192-9. presumed consent for posthumous sperm procure- 55. Baldini D, Savoia MV, Sciancalepore AG, Malvasi ment and conception. Reprod Biomed Online 2015; A, Vizziello D, Beck R, Vizziello G. High proge- 30:6-13. sterone levels on the day of HCG administration do 40. Montanari Vergallo G, Zaami S, Di Luca NM, Mari- not affect the embryo quality and the reproductive nelli E. The conscientious objection: debate on emer- outcomes of frozen embryo transfers. Clin Ter 2018; gency contraception. Clin Ter 2017; 168:e113-9. 169:e91-5. 41. Minerva F. Conscientious objection in Italy. J Med 56. Beck R, Brizzi A, Cinnella G, Raimondo P, Ethics 2015; 41:170-3. Kuczkowski KM. Anesthesia and Analgesia for Wo- 42. Ethical considerations of the new reproductive tech- men Undergoing Oocyte Retrieval. In: Malvasi A, nologies. Ethics Committee of The American Ferti- Baldini D, Eds. Pick Up and Oocyte Management. lity Society. Fertil Steril 1990; 53:1S-104S. Switzerland: Springer Cham, 2020: 99-119. 43. Simana S. Creating life after death: should posthu- 57. Ricci G, Campanozzi LL, Marinelli S, Midolo E, mous reproduction be legally permissible without Ruggeri L. The human embryo, subjectivity and le- the deceased's prior consent? J Law Biosci 2018; gal capacity. Notes in the light of art. 1 of the Italian 5:329-54. law on "medically assisted procreation". Clin Ter 44. Yoon M. The Uniform Status of Children of Assisted 2019; 170:e102-7. Conception Act: does it protect the best interests of 58. Jonlin EC. Informed Consent for Human Embryo the child in a surrogate arrangement? Am J Law Med Genome Editing. Stem Cell Reports 2020; 14:530-7. 1990; 16:525-3. 59. Marinelli S. Medically-assisted procreation and the 45. Brumback K. Judge tosses suit claiming sperm bank rise of off-center, new types of “parenthood”: it is misrepresented donor. The Associated Press. Publis- incumbent upon lawmakers to intervene. Clin Ter hed October 21st, 2015. (09 September 2020) 2019; 170:e241-4. 46. American Bar Association. National Conference of 60. Benagiano G, Filippi V, Sgargi S, Gianaroli L. Ita- Commissioners on Uniform State Laws. Uniform lian Constitutional Court removes the prohibition Parentage Act (Last Amended or Revised in 2002). on gamete donation in Italy. Reprod Biomed Online Approved by the American Bar Association Seattle, 2014; 29:662-4. Washington on February 10, 2003. https://legisla- 61. Marinelli S. No more only one mom? European ture.vermont.gov/Documents/2018/WorkGroups/ Court of Human Rights and Italian jurisprudences' House%20Judiciary/Bills/H.502/17-634%20Pa- ongoing evolution. Clin Ter 2020; 170:e36-43. rentage%20Committee%20Bill/W~Justice%20 62. Montanari Vergallo G, Zaami S, Bruti V, Signore Dooley~Uniform%20Parentage%20Act~2-23- F, Marinelli E. How the legislation on medically 2017.pdf (09 September 2020) assisted procreation has evolved in Italy. Med Law 47. Kindregan C, Snyder S. Clarifying the Law of ART: 2017; 36:5-28. The New American Bar Association Model Act Go- 63. Waler NJ, Clavijo RI, Brackett NL, Lynne CM, Ra- verning Assisted Reproductive Technology. Family masamy R. Policy on posthumous sperm retrieval: Law Quarterly 2008; 42:203-229. survey of 75 major Academic Medical Centers. Uro- 48. Katz, KD. Parenthood from the Grave: Protocols logy 2018; 113:45-51. for Retrieving and Utilizing Gametes from the Dead 64. Zinkel AR, Ankel FK, Milbank AJ, Casey CI, or Dying. University of Chicago Legal Forum: Vol. Sundheim JJ. Postmortem Sperm Retrieval in the 2006: Iss. 1, Article 11. http://chicagounbound.uchi- Emergency Department: A Case Report and Review cago.edu/uclf/vol2006/iss1/11 (09 September 2020 of Available Guidelines. Clin Pract Cases Emerg 49. Cummings BM, Paris JJ. From death to life: ethical Med 2019; 3:405-8. issues in postmortem sperm retrieval as a source of 65. Storrow RF. Judicial review of restrictions on game- new life. Camb Q Healthc Ethics 2020; 29:369-74. te donation in Europe. Reprod Biomed Online 2012; 50. Woodward v. Commissioner - 435 Mass. 536, 760 25:655-9. N.E.2d 257 (2002). https://casetext.com/case/wo- 66. Montanari Vergallo G, Zaami S, Sparic R. Medically odward-v-commissioner-of-social-security (09 Sep- Assisted Procreation: European Legislation and En- tember 2020) suing Ethical Issues. In: Malvasi A, Baldini D, Eds. 51. Kocher T, Wagner RN, Klausegger A, Guttmann- Pick Up and Oocyte Management. Switzerland: Gruber C, Hainzl S, Bauer JW, Reichelt J, Koller U. Springer Cham 2020: 361-73. Improved double-nicking strategies for COL7A1- 67. Supreme Civil Court – 1st Section. Ruling 13000/2019. editing by homologous recombination. Mol Ther https://www.altalex.com/documents/news/2019/05/20/ Nucleic Acids 2019; 18:496-507. fecondazione-omologa-post-mortem-trasmette-il-co- 52. Marinelli S, Del Rio A. Beginning of life ethics at gnome-del-padre (09 September 2020) the dawn of a new era of genome editing: are bio- ethical precepts and fast-evolving biotechnologies irreconcilable? Clin Ter 2020; 171:e407-11.

121 ORIGINAL ARTICLE

Serum total prostate-specific antigen (tPSA): correlation with diagnosis and grading of prostate cancer in core needle biopsy

Nina Čamdžić, Suada Kuskunović-Vlahovljak, Mirsad Dorić, Svjetlana Radović, Edina Lazović Salčin, Mirsad Babić

Department of Pathology, School of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina

ABSTRACT

Aim To investigate the impact of pre-treatment serum total pro- state-specific antigen (PSA) level on prevalence of prostate carci- noma detection in prostate core needle biopsy, and its correlation with established prognostic factors.

Methods Prostate needle biopsy samples of 115 patients with ava- ilable pre-treatment serum total PSA (tPSA) level were analysed. For all cases where morphology alone was insufficient, immuno- histochemistry was performed using p63, CKHMW and AMACR antibody panel in order to confirm or exclude the existence of pro- state carcinoma.

Results Statistically significant positive correlation between se- Corresponding author: rum total PSA values and prevalence of finding prostate carcinoma Nina Čamdžić in needle biopsy specimens was found (p=0.011), as well as in the Department of Pathology, case when the patients were classified into groups according to School of Medicine, tPSA levels (p=0.028). Serum total PSA values and levels (level University of Sarajevo groups) showed significant positive correlation with Gleason score Čekaluša 90, 71000 Sarajevo, (p=0.029 and p=0.036, respectively) and Grade Group of prosta- te carcinomas (p=0.044 and p=0.046, respectively). Sensitivity of Bosnia and Herzegovina the screening test by using 4 ng/mL as cut off value for tPSA was Phone: +387 33 226 478; 94.12% (CI: 80.32-99.28%), specificity 8.64% (CI: 3.55-17.00%), Fax: +387 33 666 964; positive predictive value 30.19% (CI: 21.65-39.87%) and negative E-mail: [email protected] predictive value 77.78% (CI: 39.99-97.19%). ORCID ID: https://orcid.org/0000-0002- Conclusion The increase of serum tPSA value increases the like- 0327-1151 lihood of finding prostate cancer on needle biopsy specimens. Due to such findings and its positive correlation with a grade of prostate cancer, our study indicates that tPSA can still be considered as Original submission: a useful tool both in detecting and predicting aggressiveness of 14 May 2020; prostate cancer. Revised submission: Keywords: grade group, Gleason score, screening 11 June 2020; Accepted: 24 July 2020 doi: 10.17392/1204-21

Med Glas (Zenica) 2021; 18(1):122-127

122 Čamdžić et al. PSA and prostate cancer

INTRODUCTION PATIENTS AND METHODS Prostate cancer remains a significant public he- Patients and study design alth problem, since it is the second most common male malignancy and the fifth major cause of de- In this retrospective study, 115 prostate needle bi- ath worldwide. It is responsible for 3.8% of all opsy specimens were analysed at the Department deaths caused by cancer in male population (1). of Pathology, School of Medicine, University of Although it is prostate-specific rather than di- Sarajevo in the five-year period (2015-2019). sease-specific, serum prostate-specific antigen The patients with available pre-treatment serum (PSA), a glycoprotein normally expressed by total PSA (tPSA) level were included in the stu- prostate tissue, has been a marker of choice for dy. All patients underwent digital rectal exami- early detection and follow up of patients with nation and transrectal utrasonography (TRUS). prostate carcinoma since its discovery in late Biopsies with inadequate material, other types 1980s (2). Since increased PSA serum levels can of procedures, such as transurethral resection of be found in other conditions, such as many beni- prostate (TURP) and partial or total prostatec- gn changes, urinary tract infections, or after the tomy specimens, were excluded from the study instrumentation, prostate needle biopsy repre- (despite known pre-treatment tPSA level). Pati- sents a gold standard for the diagnosis of prostate ents were divided into four subgroups according carcinoma (3). to total serum PSA level (Table 1). Despite the findings of different studies indica- Methods ting that PSA screening can help in early prostate cancer detection, there has been a lot of inconsi- Tissue specimens were fixed in 10% buffered stency about its clinical appliance as a screening formalin, paraffin-embedded, processed and sta- marker, especially in the last decade (4). Some of ined with hematoxylin and eosin. According to the reasons include a high rate of false positive current protocols, the Gleason score (7) and Gra- and negative results on needle biopsy and repea- de Group (8) were determined for all patients. ted unnecessary biopsy and delayed diagnosis (4). For all prostate biopsy specimens where morpho- Screening with serum PSA aims to detect prostate logy alone was insufficient, immunohistochemistry cancer at an early stage in order to enable adequate was performed using p63, CKHMW and AMACR treatment and to impact overall and disease-speci- antibody panel in order to confirm or exclude the fic mortality (5). Recent randomized clinical trials existence of prostate carcinoma. Tissue samples demonstrated that PSA screening has small benefit fixed in 10% formalin and embedded in paraffin in higher detection of low-risk prostate cancer, but were cut into 4-μm thick sections, mounted on co- do not support single PSA testing for population- ated slides and collected for immunohistochemical based screening (6); meta-analysis based studies staining, according to the manufacturer's protocol showed small benefit in the reduction of prostate with CKHMW (clone 34βE12, Dako; FLEX, Re- cancer specific mortality but not the overall mor- ady-to-Use, Glostrup, Denmark), p63 (clone DAK- tality, with current recommendations for clinicians p63, Dako, FLEX, Ready-to-Use, Glostrup, Den- and patients to outweigh benefits against harms of mark) and AMACR (clone 13H4, Dako, FLEX, PSA screening (5). Ready-to-Use Glostrup, Denmark). Although PSA is not a perfect marker and PSA Positive p63 staining was defined as dark brown testing has limited specificity for prostate cancer nuclear staining while positive CKHMW stai- detection, its appropriate clinical application re- ning was defined as dark brown cytoplasmic stai- mains a topic of debate (5). ning in basal cells of prostatic glands. To confirm This study is conducted to investigate a corre- foci of prostatic carcinoma, AMACR positivity lation between pre-treatment serum tPSA level was defined as dark brown cytoplasmic staining and incidence of finding prostatic carcinoma in in the absence of p63 and CKHMW positivity in prostate core needle biopsy, as well to correlate atypical glands. serum PSA levels with Gleason score and Grade All clinicopathological data are summarized in Group in cases of cancer presence. Table 1.

123 Medicinski Glasnik, Volume 18, Number 1, February 2021

Statistical analysis (range 55 to 86) compared to 81 (70.43%) pa- tients with benign prostatic hyperplasia with or Patient and clinicopathological characteristics without HGPIN lesions, 65.51±5.97 years (range were evaluated using descriptive statistics. Spear- 54 to 81) (p=0.096) (Table 1). man correlation test was used to investigate a po- ssible correlation between two variables. In cases Mean value of tPSA in patients with prostatic where normality of distribution lacked, non-para- carcinoma was 29.37 ng/mL (range 3.2-275.0), metric Spearman test was used and variables were while in patients without carcinoma it was presented by median and interquartile range. Posi- 12.82±13.51 (range 2.15-98.23) (p=0.011). tive (PPV) and negative predictive value (NPV), A difference in serum tPSA level in cancer pati- sensitivity and specificity were calculated using 4 ents compared to patients with benign prostate ng/mL as the cut-off value for total serum PSA. changes was found, i.e. the majority of patients p≤0.05 was considered statistically significant. with prostate adenocarcinoma, 20 (58.8%), had a tPSA level >10.0 ng/mL, unlike patients without RESULTS carcinoma whose tPSA level was predominantly Out of total 115 patients, in 81 (70.44%) benign between 4.0-9.99 ng/mL, 40 (49.4%). Statistically prostatic changes were found on core needle bi- significant positive correlation between serum opsy, while 34 (29.56%) patients were diagnosed tPSA levels and the prevalence of prostate ade- with prostate carcinoma. High grade prostatic in- nocarcinoma was observed (p=0.028) (Figure 1). traepithelial lesion (HGPIN) was present in ma- Sensitivity, specificity, positive and negative pre- jority of cases, both in patients with carcinoma, dictive value were calculated using tPSA cut-off 23 (20.0%), and in patients with benign prostatic changes, 58 (50.43%). Table 1. Clinicopathological characteristics of patients who underwent prostate needle biopsy No (%) of patients Variables With prostate Without prostate Total carcinoma carcinoma Prostate carcinoma 34 (29.57) 81 (70.43) 115 (100.0) Age (years) 50-59 8 (6.95) 8 (6.96) 16 (13.91) 60-69 16 (13.91) 50 (43.48) 66 (57.39) 70-79 12 (10.43) 18 (15.66) 30 (26.09) Figure 1. Difference in total prostate specific antigen (tPSA) 80-89 2 (1.74) 1 (0.87) 3 (2.61) level in patients with prostate carcinoma and patients with Mean age (years) 68.35±7.99 65.51±5.97 NA benign changes tPSA level (ng/mL) <4.0 2 (1.74) 7 (6.09) 9 (7.83) point of 4 ng/mL, showing sensitivity of 94.12% 4.0-9.99 12 (10.43) 40 (34.79) 52 (45.22) (CI= 80.32-99.28%), specificity 8.64% (CI=3.55- 10.0-19.99 7 (6.09) 22 (19.13) 29 (25.22) ≥20.0 13 (11.30) 12 (10.43) 25 (21.73) 17.00%), positive predictive value 30.19% (CI HGPIN = 21.65-39.87%) and negative predictive value Present 23 (20.0) 58 (50.43) 81 (70.43) 77.78% (CI = 39.99-97.19%). Absent 11 (9.57) 23 (20.0) 34 (29.57) Gleason score NA Gleason score was analysed in the group of pati- 3+3=6 13 (38.24) NA ents with prostatic adenocarcinoma, and showed 3+4=7 6 (17.56) NA 4+3=7 6 (17.56) NA positive correlation with serum values and levels 4+4=8 4 (11.76) NA (level subgroups of tPSA). In the group of pati- 5+5=10 5 (14.70) NA ents with Gleason score 6, mean value of tPSA Grade Group NA was 11.36 (range 3.67-37.93 ng/mL), while in 1 13 (38.24) NA 2 6 (17.65) NA patients with Gleason score 9 it was 149.61 (ran- 3 6 (17.65) NA ge 24.22-275.00) (p=0.029) (Figure 2). 4 4 (11.76) NA 5 5 (14.70) NA Gleason score 3+3=6 was the most represen- HGPIN, high grade prostatic intraepithelial neoplasia; NA, not applied ted score with most cases having tPSA below 10.0 ng/mL, while higher Gleason score 3+4=7, Mean age of the patients at the time of the diagno- 4+3=7, 4+5=9 and 5+4=9 were dominantly re- sis was 66.35±6.72 years (range 54 to 86). Pati- presented in the group of patients with tPSA level ents with prostate adenocarcinoma, 34 (29.57%) ≥20.0 ng/mL (p=0.036) (Table 2). were older with mean age of 68.35±7.99 years

124 Čamdžić et al. PSA and prostate cancer

Table 2. The incidence of different Gleason scores among different total prostate specific antigen (tPSA) level No (%) of tumours with different Gleason score tPSA level (ng/mL) 3+3=6 3+4=7 4+3=7 4+4=8 4+5=9 5+4=9 5+5=10 Total <4.0 1 (7.7) 0 0 1 (25.0) 0 0 0 2 (5.9) 4.0-9.99 7 (53.8) 1 (16.7) 2 (33.3) 1 (25.0) 0 0 1 (33.3) 12 (35.3) 10.0-19.99 4 (30.8) 1 (16.7) 1 (16.7) 0 0 0 1 (33.3) 7 (20.6) 20.0+ 1 (7.7) 4 (66.7) 3 (50.0) 2 (50.0) 1 (100.0) 1 (100.0) 1 (33.3) 13 (38.2) Total 13 (100.0) 6 (100.0) 6 (100.0) 4 (100.0) 1 (100.0) 1 (100.0) 3 (100.0) 34 (100.0) needle biopsy, whose mean age at the time of the diagnosis was 65.51±5.97 (range 54 to 81) (wit- hout statistically significant correlation between patients’ age and incidence of prostate carcino- ma). Slight differences may be caused by ethni- city, false negative or positive findings on core needle biopsy, and a small sample as well. Over the past years, serum PSA level has been a powerful tool for prostate cancer screening and early detection, especially in most Western coun- Figure 2. Values of serum total prostate specific antigen tries (10). Although an increased usage of PSA as (tPSA) according to Gleason score in patients with prostate carcinoma a screening marker has been followed by decrea- An increase of tPSA level with the increase of pro- sing prostate cancer mortality risk, it has also led state carcinoma Grade Group (GG) was found: the to over-diagnosis of many indolent tumours that highest percentage of GG 1 (61.5%) carcinomas would not have caused clinical disease (10,11). had tPSA level lower than 10 ng/mL, while 60% Despite many controversies in its clinical applian- of GG 5 carcinomas had tPSA level >20.0 ng/mL ce, PSA is one of the biomarkers with the greatest (p=0.044 and p=0.046 respectively) (Figure 3). impact on clinical practice and management (10). Results of many other studies in recent years have accumulated evidence of PSA as a predictive marker, with low PSA level (<1.0 ng/mL) having extremely low prevalence of clinically significant prostate cancer, as well as a very low risk for ad- vanced disease (12-14). In a study by Ghafoori et al. serum PSA level of 4 ng mL was found to be commonly used as an indication for prostate biop- sy (15); in our study biopsies were performed even at lower PSA values, presumably due to patients’ Figure 3. Serum total prostate specific antigen (tPSA) levels in clinical symptoms. Also, in the study by Ghafoori different Grade Groups of prostate carcinoma et al. it was found that PSA level between 4- 10 ng/ There was no statistically significant correlation mL had low sensitivity, unlike values above 10 ng/ between patients’ age and Gleason score, or pati- mL and 15 ng/mL which had much higher sensiti- ents’ age and Grade Group of prostatic adenocar- vity in detecting prostate cancer (15). Gerstenbluth cinoma (p>0.05). et al. showed that serum PSA level above 50 ng/ mL had 98.5% accuracy in predicting the presence DISCUSSION of prostate cancer in tissue biopsy (16). Our results It is well known that prostate cancer primarily are quite similar to these findings with highest can- affects elderly males with the incidence rate of cer prevalence in the group with PSA level ≥10.0 almost 60% in men older than 65 years. The pre- ng/mL (58.8%) as well as in the group with PSA valence and mortality of prostate cancer correlate level ≥ 20.0 ng/mL (38.6%). positively with age, with the average age of 66 In our study, out of 34 prostate cancer cases, only years at the time of the diagnosis (1, 9). In our 2 patients had tPSA level lower than 4 ng/mL, but study, mean age of patients with prostate ade- most of the patients without cancer also had the nocarcinoma was 68.35±7.99 (range 55 to 86) in same. These results indicate that serum total PSA comparison to patients without proven cancer on is sensitive, but not specific as a screening mar-

125 Medicinski Glasnik, Volume 18, Number 1, February 2021

ker, with the test accuracy of 33.9% when using The Grade Group system, introduced in 2013 4 ng/mL as cut-off value for total serum PSA. In and accepted later in 2014 (24) comprises 2018, the US Preventive Task Force reported that five Grade Groups (GG 1-5) that resulted in men aged 55-69 years had a potential benefit of more accurate prognosis in comparison with PSA screening due to reduced death rates (17), the Gleason system risk stratification groups. but other studies gave fewer encouraging results According to this grading system, prognostic for males over 70, for all races (18). Grade Group 1 includes all prostate cancers Gleason score, despite limitations and many with Gleason score 6 or less, which are indo- changes in the clinical and histological diagno- lent, lowest grade tumours with the best pro- sis of prostate cancer, remains one of the most gnosis. Prognostic Grade Group 4 and 5 have important predictors of biological behaviour of lower 5-year biochemical recurrence free pro- prostate carcinoma (7). Gleason scoring of pro- gression following radical prostatectomy and state carcinoma allows objective assessment of thus significantly worse prognosis (25,26). Our the degree of tumour differentiation reflecting its study showed a statistically significant positive aggressiveness and impacting a decision about correlation between serum tPSA level and GG treatment modalities (19). In our study, an addi- of prostate carcinoma, although not completely tional fact which favours PSA as a predictive linear, due to frequent high tPSA level (>20 ng/ marker is the statistically positive correlation of mL) in GG 2 prostate carcinomas. Considering tPSA level with Gleason score and Grade Group limitations such as small sample size and in- of prostate carcinoma, indicating that with the sufficiently examined “grey zone” of tumours increase of serum total PSA level, GS and GG with the medium level of serum tPSA, further of prostate carcinoma also increase. Although studies with larger sample size are imperative, the mean values were lower in Gleason score together with comparison of the efficacy of 10 (25.57 ng/mL) compared to Gleason score some other markers. 9, a statistically significant positive correlation Current recommendations indicate necessity of was noted. In our study, the most prevalent Gle- individualized approach to tPSA screening thus ason score was 6 (3+3=6) (38.24%) resulting in leaving plenty of space for new studies in this fi- 53.8% prevalence of tPSA <10.0 ng/mL, unlike eld. Our study indicates that tPSA can still be a the patients with high Gleason level 4+5=9 and useful screening marker for prostate carcinoma 5+4=9 which in 100% of cases had PSA serum in combination with digital rectal examination level of ≥20 ng/mL. Investigating a correlation and transrectal sonography (TRUS). Due to its between PSA density and features of aggressi- positive correlation with well-established pro- veness of prostate carcinoma, Kundu et al. fo- gnostic factors, serum tPSA should be seen as a und that PSA density correlated positively with continuum for recognizing the increasing risk of Gleason score and adverse pathologic features prostate malignancy and cancer aggressiveness. (20). Loeb et al. investigated PSA velocity in In conclusion, our findings confirm the importan- radical prostatectomy specimens and found pre- ce of serum tPSA in the detection of prostate car- operative PSA velocity as a significant indepen- cinoma in needle biopsy, as well as its prognostic dent predictor of Gleason score and non-organ significance along with Gleason score and Gra- confined disease (21). Our results relating to de Group. By reviewing available literature, we correlation of initial serum total PSA and Gle- have not found studies in Bosnia and Herzegovi- ason score are in contrast to the results of Nna- na which are related to tPSA levels and prostate bugwu et al. (19), who investigated correlation core needle biopsy specimens. of initial serum total PSA and Gleason score on 43 core needle biopsy specimens. Milonas et al. FUNDING (22) and Jayapradeep et al. (23) found no sta- No specific funding was received for this study. tistically significant correlation between PSA levels and Gleason score of prostate carcinomas TRANSPARENCY DECLARATION obtained on transurethral resection specimens Competing of interest: None to declare. or radical prostatectomy specimens.

126 Čamdžić et al. PSA and prostate cancer

REFERENCES 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, 15. Ghafoori M, Varedi P, Hosseini SJ, Asgari M, Sha- Jemal A. Global cancer statistics 2018: GLOBOCAN kiba M. Value of prostate-specific antigen and pro- estimates of incidence and mortality worldwide for state-specific antigen density in detection of prostate 36 cancers in 185 countries. CA Cancer J Clin 2018; cancer in an Iranian population of men. Urol J 2009; 68:394–424. 6:182-88. 2. Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS, 16. Gerstenbluth RE, Seftel AD, Hampel N, Oefelein MG, Redwin E. Prostate-specific antigen as a serum mar- Resnick MI. The accuracy of the increased prostate ker for adenocarcinoma of the prostate. N Engl J Med specific antigen level (greater than or equal to 20 ng/ 1987; 317:909-16. ml) in predicting prostate cancer: is biopsy always 3. Magi-Galluzzi C. Prostate cancer: diagnostic criteria required? J Urol 2002; 168:1990–93. and role of immunohistochemistry. Mod Pathol 2018; 17. Force USPST, Grossman DC, Curry SJ, Owens DK, 31:S12-21. Bibbins-Domingo K, Caughey AB, Davidson KW, 4. Yu W, Zhou L. Early diagnosis of prostate cancer from et al. Doubeni CA, Ebell M, Epling Jr JW, Kemper the perspective of Chinese physicians. J Cancer 2020; AR, Krist AH, Kubik M, Landefeld CS, Mangione 11:3264-73. CM, Silverstein M, Simon MA, Siu AL, Tseng C-W. 5. Ilic D, Djulbegovic M, Jung JH, Hwang EC, Zhou Screening for prostate cancer: US preventive services Q, Cleves A, Agoritsas T, Dahm P. Prostate cancer task force recommendation statement. JAMA 2018; screening with prostate-specific antigen (PSA) test: 319:1901-13. a systematic review and meta-analysis. BMJ 2018; 18. Negoita S, Feuer EJ, Mariotto A, Cronin KA, Petkov 362:k3519. VI, Hussey SK, Benard V, Henley SJ, Anderson RN, 6. Martin RM, Donovan JL, Turner EL, Metcalfe C, Yo- Fedewa S, Sherman RL, Kohler BA, Dearmon BJ, ung GJ, Walsh EI, Lane JA, Noble S, Oliver SE, Evans Lake AJ, Ma J, Richardson LC, Jemal A, Panberthy S, Sterne JAC, Holding P, Ben-Shlomo Y, Brindle P, L. Annual report to the nation on the status of cancer, Williams NJ, Hill EM, Ng SY, Toole J, Tazewell MK, part II: recent changes in prostate cancer trends and Hughes LJ, Davies CF, Thorn JC, Down E, Smith disease characteristics. Cancer 2018; 124:2801-14. GD, Neal DE, Hamdy FC. Effect of a low-intensity 19. Nnabugwu II, Udeh EI, Ugwumba FO, Ozoemena FO. PSA-based screening intervention on prostate cancer Predicting Gleason score using the initial serum total mortality: the CAP randomized clinical trial. JAMA prostate-specific antigen in Black men with sympto- 2018; 319:883-95. matic prostate adenocarcinoma in Nigeria. Clin Interv 7. Gordetsky J, Epstein J. Grading of prostatic adenocarci- Aging 2016; 11:961‐66. noma: current state and prognostic implication. Diagn 20. Kundu SD, Roehl KA, Yu X, Antenor JA, Suarez BK, Pathol 2016; 11:25. Catalona WJ. Prostate specific antigen density corre- 8. Kryvenko ON, Epstein JI. Changes in prostate can- lates with features of prostate cancer aggressiveness. cer grading: Including a new patient-centric grading J Urol 2007; 177:505–09. system. Prostate 2016; 76:427-33. 21. Loeb S, Sutherland DE, D’Amico AV, Roehl KA, 9. Ferlay J EM, Lam F, Colombet M, Mery L, Pineros M, Catalona WJ. PSA velocity is associated with Glea- Znaor A, Soerjomataram I, Bray F. Global cancer ob- son score in radical prostatectomy specimen: marker servatory: cancer today. Lyon, France: International for prostate cancer aggressiveness. 2008; Agency for Research on Cancer. https://gco.iarc.fr/ 72:1116–20. today (04 April 2020) 22. Milonas D, Smaiyse D, Jievaltas M. Factors predic- 10. Bratt O, Lilja H. Serum markers in prostate cancer ting Gleason score 6 upgrading after radical prosta- detection. Curr Opin Urol 2015; 25:59-64. tectomy. Cent European J Urol 2011; 64:205-08. 11. Fenton JJ, Weyerich MS, Durbin S, Liu Y, Bang H, 23. Jayapradeep DP, Prakash VB, Philipose TR, Pai MR. Melnikow J. Prostate-specific antigen-based screen- Histomorphological correlation of PSA levels in pro- ing for prostate cancer: evidence report and system- static carcinoma. National J Lab Med 2017; 6:28-32. atic review for the US Preventive Services Task Force 24. Pierorazio PM, Walsh PC, Partin AW, Epstein JI. JAMA 2018; 319:1914-31. Prognostic Gleason grade grouping: data based on 12. Lilja H, Ulmert D, Björk T, Becker C, Serio AM, the modified Gleason scoring system. BJU Int 2013; Nilsson JA, Abrahamsson P, Vickers AJ, Beglund G. 111:753–60. Long-term prediction of prostate cancer up to 25 ye- 25. Epstein JI, Zelefsky MJ, Sjoberg DD, Nelson JB, ars before diagnosis of prostate cancer using prostate Egevad L, Magi-Galluzzi C, Vickers AJ, Parwani AV, kallikreins measured at age 44 to 50 years. J Clin On- Reuter VE, Fine SW, Eastham JA, Wiklund P, Han col 2007; 25:431–36. M, Reddy CA, Ciezki JP, Nyberg T, Klein EA. A con- 13. Tang P, Sun L, Uhlman MA, Polascik TJ, Freedland temporary prostate cancer grading system: a valida- SJ, Moul JW. Baseline PSA as a predictor of prostate ted alternative to the Gleason score. Eur Urol 2016; cancer-specific mortality over the past 2 decades: Duke 69:428-35. University experience. Cancer 2010; 116:4711–17. 26. Barakzai MA. Prostatic adenocarcinoma: a grading 14. Orsted DD, Bojesen SE, Kamstrup PR, Nordestgaard from Gleason to the new grade-group system: A Hi- BG. Long-term prostate-specific antigen velocity in storical and Critical Review. Asian Pac J Cancer Prev improved classification of prostate cancer risk and 2019; 20:661-66. mortality. Eur Urol 2013; 64:384–93.

127 ORIGINAL ARTICLE

Scrotal trauma: interest of preoperative ultrasound in the prediction of the rupture of the tunica albuginea

Stefano Manno1,2, Antonio Cicione3, Lorenzo Bagalà2, Antonio Catricalà1, Piero Ronchi4, Simona Tiburzi5, Carolina Giannace6, Lucio Dell’Atti4

1Urology Unit, “Pugliese-Ciaccio” Hospital of Catanzaro, 2Urology Unit “Magna Graecia” University of Catanzaro; Catanzaro, 3Depart- ment of Urology, University Hospital “Ospedale Sant’Andrea”, Roma, 4Department of Urology, University Hospital “Ospedale Riuniti”, Ancona, 5Department of Anaesthesia, “Pugliese-Ciaccio” Hospital, Catanzaro, 6Public Health Institute, Section of Forensic Medicine, University ”Cattolica del Sacro Cuore”, Roma; Italy

ABSTRACT

Aim Scrotal bruises are quite frequent injuries affecting young subjects, with psychological repercussions on body image and fer- tility. The interest of ultrasound in the context of the emergency re- mains controversial. The aim of our study was to investigate clini- cal, ultrasonographic and operative features of scrotal contusions, and to evaluate the contribution of ultrasound in the description of traumatic lesions.

Methods In this retrospective and descriptive study 71 scrotal con- tusions operated from December 2015 to April 2020 were collec- Corresponding author: ted. We retrospectively analysed 26 patients (aged between 14 and Stefano Manno 79 years) of 71 who sustained a scrotal ultrasound, where the latter was positive. The primary endpoint was albuginea rupture, whose Urology Unit “Pugliese-Ciaccio” concordance between ultrasound and surgery was assessed using Hospital of Catanzaro the Kappa method. Positive and negative predictive values, sen- Viale Pio X – 88100 Catanzaro, Italy sitivity and specificity for the presence of albuginea rupture were Phone: +39 3208 384 183; evaluated for a set of ultrasound data: scrotal haematoma, haema- Fax: +39 0961 883 348; tocele, regularity of testicular contours, testicular fracture (speci- E-mail: [email protected] ficity (93%), testicular haematoma, and Doppler signal intensity. ORCID ID: https://orcid.org/.0000-0003- Results Surgical treatment was necessary in 26 (37%) patients; 0215-4698 only six orchiectomy were performed. Surgical exploration should be performed if haematocele is found in the genital examination without any ultrasound complement.

Conclusion The ultrasonography is useful, detailed and accurate when the haematocele is not clinically evident. Original submission: 05 October 2020; Key words: genital, injuries, testicular, ultrasound Revised submission: 15 December 2020; Accepted: 21 December 2020 doi: 10.17392/1286-21

Med Glas (Zenica) 2021; 18(1):128-132

128 Manno et al. Ultrasound and rupture of albuginea of testis

INTRODUCTION and April 2020 were reviewed. Demographic data, average consultation time, trauma side and injury Testicular injury as a result of scrotal trauma is a mechanism were included in the analysis. Pati- relatively uncommon occurrence. Mechanisms of ents who sustained a scrotal ultrasound in whom trauma are categorized as blunt, penetrating and US was not performed prior to exploration were degloving with more than half of all cases resul- excluded from the study (concomitant injury requ- ting from blunt trauma. Penetrating trauma to the iring urgent exploration). In accordance with the scrotum occurs less frequently and can result from European Association of Urology (EAU) guideli- a GSW (gunshot wound ultrasound), stabbing, an nes all patients with testicular trauma underwent animal attack or a self-inflicted injury (1-2). scrotal ultrasound before any surgical exploration Testicular rating injuries can often be challenging (8-9). All 71 patients with suspected testicular tra- as physical examination of the scrotum is often uma underwent ultrasound, of which 26 (36.6%) hampered by patient discomfort, or haematoma or underwent surgical treatment (Figure 1). haematocele, which may like testicular rupture (3). Testicular rupture is a surgical emergency and early diagnosis of this disease is very important (4). Ultrasound (US) and physical examination are the best diagnostic options. In front of blunt tra- uma US may preclude unnecessary surgery in the absence of findings consistent with testicular rupture (1-5). Ultrasound is readily available, inexpensive, non- invasive, and it is not associated with exposure to ionizing radiation. The clinical usefulness of the US examination depends on the skill of the techni- cian performing the examination and the physician interpreting the images. The US of testicular ruptu- re includes a finding of discontinuity of the tunica Fig. 1 Flowchart study design albuginea with loss of testicular contour and hete- US, ultrasound; rogeneity of the testicular parenchyma. Disruption The study was approved by the Ethic Committee of the tunica albuginea alone has 50% sensitivity of the Pugliese-Ciaccio Hospital, Catanzaro. and 76% specificity on ultrasound examination for testicular rupture in the setting of blunt trauma (6). Methods Rupture of the tunica albuginea may be associated with injury to the underlying tunica vaginalis and Scrotal US was performed with the 12 MHz high the testicular parenchyma, which may be apprecia- frequency (PROSOUNDSSD-3500 SX, ALOKA, ted on ultrasound as heterogeneous echogenicity Bucheon- South Corea) linear array transducer with areas of avascularity in the injured testis (7). upon patient presentation prior to proceeding to Scrotal ultrasound has demonstrated clinical use- the operating room. US findings, such as hetero- fulness in the setting of blunt scrotal trauma (7). geneous echogenicity in the testicular parenchyma and a concomitant loss of tunica albuginea con- The aim of this study was to evaluate the contri- tour, were considered a positive test. The US was bution of ultrasound in the detection of traumatic initially performed by a certified US technician or lesions according to clinical, ultrasonographic the radiologist on call and read by a radiologist at and operative data of scrotal contusions. the time of the patient presentation. All ultrasoun- PATIENTS AND METHODS ds were centrally reviewed by a single urologist in blinded fashion for the purpose of this study. Patients and study design Following US, each patient was taken to the ope- In this retrospective study records of all patients rating room for scrotal exploration within 24 hours who underwent scrotal ultrasound prior to subsequ- to the trauma. Operative findings were compared ent scrotal exploration between December 2015 with preoperative US images and records.

129 Medicinski Glasnik, Volume 18, Number 1, February 2021

Statistical analysis In 26 screened patients, in which the US findings were suggestive of a rupture of the tunica albu- The sensitivity and specificity of US were deter- ginea, in the intraoperative surgical act 20 (77%) mined in comparison to operative findings in pa- actually presented a rupture of the scrotal albugi- tients with suspected testicular trauma. The area nea. Of the 20 salvageable testicular injuries, all under the curve (AUC) was calculated from the 20 had positive US findings, but 14 (70%) were receiver operating characteristic (ROC) curve. really positive for rupture of the tunica albuginea, RESULTS while 6 (30%) cases were simply haematoma. The sensitivity of preoperative scrotal US for di- A total of 71 patients with suspected testicular agnosing testicular rupture after scrotal trauma trauma underwent ultrasound. was 66.5% and specificity was 93%. There were A total of 26 patients had positive and 45 had ne- two false-positive US results. Positive predictive gative US finding. Trauma side was on the right value was 77% and negative predictive value was testicle in 28 (40%), left side was affected in 43%. The ROC AUC was 0.75, indicating that 37 (52.2%) and bilateral trauma occurred in six preoperative scrotal ultrasound in the setting of (7.8%) patients. penetrating scrotal trauma by GSW can be consi- The study cohort consisted of 26 patients who dered a good score (Figure 2). sustained trauma of the scrotum and underwent scrotal US prior to operative exploration. Median patient age was 27.5 (14-79) years. Of the 26 pa- tients, 22 (85%) were Italian, 3 (12%) were Afri- can and 1 (3%) was of Thai origin. From the anamnestic history of the patients, 31 (44%) reported an accident or an assault, 14 (20%) testicular trauma after a sporting activity, 8 (12%) after sexual activity and 17 (24%) occurred as a re- sult of other causes (animals bite, heavy machinery, Figure 2. Receiver operating characteristic (ROC) curve of cutting/piercing instruments). Physical examinati- preoperative ultrasound on in 46 (65%) showed a haematocele, a scrotal *ROC using operative (1, positive for rupture; 0, negative) = 1 as positive level (AUC 0.74977, effect likelihood ratio test p<0.0001) haematoma in 17 (24%), testicles appeared normal The presence of a haematocele or haematoma de- in 3 (4%), a doubtful physical examination classifi- creased the sensitivity and specificity of scrotal ed as “other” in 5 (7%) patients. The average hos- US to detect albuginea rupture to 40% and 74 %, pital stay was 1.5 (1-6) days (Table 1). respectively (Table 2). Table 1. Clinical characteristics of 26 patients who sustained trauma of the scrotum and underwent scrotal ultrasound prior Table 2. Ultrasound data to operative exploration Sensibility Specificity Variable PPV NPV Variable (%) (%) Average age (years) 27.5 Haematocele 95 74 0.64 0.8 Average hospital stay (range) (days) 1.5 (1-6) Scrotal haematoma 40 48 0.51 0.36 Trauma side (No; %) Testicular fracture 66.5 93 0.77 0.43 Right 10 (40) Testicular haematoma 48 82 0.8 0.53 Left 13 (52.2) Irregularity of outlines 77 71 0.79 0.71 Bilateral 3 (7.8) Doppler alterations 40 79 0.72 0.48 Mode of injury (No; %) PPV, positive predictive value; NPV, negative predictive value; Accident 11 (44) Sports 5 (20) DISCUSSION Sexual activity 3 (12) Other* 7 (24) The EAU guidelines for urotrauma, which was *animals bite, heavy machinery, cutting/piercing instruments written in 2014 and most recently updated in Operative evaluation revealed 20 (out of 26; 2020, do not specify the optimal radiographic 77%) salvageable injuries: 14 (70%) of these 20 evaluation for high energy penetrating scrotal patients underwent surgical repair of the tuni- trauma (10). In the literature pertaining to testi- ca albuginea, and 6 (30%) underwent a simple cular injuries sustained from blunt trauma scrotal evacuation of the haematoma. Only 6 (out of 26; ultrasound is recommended to rule out testicular 23%) orchiectomies were performed. rupture, and this is supported in the guidelines

130 Manno et al. Ultrasound and rupture of albuginea of testis

(10). Although there are limited data specifically is inconclusive, testicular CT or MRI may be on scrotal ultrasound in the setting of penetra- helpful; however, these techniques did not spe- ting scrotal trauma, that recommendation is often cifically increase the detection rates of testicular extended to this setting. rupture (21-22). Of all urological injuries, 33-66% involve To our knowledge our study is among few in the the external genitalia (11). Genital trauma is literature to have evaluated ultrasound sensitivity commonly caused by blunt injuries (80%). In and specificity. Our study found 66.5%sensitivity males, blunt genital trauma frequently occurs and 93% specificity. Given the limited sensitivity unilaterally with approximately 1% presenting we would recommend that a negative scrotal ul- as bilateral scrotal or testicular injuries (12). trasound should not preclude surgical exploration Any kind of contact sport, without the use of in patients who have sustained a scrotal gunshot protective aids, may be associated with genital wound ultrasound. trauma. Off-road cycling, motor biking (especi- Testicular salvage is the objective of scrotal ally on motorbikes with a dominant petrol tank), exploration following penetrating trauma with rugby, football and hockey are all activities the aim of avoiding a missed testicular rupture associated with blunt testicular trauma (13,14). diagnosis and the potential sequelae of an undi- Penetrating injuries are most commonly caused agnosed testicular rupture like ischemia, chronic by firearms (75.8%) (15). pain and delayed orchiectomy (23). In our study Traumatic dislocation of the testicle rarely 20 of 26 cases have reported the rescue of testicle. occurs and is most common in victims of mo- Our study, a retrospective series of a cohort of pa- tor vehicle accidents (MVAs). Bilateral dis- tients who underwent scrotal ultrasound prior to location of the testes has been reported in up to operative exploration, has some limitations that 25% of cases (16). Testicular rupture is found must be acknowledged: this is a single-centre re- in approximately 50% of cases of direct blunt trospective study and a low number of patients scrotal trauma (17,18). It may occur under in- were recruited, which might result in a bias. tense compression of the testis against the in- Scrotal ultrasound may be considered a potentially ferior pubic ramus or symphysis, resulting in useful adjunct in the diagnosis of testicular injury. a rupture of the tunica albuginea. A force of The sensitivity and specificity of the examination approximately 50 kg is necessary to cause testi- remain highly questionable to date. Negative scro- cular rupture (7). Testicular rupture is associa- tal ultrasound is not sensitive enough to rule out ted with immediate pain, nausea, vomiting, and the need for surgical exploration. Therefore, nega- sometimes fainting. The hemiscrotum is tender, tive scrotal US should not be considered sufficient swollen, and ecchymotic. The testis itself may to debar prompt operative assessment (24). be difficult to palpate (18). In conclusion, it is essential to surgically explo- Ultrasound should be performed to determine in- re equivocal patients whenever imaging studies tra- and/or extra-testicular haematoma, testicular cannot exclude testicular rupture. This involves contusion or rupture (18,19). However, the lite- exploration with evacuation of blood clots and rature is contradictory as to the usefulness of US haematoma, excision of any necrotic testicular compared to clinical examination alone (7,19). tubules and closure of the tunica albuginea, usu- Lee and Bak have reported convincing findings ally with running absorbable sutures. with a specificity of up to 98.6% (20). Heterogeneous echo pattern of the testicular ACKNOWLEDGMENT parenchyma with the loss of contour definition We thank Rossana Giulietta Iannitti, PhD, for is a highly sensitive and specific radiographic editing a draft of this manuscript. finding for testicular rupture (7). Others re- ported poor specificity (78%) and sensitivity FUNDING (28%) for the differentiation between testicu- No specific funding was received for this study. lar rupture and haematocele, while accuracy is as low as 56% (7). Colour Doppler-duplex US TRANSPARENCY DECLARATION may provide useful information when used to evaluate testicular perfusion (7). If scrotal US Competing interests: None to declare.

131 Medicinski Glasnik, Volume 18, Number 1, February 2021

REFERENCES 1. Jeffrey RB, Laing FC, Hricak H, McAninch JW. So- 13. Frauscher F, Klauser A, Stenzl A, Helweg G, Amort nography of testicular trauma. AJR Am J Roentgenol B, zur Nedden D. US findings in the scrotum of extre- 1983; 141:993-5. me mountain bikers. Radiology 2001; 219:427-31. 2. Nicola R, Carson N, Dogra VS. Imaging of trauma- 14. Lawson JS, Rotem T, Wilson SF. Catastrophic injuri- tic injuries to the scrotum and penis. AJR Am J Ro- es to the eyes and testicles in footballers. Med J Aust entgenol 2014; 202:W512-20. 1995; 163:242-4. 3. Randhawa H, Blankstein U, Davies T. Scrotal trau- 15. Al-Azzawi IS, Koraitim MM. Lower genitourinary ma: A case report and review of the literature. Can trauma in modern warfare: the experience from civil Urol Assoc J 2019; 13(6 Suppl4):S67-71. violence in Iraq. Injury 2014; 45:885-9. 4. Learch TJ, Hansch LP, Ralls PW. Sonography in pa- 16. Nagarajan VP, Pranikoff K, Imahori SC, Rabinowitz tients with gunshot wounds of the scrotum: imaging R. Traumatic dislocation of testis. Urology 1983; findings and their value. AJR Am J Roentgenol. 1995 22:521-4. Oct;165(4):879-83. doi: 10.2214/ajr.165.4.7676986. 17. Cass AS, Luxenberg M. Testicular injuries. Urology PMID: 7676986. 1991; 37:528-30. 5. Buckley JC, McAninch JW. Use of ultrasonography 18. Wang Z, Yang JR, Huang YM, Wang L, Liu LF, Wei for the diagnosis of testicular injuries in blunt scrotal YB, Huang L, Zhu Q, Zeng MQ, Tang ZY. Diagno- trauma. J Urol 2006; 175:175-8. sis and management of testicular rupture after blunt 6. Guichard G, El Ammari J, Del Coro C, Cellarier D, scrotal trauma: a literature review. Int Urol Nephrol Loock PY, Chabannes E, Bernardini S, Bittard H, 2016; 48:1967-1976. Kleinclauss F. Accuracy of ultrasonography in dia- 19. Andipa E, Liberopoulos K, Asvestis C. Magnetic re- gnosis of testicular rupture after blunt scrotal trau- sonance imaging and ultrasound evaluation of penile ma. Urology 2008; 71:52-6. and testicular masses. World J Urol 2004; 22:382-91. 7. Bhatt S, Dogra VS. Role of US in testicular and scro- 20. Lee SH, Bak CW, Choi MH, Lee HS, Lee MS, Yoon tal trauma. Radiographics 2008; 28:1617-29. SJ. Trauma to male genital organs: a 10-year review 8. Isidori AM, Pozza C, Gianfrilli D, Giannetta E, of 156 patients, including 118 treated by surgery. Lemma A, Pofi R, Barbagallo F, Manganaro L, Mar- BJU Int 2008; 101:211-5. tino G, Lombardo F, Cantisani V, Franco G, Lenzi 21. Muglia V, Tucci S Jr, Elias J Jr, Trad CS, Bilbey A. Differential diagnosis of nonpalpable testicular J, Cooperberg PL. Magnetic resonance imaging of lesions: qualitative and quantitative contrast-enhan- scrotal diseases: when it makes the difference. Uro- ced US of benign and malignant testicular tumors. logy 2002; 59:419-23. Radiology 2014; 273:606-18. 22. Manno S, Cicione A, Dell'Atti L, Giudice TD. Ama- 9. Ronchi P, Manno S, Dell'Atti L. Technology meets zing result of Nivolumab in a patient with multiple carcinomas. J BUON 2019; 24:865-867. tradition: CO2 laser circumcision versus conventional surgical technique. Res Rep Urol 2020; 16:255-60. 23. Powers R, Hurley S, Park E, McArdle B, Vidal P, 10. Bryk DJ, Zhao LC. Guideline of guidelines: a re- Psutka SP, Hollowell CMP. Usefulness of preopera- view of urological trauma guidelines. BJU Int 2016; tive ultrasound for the evaluation of testicular rup- 117:226-34. ture in the setting of scrotal gunshot wounds. J Urol 11. Brandes SB, Buckman RF, Chelsky MJ, Hanno PM. 2018; 199:1546-51. External genitalia gunshot wounds: a ten-year experi- 24. Churukanti GR, Kim A, Rich DD, Schuyler KG, La- ence with fifty-six cases. J Trauma 1995; 39:266-71. vien GD, Stein DM, Siddiqui MM. Role of ultraso- 12. Monga M, Hellstrom WJ. Testicular Trauma. Ado- nography for testicular injuries in penetrating scrotal lesc Med. 1996 Feb;7(1):141-148. PMID: 10359963. trauma. Urology 2016; 95:208-12.

132 ORIGINAL ARTICLE

Workload changes during the COVID-19 pandemic and effects on the flow of cancer patients in the Maxillofacial Surgery Department

Ana Kvolik Pavić1,2, Vedran Zubčić1,2, Slavica Kvolik2,3

1Department of Maxillofacial Surgery, Osijek University Hospital, 2Faculty of Medicine, University Josip Juraj Strossmayer, Osijek, 3Department of Anaesthesiology and Critical Care, Osijek University Hospital; Osijek, Croatia

ABSTRACT

Aim A SARS Coronavirus 2 (COVID-19) pandemic drastically changed the way the health system works. In Croatia, lockdown measures to curb virus spread lasted from March to May 2020, and all non-essential medical procedures and patients’ visits have been cancelled. The study aimed to compare patients’ flow and interventions in the surgical department before, during and after the lockdown period.

Methods This cross-sectional study analysed the workload at the Maxillofacial and Oral Surgery Department (Department), Osijek University Hospital, during the COVID-19 pandemic (March-May 2020) and four subsequent months. The same period of 2019 was Corresponding author: compared as a control. The data were subtracted from hospitals’ Ana Kvolik Pavić electronic database. Department of Maxillofacial and Oral Results During COVID-19 lockdown from March to May 2020 surgery, Osijek University Hospital the number of hospitalizations (306 vs. 138), surgical procedu- J. Huttlera 4, Osijek 31000, Croatia res (306 vs. 157), and scheduled outpatient visits (2009 vs. 804), Phone: +385 31 511 466; dropped significantly as compared to 2019. The number of skin Fax: +385 31 511 462; tumour removals was halved (from 155 in 2019 to 58 in 2019) (p<0.001), and the number of emergency patients was unchanged ORCID ID: https://orcid.org/0000-0002- in the 3-month period. A significant decrease in outpatient visits 3991-6433 and hospital admissions continued after the lockdown (p<0.001).

Conclusion A decrease in the number of outpatient visits, hospita- lizations, and tumour removals may result in larger proportions of patients with advanced cancers in the future. The second wave of COVID-19 pandemic is ongoing, and special effort must be paid to reduce the number of cancer patients receiving suboptimal tre- Original submission: atment. 28 October 2020; Revised submission: Key words: head and neck neoplasms, time-to-treatment, 02 November 2020; workflow Accepted: 17 November 2020 doi: 10.17392/1308-21

Med Glas (Zenica) 2021; 18(1):133-137

133 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION the virus spread on 18 March 2020, which lasted over a month with restrictive measures gradu- In December 2019 a novel strain of SARS Coro- ally weakened in May. During the lockdown, navirus 2 (COVID-19) swept across China and all non-essential health care appointments and quickly spread to all corners of the world. The procedures were postponed or cancelled (7). Af- WHO declared a pandemic on 11 March 2020 ter the lockdown, according to the hospital re- (1). Most people infected with the virus are commendations, all patients must have a SARS- asymptomatic or present with mild respiratory CoV 2 test negative before hospital admission. symptoms, but older population and patients with comorbidities could develop severe respi- This study was approved by the Ethics Com- ratory syndrome - SARS-CoV 2 (2). This puts mittee of the Osijek University Hospital. maxillofacial and oral surgeons in an especially Methods precarious situation due to the anatomical regi- on of their interest and performed procedures The data were subtracted from the hospitals’ in- (3). By dealing with facial trauma, correction formatics system (BIS). All data were blinded, of congenital defects in maxilla and mandible, and identities of patients were not recorded. The oncologic and plastic surgery in the face and data on the number of patients in different po- neck area, medical personnel are exposed to res- liclinics of the department, the number of hos- piratory aerosols and potential infection (4). In pitalized patients, and the number of outpatient/ addition, numerous procedures in the maxillofa- inpatient surgical procedures were registered. cial surgery practice pose a great risk to health Registered outpatient procedures included small and facilitate personnel-to-patient infection (5). skin resections and reconstruction in local anae- Some patients have recognized such risk and sthesia (LA), as well as management of smaller delayed their treatment themselves (6). injuries to the face in ambulatory patients. Due to possibility of viral transmission, some Types of inpatient procedures were noted and countries delayed elective interventions reducing divided into the following groups: surgery of tu- their numbers significantly. The number of pro- mours (including resections of skin neoplasms, cedures was also reduced due to difficulties in oral, oropharyngeal, salivary gland, and bone accessing the health services and tertiary surgical tumours as well as biopsies under general anae- centres during the pandemic (6). sthesia (GA)), plastic surgery procedures (inclu- The aim of this research was to explore workload ding rhino septoplasty, blepharoplasty, etc.), change in the Department of Maxillofacial and oral surgery procedures (including planned Oral surgery during the COVID-19 pandemic in procedures such as radicular cysts, retained and 2020 year and especially during the lock-down impacted teeth and emergency procedures such measures from March-May 2020. as odontogenic abscesses, oroantral fistulas, etc.), trauma (including soft tissue defects and PATIENTS AND METHODS fractures of facial bones), surgery of salivary glands (including non-neoplastic resections and Patients and study design extraction of calculi due to sialolithiasis), and other procedures (including secondary recon- This study was a retrospective analysis com- structions of defects and scars, tracheotomies paring the types of surgical procedures in the and closure of tracheostomies, revisions of po- Department of Maxillofacial and Oral Surgery, stoperative bleeding or necrosis, etc.) University Hospital Osijek, Croatia, from Mar- ch-May 2019 and 2020 year. The same period Statistical analysis one year before the pandemic, March, April, and May 2019 was analysed as a control. Additio- Statistical difference between two periods was 2 nally, patient flow through the Department was analysed using the Pearson Chi test. All stati- compared between March-September 2020 with stical tests that were used in calculations were the same period in 2019. These months were two-sided. The level of significance was defined chosen because of the Coronavirus pandemic in as p<0.05. Croatia that began lockdown measures to curb

134 Kvolik Pavić et al. COVID-19 and maxillofacial cancer surgery

RESULTS The number of patient visits to the Department was significantly reduced during the COVID-19 lockdown. The number of emergency visits also fell in March and April, but their overall percenta- ge rose to reach as much as 60.24% of all visits in April (Figure 1). The ratio between emergency and outpatient visits in 2019 and 2020 for March did not change significantly (20.5% vs. 26.8%; p=0.23). Emergency outpatient visits grew from 17.9% and 18.3% to 60.2% and accounted for 35.5% of all outpatient examinations during April and May 2020. During the lockdown period the number of Figure 2. Comparison of the surgical procedures in general and local anaesthesia at the Department of Maxillofacial Surgery outpatient appointments and hospitalizations de- during the lockdown and four months later in 2019 and 2020 creased significantly compared to the same interval GA, general anaesthesia; LA, local anaesthesia; in 2019 (p<0.001 for both) (Figure 1). 2020, with two patients in need of revisions for postoperative bleeding and flap necroses. There were two surgical tracheostomies performed for COVID-19 positive patients (Figure 3).

Figure 1. Comparison of outpatient appointments, emergency outpatient visits to the maxillofacial policlinic, and the number of hospital admissions to the Department of Maxillofacial Sur- gery in 2019 and 2020 The total number of surgical procedures perfor- med in GA and LA decreased significantly in the observed period (p<0.001 for both GA and LA). During March, April, and May of 2019, there were Figure 3. Comparison of inpatient surgical procedures per- almost twice as many surgical procedures as in the formed during the three-month period in 2019 and 2020 same period of 2020 (306 vs. 157), with April ha- DISCUSSION ving the biggest difference (116 vs. 29). The num- ber of surgical procedures further decreased in the There has been no global medical emergency like period from June to September 2020 (Figure 2). COVID-19 since the Spanish flu of 1918. This The bulk of surgeries performed at the Depar- novel disease poses many challenges to doctors tment were excisions of neoplasms. During and nurses worldwide: how to treat this virus, COVID lockdown their number dropped signi- how to spot it, how to stop its spread. In the me- ficantly compared with the last year (p=0.005) antime, it became a challenge to continue prac- (Figure 3). The most common type of tumour ticing medicine in the COVID-19 setting while resections were resection of skin tumours, basal treating other diseases. cell carcinomas, squamous cell carcinomas, and This study has confirmed significant differences in melanomas - which fell from 109 in 2019 to 36 the workload and patients’ flow at the Department in 2020 (p<0.001). The surgical procedures for of Maxillofacial and Oral surgery during the CO- facial trauma increased from 8.5% to 14.6% ove- VID-19 lockdown period and subsequent months rall (p> 0.43). A slight increase in the surgical in 2020 compared to 2019. The first COVID-19 site complications was observed in March-May positive cases in Croatia were reported on 26 Fe-

135 Medicinski Glasnik, Volume 18, Number 1, February 2021

bruary 2020 and peaked around the end of March the unwanted consequences of the COVID-19 (7). All non-essential medical visits and consultati- crisis. The bulk of our postponed tumour remo- ons, as well as elective surgeries, were postponed. vals were premalignant or lesions of low mali- Therefore, it is not surprising that the Department gnant potential such as basocellular carcinoma, had its workload diminished by more than two and a delay in their treatment for a few months thirds. Interference and reduction of work were may not have serious consequences quod vitam. reported by other authors as well (4,6). However, there is a subgroup of patients suffe- The greatest change in the work practice in our in- ring from lesions that will have an unfavourable stitution found in our study was a lower number cosmetic outcome with delayed treatment, and a of hospitalizations and non-emergency visits; we subgroup with more aggressive tumours, i.e. me- expected to find an increase of emergency visits. lanomas, that may easily spread and metastasize If patients can not seek timely medical assistan- (14). Once admitted to the hospital in advanced ce during scheduled visits, they were expected to stage of their disease, these patients may be given seek it in emergency rooms, but this was not the suboptimal medical care with the greater cost of case. Instead we found a drop in emergency vi- treatment. These tumours must not be neglected sits. Similar trends were also reported in the USA in the COVID-19 pandemic (15). (8,9), Austria (10), Italy (11). The number of facial Maxillofacial surgery is a specialty that is par- traumas noticed in our study requiring emergency ticularly vulnerable in the setting of respiratory surgery did not change significantly, suggesting transmitted virus (16). Due to the nature of physi- that trauma cases were not affected by stay-at-ho- cians’ work every examination and procedure me measures and a prohibition of intercity tran- performed at the patients’ side present a risk for sit and public transport. However, the number of viral transmission (16,17). Specific airway pro- emergency visits increased in May 2020, when cedures, such as surgical tracheostomies in CO- the lockdown measures ceased. A phenomenon of VID-19 positive patients, pose a significant risk using emergency visits for non-urgent examinati- to all medical staff involved in the treatment due ons has been widely reported, with one review ar- to the aerosol dispersion (5). Operating in CO- ticle stating that non-urgent visits make up to 37% VID-19 operating rooms poses several challen- of all emergency visits across the literature (12). ges, with low mobility, impaired communication, A recent COVID Surg Collaborative study that and poor visibility for the personnel wearing gla- also involved Croatian surgical patients predic- sses. Shortages of PPE as well as injuries due to ted that the overall 12-week cancellation rate PPE are another risk to the health care professio- worldwide would be 72.3%; if countries increased nals (15, 18-20) their normal surgical volume after that 3-month To avoid endangering either patients or personnel period by 20%, the authors estimated that it would it was necessary to think outside the box when take a median of 45 weeks to clear the backlog working in the pandemic setting. Zimmerman of operations resulting from COVID-19 disrupti- and Nkenke proposed dividing the workload on (13). In our institution, due to heightened sa- between overlapping specialties (oral surgery/ fety measures (such as negative COVID-19 test and ENT) and concentrating on onco- not older than 2 days and negative epidemiologic logic surgery and trauma (3). The applicability of anamnesis before hospital admissions and surgical these recommendations depended heavily on the procedures) and fear of infection, patient flow was organization in each health care system. reduced even for four months after the lockdown. A weak point of this study is its retrospective cha- Our number of surgical procedures is still conti- racter. Reasons for treatment postponement and nuously lower than in 2019. This means that our cancellations of hospital admissions were not re- backlog is continuously rising. gistered. These reasons could be a key factor in re- The type and proportions of surgical procedu- ducing waiting lists and resolving healthcare pro- res performed in our institution during the CO- blems that will deteriorate after prolonged delays. VID-19 pandemic had also changed. The number In conclusion, this analysis of workload during of tumour removals has decreased, and unfavou- March, April, and May 2020 and four subsequent rable outcomes of these patients may be one of months confirmed a significant decrease in the

136 Kvolik Pavić et al. COVID-19 and maxillofacial cancer surgery

number of patients treated at the Department of nals, including both maxillofacial surgeons and Maxillofacial and Oral Surgery of our instituti- primary care practitioners, should try to facilitate on. A decrease in the number of outpatient visits, their timely access to healthcare services while hospitalizations, and tumour removals registered protecting themselves and patients. will inevitably result in an increase of patients presenting with advanced cancers. Since the se- FUNDING cond wave of SARS Coronavirus 2 pandemic is No specific funding was received for this study. ongoing, special effort must be paid to reduce the number of cancer patients with suboptimal TRANSPARENCY DECLARATION early interventions. All participating professio- Conflict of interests: None to declare

REFERENCES 1. WHO Timeline - COVID-19 https://www.who.int/ 12. Uscher-Pines L, Pines J, Kellermann A, Gillen E, news-room/detail/27-04-2020-who-timeline---co- Mehrotra A. Emergency department visits for nonu- vid-19 (11 June 2020) rgent conditions: systematic literature review. Am J 2. Wu Z, McGoogan JM. Characteristics of and impor- Manag Care 2013; 19:47–59. tant lessons from the coronavirus disease 2019 (CO- 13. COVIDSurg Collaborative. Elective surgery VID-19) outbreak in China: summary of a report of cancellations due to the COVID-19 pandemic: glo- 72314 cases from the Chinese Center for Disease bal predictive modelling to inform surgical recovery Control and Prevention. JAMA 2020; 323:1239–42. plans. Br J Surg 2020; 10.1002/bjs.11746. 3. Zimmermann M, Nkenke E. Approaches to the ma- 14. Ho AS, Kim S, Tighiouart M, Mita A, Scher KS, Ep- nagement of patients in oral and maxillofacial sur- stein JB, Laury A, Prasad R, Ali N, Patio C, Mallen- gery during COVID-19 pandemic. J Craniomaxillo- St-Claire J, Zumsteg ZS. Quantitative survival im- fac Surg 2020; 48:521–6. pact of composite treatment delays in head and neck 4. Zhao Z, Gao D. Precaution of 2019 novel coronavi- cancer. Cancer 2018; 124:3154–62. rus infection in department of oral and maxillofacial 15. Bartlett DL, Howe JR, Chang G, Crago A, Hogg surgery. Br J Oral Maxillofac Surg 2020; 58:250–3. M, Karakousis G, Levine E, Maker A, Mamounas 5. Mick P, Murphy R. Aerosol-generating otolaryngo- E, McGuire K, Merchant N, Shibata D, Sohn V, logy procedures and the need for enhanced PPE du- Solorzano C, Turaga K, White R, Yang A, Yoon S. ring the COVID-19 pandemic: a literature review. J Management of cancer surgery cases during the CO- Otolaryngol Head Neck Surg 2020; 49:29. VID-19 pandemic: considerations. Ann Surg Oncol 6. Gallo O, Locatello LG, Orlando P, Martelli F, Bruno 2020; 27:1717–20. C, Cilona M, Fancello G, Mani G, Vitali D, Bian- 16. Kowalski LP, Sanabria A, Ridge JA, Ng WT, de Bree co G, Trovati M, Tomaiuolo M, Maggiore G. The R, Rinaldo A, Takes RP, Mäkitie AA, Carvalho AL, clinical consequences of the COVID-19 lockdown: Bradford CR, Paleri V, Martl DM, Vander Poorten a report from an Italian referral ENT department. V, Nixon IJ, Lacy PD, ROdrigo JP, Guntinas-Lichi- Laryngoscope Investig Otolaryngol 2020; 5:824–31 us O, Mendenhall WM, D'Cruz A, Lee AW, Ferlito 7. KORONAVIRUS.HR. https://www.koronavirus.hr/ A. COVID-19 pandemic: effects and evidence-ba- en (11 June 2020) sed recommendations for otolaryngology and head 8. Hartnett KP, Kite-Powell A, DeVies J, Coletta MA, and neck surgery practice. Head and Neck 2020; Boehmer TK, Adjemian J, Guandlapalli AV. Impact 42:1259–67. of the COVID-19 pandemic on emergency depar- 17. Practitioners specialized in oral health and corona- tment visits - United States, January 1, 2019-May virus disease 2019: Professional guidelines from the 30, MMWR 2020; 69:699–704. French society of stomatology, maxillofacial surgery 9. Wong LE, Jessica E. Hawkins JE, Langness S, and oral surgery, to form a common front against Murrell KL, Iris P, Sammann A. Where are all the the infectious risk. J Stomatol Oral Maxillofac Surg patients? Addressing Covid-19 fear to encourage 2020;121:155–8. sick patients to seek emergency care. NEJM Catal 18. Garcia Godoy LR, Jones AE, Anderson TN, Fisher 2020;1–12. CL, Seeley KML, Beeson EA, Zane HK, Petersom 10. Metzler B, Siostrzonek P, Binder RK, Bauer A, Re- JW, Sullivan PD. Facial protection for healthcare instadler SJ. Decline of acute coronary syndrome workers during pandemics: a scoping review. BMJ admissions in Austria since the outbreak of CO- Global Health 2020; 5:e002553. VID-19: the pandemic response causes cardiac co- 19. Gefen A, Ousey K. Update to device-related pre- llateral damage. Eur Heart J 2020; 41:1852–3 ssure ulcers: SECURE prevention. COVID-19, 11. Lazzerini M, Barbi E, Apicella A, Marchetti F, Car- face masks and skin damage. J Wound Care 2020; dinale F, Trobia G. Delayed access or provision of 29:245–59. care in Italy resulting from fear of COVID-19. Lan- 20. D’Cruz L. PPE or not PPE - that is the question. Br cet Child Adolesc Heal 2020; 4:10–1. Dent J 2020; 228:753–4.

137 ORIGINAL ARTICLE

Brainstem haemorrhage as a rare complication of burr hole craniostomy

Rodolfo Corinaldesi1, Corrado Filippo Castrioto1, Francesca Romana Barbieri2, Luciano Mastronardi3, Umberto Ripani4

1Department of Neurosurgery, Ospedale Santa Maria della Misericordia, Perugia, 2Department of Neurosurgery, Ospedale di Belcolle, Viterbo; 3Department of Neurosurgery, Ospedale San Filippo Neri; Roma, 4Pain Therapy Centre, Division of Anaesthesia, Analgesia and Intensive Care, Emergency Department, Ospedali Riuniti di Ancona, Ancona; Italy

ABSTRACT

Aim Evacuation through burr hole craniostomy is the most common type of chronic subdural hematoma surgical treatment, with a morbidity rate of 0-9%.

Methods Here we present a case of 66-year-old Caucasian wo- man with bilateral hemispheric chronic subdural hematoma and left transtentorial uncal herniation. Bilateral burr hole craniostomy with gradual and simultaneous evacuation was performed and sub- dural drains were placed with daily strict monitoring of drained fluid. Corresponding author: Results Despite immediate prompt neurological improvement, on Rodolfo Corinaldesi the second postoperative day bilateral ptosis and left medial rectus Department of Neurosurgery, Ospedale weakness occurred, with no signs of consciousness deterioration. Santa Maria della Misericordia Radiological exams revealed a 9 x 6 mm haemorrhage of the te- Piazzale Giorgio Menghini, 1, gmentum mesencephali. In the next day progressive neurological 06129 Perugia, Italy improvement occurred and a follow-up at 1 month revealed per- Phone: + 39 0755782258; sistence of bilateral ptosis with almost complete regression of the left medial rectus weakness. Fax: +39 0755782258; E-mail rodolfo.corinaldesi@ospedale. Conclusion Although burr hole craniostomy is considered a minor perugia.it procedure, rare but fatal complications like brainstem haemorrha- ORCID: http://orcid.org/0000-0001-8618- ge may occur. Bilateral simultaneous and gradual drainage, strict monitoring of drained fluid and blood pressure in the perioperative 3239 period and frequent neurological with prompt radiological asse- ssment in case of clinical worsening, should be the mainstay of a correct management of chronic subdural hematoma (particularly if Original submission: bilateral) in order to avoid potentially fatal complications. 22 October 2020; Revised submission: Key words: bleeding, chronic subdural hematoma, clinical worse- 11 November 2020; ning, post-surgical issue, surgical procedure. Accepted: 16 November 2020 doi: 10.17392/1299-21

Med Glas (Zenica) 2021; 18(1):138-142

138 Corinaldesi et al. Complication of burr hole craniostomy

INTRODUCTION any significant neurological impairment, except for persistent headache. However, the next day Chronic subdural hematoma (CSDH) generally persistent and severe headache associated with occurs in the elderly, with a mortality rate ran- progressive neurological deterioration occurred. ging from 0.5 to 4% (1,2). At the admission the laboratory exams showed a Evacuation through burr hole craniostomy is the normal clotting profile, the CT scan of the brain most common type of surgical treatment, with showed bilateral hemispheric subdural hemato- a morbidity rate of 0-9% (1, 3-5). Others inclu- mas thicker on the left side. Magnetic resonance de twist-drill craniostomy (6-8) and craniotomy imaging (MRI) scans, at the admission, showed with membranectomy (2). increase of parenchymal compressive effect and Subdural fluid and blood reaccumulation, - cere inferomedial displacement of the left temporal lobe bral edema, tension pneumocephalus, seizures, (transtentorial uncal herniation) (Figure 2 A-B). subdural empyema and intracerebral haemorrha- ge represent possible post-surgical complications (9-11). Among these, intracerebral haemorrhage is rare, occurring with a reported incidence of 0.7–4.0% (9, 12-14). According to the literature, even more uncommon is brainstem haemorrhage and its mechanism still remains unclear. Here we present a rare case of a brainstem hae- morrhage following the evacuation of bilateral CSDH. The possible physiopathogenetic mecha- Figure 2. A) Pre-operative axial and B) coronal T2-weighted nisms and strategies aimed at preventing this MRI scans showing bilateral hemispheric subdural hema- complication are discussed along. tomas thicker on the left side with fluid level. The left uncus is displaced infero-medially to the crural cistern, suggesting PATIENT AND METHODS transtentorial herniation (Department of Neuroradiology, Os- pedale Santa Maria della Misericordia of Perugia, 2017) Methods Patient and study design The patient underwent urgent surgical evacuation A 66-year-old Caucasian woman was admitted of the hematomas. Under the local anaesthesia, in the Department of Neurosurgery, Ospedale bilateral parietal burr holes were performed con- Stanta Maria della Misericordia of Perugia with secutively; dura mater and outer membrane were persistent and severe headache that started 4 days exposed and opened bilaterally at the same time. before and worsened in the last 24 hours. Despite the high pressure of the hematomas, eva- Clinical history did not reveal significant comor- cuation occurred gradually in order to avoid too bidities, except for mild traumatic brain injury 2 fast decompression. Bilateral subdural drains wit- months before and intake of high doses of non- hout vacuum bulb were placed and reservoirs gra- steroidal anti-inflammatory drugs (NSAID). Neu- dually lowered with daily strict monitoring of dra- rological examination on admission did not reveal ined fluid. The patient was left supine in bed at 0°.

RESULTS The patient experienced prompt clinical and neu- rological improvement, with the resolution of the preoperative symptoms related to brain compre- ssion. Blood pressure (BP) monitoring did not re- veal significant alterations. Nevertheless, on the second postoperative day, bilateral ptosis and left medial rectus weakness occurred, with no signs Figure 1. A, B) Pre-operative axial CT scan showing bilateral of consciousness deterioration. hemispheric subdural hematomas thicker on the left side (De- partment of Neuroradiology, Ospedale Santa Maria della Miseri- Radiological evaluation with CT scans revealed cordia of Perugia, 2017) a 9 x 6 mm haemorrhage located at tegmentum

139 Medicinski Glasnik, Volume 18, Number 1, February 2021

mesencephali (Figure 3), subsequently confirmed by MRI performed 5 days after the burr hole cra- niostomy (Figure 4 A-C). In the next day progre- ssive neurological improvement occurred and the patient was discharged home. A follow-up at 1 month showed further improvement and the last neurological examination performed at 1 year revealed complete regression of the previously described symptoms.

DISCUSSION Intracerebral haemorrhage following CSDH drainage is rare and usually ipsilateral to CSDH itself. Only few cases of remote bleeding are des- cribed in the literature (15-20).

Figure 3. Post-operative CT scan showing a 9x6 mm brainstem Among these, brainstem haemorrhage is an haemorrhage of new onset located at tegmentum mesenceph- extremely rare complication of CSDH drainage ali (Departement of Neuroradiology, Ospedale Santa Maria della and only three cases are described in the literatu- Misericordia of Perugia, 2017) re (Table 1) (15,16,18) two of which detected at autopsy (15,16). Despite several theories, patho- genesis still remains unclear. Cohen et al. (19) assumes an association between postoperative cerebrospinal fluid (CSF) over dra- inage through a closed system drainage and re- mote intracranial haemorrhage, resulting in exce- ssive tearing and stretching of bridging veins. Mechanical compression, as in transtentorial herniation, may have an important role as well, as blood vessels near the brainstem might be stretched and distorted (18). Park et al. (18) describe a case of brainstem hae- morrhage following burr hole drainage of CSDH in which asymmetrical evacuation and rapid de- compression occurred. As in the present case, preoperative CT and MRI scans showed transten- torial herniation. However, unlike Park et al., in Figure 4. A) Post-operative axial fluid attenuated inversion the presented patient bilateral drainage was gra- recovery (FLAIR), B) axial and C) coronal T2-weighted MRI scans confirming the 9 x 6 mm brainstem haemorrhage lo- dual and simultaneous. So further unknown fac- cated at tegmentum mesencephali (Department of Neuroradiol- tors may have a role in this type of complication. ogy, Ospedale Santa Maria della Misericordia of Perugia, 2017) According to Ogasawara et al. (21), rapid decom- pression of CSDH frequently results in a sudden

Table 1. Cases of brainstem haemorrhage as a complication of subdural hematoma’s evacuation reported in the literature Patients data Study (year) (reference Age Subdural Time to compli- number) Gender Surgical treatment Treatment Follow up (months) (years) hematoma cation (hours) McKissock and Bloom NA NA NA NA NA Conservative NA (autopsy finding) (1960) (15) Robinson RG (1984) (16) NA 59 NA NA 0 Conservative NA (autopsy finding) Park et al. (2009) (18) M 76 Bilateral, subacute Single bilateral burr hole 48 Conservative 0.5 Presented case F 66 Bilateral, chronic Single bilateral burr hole 48 Conservative 12 NA, not available; M, male; F, female;

140 Corinaldesi et al. Complication of burr hole craniostomy

transient hyperperfusion in the cerebral cortex be- again impaired vascular autoregulation the pi- neath the hematoma due to impaired vascular au- votal factor and labile hypertension an important toregulation for long-term brain compression by cofactor of this type of complication. CSDH itself, leading to possible complications as Although CSDH drainage through burr hole cra- seizures, temporary acute agitated delirium and, in niostomy is considered a minor procedure, rare the most severe cases, intracerebral haemorrhage but fatal complications like brainstem haemorr- (22,23). The authors show how hyperperfusion hage may occur. Rapid brain decompression and observed 1 hour after CSDH drainage persisted, excessive amount of fluid drainage, hypertensi- albeit diminished, until 24 hours postoperatively on, cerebral amyloid angiopathy and coagulo- and disappeared on the third postoperative day. In pathies are the most likely factors related to this this setting, mean BP seemed to promote hyper- uncommon complication. Bilateral simultaneous perfusion during CSDH drainage, pointing out the and gradual drainage, strict monitoring of drai- importance of BP monitoring, particularly in the ned fluid and BP in the perioperative period and first 24 postoperative hours. frequent neurological assessment with prompt Indeed, due to reduced vascular compliance, labi- radiological assessment in case of clinical worse- le hypertension and increased small blood vessels ning should be the mainstay of a correct manage- fragility (in some cases related to amyloid angio- ment of CSDH (particularly if bilateral) in order pathy (21), elderly patients are more likely to de- to avoid potentially fatal complications. velop postoperative intracranial haemorrhage. FUNDING In the presented patient all precautions described were carefully observed during surgical procedu- No specific funding was received for this study. re and in the postoperative period. Nevertheless, perioperative BP peak occurred, making once TRANSPARENCY DECLARATION Competing interests: None to declare. REFERENCES 1. Richter HP, Klein HJ, Schäfer M. Chronic subdural 10. Rohde V, Graf G, Hassler W. Complications of burr- hematomas treated by enlarged burr-hole craniotomy hole craniotomy and closed-system drainage for chro- and closed system drainage. Retrospective study of nic subdural hematomas: a retrospective analysis of 120 patients. Acta Neurochir (Wien) 1984; 71:179– 376 patients. Neurosurg Rev 2002; 25:89-94. 88. 11. Weigel R, Schmiedek P, Krauss JK. Outcome of con- 2. Sambasivan M. An overview of chronic subdural hema- temporary surgery for chronic subdural hematoma: toma: experience with 2300 cases. Surg Neurol 1997; evidence based review. J Neurol Neurosurg Psychia- 47:418-22. try 2003; 74:937-43. 3. Harders A, Eggert HR, Weigel K. Treatment of chronic 12. Hyam JA, Turner J, Peterson D. Cerebellar haemorr- subdural hematoma by closed external drainage. Ne- hage after repeated burr hole evacuation for chronic urochirurgia 1982; 25:147–52. subdural haematoma. J Clin Neurosci 2007; 14:83-6. 4. Kalff R, Braun W. Chronic subdural hematoma – ope- 13. Liang CL, Rau CS, Lu K, Chen HJ. Contralateral acu- rative treatment by burr-hole trepanation. Zentralbl te subdural hematoma after burr-hole for chronic sub- Neurochir 1984; 45:210–8. dural hematoma. Injury 2001; 32:499-500. 5. Markwalder TM, Steinsiepe KF, Rohner M, Reiche- 14. Panourias IG, Skandalakis PN. Contralateral acute nbach W, Markwalder H. The course of chronic epidural hematoma following evacuation of a chronic subdural hematomas after burr-hole craniotomy and subdural hematoma with burr-hole craniostomy and closed-system drainage. J Neurosurg 1981; 55:390–6. continuous closed system drainage: a rare complicati- 6. Camel M, Grubb RL. Treatment of chronic subdural on. Clin Neurol Neurosurg 2006; 108:396-9. hematoma by twist-drill craniotomy with continuous 15. McKissock WRA, Bloom WH. Subdural hematoma. catheter drainage. J Neurosurg 1986; 65:183–7. A review of 389 cases. Lancet 1960; 1:1365-9. 7. Carlton CK, Saunders RL. Twist drill craniotomy and 16. Robinson RG. Chronic subdural hematoma: surgi- closed system drainage of chronic and subacute sub- cal management in 133 patients. J Neurosurg 1984; dural hematomas. Neurosurgery- 1983; 13:152–9. 61:263-8. 8. Tabbador K, Shulman K. Definitive treatment of chro- 17. Dinc C, Iplikcioglu AC, Bikmaz K, Navruz Y. Intrace- nic subdural hematoma by twist-drill craniotomy and rebral hemorrhage occurring at remote site following closed system drainage. J Neurosurg 1977; 46:220–6. evacuation of chronic subdural hematoma. Acta Neu- 9. D'Avella D, De Blasi F, Rotilio A, Pensabene V, Pando- rochir (Wien) 2008; 150:497-9. lfo N. Intracerebral hematoma following evacuation 18. Park KJ, Kang SH, Lee HK, Chung YG. Brain stem of chronic subdural hematomas. Report of two cases. hemorrhage following burr hole drainage for chronic J Neurosurg 1986; 65:710-2. subdural hematoma. Case report. Neurol Med Chir (Tokyo) 2009; 49:594-7.

141 Medicinski Glasnik, Volume 18, Number 1, February 2021

19. Cohen-Gadol AA. Remote contralateral intrapa- 21. Ogasawara K, Koshu K, Yoshimoto T, Ogawa A. renchymal hemorrhage after over drainage of a chro- Transient hyperemia immediately after rapid decom- nic subdural hematoma. Int J Surg Case Rep 2013; pression of chronic subdural hematoma. Neurosur- 4:834-6. gery 1999; 45:484-8. 20. Rusconi A, Sangiorgi S, Bifone L, Balbi S. Infrequ- 22. Bernstein M, Fleming JF, Deck JH. Cerebral hyper ent Hemorrhagic Complications Following Surgical perfusion after carotid endarterectomy: a cause of ce- Drainage of Chronic Subdural Hematomas. J Korean rebral hemorrhage. Neurosurgery 1984; 15:50-6. Neurosurg Soc 2015; 57:379-85. 23. Kieburtz K, Ricotta JJ, Moxley RT 3rd. Seizures following carotid endarterectomy. Arch Neurol 1990; 47:568-70.

142 ORIGINAL ARTICLE

Application of a personal Santini technique in the resolution of a complex celiac trunk aneurysm - endovascular treatment

Gianpaolo Santini1, Pasquale Quassone2, Luca Tarotto1, Francesco Arienzo1, Giuseppe Sarti1

1Vascular and Interventional Unit-P.O. Ospedale del Mare, ASL NA1 Centro, viale delle Metamorfosi, 2Department of Precision Medicine, University of Campania "L. Vanvitelli"; Naples, Italy

ABSTRACT

Aim Vascular have been already explored for the most of their aspects. It is a group of pathologies with unclear ethology and with an evolution in time not easy to forecast. Treatment gu- idelines are conflicting. The aim of this study was to describe cases in their most practical and technical aspect, especially in compli- cated conditions.

Methods This was a descriptive case report of a patient with a Corresponding author: hepatic artery aneurysm complicated by a dissection leading up Pasquale Quassone to the splenic artery, and how the team had invented a planned Department of Precision Medicine, treatment for the patient using a minimally invasive approach. The University of Campania "L. Vanvitelli" experience was born with the intention of showing how the endo- Via Vittorio Veneto 26, Marcianise, vascular approach is at least as safe as the traditional one despite Caserta, 81025, Naples, Italy the complexity of our case. Phone: +39 388 650 8712; Results The procedure was completed without any complications. Gianpaolo Santini ORCID: https://orcid. After a stay in long day surgery, the patient returned home. org/0000-0001-9573-6042 Conclusion Using a minimally invasive technique allows to redu- ce the patient's post-operative suffering and the economic burden on the health system.

Key words: aneurism, dissection, endovascular, hepatic artery, splenic artery Original submission: 23 October 2020; Revised submission: 06 November 2020; Accepted: 08 November 2020 doi: 10.17392/1302-21

Med Glas (Zenica) 2021; 18(1):143-147

143 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION PATIENT AND METHODS Visceral artery aneurysm (VAA) is an uncommon Patient and study design form of vascular pathology (1). Patients with VAAs present various symptoms up to sympto- A 56-years patient, male, arrived to our obser- maticity with acute rupture and abdominal pain, vation at the Emergency Department of the P.O. and haemorrhagic shock (1,2). Thanks to the Ospedale del Mare, Naples, during November increased usage of imaging such as ultrasound, 2019 for a violent nocturnal abdominal angina. CT and MRI, nowadays it is also possible to indi- Laboratory test revealed Hb 12g/dL, normal va- viduate VAAs as occasional reports (1,3). lues of AST/ALT, bilirubin 1.3mg/dL. Abdominal The VAAs have multiple aetiologies. They can be CT with contrast showed the presence of a celiac caused by trauma (2), inflammatory or infectious trunk dissection and an aneurysmal dilation of the conditions (2), and some iatrogenic post-surgi- hepatic artery, greater than 2cm; therefore, he was cal causes are also reported (2). In particular, an amenable to surgical treatment (Class IIa, level C, increased incidence of splenic artery aneurisms Società Italiana di Chirurgia Vascolare ed Endo- has been described in pregnant women, because vascolare - SICVE guidelines) (7) even though of an increased blood flow to the spleen (4). complicated by the splenic artery originated from the false lumen of the celiac trunk (Figure 1). Macroscopically, VAAs can be associated with splenomegaly, multiple pregnancies, portal hypertension, pancreatitis, coronary artery dise- ase, peptic ulcer or gastritis, obesity. The male to female ratio is 1:2 (4). Unlike the other VAAs, hepatic artery aneurism occurs most commonly in males rather than females, probably because of the differences in the pathogenesis of this form (4). Moreover, the prevalence of hepatic artery aneurism has increased due to the increase in the Figure 1. Angio-computed tomography (CT) cross sections show celiac trunk dissection (left); celiac trunk aneurysmal number of diagnostic and therapeutic procedures sac (right) (P.O. Ospedale del Mare, ASL NA1 Centro, 2019) in the biliary tract (5). After a consultation with vascular surgeons, it The vessels most frequently involved are: sple- was decided to perform a tailored endovascular nic (60%), hepatic (20%), superior mesenteric treatment for this patient. (5%) and celiac (4%) arteries; rarely gastroduo- denal, renal, pancreatic duodeal, jejunal, ileoco- The patient was informed in a clear and com- lic and inferior mesenteric arteries (1). In asso- prehensive way of three types of treatments and ciation with the dilatation of the vessel, there other possible surgical and conservative alterna- are some typical pathophysiological alterations: tives. A surgical consent confirmed that clinical fybrodysplasia, intimal thickening, sclerosis, data can be used for scientific studies but remain hypercholesterolemia (2). anonymous. In case of rupture, the mortality rate is between Methods 21% and 100% (from hepatic artery aneurysm The “Santini” technique - description. Throu- rupture to celiac one) (1). gh the same arterial access, two guides were in- Because of the VAAs discovering at autopsy, serted and then landed separately in hepatic and it is necessary to have a more aggressive dia- splenic arteries. gnostic and treatment approaches (1), and pa- Local anaesthesia (10 mL carbocaine 2%) was tients with a high risk of rupture such as pre- practiced in the groin before the femoral puncture. gnant or symptomatic, should be treated (3). The aim of this study was to report endovascu- An introducer (8F, Cook, Flexor, Germany) was lar surgical procedure of a patient presenting an positioned through the right femoral artery. Then, aneurysm of the hepatic artery complicated by a a selective arteriography of the celiac trunk was dissection of the splenic artery. executed and it showed a dissection at the origin

144 Santini et al. Celiac trunk aneurysm - endovascular treatment

of the trunk and the presence of a double lumen: a true one and a false one. From the false one, the splenic artery originated and the true one fed the aneurysmal sac of the hepatic artery (Figure 2).

Figure 4. A) Aneurysmal sac embolization with controlled re- leasing spirals; B) self-expanding covered stent positioning (P.O. Ospedale del Mare, ASL NA1 Centro, 2019)

RESULTS Figure 2. Pre-treatment digital subtraction angiography (DSA) angiographic control: contrast opacification of celiac trunk, Procedural angiographic control showed total splenic artery and hepatic artery aneurysm (left); lateral view exclusion from the circle of the aneurysmal sac of the contrast opacification of the aneurysm sac and a guide and the patency of the splenic artery (Figure 5). positioned in the splenic artery (right) (P.O. Ospedale del Mare, The procedural angiographic control showed an ASL NA1 Centro, 2019) opacification of the splenic artery that contained The false lumen and the splenic artery were the covered stent, the celiac tripod and the total catheterized and Supracore guide was introduced exclusion of the aneurysmal sac that appeared with the distal end at the splenic hilum (Figure embolized by the spirals (Figure 5). Then a ma- 3A); the aneurysmal sac was catheterized by nual compression manoeuvre was performed and micro-catheter (Progreat Terumo, USA) (Figu- a compression bandage applied in the inguinal re 3B). The aneurysmal sac was then embolized right area; after 24h under observation, the pati- with controlled realising spirals (Interlock, BS) ent was discharged, scheduling an angiographic and free spirals (Vortex, BS, USA) until a total CT control at one month. filling and exclusion from the circle (Figure 4A). The subsequent check-up showed the perfect succe- Afterwards, a self-expanding covered stent (8x60 ss of the operation, which was intended precisely not mm, Covera, Bard, USA) was hooked to the ori- to allow the traumatic lesions to expand (Figure 5). gin of the celiac trunk until the origin of the sple- Furthermore, by landing the covered stent before nic artery (Figure 4B). the birth of the left gastric artery, it was saved (Fi- gures 5).

DISCUSSION The VAA is a rare but potentially life-threatening disease entity. Early diagnosis and treatment are critical for the prevention of rupture. In the past years the increased use of ultrasound, CT and MRI and improved definition of the image have allowed the discovery of many cases of asymp- tomatic aneurysms (1). Despite a definitive dia- gnostic gold standard is not univocal, CT–angi- ography is highly accurate in the diagnosis and helps in determining the most correct approach, thanks to multiplanar CT reconstructions (8). Figure 3. A) Supracore guide at the splenic hilum; B) aneu- rysm micro-catheterization with Progreat Terumo (P.O. Osped- An indication for the treatment should derive ale del Mare, ASL NA1 Centro, 2019)

145 Medicinski Glasnik, Volume 18, Number 1, February 2021

from existing symptoms, or in cases of asympto- matic patients it can be determined based on the risk of rupture that corresponds with the diameter of the aneurysm (5). In the patient described above it was necessary to intervene with this new technique due to the presence of a double pathology: dissection and aneurysm on the celiac trunk with the splenic ar- tery originating from the false lumen. Some studies state that an aggressive open surgi- cal approach is justified despite the VAAs could be asymptomatic, because of low morbidity and mortality, and the endovascular approach has to be reserved for some cases not better specified (6). Due to considerable surgical trauma asso- ciated with a conventional open procedure and a surgical mortality rate that is not to be undere- stimated, the endovascular treatment of visceral artery aneurysms has significantly gained the im- portance (1). The indications for the elective treatment of vis- ceral artery aneurysm are made from a diameter of 2 cm (5). In case of isolated visceral artery dissection in a hemodynamically stable patient, medical therapy is primarily considered; in case of unstable lesi- ons, the surgical approach is preferred (6). This approach is supported by many authors who de- monstrated that hemodynamically unstable dis- sections gradually increase in length and tend to involve other visceral arterial vessels with a signi- ficant increase of mortality, with phenomena of liver thrombosis, rupture of arterial vessels, the- refore, the intervention is strongly suggested (5). In literature, due to the rarity of this pathology, there is a lack of studies comparing the outco- me between open or endovascular technique, although the interventional radiologic approach is increasingly chosen (3), not only as an emer- gency choice in the first instance, but also with, as in this case, a tailored treatment and programmed technique. The endovascular treatment of visce- ral aneurysm is already the treatment of choice in polytraumatized patients (6). In our case, an interventional radiological approach was chosen Figure 5. A) Post treatment angio-computed tomography (CT): the white arrow indicates the presence of the covered stent in accordance with recent guidelines (7). from celiac trunk to splenic artery; B) 3-D reconstruction of From the technical approach point of view, du- the post procedural angio-CT; C) Post treatment angiography shows the stent positioning and the spirals presence (P.O. Os- ring the setting, first the hepatic artery was embo- pedale del Mare, ASL NA1 Centro, 2019) lized through spirals. This approach was possible

146 Santini et al. Celiac trunk aneurysm - endovascular treatment

for two main reasons that consented to preserve cases for kicking a stream of sharing. Secondly, hepatic vascularization: the hepatic parenchyma to propose a new and never seen approach in tre- is supplied for 70% by the venous system and ating vascular pathologies using the mini-invasi- therefore it should not suffer of the lack of arte- ve approach and all of its advantages, thanks to rial supply, and we also performed a meticulous the endovascular surgery. Stetting graph offers a rescue of the left gastric artery allowing the liver potential benefit of maintaining splenic perfusion to be supplied by the gastro-duodenal circulation. while excluding the aneurysm, thereby elimina- The “Santini” technique was born “from the ting the risk of rupture (5). Moreover, because of need” to resolve the dissection of celiac-splenic the high mortality risk in case of rupture of VAAs relevance with the exclusion of the aneurysm. and dissections, the treatment by embolization Although the goal of our intervention was firstly or stenting is preferable as the first choice. Na- not focused on the treatment of splenic artery dis- turally, the support of vascular surgeons remains section, the treatment of tripod aneurysm allowed essential in case of complications, even during the resolution of both lesions. the endovascular procedure. All of the visceral aneurysms (VAAs) are an un- FUNDING common type of pathology, but to find a hepatic artery aneurysm complicated by a dissection of No specific funding was received for this study. the splenic artery, let us say it, is pretty rare. TRANSPARENCY DECLARATION The purpose of our case, as of all the literature about rare events, was initially to collect and show Conflict of interest: None to declare.

REFERENCES 1. Laganà D, Carrafiello G, Mangini M, Dionigi 6. Chiesa R, Astore D, Guzzo D, Frigerio S, Tshomba G,Caronno R, Castelli P, Fugazzola C. Multimodal Y, Castellano R, Liberato de Moura MR, Melissano approach to endovascular treatment of visceral ar- G. Visceral artery aneurisms. Ann Vasc Surg 2005; tery aneurysms and pseudoaneurysms. Eur J Radiol 19:42-8. 2006; 59:104-11. 7. Stillo F, Ebner H, Lanza G, Agus GB, Apperti M, 2. Abbas M, Stone W, Fowl R, Gloviczki P, Oldenburg Bernardini E, Bianchini G, Camparini S, Crespi A, W, Pairolero P, Hallett J, Bowe T, Panneton J, Cherry De Fiores A, Dorigo W, Emanuelli G, Ferrara F, Ge- K. Splenic artery aneurysms: two decades experience novese G, Giacomelli E, Giannasio B, Gossetti B, at Mayo Clinic. Ann Vasc Surg 2002; 16:442-9. Musiani A, Quarto G, Sellitti A, Spinelli GM. Linee 3. Tulsyan N, Kashyap V, Greenberg R, Sarac T, Clair guida sicve-sif societá italiana di chirurgia vascolare D, Pierce G, Ouriel K. The endovascular manage- ed endovascolare e societá italiana di flebologia [The ment of visceral artery aneurysms and pseudoaneu- 2016 Guidelines of the Italian Society for Vascular rysms. J Vasc Surg 2007; 45:276-83. and Endovascular Surgery (SICVE) and Italian So- 4. Panayiotopulps Y, Taylor P, Assadourian R. Aneu- ciety of Phlebology (SIF)] [In Italian] Italian Journal rysm of the visceral and renal arteries. Ann R Coll of Vascular and Endovascular Surgery 2016; 23:1-45. Surg Engl 1996, 78:412-9. 8. Sun G, Ding J, Lu Y, Li M, Li L, Li G, Zhang X. 5. Pitton M, Dappa E, Jungmann F, Kloeckner R, Comparison of standard and low tube voltage 320 Schotten S, Wirth G, Mittler J, Lang H, Mildenber- detector row volume CT angiography in detection ger P, Kreitner K, Oberholzer K, Dueber C. Visce- of intracranial aneurysms with digital subtraction ral artery aneurysms: Incidence, management, and angiography as gold standard. Acad Radiol 2012; outcome analysis in a tertiary care center over one 19:281-8. decade. Eur Radiol 2015; 25:2004-14.

147 ORIGINAL ARTICLE

Current status of localized submental fat treatment with sodium deoxicolate (ATX-101)

Cristina Ibáñez-Vicente1, Miguel Carrato-Gomez2, Luigi Meccariello3, Umberto Ripani3; Michele Bisaccia4

1Department of General Medicine, "Hospital de Getafe”, Madrid, Spain; 2Division of Orthopaedics and , “Complejo Hos- pitalario Universitario de Toledo", Toledo; Spain; 3Division of Anaesthesia, Analgesia and Intensive Care and Pain Therapy, Department of Emergency and Major Trauma, "Ospedali Riuniti di Ancona", Ancona, (Italy); 4Orthopedics and Traumatology Unit, Department of Surgical and Biomedical Science, S.M. Misericordia Hospital, University of Perugia, Sant'Andrea delle Fratte, Perugia; Italy

ABSTRACT

Aim Facial aesthetics is at present a concept intricately linked to the degree of self-esteem. Unwanted submental fat (SMF) leads to an unattractive submental profile. Sodium deoxicolate (ATX) -101 is the only injectable drug approved to decrease submental fat of moderate to severe intensity.

Methods We carried out a bibliographic review in PubMed using the key words: deoxycholic acid, ATX-101, and submental fat. Only complete articles published between 2009 and 2019, and focused on submental fat were reviewed, excluding those articles Corresponding author: relating to that spoke of deoxycholate in the treatment of fat in Michele Bisaccia other locations or in which deoxycholate was associated with other Orthopaedics and Traumatology Unit, drugs. Department of Surgical and Biomedical Results In several phase III clinical trials, injection of 2 mg/cm2 Science, S.M. Misericordia Hospital, deoxycholic acid in SMF has reduced moderate-severe fullne- University of Perugia, Sant'Andrea delle ss compared to the placebo group. These results were maintai- Fratte. Italy ned in most cases during a long follow-up period. Injections of Piazzale Menghini 1, 06100, Perugia, Italy deoxycholic acid are generally well tolerated, with limited adverse Phone +39 34 9352 4581; effects in the treatment area, with a mild and complete resolution without sequelae. However, not all patients with SMF are suitable E-mail: [email protected] for deoxycholic acid therapy, and therefore a proper selection is

Cristina Ibáñez-Vicente ORCID ID: https:// very important to achieve the desired aesthetic results. orcid.org/0000-0003-3391-8697 Conclusion Deoxycholic acid injections are effective and are a ge- nerally well-tolerated, minimally invasive option for the treatment Original submission: of moderate to severe intensity SMF in selected adults. 03 December 2020; Key words: adipocitolysis, double chin, lipolysis Revised submission: 04 December 2020; Accepted: 06 December 2020 doi: 10.17392/1328-21

Med Glas (Zenica) 2021; 18(1):148-152

148 Ibáñez-Vicente et al. Treatment of submental fat with ATX-101

INTRODUCTION a maximum of 50 per session). This can be done up to a maximum of 6 sessions, with a minimum In aesthetic medicine, many treatments are availa- separation of at least 4 weeks. Injections should ble as a result of the high demand of the popula- always be made at least 1 cm below the lower bor- tion related to dissatisfaction with their body ima- der of the mandible (from the angle of the mandi- ge. Likewise, there are numerous investigations ble to the chin) and in specifically marked areas with the objective to maintain standards of facial where submental fat accumulates (11). Histologi- symmetry and ideal body contours, such as the in- cal studies of tissues treated with DC show that troduction of new materials and techniques, espe- adipocyte lysis occurs on day 1; on day 3 there is cially those aimed to eliminate localized fat (1). evidence of acute local neutrophilic inflammation Specifically, at facial level, the ideal youthful face and macrophage infiltration occurs on day 7. Local shape has been described as an inverted triangle, inflammation resolves on day 28. All of these data being wide at the top and progressively narrower show that DC produces adipocyte lysis, causing a from the middle of the face to the chin. The shape histological phenomenon known as fat necrosis, and contour of the chin and neck play an important with massive release of triglycerides into the inter- role in facial aesthetics and the accumulation of stitium that consequently unleashes an inflamma- fat in these areas can make an individual appear tory response with infiltration of macrophages. It to be overweight and look older, causing low self- has been shown that DC does not cause the de- esteem (1). For this reason, regardless of age, an struction of other cells other than adipocytes (2,5). excess of localized fat in the submental area can Another cause of concern related to its use is any represent an aesthetic problem in women and men possible inflammatory response and damage cau- (2,3) since it can occur as a consequence of aging, se to other vital tissues, such as muscles or ner- genetic predisposition or be related to poor healthy ves. In clinical trials, the inflammatory response lifestyles, which, in addition, generally do not tend after DC infiltration (swelling, erythema, and to improve with weight reduction (1,2). bruising) has been shown to lead to repeated in- Sodium deoxycholate (ATX) -101was approved in flammation and thus potentially fibrosis (2). That 2015 by Health Canada and the US Food and Drug is why it is recommended that ATX-101 should Administration as an injectable drug for cosmetic be administered within 28-day intervals to allow purposes in the reduction of submental fat. The de- the resolution of the induced inflammation. velopment of ATX-101 as a pharmacological tre- Generally, the adverse effects observed in clini- atment for fat reduction was based on the findings cal trials were mostly minor, of small intensity of Rotunda et al. (4). From their study, deoxycholic and duration, and resolved without sequelae (2). acid (DC) was identified as the most active compo- To minimize adverse effects, it is very important nent responsible for the reduction of adipose tissue to use a strict ATX-101 injection protocol. (5-7), (8,9) and it is used for the reduction of mode- rate or severe submental fat in adults. In order to examine the current status of ATX- 101 in the treatment of localized adiposities in Deoxycholic acid is a natural constituent of bile the submental area, this paper will address, thro- salts in humans. Its detergent action derives from ugh a bibliographic review, the satisfaction and its ability to intercalate the hydroxyl residues clinical efficacy of treatments with ATX-101, as (-OH) in the hydrophobic zone of the lipid bilayer well as its safety and possible adverse effects. of the cell´s membrane, causing its disorganizati- on. Specifically, in vitro observations showed that It is convenient to clarify some scales that will be its administration on adipose tissue cells caused an mentioned in the results and that served as varia- alteration of the phospholipid bilayer in the cell bles in different studies of the European Union. membrane and led to the lysis of adipocytes [10]. These scales are: the Clinician-Reported Submen- tal Fat Rating Scale (CR-SMFRS) (Figure 1) that The administration of the DC solution must be measures the severity of submental fat and is sco- carried out in the submental area, ensuring that red by a physician; the Subject Self Rating Scale there is a sufficient amount of accumulated fat (SSRS) (Table 1) or the assessment of patient sa- between the dermis and the platysma muscle (pre- tisfaction, and the submental fat score reported by platysmal fat, more superficial). It needs to be 1 the patients with the Patient-Reported Submental cm of separation between each injection site (up to

149 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 1. Subject Self Rating Scale (SSRS) for the assessment observed that the maximum plasma concentrati- of patient satisfaction* on of DC rose rapidly, falling back to baseline en- Score† Patient satisfaction dogenous values at 24 hours. The most common 0 Extremely dissatisfied 1 Dissatisfied adverse events were pain, oedema, erythema, and 2 Slightly dissatisfied hematoma at the injection site, which appeared 3 Neither satisfied not dissatisfied on the same day of the injection and were mild. 4 Slightly satisfied 5 Satisfied In another phase I, multicentre study, Humphrey et 6 Extremely satisfied al. (15) supported this idea regarding the mecha- * Overall satisfaction with facial appearance evaluated by 7-point scale (12); †Patients with a score of 4 or higher are considered responders nism of action of DC. Before and after the abdo- minoplasty, the authors treated abdominal fat with Table 2. Submental fat score reported by the patients with the Patient-Reported Submental Fat Rating Scale (PRSMFRS) (13) ATX-101 and microscopically examined tissue Score Amount of submental fat biopsies. The authors found that ATX-101 exerts 0 No chin fat at all its primary effect, adipocytolysis, from day 1. Su- 1 A slight amount of chin fat bsequently, neutrophils invade the tissue (day 3), 2 A moderate amount of chin fat 3 A large amount of chin fat followed by macrophages (day 7) and followed by 4 A very large amount of chin fat fibroblasts (day 28). On day 28, the inflammati- on resolves, which justifies the approved 1-month Fat Rating Scale (PRSMFRS) was also assessed time interval between each treatment. (Table 2) (12,13) to examine the current status Two multicentre, randomized, double-blind, pla- of treatment of localized fat at the submental le- cebo-controlled studies (16,17) demonstrated vel with sodium deoxycholate (ATX1010) and to that a 2 mg/cm2 dose of ATX-101 had a consi- know the safety and most frequent adverse events stently higher efficacy over a 1 mg/cm2 dose. in clinical practice with ATX101. Furthermore, these studies showed that a dose greater than 4 mg/cm2 does not produce greater MATERIAL AND METHODS efficacy and produces more frequent and serio- A literature review was conducted through us adverse effects. In these studies, the 2 mg/cm2 PubMed. Key words used included deoxycholic dose was delivered by injections into the 0.2 mL acid, ATX-101, and submental fat. Only comple- submental fat pad, spaced 1 cm apart. Today, this te papers published between 2009 and 2019 and is the approved treatment protocol for ATX-101. focusing on submental fat were reviewed, exclu- The REFINE-1 study included 506 patients with ding those articles relating to deoxycholate in the moderate to severe submental fat, randomized and treatment of fat in other locations or in which treated with ATX-101 or placebo, for a minimum deoxycholate was associated with other drugs of 6 sessions. The subjects were mostly white, fe- (such as phosphatidylcholine). male, with ages around 50 years and an average Twenty sources were originally selected for the re- BMI of 29 kg/m2. The authors found that at 12 view, and additional sources from the original bi- weeks most patients treated with ATX-101 achie- bliographies were used to supplement this review. ved an improvement of ≥1 point in 5 categories of the CR-SMFRS PR-SMFRS scales (Figure 1, Ta- RESULTS ble 2) compared to those treated with placebo (70% Analysing the articles with ATX101, two phase I trials, two phase II trials and four phase III clinical trials were identified that were used for the analysis. Walker et al. (2015) (14) investigated the safety and pharmacokinetics of the maximum therapeu- tic dose of ATX-101 (100 mg). In this study, 24 patients, with previous measurements of endo- genous DC plasma levels, received subcutaneo- us injections of ATX-101 (2 mg / cm2) into the submental fat. Throughout 24 hours, the pharma- Figure 1. Clinician-Reported Submental Fat Rating Scale (CR- cokinetics were periodically reviewed and it was SMFRS) (13) (with the permission of Humphrey S)

150 Ibáñez-Vicente et al. Treatment of submental fat with ATX-101

vs. 18.6%; p<0.001). Similarly, the majority of pa- the common adverse effect in both groups, with tients treated with ATX-101 2 mg/cm2 achieved others being variable, such as swelling, numbne- an improvement of ≥2 points on the CR-SMFRS/ ss, erythema, and bruising. PR-SMFRS scales compared to those treated with In phase III, double-blind trial by Ascher et al. placebo (13.4% vs. 0%; p<0.001). Of these, 224 (19) randomized 360 patients with submental fat patients were reviewed by magnetic resonance and (moderate to severe) to receive ATX-101 (1 or it was observed that the number of patients trea- 2 mg/cm2) or placebo injections for 4 sessions. ted with ATX-101 who achieved 10% fat reducti- The main objective was the same as the study by on was eight times higher than those treated with Rzany et al. (18). At 12 weeks, 58.3% of the pa- placebo (46.3% vs. 5.3%; p<0.001). Regarding the tients treated with ATX-101 1 mg/cm2 and 62.3% adverse effects detected, they were mainly local re- of the patients treated with 2 mg/cm2 reached the actions (84.3% ATX-101 group vs. 69.0% placebo target on the CR-SMFR scale (compared to the group); more frequent in the first sessions that de- 34.5% of patients treated with placebo). creased with subsequent sessions (12). On the SSRS scale, 68.3% of patients treated The REFINE-2 study included 516 randomized with ATX-101 1 mg/cm2 and 64.8% of 2 mg/cm2 2 patients treated with ATX-101 2 mg/cm or pla- achieved the primary endpoint (compared with cebo, for a minimum of 6 sessions. Also, in this 29.3% of placebo). In the study of Rzany (2018) case, the patients were mostly white, women, the measurement of the caliber of submental fat around 50 years old and with an average BMI was not included. Adverse effects were similar to of 29.3 kg/m2. The authors found that a greater previous studies and included injection site pain, number of patients treated with ATX-101 achie- swelling, numbness, bruising, and induration. ved a ≥1-point improvement on the CR-SMFRS/ These were more common in the ATX-101 group PR-SMFRS scales after 12 weeks, compared to (99.2%) than the placebo group (78.9%) (19). patients treated with placebo (66.5% vs. 22.5%; In the REFINE-2 trial (13), mandibular marginal p<0.001). A total of 225 patients underwent MRI nerve palsy occurred in 4.3% of patients treated and also in this case, a 10% reduction in fat was with ATX-101 (compared with 0.8% of placebo), observed in most patients treated with ATX-101 with recovery of 7 to 61 days. To prevent marginal compared to placebo (40.2% vs. 5.2%; p<0.001). injury to the mandibular nerve, it is recommended Adverse events were mainly local reactions to avoid injections of ATX-101 over a line drawn (85.7% ATX-101 group vs. 76.9% placebo gro- 1.0-1.5 cm below the lower mandibular border. up); most resolved within 14 days and almost all Another potential complication is injection volu- resolved by the end of the study (13). me-related dysphagia. In the REFINE-2 trial, this Rzany et al. (2014) (18) treated 363 patients with occurred in 2.3% of patients treated with ATX-101 2 ATX-101 (1 or 2 mg/cm ) submental fat (mode- (compared with 0.4% of placebo). rate to severe grade), compared to placebo injec- tions, for 4 sessions. The primary endpoint was DISCUSSION an improvement of ≥ 1 point in the CR-SMFR To date, there are several articles that have de- and ≥4 points in the SSRS (subject self-rating monstrated the efficacy of non-surgical procedu- scale) (Table 1). The secondary objective was res to reduce submental fat deposits through the the reduction of the calibre of the submental fat. infiltration of substances with an adipocytolytic After a 12-week follow-up, 59.2% of patients effect. Reeds et al. in 2013 (20) states that injecti- 2 treated with ATX-101 1 mg/cm and 65.3% of ons of phosphatidylcholine and deoxycholate can 2 patients treated with 2 mg/cm achieved the pri- reduce the volume of abdominal fat and do not mary endpoint (compared with 23.0% placebo); appear to increase inflammation markers or affect 53.3% of patients treated with ATX-101 1mg/ glucose and lipid metabolism. The main advanta- 2 2 cm and 66.1% of patients treated with 2mg/cm ges of non-surgical fat reduction over the surgical reached the objective on the SSRS (compared technique are that it does not require anaesthe- to 28.7 % placebo). Furthermore, submental fat sia or hospitalization, it is cheaper, it produces caliper measurements showed a statistically si- fewer scars and it is a simple and fast procedure gnificant reduction. Pain at the injection site was (21). The ATX-101 was approved in 2015 as an

151 Medicinski Glasnik, Volume 18, Number 1, February 2021

injectable drug for cosmetic purposes in the re- effective in reducing unwanted localized adiposi- duction of submental fat (13). The development ties, demonstrating results from an objective po- of ATX-101 as a pharmacological treatment for int of view (use of submental circumference me- fat reduction was based on the findings of Rotun- asurements and through the use of ultrasound), da et al. (4), who demonstrated localized fat re- as well as from a subjective point of view from duction using an injectable phosphatidylcholine treated patients, using the SSRS scale. formulation. From their study, DC was identified Efficacy in the reduction of adipose tissue after as the most active component responsible for the the treatment with ATX-101 is maintained over reduction of adipose tissue (4,22,23). time (more than 5 years after treatment), not In clinical trials, the inflammatory response after requiring maintenance treatments to sustain the DC infiltration (swelling, erythema, and bruising) long-term effect or to reduce laxity of the skin. has been shown to lead to repeated inflammation The main adverse effects of ATX 101 are mild and thus potentially fibrosis (6,13,14). Therefore, it and transitory, and usually localized at the injec- is recommended that ATX-101 be administered at tion site (pain, swelling, oedema, and bruising). 28-day intervals to allow resolution of the induced They are more likely to appear in the first tre- inflammation. Furthermore, injection into tissues atment sessions and decrease in the following other than adipose tissue can lead to tissue necrosis sessions. Serious adverse effects are rare, inclu- (4,5). In general, the side effects observed in clini- ding superficial skin ulceration and mandibular cal trials were mostly minor, of small intensity and marginal nerve palsy, probably as a result of an duration, and resolved without sequelae (5,7,13). inappropriate injection technique or a high dose. To minimize adverse effects, it is very important to Therefore, to minimize the percentage of adverse use a strict ATX-101 injection protocol. effects, a good selection of patients, good tech- In conclusion, based on the bibliographic review, nique performance and the administration of an ATX-101 is the only drug currently authorized adequate dose are important. for the treatment of moderate to severe submental FUNDING fat. This treatment offers an alternative to invasi- ve measures such as liposuction, which presents No specific funding was received for this study. a greater number of complications, requiring hos- pitalization and anaesthesia. These aspects of the TRANSPARENCY DECLARATION liposuction turn out being unattractive for pati- Conflict of interest: None to declare. ents. Its use as an adipocytolytic agent is highly

REFERENCES 1. Schlessinger J, Weiss SR, Jewell M, Narurkar 5. Bechara FG, Sand M, Hoffmann K, Sand D, V, Weinkle S, Gold MH, Bazerkanian E. Percep- Altmeyer P, Stucker M. Fat tissue after lipolysis of tions and practices in submental fat treatment: a lipomas: a histopathological and immunohistoche- survey of physicians and patients. Skinmed 2013; mical study. J Cutan Pathol 2007; 34:552–7 11:27-31. 6. Kim JY, Kwon MS, Son J, Kang SW, Song Y. Se- 2. Georgesen C, Lipner SR. The development, eviden- lective effect of phosphatidylcholine on the lysis of ce, and current use of ATX-101 for the treatment of adipocytes. PLoS One 2017; 12:e0176722. submental fat. J Cosmet Dermatol 2017; 16:174–9. 7. Dayan SH, Humphrey S, Jones DH, Lizzul PF, Gro- 3. Honigman R, Castle DJ. Aging and cosmetic enhan- ss TM, Stauffer K, Beddingfield FC rd3 . Overview of cement. Clin Interv Aging 2006; 1:115-9. ATX-101 (deoxycholic acid injection): a nonsurgical 4. Rotunda AM, Suzuki H, Moy RL, Kolodney MS. approach for reduction of submental fat. Dermatol Detergent effects of sodium deoxycholate are a ma- Surg 2016; (Suppl 1):S263–70. jor feature of an injectable phosphatidylcholine for- 8. U. S. Food and Drug Administration. Kybella mulation used for localized fat dissolution. Dermatol (deoxycholic acid) injection. Surg 2004; 30:1001–8

152 ORIGINAL ARTICLE

Does low intensity direct current affect open fracture wound healing?

Yoyos Dias Ismiarto, Kemas Abdul Mutholib Luthfi, Mahyudin Mahyudin, Adriel Benedict

Department of Orthopaedics and Traumatology, Faculty of Medicine, Universitas Padjadjaran, Indonesia

ABSTRACT

Aim To explore the effects of a molecular pathway from the appli- cation of low-intensity direct current (LIDC) for wound healing through the pathway signalling growth factor and initiation of fi- broblast activation.

Methods This randomized clinical trial included 32 patients with open fracture wounds who came to Hasan Sadikin Hospital in Bandung, Indonesia. The patients were divided in the control and the treatment group. Extensive assessment of wound contractions, FGF2 and FGF7 levels, and fibroblast expression were evaluated before and after the treatment.

Corresponding author: Results This study showed a better wound area repair in the treat- ment group than the standard group, 3.17±0.11 and 0.78±0.07, re- Yoyos Dias Ismiarto spectively. The increase of FGF-2 level (42.69±3.5 and 15.09±1.8, Department of Orthopaedics and respectively), FGF-7 level (42.99±3.55 and 14.67±1.9, respec- Traumatology, Faculty of Medicine, tively), and fibroblast group expression (7.62±0.79 and 3.54±0.6, Universitas Padjadjaran respectively) were found to be higher in the treatment group (p Jl. Pasteur No.38, Pasteur, Sukajadi, <0.05). Bandung, West Java, Indonesia 40161 Conclusion Low-intensity direct current accelerates wound hea- Phone: +62 819 4958 1088; ling through the increase of growth factor and fibroblast activation. E-mail: [email protected] ORCID ID: https://orcid.org/0000-0003- Key words: fibroblast, fibroblast growth factor 2, fibroblast growth factor 7, open fracture, wound healing 2179-0104

Original submission: 24 July 2020; Revised submission: 09 September 2020; Accepted: 16 September 2020 doi: 10.17392/1241-21

Med Glas (Zenica) 2021; 18(1):153-157

153 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION current (AC), low voltage pulse current (LVPC) and transcutaneous electrical nerve stimulation Open wounds that are common in cases of open (TENS) (10,12). The LIDC is preferred because fracture trauma are infection-prone conditions (1). it has a better effect than other electric currents, Infection that occurs in these wounds will impact but until now there is no clear biomolecular res- the elongation of the inflammatory phase and inhi- ponse that can explain the mechanism of healing bit the wound healing process (2,3). The incidence of infected wounds by using LIDC (13-16). of infection in open wounds due to open fracture trauma is quite high at around 42.6% (1,2). As a This study seeks to explore therapeutic modali- result of an infection that may occur in these open ties electric in the process of accelerating open wounds causes slower wound healing, which will fracture open wound healing, and the effects of a further increase the risk of further and more severe molecular pathway from the application of LIDC infection to the extent that delays in the manage- for wound healing through pathway signalling ment will cause sepsis and death in patients (3). growth factors (FGF2 and FGF7) and initiation This condition shows that it is essential to explo- of fibroblast activation. This research is increa- re more comprehensive therapeutic modalities to singly important because the exploration con- accelerate the healing of open wounds in open ducted in this study is in clinical trials in human fracture cases so that the severe effects of the in- subjects, where exploration is still minimal. fection can be prevented (1,3). PATIENT AND METHODS Current therapies only focus on wound cleansing, and broad-spectrum antibiotics, because the most Patients and study design common cause of the infection in open wounds is gram-positive and harmful bacteria, especially This randomized clinical trial included 32 patients Staphylococcus aureus and Pseudomonas (3). with open fracture wounds, who came to Hasan Sadikin Hospital in Bandung, Indonesia, during The steps of healing of infected wound can be cla- the period between June and November 2019. ssified into three stages: the inflammation phase, the phase of cell addition, and wound healing (4). Initial assessments by physicians had been carried The infection will prolong the inflammatory pha- out to determine the patients with fracture injuries. se, and inhibit the transition into the cell addition Inclusion criteria for the patients were: over the phase, so that wound healing does not occur (3-5). age of 20 years, with infected wounds, had not In the inflammatory phase, macrophages and plate- received definitive therapy for open fracture ma- lets release several important mediators or growth nagement, and agreed to participate in the study factors, including FGF-2 protein and FGF-7 prote- by signing an informed consent. Exclusion crite- in (5,6). Both proteins play an essential role in the ria were: patients with severe chronic disorders healing process of infected wounds since they have such as diabetes mellitus, dyslipidaemia disor- broad-spectrum mitogenic abilities and help regu- ders, blood clotting disorders, immunocompro- late migration and target cell changes (7-9). mised disorders, and autoimmune disorders, and The proliferation of fibroblasts is essential in the patients who are pregnant or breastfeeding. process of wound healing (6,7). When granula- The patients were randomly divided (without tion tissue forms in dermal wounds, platelets, stratification) into two groups: a control group monocytes, and other cellular blood elements including the patients who had only got stan- release various growth factors to stimulate fi- dard antibiotic therapy for open wound of open broblasts that will migrate to the wound area and fracture, and a treatment group, i.e. patients who proliferate, to repair various connective tissue had got standard antibiotic therapy and LIDC components (10-12). Electromagnetic pulsation application. Extensive assessment of wound con- can help wound healing by enhancing new blood tractions, FGF2 and FGF7 levels, and fibroblast vessels, which will form new tissue to accelerate expression was evaluated on day one and day 14. wound healing (8,9). Ways of inducing electric The study was approved by the Hasan Sadi- current for wound healing to date are four types: kin Hospital Bandung Ethics Commission No. low-intensity direct current (LIDC), alternating LB.02.01/X.6.5/189/2019.

154 Ismiarto et al. LIDC affect wound healing

Randomization was carried out using compu- next stage, retrieval antigen was carried out with ter-generated random sequences to ensure equal the HIER (Heat-Induced Epitope Retrieval) met- allocation between the two groups carried out by hod (18), where the slides were put into a citrate the independent data centre's statistician at Hasan buffer solution, then heated at the temperature of Sadikin Hospital in Bandung, Indonesia. 95 oC for 60 minutes. Then, 1:700 (Cloud Clone, Hangzhou, China) fibronectin antibody was pain- Methods ted, followed by overnight incubation at 4oC. The The antibiotic used in this study was cefazolin next stage was to paint with a secondary anti- (the first-generation cephalosporin). body, Biotinylated-HRP (Horseradish Peroxida- The LIDC is a method of electromagnetic appli- se), incubation for 1 hour, at room temperature. cation by providing electrical current to an area Next, the administration of the chromogen was of 500uA using electrical stimulation ITO 320, carried out. Next, the dehydration process was which is applied to both electrodes continuously carried out again, using concentration and xylene within 2 hours using a DC flow battery (10,11) alcohol. Furthermore, mounting and evaluating fibronectin expression using ImageJ Software, Wound contraction rate. Aa assessment of rate of the percentage of fibronectin expression as a fi- wound contraction was done by the Gillman pro- broblast marker would be obtained. cedure (17), where wound area was measured by a digital calliper (Krisbow, Jakarta, Indonesia) so that Statistical analysis the area of the wound was obtained in mm2. Mea- Data processing began with the process of in- surements were made every day of observation. putting, editing, clearing, and coding. First, a Levels of growth factors. The assessment of descriptive and univariate analysis was per- FGF2 and FGF7 growth factor levels was carried formed, followed by a bivariate analysis to see out using the ELISA (Enzyme-Linked-Immuno- differences in the average growth factor levels, sorbent Assay) sandwich method according to differences in the average expression of fibrobla- the procedures contained in the FGF 2 and FGF sts, and the average extent of wound contracti- 7 Human ELISA Kit (Cloudclone, Hangzhou, ons. Bivariate analysis was performed using the China) manuals. Briefly, 50 μL of standard dilu- T-test, with p=0.05. ent or serum samples were added to wells that have been coated with anti-serotonin and incu- RESULTS bated at 37 °C for 30 minutes. After the plates This randomized clinical trial including 32 pati- were washed, 100 µL of biotinylated antibody ents with open fracture wounds and dividing into solution was added and incubated for 30 minutes the control group (the patients who only recei- at 37 °C. After three times washing, 50 uL avi- ved standard antibiotic therapy for open wound din-peroxidase complex solution was added and of open fracture), and the treatment group (pa- incubated for 15 minutes at 37 °C. Next step, 50 tients who received standard antibiotic therapy μL of tetramethylbenzidine colour solution was with LIDC application) were evaluated for wo- added and incubated in the dark for 15 minutes at und contractions, FGF2 and FGF7 levels, and fi- 37 °C. Finally, 50 uL stop solutions were added broblast expression on day one and day 14. to stop the reaction. Optical density (OD) values were measured using an ELISA reader (Biorad, The wound contraction area was more exten- Singapore), wavelength 450 nm. sive in the treatment group than in the control group (3.17±0.11 and 0.78±0.07, respectively) Fibroblast expression. Wound tissue samples (p<0.05). The addition of the area of wound con- were inserted into the next fixation fluid, dehydra- traction in the treatment group was significantly ted using alcohol and xylene, then paraffinized different compared to the control group (p<0.05) and cut as thick as 5 um using a rotary microtome (Table 1). (Leica, Illinois, USA). The following tissue was placed on the coated-object glass. Then, rehydra- The FGF-2, FGF-7 level, as well as fibroblast tion was carried out on the tissue using xylene expression, all were significantly increased after and alcohol with a concentration of 96%, 90%, the treatment in the treatment group comparing 80%, and 70% and rinsed with tap water. In the to the control group: for 42.69±3.5 and 15.09±1.8

155 Medicinski Glasnik, Volume 18, Number 1, February 2021

pg/mL (p<0.05), 42.99±3.55 and 14.67±1.9 pg/ the wound's edge with the central wound, which mL (p<0.05), and 7.62±0.79 and 3.54±0.6 mm2 ultimately accelerates wound closure (23-24). (p <0.05), respectively (Table 1). The use of the LIDC application is also believed to increase blood flow to the injured area through Table 1. Comparison of wound contraction area, FGF-2, FGF- the effect of heat regulation on the skin around the 7, fibroblast expression before and after the treatment wound (23). Adequate blood flow will accelerate Control group Treatment group the inflammatory process in the wound area by Variable Before After Before After treatment treatment treatment treatment accelerating the migration of various inflamma- Contraction area tory cells; both neutrophils and macrophages will 0.430.02 1.210.09 0.450.01 3.620.12 SD (mm2) produce various growth factors, including FGF FGF-2 16.3482 31.4362 16.4372 59.1222 SD (pg/mL) (23). Increased levels of growth factors will ini- FGF-7 tiate the process of vascularity and angiogenesis. 17.6582 32.3272 17.2244 60.2199 SD (pg/mL) The angiogenesis process will be followed by the Fibroblast expression 3.1122 7.6582 3.2523 10.8702 SD (%) activation of fibroblasts, where fibroblasts are FGF: Fibroblast Growth Factor precursor cells that play an essential role in pro- ducing collagen. Collagen produced by fibrobla- DISCUSSION sts will act as the primary substance that plays a This study shows that the application of LIDC in role in wound closure (25). open fracture open wounds had a very positive However, this study has limitations because it impact on the wound healing process. The LIDC used a minimal number of samples. application can increase wound healing accelera- In conclusion, low-intensity direct current was tion through increased production of growth fac- effective to accelerate wound healing through tors, FGF-2, and FGF-7. The fibroblast activation increased growth factor and fibroblast activation. process begins with an increase in growth factor Further research needs to be done with a larger production, which then spurs an increase in co- sample, so it is expected that low-intensity direct llagen production. This collagen will later act as current can be used as an additional therapy for a wound covering by forming a layer of web and wound healing. thread on the damage (19). The use of LIDC to initiate growth factor produc- ACKNOWLEDGEMENT tion is an exciting subject to study further. Some The authors would express their gratitude to the studies show that the use of electrical devices can Medical Research Unit of the Faculty of Medici- influence the electronegativity of cells (20-23). ne, Universitas Padjadjaran, Bandung, Indonesia. Changes in electronegativity in cells will stimu- late the release of sodium to extracellular in FUNDING the epidermis via the Na / K-ATPase pump. The No specific funding was received for this study. epidermis located on the central wound's edge becomes an electropositive area, while the cen- TRANSPARENCY DECLARATION tral wound area is electronegative. This condition will trigger the traction contraction force between Conflict of interest: None to declare.

REFERENCES 1. Ryan SP, Pugliano V. Controversies in initial ma- 5. Ashrafi M, Alonso-Rasgado T, Baguneid M, Bayat nagement of open fractures. Scand J Surg 2015; A. The efficacy of electrical stimulation in lower 103:132-7. extremity cutaneous wound healing: a systematic 2. Saldanha V, Tiedeken N, Godfrey B, Ingalls N. War- review. Exp Dermatol 2017; 26:171-8. time soft tissue coverage techniques for the deployed 6. Kai H, Yamauchi T, Ogawa Y, Tsubota A, Magome surgeon. Mil Med 2018; 183:247-54. T, Miyake T, Yamasaki K, Nishizawa M. Accelera- 3. Patzakis MJ, Levin LS, Zalavras CG, Marcus RE. ted wound healing on skin by electrical stimulation Principles of open fractures management. Instr with bioelectric plaster. Adv Heathc Mater 2017; 6: Course Lect 2018; 67:3-18. 22-5. 4. Hunckler J, Mel A. A current affair: electrotherapy in 7. Cullum, N, Liu Z. Therapeutic ultrasound for ve- wound healing. J Multidiscip Healthc 2017; 10:179- nous leg ulcers. Cochrane Database Syst Rev 2017; 94. 5:CD001180.

156 Ismiarto et al. LIDC affect wound healing

8. Rastogi A, Bhansali A, Ramachandran S. Efficacy 17. Vidal A, Zeron HM, Giacaman I, Romero SC, Lopez and safety of low-frequency, noncontact airborne SP, Oreliana SL, Concha M. A simple mathematical ultrasound therapy (Glibetac) for neuropatic diabetic model for wound closure evaluation. J Am Coll Clin foot ulcers: a randomized, double-blind, sham-con- Wound Spec 2015; 7:40-9. trol study. The International Journal of Lower Extre- 18. Vinod KR, Jones D, Udupa V. A simple and effecti- mity Wounds 2019; 18:81-8. ve heat induced antigen retrieval method. MethodsX 9. Greaves NS, Benatar B, Baguneid, M, Bayat, A. Sin- 2016; 3:315-9. gle-stage application of a novel decellularized der- 19. Thakral G, Lafontaine J, Najafi B, Talal TK, Kim P, mis for treatment-resistant lower limb ulcers: posi- Lavery LA. Electrical stimulation to accelerate wo- tive outcomes assessed by siascopy, laser perfusion, und healing. Diabet Foot Ankle 2013; 4. and 3D imaging, with sequential timed histological 20. Atalay C, Yilmaz KB. The effect of transcutaneo- analysis. Wound Repair Regen 2015; 21:813–22. us electrical nerve stimulation on postmastectomy 10. Petrofsky J, Lawson D, Prowse M, Suh HJ. Effects skin flap necrosis. Breast Cancer Res Treat 2019; of a 2-, 3- and 4-electrode stimulator design on cu- 117:611–4. rrent dispersion on the surface and into the limb du- 21. Blount AL, Foster S, Rapp DA, Wilcox R. The use ring electrical stimulation in controls and patients of bioelectric dressings in skin graft harvest sites: with wounds. J Med Eng Technol 2018; 32:485–97. a prospective case series. J Burn Care Res 2017; 11. Jünger M, Arnold, A, Zuder D, Stahl HW, Heising, S. 33:354–7. Local therapy and treatment costs of chronic, venous 22. Wirsing PG, Habrom AD, Zehnder TM, Friedli S, leg ulcers with electrical stimulation (Dermapulse): Blatti M. Wireless micro current stimulation—an a prospective, placebo controlled, double blind trial. innovative electrical stimulation method for the tre- Wound Repair Regen 2018; 16:480–7. atment of patients with leg and diabetic foot ulcers. 12. Isseroff RR, Dahle SE. Electrical stimulation the- Int Wound J 2015; 12:693-8. rapy and wound healing: where are we now? Adv 23. Ud-Din S, Perry D, Giddings P, Colthurst J, Zaman Wound Care 2016; 1:238–43. K, Cotton S, Whiteside S, Morris J, Bayat A. Electri- 13. Hoare JI, Rajnicek AM, McCaig CD, Barker RN, cal stimulation increases blood flow and haemoglo- Wilson HM. Electric fields are novel determinants of bin levels in acute cutaneous wounds without affec- human macrophage functions. J Leukoc Biol 2016; ting wound closure time: evidenced by non-invasive 99:1141–51. assessment of temporal biopsy wounds in human 14. Kim MS, Lee MH, Kwon BJ, Koo MA, Seon GM, volunteers. Exp Dermatol 2016; 2:758–64. Park JC. Golgi polarization plays a role in the di- 24. Santamato A, Panza F, Fortunato F, Portincasa A, rectional migration of neonatal dermal fibroblasts in- Frisardi V, Cassatella G, Valente M, Seripa D, Ra- duced by the direct current electric fields. Biochem nieri M, Fiore P. Effectiveness of the frequency Biophys Res Commun 2015; 460:255–260 rhythmic electrical modulation system for the tre- 15. Rouabhia M, Park H, Meng S, Derbali H, Zhang Z. atment of chronic and painful venous leg ulcers in Electrical stimulation promotes wound healing by older adults. Rejuvenation Res 2015; 15:281–7. enhancing dermal fibroblast activity and promoting 25. Felician FF, Yu R, Li M, Li C, Chen Q, Jiang Y, Tang myofibroblast transdifferentiation. PLoS One 2015; T, Qi W, Xu H. The wound healing potential of co- 8:e71660. llagen peptides derived from the jellyfish Rhopilema 16. Young S, Hampton S, Tadej M. Study to evaluate the esculentum. Chin J Traumatol 2019; 22:12-20. effect of low-intensity pulsed electrical currents on levels of oedema in chronic non-healing wounds. J Wound Care 2015; 20:370–3.

157 ORIGINAL ARTICLE

Intermittent traction therapy in the treatment of chronic low back pain

Edina Tanović1, Damir Čelik1, Āemil Omerović2, Vanda Zovko Omeragić3, Amila Jaganjac4, Hadžan Konjo4, Emina Rovčanin5, Hana Omerović6

1Clinic for Physical Medicine and Rehabilitation, 2Ortopaedics and Traumatology Clinic; Clinical Centre, University of Sarajevo, Sarajevo, 3Faculty of Health Studies, University of Mostar, Mostar, 4Faculty of Health Studies, University of Sarajevo, Sarajevo, 5Public Health Institution Pharmacies of Sarajevo, 6Private Health Institution Verdant Pharmacies, Sarajevo; Bosnia and Herzegovina

ABSTRACT

Aim To investigate the effect of intermittent traction therapy (ITT) on pain in patients with chronic low back pain (CLBP).

Methods A total of 81 patients with CLBP were included: expe- rimental group received ITT (n=40) and control group received conservative physical treatment (n=41) 10 times for two weeks. A visual analogue scale (VAS) was used for measuring low back pain.

Corresponding author: Results In the experimental group, 26 (out of 40; 65.0%) patients Edina Tanović were females, in the control group 20 (out of 41; 48.8%) were Clinic for Physical Medicine and females (p=0.141). In a within-group comparison, median of VAS Rehabilitation, value was significantly decreased in both groups after ITT. A comparison of pre-intervention and post-intervention VAS value Clinical Centre of University of Sarajevo showed no statistically significant difference. Females from the Bolnička 25, 71 000 Sarajevo, experimental group had a significantly greater decrease of VAS Bosnia and Herzegovina compared with females from the control group. Patients in the ITT Phone: +387 33 278 465; group with L5/S1 level of hernia had lower estimated marginal Fax: +387 33 297 803, mean of VAS scale compared to the control group, as well the E-mail: [email protected] patients with left side of leg pain. ORCID ID: https://orcid.org/0000-0002- Conclusion: Intermittent traction therapy is an effective treatment 9862-5003 for pain reduction in patients with chronic low back pain.

Key words: physical therapy, traction, visual analogue scale Original submission: 14 August 2020; Revised submission: 23 September 2020; Accepted: 23 October 2020 doi: 10.17392/1252-21

Med Glas (Zenica) 2021; 18(1):158-163

158 Tanović et al. Intermittent traction therapy

INTRODUCTION bone structure and relaxation, that also helps to relieve pain due to spinal dysfunction (13). Low back pain (LBP) is a health disorder of high epidemiological, medical and economical impor- Some systematic reviews have shown that lum- tance. LBP is defined as pain, muscle tension bar traction has little or no value on clinical and stiffness (1). Low back is the location where outcome of pain intensity and it does not appear the highest incidence of musculosceletal pain is to lead to quicker return to work among people observed (2). Approximately 80% of individu- with LBP with or without sciatica (14,15). These als experienced LBP during a part of their acti- conclusions show a position of lumbar traction ve lives (3). In most cases, patients’ back pain is in the current clinical practice (16). relieved within two weeks, but 20% of patients Mechanical traction in combination with exten- suffer from continuous pain that does not respond sion exercises was investigated by Fritz et al. to therapy, which is known as chronic low back and the results showed significant improvement pain (CLBP). Global CLBP incidence is 9442.5 compared to extension exercises alone for pati- per 100,000 (9%) of population (4). ents with acute LBP (17). Researches Prasad et CLBP is a common, long-lasting and disabling al. compared inversion traction in combination condition with high costs for a society (5). Direct with physical therapy and physical therapy alone healthcare costs are usually connected with sear- in patients who were waiting for surgery of disc ches for pain treatment. Indirect or societal costs herniation, and found that combined therapy in are usually secondary consequences of CLBP 77% of patients helped to avoid surgery, while that include morbidity or disability (6,7). Althou- 22% of patients that had only physical therapy gh direct healthcare costs such as medical specia- avoided surgery (18). list care and hospital costs are high, indirect costs Scientists also noticed that efficacy of lumbar are the highest cost factor for CLBP (6). traction therapy and physical therapy in patients The location of pain helps differ chronic disco- with LBP has been questioned (13). The CLBP genic low back pain (CDP) in clinical practice has not been investigated precisely in Bosnia and from other CLBP patients, which is more axial Herzegovina. and pain is severe (8). There is evidence that The aim of this study was to investigate the effect CDP more often starts at a younger age than other of intermittent traction therapy (ITT ) on pain in types of chronical pain (9). patients with CLBP and to investigate time and Traction is one of the physical therapy modali- traction power parameters on CLBP. ties used in the treatment of lumbar disc hernia (LDH), which can also be combined with other PATIENTS AND METHODS modalities (10). Lumbar traction is one of the oldest treatment modalities for LBP (10). Trac- Patients and study design tion in physiatry practice is used on the neck and A clinical prospective study included 81 patients back spine, and it can ensure to achieve separa- with CLBP admitted to the Clinic for Physical Me- tion of the joint surfaces, decreased disc protru- dicine and Rehabilitation, Clinical Centre of the sion, elongation in the soft tissues, relaxation in University of Sarajevo, during the period betwe- muscles, and mobilization in the joints (11). en September 2019 and March 2020. All patients As a result of separation of the joint surfaces, gave and signed their consent to be included in this the compression in the surrounding tissues can investigation. Inclusion criteria were the patients be removed. Investigating magnetic resonance with CLBP associated with lumbar disc herniation images of lumbar spine before and after 30 min (LDH) for more than three months, without lum- of horizontal traction of 42% body weight in bar spinal injection or lumbar surgery history, and participants without any LBP history, Chow et without previous physical therapy and rehabilita- al. showed that horizontal traction was effective tion session during the past six months. Exclusion treatment that increased the disc height of lower criteria were: cognitive dysfunction, neurological lumbar levels, particularly in the posterior regi- deficits, extruded and/or sequestrated LDH, spinal ons of the discs (12). Positive outcomes that can fusion, pregnancy, malignancy, spinal compression be achieved with ITT include improvement in the fracture, spondylolisthesis, aortic aneurysms, seve-

159 Medicinski Glasnik, Volume 18, Number 1, February 2021

re peripheral neuropathy, vertebral infection, rheu- RESULTS matic diseases, and moderate to severe depression. In the experimental group, 26 (out of 40; 65.0%) Methods were females, in the control group 20 (out of 41; 48.8%) were females (p=0.141). Patients were divided in two groups: experimen- There was no statistically significant difference tal group who received ITT (n=40) and control in the mean of age between the ITT and control group who received conservative physical tre- group, 51.9±13.2 and 50.0±10.5 years, respecti- atment (n=41). vely (p=0.099) (Table 1.). In the experimental group, thermotherapy (Solux, The most common hernia was at L5/S1 (Table 1, 2). bulb for optimal daylight illumination) was appli- ed five minutes before traction therapy and after In a within-group comparison, median of VAS ITT each patient rested for 15 minutes in a supine value was significantly decreased in the both gro- position. In the control group, isometric exercises ups (p<0.001) (Table 1) and electrotherapy were administered. Both grou- The direction of hernia that was the most effec- ps received therapy 10 times for two weeks. tive in both of groups was protrusion, in 78 The visual analogue scale (VAS) was used to (97.5%) patients. (Table 1). measure participants’ back pain at start and after After adjustment for pre-intervention VAS value, therapy. A 10 cm Visual Analog Scale (VAS) was there was no statistically significant difference in used to evaluate the pain severity (19). The pati- post-intervention VAS value between the two tre- ents were asked to mark the score corresponding atments, F (2, 78) = 2.893 (p=0.093; partial η2 = to their pain level on the pain scale, which was 0.036) (Table 1). between 0=no pain and 10=most severe pain be- Table 1. Demographic and clinical characteristics of patients fore and after therapy. No (%) of patients in the group Variable The patients’ body weights were taken with a ITT (n=40) Control (n=41) Males/females 14/26 21/20 weighing scale before the treatment. Traction was Age (years) applied to the patient lying on the table in the supi- Males 52.0 (IQR=45.0 to 63.0) 48.0 (IQR=40.0 to 58.0) ne position. A chest strap was fitted over the lower Females 51.5 (IQR=39.8 to 64.3) 51.5 (IQR=43.5 to 56.0) Level of hernia ribs, and a waist strap on anterior iliac crests. A sto- L1/L2 1 (2.1) 0 (0.0) ol was placed below the patient’s legs in such a way L2/L3 2 (4.2) 4 (8.2) that the hip and knees flexed to 90 degrees to reduce L3/L4 6 (12.8) 4 (8.2) L4/L5 17 (36.2) 23 (46.9) the patient's lumbar lordosis. Traction power on the L5/S1 21 (44.7) 18 (36.7) BTL-16 Plus traction device (Madrid, Spain) star- Multiple levels of hernia ted at the initial level of 15 kg and increased gradu- 1 30 (75.0) 34 (82.9) ally at a certain rate of 30% of body weights. The 2 9 (22.5) 7 (17.1) 3 1 (2.5) 0 (0.0) ratio between hold time and rest time was set at 2:1. Type of hernia Protrusion 39 (97.5) 39 (95.1) Statistical analysis Prolapse 1 (2.5) 0 (0.0) Extrusion 0 (0.0) 2 (4.9) Data are presented as mean±standard deviation Direction of hernia (N) or median with interquartile range (IQR, 25th Central/Other 6/34 18/23 The side of leg pain to 75th percentiles) dependent on normality of Right 9 (22.5) 12 (29.3) variables distribution. The Kolmogorov– Smir- Left 15 (37.5) 8 (19.5) nov test with a Lilliefors significance level was Both 16 (40.0) 21 (51.2) used for testing normality of distribution. In the ITT, intermittent traction therapy; case of categorical variables, absolute numbers Post-intervention VAS values in females were sta- and percentages were reported. ANCOVA was tistically significantly lower in the ITT group vs used to determine the effect of a difference in the the control group: mean difference of -1.229 (95% treatment on post-intervention of VAS after con- CI -2.026 to -0.431) mmol/L (p =0.003) F(1, 43) = 2 trolling for pre-intervention VAS value of pain 9.660 (p=0.003; partial η = 0.183) (Table 1). that was measured. p<0.05 was considered as si- There was no significant difference in VAS value gnificant. VAS scale was presented in MS Excel. for hernia L1 to L4, and hernia L4/L5. (Table 2).

160 Tanović et al. Intermittent traction therapy

Table 2. Adjusted and unadjusted intervention mean and vari- Patients in the ITT group with L5/S1 level of her- ability for post-intervention visual analogue scale (VAS) value nia had lower estimated marginal means of VAS with pre-intervention VAS value as a covariate by level of hernia scale compared to the control group (p=0.048). Level of Group VAS intervention mean p hernia (No of patients) (standard error) (Figure 2). ITT (9) 2.56 (0.44) 2.97 (049) L1 to L4 0.119 Control (8) 4.63 (0.51) 4.17 (052) 4.5 ITT (17) 2.7 (0.32) 3.06 (037) L4/L5 0.721 Control (21) 3.52 (0.34) 3.24 (034) 4.0 ITT (14) 2.36 (0.30) 2.69 (029) L5/S1 0.048 Control (12) 4.01 (032) 3.61 (032) 3.5

M, mean, SE, standard error; ITT, intermittent traction therapy; AS scale

V

Patients in the ITT group with L5/S1 level of her- 3.0 nia had lower estimated marginal means of VAS Mean of scale compared to the control group (p=0.048). 2.5 Patients in the ITT group with left side of leg pain 2.0 had lower estimated marginal means of VAS scale ITT group Control group compared to the control group (p=0.038) (Table 3) Observed Grand Mean Pain in leg was most often on the left side and Figure 2. Mean of Visual Analogue Scale (VAS) with 95% showed the highest decrease of VAS value after confidence interval in patients with L5/S1 level of hernia with intermittent traction therapy (ITT) and controls ITT (Table 3). *Covariates appearing in the model are evaluated at the VAS at start = 6.77 Table 3. Adjusted and unadjusted intervention mean and variability for post-intervention visual analogue scale (VAS) DISCUSSION values with pre-intervention VAS values as a covariate by side of leg pain In our study, post-intervention VAS values in Side of leg Group Mean (standard error) p females were statistically significantly lower in pain (No of patients) Unadjusted Adjusted the ITT group vs. the control group. In the ITT ITT (9) 1.67 (0.53) 2.45 (0.56) Right 0.627 Control (12) 3.42 (0.44) 2.83 (0.48) group, patients with L5/S1 level of hernia and ITT (15) 2.47 (0.30) 2.79 (0.32) patients with left side of leg pain had lower esti- Left 0.038 Control (8) 4.63 (0.40) 4.01 (0.44) mated marginal means of VAS scale compared to ITT (16) 3.12 (0.35) 3.42 (0.39) Both 0.682 the control group. Control (21) 3.86 (0.30) 3.63 (0.34) Chronic LBP is a complex disorder that must Females in the intermittent traction therapy (ITT) be managed with a multidisciplinary approach group had significantly greater decrease of VAS including physical and socioeconomic aspects values compared to the control group (p=0.003) of the illness (14). The leading causes of work (Figure 1). absence, unproductivity and disability before the age of 45 are LBP and sciatica in many countries. 10 Therefore, we did not include patients over the age of 65 in the study, which is similar to the re- 8 sults from other studies (20). Traction is a technique used to stretch soft tissues 6 and separate joint surfaces or bone fragments by the use of a pulling force (21). Many physicians

AS scale V 4 recommended the technique of traction for condi- tions such as protruded intervertebral discs, spinal 2 muscle spasm, and general pain and stiffness (19). Recent research and systematic reviews indicated 0 small samples of respondents and a general lack ITT (n=26) Control group (n=20) of high-quality studies. Traction for patients with VAS at start VAS after two weeks of therapy sciatica cannot be judged effective at present Figure 1. Visual Analogue Scale (VAS) values at start and af- either, due to inconsistent results and methodolo- ter two weeks of therapy in females with intermittent traction therapy (ITT) and controls gical problems in most studies (20).

161 Medicinski Glasnik, Volume 18, Number 1, February 2021

In a randomized-controlled study experimental expenses are even greater if LBP is a chronical group received ITT, exercises and ice packs, while problem (28, 29). Different physical therapy pro- the control group received only exercises and ice grams are often included in patients with chronic packs, the authors concluded that intermittent trac- LBP and they require great different implementati- tion had a big impact on pain reduction, although on costs. The key role in therapy of chronic LBP is this effect was not statistically significant (22). that it must be treated optimally in order to reduce The results of retrospective study conducted by this high financial and human cost (30). Macario et al. included 94 patients with chronic Research conducted at several orthopaedic cli- discogenic LBP and measured verbal numerical nics showed that lumbar traction as a therapy for pain intensity rating (NRS) by the scale using ITT patients with chronic LBP enables an immedia- and decompression (all patients received physical te positive response after traction (31). In other therapy that included hot pack application before studies, also conducted on several orthopaedic the treatment and ice application and stretching clinics, various traction delivery modes/parame- exercises after the treatment) showed that VAS ters in combination with multiple interventions scores were reduced from 6.1±2.3 to 0.9±1.2, and showed best results (32). the amount of analgesics was also decreased, whi- Professional characteristics that include educati- le every day activities were improved (23). on level and clinical specialist credentialing have Medication and physical therapy methods inclu- an important role in traction usage (32). ding traction have proven to be useful in pain re- Patients are important as well because the key lief. They also have a large impact on every day role in education and home-based exercise de- activities that include exercises and education pends on them. This is one of the most cost effec- that promotes functional restoration (14,23). tive approaches that was considered (33). Lumbar traction has been used previously for spi- In conclusion, our results provide evidence for nal disorders. Its mechanism of action is based on additional effects of traction compared with tra- relieving pain with separating the vertebrae. It re- ditional physical therapy in patients with persi- moves pressure or contact forces from injured ti- stent, nonspecific LBP. We suggest additional ssue, while it increases peripheral circulation by a different focus on back education and exercise massage effect, and reduces muscle spasm (24,25). therapy in the management of patients suffering The treatment for patients with low back pain is not from persistent LBP. These methods are effec- a static process, but rather a fluid one that changes tive and they include less burden on the health together with a clinical status of the patients. This care system. All patients should have instructions approach with traction therapy is supported with about correct posture principles in their daily ac- clinical experience and indirect evidence (26). tivities along with descriptions of recommended In our study, patients with LBP reported feeling therapeutic exercises. better as a result of both therapy programs, con- Further evidence of the effects of traction of servative physical therapy and ITT, but they still different modes, magnitudes and duration is experienced recurrence of pain at follow-up. Other required for a proper control of traction applied factors such as psycho-social or environmental are to different disc levels. not included in this study, although they may have an effect on perceiving chronic pain (27). FUNDING International studies have estimated that back pain No specific funding was received for this study. affects 65% to 80% of the population during the- ir life at least once. Costs of LBP are among the TRANSPARENCY DECLARATION greatest total costs of a health care system. The Competing interests: None to declare.

REFERENCES 1. Ay BK, Berk E, Demirel A, Nacitarhan V. The 2. Koçak FA, Tunç H, Sütbeyaz ST, Akkuş S, Köseoğlu correlation between neuropathic pain incidence and BF, Yılmaz E. Comparison of the short-term effects vitamin D levels in patients with chronic low back of the conventional motorized traction with non- pain. Ann Med Res 2019; 26:3037-41. surgical spinal decompression performed with a

162 Tanović et al. Intermittent traction therapy

DRX9000 device on pain, functionality, depression, on Board Review. New York: Springer Publishing and quality of life in patients with low back pain as- Company 2019:55-100. sociated with lumbar disc herniation: a single-blind 19. Petit A, Roquelaure Y. Low back pain, intervertebral randomized-controlled trial. Turk J Phys Med Re- disc and occupational diseases. Int J Occup Saf Er- hab 2018; 64:17-27. gon 2015; 21:15-9. 3. O'Sullivan SB, Schmitz TJ, Fulk G. Physical rehabili- 20. Arora S, Erosa S, Danesh H. Physical medicine and tation. Philadelphia: FA Davis Company, 2019; 1-20. rehabilitation. New York: Springer, Cham 2019: 4. Global Burden of Disease Study 2013 Collaborators. 143-51. Global, regional, and national incidence, prevalence, 21. El-Gendy SR. Impact of spinal decompression on and years lived with disability for 301 acute and pain in patients with chronic lumbar disc prolapse. chronic diseases and injuries in 188 countries, 1990– Int J Physiother 2015; 2:819-23. 2013: a systematic analysis for the Global Burden 22. Zhao L, Manchikanti L, Kaye AD, Abd-Elsayed of Disease Study 2013. Lancet 2015; 386:743–800. A. Treatment of discogenic low back pain: current 5. Hartvigsen J, Hancock MJ, Kongsted A, Louw Q, treatment strategies and future options—a literature Ferreira ML, Genevay S, Smeets RJ. What low back review. Curr Pain Headache Rep 2019; 23:84-6. pain is and why we need to pay attention. Lancet 23. Wong JJ, Côté P, Sutton DA, Randhawa K, Yu H, 2018; 391:2356-67. Varatharajan S, Goldgrub R, Nordin M, Gross DP, 6. Itz CJ, Ramaekers BLT, Van Kleef M, Dirksen Shearer M, Carroll LJ, Stern PJ, Ameis A, Shouterst CD. Medical specialists care and hospital costs S, Mior S, Stupar M, Varajtharajan T, Taylor Vaisey for low back pain in the Netherlands. Eur J Pain A. Clinical practice guidelines for the noninvasive 2017; 21:705-715. management of low back pain: A systematic review 7. Carvalho AR, Ribeiro Bertor WR, Briani RV, Za- by the Ontario Protocol for Traffic Injury Manage- nini GM, Silva LI, Andrade A, Peyré-Tartaruga L. ment (OPTIMa) Collaboration. Eur J Pain 2017; A. Effect of nonspecific chronic low back pain on 21:201-16. walking economy: an observational study. J Motor 24. Afolabi OT, Egwu MO, Mbada C, Afolabi AD. Behav 2016;48: 218-26. Comparative effectiveness of lumbar stabilisa- 8. Isaikin AI, Ivanova MA, Kavelina AV, Chernenko tion exercises and vertical oscillatory pressure in OA. Lumbar discogenic pain. Neurol, Neuropsych, the management of patients with chronic low back Psychosom 2016; 8 88-94. pain. Int J Phys Med Rehabil 2018; 6:2. 9. Yun YH, Lee BK, Yi JH, Seo DK. Effect of nerve 25. Alrwaily M, Timko M, Schneider M, Stevans J, Bise mobilization with intermittent cervical segment trac- C, Hariharan K, Delitto A. Treatment-based classi- tion on pain, range of motion, endurance, and disa- fication system for low back pain: revision and up- bility of cervical radiculopathy. Phys Therapy Rehab date. Phys Ther 2016; 96:1057–66. Science 2020; 9:149-54. 26. Ekediegwu EC, Chuka C, Nwosu I, Uchenwoke C, 10. Cheng YH, Hsu CY, Lin YN. The effect of mechani- Ekechukwu N. A Case series of non-surgical spinal cal traction on low back pain in patients with hernia- decompression as an adjunct to routine physiothera- ted intervertebral disks: a systemic review and meta- py management of patients with chronic mechanical analysis. Clinical Rehab 2020; 34:13-22. low back pain. J Spine 2019; 8:2. 11. Chow DHK, Yuen EMK, Xiao L, Leung MCP. Me- 27. Traeger A, Buchbinder R, Harris I, Maher C. Diag- chanical effects of traction on lumbar intervertebral nosis and management of low-back pain in primary discs: A magnetic resonance imaging study. Muscu- care. CMAJ 2017; 189: E1386-E1395. loskelet Sci Pract 2017; 29:78-83. 28. Foster NE, Anema JR, Cherkin D, Chou R, Cohen 12. Dupuis M, Duff E. Chronic low back pain: eviden- SP, Gross DP, Ferreira PH, Fritz JM, Koes BW, Peul ce-informed management considerations for nurse W, Turner JA, Maher CG. Prevention and treatment practitioners. J Nurse Pract 2019; 15:583-7. of low back pain: evidence, challenges, and promis- 13. Hall JA, Konstantinou K, Lewis M, Oppong R, ing directions. Lancet 2018; 391: 2368-83. Ogollah R, Jowett S. Systematic review of decision 29. De Oliveira IO, De Vasconcelos RA, Pilz B, Teixeira analytic modelling in economic evaluations of low PEP, De Faria Ferreira E, Mello W. Grossi DB. Pre- back pain and sciatica. Appl Health Econ Health Po- valence and reliability of treatment-based classifica- licy 2019:1-25. tion for subgrouping patients with low back pain. J 14. Alrwaily M, Almutiri M, Schneider M. Assessment Man Manip Ther 2018; 26:36-42. of variability in traction interventions for patients 30. Shigeru T, Hideki T, Sadao A, Keiji F, Tokuhide with low back pain: a systematic review. Chiropr D, Masami A. Lumbar mechanical traction: a bio- Man Therap 2018; 26:35. mechanical assessment of change at the lumbar spi- 15. Alrwaily M, Timko M, Schneider M, Stevans J, Bise ne. BMC Musculoskelet Disord 2019; 20:1-12. C, Hariharan K, Delitto A. Treatment-based classi- 31. Tadano S, Tanabe H, Arai S, Fujino K, Doi T, Akai fication system for low back pain: revision and up- M. Lumbar mechanical traction: A biomechanical date. Phys Ther 2016; 96:1057–66. assessment of change at the lumbar spine. BMC 16. Thackeray A, Fritz JM, Childs JD, Brennan GP. The Musculoskelet Disord 2019; 20:155. effectiveness of mechanical traction among subgro- 32. Madison JT, Hollman HJ. Lumbar traction for ma- ups of patients with low back pain and leg pain: a naging low back pain: a survey of physical therapists randomized trial. JOSPT 2016; 46:144-54. in the United States. JOSPT 2015; 45:586-95. 17. Kanji G, Menhinick P. Inversion therapy in patients 33. Şahin N, Karahan AY, Albayrak İ. Effectiveness of with pure single level lumbar discogenic disease: physical therapy and exercise on pain and function- a pilot randomized trial. Austral Musculo Med al status in patients with chronic low back pain: a 2017; 21:39. randomized-controlled trial. Turk J Phys Med Rehab 18. Cuccurullo SJ. Physical Medicine and Rehabilitati- 2018; 64:52.

163 ORIGINAL ARTICLE

Efficacy of Zhu’s acupuncture techniques to improve muscle strength of motion limbs in stroke patients

Indri Seta Septadina, Erial Bahar

Department of Anatomy, Faculty of Medicine Universitas Sriwijaya, Indonesia

ABSTRACT

Aim. To investigate the efficacy of Zhu acupuncture in motoric ability improvement of stroke patients. This research is a preli- minary study in an effort to test the efficacy of Zhu's acupuncture technique in motor improvement in stroke patients.

Methods. This quasi-experimental study, with the pretest and po- sttest approach without control group design was conducted from June to December 2019 at Holistica Medical Centre Complemen- tary Clinic Palembang, Indonesia. Manual Muscle Testing (MMT) was used before and after the intervention. Zhu’s acupuncture was given five times a week for four weeks. Corresponding author: Results Eight patients (four males and four females), who were in Department of Anatomy, the recovery phase (within a period of recovery less than 1 year) Faculty of Medicine, after stroke attack were involved. Seven patients experienced im- Universitas Sriwijaya, Indonesia provements in mild and moderate grades for shoulder joint move- Jl. Dr. Moh Ali KM 3.5 Sekip Madang, ment after the intervention. Regarding the elbow joint movement, 30162 Palembang, Indonesia the majority of patients (seven) experienced mild and moderate Phone: +62 878 371 608 09; improvements. Of the total patients who experienced improve- ment, the majority had mild grade improvements. E-mail: [email protected] ORCID ID: https://orcid.org/0000-0002- Conclusion Zhu’s acupuncture technique was effective in impro- 7024-4524 ving muscle strength of motion limbs on stroke patients.

Key words: acupuncture therapy, movement, muscle strength, stroke

Original submission: 28 February 2020; Revised submission: 28 May 2020; Accepted: 17 August 2020 doi: 10.17392/1152-21

Med Glas (Zenica) 2021; 18(1):164-169

164 Septadina IS and Bahar E. Efficacy of Zhu’s acupuncture in stroke

INTRODUCTION proving motor movements of the upper and lower extremities of patients who had a stroke. Also, Stroke is the second leading cause of death and this research is a preliminary study in an effort to the third highest cause of disability in the world. test the efficacy of Zhu's acupuncture technique In the past two decades, 16.7 million people have in motor improvement in stroke patients. suffered the first stroke attack, 33 million stroke survivors and 5.9 million deaths from strokes, PATIENTS AND METHODS with epidemiological numbers increasing every day (1). Data from the American Heart Associa- Patients and study design tion state that as many as 795,000 people expe- This research was a quasi-experimental study, with rience the first stroke or recurrent stroke every the pretest and posttest approach without control year (2). Stroke is not only life threatening, but group design. This research was conducted from also gives burdens for post-stroke recovery for June to December 2019 at Holistica Medical Cen- survivors. As many as 70% of stroke survivors ter Complementary Clinic Palembang, Indonesia. have functional disabilities and motor dysfunc- tion (3). The ability to interact socially and the The inclusion criteria of this study were stroke ability of daily living activities is limited due to patients aged 40-70 years, stroke diagnosed ba- the occurrence of motor dysfunction (3,4). Certa- sed on cerebral arterial thrombosis criteria in inly, this causes a large impact on the quality of Western medicine (7), stroke ischemia confirmed life of patients and impacts social activities in the by brain computed tomography (CT) or magne- community, which in the end does not only be- tic resonance imaging (MRI) scans followed by come a health problem but a social problem that a stable and good condition of the patient, the needs serious management (3). patient had just a stroke within 1-6 months after The conventional intervention of Western medici- the onset of the attack, stroke with motor limb ne in stroke patients is in the form of pharmacolo- dysfunction, patients had sufficient cognitive, gical management, surgical operation and multi- with a Mini-Mental State Examination (MMSE) professional rehabilitation (5). Management with score >24 (9), and willing to participate in the a multidisciplinary approach is carried out to im- study by signing an informed consent. prove functional disability, prevent complications Exclusion criteria were stroke with decreased and reduce the risk of stroke re-attacks (5,6). consciousness or experiencing serious cognitive Acupuncture is one of the alternative or comple- impairment, patients with other chronic disorders mentary therapies originating from China, where (Parkinson's disease, cardiac disease, cancers, alco- the use of this technique in the health world is holism or epilepsy), patients with kidney and liver increasing (6,7). It is believed that acupuncture is disorders, patients at risk of bleeding, oversensiti- effective in the management of motor dysfuncti- vity to acupuncture, participation in other research. on in stroke patients; even the National Institutes Patients were informed that participation in of Health (NIH) has recommended acupuncture the study was voluntary and the patients could as a complementary therapy for the management withdraw from the study at any time. In case of of motor dysfunction in stroke patients (7-9). The patients’ withdrawal from the study, the data will motor area of Zhu's​​ acupuncture or anterior oblique not be deleted, i.e. the patient’s data will still be line of the vertex -Temporal scalp acupuncture is a recorded as a medical record. An approval by in- technique commonly used in acupuncture therapy formed consent was made by each patient before in stimulating motoric dysfunction in stroke pati- the study start. This study was approved by the ents (10). The motor area of Zhu’s​​ acupuncture is Medical Research Unit Committee of the Faculty equivalent to the structure of the precentral cere- of Medicine, Universitas Sriwijaya, Indonesia. bral cortex gyrus on the scalp (10,11). Methods The aim of this study was to uncover the efficacy of acupuncture as a Chinese complementary me- A total of eight patients were examined by Manu- dicine technique in motoric ability improvement al Muscle Testing (MMT) before and after the in- of stroke patients. This study seeks to test the tervention. The intervention in the form of Zhu’s efficacy of Zhu’s acupuncture techniques in im- acupuncture was given to the patients five times

165 Medicinski Glasnik, Volume 18, Number 1, February 2021

a week for four weeks. During the intervention The procedure of the MTT was the following (12). with Zhu's acupuncture, all patients were still gi- For each muscle tested, the examiner stood to ven conventional rehabilitation treatment in the the side being tested, and the patient was sitting form of physical and occupational therapy. We- upright and in positions to allow full movement stern medicine therapy (11) was still permitted of the joint against gravity. The examiner demon- to be given in relation to symptomatic treatment, strated the desired movement against gravity. The such as antihypertensive drugs, lipid-lowering examiner then requested the patient to repeat the drugs and platelet aggregation inhibitors. The use motion. If the patient could move through the de- of herbal medicines was prohibited during the re- sired range of motion against gravity, the examiner search process. attempted to apply resistance in the testing posi- Zhu's acupuncture technique. Acupuncture in- tion while stating "Hold it, do not let me push it terventions follow the standard for reporting in- down" or "Hold it, do not let me bend it". Suppo- terventions in clinical trials of acupuncture guide- sing the patient tolerated no resistance, the muscle lines (10). Two reference parameters of the Zhu’s score is in Grade 3, if the patient tolerated some acupuncture technique were applied. 1) The area resistance, the score is Grade 4, and full resistan- of ​​intervention was the over anterior central con- ce, Grade 5. If the patient could not move against volution of the cerebral cortex. This point was gravity, the patient was repositioned to allow mo- a line starting from the point (known as the up- vement of the extremity with gravity eliminated. per point of the motor area) 0.5 cm posterior to If supporting the limb, the examiner provided the midpoint of the anterior-posterior midline of neither assistance nor resistance to the patient's the head and stretches diagonally to the junction voluntary movement. This gravity-eliminated po- between the brow-occipital line and the anterior sitioning was varied for each muscle tested. If the border. Next from the angle of the temporal hair- patient could not complete at least a partial range line, draw a vertical line up from the midpoint of of motion with gravity eliminated, the muscle or the zygomatic curve to the brow-occipital line; tendon was observed and palpated for contraction. the intersection of two lines is the projection Statistical analysis of the motor area. The motor area was divided into five equal parts: the top fifth is the area of​​ An univariate analysis was performed with pre- the lower limb motor area, the middle two-fifths sentation of data frequency distribution, bivariate were the upper limb motor area, and the bottom analysis to compare MMT values before​​ and after fifth was the face motor area. 2) Acupuncture the intervention with Student's T dependent test. interventions with disposable stainless steel nee- Statistical significance was adjusted at p<0.05. dles (0.25 mm×40 mm) (Huatuo, Suzhou Medi- cal Appliance, Suzhou, Jiangsu Province, China) RESULTS were manually inserted at an angle of about 15° The average age of eight patients involved in this to a depth of 1.0-1.5 cm along the top and middle study was 54.7 years, and they were still classified points of the motor area on the scalp. To over- as working age. All patients followed the research come motor dysfunction, the needle was rotated intervention process from beginning to end. All at least 200 revolutions per minute for 1 minute patients were employees both as civil servants and every 10 minutes for a total of 60 minutes. Scalp private employees. The majority of patients, six, acupuncture treatment was carried out by an in- had high education (bachelor degree) and two pa- dependent certified practitioner (acupuncturist) tients had low education (high school graduates). with 5 years of clinical experience. All patients were married and all spouses of pati- Outcome measure. The outcome assessment ents were still alive. All patients were with vital included a demographic status (age, gender, edu- signs (good condition), except two patients with cational background, marital status) and vital signs systolic blood pressure of 140 mmHg, but both in the form of pulse rate, respiratory rate, tempera- had taken antihypertensive drugs (Table 1). ture and blood pressure, as well as Manual Muscle The majority of patients, seven, experienced im- Testing (MMT) (11) in order to quantify the stren- provements in mild and moderate grades for sho- gth of the motor movements of the limbs. ulder joint movement after the intervention. Re-

166 Septadina IS and Bahar E. Efficacy of Zhu’s acupuncture in stroke

Table 1. Baseline characteristics the patients Shoulder joint movement showed statistically Characteristics Frequency significant improvement in the MMT score aro- Age (Mean ±SD) (years) 54.7 und 1.25 (p=0.015). The elbow joint movement Gender (No, %) Male 4 (50) showed an improvement in the score of around Female 4 (50) 1.25 points (p=0.016). The wrist joint of about Education (No, %) 2.25 (p=0.016), hip joint by about 1.5 points Low 2 (25) High 6 (75) (p=0.016), the knee joint by about 1.3 points Married (No, %) 8 (100) (p=0.016), and ankle joint movement improved Employement (No, %) score by 1.62 points (p=0.009) (Table 3). Civil servant (No, %) 6 (75) Private (No, %) 2 (25) Table 3. Manual Muscle Testing (MMT) score change Vital signs (Mean±SD) Muscle strength Pulse (Mean±SD 88.7 Joint type Motion p (Mean±SD) Respiratory rate 20.7±1.4 Abduction before 2,25 ± 1.16 Temperature 36.7±1.8 0.015 Sistolic blood pressure 130.7±10.2 Abduction after 3.50 ± 1.51 Adduction before 2.25 ± 1.16 Diastolic blood pressure 86.7±7.9 0.015 Adduction after 3.50 ± 1.51 Shoulder joint Flexion before 2.25 ± 1.16 0.015 garding the elbow joint movement, seven patients Flexion after 3.50 ± 1.51 Extension before 2.12 ± 1.12 experienced mild and moderate improvements 0.016 but of the total seven patients who experienced Extension after 3.43 ± 1.54 Flexion before 2.50 ± 1.30 0.016 improvement, the majority were of mild grade Flexion after 3.93 ± 1.65 Elbow joint Extension before 2.50 ±1.30 improvements. Regarding the improvement of 0.016 hip joint movement, seven patients experienced Extension after 3.75 ± 1.62 Flexion before 1.25 ± 1.58 0.016 mild and moderate improvements. The knee joint Flexion after 3.43 ± 1.45 Wrist joint movement also improved, where seven experi- Extension before 1.25 ±1.58 0.016 enced mild and moderate improvement. Ankle Extension after 3.43 ±1.45 Bridging before 2.75 ± 0.88 0,010 joint movement showed significant improvement Bridging after 4.37 ± 1.06 (p=0.009) where eight patients experienced mo- Abduction before 2.37 ± 1.30 0.016 derate grade improvement, and no patients wit- Abduction after 4.00 ± 1.77 Adduction before 2.37 ± 1.30 Hip joint 0.016 hout any improvement (Table 2). Adduction after 4.00 ± 1.77 Flexion before 2.37 ± 1.30 0.016 Table 2. Result of Manual Muscle Testing (MMT) in eight Flexion after 4.00 ± 1.77 patients Extension before 2.37 ± 1.30 0.016 Joint type and No (%) of patients with Extension after 4.00 ± 1.77 motion No improvement Mild (<2) Moderate (2-3) Flexion before 2.62 ± 1.18 0.016 Flexion after 4.00 ± 1.69 Shoulder joint movement Knee joint Extension before 2.50 ± 1.19 Abduction 1 (12.5) 4 (50) 3 (37.5) 0.016 Adduction 1 (12.5) 4 (50) 3 (37.5) Extension after 3.87 ± 1.72 Dorsiflexion before 2.00 ± 1.19 Flexion 1 (12.5) 4 (50) 3 (37.5) 0.009 Dorsiflexion after 3.62 ± 1.30 Extension 1 (12.5) 4 (50) 3 (37.5) Ankle joint Plantarflexion before 2.25 ± 1.16 Elbow joint movement 0.010 Flexion 1 (12.5) 5 (62.5) 2 (25) Plantarflexion after 3.75 ± 1.28 Extension 1 (12.5) 6 (75) 1 (12.5) Wrist joint movement DISCUSSION Flexion 1 (12.5) 1 (12.5) 5 (62.5) Extension 1 (12.5) 1 (12.5) 5 (62.5) Zhu's acupuncture technique is a contemporary Hip joint movement acupuncture technique that combines basic acu- Bridging 0 5 (62.5) 3 (37.5) Abduction 1 (12.5) 3 (37.5) 4 (50) puncture techniques and modern medical science Adduction 1 (12.5) 3 (37.5) 4 (50) by understanding the acupuncture points of the Flexion 1 (12.5) 3 (37.5) 4 (50) cerebral cortex area on the human scalp (13). The Extension 1 (12.5) 4 (50) 3 (37.5) Knee joint movement application of acupuncture in different treatments Flexion 1 (12.5) 4 (50) 3 (37.5) is growing, even now more and more physicians Extension 1 (12.5) 4 (50) 3 (37.5) are developing modern neurophysiology that Ankle joint movement Dorsal flexion 0 3 (37.5) 5 (62.5) utilizes acupuncture techniques and seeks to Plantarflexion 0 4 (50) 4 (50) explore the relationship of brain and human body

167 Medicinski Glasnik, Volume 18, Number 1, February 2021

(14). The Zhu acupuncture technique seeks to moving large muscles that move large joints (13- harmonize the brain and body (14,15). Based on 17). Likewise, it happens in the improvement of the principles of , there are lower motion where visible gradation differences meridian points that act as regulators of vario- in motion joint repair. us organs and limbs (10). Meridian is an energy Improved ankle joint MMT score was better than transfer circuit and pathway that connects vario- knee and hip joint motion repair in this study. us organs in the body (14). Stimulation of vario- However, there is also a difference in the repair us meridian points will trigger the activation of of the ankle and the wrist joint in the periphery, energy transfer, which will drive electrical acti- where the wrist joint has a much better improve- vity in the target organs connected to the meridi- ment than the ankle joint (13-17). This is caused an pathway (10-14). The meridians also connect by the muscles that build the wrist joint smaller peripheral organs such as the upper and lower than the muscles that make up the ankle joint. limbs to the cerebrum via the meridian pathway, The small muscles that build joint motion will or in modern medical science known as the spinal be more optimal because energy transfer through cord pathway (15). The GV 20 meridian is loca- the meridian points can be more optimal, and jo- ted in the vertex, it is believed that, if it is able int motion will be better than before acupuncture to stimulate, it will produce energy to the central (13-17). Several studies that are in line with this nervous system in the brain and be able to trigger study have shown the efficacy of acupuncture in electroactivity at motor points in the brain's pre- improving body function and quality of life in central gyrus. Electroactivity at the motor point stroke patients (18-22). at the centre of the motor in the cerebrum will However, this study has a limitation because it trigger the transfer of impulses to the peripheral included a small number of patients. upper and lower limbs (13). In conclusion, Zhu’s acupuncture technique was The results of this study showed that interventi- effective in improving muscle strength of motion on in stroke patients with the Zhu’s acupuncture limbs on stroke patients. Further research needs technique can improve the motion of various jo- to be done with a larger sample, so it is expected ints of the upper and lower limbs. Based on the that acupuncture can be used as additional the- results of this study it can be seen that there is a rapy for post stroke repair. gradation in the improvement of the motion stren- gth of the upper limbs between shoulder, elbow ACKNOWLEDGEMENT and wrist joints. The more peripheral location of The authors would like to express their sincere the joint motion, the better the joint motion im- gratitude to the Clinical Research Unit of Fa- provement was achieved. The improvement in culty of Medicine, Universitas Sriwijaya, Palem- wrist joint motion was far better than the repair bang, Indonesia, for the permission (No. 339/ in the elbow or shoulder joint. This can be related kptfkunsri-rsmh/2019). to the effectiveness and optimization of energy transfer through the meridian pathway. Periphe- FUNDING ral joints, wrist joints, are smooth motion joints that are moved by small motion muscles and con- No specific funding was received for this study. sequently, because they are moved by small mo- TRANSPARENCY DECLARATION tors, the transfer of energy given to move smooth motion muscles in peripheral joints is less than Conflict of interest: None to declare.

REFERENCES 1. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya 2. Murray CJL, Vos T, Lozano R, Naghavi M, Flaxman K, Aboyans V. Global and regional mortality from A, Michaud C, Ezzati M. Disability-adjusted life- 235 causes of death for 20 age groups in 1990 and years (DALYs) for 291 diseases and injuries in 21 2010: a systematic analysis for the Global Burden regions, 1990–2010: a systematic analysis for the of Disease Study 2010. Lancet 2012; 380:2095–128. Global Burden of Disease Study 2010. Lancet 2012; 380:2197–223.

168 Septadina IS and Bahar E. Efficacy of Zhu’s acupuncture in stroke

3. Feigin VL, Forouzanfar MH, Krishnamurthi R, 13. Bai L, Tian J, Zhong C, Xue T, You Y, Liu Z, Chen Mensah GA, Connor M, Bennett DA, Moran AE, P, Gong Q, Ai L, Qin W, Dai J, Liu Y. Acupuncture Sacco RL. Global and regional burden of stroke du- modulates temporal neural responses in wide bra- ring 1990– 2010: findings from the Global Burden in networks: evidence from fMRI study. Mol Pain of Disease Study 2010. Lancet 2014; 383:245–54. 2010; 61:73. 4. Roger VL, Go AS, Lloyd-Jones DM, Adams RJ, 14. Li L, Zhang H, Meng SQ, Qian HZ. An updated me- Berry JD, Brown TM, Carnethon MR, Dai S, Si- ta-analysis of the efficacy and safety of acupuncture mone G. Heart disease and stroke statistics—2011 treatment for cerebral infarction. PLoS One 2014; update: a report from the American Heart Associati- 9:e114057. on. Circulation 2011; 123:e18–209. 15. Wang HQ, Wang F, Liu JH, Dong GR. Introduction 5. He J, Gu D, Wu X, Reynolds K, Duan X, Yao C, on the schools of the scalp acupuncture for treatment Wang J, Chen CS, Chen J, Wildman RP, Klag M, of the stroke hemiplegia. Zhongguo Zhen Jiu 2010; Whelton PK. Major causes of death among men and 30:783–6. women in China. N Engl J Med 2005; 353:1124–34. 16. Xu XH, Zheng P, Wang FC. The comparison and 6. Zhao D, Liu J, Wang W, Zeng Z, Cheng J, Sun J, analysis of curative effect for three scalp acupunctu- Wu Z. Epidemiological transition of stroke in China: re school. Jilin J Tradit Chin Med 2007; 4:47–8. twenty-one-year observational study from the Sino- 17. Sze FK-H, Wong E, Kevin KH, Lau J, Woo J. Does MONICA-Beijing Project. Stroke 2008; 39:1668–74. acupuncture improve motor recovery after stroke? A 7. Liu M, Wu B, Wang WZ, Lee LM, Zhang SH, Kong meta-analysis of randomized controlled trials. Stro- LZ. Stroke in China: epidemiology, prevention, ke 2002; 3:2604–19. and management strategies. Lancet Neurol 2007; 18. Kong JC, Lee MS, Shin BC, Jang I, Park JJ. Acu- 6:456–64. puncture for function recovery after stroke: a syste- 8. Jia Q, Liu LP, Wang YJ. Stroke in China. Clin Exp matic review of sham-controlled randomized clini- Pharmacol Physiol 2010; 37:259–64. cal trials. CMAJ 2010; 82:1723–9. 9. Liu L, Wang D, Wong KS,Wang J. Stroke and stro- 19. Park J, White AR, James MA, Hemsley AG, Johnson ke care in China: huge burden, significant workload, P. Acupuncture for subacute stroke rehabilitation: a and a national priority. Stroke 2011; 42:3651–4. sham-controlled, subject- and assessor-blind, rando- 10. Jiao SF. Head Acupuncture. Beijing: Foreign Lan- mized trial. Arch Intern Med 2005; 165:2026–31. guages Press, 1993: 17–22 20. Wu P, Mills E, Moher D. Acupuncture in poststroke 11. Gu J, Wang Q, Wang XG, Li H, Gu M, Ming H, rehabilitation: a systematic review and meta-analysis Dong X, Yang K, Wu H. Assessment of registrati- of randomized trials. Stroke 2010; 41:e171–9. on information on methodological design of acu- 21. Lee SJ, Shin BC, Lee MS, Han CH, Kim JI. Scalp puncture RCTs: a review of 453 registration records acupuncture for stroke recovery: a systematic review retrieved from WHO International Clinical Trials and meta-analysis of randomized controlled trials. Registry Platform. Evid Based Complement Alternat Eur J Integr Med 2013; 2:87–99. Med 2014; 2014:614850. 22. Wang Y, Shen JG, Wang XM, Fu DL, Chen CY. 12. Ciesla N, Dinglas V, Fan E, Kho M, Kuramoto J, Scalp acupuncture for acute ischemic stroke: a meta- Needham D. Manual Muscle Testing: A method of analysis of randomized controlled trials. Evid Based measuring extremity muscle strength applied to cri- Complement Alternat Med 2012; 2012:480950. tically ill patients. J Vis Exp 2011; 2632.

169 ORIGINAL ARTICLE

The importance of education in patients with metabolic syndrome with regard to their knowledge and attitudes about the disease, and the impact of education on laboratory parameters

Azra Bureković, Elvira Āozo, Anida Divanović

Department of and Endocrinology, Clinical Centre, University of Sarajevo, Sarajevo, Bosnia and Herzegovina

ABSTRACT

Aim A metabolic syndrome (MetS) increases the risk of heart di- sease and diabetes mellitus type 2, thus the aim of this study is to correlate the clinical and laboratory parameters in patients suffe- ring from MetS who have previously had education compared to patients who have not had any education about a healthy lifestyle, and to check their knowledge and attitudes about healthy lifestyle.

Methods The study included patients of both genders aged 18 - 70 years, diagnosed with MetS, who are members of the Association Corresponding author: of Diabetics in Canton of Sarajevo. It used anthropometric para- Azra Bureković meters, laboratory findings, and an original, structured questionna- Department of Nuclear Medicine and ire about diet and frequency of physical activities. Endocrinology, Clinical Centre, Results Clinical and laboratory parameters did not differ signi- University of Sarajevo ficantly between educated and uneducated patients, as well as Bolnička 25, 71000 Sarajevo, habits in diet, physical activity, and knowledge about metabolic Bosnia and Herzegovina syndrome. The questions from the questionnaire have shown a Phone: +387 33 297 303; good value of variance, suggesting that the questionnaire can be Fax: +387 33 298 246; considered reliable. E-mail: [email protected] Conclusion This study showed that people with metabolic syn- ORCID ID: https://orcid.org/0000-0002- drome are not educated and motivated enough to change their life- 1158-510X style. The need for education of such patients is necessary. Key words: cardiovascular disease, dyslipidemia, type 2 diabetes mellitus Original submission: 16 October 2020; Accepted: 19 November 2020 doi: 10.17392/1291-21

Med Glas (Zenica) 2021; 18(1):170-175

170 Bureković et al. Education in patients with MetS

INTRODUCTION diet and exercise to the patient (12). If the patient has high blood pressure, it is recommended that Metabolic syndrome (MetS) is a group of dise- the blood pressure is about 140/90 mmHg, whi- ases that together increase the risk of metabolic le in patients with diabetes about 130/80 mmHg disorders such as insulin resistance, and diabetes (12). In patients older than 60 years, the goal sho- mellitus, atherosclerotic cardiovascular disease, uld be less than 150/90 mmHg (12). If the patient and cerebrovascular accident (1). To establish the has high lipids, it is necessary first to recommend diagnosis, it is necessary to meet at least three a diet, and if the diet has minimal effect, it is ne- criteria according to the International Diabetes cessary to start a drug treatment. The first line in Federation (IDF) (2). the treatment of hyperlipidemia is statins, however It is estimated that about 12–37% of the population fibrates, niacin, and omega acids are also availa- of Asia and 12–26% of the population of Europe ble in the treatment (13). Bariatric surgery is re- suffer from the metabolic syndrome (3,4). The pat- commended in patients with severe obesity. The hophysiological mechanisms of MetS are complex indication for bariatric surgery is a body mass in- and insulin resistance, neurohormonal activation, dex (BMI) ≥ 40 kg/m2 or in those with a BMI ≥ 35 and chronic inflammation are thought to be the kg/m2 and other comorbidities (14). main triggers in the progression of the metabolic The consequences of MetS on the health of pa- syndrome (5). Furthermore, genetic and acquired tients as well as on the entire health care system factors can influence the occurrence as well as the has not been sufficiently researched in Bosnia progression of MetS (6). Proinflammatory cytoki- and Herzegovina (B&H). nes released from adipose tissue are responsible for the development of atherosclerosis (6). Furthermo- The aim of this study was to determine the frequ- re, metabolic syndrome can also cause processes ency of education about healthy lifestyle (proper that cause liver damage by causing steatosis that diet and physical activity) in people suffering from can progress to non-alcoholic steatohepatitis, fibro- MetS, to correlate the clinical and laboratory pa- sis, cirrhosis, and hepatocellular carcinoma (7). rameters in the patients suffering from MetS who had previously had education compared to the To establish a diagnosis, it is first necessary to obta- patients who had not had any education about a in data from medical history. Furthermore, chec- healthy lifestyle, and to check knowledge and king the vital signs and appearance of the patient attitudes of the patients about the healthy lifestyle may lead physicians to suspect hypertension, dysli- through a pilot project analysis of a questionnai- pidemia, or other metabolic diseases. It is nece- re (about diet, and physical activity) by using the ssary to examine the patient about life habits with Likert scale in order to point out the problem of special reference to diet and physical activity (8). educating patients with MetS. This work would be Family history examination is necessary given the useful in the process of adopting a program to pro- strong influence of genetics on MetS. Moreover, a mote physical activity and proper nutrition. detailed physical examination is necessary, becau- se peripheral neuropathy, retinopathy, acanthosis PATIENTS AND METHODS nigricans may indicate metabolic disorders (9). A thorough medical history and physical examinati- Patients and study design on are the basis for establishing a MetS diagnosis. The study included 40 patients of both genders After medical history and physical examination, diagnosed with MetS, aged 18 - 70 years, and laboratory tests should be performed to confirm all are members of the Association of Diabetics the diagnosis. Blood pressure, lipid profile, and in Canton of Sarajevo. Patients were chosen by blood sugar should be determined first. Also, it is using random selection during the period from desirable to determine liver and kidney enzymes. September 2019 to January 2020. In patients in whom atherosclerotic changes are suspected, it is necessary to evaluate patients for Out of 40 patients, 29 previously had education signs of cardiac ischemia, infarct (10,11). about lifestyle by doctors and nurses, via leaflets, media, internet, books, and 11 patients previously MetS treatment should focus on treating the cause. had no education. All patients gave a written con- If the cause is obesity, it is necessary to recommend sent for participation in the study. The consent of

171 Medicinski Glasnik, Volume 18, Number 1, February 2021

the Board of Directors and the Ethics Committee Table 2. Questionnaire about diet and physical activity of the Association of Diabetics of Sarajevo Can- Claim* ton was obtained. Proper nutrition is characterized by moderation, diversity and balance All types of food can be an integral part of proper and a balanced Methods diet, provided that portion size is adapted to gender, age and type physical activity During the trainings at the Association of Diabe- Proper nutrition maintains good health, meets energy needs and tics in Canton of Sarajevo, the frequency of MetS enables performing physical activity criteria was determined according to the IDF de- Any increase in BW (body weight) increases the risk of dyslipidemia, high BP, cardiovascular diseases, carcinomas finition (2): waist circumference as a mandatory Proper nutrition can improve health criterion, whose values vary depending on ethni- With proper nutrition, they can reduce excess weight city (for Europeans it is ≥94 cm for males or ≥80 Reduced salt intake can reduce cardiovascular risk cm for females), and at least two additional criteria Reduced fat intake can reduce cardiovascular risk such as fasting blood glucose ≥5.6 mmol/L or ta- The increase in BW is due to the entry of a larger number of /more than needed king medications for hyperglycaemia, blood pre- Regular physical activity is a good way of calorie consumption ssure ≥130/85 mmHg, or taking medications for Physical activity has a positive effect on your health and helps you hypertension, triglycerides ≥1.7 mmol/L or taking feel good medications for hypertriglyceridemia, high-den- Cigarette consumption has a detrimental effect on your health Balanced and healthy diet, enough physical activities, stress-free life, sity lipoproteins <1.03 mmol/L for males or <1.29 avoiding smoking and alcohol, represents a healthy lifestyle mmol/L for females or taking medications. Lifestyle changes need to be introduced gradually and should become a part of everyday life The study used anthropometric parameters Offered answers were used for the evaluation according to the Likert (age, body weight, body height, body mass scale: 1- do not agree at all; 2 - disagree; 3 - neither agree nor disa- index, waist circumference, blood pressure), gree; 4 - agree; 5 - totally agree laboratory findings (sugar, cholesterol, high- density lipoproteins, low-density lipoproteins, Statistical analysis triglycerides) and the original, structured que- The χ2 test was used for statistical processing. stionnaires about diet (nutrition questionnaire), Cronbach’s alpha was used in order to assess the frequency of physical activities (questionnaire reliability. T-tests were used for the comparison about physical activity), knowledge and under- of independent samples between groups. The standing of patients, constructed for this rese- p<0.05 was used for statistical significance. arch. The Likert scale (15) with offered answers was used for the evaluation of answers: 1- do RESULTS not agree at all; 2 - disagree; 3 - neither agree nor disagree; 4 - agree; 5 - totally agree (Table There was no statistically significant difference 1, Table 2) in the patients’ age and gender between the grou- ps, without and with the education (p=0.1307 and p=0.3992, respectively). Table 1. Nutrition questionnaire for patients with and without education No statistically significant difference was recor- Question ded in the values of systolic and diastolic blo- How often do you eat simple carbohydrates (CH) (cakes, chocolate, od pressure in the group without the education honey, jam, other sweets, juices) in quantities greater than 100g? in relation to the group that had the education How often do you eat complex CHs (bread, pastries, potatoes, rice, pie, beans, pasta) in an amount greater than 300g? (p=0.9631 and p=0.8043, respectively), as well How often do you eat more than 300g of protein (lean meat, fish, lean as comparing the values of blood sugar, chole- cheese, eggs, skim milk)? sterol, high-density lipoproteins and low-density How often do you eat more than 100g of fat (oil, olive oil, butter, lipoproteins (Table 3). cream, greasy cheese, greasy milk, fatty meat)? How often do you eat more than 300g of fruit (all types of fruit)? The patients in the group with no previous edu- How often do you eat more than 300g of vegetables (except potatoes cation had higher waist circumference and BMI and beans)? compared to the group with previous education How often do you consume more than 5g of salt? How often do you drink more than one alcoholic beverage (1.5 dl of (p=00104 and p=0.0472, respectively) (Table 3) wine, 3 dl of beer and 0.3 dl of spirits)? Triglyceride values and excess body weight that *offered answers: not at all, every day, two times per week, once patients should lose in the group of patients who per month

172 Bureković et al. Education in patients with MetS

Table 3. Distribution of patients with and without education No statistically significant difference was found with clinical and laboratory parameters in the distribution of the patients' answers to Without education With education Para- the question on low intensity physical activity (11 patients) (29 patients) p meter (p=0.0918), moderate (p=0.0614), high intensity Min. Max. Mean SD Min. Max. Mean SD Age physical activity (p=0.6358) (questionnaire about 41 64 53.64 7.06 40 70 58.00 8.28 0.1307 (years) physical activity). WC 98 139 117.82 14.04 90 127 107.93 8.67 0.0104 (cm) The Cronbach’s alpha correlation matrix was 0.7834, while the 95% lower confidence limit BW (kg) 82 155 110.41 22.76 70 138 93.57 14.83 0.009 was 0.6916. The questions from the questionna- BH (m) 1.59 1.93 1.73 0.12 1.52 1.91 1.69 0.09 0.2821 ire showed a satisfactory correlation and had a BMI good value of variance, suggesting that the que- 27.4 50 37.01 7.90 25.4 45 32.66 5.15 0.0472 (kg/m2) stionnaire can be considered reliable. SBP 110 170 142.73 19.28 90 190 143.10 23.92 0.9631 (mmHg) DISCUSSION DBP 70 100 86.82 11.24 60 110 85.69 13.28 0.8043 (mmHg) This study is among a few studies showing the BP 4.5 21 10.77 4.60 4.3 28.8 9.68 6.30 0.6036 (mmHg) impact of education on MetS. CHL 3.3 13 6.11 2.88 2.5 7.1 5.21 1.01 0.1451 Our results showed that the mean age of patients (mmol/l) who had been previously educated was 58, while HDL 0.66 1.52 0.97 0.22 0.73 1.87 1.19 0.34 0.0566 (mmol/l) in patients who had not, it was 53.64. The study LDL 1.5 12.11 3.61 3.12 0.7 4.1 2.88 0.79 0.2465 conducted in China in 2018 showed that MetS (mmol/l) was more common in people over the age of 65 TGL 1.12 29.41 6.60 9.32 0.82 5.36 2.35 1.09 0.0183 (mmol/l) (16). Also, the study conducted in March 2020 EW (kg) 15 83 42.23 21.80 9 66 29.52 13.97 0.0348 showed that the prevalence of MetS was associa- Min., minimum; Max., maximum; WC, waist circumference; BW, ted with older age (17). body weight; BH, body height; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; BP, blood pressure; Waist circumference in educated patients in our CHL, cholesterol; HDL, high-density lipoprotein; LDL, low-density study was lower than in non-educated. Accor- lipoprotein; TGL, triglycerides; EW, excess weight ding to Fisher et al., higher WC will lead to had not had education were higher comparing to more serious MetS conditions (18). In both edu- the group that had had one earlier (p=0.0183 and cated and uneducated patients, the average BMI 2 p=0.0348, respectively) (Table 3). was above 30 kg/m . Also, a study conducted by Gierach et al. showed that BMI had a strong Out of 40 (100%), 29 patients (72.5%) had previo- linear correlation with WC, and that obesity in usly had education about lifestyle change and 11 males and even normal body weight in females patients (27.5%) had not undergone any education. corresponded to an increased volume of visceral There was no statistically significant differen- tissue in the abdomen (19). ce in the distribution of the patients' answers No significant difference in the distribution of the to the question whether they needed education patients' answers to the question of whether they about nutrition and physical activity (p=0.5921), needed education about nutrition and physical ac- whether patients intended to start "living healt- tivity was found in our study. On the other hand, a hily" (p=0.7473), about the intake of simple car- questionnaire study on knowledge and awareness bohydrates (p=0.4759), complex carbohydrates of the metabolic syndrome conducted in under- (p=0.5976), more than 300g proteins (p=0.8576), graduate medical students at entry-level, showed more than 300g fruits (p=0.2308) and more than that they were educated about the impact of physi- 300g vegetables (p=0.5454), consumption of cal activity and diet on MetS (20); however, this more than 5g of salt (p=0.95) and drinking more study included subjects who were much younger than one alcoholic beverage (p=0.941) (nutrition than our patients. Furthermore, a study examining questionnaire). knowledge about MetS syndrome in 204 hospita- A statistically significant difference between the lized patients showed that adults with cardiometa- groups was found only in the intake of more than bolic risk factors were at risk of developing MetS; 100g of fat (p=0.0148). they were with a low level of knowledge (21).

173 Medicinski Glasnik, Volume 18, Number 1, February 2021

Our patients with MetS are insufficiently edu- ted and uneducated patients, as well as habits in cated about their condition and they do not un- diet, physical activity, knowledge and attitudes. derstand the consequences of MetS. Similarly, a The questions from the questionnaire showed a MetS knowledge questionnaire was conducted satisfactory correlation with each other and had a among adult Chinese population, and proved that good value of variance, suggesting that the que- the population between the age of 18 and 65 was stionnaire can be considered reliable. The need not sufficiently familiar with MetS (22). for education of such patients is necessary. Edu- There are several limitations as well as benefits cation should be conducted by doctors and other regarding our study. The limitation is older age medical experts, media, as well as by care of the of the patients, and a short period of investigation entire community, because changing attitudes time. One of the greatest benefits is that it is the and adopting a healthy lifestyle will prevent high first study to examine the impact of educated and cardiovascular risk, premature morbidity, and uneducated patients on MetS in Bosnia and Her- mortality from cardiovascular diseases. zegovina, and to develop questionnaires that will FUNDING be useful for further clinical practice. In conclusion, patients diagnosed with MetS are No specific funding was received for this study. not sufficiently educated or motivated to change TRANSPARENCY DECLARANTIONS: their lifestyle. Clinical and laboratory parame- ters did not differ significantly between educa- Competing interests: None to declare. REFERENCES 1. van der Pal KC, Koopman ADM, Lakerveld J, van der 9. Goh VHH, Hart WG. Excess fat in the abdomen but Heijden AA, Elders PJ, Beulens JW, Ruters F. The not general obesity is associated with poorer meta- association between multiple sleep-related charac- bolic and cardiovascular health in premenopausal teristics and the metabolic syndrome in the general and postmenopausal Asian women. Maturitas 2018; population: the New Hoorn study. Sleep Med 2018; 107:33-8. 52:51-7. 10. Klimova B, Kuca K, Maresova P. Global view on 2. Ford ES. Prevalence of the metabolic syndrome defi- Alzheimer's disease and diabetes mellitus: threats, ned by the International Diabetes Federation among risks and treatment Alzheimer's disease and diabetes adults in the U.S. Diabetes Care 2005; 28:2745-9. mellitus. Curr Alzheimer Res 2018; 15:1277-82. 3. Sigit FS, Tahapary DL, Trompet S, Sartono E, Willems 11. Chiarelli F, Mohn A. Early diagnosis of metabolic van Dijk K, Rosendaal FR, de Mutstret R. The pre- syndrome in children. Lancet Child Adolesc Health valence of metabolic syndrome and its association 2017; 1:86-8. with body fat distribution in middle-aged individuals 12. Jung JY, Oh CM, Choi JM, Ryoo JH, Chung PW, from Indonesia and the Netherlands: a cross-sectional Hong HP, Park SK. Levels of systolic and diastolic analysis of two population-based studies. Diabetol blood pressure and their relation to incident metabolic Metab Syndr 2020; 12:2. syndrome. Cardiology 2019; 142:224-31. 4. Ranasinghe P, Mathangasinghe Y, Jayawardena R, 13. Jung JY, Oh CM, Ryoo JH, Choi JM, Choi YJ, Ham Hills AP, Misra A. Prevalence and trends of metabolic WT, Park SK. The influence of prehypertension, syndrome among adults in the asia-pacific region: a hypertension, and glycated hemoglobin on the deve- systematic review. BMC Public Health 2017; 17:101. lopment of type 2 diabetes mellitus in prediabetes: the 5. Rochlani Y, Pothineni NV, Kovelamudi S, Mehta JL. Korean Genome and Epidemiology Study (KoGES). Metabolic syndrome: pathophysiology, management, Endocrine 2018; 59:593-601. and modulation by natural compounds. Ther Adv 14. Cordero P, Li J, Oben JA. Bariatric surgery as a tre- Cardiovasc Dis 2017; 11:215-25. atment for metabolic syndrome. J R Coll Physicians 6. Matsuzawa Y, Funahashi T, Nakamura T. The concept Edinb 2017; 47:364-8. of metabolic syndrome: contribution of visceral fat 15. Sullivan GM, Artino AR, Jr. Analyzing and interpre- accumulation and its molecular mechanism. J Athe- ting data from likert-type scales. J Grad Med Educ roscler Thromb 2011; 18:629-39. 2013 ;5:541-2. 7. Chen S, Zhao X, Ran L, Wan J, Wang X, Qin Y, Shu 16. Jiang B, Zheng Y, Chen Y, Chen Y, Li Q, Zhu C, Wang F, Gao Y, Yuan L, Yhang Q, Mi M. Resveratrol im- N, Han B, Zhai H, Lin D, Lu Y. Age and gender-spe- proves insulin resistance, glucose and lipid metaboli- cific distribution of metabolic syndrome components sm in patients with non-alcoholic fatty liver disease: in East China: role of hypertriglyceridemia in the a randomized controlled trial. Dig Liver Dis 2015; SPECT-China study. Lipids Health Dis 2018; 17:92. 47:226-32. 17. Stephens CR, Easton JF, Robles-Cabrera A, Fossion 8. Kwon H, Kim D, Kim JS. Body fat distribution and the R, de la Cruz L, Martínez-Tapia R, Barajas-Martínez risk of incident metabolic syndrome: a longitudinal A, Hernández-Chávez A, López-Rivera JA, Rive- cohort study. Sci Rep 2017; 7:1-8. ra AL. The impact of education and age on metabolic disorders. Front Public Health 2020; 8:180.

174 Bureković et al. Education in patients with MetS

18. Fisher E, Brzezinski RY, Ehrenwald M, Shapira I, 20. Vemuri S, Desai K, Neha C, Reddy S. A questionnaire Zeltser D, Berliner S, Marcus Y, Shefer G, Naftali study on knowledge and awareness of metabolic syn- Stern N, Rogowski O, Halperin E, Rosset S, Shen- drome and it’s components in undergraduate medical har-Tsarfaty S. Increase of body mass index and wa- students at entry level. Res Pharm Sci 2020; 11:3645-56. ist circumference predicts development of metabolic 21. Wang Q, Chair SY, Wong EM-L, Taylor-Piliae RE, syndrome criteria in apparently healthy individuals Qiu XCH, Li XM. Metabolic syndrome knowledge with 2 and 5 years follow-up. Int J Obes (Lond) 2019; among adults with cardiometabolic risk factors: a 43:800-7. cross-sectional study. Int J Environ Res Public Health 19. Gierach M, Gierach J, Ewertowska M, Arndt A, Junik 2019; 16:159. R. Correlation between body mass index and waist 22. Lo SWS, Chair SY, Lee IFK. Knowledge of metabolic circumference in patients with metabolic syndrome. syndrome in Chinese adults: implications for health ISRN Endocrinology 2014; 2014:514589. education. Health Educ J 2015; 75:589-99.

175 ORIGINAL ARTICLE

Effects of semi-refined carrageenan (food additive E407a) on cell membranes of leukocytes assessed in vivo and in vitro

Anton Tkachenko1,2, Anatolii Onishchenko1.2, Alexander Roshal3, Oksana Nakonechna1, Tetyana Chu- machenko2, Yevgen Posokhov4

1Department of Biochemistry, Kharkiv National Medical University, 2Department of Epidemiology, Kharkiv National Medical University, 3Institute of , V.N. Karazin Kharkiv National University, 4Department of Organic Chemistry, Biochemistry and Microbiology, The National Technical University “Kharkiv Polytechnic Institute”; Kharkiv, Ukraine

ABSTRACT

Aim To assess the impact of semi-refined carrageenan (E407a) on hydrophobic regions of phosphololipid bilayer in cell membranes of leukocytes collected from rats orally administered this food additive and white blood cells incubated with E407a.

Methods Fluorescent probes (ortho-hydroxy derivatives of 2,5-diaryl-1,3-oxazole) were used to estimate the state of lipid bi- layer in leukocytes obtained from rats orally exposed to the food additive E407a and in white blood cells incubated with E407a.

Results No noticeable changes in the physico-chemical properties Corresponding author: were observed in the lipid membranes of leukocytes in the region Anton Tkachenko where the probes locate in response to oral intake of semi-refined Department of Biochemistry, carrageenan. Incubation of leukocytes with E407a solutions resul- Kharkiv National Medical University ted in a decrease in polarity and proton-donor ability of leukocytes Nauky av. 4, 61022 Kharkiv, Ukraine in the area of carbonyl groups of phospholipids and in the area of hydrocarbon chains of phospholipids near the polar region of the Phone: +380 50 109 45 54; bilayer. Fax: +380 57 700 41 32; E-mail: [email protected] Conclusion Membrane fluidity abnormalities found in cells expo- ORCHID ID: http://www.orcid.org/0000- sed to E407a are similar to those observed in patients with IBD suggesting that contribution of carrageenan to IBD development 0002-1029-1636 may be partially explained by leukocyte membrane modifications.

Key words: fluorescence, membrane fluidity, phospholipids, polysaccharides, rats

Original submission: 26 June 2020; Revised submission: 13 July 2020; Accepted: 17 August 2020 doi: 10.17392/1213-21

Med Glas (Zenica) 2021; 18(1):176-183

176 Tkachenko et al. Effects of E407a on leukocyte membranes

INTRODUCTION supports the view on IBD as a disease developing as a result of impaired intestinal homeostasis. Both animal and epidemiological studies have Furthermore, there is accumulating evidence that provided compelling evidence that dietary habits environmental exposures contribute to IBD patho- influence inflammatory bowel disease (IBD) de- genesis via modifying intestinal microbiota (16). velopment, including Crohn’s disease (CD) and ulcerative colitis (UC), and intensity of intestinal It is worth noting that IBD prevails in urban We- inflammation (1,2). The CD and UC are debili- sternized societies where the Western-style diet tating chronic inflammatory disorders of the gut characterized by high intake of pro-inflammatory clinically characterized by fatigue, diarrhoea, processed foods is common. This type of diet is pain in the abdomen, loss of weight, and rectal associated with consumption of food rich in satu- bleeding (3,4). Inflammation in UC is usually li- rated fats, carbohydrates with high glycaemic in- mited to the large bowel and affects mucosal and dex, and xenobiotics such as food additives (17). submucosal layers. CD, in its turn, can affect any One of such food additives that have drawn a lot region of the gut transmurally (5). Europe and of attention recently is carrageenan, which is a li- North America have a higher prevalence of IBD near polymer of carbohydrate nature isolated from compared to other regions of the world. However, marine red algae, primarily Kappaphycus alvarezii the trend towards an increase in the number of (18). Structurally, carrageenans are composed of CD and UC cases has been observed worldwide. D-galactose and 3,6-anhydrogalactose monosacc- In particular, the average prevalence rate of IBD haride units linked with α1,3- and β1,4-glycosidic increased from 79.5/100,000 persons in 1990 to bonds. There are three major types of carragee- 84.3/100,000 persons in 2017 (6). nans (kappa, lambda and iota). They differ in the number of sulfate groups and sulfate sites (19). Of note, IBD etiopathogenesis is complex. It com- Carrageenans in food industry are available as prises a combination of genetic predisposition with E407 (refined food-grade carrageenan) and E407a bacterial and environmental (primarily dietary) (semi-refined carrageenan). Both food additives are factors. In a simplified way, IBD can be considered used primarily to improve the texture of processed an alteration of gut homeostasis (7). Recent geno- foods, as well as thickeners and emulsifiers (18,19). me-wide association studies (GWAS) have shed According to WHO and FDA, the consumption light on genetically determined factors that might of carrageenans should be limited to 75 mg/kg of increase risks for IBD development. IBD suscep- weight daily, i.e. 5.25 g per an average 70-kg adult tibility single nucleotide polymorphisms (SNPs) (20,21). This temporary limitation has been recently have been identified and allowed confirming the introduced due to a number of papers reporting on involvement of autophagy, IL-17, IL-23, and type intestinal and extraintestinal inflammatory effects 3 innate lymphoid cells in IBD development (8). of orally consumed E407 and E407a (19,22,23). However, even prior to the implementation of There is accumulating evidence that TLR4 and GWAS approach that has revolutionized our un- Bcl10 pathways are involved in carrageenan-in- derstanding of associations between particular loci duced inflammation (24,25). The possible role of and diseases, SNPs in the nucleotide-binding oligo- carrageenans in IBD is confirmed by the simila- merization domain-containing protein 2 (NOD2) rity of intestinal morphological alterations caused gene were associated with susceptibility to IBD by the intake of carrageenan and IBD (17,26). In (9-11). NOD2 is an intracellular protein receptor addition, a recently conducted randomized trial has activated upon exposure to bacterial components demonstrated that carrageenan-free diet maintains and its downstream effects are activation of NF- remission in IBD (27). κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and release of cytokines (12,13). Another factor that may affect the course of IBD The NOD2 protein is expressed in monocyte/ma- is impaired enterocyte transcytosis that may pro- crophage lineage cells, Paneth cells and intestinal mote the transfer of luminal antigenic molecules epithelial cells playing a crucial role in the main- into the sterile lamina propria contributing to tenance of gut homeostasis and intestinal micro- antigenic overload. Cell membrane abnormaliti- biota-host interactions (12,14,15). The recognized es have been reported to alter transcytosis (28). impact of NOD2 gene mutations in IBD aetiology Surprisingly, data on cell membrane alterations

177 Medicinski Glasnik, Volume 18, Number 1, February 2021

in IBD is extremely scarce. It has been shown to the corresponding test tubes for a 4h incubation. that lipid composition of cell membranes in red All experimental procedures, accommodation blood cells (RBCs) and colonic mucosa even and care of animals were conducted in accordan- in uninflamed tissue in IBD is affected (29,30). ce with the European Convention for the Protec- Furthermore, cell membrane fluidity in erythro- tion of Vertebrate Animals used for Experimental cytes has been reported to be reduced in CD and other Scientific Purposes (EST 123), Direc- (30). Carrageenan has been also demonstrated tive 2010/63/EU for the Protection of Animals to affect cell membrane lipid order in RBCs and Used for Scientific Purposes, and Recommen- enterocytes upon oral exposure by experimental dation 2007/526/EC regarding guidelines for animals (31,32). However, there are no reports the accommodation and care of animals used concerning the effects of carrageenan intake on for experimental and other scientific purposes. phospholipid bilayer of leukocytes. The study design was approved by the Bioethics The aim of this study was to investigate the Committee of the Kharkiv National Medical Uni- effects of carrageenan oral intake on cell mem- versity (Kharkiv, Ukraine). branes of leukocytes using fluorescent probes. Methods MATERIALS AND METHODS Lyse/wash protocol. To analyse the state of le- ukocyte membranes by fluorescent probes, leu- Study design kocyte suspensions were prepared from freshly The study was performed at Kharkiv National collected blood samples in accordance with the Medical University and V.N. Karazin Kharkiv lyse/wash protocol provided by Becton Dickin- National University (Kharkiv, Ukraine) in De- son (2002). Briefly, 2 mL 1x FACSLyse soluti- cember 2019. on (Becton Dickinson, USA) was added to 100 A total of 24 female WAG rats weighing 160- μL blood placed in capped test tubes. To provide 190 g. were used, 16 of which were randomly lysis of erythrocytes, incubation for 15 minutes divided in into two groups: experimental (n=8) at 24 °C was performed. After centrifugation at and control (n=8). The animals from the experi- 500g for 5 minutes, the supernatant was discar- mental group ingested a solution of semi-refined ded. Then solutions were washed twice with 2 carrageenan at a final concentration of dry com- mL PBS. Cell pellets were resuspended in PBS ponents of 140 mg/kg of weight. The control ani- to form suspensions to which fluorescent probes mals received no semi-refined carrageenan. The O6O and PH7 were added. access to food and drinking water was ad libitum. Fluorescent probes O6O and PH7. The cells The rodents were housed in standard conditions. were fluorescently labelled by the same procedu- Acclimatization period lasted for 2 weeks. re: an aliquot of the probe stock solution in aceto- When animals were anesthetized and sacrificed, nitrile was added to the white blood cell (WBC) their blood was collected into sterile dipotassi- suspension to achieve a final probe concentration um ethylenediaminetetraacetate (EDTA)-conta- of ~5×10–6 mol/L. Lipid-to-probe molar ratio was ining vacutainer tubes; 100 mL blood from each ~200:1. Before fluorescence measurements, the animal of experimental and control groups was cell suspensions were incubated with the probe at lysed and washed in accordance with a protocol room temperature for 1 hour. (described below) to obtain leukocyte suspen- The fluorescence spectra for leukocyte suspensi- sions. In addition, blood was incubated with se- ons obtained from blood extracted from animals mi-refined carrageenan. Briefly, 2 mL blood was treated and untreated with E407a were recorded collected into the K2 EDTA vacutainer tubes to on a Thermo Scientific Lumina fluorescence prevent clotting. This volume obtained from each spectrometer (Thermo Fisher Scientific, Waltham, rat was equally distributed between four capped USA) in the range of 350-630 nm, with an incre- polystyrene test tubes, i.e. 500 μL blood per sam- ment of 0.1 nm. Data were collected with 0.02 s ple; 100 μL of phosphate buffered saline (PBS, pH interval. The slits on the excitation and emission 7.4) (Becton Dickinson, USA), 1%, 2% and 5% monochromators were 5 and 10 nm, respectively. solutions of semi-refined carrageenan were added The excitation wavelength was 330 nm.

178 Tkachenko et al. Effects of E407a on leukocyte membranes

For WBC suspensions incubated with semi-re- nvironment: e.g. with growth of polarity and/or fined carrageenan, the fluorescence spectra were proton-donor ability of the media, the ratio IT*/IN* measured on a fluorometer (Hitachi F850, Tokyo, decreases (34-36). Japan) in the range of 350-630 nm, with an incre- Since the increase in the hydration of the lipid bi- ment of 2 nm. The excitation wavelength was 330 layer leads to an increase in proton-donor ability and nm. The excitation and emission slits were 5 nm. polarity of the membrane medium (37,38), the pro- Fluorescent probes O6O (2-(2'-hydroxy- bes can be used to detect the changes in the hydra- phenyl)-5-(4'-biphenyl)-1,3-oxazole) and PH7 tion of lipid membranes (33). Taking into account (2-(2'-hydroxy-phenyl)-phenanthro[9,10-d]-1,3- that the changes in membrane hydration in their turn oxazole) were used, since its fluorescence para- are linked with the changes of the membrane lipid meters depend upon the polarity and proton-donor order (39-41), the probes can indicate the latter. ability of the microenvironment (33-36). Statistical analysis Probe O6O is located (Figure 1) in the area of carbonyl groups of phospholipids and in the area An assessment of the normality of data was per- of hydrocarbon chains of phospholipids near the formed by Shapiro-Wilk test. Based on its results, polar region of the bilayer, while probe PH7 is non-parametric tests were used to compare inde- localized (Figure 1) in the area of hydrocarbon pendent groups of variables. To analyse the results chains of phospholipids closer to the centre of the of an in vivo study, the Mann-Whitney U test was lipid bilayer (36). used. To evaluate differences between four inde- pendent groups in an in vitro study, the Kruskal– Wallis one-way analysis of variance was selected. It was followed by the Dunn’s multiple compari- sons test. p<0.05 was statistically significant.

RESULTS The spectra of fluorescent probe PH7 in leukocyte cell membranes differed negligibly between leu- kocytes treated and untreated with semi-refined carrageenan (Figure 2). Comparison of numerical

values of IT*/IN* ratios demonstrated no statistically significant difference (p>0.05) (Table 1) indica- ting that incubation of blood with E407a did not Table 1. The ratio of the fluorescence intensities of the photo Figure 1. Localization and orientation of fluorescent probes tautomer and normal forms (IT*/IN*) of probes O6O and PH7 in O6O and PH7 in phospholipid membranes. Two molecules of cell membranes of intact leukocytes incubated with different phosphatidylcholine from the outer leaflet are shown to de- concentrations of E407a note the localization of the probe (Adapted from Posokhov and Probe 060 Probe PH7 Kyrychenko) (36)

IT*/IN* IT*/IN* Animal group ratio (median; ratio (median; When the probes O6O and PH7 are in the exci- p* p* (interquartile (interquartile ted state, the excited state proton transfer reaction range) range) occurs (33-36). As a result of this reaction, photo Intact leukocytes untreated with semi- tautomer form (T*) is formed. The photoproduct 3.9 (3.6-4.2) 3.4 (3.0-3.8) refined carrageenan is fluorescent in significantly longer wavelengths (n=8) (control) in comparison with the initial (or so-called “nor- Intact leukocytes tre- mal”) form (N*) (33-36). ated with 1% E407a 5.5 (5.1-5.7) p<0.0001 3.6 (3.0-3.9) p>0.05 solution (n=8) The presence of two-band fluorescence enables Intact leukocytes us to conduct ratiometric measurement, i.e. to treated with 2% 4.8 (4.7-5.0) p<0.05 3.5 (3.30-3.7) p>0.05 use the ratio of the photo tautomer form and E407a solution (n=8) the initial form fluorescence intensities (I /I ) Intact leukocytes T* N* treated with 5% 5.8 (5.4-6.1) p<0.0001 3.7 (3.1-3.9) p>0.05 as a parameter for estimation of the changes in E407a solution (n=8) physical and chemical properties of the microe- *p as comparing to control group;

179 Medicinski Glasnik, Volume 18, Number 1, February 2021

Figure 2. Representative fluorescence spectra of A) probes Figure 3. Representative fluorescence spectra of probes O6O O6O and B) PH7 in leukocyte suspensions for the control group (panel A) and PH7 (panel B) in leukocyte suspensions: A) of rats (solid line), white blood cells exposed to the 1% solu- control group of rats (solid line), B) animals orally exposed to tion of E407a (dashed line), leukocytes treated with the 2% E407a during two weeks (dashed line). For better comparison, E407a solution and white blood cells incubated with the 5% the spectra were normalized to the fluorescence intensity of E407a solution (dash-dot line) the normal form affect the polarity and the proton-donor ability in difference between the ratios of the fluorescen- rat WBC cell membranes in the membrane area, ce intensities of the photo tautomer and normal where probe PH7 locates, i.e. in the hydrophobic forms (IT*/IN*) for probe O6O was found to be region closer to the centre of the lipid bilayer. statistically insignificant (Table 2). This suggests In the case of probe O6O, statistically significant that oral intake of E407a promoted no changes changes in the spectra of the probe were detec- in the polarity and the proton-donor ability in the lipid membranes of WBCs in the regions, where ted (Figure 2). The IT*/IN* ratios of probe O6O for WBC suspensions treated with E407a solutions probe O6O locates.

were statistically significantly higher compared Table 2. The ratio of the fluorescence intensities of the pho- with the control samples (Table 1). Any concen- totautomer and normal forms (IT*/IN*) of probes O6O and PH7 tration of semi-refined carrageenan used for in- in cell membranes of leukocytes obtained from rats orally cubation affected the fluorescence of probe O6O. administered semi-refined carrageenan Thus, our in vitro experiment demonstrated that Probe 060 Probe PH7 I */I * (medi- I */I * (medi- Animal group T N T N E407a influenced the state of phospholipid bi- an; (interquar- p* an; (interquar- p* layer in the regions, where probe O6O locates: tile range) tile range) i.e. in the area of carbonyl groups of phospho- 10.1 2.5 Control group (n=8) lipids and in the area of hydrocarbon chains of (9.4-10.5) (2.3-2.6) Rats orally exposed to 2.3 phospholipids near the polar region of the bilayer. 9.9 E407a during 2 weeks p>0.05 (2.2-2.5) p>0.05 (8.9-11.2) The spectra of fluorescent probe O6O bound to (n=8) the WBCs extracted from the rats treated with *p as comparing to control group; semi-refined carrageenan during two weeks co- The same trend was observed for probe PH7. The incided with the corresponding spectrum for the difference between the spectra of the probe bo- control group of animals (Figure 3). Hence, the und to the WBCs obtained from the rats orally

180 Tkachenko et al. Effects of E407a on leukocyte membranes

administered E407a during a fortnight was found of hydrocarbon chains of phospholipids near the to be negligible from the corresponding spectra polar region of the bilayer. The dehydration, in of the probe embedded in the cell membranes its turn, indicates an increase in the lipid order of leukocytes from the control group (Figure 3). (i.e. a decrease in fluidity) of WBC membranes In the case of probe PH7, the ratios of the flu- exposed to high doses of E407a. We believe that orescence intensities of the photo tautomer and the increased lipid order of membranes in WBCs normal forms (IT*/IN*) did not differ significantly treated with high concentrations of E407a might (p>0.05) from the corresponding ratios calcu- be attributed to direct electrostatic interactions lated for the control group (Table 2). Thus, in of negatively charged carrageenan molecules, comparison with the control group, no significant which are characterized by the high level of changes in the polarity and the proton-donor abi- sulfate ion with outer leaflets of cell membra- lity were observed in the experimental group in nes. Furthermore, our observation of membrane the membrane area, where probe PH7 locates. dehydration and, thus, the increase in the mem- An incubation of WBCs with E407a induced brane lipid order, may develop in response to li- changes in cell membranes in the regions where pid peroxidation (46,47), since polyunsaturated probe O6O is located without affecting those areas fatty acids (PUFAs) found in cell membranes are where probe PH7 locates, while oral consumpti- prone to free radical oxidation caused by reactive on of this food additive produced no effect on the oxygen species (ROS) whose overgeneration by corresponding regions of leukocyte lipid bilayer. neutrophils under the influence of carrageenan has been reported (48). DISCUSSION Our experimental results in an in vivo study indi- Cell membranes are complex lipid-protein structu- cate that no changes in the membrane hydration res acting as semi-permeable barriers. However, were detected in the hydrophobic area adjacent in addition to the barrier function, they perform to the polar region and the hydrophobic region a crucial role in cells mediating interaction and closer to the centre of the lipid bilayer of the cell communication with the extracellular envi- membranes of leukocytes in the rats orally expo- ronment. Membrane fluidity whose maintenance sed to the common food additive E407a during is of paramount importance for such interactions two weeks. The discussed lack of the changes in and, thus, modulation of cellular functions is de- the membrane hydration points to the absence of termined by a number of factors, including fatty changes in the lipid order of the hydrophobic re- acid composition of phospholipids in bilayer and gion of the leukocyte membrane. the percentage of cholesterol (42). It is important However, it is worth mentioning that the absence to note that over 30% of all cellular proteins are of structural changes in rather hydrophobic regi- membrane-embedded. Thus, changes in membra- ons of leukocyte lipid bilayer in rats orally expo- ne fluidity alter lateral mobility of these proteins, sed to the common food additive E407a during protein-protein interactions and protein diffusi- two weeks does not exclude the impact of oral vity, which affects signalling from membrane- carrageenan consumption on leukocyte membra- embedded receptors and, hence, response of cells nes. Earlier we demonstrated that oral admini- to environmental challenges (42,43). Experimen- stration of E407a affected less hydrophobic re- tal evidence also supports the role of membrane gions of leukocyte cell membranes (Tkachenko fluidity in leukocytes. In particular, cell membra- AS, personal communication 2020): the increase nes are involved in extravasation of neutrophils, in the polarity and the proton-donor ability of including interactions with vascular endothelium, the microenvironment of fluorescent probe O1O rolling, adhesion, and diapedesis (44). In addition, (2-(2'-hydroxy-phenyl)-5-phenyl-1,3-oxazole) there is evidence that changes in membrane lipid pointed to an increase in hydration in the area of order (membrane fluidity) reduce leukocyte moti- glycerol backbones of phospholipids and thus, su- lity and, thus, chemotaxis (45). ggested the decrease of the membrane lipid order. Thus, our findings suggest a higher dehydration In conclusion, E407a has impact on leukocyte of leukocyte membranes of rats in the area of membrane both in vivo and in vitro. It should carbonyl groups of phospholipids and in the area be noted that in the case of direct exposure to

181 Medicinski Glasnik, Volume 18, Number 1, February 2021

carrageenan more hydrophobic regions are affec- FUNDING ted compared with the effects of orally consumed The study was funded by the Ministry of Health carrageenan. Cell membrane fluidity abnormali- of Ukraine using the funds provided by the state ties found in this study are similar to those obser- budget as a fragment of a research titled “Scien- ved in patients with IBD suggesting that the con- tific Substantiation of the Complex of Measures tribution of carrageenan to the IBD development for the Prevention of Nutrition-related Disease may be explained by leukocyte membrane modi- in Schoolchildren in Ukraine’’ (state registration fications. However, further experimental and cli- number 0118U000943). nical studies are required to elucidate the role of carrageenan in IBD etiopathogenesis. TRANSPARENCY DECLARATION ACKNOWLEDGEMENT Conflicts of interest: None to declare Authors want to express their sincere gratitude to Prof. A.O. Doroshenko for gifting us with ortho- hydroxy derivatives of 2,5-diaryl-1,3-oxazole.

REFERENCES

1. Lewis JD, Abreu MT. Diet as a trigger or therapy 12. Negroni A, Pierdomenico M, Cucchiara S, Stronati for inflammatory bowel diseases. Gastroenterology L. NOD2 and inflammation: current insights. J In- 2017; 152:398-414.e6. flamm Res 2018; 11:49-60. 2. Knight-Sepulveda K, Kais S, Santaolalla R, Abreu 13. Boyle JP, Parkhouse R, Monie TP. Insights into the MT. Diet and inflammatory bowel disease. Gastro- molecular basis of the NOD2 signalling pathway. enterol Hepatol (N Y) 2015; 11:511-20. Open Biol 2014; 4:140178. 3. Seyedian SS, Nokhostin F, Malamir MD. A review 14. Ferrand A, Al Nabhani Z, Tapias NS, Mas E, Hugot of the diagnosis, prevention, and treatment methods JP, Barreau F. NOD2 expression in intestinal epithe- of inflammatory bowel disease. J Med Life 2019; lial cells protects toward the development of in- 12:113-22. flammation and associated carcinogenesis. Cell Mol 4. Perler B, Ungaro R, Baird G, Mallette M, Bright R, Gastroenterol Hepatol 2019; 7:357-69. Shah S, Shapiro J, Sands BE. Presenting symptoms 15. Sidiq T, Yoshihama S, Downs I, Kobayashi KS. in inflammatory bowel disease: descriptive analysis Nod2: A critical regulator of ileal microbiota and of a community-based inception cohort. BMC Ga- Crohn’s disease. Front Immunol 2016; 7:367. stroenterol 2019; 19:47. 16. Vedamurthy A, Ananthakrishnan AN. Influence of 5. Qin X. Why is damage limited to the mucosa in ul- environmental factors in the development and outco- cerative colitis but transmural in Crohn’s disease? mes of inflammatory bowel disease. Gastroenterol World J Gastrointest Pathophysiol 2013; 4:63-4. Hepatol (N Y) 2019; 15:72-82. 6. GBD 2017 Inflammatory Bowel Disease Collabo- 17. Rizzello F, Spisni E, Giovanardi E, Imbesi V, Salice rators. The global, regional, and national burden of M, Alvisi P, Valerii MC, Gionchetti P. Implications inflammatory bowel disease in 195 countries and of the westernized diet in the onset and progression territories, 1990-2017: a systematic analysis for the of IBD. Nutrients 2019; 11:1033. Global Burden of Disease Study 2017. Lancet Ga- 18. Bui TNTV. Structure, rheological properties and stroenterol Hepatol 2020; 5:17-30. connectivity of gels formed by carrageenan extrac- 7. Abdel Hadi L, Di Vito C, Riboni L. Fostering in- ted from different red algae species. Organic Chemi- flammatory bowel disease: sphingolipid strategies to stry. Mans, France: Université du Maine, 2019. join forces. Mediators Inflamm 2016; 2016:3827684. 19. Necas J, Bartosikova L. Carrageenan: a review. Ve- 8. Verstockt B, Smith KG, Lee JC. Genome-wide asso- terinarni Medicina 2013; 58:187-205. ciation studies in Crohn’s disease: past, present and 20. Heikenwälder H, Heikenwälder M. Krebs - Lifestyle future. Clin Transl 2018; 7:e1001. und Umweltfaktoren als Risiko. Berlin, Germany:

9. Yamamoto S, Ma X. Role of NOD2 in the deve- Springer-Verlag, 2019. lopment of Crohn’s disease. Microbes Infect 2009; 21. Younes M, Aggett P, Aguilar F, Crebelli R, Filipič 11:912-18. M, Frutos MJ, Galtier P, Gott D, Gundert-Remy U, 10. Hugot JP. CARD15/NOD2 mutations in Crohn’s di- Kuhnle KK, Lambre C, Leblanc J-C, Lillegaard IT, sease. Ann N Y Acad Sci 2006; 1072:9-18. Moldeus P, Mortensen A, Oskarsson A, Stankovic I, 11. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen Waalkens-Berendsen I, Woutersen RA, Wright M, FF, Ramos R, Britton H, Moran T, Karaliuskas R, Brimer L, Lindtner O, Mosesso P, Christodoulidou Duerr RH, Achkar JP, Brant SR, Bayless TM, Kirsch- A, Ioannidou S, Lodi F, Dusemund B. Re-evaluati- ner BS, Hanauer SB, Nuñez G, Cho JH. A frameshift on of carrageenan (E407) and processed Eucheuma mutation in NOD2 associated with susceptibility to seaweed (E407a) as food additives. EFSA J 2018; Crohn’s disease. Nature 2001; 411:603-06. 16:e05238.

182 Tkachenko et al. Effects of E407a on leukocyte membranes

22. Feferman L, Bhattacharyya S, Oates E, Haggerty N, 34. Doroshenko AO, Posokhov EA, Verezubova AA, Wang T, Varady K, Tobacman JK. Carrageenan-free Ptyagina LM, Skripkina VT, Shershukov VM. Ra- diet shows improved glucose tolerance and insulin diationless deactivation of excited phototautomer signaling in prediabetes: a randomized, pilot clinical form and molecular structure of ESIPT- compounds. trial. J Diabetes Res 2020; 2020:8267980. Photochem Photobiol Sci 2002; 1:92-9. 23. Tobacman JK. Review of harmful gastrointestinal 35. Doroshenko AO, Posokhov EA, Shershukov VM, effects of carrageenan in animal experiments. Envi- Mitina VG, Ponomarev OA. Intramolecular proton- ron Health Perspect 2001; 109:983–94. transfer reaction in an excited state in a series of ort- 24. Bhattacharyya S, Gill R, Chen ML, Zhang F, Linhar- ho-hydroxy derivatives of 2,5-diaryloxazole. High dt RJ, Dudeja PK, Tobacman JK. Toll-like receptor Energy Chemistry 1997; 31:388-94. 4 mediates induction of the Bcl10-NFkappaB-inter- 36. Posokhov Y, Kyrychenko A. Location of fluorescent leukin-8 inflammatory pathway by carrageenan in probes (2-hydroxy derivatives of 2,5-diaryl-1,3- human intestinal epithelial cells. J Biol Chem 2008; oxazole) in lipid membrane studied by fluorescence 283:10550-8. and molecular dynamics simulation. 25. Borthakur A, Bhattacharyya S, Dudeja PK, Tobac- Biophys Chem 2018; 235:9-18. man JK. Carrageenan induces interleukin-8 pro- 37. Kurad D, Jeschke G, Marsh D. Lipid membrane po- duction through distinct Bcl10 pathway in normal larity profiles by high-field EPR. Biophys J 2003; human colonic epithelial cells. Am J Physiol Gastro- 85:1025–33. intest Liver Physiol 2007; 292:829-38. 38. Bartucci R, Guzzi R, Marsh D, Sportelli L. Intra- 26. Martino JV, Van Limbergen J, Cahill LE. The role of membrane polarity by electron spin echo spectros- carrageenan and carboxymethylcellulose in the de- copy of labeled lipids. Biophys J 2003; 84(2 Pt velopment of intestinal inflammation. Front Pediatr 1)1025–30. 2017; 5:96. 39. Ho C, Slater SJ, Stubbs CD. Hydration and order in 27. Bhattacharyya S, Shumard T, Xie H, Dodda A, Va- lipid bilayers. Biochemistry 1995; 34:6188–95. rady KA, Feferman L, Halline AG, Goldstein JL, 40. Binder H, Gawrisch K. Effect of unsaturated lipid Hanauer SB, Tobacman JK. A randomized trial of chains on dimensions, molecular order and hydration the effects of the no-carrageenan diet on ulcerative of membranes. J Phys Chem B 2001; 105:12378–90. colitis disease activity. Nutr Healthy Aging 2017; 41. Noethig-Laslo V, Šentjurc M. Transmembrane pola- 4:181-92. rity profile of lipid membranes. Advances in Planar 28. Pravda J. Crohn’s disease: evidence for involvement Lipid Bilayers and Liposomes. Academic Press, El- of unregulated transcytosis in disease etio-pathoge- sevier, 2006:365-415. nesis. World J Gastroenterol 2011; 17:1416-26. 42. Levental KR, Malmberg E, Symons JL, Fan YY, 29. Bühner S, Nagel E, Körber J, Vogelsang H, Linn Chapkin RS, Ernst R, Levental I. Lipidomic and T, Pichlmayr R. Ileal and colonic fatty acid profiles biophysical homeostasis of mammalian membranes in patients with active Crohn’s disease. Gut 1994; counteracts dietary lipid perturbations to maintain 35:1424–8. cellular fitness. Nat Commun 2020; 11:1339. 30. Aozaki S. Decreased membrane fluidity in erythro- 43. Desai AJ, Miller LJ. Changes in the plasma mem- cytes from patients with Crohn’s disease. Gastroen- brane in metabolic disease: impact of the membrane terol Jpn 1989; 24:246–54. environment on G protein-coupled receptor structure 31. Tkachenko A, Marakushyn D, Kalashnyk I, Kor- and function. Br J Pharmacol 2018; 175:4009-25. niyenko Y, Onishchenko A, Gorbach T, Nakonechna 44. Seely AJ, Pascual JL, Christou NV. Science review: O, Posokhov Y, Tsygankov A. A study of enterocyte Cell membrane expression (connectivity) regulates membranes during activation of apoptotic processes neutrophil delivery, function and clearance. Crit in chronic carrageenan-induced gastroenterocolitis. Care 2003; 7:291-307. Med Glas (Zenica) 2018; 15:87-92. 45. Kantar A, Oggiano N, Giorgi PL, Fiorini R. Mem- 32. Tkachenko AS, Marakushyn DI, Rezunenko YK, brane fluidity of polymorphonuclear leukocytes Onishchenko AI, Nakonechna OA, Posokhov YO. from children with primary ciliary dyskinesia. Pe- A study of erythrocyte membranes in carrageenan- diatr Res 1993; 34:725-8. induced gastroenterocolitis by method of fluorescent 46. Gaschler MM, Stockwell BR. Lipid peroxidation in probes. HVM Bioflux 2018; 10:37-41. cell death. Biochem Biophys Res Commun 2017; 33. Posokhov YO, Kyrychenko A, Korniyenko Y. De- 482:419-25. rivatives of 2,5-diaryl-1,3-oxazole and 2,5-diaryl- 47. Chen JJ, Yu BP. Alterations in mitochondrial mem- 1,3,4-oxadiazole as environment-sensitive fluores- brane fluidity by lipid peroxidation products. Free cent probes for studies of biological membranes. In: Radic Biol Med 1994; 17:411-8. Geddes C. Reviews in Fluorescence 2017. Chapter 48. Sokolova EV, Karetin Y, Davydova VN, Byankina 9. Cham, Switzerland: Springer Nature Switzerland AO, Kalitnik AA, Bogdanovich LN, Yermak IM. AG, 2018: 199-230. Carrageenans effect on neutrophils alone and in combination with LPS in vitro. J Biomed Mater Res A 2016; 104:1603-9.

183 Medicinski Glasnik, Volume 18, Number 1, February 2021 UNIQA “Sigurna profesija” Program prevencije i zaštite pripadnika medicinskih i srodnih djelatnosti

Želimo Vas uvesti u svijet zaštite, Vas i Vaše medicinske profesije. Vođeni tom idejom naš cjelokupni program zaštite medicinske profesije objedinili smo pod nazivom UNIQA Sigurna profesija - najbolje rješenje za sve neželjene okolnosti na koje nemate uticaja.

Kome je “Sigurna profesija” namijenjena? “Sigurna profesija” je program namijenjen isključivo za pripadnike medicinske profesije: ljekare, doktore stoma- tologije, inženjere medicinske biohemije, farmaceute i Osiguranje finansijskih gubitaka zdravstvene radnike. Osiguran je finansijski gubitak uzrokovan prekidom rada osigurane djelatnosti ili ordinacije, te dio izgubljenog UNIQA je prva i jedina osiguravajuća kuća u Bosni prihoda zdravstvenog radnika kao posljedice bolesti i Hercegovini koja nudi jedinstven model podrške ili nesretnog slučaja osigurane osobe, kao i karantene za pripadnike medicinske profesije. uvedene zbog zaraze ili epidemije. Program “Sigurna profesija” omogućava da djelujete unaprijed i zaštitite svoju karijeru pomoću specijaliziranih osiguranja profesionalnih rizika. Osiguranje pravne zaštite u krivičnom i prekršajnom postupku Šta sadrži program Osiguranje pravne zaštite ima za cilj pružanje pravne “Sigurna profesija”? zaštite zdravstvenim radnicima i naknadu troškova koji nastanu kao posljedica radnji i propusta u obavljanju Osiguranje pokriva materijalne i nematerijalne medicinske djelatnosti kao profesionalne djelatnosti. štete počinjene trećim licima prilikom obavljanja medicinske djelatnosti kao profesionalne djelatno- sti, kao i pokriće za troškove pravnog savjetovanja, advokata i vještačenja u disciplinskom, prekršaj- Osiguranje od nom ili krivičnom postupku, te isplatu dnevne na- profesionalne odgovornosti knade za osiguranu osobu na ime izgubljenog pri- Obuhvata rizike koji mogu nastati tokom obavljanja hoda za svaki dan koji osigurano lice ne radi. poslova iz djelokruga osigurane, profesionalne Program sadrži: djelatnosti, za koju je zdravstveni radnik stručno i znanstveno osposobljen unutar ustanove u kojoj djeluje.  osiguranje finansijskih gubitaka  osiguranje pravne zaštite  osiguranje od profesionalne odgovornosti

Više informacija putem besplatnog info telefona 184 080 02 02 51 ili putem www.uniqa.ba UNIQA “Sigurna profesija” Program prevencije i zaštite pripadnika medicinskih EDITORIAL i srodnih djelatnosti What is a new in the world of orthopaedics and trauma? Želimo Vas uvesti u svijet zaštite, Vas i Vaše medicinske profesije. Vođeni tom idejom naš cjelokupni program zaštite medicinske profesije objedinili smo pod nazivom UNIQA Sigurna profesija - najbolje rješenje za sve neželjene okolnosti na koje nemate uticaja. The previous 10 years have truly opened a new era of orthopaedic trauma care. Rapid advances in the development of systems for internal and external fixation have been made like the support of other specialties. Improvements in technology and surgical technique have allowed fracture reduction and Kome je “Sigurna profesija” fixation to be achieved with less-invasive surgical approaches or the reduction of pain or dysfunction. namijenjena? This has reduced postoperative morbidity, decreased hospitalization, and expedited the recovery of function. A new understanding of what you are looking for in orthopaedics, traumatology, anaesthesia, “Sigurna profesija” je program namijenjen isključivo za plastic surgery, urology, neurosurgery etc. offers the possibility for an update of everything that revolves pripadnike medicinske profesije: ljekare, doktore stoma- around orthopaedics and traumatology. tologije, inženjere medicinske biohemije, farmaceute i Osiguranje finansijskih gubitaka zdravstvene radnike. The aim of this thematic issue was to report on original researches or literature review about the new Osiguran je finansijski gubitak uzrokovan prekidom rada clinical experience in orthopaedics and trauma. osigurane djelatnosti ili ordinacije, te dio izgubljenog UNIQA je prva i jedina osiguravajuća kuća u Bosni prihoda zdravstvenog radnika kao posljedice bolesti Luigi Meccariello, MD, Guest Editor i Hercegovini koja nudi jedinstven model podrške ili nesretnog slučaja osigurane osobe, kao i karantene Prof. Selma Uzunović, MD, MA, PhD, Editor-in-Chief, Medicinski Glasnik za pripadnike medicinske profesije. uvedene zbog zaraze ili epidemije. Program “Sigurna profesija” omogućava da djelujete unaprijed i zaštitite svoju karijeru pomoću specijaliziranih osiguranja profesionalnih rizika. Osiguranje pravne zaštite u krivičnom i prekršajnom postupku Šta sadrži program Osiguranje pravne zaštite ima za cilj pružanje pravne “Sigurna profesija”? zaštite zdravstvenim radnicima i naknadu troškova koji nastanu kao posljedica radnji i propusta u obavljanju Osiguranje pokriva materijalne i nematerijalne medicinske djelatnosti kao profesionalne djelatnosti. štete počinjene trećim licima prilikom obavljanja medicinske djelatnosti kao profesionalne djelatno- sti, kao i pokriće za troškove pravnog savjetovanja, advokata i vještačenja u disciplinskom, prekršaj- Osiguranje od nom ili krivičnom postupku, te isplatu dnevne na- profesionalne odgovornosti knade za osiguranu osobu na ime izgubljenog pri- Obuhvata rizike koji mogu nastati tokom obavljanja hoda za svaki dan koji osigurano lice ne radi. poslova iz djelokruga osigurane, profesionalne Program sadrži: djelatnosti, za koju je zdravstveni radnik stručno i znanstveno osposobljen unutar ustanove u kojoj djeluje.  osiguranje finansijskih gubitaka  osiguranje pravne zaštite  osiguranje od profesionalne odgovornosti

Više informacija putem besplatnog info telefona 080 02 02 51 ili putem www.uniqa.ba 185 REVIEW

Biological augmentation strategies in rotator cuff repair

Erdi Özdemir1, Dogac Karaguven2, Egemen Turhan1, Gazi Huri1

1Department of Orthopaedics and Traumatology, Hacettepe University Faculty of Medicine, 2Department of Orthopaedics and Traumatol- ogy, Ufuk University Faculty of Medicine; Ankara, Turkey

ABSTRACT

Rotator cuff tears (RCT) are a common problem encountered by orthopaedic surgeons. The incidence of re-tears (up to 94%) following surgical repair of RCTs renders the management of RCTs challenging. This higher re-tear rate has been attributed to the failure of healing at the tendon-bone junction. Biological augmentation methods such as growth factors, stem cell thera- pies, and biomaterials have been developed to promote the he- aling at the tendon-bone junction. Growth factors and stem cell therapies have been intensively studied in mid to large RCTs. Biomaterials have been generally utilized for large or massive RCTs. However, these newly generated biological augmentation Corresponding author: strategies are mostly studied in animal models. The efficacy and Gazi Huri safety of the biological augmentation methods in humans need Department of Orthopaedics and Trau- further investigation. In this review, we aimed to highlight the matology, Hacettepe University Faculty most recent advancements in RCT surgical repair with biologi- cal augmentation. of Medicine 06230 Ankara, Turkey Key words: platelet-rich plasma, rotator cuff injuries, stem cells, Phone: +90 53 2486 9155; fax: +90 tissue engineering 312 310 0161; E-mail: [email protected] Erdi Özdemir's Orcid ID: 0000-0002- 3147-9355 Gazi Huri's Orcid ID: 0000-0002-7036- 8455

Original submission: 24 October 2020; Revised submission: 07 November 2020; Accepted: 09 November 2020 doi: 10.17392/1305-21

Med Glas (Zenica) 2021; 18(1):186-191

186 Özdemir et al. Rotator cuff repair and biological solutions

INTRODUCTION normal concentration. Some growth factors such as platelet-derived growth factor (PDGF), insu- Rotator cuff tears (RCT) are a frequent problem lin-like growth factor-1 (IGF-1), transforming encountered in daily orthopaedics practice. There growth factor-β (TGF-β), and vascular endo- has been an increase in the incidence of patients thelial growth factor (VEGF) could be released undergoing surgery due to RCT since 2001 (1). by platelets and these growth factors have been It has been reported that more than 16000 RCT shown to enhance tendon healing (11). Owing to repairs were performed only in New York State the potential effects of PRP on soft tissue rege- in 2009, and the incidence of surgical procedures neration, PRP has gained popularity during the for RCTs has an upward trend (2). surgical repair of soft tissues (11,12). Patients with RCT often complain of shoulder The augmentation of RCT with PRP has been in- pain and disability. Thus, surgical repair of RCTs tensively studied in the last decade with animal aims to alleviate the shoulder pain (3). Although studies as well as clinical trials (12,13). There are surgical repair of RCTs commonly resolves the controversial results regarding the effect of PRP on shoulder pain, re-tear following RCT repair is a RCT repairs. Dolkart et al. reported that a single major concern for orthopaedic surgeons. Re-tear dose of PRP during surgical repair of a rat's supras- rate after RCT repair has been reported to range pinatus tendon enhanced the histological parame- up to 40% for small to medium tears and up to ters of tendon healing, resistive strength to load and 94% for large and chronic tears (4). The etiology tendon stiffness (13). Despite the promising effect of re-tears has been investigated and it has been of PRP on RCT repair in animal models, many cli- reported that initial biomechanical strength of the nical trials failed to demonstrate its positive effect repair, tear size, tissue quality of the tendon were in the re-tear rate following RCT repair (12,14,15). strongly associated with re-tear rates (5). On the other hand, in a recent systematic review The tendon to bone healing following RCT repa- with meta-analysis Cavendish et al. reported that ir is quite different than the original structure of perioperative augmentation of RCTs with PRP re- tendon-bone junction. The native tissue of ten- duces the re-tear risk; however, the authors were don insertion to the bone is composed of mostly unable to make a specific recommendation due to type I collagen fibres. On the other hand, repa- variable PRP preparation procedures (11). ired RCT does not regenerate and tendon-bone interface is made up of a fibro vascular scar tissue Other growth factors containing predominantly type III collagen fibres Growth factors were up-regulated in the injury site (6). In addition, these type III collagen fibres are during tendon healing until the establishment of less organized and have reduced tensile strength the tissue repair (16). In contrast to the high data than the original structure of tendon insertion (7). volume regarding PRP on tendon healing, several As the biology of tendon to bone healing in the studies have investigated a single growth factor or surgical repair of RCT has been enlightened in a mixture of growth factors mostly in animal mo- more detail (6,7), biological augmentation became dels (17-19). Bone morphogenetic protein (BMP)- an encouraging method to improve the healing of 7 has been reported to improve enthesis matrix repaired RCT (5). Current literature has focused production in a rat RCT model (20). Lamplot et on the biological solutions for decreasing the re-te- al. demonstrated that BMP-13 yielded higher ar rate following the surgical repair of RCT (8-11). mechanical strength than PRP in rat supraspinatus In this review, we will discuss the current biological tendon insertion model (17). During the revascula- augmentation strategies in the treatment of RCT. rization of the injured tendon, VEGF expression in the endothelial cells increases. In addition, VEGF GROWTH FACTORS has been reported to improve tendon healing via inhibiting microRNA-205-5p expression in a rat Platelet-rich plasma (PRP) model (18). The VEGF is a major growth factor Platelet-rich plasma (PRP) is an autologous con- in tendon healing by promoting vascularization; centration of the patients' blood to enrich the however, excessive vascularization could lead to platelet level. The platelet concentration of PRP proteolysis of the extracellular matrix (21). Ro- has been reported to be three or five folds of the deo et al. investigated a mixture of growth fac-

187 Medicinski Glasnik, Volume 18, Number 1, February 2021

tors including TGF-β1, TGF-β2, and TGF-β3; tendon, bursa derived have been used in pre-cli- fibroblast growth factor (FGF); and BMP-2 thro- nical studies for the augmentation of RCT repairs ugh 7 utilizing a type I collagen sponge in a sheep (26). However, two autologous sources of MSCs, infraspinatus tear model. The experimental group bone marrow-derived and adipose-derived, are cu- had higher fibrocartilage formation, better mecha- rrently available for commercial use (5). nical strength than the control group (19). The first study reporting the results of biologic au- Growth factor levels have a fluctuating concen- gmentation with bone marrow-derived MSCs du- tration during rotator cuff healing. It has been de- ring RCT surgery was conducted by Ellera Gomez monstrated in rat and rabbit studies, their levels et al. in 2011. The authors repaired 14 patients' rise and fall in two weeks starting from the injury RCT with trans osseous stitches through mini- time (22). Thus, a single bolus of injection during open incision and injected bone marrow-derived the surgical repair of RCT may not be the optimal MSCs to the tendon borders which were obtained method for RCT augmentation. A protocol mimic- from iliac crests. MRI was obtained from each king the natural healing period by augmentation patient after 12 months and revealed tendon inte- with growth factors needs further investigations. grity in all patients. At a minimum 12 months of follow-up, patients had significant improvements STEM CELLS in the UCLA scores except for one patient (27). Considering the importance of the biological envi- Since then only one study has reported utilizing ronment during tendon healing, stem cell therapies bone marrow-derived MSC for the biological au- have gained popularity in recent years (9). Me- gmentation for RCT. Hernigou et al. compared the senchymal stem cells (MSC) are commonly used outcomes of 90 patients who underwent arthrosco- for the biological augmentation of soft tissue repa- pic single-row RCT repair with (n=45) or without irs due to their secretory capability of trophic fac- augmentation (n=45) with bone marrow-derived tors in wound healing, inflammation and fibrocar- MSCs aspirated from anterior iliac crest. Injec- tilage formation (23). Promising results have been tion of MSCs was performed to the tendon-bone reported with the utility of MSCs in animal RCT junction and to the footprint. The most important models. Omi et al. reported that bone marrow- finding was that the augmentation of RCT surgical derived MSCs increased the healing strength and repair with MSCs reduced re-tear rate. At the most stiffness following RCT repair in a rat model (24). recent follow up, 87% of the augmented group had Kaizawa et al. augmented the RCT repair with adi- intact rotator cuff while the non-augmented group pose-derived MSCs in a rat chronic supraspinatus had 44% (28). tear model: at the eighth week, adipose-derived In the two published studies regarding augmen- stem cell augmentation group revealed better bone tation of RCT with bone marrow-derived MSCs, morphometry at the supraspinatus insertion on the cells were obtained from ilium before the surgery humerus than the non-augmented control group (27, 28). However, Otto et al. reported that proxi- (8). Morton-Gonzaba et al. recently conducted me- mal humerus is a reliable source for bone marrow- ta-analysis on the application of MSCs to rotator derived MSCs as ilium during arthroscopic sur- cuff pathologies including 18 pre-clinical studies. gery (29). Considering the positive effect of bone Their analysis revealed that biologic augmentation marrow-derived MSCs on RCT repair and easy with MSCs improved biomechanical failure loads, access through proximal humerus during shoulder bone mineral densities, and stimulated fibrocarti- arthroscopy, biological augmentation may turn to lage formation. Despite the promising outcomes be a routine procedure. On the other hand, the pre- with MSC augmentation, the authors emphasized sence of limited data with only two reported studi- the requirement for optimizing MSCs for standard es with bone marrow-derived MSC augmentation protocols (9). does not allow strong recommendation. Owing to the encouraging results with the utility of Administration of adipose-derived MSCs on RCT MSCs in RCT surgical repairs in animals, MSCs is mostly centred around intratendinous injections have started to be used for biological augmen- in the literature. The first human trial was conduc- tation in humans as well (25). Different sources ted by Jo et al. in 2018 with adipose-derived MSCs of MSCs such as bone marrow, adipose, muscle, on RCT. The authors investigated three different

188 Özdemir et al. Rotator cuff repair and biological solutions

injection doses of adipose-derived MSCs, the low histological behaviour of highly porous reconstitu- dose (1.0×107 cells), mid dose (5.0×107 cells), and ted bovine collagen implants in seven patients at an high dose (1.0×108 cells). Arthroscopic examinati- average of 3 months (5 weeks to 6 months) after on at sixth month revealed that the size of RCT de- surgery; histology sections revealed aligned linear fects decreased 83% in mid doses and 90% in high orientation of the cells within the collagen implant doses (30). The authors also followed the same pa- structure (35). Due to the small number of studies tients for 2 years and MRIs of the patients in high with small patient population, further human stu- dose group at first-year demonstrated that bursal dies are needed before the wide use of xenografts. side tears almost disappeared and did not recur in the second year (31). Only a single study by Kim et Allografts al. has reported the biologic augmentation of RCT Allografts that are harvested from tensor fascia lata repair with adipose-derived MSCs in humans. The or skin tissue have been used for the biological au- authors compared arthroscopic double-row repair gmentation of the RCT repairs. Agraval et al. used technique with or without augmentation with adi- acellular human dermal graft in patients with large, pose-derived MSCs and reported that re-tear rate massive and re-teared RCT; MRIs of the patients with MRI evaluation at minimum 12 months after demonstrated 85.7% intact rotator cuff in addition surgery was 28.5% in the non-augmented group to the favourable functional outcomes (36). Barber and 14.3% in the augmented group. Further studi- et al. investigated the effect of dermal grafts pre- es are warranted evaluating the biologic augmen- pared from epidermal and dermal layers of human tation of RCT repair with adipose-derived MSCs skin in a prospective, randomized controlled trial because of limited evidence in the literature. including patients with massive and two-tendon RCT and found that MRI scans showed 85% intact BIOMATERIALS rotator cuffs in the augmented group but 40% in High failure rates especially following large RCT the non-augmented group (37). In the Hohn et al. have promoted seeking new strategies to reduce study with the minimum 2 years follow-up, 69% of re-tear rates. Improvements in tissue enginee- patients who underwent revision RCT repair sur- ring studies have allowed the use of scaffolds gery with the use of acellular human dermal matrix that maintain cellular ingrowth while providing allograft showed intact repair constructs in MRI or mechanical support until healing. Various types ultrasonography (38). Further research is required of biomaterials including xenografts, allografts, with larger patient populations to confirm the fin- and synthetic grafts have been used to augment dings of existing literature regarding the allografts. healing after RCT repair (10). Synthetic grafts Xenografts Synthetic grafts could be synthesized from varia- Xenografts originating from different species of ble polymers such as polyester, polyacrylamide, different tissues have been developed in recent polypropylene, dacron, silicon, carbon, or nylon. years. Using an acellular sheet of cross-linked Synthetic grafts have drawbacks mainly due to porcine dermis for the augmentation of massive foreign body reactions although they are bio- RCTs Cho et al. found that the MRI of the pati- mechanically superior to the biologic grafts (39). ents at eight months follow-up demonstrated 80% Investigating polycarbonate polyurethane scaffold integrity of rotator cuff (32). However, Soler et al. in the open repair of full thickness RCTs of ten reported 100% inflammatory reaction in their small patients, Escalada-Diaz et al. reported 10% failure case series including four patients with using the rate at first-year follow-up (40). Proctor reported same acellular sheet of cross-linked porcine dermis the long-term outcomes of 18 patients with large (33). Gupta et al. used porcine dermal tissue matrix to massive RCT with augmentation via poly-l-lac- xenograft in 27 shoulders with massive or two-ten- tic acid synthetic patch; 83% of the patients had don RCT: a total of 73% of the patients had visible intact rotator cuff at the annual follow-up and 78% intact rotator cuff on ultrasonography at the most had intact rotator cuff at a mean 42 months after recent follow-up and only one patient had compla- surgery (41). Ciampi et al. conducted a controlled ined of re-tear (34). Arnocky et al. investigated the study on massive RCT and compared the conven-

189 Medicinski Glasnik, Volume 18, Number 1, February 2021

tional repair with polypropylene patch augmenta- can occur later because of insufficient healing at tion: the polypropylene patch group had 17% re- the tendon-bone junction. Biological augmentati- tear rate at 3 years follow-up while control group on strategies aim to promote the repair site loca- had 41% (42). Renebo et al. reported long-term ted at tendon-bone junction. Most of the studies results of a synthetic graft made of Dacron; nine were performed on animal models in addition to of 12 patients had rotator cuff arthropathy after a the few human studies without control groups. As mean 17-year follow-up (43). Biologic augmenta- the results of biologic augmentation of RCT are tion with synthetic grafts appears to reduce the re- promising, further controlled studies with large tear rate, however, a study by Renebo et al.(43) ra- patient population would be beneficial to transla- ised questions regarding the success in long-term. te previous literature to routine clinical use. In conclusion, the surgical repair of RCTs has FUNDING successful outcomes. On the other hand, re-tear of the repaired RCT is a disappointing complication No specific funding was received for this study. which could be observed up to 94%. Re-tear of the repaired RCT may occur due to a mechanical TRANSPARENCY DECLARATION failure at suture-tendon site at the short term or it Conflicts of interest: None to declare

REFERENCES 1. Hakimi O, Mouthuy PA, Carr A. Synthetic and degra- 11. Cavendish PA, Everhart JS, DiBartola AC, Eike- dable patches: an emerging solution for rotator cuff nberry AD, Cvetanovich GL, Flanigan DC. The effect repair. Int J Exp Pathol 2013; 94:287-92. of perioperative platelet-rich plasma injections on po- 2. Ensor KL, Kwon YW, Dibeneditto MR, Zuckerman stoperative failure rates following rotator cuff repair: JD, Rokito AS. The rising incidence of rotator cuff a systematic review with meta-analysis. J Shoulder repairs. J Shoulder Elbow Surg 2013; 22:1628-32. Elbow Surg 2020; 29:1059-70. 3. McElvany MD, McGoldrick E, Gee AO, Neradilek 12. Flury M, Rickenbacher D, Schwyzer HK, Jung C, MB, Matsen FA, 3rd. Rotator cuff repair: published Schneider MM, Stahnke K, Goldhahn J, Audigé L . evidence on factors associated with repair integrity Does pure platelet-rich plasma affect postoperative and clinical outcome. Am J Sports Med 2015; 43:491- clinical outcomes after arthroscopic rotator cuff repa- 500. ir? A randomized controlled trial. Am J Sports Med 4. Le BT, Wu XL, Lam PH, Murrell GA. Factors pre- 2016; 44:2136-46. dicting rotator cuff retears: an analysis of 1000 con- 13. Dolkart O, Chechik O, Zarfati Y, Brosh T, Alhajaj- secutive rotator cuff repairs. Am J Sports Med. 2014; ra F, Maman E. A single dose of platelet-rich plasma 42:1134-42. improves the organization and strength of a surgically 5. Mirzayan R, Weber AE, Petrigliano FA, Chahla J. repaired rotator cuff tendon in rats. Arch Orthop Tra- Rationale for biologic augmentation of rotator cuff uma Surg 2014; 134:1271-7. repairs. J Am Acad Orthop Surg 2019; 27:468-78. 14. Chahal J, Van Thiel GS, Mall N, Heard W, Bach BR, 6. Thomopoulos S, Genin GM, Galatz LM. The deve- Cole BJ, Nicholson GP, Verma NN, Whelan DB, Ro- lopment and morphogenesis of the tendon-to-bone meo AA. The role of platelet-rich plasma in arthros- insertion - what development can teach us about hea- copic rotator cuff repair: a systematic review with qu- ling. J Musculoskelet Neuronal Interact 2010; 10:35- antitative synthesis. Arthroscopy 2012; 28:1718-27. 45. 15. Saltzman BM, Jain A, Campbell KA, Mascarenhas R, 7. Galatz LM, Ball CM, Teefey SA, Middleton WD, Romeo AA, Verma NN, Cole BJ. Does the use of pla- Yamaguchi K. The outcome and repair integrity of telet-rich plasma at the time of surgery improve clini- completely arthroscopically repaired large and ma- cal outcomes in arthroscopic rotator cuff repair when ssive rotator cuff tears. J Bone Joint Surg Am 2004; compared with control cohorts? A systematic review 86:219-24. of meta-analyses. Arthroscopy 2016; 32:906-18. 8. Kaizawa Y, Franklin A, Leyden J, Behn AW, Tulu US, 16. Thomopoulos S, Parks WC, Rifkin DB, Derwin KA. Sotelo Leon D, Wang Z, Abrams GD, Chang J, Fox Mechanisms of tendon injury and repair. J Orthop Res PM. Augmentation of chronic rotator cuff healing 2015; 33:832-9. using adipose-derived stem cell-seeded human ten- 17. Lamplot JD, Angeline M, Angeles J, Beederman don-derived hydrogel. J Orthop Res 2019; 37:877-86. M, Wagner E, Rastegar F, Scott B, Skjong C, Mass 9. Morton-Gonzaba N, Carlisle D, Emukah C, Chorath D, Kang R, Ho S, Shi LL. Distinct effects of plate- K, Moreira A. Mesenchymal stem cells and their let-rich plasma and BMP13 on rotator cuff tendon application to rotator cuff pathology: a meta-analysis injury healing in a rat model. Am J Sports Med 2014; of pre-clinical studies. Osteoarthritis and Cartilage 42:2877-87. Open 2020; 2:100047. 18. Xu Q, Sun WX, Zhang ZF. High expression of 10. Karuppaiah K, Sinha J. Scaffolds in the management VEGFA in MSCs promotes tendon-bone healing of of massive rotator cuff tears: current concepts and li- rotator cuff tear via microRNA-205-5p. Eur Rev Med terature review. EFORT Open Rev 2019; 4:557-66. Pharmacol Sci 2019; 23:4081-8.

190 Özdemir et al. Rotator cuff repair and biological solutions

19. Rodeo SA, Potter HG, Kawamura S, Turner AS, Kim 31. Jo CH, Chai JW, Jeong EC, Oh S, Yoon KS. Intraten- HJ, Atkinson BL. Biologic augmentation of rotator dinous injection of mesenchymal stem cells for the cuff tendon-healing with use of a mixture of osteoin- treatment of rotator cuff disease: a 2-year follow-up ductive growth factors. J Bone Joint Surg Am 2007; study. Arthroscopy 2020; 36:971-80. 89:2485-97. 32. Cho CH, Lee SM, Lee YK, Shin HK. Mini-open su- 20. Kabuto Y, Morihara T, Sukenari T, Kida Y, Oda R, ture bridge repair with porcine dermal patch augmen- Arai Y, Sawada K, Matsuda K-I, Kawata M, Tabata tation for massive rotator cuff tear: surgical technique Y, Fujiwara H, Kubo T. Stimulation of rotator cuff re- and preliminary results. Clin Orthop Surg 2014; pair by sustained release of bone morphogenetic pro- 6:329-35. tein-7 using a gelatin hydrogel sheet. Tissue Eng Part 33. Soler JA, Gidwani S, Curtis MJ. Early complications A 2015; 21:2025-33. from the use of porcine dermal collagen implants 21. Savitskaya YA, Izaguirre A, Sierra L, Perez F, Cruz (Permacol) as bridging constructs in the repair of F, Villalobos E, Almazan A, Ibarra C. Effect of angi- massive rotator cuff tears. A report of 4 cases. Acta ogenesis-related cytokines on rotator cuff disease: the Orthop Belg 2007; 73:432-6. search for sensitive biomarkers of early tendon dege- 34. Gupta AK, Hug K, Boggess B, Gavigan M, Toth AP. neration. Clin Med Insights Arthritis Musculoskelet Massive or 2-tendon rotator cuff tears in active pati- Disord 2011; 4:43-53. ents with minimal glenohumeral arthritis: clinical and 22. Kobayashi M, Itoi E, Minagawa H, Miyakoshi N, Ta- radiographic outcomes of reconstruction using der- kahashi S, Tuoheti Y, Okada K, Shimada Y. Expressi- mal tissue matrix xenograft. Am J Sports Med 2013; on of growth factors in the early phase of supraspina- 41:872-9. tus tendon healing in rabbits. J Shoulder Elbow Surg 35. Arnoczky SP, Bishai SK, Schofield B, Sigman S, Bus- 2006; 15:371-7. hnell BD, Hommen JP, Van Kampen C. Histologic 23. Caplan AI, Dennis JE. Mesenchymal stem cells as evaluation of biopsy specimens obtained after rotator trophic mediators. J Cell Biochem 2006; 98:1076-84. cuff repair augmented with a highly porous collagen 24. Omi R, Gingery A, Steinmann SP, Amadio PC, An implant. Arthroscopy 2017; 33:278-83. KN, Zhao C. Rotator cuff repair augmentation in a rat 36. Agrawal V. Healing rates for challenging rotator cuff model that combines a multilayer xenograft tendon tears utilizing an acellular human dermal reinforce- scaffold with bone marrow stromal cells. J Shoulder ment graft. Int J Shoulder Surg 2012; 6:36-44. Elbow Surg 2016; 25:469-77. 37. Barber FA, Burns JP, Deutsch A, Labbé MR, Litchfi- 25. Berebichez-Fridman R, Gómez-García R, Granados- eld RB. A prospective, randomized evaluation of Montiel J, Berebichez-Fastlicht E, Olivos-Meza A, acellular human dermal matrix augmentation for Granados J, Velasquillo C, Ibarra C. The holy grail arthroscopic rotator cuff repair. Arthroscopy 2012; of orthopedic surgery: mesenchymal stem cells-their 28:8-15. current uses and potential applications. Stem Cells Int 38. Hohn EA, Gillette BP, Burns JP. Outcomes of arthros- 2017; 2017:2638305. copic revision rotator cuff repair with acellular human 26. Patel S, Gualtieri AP, Lu HH, Levine WN. Advances dermal matrix allograft augmentation. J Shoulder in biologic augmentation for rotator cuff repair. Ann Elbow Surg 2018; 27:816-23. N Y Acad Sci 2016; 1383:97-114. 39. McCormack RA, Shreve M, Strauss EJ. Biologic 27. Ellera Gomes JL, da Silva RC, Silla LM, Abreu MR, augmentation in rotator cuff repair--should we do it, Pellanda R. Conventional rotator cuff repair comple- who should get it, and has it worked? Bull Hosp Jt Dis mented by the aid of mononuclear autologous stem 2014; 72:89-96. cells. Knee Surg Sports Traumatol Arthrosc 2012; 40. Encalada-Diaz I, Cole BJ, Macgillivray JD, Ruiz-Su- 20:373-7. arez M, Kercher JS, Friel NA, Valero-Gonzalez F. Ro- 28. Hernigou P, Flouzat Lachaniette CH, Delambre J, tator cuff repair augmentation using a novel polycar- Zilber S, Duffiet P, Chevallier N, Rouard H. Biologic bonate polyurethane patch: preliminary results at 12 augmentation of rotator cuff repair with mesenchymal months' follow-up. J Shoulder Elbow Surg 2011; stem cells during arthroscopy improves healing and 20:788-94. prevents further tears: a case-controlled study. Int 41. Proctor CS. Long-term successful arthroscopic repair Orthop 2014; 38:1811-8. of large and massive rotator cuff tears with a functio- 29. Otto A, Muench LN, Kia C, Baldino JB, Mehl J, nal and degradable reinforcement device. J Shoulder Dyrna F, Voss A, McCarthy MB, Nazal MR, Martin Elbow Surg 2014; 23:1508-13. SD, Mazzocca AD. Proximal humerus and ilium are 42. Ciampi P, Scotti C, Nonis A, Vitali M, Di Serio C, Pe- reliable sources of bone marrow aspirates for biologic retti GM, Fraschini G. The benefit of synthetic versus augmentation during arthroscopic surgery. Arthrosco- biological patch augmentation in the repair of poste- py 2020; 36:2403-11. rosuperior massive rotator cuff tears: a 3-year follow- 30. Jo CH, Chai JW, Jeong EC, Oh S, Kim PS, Yoon JY, up study. Am J Sports Med 2014; 42:1169-75. , Yoon KS. Intratendinous injection of autologous 43. Ranebo MC, Björnsson Hallgren HC, Norlin R, Ado- adipose tissue-derived mesenchymal stem cells for lfsson LE. Long-term clinical and radiographic outco- the treatment of rotator cuff disease: a first-in-human me of rotator cuff repair with a synthetic interposition trial. Stem Cells 2018; 36:1441-50. graft: a consecutive case series with 17 to 20 years of follow-up. J Shoulder Elbow Surg 2018; 27:1622-8.

191 ORIGINAL ARTICLE

Arthroscopic labral repair with all-suture anchors: a magnetic resonance imaging retrospective study with a 2.5-year follow-up

Federico Sacchetti1,2, Martina Di Meglio3,4, Nicola Mondanelli3,4, Nicola Bianchi1,2, Vanna Bottai1,2, Fed- erico Cartei5,6, Fabio Cosseddu1,2, Rodolfo Capanna1,2, Stefano Giannotti3,4

1Department of Orthopaedic and Trauma Surgery, University of Pisa, 2Department of Orthopaedic and Trauma Surgery, Azienda Ospedaliero Universitaria Pisana; Pisa, 3Section of Orthopaedics and Traumatology, Department of Medicine, Surgery and Neurosci- ences, University of Siena, 4Section of Orthopaedics and Traumatology, Azienda Ospedaliera Universitaria Senese; Siena, 5Department of Radiology, University of Pisa, 6Department of Radiology, Azienda Ospedaliero Universitaria Pisana; Pisa, Italy

ABSTRACT

Aim To evaluate radiological and clinical outcomes of a case seri- es of patients affected by glenohumeral instability (Bankart lesion) or superior labrum tear from anterior to posterior (SLAP) lesions treated by arthroscopic repair using all-suture anchors.

Methods Patients were operated by a single surgeon at a single Institution. Exclusion criteria were chondral lesions of the glenoid, rotator cuff lesions, previous surgery at the index shoulder, or a bony Bankart lesion. Position and numbers of anchors used depen- ded on the dimension and type of lesion. The DASH (Disability Corresponding author: of the Arm, Shoulder and Hand) and Constant scores were used Nicola Mondanelli for subjective and clinical evaluation at follow-ups (FUs); also, at Section of Orthopaedics, 1-year FU, MRI scan was obtained to evaluate bone reaction to the implanted devices. Department of Medicine, Surgery and Neurosciences, Results Fifty-four patients were included. A mean of 2.7 devices University of Siena per patient (145 in total) were implanted. Mean FU was 30 (ran- Viale Mario Bracci 16, 53100 Siena, Italy ge 12 – 48) months. No patient reported recurrent instability, nor hardware-related complications were registered. MRI analyses E-mail: [email protected] showed that 119 (82%) implants did not alter surrounding bone Phone +39 0577 585675; fax +39 0577 (grade 0), 26 (18%) implants were surrounded by bone oedema 233400; (grade 1), while no bone tunnel enlargement nor a bone cyst (grade Federico Sacchetti ORCID ID: https:// 2 or 3, respectively) were registered. orcid.org/ 0000-0003-1276-0624 Conclusion This study confirmed the efficacy and safety of a spe- cific all-suture anchor system in the arthroscopic repair of the gle- noid labrum for glenohumeral instability or a SLAP lesion. In the Original submission: short- and mid-term period, these devices were associated with 23 November 2020; good clinical and radiological outcomes without clinical failures Accepted: or reaction at bone-device interface. 28 November 2020 Key words: Bankart lesion, bone-implant interface, hardware doi: 10.17392/1320-21 complication, shoulder, SLAP lesion

Med Glas (Zenica) 2021; 18(1):192-195

192 Sacchetti et al. All-suture anchors labral rep

INTRODUCTION All patients gave their written consent to the tre- atment and anonymous use of data and images for In recent years, several improvements have been research and academic purposes. At our Instituti- introduced in the techniques for glenoid labral ar- ons, no Ethical Committee nor Institutional Review throscopic repair in patients affected by recurrent Board approval are needed for retrospective studies. glenohumeral instability (1,2). The main techno- logical drive to these developments was the in- Methods troduction of more suitable implants and surgi- cal tools. However, it is still under debate which Patients underwent surgery in the contralateral implant is to be considered as the gold standard lateral decubitus position, with traction applied to for these repairs. Recently, some concerns have the involved upper limb with 60°-70° of shoulder been posed on the repair with anchors, as several abduction and 15°-20° of anterior flexion. For each studies have underlined the relatively high rate of patient, standard posterior and standard antero-su- implant-related complications such as iatrogenic perior portals were used, plus appropriate supple- cartilage damages, formation of bone cysts and mentary portals as needed. After evaluation of the implant migrations (3–6). lesion and preparation of the tissues (scar tissue removal to favor bleeding and subsequent tissue Different anchoring systems that are not made of healing), a 13-mm deep hole was drilled through rigid materials have been studied to reduce rates a dedicated pointer with a drill bit. The all-suture of implant-related complications (1,2,4). All-sutu- anchors were positioned through the pointer, the re devices have been proposed as a new anchoring suture pulled and so the sleeve was cinched up to system and they have been vastly used in surgical compress against the bone creating an anchoring practice (1,6). However, there is a lack of informati- ball, then the sutures were passed into the soft on about clinical and especially radiological outco- tissues and a knot was tied (Figure 1). Position mes in patients treated with all-suture anchors (7). and numbers of anchors depended on the dimen- The aim of this study was to evaluate the radiologi- sion and type of lesion. After surgery, patients cal and clinical outcome of a case series of patients were immobilized in a sling for 4 weeks allowing affected by glenohumeral instability with Bankart only passive motion of the involved shoulder. At lesion or by superior labrum tear from anterior to 4 weeks post-operatively, physical therapy con- posterior (SLAP) lesions treated by arthroscopic tinued with active range of motion exercises and repair using an all-suture anchor system. muscular strengthening exercises. PATIENTS AND METHODS All patients were retrospectively evaluated su- bjectively and clinically, using the Disability of Patients and study design the Arm, Shoulder and Hand (DASH) score (0 All patients included into the study were treated with arthroscopic labral repair of a Bankart lesion, a SLAP lesion, or both, for shoulder instability at the Azienda Ospedaliera Universitaria Pisana – University of Pisa (Pisa, Italy) and Azienda Ospe- daliera Universitaria Senese – University of Siena (Siena, Italy), between July 2016 and June 2019. All patients were operated by the same surgeon (SG) (he moved during the study period from a hospital to the other), and in all patients the same all-suture anchor system was used (Y-Knot PRO Flex, ConMed Inc, Utica, NY, USA). Exclusion criteria were the presence of cartilaginous or bone lesions of the scapular glenoid, rotator cuff lesi- Figure 1. Arthroscopic labrum repair with all-suture anchor ons, history of previous surgery on the involved system. A) preparation of the torn labrum; B) insertion with the shoulder, and bony Bankart lesions. pointer of the all-suture anchor after having drilled the bone through the same guiding pointer; C) the suture is passed over the labrum and D) tied (Sacchetti F, 2019)

193 Medicinski Glasnik, Volume 18, Number 1, February 2021

is best, 100 is worst) (8), and the Constant score excellent to the Constant grading with respect to (100 is the best, 0 is worst) (9) and grading (10). the normal contralateral side. The DASH and Constant scores were administe- The MRI scan was performed in all patients at red to all patients before surgery, at six-months 12 to 15 (mean 13) months from the surgery. follow up (FU) and then annually; the final score MRI evaluation showed that 119 (82%) implants was considered the one at the last available FU. did not alter the surrounding bone (grade 0), 26 A magnetic resonance imaging (MRI) scan was (18%) implants were surrounded by bone oede- obtained for all patients at 1-year FU (Figure ma (grade 1), no implant was surrounded by a 2). Sagittal T1-weighted, axial gradient-echo, bone tunnel enlargement bigger than 2 mm or by oblique coronal and sagittal T2-weighted, coro- a bone cyst (grade 2 or 3, respectively) (Table 1). nal fat-suppressed images were acquired using Table 1. Bone reaction to glenoid implant on magnetic resonance a high-field (3 Tesla) scanner. Images were re- imaging (MRI) based on Willemot et al. (7) grading system No of cases (total 145 viewed by a single radiologist (FC) with more Willemot et al. (7) MRI grading than 10 years of experience in musculoskele- anchors in 54 patients) Grade 0 No bone reaction 119 tal radiology. The scoring system suggested by Grade 1 Bone oedema 26 Willemot et al. (7) was used to assess the varia- Grade 2 Tunnel widening > 2 mm 0 tion in the bone tissue near the anchors conside- Grade 3 Bone cyst 0 ring: normal bone aspect (grade 0), presence of local bone oedema (grade 1), enlargement of the DISCUSSION bone tunnel of more than 3 mm (grade 2) or pre- The main finding of this retrospective, single-sur- sence of a bone cyst (grade 3). geon study is the absence of hardware complica- tions nor bone reaction to the anchors, confirming previous findings about the safety and efficacy of all-suture anchor systems in the arthroscopic tre- atment of shoulder instability. Willemot et al. (7) already showed excellent results out of 58 all-sutu- re anchors in their original work. The present study was conducted on a 2.5-fold larger case series of patients and implanted devices as well, therefore Figure 2. Magnetic reso- nance imaging of a Bankart with a stronger statistical power and significance. + superior labrum tear from Also, a 3T-MRI scan was used in the present study, anterior to posterior (SLAP) with theoretically better imaging than 1.5T-MRI lesion repaired with 3 an- chors in A) the sagittal, B) scan used by Willemot et al. To our knowledge, axial and C) coronal planes to date no other studies in the English literature (Cartei F, 2019) evaluated the bone-implant interface with MRI in all-suture devices in the glenoid, while only other RESULTS studies exist, and it is about rotator cuff repair (11), Fifty-four patients fulfilled the inclusion crite- with slightly worse results. Bone density plays a ria and were therefore included into the study. major role in implant stability (integration / loose- Thirty-two patients were male and 22 females, ning), and proximal humerus is less compact and mean age at surgery was 26 (range 15–45) years. dense than the glenoid, leading in our opinion to A mean of 2.7 devices per patient (145 in total) possible easier reaction to implants (intravasation were implanted. of synovia fluid, micromotion of the anchors) than Mean subjective and clinical FU was 30 (range dense bone as in the glenoid. 12 – 48) months. This study has several limitations such as the re- No patient reported recurrent instability nor trospective nature of the analysis and lack of a hardware-related complications were registe- blind evaluation of the clinical outcomes. Also, red. Mean DASH score was 15.3 points (range lack of a control group represents another impor- 5–25.8) and mean Constant score was 92.3 out of tant bias. However, radiological findings are not 100 (range 86–100); all patients but one graded affected by such bias of the study. In this case se-

194 Sacchetti et al. All-suture anchors labral rep

ries, bone reaction to implants was absent or low- suture anchors compared to the ones required to grade on MRI in all cases, and clinical outcomes implant a traditional anchor (usually 2.5 to 3 mm were satisfactory. The results of this study confirm drill bit). Less aggressive drillings reduce risk of the optimal biocompatibility with the host bone of glenoid fractures and anchor pull-out, and even in all-suture devices in the short period, and excellent the case of anchors migration, the soft materials of clinical results, as shown in recent review (12). The the all-sutures devices limit the risk of secondary prevalence and number of bone reactions was pre- joints damages. The absence of recurrent and the dictable since the main drive to the development excellent subjective and clinical outcomes asse- of the all-suture anchors devices was to reduce it, ssed by DASH and Constant scores underline the improving the biocompatibility of the anchoring efficacy of such all-suture anchors systems. systems. As for the main concern about all-suture In conclusion, this study confirms the efficacy anchors, their fixation strength, no clinical failures and safety of this specific all-suture anchor were registered in this series. Biomechanical stu- system in the arthroscopic repair of the glenoid dies have shown that the ultimate load to failure labrum for glenohumeral instability or a SLAP is higher in all-suture anchor constructs compared lesion. In the short- and mid-term period, these to standard metallic anchors (13,14). On the other devices are associated with good clinical and ra- hand, some animal models have shown the for- diological outcomes (no recurrences, no reaction mation of bone cyst around the sutures that could at bone-implant interface). lead to loosening and early failure of the construct (15). In this series, no clinical failure of the suture FUNDING and no recurrence of instability were reported, and No specific funding was received for this study. no anchor loosening was evident at MRI. This can also be explained by the small dimension of the TRANSPARENCY DECLARATION holes (1.3 mm drill bit) needed to implant the all- Conflict of interest: None to declare.

REFERENCES 1. Barber FA, Herbert MA, Beavis RC, Barrera Oro F. 9. Constant CR, Murley AH. A clinical method of functio- Suture anchor materials, eyelets, and designs: update nal assessment of the shoulder. Clin Orthop Relat Res 2008. Arthroscopy 2008; 24:859–67. 1987; 214:160–4. 2. Castagna A, Markopoulos N, Conti M, Delle Rose G, 10. Fabre, Piton C, Leclouerec G, Gervais-Delion F, Du- Papadakou E, Garofalo R. Arthroscopic bankart su- randeau A. Entrapment of the suprascapular nerve. J ture-anchor repair: radiological and clinical outcome Bone Joint Surg Br 1999; 81-B:414–9. at minimum 10 years of follow-up. Am J Sports Med 11. Van der Bracht H, Van den Langenbergh T, Pouillon 2010; 38:2012–6. M, Verhasselt S, Verniers P, Stoffelen D. Rotator cuff 3. Banerjee S, Weiser L, Connell D, Wallace AL. Gle- repair with all-suture anchors: a midterm magnetic noid rim fracture in contact athletes with absorba- resonance imaging evaluation of repair integrity and ble suture anchor reconstruction. Arthroscopy 2009; cyst formation. J Shoulder Elb Surg 2018; 27:2006– 25:560–2. 12. 4. Dhawan A, Ghodadra N, Karas V, Salata MJ, Cole BJ. 12. Ergün S, Akgün U, Barber FA, Karahan M. The Cli- Complications of bioabsorbable suture anchors in the nical and biomechanical performance of all-suture shoulder. Am J Sports Med 2012; 40:1424–30. anchors: a systematic review. Arthrosc Sport Med 5. Goeminne S, Debeer P. Delayed migration of a metal Rehabil 2020; 2:e263–75. suture anchor into the glenohumeral joint. Acta Ort- 13. Barber FA, Herbert MA, Hapa O, Rapley JH, Bar- hop Belg 2010; 76:834–7. ber CAK, Bynum JA, Hrnack SA. Biomechanical 6. Nho SJ, Provencher MT, Seroyer ST, Romeo AA. analysis of pullout strengths of rotator cuff and gleno- Bioabsorbable anchors in glenohumeral shoulder sur- id anchors: 2011 update. Arthroscopy 2011; 27:895– gery. Arthroscopy 2009; 25:788–93. 905. 7. Willemot L, Elfadalli R, Jaspars KC, Awh MH, Pee- 14. Mazzocca AD, Chowaniec D, Cote MP, Fierra J, ters J, Jansen N, De Clerq G, Verborgt O. Radiolo- Apostolakos J, Nowak M, Arciero RA, Beitzel K. gical and clinical outcome of arthroscopic labral re- Biomechanical evaluation of classic solid and novel pair with all-suture anchors. Acta Orthop Belg 2016; all-soft suture anchors for glenoid labral repair. Ar- 82:174–8. throscopy 2012; 28:642–8. 8. Hudak PL, Amadio PC, Bombardier C. Development 15. Pfeiffer FM, Smith MJ, Cook JL, Kuroki K. The hi- of an upper extremity outcome measure: the DASH stologic and biomechanical response of two commer- (disabilities of the arm, shoulder and hand) (correc- cially available small glenoid anchors for use in la- ted). The Upper Extremity Collaborative Group bral repairs. J Shoulder Elb Surg 2014; 23:1156–61. (UECG). Am J Ind Med 1996; 29:602–8.

195 ORIGINAL ARTICLE

Distal biceps tendon repair and posterior interosseous nerve injury: clinical results and a systematic review of the literature

Silvio Chiossi, Marco Spoliti, Pasquale Sessa, Valerio Arceri, Attilio Basile, Francesca Romana Rossetti, Riccardo Maria Lanzetti

Orthopedics and Traumatology Unit, Department of Emergency and Acceptance, San Camillo - Forlanini Hospital Rome, Italy

ABSTRACT

Aim To report clinical, functional and radiographic results of one- incision distal biceps tendon repair with Toggle Loc (Zimmer-Bi- omet, Warsaw, Indiana, USA) at an average 4-year follow-up and to assess posterior interosseous nerve injury complications after reconstruction.

Methods We conducted a retrospective review of 58 consecutive distal biceps tendon repairs performed at our department between 2010 and 2018. Disabilities of Arm, Shoulder and Hand (DASH) score, Visual Analogue Scale (VAS) scale and elbow range of motion (ROM) were recorded at each follow-up and an ultraso- und examination was also performed to assess the repaired biceps Corresponding author: brachii tendon. Silvio Chiossi Orthopedics and Traumatology Unit, Results Clinical evaluation showed good and excellent results at Department of Emergency and medium- and long-term follow-up. A temporary posterior intero- sseous nerve (PIN) palsy developed in four (6.81%) patients and Acceptance, always resolved in 8 weeks. PIN palsy prevalence is in accordance San Camillo - Forlanini Hospital with the results of the previous studies. Circonvallazione Gianicolense 87, 00153, Rome, Italy Conclusion Distal biceps tendon repair with Toggle Loc is an Phone: +39 06 58703171; effective surgical procedure. PIN injury is a relatively rare compli- cation after one-incision anterior repair. Our complication rate did Fax: +39 06 58704659; not differ significantly from other studies that have used cortical E-mail: [email protected] button fixation, reported in current literature. Our results confirm ORCID ID: https://orcid.org/0000-0002- that accidental injury of PIN may also happen to experienced sur- 9383-1000 geons and suggest extreme care and an appropriate surgical tech- nique to reduce this iatrogenic risk. Original submission: Key words: radial nerve lesion, surgical repair, sport trauma, ten- 24 October 2020; Revised submission: don avulsion 02 November 2020; Accepted: 23 November 2020 doi: 10.17392/1303-21

Med Glas (Zenica) 2021; 18(1):196-201

196 Chiossi et al. A single anterior incision repair technique

INTRODUCTION San Camillo-Forlanini Hospital (Rome) between January 2010 and December 2018. Rupture of distal biceps tendon is a relatively rare injury with an incidence of 1.2 per 100,000 pati- The diagnosis was based on clinical history and ents per year; injuries are most commonly seen in patient physical examination. X-ray and ultraso- the dominant elbow of males (86%) with an ave- und examination of the affected elbow were obta- rage age of 47 years. Smoke, steroids and statins ined before surgery. A Magnetic Resonance Ima- are associated to an increased risk of lesion (1,2). ging study (MRI) was done in 35 out of 58 (60%) patients. An ultrasound examination was perfor- Non-operative treatment leads to a functional med after the surgical procedure in each patient. loss of supination and flexion strength and en- durance; this is reserved for older and sedentary The injury mechanism was recorded: the most patients with elevated surgical risks (3,4). common mechanism was a forceful eccentric exten- sion of a flexed elbow as in lifting heavy objects. There are multiple options regarding the surgical technique of biceps tendon repair (one or two- Surgical procedures were all performed with incision techniques) and fixation devices (suture Toggle Loc repair (Zimmer-Biomet, Warsaw, anchors, interference screws, cortical buttons, Indiana, USA) by the senior and experienced bone tunnels) (5,6). Complications accompany elbow surgeon. Distal biceps tendon repair was both approaches and involve a spectrum of ner- always performed within 10 days from the injury. ve injuries, as well as heterotopic ossification, Patients with previous surgical procedures on the radioulnar synostosis, loss of forearm rotation affected elbow, systemic disease (diabetes, rheu- and wound infection (7,8). Fixation with cortical matoid arthritis) and previous local corticosteroid button provided the highest load to failure (584 injections were excluded from this study. Con- N) compared to suture anchors (253 N) and bone trols were performed at 6 and 12 months for the tunnels (173 N) (9). first year, and then yearly. Evaluation consisted of patient’s physical examination with an asse- Injury to the radial nerve is well-known and frequ- ssment of elbow range of motion (ROM), pain, ently described publication after distal biceps ten- quantified using the Visual Analogue Scale (VAS) don repair (7,8). Historically, repair of the distal (0 indicating absence of pain and 10 maximum biceps tendon was complicated by injury to the pain); standard radiographs in antero-posterior posterior interosseous nerve (PIN) in 10% to 15% (AP) and lateral (LL) views; Disabilities of Arm, of patients. More recent literature has suggested Shoulder and Hand (DASH score) questionnaires that injury to the PIN has decreased to less than were also obtained, with 0 reflecting no disability 10%, with rates as low as 1% (10). and 100 reflecting major disability. The aim of the present study was to report clini- Ultrasound records were performed at the final cal, functional and radiographic results of distal follow-up. biceps tendon repair with Toggle Loc (Zimmer- According to the current law of our country, no Biomet, Warsaw, Indiana, USA) at 4-year mean ethical review board was required due to the follow-up, PIN injury complications and to eva- retrospective nature of the study and it was con- luate the current literature to better quantify this ducted in accordance with the principles of the complication in a larger population of patients. Declaration of Helsinki and its amendments. We Such data could be potentially useful in preven- fully informed all the patients about the charac- ting these lesions. teristics of the study and they gave their consents. PATIENTS AND METHODS Methods Patients and study design Surgical technique. The surgical procedure con- sisted in a single anterior incision 1 cm distal to the We performed a retrospective analysis of 58 con- elbow skin crease extended longitudinally for 3-4 secutive patients who underwent a surgical repair cm, in correspondence with the radial tuberosity. of the distal biceps tendon due to a traumatic (i.e. The retracted distal biceps tendon was identified non spontaneous) tendon rupture at the Depar- and secured with a # 2 Orthocord (DePuy-Mitek, tment of Orthopaedics and Traumatology of the Raynham, MA, USA), placed in a whip stitch

197 Medicinski Glasnik, Volume 18, Number 1, February 2021

fashion. The dissection was carried down toward study: three because of an age <18 years, seven the radial tuberosity between the brachioradialis did not give their informed consent to the study laterally and the pronator-teres muscles medially. and one was lost to follow up, leaving 58 patients During this exposure the lateral antebrachial cuta- eligible for the present study. neous nerve and the deeper recurrent branches The mean age at the time of injury was 39 (range of the radial artery were identified and protected. 18-55) years. The surgical repair was performed, With the elbow in full extension and supination on average, at 5 days from the trauma. The mean the radial bicipital tuberosity was exposed. With follow-up was 49 months. the forearm in a maximally supinated position, a At clinical examination none of the patients lost guide wire was inserted through the bicipital tube- more than 5° in the flexion-extension or prona- rosity in an anterior to posterior direction, aiming tion-supination arc respect to the non-operated just slightly distal and ulnarly. Straight-cannulated limb; five patients had a slight loss of extension drill bits were then advanced over the guide wire: (4°), pronation (3°) and supination (5°) respect a 4,5 mm for the far, posterior cortex and usually 8 to the non-operated limb; at clinical examinati- mm, based on the size of the tendon, for the anteri- on ROM measurements were comparable in the or cortex. A Biomet Toggle Loc (Zimmer-Biomet, operated limbs in all patients (p>0.05). Warsaw, Indiana, USA) was secured to the distal biceps; the cortical button was pulled through the The 6-month follow up mean DASH score was radius using a pin to pass the suture and engaged 21.5 (±10.6) with 75% of excellent results and to the opposite radial cortex. The biceps tendon 25% of good results; at 12-month follow up 18.0 was then mobilized and brought into the prepared (±9.9) with 78% of excellent results and 22% drill hole on the radial tuberosity by shortening the of good results; at the final follow up it was 16 Zip loop. The fixation was tested and intraopera- (±10.3) with 80% of excellent results and 20% of tive fluoroscopy was used to confirm the correct good results. The statistical analysis showed no position of cortical button. The passing suture was statistical difference (p>0.05) between the clinical removed. scores at 6, 12 months and the final follow up. The mean VAS score was 2.1(±2) at 6-month follow Rehabilitation protocol. The arm was immo- up, 1.8 (±1.2) at 12-month follow up; 1.6 (±0.9) bilized at 90° flexion with a sling for 2 weeks; at the final follow up; no statistically significant passive movements were started immediately po- differences were found between the VAS score re- stoperatively with restricted extension that was sults at each follow-up (p>0.05) (Table 1). sequentially increased with a full extension at 6 weeks. Active ROM was allowed at 6 weeks Table 1. Mean DASH and VAS clinical scores Variable 6-month 12-month Last and gradual loading was applied to the arm until p (mean±SD) follow-up follow-up follow-up the fifth month from surgery. Patients performing DASH 21.5 (±10.6) 18 (±9.9) 16 (±10.3) >0.05 sports, returned to full athletic activity 10 months VAS 2.1 (±2) 1.8 (±1.2) 1.6 (±0.9) >0.05 postoperatively. DASH, Disabilities of the Arm, Shoulder and Hand; VAS, Visual Analogical Scale Statistical analysis Ultrasound and radiological examination did not Continuous variables were compared using the reveal patients of biceps tendon re-rupture or Student t-test. Results were analysed and the signs of heterotopic ossification or synostosis. study groups were compared with each other. No postoperative wound problems or infectious Continuous variable was described using the complications were observed. One patient re- mean ±SD. The level of significance was set ported mild pain during maximal biceps brachii at p= 0.05. contraction and three patients described sensory problems like paraesthesia long the surgical in- RESULTS cision. One patient reported complaints (tender- In the considered study period, 69 potentially ness) in the soft tissue of the surgical site. The eligible patients who underwent an acute surgi- most frequent complication was a transient palsy cal repair of the distal biceps tendon lesion were of the posterior interosseous nerve: four patients found. Eleven patients were excluded from the presented during their postoperative visit with

198 Chiossi et al. A single anterior incision repair technique

inability to extend the wrist, thumb and fingers with cortical button repair was used (9, 22-26) and were placed in a dynamic digital extension although one retrospective study (27) on 280 pa- splint, starting early active flexion hand exerci- tients found an incidence of transient PIN palsy ses. At 4 weeks postoperatively, the patients had of 3.2% (nine patients) when suture anchors (3 a slight improvement of extensor functions and cases) and cortical button (6 cases) were used as at 8 weeks they regained PIN function (Table 2). a fixation technique in a single surgical approach technique. In a recent systematic review of the Table 2. Postoperative complications literature regarding fixation methods and compli- Complication N (%) of patients Sensory disturbances 1 (1.7) cations Chavan et al. (28) found that end button Flexion tenderness 1 (1.7) repairs exhibited the best performance in com- Transient posterior interosseous nerve palsy 4 (6.9) parative biomechanical studies and reported that both transient and permanent nerve palsies were DISCUSSION the most common complication in a single in- Injury to the radial nerve is a well-known and frequ- cision group in 13%. Di Raimo et al. (29) was ently described complication after distal biceps ten- the first to investigate the use of Toggle Loc Zip don repair. The original incision technique of repair Loop and reported only one transient superficial described by Dobbie resulted in a high rate of radi- sensory radial nerve palsy on a series of 4 pati- al nerve injury (11). Two incision techniques were ents. Kodde et al. (30) reported a dysfunction of later developed by Boyd and Anderson in order to PIN in 2 patients of 22 (9%), which was transient decrease the high rate of nerve injury; however, in both patients; in this study Toggle Loc fixation this repair technique required two large incisions was used in 14 elbows: guide wire was drilled and was complicated by heterotopic ossification aiming just slightly distal and ulnarly. and/or radioulnar synostosis (12). The introduction These studies show that PIN palsy is a relatively of suture anchors, cortical buttons, including Endo- rare but serious complication after biceps repair button and interference screws, allowed repair of using cortical button fixation and may occur du- the distal biceps through a smaller single anterior ring dissection along the proximal radius, drill bit incision but also seemed to carry an increased risk placement or entrapment under a cortical button of injury to the superficial radial nerve, the lateral (31,32). The drill bit trajectory across the radi- antebrachial cutaneous nerve and the posterior in- us can be influenced by the skin incision used terosseous nerve (13-15). Therefore, injury to the to expose the biceps tuberosity of the radius; a PIN can occur both with one- incision and two-in- longitudinal incision placed over the biceps tube- cision approach techniques. In a systematic review, rosity of the radius results in the drill passing the Amin et al. (16) reported a complication rate of radius perpendicular to its longitudinal axis with PIN palsy of 2.7% (13/785) in the single incision an exit dorsal to the biceps tuberosity of the radi- procedure versus 0.2% (1/498) in the double inci- us. Two studies on fresh frozen cadavers showed sion procedure: this finding was most likely related that the mean distance of the button from the PIN to the need for more extensive anterior dissection was 9.3 mm (9) and 11.6 mm (33). to obtain an anatomic repair. Bain et al. (23) found that drilling anterior to po- In a retrospective cohort study of distal biceps sterior is safer than drilling radially: the distance tendon repair, Dunphy et al. (17) reported that from the tip of the Steinman pin advanced thro- single incision repair undergoes lower rates of re- ugh the bicipital tuberosity where it exited the operation, PIN palsy and heterotopic ossification posterior cortex ranged from 10 to 18 mm with (HO), but a significantly higher rates of transient 0 angulation and from 0 to 13 mm with 45° po- sensory nerve palsy compared to the double in- sterolateral angulation. Lo et al. (34) observed an cision technique. The overall rate of PIN palsy average distance of 11.2 mm from the PIN in the reported by this study was 1.3%. This value was anterior to posterior trajectory from the radial tu- lower than that previously reported by other stu- berosity, compared with 4.2 mm in the radial tra- dies (17-22) ranging from 2% to 14.8%. jectory; aiming the guide pin 30° ulnarly resulted Indeed, several studies reported no cases of PIN in the greatest distance from the PIN (16.7 mm), palsy when a single anterior surgical approach but such an ulnar angulation risks an impinge-

199 Medicinski Glasnik, Volume 18, Number 1, February 2021

ment of the cortical button on the ulna in supi- of iatrogenic injury. When an anterior approach nation. A distal drill trajectory across the radius is chosen, the arm should be supinated to protect resulted in the greatest risk of iatrogenic injury the posterior interosseous nerve and when pa- of the PIN (2.0 mm average distance between the ssing pins or drilling; oscillation or tapping of the drill bit and the PIN). slotted passing pin is recommended to prevent Such data were confirmed by two further studies entrapment of the posterior soft tissues. In some (31,35), reporting that drilling 30° ulnarly with cases, a small posterior-lateral incision could be the forearm in maximum supination results in the made to ensure that the PIN is not under the corti- greatest distance from the PIN, compared with cal button. Intraoperative imaging may be used to drilling anterior to posterior, with no decrease in confirm an appropriate position of the button on the bony tunnel length available for implants. the radial tuberosity; the cortical button should be deployed just as it exits the posterior cortex, to A recent study of Becker (36) showed that the avoid soft tissue interposition (20). PIN travels from an anterior position on the ra- dius when measuring 1 cm proximal on the bici- Identification of the radial nerve and its branches pital tuberosity to a lateral position on the radius can be important in protecting it from injury: if the at the level of the bicipital tuberosity prominence radial nerve and its PIN branches are isolated in- on the contralateral cortex to a slightly more po- traoperatively, the incidence of injury to these im- sterior position on the radius 1 cm distal to the portant structures can theoretically be minimized bicipital tuberosity; typically the PIN sits directly (37). We also advise extreme care in the exposition opposite the biceps tuberosity on the cortex of the of the radial tuberosity and recommend the use of radius in full supination and, therefore, perpendi- hand-held right-angle retractors (skin hooks) in- cular drilling starting at the bicipital tuberosity stead of Hohmann retractors at both sides of the should be avoided for the risk of iatrogenic PIN radius, that may lead to neurovascular complicati- injury; a more proximal and ulnar drilling angle ons caused by their increased force. is recommended; defining a safe zone for an ante- In conclusion, this study showed that “single- rior approach seems to be clinically unhelpful for incision” Toggle loop repair (Zimmer-Biomet, the high anatomical variability that exists for the Warsaw, Indiana, USA) of distal biceps tendon position of the PIN around the proximal radius. ruptures is a “reproducible” operation with good In the current study we inserted a guide wire clinical/functional results and a relatively rare in- through the bicipital tuberosity in an anterior to cidence of PIN palsy: this complication can be posterior direction, aiming just slightly distally reduced with appropriate surgical technique. and ulnarly: this distal drilling could potenti- FUNDING ally explain our cases of PIN neuroapraxia. Our experience confirms that accidental lesion of the No specific funding was received for this study. PIN may also happen to experienced surgeons; therefore, we advise extreme care and suggest an TRANSPARENCY DECLARATION appropriate surgical technique to reduce the risk Conflict of interest: None to declare.

REFERENCES

1. Safran MR, Graham SM. Distal biceps tendon ruptures: 4. Quach T, Jazayeri R, Sherman OH, Rosen JE. Distal incidence, demographics, and the effect of smoking. biceps tendon injuries-current treatment options. Bull Clin Orthop Relat Res 2002; 404:275-83. NYU Hosp Jt Dis 2010; 68:103-11. 2. Kelly MP, Perkinson SG, Ablove RH, Tueting JL. Distal 5. Lang NW, Bukaty A, Sturz GD, Platzer P, Joestl J. Tre- biceps tendon ruptures: an epidemiological analysis atment of primary total distal biceps tendon rupture using a large population database. Am J Sports Med using cortical button, transosseus fixation and suture 2015; 43:2012-7. anchor: a single center experience. Orthop Traumatol 3. Chillemi C, Marinelli M, De Cupis V. Rupture of the Surg Res 2018; 104:859-63. distal biceps brachii tendon: conservative treatment 6. Tjoumakaris FP, Bradley JP. Distal Biceps Injuries. versus anatomic reinsertion-clinical and radiological Clin Sports Med 2020; 39:661-672. evaluation after 2 years. Arch Orthop Trauma Surg 7. Stoll LE, Huang JI. Surgical treatment of distal biceps 2007; 127:705-8. ruptures. Orthop Clin North Am 2016; 47:189-205.

200 Chiossi et al. A single anterior incision repair technique

8. Ford SE, Andersen JS, Macknet DM, Connor PM, Lo- 24. Spencer EE Jr, Tisdale A, Kostka K, Ivy RE. Is the- effler BJ, Gaston RG. Major complications after distal rapy necessary after distal biceps tendon repair? Hand biceps tendon repairs: retrospective cohort analysis of (NY) 2008; 3:316-9. 970 cases. J Shoulder Elbow Surg 2018; 27:1898-906. 25. Peeters T, Ching-Soon NG, Jansen N, Sneyers C, 9. Greenberg JA, Fernandez JJ, Wang T, Turner C. En- Declercq G, Verstreken F. Functional outcome after doButton-assisted repair of distal biceps tendon rup- repair of distal biceps tendon ruptures using the en- tures. J Shoulder Elbow Surg 2003; 12:484-90. dobutton technique. J Shoulder Elbow Surg 2009; 10. Garon MT, Greenberg JA. Complications of distal 18:283-7. biceps repair. Orthop Clin N Am 2016; 47: 435-444. 26. Dillon MT, Bollier MJ, King JC. Repair of acute and 11. Dobbie RP. Avulsion of the lower biceps brachii ten- chronic distal biceps tendon ruptures using the Endo- don. Am J Surg 1941; 51: 662-83. Button. Hand (NY) 2011; 6:39-46. 12. Boyd HB, Anderson LD. A method for reinsertion of 27. Nigro PT, Cain R, Mighell MA. Prognosis for reco- the distal biceps brachii tendon. J Bone Joint Surg very of posterior interosseous nerve palsy after distal 1961; 43:1041-3. biceps repair. J Shoulder Elbow Surg 2013; 22:70-3. 13. Kelly EW, Morrey BF, O’Driscoll SW. Complications 28. Chavan PR, Duquin TR, Bisson LJ. Repair of the rup- of repair of the distal biceps tendon with the modified tured distal biceps tendon: a systematic review. Am J two-incision technique. J Bone Joint Surg Am 2000; Sports Med 2008; 36:1618-24. 82:1575-81. 29. DiRaimo MJ Jr, Maney MD, Deitch JR. Distal biceps 14. Miyamoto RG, Elser F, Millett PJ. Distal biceps ten- tendon repair using the toggle loc with zip loop. Ort- don injuries. J Bone Joint Surg Am 2010; 92:2128-38. hopedics 2008; 31. 15. Grewal R, Athwal GS, MacDermid JC, Faber KJ, Dro- 30. Kodde IF, van den Bekerom MP, Eygendaal D. Re- sdowech DS, El-Hawary R, King GJ. Single versus construction of distal biceps tendon ruptures with a double-incision technique for the repair of acute distal cortical button. Knee Surg Sports Traumatol Arthrosc biceps tendon ruptures: a randomized clinical trial. J 2015; 23:919-25 Bone Joint Surg Am 2012; 94:1166-74. 31. Thumm N, Hutchinson D, Zhang C, Drago S, Tyser 16. Amin NH, Volpi A, Lynch TS, Patel RM, Cerynik AR. Proximity of the posterior interosseous nerve DL, Schickendantz MS, Jones MH. Complications of during cortical button guidewire placement for distal distal biceps tendon repair: a meta-analysis of single- biceps tendon reattachment. J Hand Surg Am 2015; incision versus double-incision surgical technique. 40:534-6. Orthop J Sports Med 2016; 4:2325967116668137. 32. Lo EY, Li CS, Van den Bogaerde JM. The effect of 17. Dunphy TR, Hudson J, Batech M, Acevedo DC, Mir- drill trajectory on proximity to the posterior interos- zayan R. Surgical treatment of distal biceps tendon seous nerve during cortical button distal biceps repair. ruptures: an analysis of complications in 784 surgical Arthroscopy 2011; 27:1048-54. repairs. Am J Sports Med 2017; 45:3020-9. 33. Tat J, Hart A, Cota A, Alsheikh K, Behrends D, Mar- 18. Cain RA, Nydick JA, Stein MI, Williams BD, Poli- tineau PA. Distal biceps repair with flexible instru- kandriotis JA, Hess AV. Complications following dis- mentation and risk of posterior interosseous nerve in- tal biceps repair. J Hand Surg Am 2012; 37:2112-7. jury: a cadaveric analysis. Orthop J Sports Med 2018; 19. Carroll MJ, DaCambra MP, Hildebrand KA. Neurolo- 6 :2325967118810523. gic complications of distal biceps tendon repair with 34. Lo EY, Li CS, Van den Bogaerde JM. The effect of 1-incision endobutton fixation. Am J Orthop (Belle drill trajectory on proximity to the posterior interos- Mead NJ) 2014; 43:E159-62. seous nerve during cortical button distal biceps repair. 20. Garon MT, Greenberg JA. Complications of distal bi- Arthroscopy 2011; 27:1048-54. ceps repair. Orthop Clin N Am 2016; 47: 435-44. 35. Saldua N, Carney J, Dewing C, Thompson M. The ef- 21. McKee MD, Hirji R, Schemitsch EH, Wild LM, Wad- fect of drilling angle on posterior interosseous nerve dell JP. Patient-oriented functional outcome after safety during open and endoscopic anterior single-in- repair of distal biceps tendon ruptures using a sin- cision repair of the distal biceps tendon. Arthroscopy gle-incision technique. J Shoulder Elbow Surg 2005; 2008; 24:305-10. 14:302-6. 36. Becker D, Lopez Marambio FA. How to avoid poste- 22. Banerjee M, Shafizadeh S, Bouillon B, Tjardes T, Wa- rior interosseous nerve injury durino single-incision faisade A, Balke M. High complication rate following distal biceps repair drilling. Clin Orthop Relat Res distal biceps refixation with cortical button. Arch Ort- 2019; 477: 424-31. hop Trauma Surg 2013; 133:1361-6. 37. Mokhtee DB, Brown JM, Mackinnon SE, Tung TH. 23. Bain GI, Prem H, Heptinstall RJ, Verhellen R, Paix Reconstruction of posterior interosseous nerve injury D. Repair of distal biceps tendon rupture: a new tech- following biceps tendon repair: case report and cada- nique using the Endobutton. J Shoulder Elbow Surg veric study. Hand (NY) 2009; 4:134-9. 2000; 9:120-6.

201 ORIGINAL ARTICLE

A new technique of flexor carpi ulnaris transfer in multilevel surgery for upper extremity deformities in spastic cerebral palsy

Georgy Chibirov, Mairbek Pliev, Dmitry Popkov

Ilizarov National Medical Research Centre for Traumatology and Orthopaedics, Kurgan, Russian Federation

ABSTRACT

Aim To assess treatment outcomes of cerebral palsy (CP) patients who underwent upper limb surgical treatment including new tech- nique of flexor carpi ulnaris (FCU) transfer.

Methods The study included an outcome of orthopaedic surgeries in 30 upper limbs of 25 CP patients aged 10 to 24 years (mean age of 15.1 years). In addition to standard orthopaedic assessment, we used the integral scales of the Gross Motor Function Classificati- on System (GMFCS) and Manual Ability Classification System (MACS). Functional disorders of the upper limb were also evalua- ted with classifications of Van Heest, House, Gshwind and Tonkin.

Corresponding author: Results A total of 30 surgical interventions were performed. In Dmitry Popkov seven patients with hemiparesis, surgical treatment was accom- panied by simultaneous intervention on the lower limb. Improve- Ilizarov National Medical Research Centre ment of the functional capabilities and cosmetic appearance was for Traumatology and Orthopaedics noted in all cases in a follow-up over 12 months, as evidenced by M. Ulyanova Street 6, 640014 Kurgan, an improvement in the functional class according to Van Heest Russian Federation classification. Phone: +7 3522 454747; Conclusion A new technique of FCU transfer to the radius showed Fax: +7 3522 454060; to be an effective method to address pronation contracture of the E-mail: [email protected] forearm joints and can be used in combination with other elements Georgy Chibirov ORCID ID: https://orcid. of surgical intervention for elbow and thumb contractures. The org/0000-0001-6925-6387 FCU rerouting and transfer to distal radius is a good option in the absence of active supination. Distal release of FCU weakening flexion forces with a simultaneous procedure restoring active wrist extension provides satisfactory outcomes in the treatment of asso- Original submission: ciated flexed wrist contracture. 11 November 2020; Key words: cerebral palsy, flexor carpi ulnaris transfer, pronation Revised submission: contracture 13 November 2020; Accepted: 27 November 2020 doi: 10.17392/1313-21

Med Glas (Zenica) 2021; 18(1):202-207

202 Chibirov et al. Forearm surgery in cerebral palsy

INTRODUCTION Kurgan, Russian Federation. Limited articular function and deformities of upper limb of diffe- Cerebral palsy (CP) is a neurological, non-pro- rent severity was the main complaint of all pati- gressive disorder that affects the central nervous ents. Patients were divided into two groups after system. It is a heterogenic group of clinical syndro- physical examination: group 1 with spastic diple- mes describing impaired motion and posture, cha- gia (n=9) and group 2 with hemiparesis (n=16). racterized by pathological muscular tone, impaired control of movements and body position (1,2). In addition to standard orthopaedic assessment of the outcomes, the integral scales of the Gross Mo- Classically, CP patients with upper limb spasticity tor Function Classification System (GMFCS) (2) present with a flexed wrist, thumb-in-palm, and and the Manual Ability Classification System for forearm pronation deformity (3,4). Depending on children with Cerebral Palsy MACS were used the severity, the wrist and hand contracture may (6). For a differentiated evaluation of various produce a hygienic and cosmetic problems, and/ motor and functional disorders of the upper limb, or functional disability (4-6). Contractures of the the classifications of Van Heest for hand function upper limbs, dislocated joints, deformities in pa- disorders (17), House classification (4), Gshwind tients with CP developing due to muscle retracti- and Tonkin classification were used (10). on and causing serious functional limitations are known to be indications to operative orthopaedic The House classification (4) was used to assess treatment (3-6). Disturbed physical appearance is position and function of the thumb. This classifi- also an important consideration for surgery. The cation grouped patients depending on the type and wrist and hand contracture make grasp, pinch, severity of contracture of the first metacarpal-pha- and release activities difficult or even to impossi- langeal and interphalangeal joints. Gshwind and ble to perform and maintain (4, 7-13). Tonkin classification (10) was used to assess the severity of pronation contracture of the forearm Pronation contracture is observed in 48-50% of that was classified into four types of contracture: patients with upper limb injuries (4) with pre- type 1 included possibility to supinate actively valence up to 86% in hemiparetic CP associated beyond neutral, type 2 included possibility to su- with severely impaired functional activity in the pinate actively to get neutral position or less, type affected side (7-10). The wrist flexion contractu- 3 showed no active supination with the possibility re determines the appearance of the limb and si- of passive forearm supination to be achieved, and gnificantly limits the implementation of various type 4 had no active and passive supination. types of grip (6,8). The thumb-in-palm deformiti- es represent one more special condition requiring This research was approved by the Ilizarov Cen- surgical correction (7,9,14). tre Review Board. The study complies with the Declaration of Helsinki statement on the medical A multi-level surgery in a single procedure is pre- protocol and ethics. Representatives of all pati- ferable to many small procedures (15,16). The ents enrolled in the study provided an oral and aim of this study was to investigate a correction written informed consent. of forearm pronation, wrist flexion and thumb-in- palm deformities performed throughout single- Methods event procedure in CP patients and to assess the outcomes of operative procedures in their various Surgical technique. Various combinations of combination on upper limbs in CP patients where surgical elements were used during interventi- new technique of flexor carpi ulnaris was applied. ons depending on the presence and severity of forearm orthopaedic disorders. As a retrospecti- PATIENTS AND METHODS ve cohort, there were no criteria for the choice of surgery, and the decision was made by each The study included outcome of orthopaedic sur- surgeon based on his experience: procedures geries in upper limbs of 25 CP patients aged 10 involving the pronator teres muscle; release at to 24 years (mean age of 15.1 years). A total of distal ends; transposition of the pronator teres 30 operative interventions were performed in the to the extensor’s carpi radialis; myotomy of the period 2013-2019 in the National Ilizarov Medi- pronator quadratus muscle; radial (rotational) cal Centre for Traumatology and Orthopaedics, osteotomy; adductor and/or first dorsal intero-

203 Medicinski Glasnik, Volume 18, Number 1, February 2021

sseous releases; first web Z-plasty; flexor pollicis third of the forearm. The tendons of the abductor longus lengthenings; fractional or Z-lengthening pollicis longus, extensor pollicis brevis muscles of flexors digitorum; shortening of the extensor and the distal tendon of the brachioradialis muscle pollicis longus and the extensor pollicis brevis being adjacent to the radius periosteum at the level tendons; flexor carpi ulnaris (FCU) rerouting and where they were exposed. The canal in the radius transposition to distal radius. was produced in the radial shaft distal third or in The surgical techniques for the first six groups of the distal metadiaphysis. Trajectory of the canal procedures were standard ones (5, 7-12). But, the was arranged in a blunt manner in oblique des- last type of the procedure was specific (Figures cending way under the posterior forearm muscles 1,2). M. flexor carpi ulnaris was exposed using to drag the M. flexor carpi ulnaris tendon. Ulnar medial approach in the distal and middle third flexor threads and a portion of the tendon run thro- of the forearm and sutured distally at the site of ugh the canal, were sutured back to the tendon, attachment with tenotomy and mobilization pro- and to the tendon of the brachioradialis muscle duced with two thirds. Radius was exposed subpe- and periosteum applying interrupted sutures. The riosteally using S-shaped approach in the lower forearm and the hand were secured in a maxi- mally supinated position while suturing. It should be emphasized that the transfer of the FCU to the distal radius provides a supination moment arm, which is greatest if the FCU is released two-thirds of the length of the forearm as it wraps around the ulna onto the dorsum of the wrist. One of the variations of the above-mentioned manipulation implies attachment the tendon to the radius using an anchor. Torsion of the forearm bones and severely rigid contractures of the jo- ints were found in the upper limbs in two patients with no active and passive supination. Excessive pronation value was of more than 50° in both pa- tients. A derotation radius osteotomy (Figure 3) has been done to correct tortional deformity in both patients. A locked titanium plate was used for osteosynthesis. The Z-shaped skin plasty of the first interdigital space was performed in combination with teno-

Figure 1. Schema of the surgical technique. A) FCU exposed, distally sutured and mobilized by tenotomy (front view); B) FCU tendon run through the oblique descending canal towards the distal radius (back view); C) FCU sutured to the anchor and dis- tal tendon of the brachioradialis (lateral view) (Chibirov G, 2020)

Figure 2. Photo of the approach and FCU tendon attachment, Figure 3. Radiographs of forearm. A) Radius with inserted an- arrows point out: 1 – distal portion of FCU tendon; 2 – anchor chor; B) forearm before surgery; C) anchor into radius, derotation inserted into distal radius; 3 – distal radius (Popkov D, 2020) osteotomy, osteosynthesis with locked plate (Chibirov G, 2018)

204 Chibirov et al. Forearm surgery in cerebral palsy

tomy of the transverse portion of the adduction upper limb, cosmetic appearance and ease of use pollicis muscle, aponeurotomy of the flexor polli- were noted in all cases, as evidenced by an im- cis longus muscle to address impaired function of provement in the functional class with regard to the thumb if indicated. Van Heest classification (Table 2).

Statistical analysis Table 2. Functional ability of the patients (by Van Heest clas- sification) The statistical values were described as the mean No of patients in the group Van Heest Group 1 Group 2 and standard deviation. classification level Preoperati- Postoperati- Preoperati- Postoperati- RESULTS vely vely vely vely 2 1 4 In the group 1 (spastic diplegia) four patients 3 5 5 4 3 2 7 8 were classified as GMFCS level II, four as 5 5 5 GMFCS level III and one patient as GMFCS le- 6 2 3 vel IV; three patients were classified as MACS After a mean follow-up of more than 12 months level II, four as MACS level III, and two as (12-23 months), the whole patient cohort was fo- MACS level IV. In the group 2 (spastic hemipa- und to have had significant improvement in their resis) 11 patients were classified as GMFCS level ranges of motion from a mean preoperative negati- II, five as GMFCS level III; nine were classified ve active supination of -11° to a mean postoperati- as MACS level II, four as MACS level III and ve supination of 29° in the group of spastic diplegia three as MACS level IV. and 34° in hemiparesis patients (p<0.03) (Figure The House classification evaluated the use of pa- 4). No statistically significant difference between tients' hand as type 1 (n=2), type 2 (n=2), type 3 (n=16) and type 4 (n=5). Pronation deformity cla- ssified by Gshwind and Tonkin was evaluated as type 2 (n=2), type 3 (n=18) and type 4 (n=5). There was clinical correlation between severity of pronati- on contracture and impaired function of the thumb. A total of 30 surgical interventions were perfor- med (including five patients with spastic -diple gia, sequentially on both upper limbs). In seven patients with hemiparesis, surgical treatment was accompanied by simultaneous intervention on the lower limb (Strayer procedure and guided Figure 4. A female patient aged 36 years, left-side spastic growth by temporary epiphysiodesis). hemiplegia. A) before surgery; B) active supination and dorsal flexion restored (Dr.Pliev M, 2016-2017) Multilevel surgery led to better hand usage and the groups regarding active supination and prona- better overall upper limb function (Table 1). Im- tion movement was found. Probably this homoge- provement of the functional capabilities of the neity of results was related to mild neurologic dis- Table 1. Mean values of active ROM (Range of Motion) in orders classified as GMFCS level I-III in the whole the joints of the upper limbs preoperatively and in a long- term follow-up cohort. Regarding maximal active dorsiflexion, af- Postoperative period* ter the surgery, there was a statistically significant Pre-operati- improvement. The group 2 (spastic hemiparesis Segment Motion Group 1 Group 2 ve period (spastic (spastic he- patients) showed better results in wrist extension. diplegia) miparesis) Elbow flexion/extension 148º/39º/0 147º/14º/0 152º/18º/0 We found no overcorrection complications in our supination/ cohort. Forearm 0/11º/81º 29º/0/50º 34º/0/52º pronation dorsiflexion /palmar Wrist 0/54°/86º 18º/0/69º 32º/0/57º DISCUSSION flexion adduction/abduction 45º/29º/0 31º/0/17º 25º/0/29º The significance of forearm pronation deformi- Thumb flexion/extension 76º/48º/0 54º/0/16º 50º/0/18º ties in CP patients is not small. Aside from the abduction/adduction 0/14º/56º 31º/0/36º 35º/0/33º *Follow-up over 12 months aesthetics, a pronated forearm position associa-

205 Medicinski Glasnik, Volume 18, Number 1, February 2021

ted with flexed wrist interferes with normal upper difficult to control and there is a high risk of either limb function. This excludes many important so- under lengthening or overcorrection (18,26,28). cial and functional activities including handsha- This risk is particularly higher for wrist flexor len- king, face washing, clapping and causes compen- gthening in contrast to Z-lengthening of adductor sate bizarre postures and other body and shoulder pollicis longus and skin plasty (14). movements (3,6,8). The orthopaedic surgery for This study has shown that the new technique of contractures and deformities in upper limbs in FCU transfer is highly effective in the manage- CP patients aims to improve functional abilities, ment of pronation deformities of the forearm hygienic care and cosmetic appearance (9,17,18). and flexed wrist in patients with cerebral palsy. There are various orthopaedic conditions in Furthermore, this surgical technique not only re- upper limbs justifying reconstructive surgery leases the M. flexor carpi ulnaris as a deforming (3,6,8,11,19,20). Pronation forearm contracture wrist flexion and cubital deviation force, but also due to retraction of M. pronator teres and M. pro- transfers the M. flexor carpi cubitalis to the radius nator quadratus is a common issue in CP patients as a supinator force. The physiological reason of (20-23). Restoration of active supination is reco- this transfer is related to the fact that CP patients gnized as an objective for orthopaedic surgery (13, are likely to actively use the flexor carpi ulnaris 20-24). There is a variety of techniques to address for functional needs (13,29). In comparison with the contracture including tendon and muscle rele- our previous series (30), this study refined indi- ases, transposition of flexor and pronator tendons cations for FCU transfer: highly restricted or lost to extensors carpi, corrective detorsion osteotomi- active forearm supination, total M. supinator im- es (8,18,20). But there is no clear consensus over pairment. On the other hand, FCU release in com- how to optimally manage pronation deformities of bination with shortening of the extensor pollicis the forearm in cerebral palsy patients yet. longus and the extensor pollicis brevis tendons The use of transfer of FCU to M. extensor carpi ensured active wrist extension. Results of our seri- radialis brevis or M. pronator teres transfer rema- es justify surgical approach to address contracture ins controversial in the treatment for flexed wrist. and deformities in upper limbs in CP patients to Transposition of the flexor carpi ulnaris muscle improve functional abilities. The choice of tech- can be advocated to improve dorsiflexion of the nique and prognosis of the results rely on the level hand. The surgery was first described by Green of neurological deficiency and motivation of a pa- (25) and was found efficient enough (9,22,23,26). tient to use the operated limb (2,3,8,19). Unfortu- According to Green, M. extensor carpi radialis nately, a satisfactory functional outcome cannot be brevis/longus is an optimal site for transposition expected in patients with an IQ less than 70 (27). of the M. flexor carpi ulnaris to ensure correction Limitations of this study are the small number of of the wrist flexion and ulnar deviation of the hand patients and relative heterogeneity of the series. (25). On the other hand, Patterson et al. (26) repor- The new technique of FCU transfer to the radi- ted occurrence of postoperative extension defor- us has been shown to be an effective method to mity of the wrist after this transfer. address pronation contracture of the forearm jo- Flexion contractures of the wrist and fingers are ints and can be used in combination with other often present in CP patients (11). Flexion and ad- elements of surgical intervention for elbow and duction of the thumb coupled with flexion con- thumb contractures. The FCU rerouting and tran- tracture of the wrist cause more serious functional sfer to distal radius is a good option in the absen- concerns (7,14). Surgical treatment is primarily ce of active supination. Distal release of FCU aimed at the correction of fixed contractures and weakening flexion forces with a simultaneous establishment of a functional balance betwe- procedure restoring active wrist extension pro- en spastic flexors and weak extensors (21). The vides satisfactory outcomes in the treatment of transfer of the flexor carpi ulnaris solely fails to associated flexed wrist contracture. address flexion in the radiocarpal joint related to other shortened flexors of the wrist. Surgical opti- ACKNOWLEDGMENTS ons addressing the condition include aponeurotic The authors wish to thank Luigi Meccariello, release of short muscles and their Z-lengthening M.D., and prof. Selma Uzunović, MD, MA, PhD (11,13,27). Reported studies show related com- for their kind invitation to participate in the spe- plications of surgical treatment. Length gain is cial volume of Medicinski Glasnik.

206 Chibirov et al. Forearm surgery in cerebral palsy

FUNDING TRANSPARENCY DECLARATION No specific funding was received for this study. Competing interests: None to declare.

REFERENCES 1. Stavsky M, Mor O, Mastrolia SA, Greenbaum S, Than 15. Smitherman JA, Davids JR, Tanner S, Hardin JW, Wa- NG, Erez, O. Cerebral palsy -trends in epidemiology gner LV, Peace LC, Gidewall MA. Functional outco- and recent development in prenatal mechanisms of di- mes following single-event multilevel surgery of the sease, treatment, and prevention. Front Pediatr 2017; upper extremity for children with hemiplegic cerebral 13:21. palsy. J Bone Joint Surg Am 2011; 93:655-61. 2. Paulson A, Vargus-Adams J. Overview of four functio- 16. Chibirov GM, Dolganova TI, Dolganov DV, Popkov nal classification systems commonly used in cerebral DA. Analysis of the causes of pathological patterns of palsy. Children (Basel) 2017; 4:30. the kinematic locomotor profile based on the findings 3. Klevberg GL, Elvrum AG, Zucknick M, Elkjaer S, of computer gait analysis in children with spastic CP Østensjø S, Krumlinde-Sundholm L, Kjeken I, Jahn- types. Genij Ortopedii 2019; 25:493-500. sen R. Development of bimanual performance in 17. Van Heest AE, House JH, Cariello C. Upper extre- young children with cerebral palsy. Dev Med Child mity surgical treatment of cerebral palsy. J Hand Surg Neurol 2018; 60:490-7. (Am) 1999; 24:323-30. 4. House JH, Gwathmey FW, Fidler MO. A dynamic 18. Gharbaoui I, Kania K, Cole P. Spastic paralysis of the approach to the thumb-in-palm deformity in cerebral elbow and forearm. Semin Plast Surg 2016; 30:39-44. palsy. J Bone Joint Surg Am 1981; 63:216-25. 19. Simon-Martinez C, Jaspers E, Mailleux L, Desloovere 5. Tranchida GV, Van Heest A. Preferred options and evi- K, Vanrenterghem J, Ortibus E, Molenaers G, Feys dence for upper limb surgery for spasticity in cerebral H, Klingels K. Negative influence of motor impair- palsy, stroke, and brain injury. J Hand Surg Eur Vol. ments on upper limb movement patterns in children 2020; 45:34-42. with unilateral cerebral palsy. a statistical parametric 6. Park ES, Sim EG, Rha DW. Effect of upper limb de- mapping study. Front Hum Neurosci 2017; 5:482. formities on gross motor and upper limb functions in 20. Gschwind CR. Surgical management of forearm pro- children with spastic cerebral palsy. Res Dev Disabil nation. Hand Clin 2003; 19:649-55. 2011; 32:2389-97. 21. Zancolli EA, Zancolli ER Jr. The infantile spastic 7. Van Heest AE. Surgical technique for thumb-in-palm hand. Surgical indications and management. Ann deformity in cerebral palsy. J Hand Surg (Am) 2011; Chir Main 1994; 3:66-75. 36:1526-31. 22. Veeger H, Kreulen M, Smeulders M. Mechanical eva- 8. Leafblad ND, Van Heest AE. Management of the spa- luation of the pronator teres rerouting tendon transfer. stic wrist and hand in cerebral palsy. J Hand Surg J Hand Surg (Br) 2004; 29:259-64. (Am) 2015; 40:1035-40. 23. Bunata R. Pronator teres rerouting in children with 9. Seruya M, Dickey RM, Fakhro A. Surgical Treatment cerebral palsy. J Hand Surg (Am) 2006; 31:474-82. of Pediatric Upper Limb Spasticity: The Wrist and 24. Tranchida GV, Van Heest AE. Outcomes After Surgi- Hand. Semin Plast Surg. 2016; 30:29-38. cal Treatment of Spastic Upper Extremity Conditions. 10. Gschwind C, Tonkin M. Surgery for cerebral palsy: Hand Clin. 2018; 34:583-591 part 1. Classification and operative procedures for 25. Green WT, Banks HH. Flexor carpi ulnaris transplant pronation deformity. J Hand Surg (Br) 1992; 17:391- and its use in cerebral palsy. J Bone Joint Surg (Am) 5. 1962; 44:1343-430. 11. Van Heest AE, Ramachandran V, Stout J, Wervey R, 26. Patterson JM, Wang AA, Hutchinson DT. Late defor- Garcia L. Quantitative and qualitative functional eva- mities following the transfer of the flexor carpi ulnaris luation of upper extremity tendon transfers in spastic to the extensor carpi radialis brevis in children with hemiplegia caused by cerebral palsy. J Pediatr Orthop cerebral palsy. J Hand Surg (Am) 2010; 35:1774-8. 2008; 28:679-83. 27. Čobeljić G, Rajković S, Bajin Z, Lešić A, Bumbašire- 12. Van Heest AE, Bagley A, Molitor F, James MA. Ten- vić M, Aleksić M, Atkinson HD. The results of surgi- don transfer surgery in upper-extremity cerebral palsy cal treatment for pronation deformities of the forearm is more effective than botulinum toxin injections or in cerebral palsy after a mean follow-up of 17.5 years. regular, ongoing therapy. J Bone Joint Surg (Am) J Orthop Surg Res 2015; 10:106. 2015; 97:529-36. 28. Bisneto Ede N, Rizzi N, Setani EO, Casagrande L, 13. Fitoussi F, Diop A, Maurel N, Laasel M, Ilharreborde Fonseca J, Fortes G. Spastic wrist flexion in cerebral B, Penneçot GF. Upper limb motion analysis in chil- palsy. Pronator teres versus flexor carpi ulnaris tran- dren with hemiplegic cerebral palsy: proximal kine- sfer. Acta Ortop Bras 2015; 23:150-3. matic changes after distal botulinum toxin or surgical 29. Hoffer MM. The use of the pathokinesiology labo- treatments. J Child Orthop 2011; 5:363-70. ratory to select muscles for tendon transfers in the 14. Alewijnse JV, Smeulders MJ, Kreulen M. Short-term cerebral palsy hand. Clin Orthop Relat Res 1993; and long-term clinical results of the surgical correcti- 288:135-8. on of thumb-in-palm deformity in patients with cere- 30. Chibirov GM, Leonchuk SS, Ezhova KS, Gubina EB, bral palsy. J Pediatr Orthop 2015; 35:825-30. Pliev MK, Lascombes P, Popkov DA. Operative tre- atment of orthopedic complications in upper limb in children and adults with cerebral palsy. Genij Ortope- dii 2018; 24:312-20.

207 ORIGINAL ARTICLE

The challenge of the surgical treatment of paediatric distal radius/ forearm fracture: K wire vs plate fixation - outcomes assessment

Salvatore Di Giacinto1, Giuseppe Pica2, Alessandro Stasi3, Lorenzo Scialpi3, Alessandro Tomarchio4, Alberto Galeotti5, Vlora Podvorica6, Annamaria dell’Unto7, Luigi Meccariello2

1Department of Pediatrics Orthopedics and Traumatology, Meyer University Children's Hospital, Florence, 2Department of Orthopedics and Traumatology, AORN San Pio, Benevento, 3Department of Orthopedics and Traumatology, Santissima Annunziata Hospital, Taranto, 4Department of Orthopedics and Traumatology, S. Croce e Carle Cuneo Hospital, Cuneo, 5Orthopaedic Traumatology Centre, Careggi University Hospital, Florence; Italy, 6Pediatric Orthopedic and Trauma Unit, University Clinical Center, Prishtina, Kosovo, 7Department of Orthopedics, Sapienza - University of Roma La Sapienza, Roma, Italy

ABSTRACT

Aim Distal radius/forearm fractures in adolescent patients remain challenging injuries to treat. Distal radius/forearm bony anatomy is not completely restored with intramedullary K wire fixation. The aim of this study was to compare radiographic and functional outcomes obtained using intramedullary K wire fixation and open reduction and internal fixation in the treatment of distal radius/ forearm fracture.

Methods A total of 43 patients who presented with distal radius/ forearm fractures were enrolled and divided into two groups: 23 Corresponding author: patients treated with K-wire (IMNK) and 20 patients treated with Salvatore Di Giacinto plate and screws (ORIF). The evaluation criteria were: fracture he- Department of Paediatrics, aling time, objective quality of life measured by the Mayo wrist Orthopaedics and Traumatology, score (MWS) and quick disabilities of the arm, shoulder and hand score (QuickDash), length time of surgery, complications, sport or Meyer University Children's Hospital play return, forearm visual analogic pain (FVAS), bone healing by Viale Gaetano Pieraccini, 24, 50139 radius union scoring system (RUSS). Firenze, Italy Phone: +39 329963738; Results In both groups the results obtained were comparable in Fax: +39 0555662908; terms of functional, pain and return to play/sport after the third month after surgery. Bone healing was faster in IMNK than ORIF E-mail: [email protected] but without significance (p>0.05). There was less complication in ORCID ID: https://orcid.org/0000-0003- ORIF than IMNK (p<0.05). 2569-307X Conclusion The treatment of adolescent distal radius or forearm fractures remains challenging. One challenge facing the physician Original submission: is the choice of surgical technique and fixation method, which will be influenced by individual experience and preference. 13 November 2020; Accepted: The question of distal radius or forearm fractures in adolescents 17 November 2020 would be best answered with a prospective randomized study. doi: 10.17392/1315-21 Key words: paediatric, pain, wrist

Med Glas (Zenica) 2021; 18(1):208-215

208 Di Giacinto et al. K wire vs plate in radius/forearm fracture

INTRODUCTION The rapid growth feature increases fracture ten- dency at the lower end of the radius, because dis- Distal radius fractures are the most common fractu- tal metaphysis is relatively weak due to continuo- res in children, amounting to around a quarter to a us remodelling. Displaced distal radius fractures third of all paediatric fractures (1). Annual inciden- are usually treated with closed reduction and in- ces of 30/10,000 children (aged 0 to 17 years) have tramedullary nail K wires (IMNK) (Figure 1) (4). been reported in the US during 2009 (2). The mean age of children (aged up to 16 years) presenting Prevention of the reduction loss is the main issue with these injuries in 2000 at two Edinburgh hos- in conservative treatment (5). Distal radius/fore- pitals was 9.9 years and 55% were boys (3). The arm fractures in adolescent patients remain chall- distribution of fractures is unimodal for both gen- enging injuries to treat. Distal radius/forearm ders (3). In 2010, Hedström et al. reported peaks at bony anatomy is not completely restored with 11 years for girls and 14 years for boys. Fractures IMNK. Results suggested an association towar- are seen especially in pubertal growth ages (11–14 ds increased complication rates and complication years in males, 8–11 years in females) and in spring severity with intramedullary fixation (6). Nowa- and summer months when physical activity incre- days open reduction and internal fixation with ases (3). Distal radius fractures most commonly plates and screws (ORIF) are commonly used in result from a fall on an outstretched hand (3). the pubertal growth age (7) (Figure 2).

Figure 1. 12-year-old female with bike fall trauma to her left wrist. A, B) Preoperative X-rays showed the complete fracture of distal radius and ulna; C, D) post-surgery X-rays showed the intramedullary K wires fixation of only distal radius; E, F) the X-rays showed the bone healing after only one month from the surgery (Di Giacinto S, 2019)

Figure 2. 13-year-old male, sports trauma to the right wrist. A, B) Preoperative X-rays showed the complete fracture of distal radius and ulna; C, D) post-surgery X-rays showed the plate fixation on the radius and ulna; E, F) the X-rays showed the bone healing after only one month from the surgery (Di Giacinto S, 2019)

209 Medicinski Glasnik, Volume 18, Number 1, February 2021

The aim of this study was to compare the radio- ring System (NUSS) was used (9). The criteria to graphic and functional outcomes obtained using evaluate the two groups during the follow-up were: IMNK and ORIF in the treatment of distal radius/ the objective quality of life and the wrist function forearm fracture. measured by the Mayo wrist score (MWS) (3), the subjective quality of life and the wrist function me- PATIENTS AND METHODS asured by quick disabilities of the arm, shoulder and hand score (QuickDASH) (3), length of sur- Patients and study design gery, complications, sport or play return, forearm Among a total of 123 radius/forearm fractures of visual analogue pain (VAS) (3), bone healing by the patients admitted and treated at one Level I tra- radius union scoring system (RUSS (10). Union of uma Centre of Meyer University Children's Hos- each of the 4 cortices was graded on a 3-point sca- pital, Florence and one Level II Trauma Centre of le: 0 - fracture line visible with no callus; 1 - callus AORN San Pio, Benevento, from January 2017 to formation but fracture line present, 2 - cortical December 2019, 43 patients with distal/forearm bridging without clear fracture line. Reviewers also fractures were finally included. Inclusion criteria recorded their overall impression of fracture uni- were: patients admitted to our Centre for surgical on (united or not united). Regarding radiographic treatment, patients fit for surgery, age between 12- measurements as volar tilt, radial inclination, radial 14 years, bayonetting >1 cm, angulations >10°, length and others, standard posteroanterior, and la- malrotation >30°, dorsal angulation >20 degrees. teral radiographs were used. The evaluation endpo- Exclusion criteria were: haematological or oncolo- int was set at 12 months after surgery. gical patients, acute or chronic infections, previous Rehabilitation protocol. Casting historically lower limb trauma, nerve injuries, segmental con- consisted of a long arm cast for 6-8 weeks with tralateral fracture, vessels injuries, age <12 and >14 the possibility of conversion to a short arm cast years old, conservative treatment, fracture older after 2-4 weeks depending on the type of fracture than 21 days, distal physis, diaphysis, bayonetting and healing response; may cast for shorter pe- <1 cm, angulations <10°, malrotation <30°, dorsal riods, 3-4 weeks, depending on child's age and angulation <20 degrees, non 23-M/3 complete or healing on imaging; multiple high quality studies 23r-M/3 complete type of fracture according to AO show fractures of distal third may be immobili- (Arbeitsgemeinschaft für Osteosynthesefragen) zed with a properly moulded short arm cast (4,6); Classification (8), bone metabolism diseases, ske- special case of fractured distal radius with intact letal immaturity, mental or neurologic disorder. ulna: extreme ulna deviation of wrist helps keep All patients’ parents were informed in a clear and radius fracture out to length. comprehensive way of two types of treatments During the cast period children may use the inju- and other possible surgical and conservative al- red hand for light activities only and they sho- ternatives. Patients were treated according to the uld move their wrist and fingers within their pain Ethical Standards of the Helsinki Declaration, tolerances straight away. It is advisable to take and were invited to read, understand, and sign an pain killers as prescribed to enable your child to informed consent form. complete the exercises. Azienda Ospedaliera Mayer Firenze/Italy Ethical Say to parents: It is common for the wrist to ache Committee approved this research. and sometimes be painful for further 3-6 months after the injury. Methods Stage 1 (after 4 weeks to 5 weeks after the sur- All fractures were classified according to the AO gery): finger and wrist flexion and extension, Classification (8) Forty-three patients were divi- elbow bend and straighten, forearm rotations. ded in two groups: 20 patients treated with ORIF Another advice for the patient was: cold pack (ice with plate and 23 patients treated with intrame- pack or frozen peas wrapped in a damp towel) dullary K wire (IMNK) (Table 1). can provide short-term pain relief. Apply this Both groups underwent the same rehabilitation to the sore area for up to 15 minutes every few protocol (see rehabilitation protocol). To study the hours, ensuring the ice is never in direct contact bone healing on radiographs, the Non-Union Sco- with the skin.

210 Di Giacinto et al. K wire vs plate in radius/forearm fracture

Stage 2 (after 5 weeks): 3-4 times a day, wrist (and the range) of the patients was rounded at flexion stretch, wrist extension stretch (prayer the closest year. The predictive score of out- exercise), sideways wrist stretches. come and quality of life and the range were Grip strength exercises. It is advisable to take approximated at the first decimal, while the pain killers as prescribed to enable your child to Pearson correlation coefficient was approxi- complete the exercises. Cold packs: can provide mated at the second decimal (r). Cohen's kappa short-term pain relief. Apply this to the sore area coefficient (κ) was used to measure inter-rater for up to 15 minutes every few hours, ensuring agreement for qualitative (categorical) items; the ice is never in direct contact with the skin. through this parameter we calculated the con- cordance between different qualitative values Statistical analysis of the outcomes and the bone healing, the an- atomical and biomechanical axis of the distal Descriptive statistics were used to summarize forearm from the radiological point of view. the characteristics of the study group and sub- groups, including mean and standard deviation RESULTS of all continuous variables. The t-test was used to compare continuous outcomes. The χ2 test or There was no statistically significant difference Fisher’s exact test (in subgroups smaller than between the two populations according to age, 10 patients) were used to compare categorical gender, type of fracture, NUSS, etc. (Table 1). variables. The statistical significance was de- In 12 of 23 (52.17%) of IMNK group, it had to be fined as p<0.05. The Pearson correlation coef- open to reduce the fracture (Table 1). ficient (r) was used to compare the predictive The surgery lasted for an average of 32.6 (±11.6; score of outcomes and quality of life. Mean age range 15-42) minutes in IMNK (p<0.05), while

Figure 3. 13-year-old male with sports trauma to the right wrist. A, B) Preoperative X-rays showed the complete fracture of distal radius and ulna; C, D) post-surgery X-rays showed intramedullary K wires fixation on the radius and ulna; E, F) the X-rays showed non-union/malunion with dorsal angulation, bayoneting, and radial angulation of distal forearm. Healing after only one month from the remotion of IMNK; G, H) X-rays post re-surgery showed the anatomic reduction with plate and screws on radius and ulna, after the debridement and calloclasy of non-union focus; I, J) bilateral bone healing with exuberant callus on all cortices just one month after re-surgery (Meccariello L, 2019)

211 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 1. Characteristics of the intramedullary K wire fixation Table 2. The comparison of the patients treated with intramed- (IMNK) and open reduction internal fixation (ORIF) patient’s ullary nail K wire (IMNK) and open reduction and internal fixa- groups tion with plate and screws (ORIF) Characteristic IMNK (n=23) ORIF (n=20) Variable IMNK (n=23) ORIF (n=20) p 32.6 54.8 Average age, years (standard Average follow up, length of 12.86 (±0.64) 13.02 (±11.77) (±11.6; (±17.9; <0.05 deviation, SD) time of surgery (SD; range) Age range (years) 12-14 12-14 range 15-42) range 37-74) Average follow up, month (SD; 16.86 16.37 Gender ratio (No) (male:female) 1.875 (15:8) 1.857 (13:7) >0.05 Previous type of accident (No, %) range) (±5.64;12-36) (±5.77;12-36) Average bone healing, month 2.6 2.8 Fall from height 8 (34.78) 5 (20) >0.05 Bike accident 4 (17.39) 5 (20) (SD; range) (±1.22;1-4) (±1.34;1-4) Average RUSS at the moment Motorcycle accident 4 (17.39) 5 (20) 29.4 29.8 of bone healing, points (SD; >0.05 Sport accident 7 (30.44) 5 (20) (±1.22;29-30) (±0.1;29-30) range) Previous type of femoral shaft fractures according to AO (8) (No, %) Average VAS at the moment of 0.8 0.7 Non 23-M/3 complete 16 (69.56) 12 (60) bone healing, points (±0.2; (±0.3; >0.05 23r-M/3 complete 7 (30.44) 8 (40) (SD; range) range 0-1) range 0-1) Type of fracture (No, %) Cohen’s kappa (k) 0.89 0.91 Closed 23 (100) 23 (100) <0.05 (SD; range) (±0.11: 0.78-1) (±0.09: 0.90-1) Open 0 0 Pre-operative radiological characteristics of fracture Injured upper limb side (No, %) Average bayonetting (SD; 1.56 1.67 Right 8 (34.78) 7 (35) >0.05 range) (cm) (±0.32;1-3) (±0.48;1-3) Left 15 (65.22) 13 (65) Average angulation (SD; range) 36.4 35.8 Average non-union scoring >0.05 4.89 (±1.33) 4.94 (±1.47) (degrees) (±8.77;29-52) (±7.89;29-52) system (SD) Average maltrotation (SD; 33.7 34.1 Range non-union scoring >0.05 0-15 0-15 range) (degrees) (±2.55;30.1-36)(±3.27;30.1-38) system Average dorsal angulation (SD; 24.6 22.3 Open to reduce the fracture >0.05 12 (52.17) 20 (100%) range) (degrees) (±2.55;20.1-27)(±2.16;20.1-28) (No, %) Post bone healing radiological characteristics of fracture AO, Arbeitsgemeinschaft für Osteosynthesefragen Average Bayonetting, (SD; 0.47 0.36 >0.05 54.8 (±17.9; range 37-74) minutes for ORIF range) (cm) (±0.23;0.3-0.8) (±0.12;0.3-0.7) Average angulation (SD; range) 4.23 3.48 >0.05 (Table 2). (degrees) (±1.37;0-8) (±0.12;0-8) Average maltrotation (SD; 2.64 2.21 The mean of follow-up was 16.86 (±5.64;12- >0.05 range) (degrees) (±1.41;0-6) (±1.57;0-7) 36) months for IMNK and 16.37 (±5.77;12-36) Average dorsal angulation (SD; 15.64 14.96 >0.05 months for ORIF (p>0.05) (Table 2). range) (degrees) (±3.82;0-20) (±4.01;0-20) Complication (No, %) In both groups, patients demonstrated appropria- Skin Infection 3(13.04) 2 (10) >0.05 te wound healing within 21 days. Non-union 1 (4.34) 0* <0.05 The average time of bone healing was 2.6 Malunion 1 (4.34) 0* <0.05 Re-fractured 2 (8.69) 0* <0.05 (±1.22;1-4) months after the surgery in IMNK, Daily outcomes (No, %) while it was 2.8 (±1.34;1-4) months for ORIF Return to sport 23 (100) 20 (100) 1.00 (p>0.05) (Table 2). The average time of bone he- RUSS, radius union scoring system; VAS, Visual analogic pain; SD, Standard Deviation aling in re-operated IMNK cases was 2.2 (±0.4; 1 -3) months. one case and 2 in the other case. At average day At average day of the bone healing the RUSS of the bone healing in the ORIF re-operated gro- was of 29.4 (±1.22;29-30) points in IMNK, while up, the VAS was 2.3 (±0.57; range 2-3). it was 29.8 (±0.1;29-30) in ORIF (p<0.05) (Table At average day of bone healing the regressi- 2). At the last X-ray control before the breakage on between RUSS and VAS scores showed of the callus associated to malunion or non-uni- a p=0.068 in IMNK, while p=0.043 in ORIF on the RUSH was of 29.2 points in one case and (p<0.05) for ORIF; at average day in IMNK re- 29.3 in the other case. At average day of the bone operated group of bone healing, the regression healing in the IMNK reoperated group, the RUSS between RUSS and VAS scores showed p=0.047. was of 29.64 (±0.27; range 29.2-30). The average correlation of clinical-radiographic In the average day of the bone healing the VAS results and patients’ outcomes was high accor- was 0.8 (±0.2; range 0-1) point in IMNK, whi- ding Cohen κ: 0.89 (±0.11; range 0.78-1) for le it was 0.7 (±0.3; range 0-1) in ORIF (p>0.05) IMNK, while κ: 0.91 (±0.09; 0.90-1) for ORIF (Table 2). At the last follow up review before the (p<0.05) (Table 2). breakage of the plates, the VAS was 1 point in

212 Di Giacinto et al. K wire vs plate in radius/forearm fracture

During the follow up no complications were te and screw fixation (4-6). To our knowledge, noticed in ORIF group; instead, there were two there are three previous studies comparing IMNK non-union or malunion after the remotion of K fixation and ORIF in the treatment of both bone wires (p<0.05) for ORIF (Table 2). The time of forearm fractures in adolescent patients (11–13). callus breakage or malunion with respect to sur- Two recent systematic reviews evaluating the tre- gery was: 47 days in one case and 61 days in the atment of distal radius or distal both-bone forearm second case. fractures in children of all ages have highlighted All the ORIF cases were re-operated using com- the lack of high-level evidence guiding treatment pression locking plate screws. All these surgeries of these common injuries (14-16). For children were successfully performed and were uneventful. with a displaced distal radius fracture, the presen- There was no statistically significant difference ce of a both-bone fracture, complete displacement in pre- and post-surgery X-rays between the two of the distal radius and non-anatomical reduction populations according to bayonetting, angulations, are risk factors for re-displacement after the reduc- malrotation, dorsal angulation degrees (Table 2). tion of their initially displaced distal radius fractu- re. Children with one or more of these risk factors The objective quality of life and wrist function in probably benefit most of the reduction combined IMNK’s group before the trauma, measured by with primary K-wire fixation (16). Two studies MWS, was about 100 points, while the quite qua- included in the recent meta-analysis (16) reported lity of life before the trauma, measured by MWS, re-displacement rates between 9.7% and 35% after was about 100 points in ORIF (p=1.00). At the the reduction and cast immobilization of displa- moment of trauma, in IMNK group the MWS ced distal radius fractures in children: only 61% was 14.3 (±2.4; range 0-26) in the same moment of 313 re-displaced fractures received secondary in ORIF group the MWS was 15.1 (±2.4; range treatment, 19.0% patients were considered to have 0-26) (p>0.05). After 1 month from the surgery enough potential for remodelling and received no the MWS score was 82.5 (±12.4; range 72-100) further treatment after re-displacement; for the re- for IMNK and 92.4 (±5.4range 86-100) in ORIF maining 20.0% with a re-displaced fracture, it was (p<0.05) for ORIF. Also, the third month after the not explicitly reported why secondary treatment surgery (p>0.05), MWS score was 100 in IMNK was not deemed necessary. A reason might be that and 100 in ORIF, as well as the sixth month of the definition for re-displacement and the indica- follow-up and at twelve months. tions for the secondary treatment were not similar The subjective quality of life and wrist function in all studies. Also, wait and see policies are pro- of IMNK’s group before the trauma, measured by bably also based on the expectation that there is QuickDASH, was about 100 points, while the qu- sufficient growth and the re-modelling potential in ite quality of life before the trauma was about 100 the injured bone in children (16). Finally, the fact points in ORIF (p=1.00). At the moment of trau- that an association of repeat reduction with growth ma, in the IMNK group the QuickDASH was 15.3 disturbances and worse functional outcome has (±3.7; range 0-26) and in the same moment ORIF, been described, may have contributed to a reser- the QuickDASH was 15.6 (±3.6; range 0-26), ved attitude towards repetitive reduction (17,18). p>0.05. After 1 month from the revision surgery The choice of intervention is influenced primarily the QuickDASH score was 80.4 (±9.2; range 70- by an assessment of the stability and the degree 100) in IMNK and 88.7 (±8.4; range 78-100) in of displacement of the distal radius fracture taking ORIF (p<0.05). Also, the third month after the into account the age of the child and the potential surgery (p>0.05), QuickDASH score was 100 in for re-modelling. In particular, the concept of tole- IMNK and 100 in ORIF, as well as the sixth month rable displacement (angulation or linear displace- of the follow-up and the twelfth month. ment, or both) is useful in children’s fracture prac- DISCUSSION tice; it describes an amount of displacement that will reliably remodel to a normal shaped and sized Surgical treatment of distal radius or both dis- bone for stable fractures, predominantly buckle tal bone forearm fractures is based on surgeon’s fractures. The main aim of the treatment is pain re- experience and preference with success docu- lief and protection, including forearm-injury (19). mented with both intramedullary nailing and pla-

213 Medicinski Glasnik, Volume 18, Number 1, February 2021

The complication rate among our patients in the in post-operative fracture angulation that was not IMNK group was 29% compared to 12% in the statistically different. ORIF resulted in almost no ORIF group. There were no major complications post-operative displacement at the fracture site (6). in the ORIF group and 11 major complications In conclusion, the treatment of adolescent distal (55%) in the IMNK group. Shah et al. noted a radius or forearm fractures remains challenging 20% complication rate in the IMNK group, all as highlighted by the relatively high complication of which were minor (18); the ORIF group had rate across multiple studies. One challenge facing an overall complication rate of 30%. Five of 13 the physician is the choice of surgical technique complications (38%) in the ORIF group were and fixation method, which will be influenced by considered major complications. In contrast, individual experience and preference. In our stu- Reinhardt et al. had a similar complication rate dy, plate and screw fixation more closely restores in both patient groups (ORIF 66.6% vs. IMN anatomy and has a trend towards the lowest re- 63.2%) (11). In their study, there was no differen- operation rate when compared to intramedullary ce in the rate of major complications between the k wire fixation (IMNK) in our adolescent age groups. Baldwin et al. performed a meta-analysis group. There is insufficient data to recommend as of children of all ages and found the complicati- a gold standard the ORIF with plate and screws, on rate after ORIF to be 16.5% and 18.7% after although open reduction and internal fixation IMNK fixation (14) and reported no difference may be preferred as patients approach skeletal in major complication rates and when they eva- maturity. The question of distal radius or forearm luated overall clinical outcome, the rates of poor fractures in adolescents would best be answered outcomes were 13.2% for IMNK and 3.6% for with a prospective randomized study. ORIF; IMNK type, patient age, open fractures, and fracture location were not found to be asso- FUNDING ciated with the likelihood of a complication. No specific funding was received for this study. The evaluated radiographic parameters included the post- operative fracture angulation, post-ope- TRANSPARENCY DECLARATION rative fracture displacement, and post-operative Conflict of interest: None to declare. radial bow. Both IMNK fixation and ORIF resulted

REFERENCES 1. Hedström EM, Svensson O, Bergström U, Michno 7. Freese KP, Faulk LW, Palmer C, Baschal RM, Sibbel P. Epidemiology of fractures in children and adoles- SE. A comparison of fixation methods in adolescent cents: increased incidence over the past decade: a patients with diaphyseal forearm fractures. Injury population-based study from northern Sweden. Acta 2018; 49:2053-7. Orthopaedica 2010; 81:148–53. 8. Cha SM, Shin HD. J Pediatr Orthop B. Buttress 2. Karl JW, Olson PR, Rosenwasser MP. The epidemi- plating for volar Barton fractures in children: Sal- ology of upper extremity fractures in the United Sta- ter-Harris II distal radius fractures in sagittal plane. tes, 2009. J Orthop Trauma 2015; 29:e242–4. 2019; 28:73-8. 3. Handoll HH, Elliott J, Iheozor-Ejiofor Z, Hunter J, 9. Joeris A, Lutz N, Blumenthal A, Slongo T, Audigé Karantana A. Interventions for treating wrist fractu- L. The AO Pediatric Comprehensive Classification res in children. Cochrane Database Syst Rev 2018; of Long Bone Fractures (PCCF). Acta Orthop 2017; 12:CD012470. 88:129-32. 4. Schneidmueller D, Kertai M, Bühren V, von Rüden 10. Calori GM, Colombo M, Mazza EL, Mazzola S, C. Kirschner wire osteosynthesis for fractures in Malagoli E, Marelli N, Corradi A. Validation of the childhood: bury wires or not? Results of a survey Non-Union Scoring System in 300 long bone non- on care reality in Germany. Unfallchirurg 2018; unions. Injury 2014; 45(Suppl 6):S93-7. 121:817-4. 11. Patel SP, Anthony SG, Zurakowski D, Didolkar 5. Akar D, Köroğlu C, Erkus S, Turgut A, Kalenderer MM, Kim PS, Wu JS, Kung JW, Dolan M, Rozental Ö. Conservative follow-up of severely displaced dis- TD. Radiographic scoring system to evaluate union tal radial metaphyseal fractures in children. Cureus of distal radius fractures. J Hand Surg Am 2014; 2018; 10:e3259. 39:1471-9. 6. Akar D, Köroğlu C, Erkus S, Turgut A, Kalenderer 12. Reinhardt KR, Feldman DS, Green DW, Sala DA, Ö. Conservative follow-up of severely displaced dis- Widmann RF, Scher DM. Comparison of intrame- tal radial metaphyseal fractures in children. Cureus dullary nailing to plating for both-bone forearm 2018; 10:e3259. fractures in older children. J Pediatr Orthop 2008; 28:403–9.

214 Di Giacinto et al. K wire vs plate in radius/forearm fracture

13. Fernandez FF, Egenolf M, Carsten C, Holz F, Sch- 17. Sengab A, Krijnen P, Schipper IB. Risk factors for neider S, Wentzensen A. Unstable diaphyseal fractu- fracture redisplacement after reduction and cast res of both bones of the forearm in children: Plate immobilization of displaced distal radius fractures in fixation versus intramedullary nailing. Injury 2005; children: a meta-analysis. Eur J Trauma Emerg Surg 36:1210–6, 2020; 46:789-800. 14. Carmichael KD, English C. Outcomes assessment of 18. Lee BS, Esterhai JL, Jr, Das M. Fracture of the dis- pediatric both-bone forearm fractures treated opera- tal radial epiphysis. Characteristics and surgical tre- tively. Orthopedics 2007; 30:379–83. atment of premature, post-traumatic epiphyseal clo- 15. Baldwin K, Morrison MJ 3rd, Tomlinson LA, Ra- sure. Clin Orthop Relat Res 1984; 185:90–6. mirez R, Flynn JM. Both bone forearm fractures in 19. Zimmermann R, Gabl M, Angermann P, Lutz M, Re- children and adolescents, which fixation strategy is inhart C, Kralinger F, Pechlaner S. Late sequelae of superior - plates or nails? A systematic review and fractures of the distal third of the forearm during the meta-analysis of observational studies. J Orthop Tra- growth period. Handchir Mikrochir Plast Chir 2000; uma 2014; 28:e8-e14. 32:242–9. 16. Sengab A, Krijnen P, Schipper IB. Displaced distal 20. Schneidmüller D, Röder C, Kraus R, Marzi I, Ka- radius fractures in children, cast alone vs additio- iser M, Dietrich D, von Laer L.. Development and nal K-wire fixation: a meta-analysis. Eur J Trauma validation of a paediatric long-bone fracture classifi- Emerg Surg 2019; 45:1003-11. cation. A prospective multicentre study in 13 Euro- pean paediatric trauma centres. BMC Musculoskelet Disord 2011;12:89.

215 ORIGINAL ARTICLE

Modalities of extensor tendon repair related to etiological factors and associated injuries

Sanela Salihagić1, Zlatan Zvizdić2, Dženana Hrustemović3, Redžo Čaušević4, Ahmad Hemaidi1

1Clicnic of Reconstructive and Plastic Surgery, 2Clicnic of Paediatric Surgery; Clinical Centre of Sarajevo University, Sarajevo; 3Faculty of Pharmacy and Health Travnik, Travnik, 4Faculty of Health Studies, University of Sarajevo, Sarajevo; Bosnia and Herzegovina

ABSTRACT

Aim To evaluate modalities of extensor tendons repair of hand and forearm in specific anatomical zones with regard to etiological factors and presence of associated injuries of adjacent anatomical structures.

Methods This cross-sectional study included 279 patients referred for extensor tendon repair of hand and forearm in specific anatomi- cal zones. Available treatment modalities were evaluated concer- ning etiological factors, anatomical zones, and associated injuries. Statistical significance was analysed in the occurrence of early and Corresponding author: late postoperative complications according to anatomical zones. Sanela Salihagic, Clinic of Reconstructive Results Direct repair of extensor tendon lesions was found to and Plastic Surgery, be the most common modality of reconstruction, 230 (93.5%), Clinical Centre of the University of of which blade injuries were predominant, 120 (48.7%). Direct Sarajevo tendon repair was mostly indicated in Zone VI and Zone III, in Bolnička 25, 71000 Sarajevo, 55 (23.9%) and 42 (18.3%) patients, respectively. Statistically, a Bosnia and Herzegovina significant correlation was confirmed between treatment modali- ties, injuries in specific anatomical zones, and type of etiological Phone: +387 33 297 024, factor (p<0.0001). Statistical correlation was confirmed between Fax: +387 33 297 819; zones of injuries and the occurrence of early and late complicati- E-mail: [email protected] ons (p=0.002). ORCID ID: https://orcid.org/0000-0002- Conclusion Successful postoperative recovery was correlated 8137-0315 with the recognition of functional failure in specific zones, asse- ssment of potential associated injuries, and selection of the most optimal modality of reconstruction. Original submission: 04 May 2020; Key words: associated injury, etiologic factors, extensor tendons, Revised submission: tendon repair 17 June 2020; Accepted: 09 September 2020 doi: 10.17392/1191-21

Med Glas (Zenica) 2021; 18(1):216-221

216 Salihagic et al. Modalities extensor tendon repair

INTRODUCTION Certain anatomical zones were related to better postoperative results regardless of treatment mo- The specifics of the extensor tendons are reflec- dality (12). ted in their superficial anatomical localization, which makes them exposed to a potentially de- Reconstruction of extensor tendons must be seen structive action of various etiological factors (1). as a reconstructive challenge (13), because the Due to the balance of the extensor and flexor type of primary trauma can potentially comple- system of the hand and forearm, which is nece- tely exclude the possibility of direct tendon re- ssary for the sophisticated function of the hand, pair and may represent an absolute indication for the reconstruction of the extensor tendons in tendon transfer or tendon grafts, which is related terms of primary reparation, tendon transposition to the etiological aspect of the injury (14) where or tenoplasty, is a complex process (2). the choice of donor's motor unit or tendon graft In acute hand trauma, treatment can be conserva- is crucial for the final functional outcome (13). tive and surgical, in terms of primary tendon re- This is particularly important in complex upper paration with the approximation of tendon ends, extremity trauma (15). or tendon transposition and tenoplasty, in the Due to anatomical complexity, the focus of re- case of significant tissue destruction (2). construction has previously been directed to the Multiple etiological factors can lead to a trauma- flexor tendons of the hand, but, as presented by tic lesion of the extensor tendons of the hand and Amirharajah et al. new concepts of acute hand forearm (3). trauma emphasize the timely reconstruction of Any trauma of the extensor system requires extensor tendons (16). appropriate surgical evaluation to select the most To optimize the treatment, it is necessary to esta- optimal reconstructive procedure based on an in- blish clinical guidelines to improve the postope- dividual reconstruction plan (4). rative functional results after conservative and The anatomical division of the extensor tendons operative treatment (17). of the hand and forearm into specific zones is The aim of this study was to evaluate the rela- a useful means of facilitated evaluation of the tionship between the etiological factor and the extent of the lesion, taking into account the spe- resulting tendon destruction as clinical guideli- cifics of each zone (5). nes in the selection of surgical modality in spe- Due to its superficial localization, the extensor cific anatomical zones of the extensor system of system is potentially exposed to trauma in all the hand and forearm, as well as the occurren- anatomical zones, with a full spectrum of va- ce of associated injuries of adjacent anatomical riations of acute and chronic injuries (6), but structures and postoperative complications in the evaluation of the extent of the injury is fa- anatomical zones, which are the result of tissue cilitated by classification into anatomical zones, destruction caused by etiological factors. which allows taking into account their specifics and the existence of possible associated injuries PATIENTS AND METHODS of neighboring anatomical structures (7,8) which Patients and study design are most often the consequence of the destructive action of certain etiological agents, with multile- The observational, cross-sectional, controlled vel lesions and difficult functional recovery (9). study involved 279 patients who were diagnosed Modern concepts of go in the direc- and treated with extensor tendon injuries of hand tion of rapid recovery and satisfactory postope- and forearm in different zones at the Clinic of Re- rative functionality because inadequately treated constructive and Plastic Surgery, Clinical Centre and unrecognized lesions lead to permanent disa- of the University of Sarajevo, during the period bility, and potential complications (10). The most 2014-2019. All patients underwent clinical exa- optimal treatment is the one that enables the best mination for functional failure and standard hand functional result, the direct reparation is most op- X-ray for assessment of associated bone injuries, timal whenever local conditions allow it, which as a diagnostic method of choice. The patients is a consequence of the relative simplicity of the previously treated in other hospital centres and procedure itself (11). the patients with unrecognized extensor lesions were excluded from this study.

217 Medicinski Glasnik, Volume 18, Number 1, February 2021

All patients signed a written consent for inclusi- struction based on the use of an available donor on in the study. tendon unit, was indicated in 13 (4.66%) patients, The study was approved by the Research Ethics of which it was most often indicated in the circu- Committee of the Clinical Centre of the Univer- lar saw machine injury, in nine (3.23%) patients. sity of Sarajevo Tenoplasty, based on the use of free tendon grafts, was indicated in three (1.08%) patients, all for Methods circular saw machine injuries due to loss of tissue According to the level of the lesion, extensor ten- continuity. Closed injuries, related to the loss of dons were classified into anatomical zones (1): tendon continuity at the level of the distal phalan- Zone I – distal interphalangeal joint, Zone II - geal joint ("mallet finger"), were mostly treated middle phalanx, Zone III – proximal interphalan- conservatively by six-week splint immobilization, geal joint, Zone IV - proximal phalanx, Zone V in 33 (11.83%) patients (Table 1) (p<0.0001). - metacarpophalangeal joint, Zone VI - metacarpal Table 1. Distribution of tendon destruction in 279 patients level, Zone VII - dorsal retinaculum, Zone VIII caused by different etiological factors according to the treat- - distal forearm, and Zone IX - mid and proximal ment modality forearm. Available treatment modalities (direct No (%) of patients Etiology Direct Tendon tran- Conservative tendon repair, tenoplasty, tendon transposition or of destruction Tenoplasty repair sposition treatment conservative treatment) were chosen according to Blade 120 (43) 0 0 0 anatomical zones, different etiological factors (a Circular sawing 69 (24.73) 3 (1.08) 9 (3.23) 0 blade, circular sawing machine, axe, and glass), machine and associated injuries (bones-extensor tendons, Axe 30 (10.75) 0 4 (1.43) 0 Glass 10 (3.58) 0 0 0 flexor-extensor tendons, peripheral nerves-exten- Closed injury 1 (0.36) 0 0 33 (11.83) sor tendons) of the adjacent anatomical structures. Total 230 (82.43) 3 (1.08) 13 (4.66) 33 (11.83) The distribution of early and late postoperative Although different etiological factor determines complications by anatomical zones was analysed. reconstruction modalities, certain types of recon- Statistical analysis struction were more represented than others in Descriptive processing of statistical data was anatomical zones (p<0.0001). Zone I, associated carried out for the significance of etiological fac- with injury of extensor aponeurosis at the level of tors on the selection of reconstruction modalities, the distal interphalangeal joint, was successfully as well as the appearance of combined lesions of treated mostly with conservative six-week immo- adjacent anatomical structures and postoperative bilization, in 33 (82.5%) patients. The direct re- complications in specific zones. The data were pair was the most common modality of recon- analysed using ꭓ2 and Fischer test. The p <0.05 struction. All injuries in Zone II, 30 (10.75%), was used as statistically significant. were treated by direct reparation. In other ana- tomical zones, the frequency of direct tendon RESULTS repair was variable, from 10 (3.58%) in Zone I The study included 279 patients (233 males and to 55 (19.7%) in Zone VI (19.7%). Tendon tran- 46 females). Mean age of male patients was 39 sposition was most common in Zone IV, in nine (range from 28 to 50 years), of female 35 (range (3.22%) patients. Tenoplasty was the least repre- from 27 to 50 years) (p=0.969). sented operative modality, in one (0.36%) patient in each Zone V and VI (Figure 1) (p<0.0001). A type of etiological factor, due to the different degrees of tissue destruction, was determined as the most optimal modality of treatment. Direct tendon reparation was the most common treatment modality, in 230 (82.43%) patients, of which 120 (43%) had blade injury, and 69 (2 4.73%) circular saw machine injury; in one (0.36%) patient with persistent deformity after the removal of splint immobilization, direct tendon repair was indica- ted. Tendon transposition, a modality of recon- Figure 1. Treatment modalities in anatomical zones

218 Salihagic et al. Modalities extensor tendon repair

Associated injuries of the adjacent structures DISCUSSION were differently distributed in anatomical zones The results of this study showed that direct repa- (p<0.0001). In Zone IV associated bone-exten- ir of the extensor tendon mechanism of the hand sor tendons injuries were relatively common, 23 and forearm has been the most common modality (8.24%). The higher possibility of the presence of reconstruction, regardless of the etiological of this type of associated injuries was found in factor and the anatomical zone of the injury. Iso- Zones V and VI, in 15 (5.37%), and 21 (7.53%) lated injuries were the most common in all zo- patients, respectively. Associated injuries of the nes, while combined extensor lesions with bone extensor tendons with flexor tendons and pe- structures were the most common type of asso- ripheral nerves were presented in smaller percen- ciated injury due to close anatomical localizati- tages. Associated extensor-flexor tendons injuries on. Associated injuries with flexor tendons, and were presented in smaller percentages, with one peripheral nerves were present in a small number (0.36%) in each Zone IV, VI and VII, and two of cases, with a specific distribution according to (0.72%) in Zone V. Associated peripheral nerves- the corresponding zones, but also as a consequ- extensor tendons injuries were minimally repre- ence of stronger destruction of specific etiolo- sented, one (0.36%) in each Zone IV-VI (Table gical factors. The distribution of early and late 2) (p<0.0001). complications was without a clear clinical corre- Table 2. Types of associated injuries according to anatomical lation with injury zones. zones The etiological factors with the resulting lesions of No (%) of patients with extensor tendon Anatomical the extensor tendons are numerous, mostly related zone Bones- Flexor- Peripheral Isolated extensor extensor nerves-extensor extensor to work activity, which was defined in 2010 as the Zone I 0 0 0 43 (15.4) Standard Occupational Classification structure. Zone II 0 0 0 30 (10.7) Injuries inflicted by machines at work and home Zone III 0 0 0 42 (15.1) Zone IV 23 (8.24) 1 (0.36) 0 16 (5.73) are very common in everyday practice. The type of Zone V 15 (5.37) 2 (0.72) 1 (0.36) 6 (2.15) etiological factor determines the so-called "injury Zone VI 21 (7.53) 1 (0.36) 1 (0.36) 33 (11.8) pattern" and the type of reconstructive procedure, Zone VII 1 (0.36) 1 (0.35) 1 (0.36) 17 (6.09) Zone VIII 0 0 0 13 (4.66) although primary tendon repair is preferred due to Zone IX 0 0 0 11 (3.94) faster postoperative recovery (18). Total 60 (21.5) 5 (1.8) 3 (1.08) 211 (75.62) The type of extensor reparation is not correlated with the anatomical zones of injury, unlike etio- Two types of early complications were found in logical factors because certain etiological factors our study group, tendon rupture, in two (0.72%) resulted in a greater degree of tissue destruction patients in each Zone IV and VI, and infection in and prevented direct and /or primary reparation. eight (2.87%) patients in each Zone IV, VI, and Primary reparation, as functionally and recon- VII. Contractures were represented in two anato- structively the most acceptable treatment moda- mical zones, seven (2.5%) in each Zone III and lity, is preferred in all anatomical zones (19). VI (p=0.002) (Table 3). Injuries of the extensor tendons can be combined Table 3. Distribution of early and late complications accord- with a lesion of bone structures, neurovascular ing to anatomical zones elements, flexor tendons, and soft tissue cove- No (%) of patients with/without complication ring, which makes postoperative recovery and Anatomical Without Rupture Infection Contracture the functional outcome more complex. Associa- zone complications ted extensor-flexor tendon and peripheral nerve I 0 0 0 43 (15.4) II 0 0 0 30 (10.8) -extensor tendon injuries represent the consequ- III 0 0 3 (1.07) 39 (13.9) ence of extensive tissue destruction. The proxi- IV 1 (0.36) 3 (1.07) 0 36 (12.9) mity of extensor tendons with phalangeal and V 0 0 0 24 (1.43) VI 1 (0.36) 2 (0.72) 4 (1.43) 49 (17.6) metacarpal bones makes this type of associated VII 0 3 (1.07) 0 17 (6.09) injury more common despite the absence of si- VIII 0 0 0 13 (4.66) gnificant tissue destruction. Associated injuries IX 0 0 0 11 (3.94) Total 2 (0.72) 8 (2.87) 7 (2.5) 262 (93.91) of extensor tendons and bone structures are most

219 Medicinski Glasnik, Volume 18, Number 1, February 2021

common due to close anatomical contact and a study by Meritt et al., thereby significantly im- high probability of combined lesions (20). proving postoperative functionality (23). Postoperative treatment is very important to pre- In this study we confirmed that the direct repair vent potential complications, which requires care- of the tendon mechanism was the most optimal ful postoperative monitoring by injury zones. The modality of reconstruction in all anatomical zo- low prevalence of early and late complications nes of injury and that the etiological factors were is the result of the implementation of antibiotic directly related to the degree of destruction. The prophylaxis protocols, postoperative mobilization, presence of associated injuries does not affect the and active cooperation with the patient (5). reconstruction modalities selection. We have pro- The study showed the importance of clinical ven the possibility of direct repair of the extensor assessment of extensor tendon injury to the aetio- tendon on all anatomical zones, regardless of the logy, the level of injury classified into anatomical associated injuries, in all cases of the direct ten- zones and to the treatment modality, which is the don approximation. ultimate treatment protocol, especially conside- In conclusion, a lesion of the extensor tendons ring the synergy with flexor tendons, as complex of the forearm and hand requires careful clinical musculoskeletal systems, necessary for the sop- assessment and recognition of functional failu- histicated hand function (11). re. The possibility of the existence of associated Intra and postoperative protocols are still subjects injuries requires the adequate clinical assessment of debates, but the importance of the appropriate of functional failure and proper treatment to pro- assessment of the lesion extent and its anatomical mote quality and rapid recovery. The implemen- level is crucial for the quality of postoperative re- tation of clinical guidelines enables appropriate covery (21). Different variations of the treatment clinical assessment consistent with the modern protocol depending on the lesion zone have been concept of hand surgery. presented in the literature, but in clinical practice, FUNDING it has been confirmed that a unique approach to the treatment is applicable in all zones (22). New No specific funding was received for this study. concepts of early active postoperative mobiliza- TRANSPARENCY DECLARATION tion have been replaced by new concepts of early active mobilization, which has been presented in Competing interests: None to declare.

REFERENCES 1. Türker T, Hassan K, Capdarest-Arest N. Extensor 10. Wilken F, Banke IJ, Hauschild M, Winkler S, Schott tendon gap reconstruction: a review. J Plast Surg K, Rudert M, Eisenhart-Rothe RV. Endoprosthetic Hand Surg 2016; 50:1-6. tumor replacement: reconstruction of the extensor 2. Yoon AP, Chung KC. Management of acute extensor mechanism and complications. Orthopade 2016; tendon injuries. Clin Plast Surg 2019; 46:383-91. 45:439-45. 3. Hassine YH, Hmid M, Baya W. Trauma of the hand 11. Sando IC, Chung KC. The use of dermal skin substi- from circular saw table: a series of 130 cases. Tunis tutes for the treatment of the burned hand. Hand Clin Med 2016; 94(Suppl 12):851. 2017; 33:269-76. 4. Schubert CD, Giunta RE. Extensor tendon repair 12. Mehdinasab SA, Pipelzadeh MR, Sarrafan N. Re- and reconstruction. Clin Plast Surg 2014; 41:525-31 sults of primary extensor tendon repair of the hand 5. Lutz K, Pipicelli J, Grewal R. Management of com- with respect to the zone of injury. Arch Trauma Res plications of extensor tendon injuries. Hand Clin 2012; 1:131-4. 2015; 31:301-10. 13. Pierrart J, Tordjman D, Otayek S, Douard R, Mahjo- 6. Skinner S, Isaacs J. Extensor tendon injuries in at- ubi L Masmejean E. Two-stage extensor tendon graft hlete. Clin Sport Med 2020; 39:259-77 using Paneva-Holevitch procedure: a new technique. 7. Dy C, Rosenblatt L, Lee S. Current methods and bi- Hand Surg Rehabil 2018; 37:12-15. omechanics of extensor tendon repairs. Hand Clin 14. Bhardwaj P, Muddappa PP , Bindesh D, Sabapathy 2013; 29:261-8. SR. Evaluation of extensor pollicis brevis as a reci- 8. Sari E. Tendon injuries of the hand in Kirikkale, Tur- pient of tendon transfer for thumb extension. Indian key. World J Plast Surg 2016; 5:160-7. J Plast Surg 2019; 52:171-77. 9. Chauan A, Jacobs B, Andoga A, Baratz ME. Exten- 15. Carty MJ, Blazar PE. Complex flexor and extensor sor tendon injuries in athletes. Sports Med Arthrosc tendon injuries. Hand Clin 2013; 29:283-93. Rev 2014; 22:45-55.

220 Salihagic et al. Modalities extensor tendon repair

16. Amirharajah M, Lattanza L. Open extensor tendon 20. Goodman AD, Got CJ, Weiss APC. Crush Injuries injuries. J Hand Surg Am 2015; 40:391-7. of the Hand. J Hand Surg Am 2017; 42:456-63. 17. Colzani G, Tos P, Battiston B, Merolla G, Porcelli- 21. Griffin M, Hindocha S, Jordan D, Saleh M, Khan ni G, Artiaco S. Traumatic extensor tendon injuries W. Management of extensor tendon injuries. Open to the hand: clinical anatomy, biomechanics, and Orthop J 2012; 36-42. surgical procedure review. J Hand Microsurg 2016; 22. Rabbani MJ, Amin M, Khalid K, Khan H, Shahzad 8:2–12. I, Rabbani A, Nasrullah M, Tarar MN, Rabba- 18. de Jong JP, Nguyen JT, Sonnema AJM, Nguyen ni S. Early active mobilization vs immobilization EC Amadio PC, Moran SL. The incidence of acute following modified Kessler repair of extrinsic exten- traumatic tendon injuries in the hand and wrist: a 10- sor tendons in zone V to VII. J Ayub Med Coll Ab- year population-based study. Clin Orthop Surg 2014; bottabad 2019; 31:320-5. 6:196-202 23. Merrit WH, Wong AL, Lalonde DH. Recent deve- 19. Desai MJ, Wanner JP, Lee DH, Gauger EM. Failed lopments are changing extensor tendon manage- extensor tendon repairs: extensor tenolysis and recon- ment. Plast Reconstr Surg 2020; 145:617-28. struction. J Am Acad Orthop Surg 2019; 27:563-74.

221 ORIGINAL ARTICLE

Traumatic bilateral scaphoid fractures

Dariush Ghargozloo1, Alessandro Tomarchio2, Mauro Ballerini1, Gianpaolo Chitoni1

1Department of Orthopaedics and Traumatology, Esine hospital, Valcamonica (Bs), 2Department of Orthopaedics and Traumatology, S. Croce e Carle Hospital, Cuneo; Italy

ABSTRACT

Aim To illustrate the surgical treatment of bilateral post-traumatic scaphoid fracture.

Methods We came across a young student, who sustained bilate- ral, undisplaced scaphoid waist fractures following a fall during a football match. Despite careful clinical and radiographic evalua- tion by four views at the Accident and Emergency (A&E) Depar- tment, we initially performed only the diagnosis of the left scapho- id fracture treating it with a percutaneous Acutrack headless screw. Eight months later this patient returned to the A&E department due to a new trauma to his right wrist with the onset of painful symptoms: cystic scaphoid non-union. No pain had been reported on the wrist in those months.

Corresponding author: Results We performed osteosynthesis with Herbert headless screw Alessandro Tomarchio through an extended volar approach placing a non-vascularized Department of Orthopaedics and cortico-spongious bone grafts taken from radius. Periodic follow up by clinical examination, X-ray and CT scan with evidence of Traumatology, S. Croce e Carle Hospital bone healing was performed. Via M. Coppino 26, Cuneo 12100, Italy Phone: +39 3206014935; Conclusion Bilateral scaphoid fractures are rarely encounte- Fax: +39 0171642208; red, mostly as stress fractures in athletes and manual workers. If left untreated, arthritis, deformity, and instability can lead to E-mail: [email protected] significant disability. Comprehensive imaging should be done

Dariush Ghargozloo ORCID ID: https:// in case of suspected scaphoid fractures, especially after a trau- orcid.org/0000-0003-1839-4362 ma, even in the presence of modest symptoms, as failure to do so may lead to missed fracture. Considering what was expo- sed, the radiographic check on the right wrist repeated about two weeks after the trauma would have avoided a missed di- agnosis, even in the absence of reported clinical symptoms. Original submission: We therefore recommend to repeat the radiographic examination 08 December 2020; in all situations like these. Revised submission: Key words: bone screws, carpal bones, delayed diagnosis, osteo- 10 December 2020; synthesis, scaphoid bone Accepted: 17 December 2020 doi: 10.17392/1332-21

Med Glas (Zenica) 2021; 18(1):222-225

222 Ghargozloo et al. Traumatic bilateral scaphoid fractures

INTRODUCTION The scaphoid is the largest bone of the proximal carpal row, serving as a bridge between the proxi- mal and the distal carpal bones; it has a major role in maintaining carpal stability (1). The primary mechanism of scaphoid fracture is hyperextension of the wrist beyond, which commonly occurs by a fall onto an outstretched hand (2,3). The waist of the scaphoid tends to be the focus of this stress, which leads to fractures at this site in approxima- tely 80% of cases of scaphoid fractures (4,5). The diagnosis is sometimes missed on initial ra- diographs done immediately: in fact x-rays are often repeated after a few days (to the persistence Figure 1. Waist fracture at the left scaphoid (Ghargozloo D, 2016) of pain) to diagnose these occult fractures. Com- puter tomography or Magnetic Resonance Ima- Herbert classification B2 type (9). A computed ging (MRI) are often used for clinically suspicio- tomography (CT) scan was done to confirm this us fractures in patients with negative X-rays (6). finding, to check the displacement and to rule out any other injury. Failure to diagnose and treat scaphoid fractures lead to complications like nonunion, avascular About 8 months after the previous trauma, the necrosis and osteoarthritis. patient suffered crushing in the right hand during a football match. Presented at our A&E Depar- The treatment generally includes conservative tment again, we performed radiographic control treatment with cast in not displaced fracture or (Figure 2), showed the fracture of the scaphoid percutaneous fixation, to open reduction and in- waist with evident shortening of the same with ternal fixation in case of displaced ones. The tre- hump back deformity and cystic non-union, cla- atment of non-unions requires, instead, an open ssified as Herbert D2 type (9). reduction, bone grafting and internal fixation. Unilateral fracture of the scaphoid is widely known (5,7,8). However, isolated bilateral fractu- res of the scaphoid are rare in occurrence.

PATIENTS AND METHODS

Patient and study design A seventeen-year-old young student, after falling on the field during a soccer match with a bilate- ral hand-wrist trauma, arrived at the Accident and Emergency (A&E) at Esine hospital, and admitted Figure 2. Presenting view with hump back deformity and cystic referring with pain to the left wrist. He had functi- non-union of the right scaphoid (Ghargozloo D, 2016) onal limitation of the wrist’s range of motion and Immediately performed, CT scan (Figure 3) con- moderate pain in correspondence to the pressure firmed this diagnosis. It was therefore a picture of of the tubercle of the scaphoid. Before presenting non-union on a previously unrecognized fracture. to us, he had only taken an analgesic medication to The patient denied previous other traumas saying lessen the pain. The physical examination showed that he had observed rest as prescribed by us after a healthy-looking young man. He did not report the surgical treatment of the left wrist. pain or functional limitation to the right wrist. Radiographic examination at both wrists throu- Methods gh four views showed a displaced waist fracture Having informed the patient, three days after at the left hand (Figure 1) that we classified as a the trauma on the left wrist we performed a vo-

223 Medicinski Glasnik, Volume 18, Number 1, February 2021

Figure 5. Six-month post-surgery CT scan view with 3.5 x 24mm Herbert headless screw through an extended volar ap- proach right wrist (Ghargozloo D, 2017)

Figure 3. CT scan of the right scaphoid (Ghargozloo D, 2016) RESULTS lar percutaneous approach osteosynthesis, with We found no complications after volar percuta- micro Acutrack Acumed headless screw 2.8mm x neous approach osteosynthesis neither on the left 24mm (Figure 4) and immobilization with short wrist nor on the right. Bilaterally, at the final cli- spica cast including first metacarpal for 30 days. nical follow-up (six month) we found the resoluti- The operating time was 50 minutes. on of pain symptoms and good radiographic bone consolidation without loosening of the hardware. The patient attended to our outpatient clinic at 30 and 90 days for an X-ray check, the joint excur- sion, the possible presence of pain and muscle strength. At 12 weeks of the follow-up, the fractures he- aled completely. At final clinical follow-up the patient confirmed the absence of pain symptoms and we found through radiographic and CT scan a good bone consolidation without loosening of the hardware or bone resorption.

Figure 4. Six-month postoperative X ray view with micro Acu- DISCUSSION track Acumed headless screw 2.8 x 24mm fixation (Ghargozloo D, 2016) Considering that scaphoid fractures are the most About 8 months after the previous trauma and af- common of all carpal bone fractures, in all pa- ter pre-operative planning of scaphoid non-union, tients with wrist injury, anatomical snuff box we performed osteosynthesis, on the right wrist, tenderness is sensitive but not specific for scap- with 3.5 mm x24 mm Herbert headless screw hoid fractures (10). Instead, scaphoid tubercle through an extended volar approach. We also and scaphoid compression tests are considered took from the ulnar portion of the radial distal more specific to scaphoid fractures (10,11). Des- epiphysis and positioned non-vascularized cor- pite X-ray examination, the initial diagnosis of tico-spongious bone grafts to restore the correct the carpal scaphoid fracture on the right side height of the scaphoid and the dorsal deviation of was missed. So, in addition to a careful clinical the surface of the lunate (dorsal intercalated se- examination, we recommend the complete radi- gment instability, DISI aspect) (Figure 5). Also, ographic examination (comprising of at least 4 an immobilization the upper limb was done, with views– PA, lateral, semipronated oblique, and short spica cast including first metacarpal for 30 scaphoid with the wrist pronated in ulnar devia- days. After that, gentle ranges of motion exerci- tion). The standard guidelines now are to treat a ses were started. The operating time was 116 mi- case clinically suggestive of scaphoid fracture as nutes and we found no complications. a fracture, even if it is not visible in any of the X- ray views. Such a patient with wrist pain should In addition, for 60 days we used a capacitively be followed up after 2 weeks with an X-ray or a coupled electric field stimulation CT scan (12,13). In our case, however, the patient (Osteobit® Terapia, IGEA)

224 Ghargozloo et al. Traumatic bilateral scaphoid fractures

does not report pain to his right wrist after the lable studies, the field of application using both injury, even weeks later. grafts for SN still remains controversial (15). On A treatment of scaphoid fractures is controver- the other hand, we know for sure that failure to sial (14,15). Regarding the performed surgical diagnose or treat these fractures adequately can treatment we decided together with the patient lead to the formation of scaphoid nonunion ad- with an acute fracture, having done a CT scan to vanced collapse (SNAC). The SNAC wrist is one confirm the fracture pattern, to do an immediate of the most frequent complications as a result of headless screw fixation. On the other hand, with scaphoid fractures; the most common treatments scaphoid non-union fracture, we were obviously are Partial Carpal Arthrodesis (17,18) or proxi- forced to perform the surgical treatment as soon mal row carpectomy (18). as the diagnosis was made. According to what In conclusion, to our knowledge this is the first had been said to Frisk (16), we performed a ra- report of traumatic bilateral scaphoid fractures dial exposure with osteotomy of the radial stylo- surgically treated and the good clinical and in- id and after reduction of the instability of the strumental result obtained testifies on good sur- carpus, the palmar radial wedge-shaped defect gical practice. of the navicular was filled in with a graft taken from the osteotornized styloid process. This is a FUNDING non-vascularized cortico-spongious bone grafts. No specific funding was received for this study. Conventional non-vascularized bone grafts as well as vascularized bone grafts are used to treat TRANSPARENCY DECLARATION scaphoid non-union (SN). Due to limited avai- Competing interests: None to declare.

REFERENCES 1. Krasin E, Goldwirth M, Gold A, Goodwin DR. Review 11. Mallee WH, Walenkamp MMJ, Mulders MAM, Go- of the current methods in the diagnosis and treatment slings JC, Schep NWL. Detecting scaphoid fractures of scaphoid fractures. Postgrad Med J 2001; 77:235–7. in wrist injury: a clinical decision rule. Arch Orthop 2. Louis S, Warwick DJ, Nagayam S. Appley’s System Trauma Surg 2020; 140:575-81. of Orthopaedics and Fractures. 8th ed. London: Anold, 12. Cooney WP, Linscheid RL, Dobyns JH. Fractures and 2001:620-8. dislocations of the wrist. In: Rockwood CA, Bucholz 3. Chapman MW. Chapman’s Orthopaedic Surgery. Phila- RW, Court-Brown CM, Heckman JD, Tornetta P, eds. delphia: JB Lipincott Co., 1996: 1307-31. Fractures in Adults. 4th ed. Philadelphia: Lippincott- 4. Borgeskov S, Christiansen B, Kjaer A, Balslev I. Raven, 1996; 745–67. Fractures of the carpal bones. Acta Orthop Scand 13. Sampson SP, Wisch D, Akelman E. Fractures and dis- 1966; 37:276-87. locations of the hand and wrist. In: Dee R. Principles 5. Amadio PC, Taleisnik J. Fractures of the carpal bones. of Orthopaedic Practice. 2nd ed. New York: McGraw In: Amadio PC, Taleisnik J, eds. Green’s Operative Hill, 1997:429–42. Hand Surgery. Oxford, UK: Elsevier, 1999: 815. 14. Tada K, Ikeda K, Okamoto S, Hachinota A, Yamamoto 6. Mallee WH, Wang J, Poolman RW, Kloen P, Maas M, D, Tsuchiya H. Scaphoid fracture--overview and con- de Vet HCW, Doornberg JN. Computed tomography servative treatment. Hand Surg 2015; 20:204-9. versus magnetic resonance imaging versus bone scin- 15. Hirche C, Xiong L, Heffinger C, Münzberg M, Fis- tigraphy for clinically suspected scaphoid fractures in cher S, Kneser U, Kremer T. Vascularized versus patients with negative plain radiographs. Cochrane non-vascularized bone grafts in the treatment of scap- Database of Syst Rev 2015; 2015:CD010023. hoid non-union. J Orthop Surg (Hong Kong) 2017; 7. Van Tassel DC, Owens BD, Wolf JM. Incidence, esti- 25:2309499016684291. mates, and demographics of scaphoid fracture in the 16.Fisk GR. Overview of wrist injuries. Clin Orthop U.S. population. J Hand Surg Am 2010; 35:1242-5. 1980; 149:137-43. 8. Hankin FM, Smith PA, Braunstein EM. Evaluation of 17.Rollo G, Bisaccia M, Irimia JC, Rinonapoli G, Pasqu- the carpal scaphoid. Am Fam Pract 1986; 34:129-32 ino A, Tomarchio A, Roca L, Pace V, Pichierri P, Gia- 9. Herbert TJ, Fisher WE. Management of the fractured racuni M, Meccariello L. The Advantages of Type III scaphoid using a new bone screw. J Bone Joint Surg Scaphoid Nonunion Advanced Collapse (SNAC) Tre- Br 1984; 66:114-23. atment With Partial Carpal Arthrodesis in the Domi- 10. Mallee WH, Henny EP, van Dijk CN, Kamminga SP, nant Hand: Results of 5-year Follow-up. Med Arch. van Enst WA, Kloen P. Clinical diagnostic evaluation 2018; 72:253-56. for scaphoid fractures: a systematic review and meta- 18. Williams JB, Weiner H, Tyser AR. Long-Term Outco- analysis. J Hand Surg Am 2014; 39:1683-91. me and Secondary Operations after Proximal Row Carpectomy or Four-Corner Arthrodesis. J Wrist Surg 2018; 7:51-6.

225 ORIGINAL ARTICLE

Impact of load on the knee in relation to a treadmill angle

Fikret Veljović1, Edin Begić2,3, Avdo Voloder1, Reuf Karabeg4, Amer Iglica5, Nedim Begić6, Alden Begić7, Adisa Chikha8

1Faculty of Mechanical Engineering, University of Sarajevo, Sarajevo, 2Department of Cardiology, General Hospital "Prim.dr. Abdulah Nakaš", 3Department of Pharmacology, School of Medicine, Sarajevo School of Science and Technology, 4Private Clinic "Karabeg", 5Department of Cardiology, Clinic for Heart, Blood Vessel and Rheumatic Diseases, Clinical Centre University of Sarajevo, 6Depart- ment of Cardiology, Paediatric Clinic, Clinical Centre University of Sarajevo, 7Department of , Clinic for Heart, Blood Vessel and Rheumatic Diseases, Clinical Centre University of Sarajevo, 8Clinic for Pathology, Clinical Centre University of Sarajevo; Sarajevo, Bosnia and Herzegovina

ABSTRACT

Aim To determine the effect of the load on the meniscus in relation to a different angle, and to present the impact of force on eventual injury of menisci.

Methods Research included 200 males with average height of 178.5 cm, mass 83.5 kg, and average age of 22 years. The simula- tion of treadmill that was used in the evaluation of ischemic heart Corresponding author: disease was made. Effects on the knee were evaluated by measu- Fikret Veljović ring at different inclinations (5°70’, 6°80’, 7°90’, 9°10’, 10°20’, Faculty of Mechanical Engineering, 11°30’ and 12°40’). University of Sarajevo Results With increasing ascent of treadmill the load on the me- Vilsonovo šetalište 9, 71000 Sarajevo, niscus also increased. Each increase in ascent after 22% (which Bosnia and Herzegovina corresponded to the angle of 12°40’ and seventh degree of load Phone: +387 33 729 800; according to the Bruce protocol) at given anthropological values was an etiological factor for meniscus injury. Fax: +387 33 653 055; E-mail: [email protected] Conclusion The seventh degree of load according to the Bruce ORCID ID: https://orcid.org/0000-0002- protocol can lead to the meniscus injury. 3722-2542 Key words: computer simulation, injuries, mechanics, meniscus

Original submission: 23 July 2020; Revised submission: 15 August 2020; Accepted: 30 August 2020 doi: 10.17392/1240-21

Med Glas (Zenica) 2021; 18(1):226-231

226 Veljović et al. Treadmill effect on knee

INTRODUCTION knee stabilizers (8,9,10). Passive stabilizers are li- gaments, while active knee stabilizers are muscles In the evaluation of ischemic heart disease, car- (8). In terms of mechanics, the knee joint is made diac stress testing is the basis of the diagnostic up of an angular and a rotating joint (10,11). The modality (1). The most commonly used are tread- knee joint contains the menisci, which are located mill and cycle ergometer (1,2). Treadmill testing between the femoral condyle and the tibial plate- according to the Bruce protocol is most common au (9). Menisci have a role in the distribution and in practice (3,4). A load level lasts 3 minutes with transfer of load in the knee when walking and stan- an increasing inclination and speed of treadmill ding, absorb shocks, serve as a secondary stabili- (initial treadmill inclination is in the first stage of zer of the knee, provide joint lubrication, nourish exercise 10% at a speed of 1.7mph or 2.7km/h, and protect articular cartilage, ensure compliance and then the inclination and speed of movement of joint surfaces, increase contact area and prevent gradually increase). Arterial pressure is measured extreme flexion and extension (10,12). every 2 minutes, with monitoring of the electro- cardiogram (ECG) (4). According to the Bruce Menisci injuries, especially those resulting from protocol, when the submaximal and/or maximum sports activities, are usually caused by rotational heart rate is achieved, the test is considered to forces (12,13). A common mechanism of injury be technically appropriate (4). Changes in heart is the action of lateral forces on the knee in flexi- rhythm, ST segment and T wave are monitored on (12). Predisposition to injury is the position in (4). During cardiac stress testing metabolic equi- which the meniscus is most retracted into the jo- valents of task (METs) are noted and represent a int (12). For the medial meniscus, this position is sign of a condition of the body (4). A value of 1 a knee flexion with the lower leg rotated outward MET is the consumption of 3.5 mL of per kilo- and the knee turned inward (lower leg abduction) gram of body mass per minute (4). (13,14). For the lateral meniscus, it is the knee flexion, inward rotation, and lower leg abduction Cardiac stress testing is often unreliable, and in a (13,14). They make up about 75% of intra-articular large percentage it is done without real indicati- pathology of the knee (15). Ruptures of the menis- on (5,6). Although there are methods to objectify cus can be different, from partial rupture to comple- whether the patient is adequately loaded during te longitudinal or transverse rupture or rupture of a the test, or whether the test is valid, the test in part of the meniscus or its grip (16,17). Chondral most cases cannot be completed due to fatigue of defects can also be in the form of micro sutures, but the patient, which is not associated with coronary they can also give clinical symptoms (18,19). circulation (6,7). Obesity, insufficient physical fitness, and especially the condition of the muscu- A large number of stress tests cannot be perfor- loskeletal system can lead to false results, which med adequately due to knee pain, and as such are then lead to immoderate spending of medical re- not adequate for the evaluation of ischemic heart sources (4,5). In the evaluation of ischemic heart disease. It was necessary to make a model that disease, the sensitivity of cardiac stress testing is can determine exact loading of the patient wit- 68%, while the specificity is 77% (4). hout consequent knee injury. No investigations were found relating to this topic. The knee is the largest joint in the human body, and also the largest joint of the musculoskeletal The aim of this research was to determine the system, which supports body weight and has the effect of the load on the meniscus in relation to biggest role in body movement (8). Due to the different angle, and to present the impact of force high load it is prone to frequent various overexer- on eventual injury of menisci. The results will be tion syndromes or chronic damage (9). In order important in everyday clinical cardiac practice. for the knee to have normal function, the correct EXAMINEES AND METHODS shape and position of the bones, strong and deve- loped muscles and strong ligaments are important Examinees and study design (9,10). The intra-articular bodies are the condyles of the femur and the tibia; additionally, the patella This prospective research was conducted at the is also part of knee joint (8-10). The movements Faculty of Mechanical Engineering, University in the knee joint are enabled by passive and active of Sarajevo, in June 2020 and included 200 male

227 Medicinski Glasnik, Volume 18, Number 1, February 2021

students. The inclusion criteria were the absence of osteomuscular deformity and negative ana- mnestic data on the existence of heart disease. The participants were volunteers. A written in- formed consent was obtained from all the par- ticipants. The average anthropometric measure- ments (average model) of the examinees were obtained (height 178.5 cm, mass 83.5 kg, average age 22 years). An ethical approval was obtained from the Ethical Committee, Faculty of Mechani- cal Engineering, University of Sarajevo.

Methods The simulation of examinees on a treadmill that was used in the evaluation of ischemic heart disease was made. Average values of anthropo- metric measures were obtained: height of 178.5 cm, mass of 83.5 kg, weight was equal to the

product of the force of the earth's gravity and Figure 2. Equilibrium of the thigh in a given position; YB, force in the knee; M , moment in the knee the mass of the examinees, and it was equal to B 819.135 Newton (N). According to the average The mass of the part of an examinee above the model, the model of the knee joint was analysed knee, when standing on one leg (total mass-mass (Figure 1, Figure 2). Measurements were obser- of the lower leg-mass of the foot) was calculated ved at different inclinations (5°70’, 6°80’, 7°90’, as the difference of total mass, lower leg mass and 9°10’, 10°20’, 11°30’ and 12°40’) and effects on foot mass, and the result for the average exami- knee were evaluated (Figure 1). The mass of the nee was 79.176 kg. The pressure on the meniscus foot was obtained from the Donskoi-Zaciorski (the force acting on the meniscus relative to the pattern, as well as the mass of the lower leg (20). surface of the meniscus) was analysed, as well as the allowable stress on the meniscus relative to the angle of the moving strips. Equilibrium of the thigh was calculated according to Equation 1: = 0.2643 × h= 0.2643 × 178.5 = 47.177 cm; α +β ≈ 60° The weight of the portion of the examinee’s mass above the knee was calculated from Equation 2: G′= m′ × g = 79.176 × 9.81 = 776.716 N The length of the upper leg was calculated from Equation 3: AB= 0.2643 × h = 0.2643 × 1.785 = 0.471

Statistical analysis The obtained anthropometric values of the mo- del were obtained from the mean value of the analysed parameters. Donskoi-Zaciorski pattern was used to analyse the mass of segments of the lower extremity. The moment of force in the knee Figure 1. Biomechanical model of a male examinee - position and the load force on the knee was calculated in during testing on treadmill; the mass of the foot was obtained relation to the angle of inclination, as well as the from the Donskoi-Zaciorski pattern, as well as the mass of the lower leg tension in the meniscus.

228 Veljović et al. Treadmill effect on knee

RESULTS DISCUSSION As the angle of the treadmill increased, the slope This research and biomechanical model indicated also increased; the moment of force acts on the that in the angle of 22% on treadmill, the knee co- knee joint, and thus on the meniscus (Table 1). mes to a position where injuries are possible, and At the slope of 10%, the moment of force on the that is risk zone for the injury of musculoskeletal knee was 213.53 Nm, and at 22% it was 246.71 system. Similar studies that have addressed this Nm (Table 1). problem have not been found in the literature. In research, male examinees were selected because of less bias due to anthropometric gender differences. Table 1. Moment in the knee (MB) related to the angle of inclination The main problem in the assessment of a pati- Slope (%) 10 12 14 16 18 20 22 ent who is on the treadmill is non-specific pain Angle β° 5.71 6.84 7.96 9.09 10.20 11.30 12.40 Angle α° 54.29 53.15 52.03 50.91 49.79 48.69 47.59 in the legs or back and abdomen, which is most MB (Nm) 213.53 219.35 225.07 230.67 236.14 241.49 246.71 often associated with osteomuscular deformiti- M (Nm), moment in the knee (Newton meter); B es; because of that, cardiologists have a problem with a clear evaluation of the patient (5). All With an increase of ascent, the load force on the this leads to a waste of resources and a burden meniscus also increased (Table 2). At the slope of on the medical system, and often the doctors do 10%, the force on the meniscus was 837.37 N, the not base their decision on objective facts, but on load on the meniscus was 0.23 N/mm2, and it gra- patient's subjective difficulties and the existence dually increased with increasing of the slope; at the of comorbidities (5). The question is often asked slope of 22%, the force on the meniscus was 967.51 whether this pain in the legs and especially in the N, while the load was 0.26 N/mm2 (Table 2). knee is still a condition associated with the con- dition of the knee joint, or the existence of dege- Table 2. Load force on the knee related to the angle of inclination nerative osteoarthritis, which is very present in Slope (%) 10 12 14 16 18 20 22 the modern population (7). Load on the treadmill Angle β0 5.71 6.84 7.96 9.09 10.20 11.30 12.40 can even lead to acute meniscus injury, which is Angle α0 54.29 53.15 52.03 50.91 49.79 48.69 47.59 something that should be avoided (7). Fmen (N) 837.37 860.02 882.64 904.59 926.05 947.03 967.51 2 σmen (N/mm ) 0.231 0.237 0.244 0.25 0.256 0.261 0.267 The meniscus is a very important part of the knee σ 2) Fmen (N), force on the meniscus (Newton); men (N/mm ), load on the joint because it has a function in transmitting of meniscus (Newton per square meter); the load, thus preventing joint injury (17,21). Ma- jewski et al. stated that the incidence of the menis- We could expect that for each increase in ascent cus injury is 12-14%, and the prevalence is 61 ca- after 22% at given anthropological values (which ses per 100,000 persons (22). The functional state corresponded to the angle of 12° 40’), the menis- of the joints is one of the most important factors cus can be damaged (in the comparison to per- contributing to the meniscus injury (23). Reid et missible stress on the meniscus, which was 0.29 al. stated that squatting, kneeling, crawling, sitting MPa (18) (Figure 3). while driving, stair climbing, lifting items are etio- logical factors that can lead to the meniscus injury (24). McMillan and Nichols list osteoarthritis as one of the most important factors that can contri- bute to the meniscus injury, and chronic repetitive knee loading, and primarily squatting and knee- ling can contribute to osteoarthritis (25). Snoeker et al. stated that the age (over 60), male gender, work-related kneeling and squatting, and climbing stairs are risk factors for the meniscus injury (26). The same authors stated, however, that running is not a risk factor for the injury itself, and that sitting Figure 3. Tension in the meniscus as a function of the angle 2 greater than 2 hours per day may reduce the risk of of inclination; σmen, tension in the meniscus (N/mm ); β, angle of inclination (%); degenerative meniscal tears (26). It is important to

229 Medicinski Glasnik, Volume 18, Number 1, February 2021

emphasize that the elderly population has a physi- the meniscus injury (32). The same authors state ological degenerative knee changes, and that older that warming-up before exercise is very important people are expected to be more prone to sudden for the prevention of the meniscus injury (32). It injuries (25,27,28). should be noted that the knee should always be a In the daily work in cardiology, cardiac stress te- neutral mechanical axis, and inappropriate loading sting is an unavoidable method for the evaluation and sudden unnatural movement lead the knee to of ischemic disease. Due to inadequate testing it is axis dysregulation, what is a predisposing factor increasingly sidelined and losing its importance, for the injury (33,34). Before cardiac stress testing, because multislice scanner coronary angiography attention should be paid to the condition of the and myocardial perfusion scintigraphy are more musculoskeletal system, although this is probably preferred methods (29-31). However, considering very difficult to know without prior diagnostics. low availability, especially in less developed co- However, overloading of the patient, and especi- untries, cardiac stress testing still represents an ally an unnecessary load, can lead to the injury. important issue in clinical practice (2). The fact is In conclusion, the seventh degree of load accor- that, according to the Bruce protocol (4,5), if there ding to the Bruce protocol during cardiac stress is no chest pain during six stages of exercise, there testing and the slope of 22% of treadmill is a zone is probably no point in forcing the patient. Howe- in which the meniscus injury can occur due to ver, this is questionable for professional athletes, overload of the meniscus. These results can help and the decision about terminating stress testing cardiologists in daily work during the evaluation should be made individually (5). Preventive pro- of ischemic heart disease. grams before stress testing must be implemented, and risk factors that can lead to possible injury FUNDING should be clearly isolated. Prevention of injury has No specific funding was received for this study. to be the aim of clinicians’ work, and modifiable factors especially noted. Ma et al. marked obesity TRANSPARENCY DECLARATION as one of the most important factors leading to Competing interests: None to declare.

REFERENCES 1. Loftin M, Sothern M, Warren B, Udall J. Compari- 8. Helito CP, Demange MK, Bonadio MB, Tírico LE, son of VO2 Peak during Treadmill and Cycle Ergo- Gobbi RG, Pécora JR, Camanho GL. Anatomy and metry in Severely Overweight Youth. J Sports Sci histology of the knee anterolateral ligament. Orthop Med 2004; 3:554-60. J Sports Med 2013; 1:2325967113513546. 2. Kharabsheh SM, Al-Sugair A, Al-Buraiki J, Al-Far- 9. Makris EA, Hadidi P, Athanasiou KA. The knee han J. Overview of exercise stress testing. Annals of meniscus: structure-function, pathophysiology, cu- Saudi medicine 2006; 26:1–6. rrent repair techniques, and prospects for regenerati- 3. Vilcant V, Zeltser R. Treadmill Stress Testing. In: on. Biomaterials 2011; 32:7411-31. StatPearls [Internet]. Treasure Island (FL): StatPe- 10. Claes S, Vereecke E, Maes M, Victor J, Verdonk P, arls Publishing, 2020. Bellemans J. Anatomy of the anterolateral ligament 4. Garner KK, Pomeroy W, Arnold JJ. Exercise stress of the knee. J Anat 2013; 223:321-8. testing: indications and common questions. Am Fam 11. Fox AJ, Bedi A, Rodeo SA. The basic science of hu- Physician 2017; 96:293-9. man knee menisci: structure, composition, and func- 5. Silva AM, Armstrong AC, Silveira FJ, Cavalcanti tion. Sports Health 2012; 4:340-51. MD, França FM, Correia LC. Prevalence and fac- 12. Doral MN, Bilge O, Huri G, Turhan E, Verdonk R. tors associated with inappropriate use of treadmill Modern treatment of meniscal tears. EFORT Open exercise stress test for coronary artery disease: a Rev 2018; 3:260-8. cross-sectional study. BMC cardiovascular disorders 13. Mordecai SC, Al-Hadithy N, Ware HE, Gupte CM. 2015; 15:54. Treatment of meniscal tears: An evidence based 6. Victor de Sousa C, Sales MM, Aguiar Sda S, Bo- approach. World J Orthop 2014; 5:233-41. ullosa DA, Rosa Tdos S, Baldissera V, Simões HG. 14. Beaufils P, Becker R, Kopf S, Matthieu O, Pujol N. Double product break point estimates ventilatory The knee meniscus: management of traumatic tears threshold in individuals with type 2 diabetes. J Phys and degenerative lesions. EFORT Open Rev 2017; Ther Sci 2016; 28:1775-80. 2:195-203. 7. Susko AM, Fitzgerald GK. The pain-relieving qua- 15. Bryceland JK, Powell AJ, Nunn T. Knee Menis- lities of exercise in knee osteoarthritis. Open Access ci. Cartilage 2017;8:99-104. Rheumatol 2013; 5:81-91.

230 Veljović et al. Treadmill effect on knee

16. Chivers MD, Howitt SD. Anatomy and physical exa- 25. McMillan G, Nichols L. Osteoarthritis and meniscus mination of the knee menisci: a narrative review of disorders of the knee as occupational diseases of mi- the orthopedic literature. J Can Chiropr Assoc 2009; ners. Occup Environ Med 2005; 62:567-75. 53:319-33. 26. Snoeker BA, Bakker EW, Kegel CA, Lucas C. Risk 17. Heckmann TP, Barber-Westin SD, Noyes FR. Me- factors for meniscal tears: a systematic review inclu- niscal repair and transplantation: indications, tech- ding meta-analysis, J Orthop Sports Phys Ther 2013; niques, rehabilitation, and clinical outcome. J Ort- 43:352-67. hop Sports Phys Ther 2006; 36:795-814. 27. Brindle T, Nyland J, Johnson DL. The meniscus: re- 18. Pellegrino M, Trinchese E, Bisaccia M, Rinonapoli view of basic principles with application to surgery G, Meccariello L, Falzarano G, Medici A, Piscitelli and rehabilitation. J Athl Train 2001; 36:160-9. L, Ferrara P, Caraffa A. Long-term outcome of grade 28. Raj MA, Bubnis MA. Knee Meniscal Tears. In: Stat- III and IV chondral injuries of the knee treated with Pearls [Internet]. Treasure Island (FL): StatPearls Steadman microfracture technique. Clin Cases Mi- Publishing, 2020. ner Bone Metab 2016; 13:237-40. 29. Fathala A. Myocardial perfusion scintigraphy: tech- 19. Rollo G, Falzarano G, Ronga M, Bisaccia M, Grubor niques, interpretation, indications and reporting. P, Erasmo R, Rocca G, Tomé-Bermejo F, Gómez- Ann Saudi Med 2011; 31:625-34. Garrido D, Pichierri P, Rinonapoli G, Meccariello L. 30. Wehrschuetz M, Wehrschuetz E, Schuchlenz H, Challenges in the management of floating knee inju- Schaffler G. Accuracy of MSCT coronary angio- ries: Results of treatment and outcomes of 224 con- graphy with 64 row CT scanner-facing the facts. Clin secutive cases in 10 years. Injury 2019; 50:S30-8. Med Insights Cardiol 2010;4:15-22. 20. Zaciorski WM. Fundamentals of sports metrology 31. Zhou T. Analysis of the biomechanical characteri- (in Russian). Moscow: Fizkultura i Sport, 1979. stics of the knee joint with a meniscus injury, He- 21. Abdelgaied A, Stanley M, Galfe M, Berry H, Ingham althcare Technology Letters 2018; 5:247-9. E, Fisher J. Comparison of the biomechanical tensi- 32. Ma JZ, Cui SF, Hu F, Lu QJ, Li W. Incidence and le and compressive properties of decellularised and characteristics of meniscal injuries in cadets at a mi- natural porcine meniscus. J Biomech 2015; 48:1389- litary school. 2013-2015. J Athl Train 2016; 51:876- 96. 9. 22. Majewski M, Susanne H, Klaus S. Epidemiology of 33. Rollo G, Pichierri P, Grubor P, Marsilio A, Bisaccia athletic knee injuries: A 10-year study. Knee 2006; M, Grubor M, Pace V, Lanzetti RM, Giaracuni M, 13:184-8. Filipponi M, Meccariello L. The challenge of no- 23. Frizziero A, Ferrari R, Giannotti E, Ferroni C, Poli nunion and malunion in distal femur surgical revi- P, Masiero S. The meniscus tear. State of the art of sion. Med Glas (Zenica) 2019; 16(2) Online ahead rehabilitation protocols related to surgical procedu- of print. res. Muscles Ligaments Tendons J 2013; 2:295-301. 34. Falzarano G, Pica G, Medici A, Rollo G, Bisaccia M, 24. Reid CR, Bush PM, Cummings NH, McMullin DL, Cioffi R, Pavone M, Meccariello L. Foot loading and Durrani SK. A review of occupational knee disor- gait analysis evaluation of nonarticular tibial pilon ders. J Occup Rehabil 2010; 20:489-501. fracture: a comparison of three surgical techniques. J Foot Ankle Surg 2018; 57:894-8.

231 ORIGINAL ARTICLE

Two-stage bone-and-strut technique in the treatment of septic non-unions in the upper limb

Luigi Meccariello1, Ante Prkić2, Vincenzo Campagna3, Alberto Serra4, Vincenzo Piccinni5, Denise Eygen- daal2,6, Michele Bisaccia7, Giuseppe Pica8, Andrea Schiavone8, Giuseppe Rollo1

1Department of Orthopaedics and Traumatology, Vito Fazzi Hospital, Lecce, Italy, 2Department of Orthopaedic Surgery, Amphia Hospital, Breda, the Netherlands, 3Department of Emergency Surgical and Medicine, 4Department of , 5Department of Orthopaedics and Traumatology; Italian Army Celio Policlinic, Rome, Italy, 6Department of Orthopaedics, AUMC, Amsterdam, the Neth- erlands, 7Orthopaedics and Traumatology Unit, Department of Surgical and Biomedical Science, S.M. Misericordia Hospital, University of Perugia, Sant’Andrea delle Fratte, Perugia, Italy, 8Department of Orthopaedics and Traumatology, AORN San Pio, Benevento, Italy

ABSTRACT

Aim To report the results of a two-stage reconstruction of septic non-unions of the upper limb using the bone-and-strut technique with a follow-up of more than two years.

Methods A total of 19 patients (12 males and seven females; age 27 to 85 years) were included in this cohort study. The evaluation endpoint was set at 24 months. Radiographic union, Quick Disa- bilities of the Arm, Shoulder and Hand (QuickDASH) scores, pain Corresponding author: and return to work were assessed. All patients were treated with Ante Prkić debridement and antibiotic therapy. At a second stage, the non- Department of Orthopaedic Surgery, union focus was filled with a cancellous bone allograft. Stability was provided using a locking plate and a bone strut. Amphia Hospital, Breda, the Netherlands Phone: +31 765951177; Results After 24 months, the QuickDASH scores improved from E-mail: [email protected] a median of 28 (interquartile range, 13 – 35 points), to a median Luigi Meccariello ORCID: https://orcid. of 78 (interquartile range, 70 – 89 points). Mean pain scores im- proved from 8.1 (range, 0.3-10) to 0.6 (range 0-2). Radiographic org/0000-0002-3669-189X and clinical union was seen in all patients. The majority of patients returned to work or previous activities when retired. A new neu- rological deficit, recurrence of infection, or other surgery-related adverse events were not observed.

Conclusion The two-stage bone-and-strut technique is a safe and effective technique in the treatment of septic non-unions of the upper limb. The union rate is high, the complication rate is accep- Original submission: table and return return-to-work is high. Recurrence of infectious 26 October 2020; sequelae during a follow-up period of at least two years was not Revised submission: seen. The patient-reported outcomes increased significantly. 29 October 2020; Key words: fractures, ununited; infections; surgical procedures, Accepted: operative 04 November 2020 doi: 10.17392/1306-21

Med Glas (Zenica) 2021; 18(1):232-238

232 Meccariello et al. Bone-and-strut for septic non-unions

INTRODUCTION PATIENTS AND METHODS Infected non-unions of long bones of the upper Patients and study design extremity are uncommon, and their successful treatment represents a great challenge to an ort- At the Vito Fazzi Hospital, Lecce, Italy (a ter- hopaedic surgeon. Bowen and Widmaier found tiary referral centre for non-unions in Italy), 19 that patients with three or more immune system patients with upper extremity septic non-unions compromising factors and an open fracture were enrolled after excluding patients with an to have an incidence of infection of 30% (1). Association for the Study and Application of the Other risk factors are bone and soft-tissue loss, Method of Ilizarov (ASAMI) nonunion classifi- inadequate fixation, poor vascularity of the cation type A or B, and the age of less than 18 ye- bony fragments, prolonged wound drainage and ars (15). Inclusion took place between 2005 and formation of a sinus, osteopenia, pre-existent 2019. Median age at presentation was 37 years osteomyelitis, adjacent joint stiffness, deformi- (interquartile range 37–47 years), and only one ties or length discrepancies, previous surgery patient was retired. The follow-up had a median on the fractured bone and resistant polybacte- duration of 32 months (range 24–156 months). rial infection (2–4). Pre-operatively, all patients were informed in a Non-unions with a concomitant infection pose clear and comprehensive way about the type of the dual challenge of eradicating infection whi- treatment and other possible surgical and conser- le trying to obtain union in an unfavourable vative alternatives possible in their specific case. environment for bone healing. Issues include Patients were treated according to the ethical soft tissue damage from open fractures and pri- standards of the Helsinki Declaration, and were or surgery, bone loss and segmental loss, insta- invited to read, understand and sign an informed bility and multiple medical comorbidities (1,3). consent form regarding their surgery. Two strategies are possible; first, to obtain uni- The Azienda Sanitaria Locale (ASL) Lecce on of the fracture followed by removal of in- (Italy) Ethical Committee approved this research. fected osteosynthesis, or second, by removing the infected material and to create optimal cir- Methods cumstances for bone healing with a two-stage First, the septic focus was debrided, and tempo- treatment (3,5,6). rary stability to the limb was provided according In the two-stage treatment of septic non-unions, to the location of the non-union: external fixati- infections are usually treated with removal of all on, a cast, a polymethylmethacrylate spacer, or a foreign material and devitalized tissue followed combination when necessary. Cultures were taken by parenteral or oral antibiotics; sometimes an- at the first debridement surgery and prophylactic tibiotic-impregnated polymethylmethacrylate antibiotics were administered to the patient after spacers are used to maintain bone length for su- a representative sample was obtained. A persona- bsequent surgeries and to achieve high doses of lized scheme of antibiotics was provided accor- antibiotics at the non-union site (2–4, 7,8). The ding to the culture results, and consisted of at le- second stage of treatment consists of obtaining ast 6 weeks of antibiotic administration. Then, an best biological and mechanical circumstances for antibiotic-free interval was used to see whether union, as in aseptic non-unions; internal fixation, the patients remained non-infectious. bone graft and soft tissue reconstruction (9–13). When clinical and laboratory results (C-reactive When this type of reconstruction is not favoura- protein, leucocyte count) remained satisfactory, ble or possible, bone transport with distraction the second surgical step proceeded where tem- osteogenesis is another possible solution (14). porary fixation was removed and the non-union The aim of this study was to report the outcomes focus was again debrided, and filled with can- of the patients treated of the two-stage recon- cellous bone allograft. Stability was provided struction of septic non-unions of the upper limb using a locking plate, connected to a bone strut with a follow-up of more than two years using on the opposite side for maximal rigidity. This the bone-and-strut technique. plate-and-strut technique is in use at our centre

233 Medicinski Glasnik, Volume 18, Number 1, February 2021

for aseptic non-unions (9,12,13). Post-operati- With regard to the microbial cause of the non- vely, the patients underwent a personalized reha- unions, staphylococcal infections were most bilitation protocol with a physiotherapist. common (Staphylococcus aureus, and Staphylo- To quantify the non-union severity the Non- coccus epidermidis in five and two patients, res- Union Scoring System (NUSS) in retrospective pectively); Pseudomonas aueruginosa in five, mode was used (16). Bone union was measured Escherichia coli and Proteus mirabilis in three using the radiographic union score as it described patients each, and Klebsiella pneumoniae in one (RUS) (17). patient. The duration of infectious symptoms va- ried between 1-24 months, with a median durati- Other aspects of the follow-up were the occu- on of 5 months (interquartile range 5–9 months). rrence of adverse events and the subjective qua- lity of life measured with the Quick Disabilities During the first stage, 16 patients received an an- of the Arm, Shoulder and Hand (QuickDASH). tibiotic-loaded polymethylmethacrylate spacer Pain was measured with the visual analogue sca- (in five patients it was combined with external le (VAS) with scores ranging from 0 (no pain at fixation for additional stability). Three patients all) to 10 (worst pain imaginable). In the case received a cast (forearm non-unions) and one pa- of humeral fractures, alignment was qualified tient received an external fixator only (humeral by rotation, alignment and length. In the case of shaft fracture). The second stage was performed elbow fractures, the trochleo-capitellar angle was after 2 to 6 months (median 3 months). Sevente- evaluated (18). For forearm fractures, alignment, en patients received an allograft, and two patients rotation and length were scored. a peroneal autograft because of the location. The peroneal autograft was used in ulna fractures to Statistical analysis promote bone healing by addition of living cells to the fracture site because of the notorious possi- The evaluation endpoint of the treatment of septic bility of ulnar non-unions. However, two patients non-unions was set at 24 months after surgery to had associated injuries of the lower extremities monitor for long-term adverse events, such as low- and an autograft was not possible without accep- grade infections. At this follow-up moment, radio- table extra morbidity. graphic union, QuickDASH scores, pain and return to work were assessed. The correlation between ra- Regarding the functional outcomes, at the 2-year diographic union and the subjective outcomes (Qu- follow-up, the QuickDASH scores improved from ickDASH score, pain, return to work yes/no) were a median of 28 points at time of non-union (in- correlated using Cohen’s kappa (κ) (19). terquartile range 13-35 points), to a median of 78 (interquartile range 70–89 points). Mean pain sco- RESULTS res were significantly reduced with a decline from In total 19 patients were enrolled, 12 males and 8.1 (range 0.3–10) during the non-union situation, 7 females. Most patients had a closed primary to 3.4 (range 0.2–6) one month after surgery and injury; five patients had an open fracture. Nine to 0.6 (range 0–2) after one year. The majority of patients had a confined upper limb injury, nine patients returned to work (or previous activities were involved in a polytrauma and one had an when retired) without limitations; 11 without limi- associated lower limb injury. The primary inju- tation, 7 with limitations. The patient with com- ries were distributed over the humerus (nine ca- bined nerve injury did not return to work, as he ses), elbow joint (three cases) and forearm (seven was retired and incapable of performing activities cases), and were most often treated with open re- of daily life. During follow-up, we did not observe duction and plate fixation (13 of 19 cases). Fa- new neurological deficits, recurrence of infection, ilure of the primary fixation was seen in 6 cases: nor other surgery-related adverse events. five times because of excessive movement over DISCUSSION the non-union site, and once because of a peri- implant fracture, which was complicated with a In this study we presented the bone-and-strut combined median, radial and ulnar nerve palsy. method as a treatment for septic non-unions in The NUSS score varied between 21 and 56, with different sites of the upper limb. All patients were a median of 27 (interquartile range 24–36 points). at first debrided for infection control, and later

234 Meccariello et al. Bone-and-strut for septic non-unions - Bacterial agent S. aureus K. pneumonie S. epidermidis P. aeuriginosa P. S. epidermidis E. coli P. aeuriginosa P. P. aeuriginosa P. P. mirabilis P. S. aureus P. aeuriginosa P. P. mirabilis P. P. aeuriginosa P. S. aureus E. coli S. aureus S. aureus E. coli P. mirabilis P. Neurogical Injury Neurogical - - Radial nerve - - Median, ulnar and radial nerve palsy - - - - Ulnar nerve ------Hardware failure Hardware Screws - Plate D Screws - Perimplant fracture - - - Second surgery plate Second surgery ------Septic symptoms (months) duration 6 5 5 5 7 2 5 2 5 1 5 10 2 11 9 8 24 12 8 Type of primary Type osteosynthesis Plate External fixator Plate Nail Plate Plate Plate Plate K wire K wires Plate Plate Plate and cerclage Plate Plate Plate Plate External fixator Plate NUSS at non-union 32 42 34 26 40 56 23 21 22 S. aureus, Staphylococcus aureus; K. Pneumonie, Klebsellia pneumonie; S. Epidermidis, Staphylococcus epidermidis; P. Aeurigi K. Pneumonie, Klebsellia pneumonie; S. Epidermidis, Staphylococcus epidermidis; P. Staphylococcus aureus; S. aureus, 38 24 27 26 34 23 37 28 23 26 Closed/open fracture Closed Open Closed Closed Closed Closed Closed Closed Open Open Closed Closed Open Closed Closed Closed Closed Open Closed Associated injury None Polytrauma None Polytrauma None None None None None None None Polytrauma Polytrauma Lower limbs Polytrauma Polytrauma Polytrauma Polytrauma Polytrauma Occupation Seller Homemaker Police officer Farmer Metal workman Retired Truck driver Truck Student Building workman Building workman Retired Plumber Building workman Carpenter Student Building workman Teacher Plumber Carpenter Follow-up Follow-up (months) 60 48 37 29 24 32 28 24 24 144 24 24 36 32 24 24 156 48 33 Site of fracture Humerus S Humerus S Humerus S Humerus S Homerus S Humerus D Humerus D Humerus D Humerus D Elbow Elbow Elbow Ulna P Ulna P Ulna P Ulna S Radius D Radius D Radius D Side R L L L R L L R L L R R L R L R L R L Age (years) 56 56 48 27 48 85 47 27 38 31 46 47 33 36 58 56 43 55 56 Gender M F M M F F F M M M F M M M F M F M M Patient Table 1. Characteristics of 19 patients treated the two-stage reconstruction septic non-unions upper limb proximal, S, shaft; female; M, male; L, Left; R, Right; D, distal; P, NSSU, Non-Union Scoring System; F, nosa, Psedomonas aeuriginosa; E. coli , Escherichia coli; P. Mirabilis, Proteus mirabilis; Mirabilis, Proteus nosa, Psedomonas aeuriginosa; E. coli , Escherichia coli; P. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 (Figure 1) 18 19

235 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 2. Surgical and follow-up outcome for 19 patients treated of the two-stage reconstruction of septic non-unions of the upper limb

Correlation NUSS Months QDASH QDASH Type of Bone QDASH between after between Radiologic before one year Patient Type of first step second healing with septic Return to work clinical and first first and reduction initial after se- step (months) non-union radiographic step second step trauma cond step results 1 Cemented spacer 62 3 P+Al Anatomic 6 100 10 88 With limitations 0.81 2 Cemented spacer 72 2 P+Al Anatomic 5 100 12 92 Fully 0.86 3 Cemented spacer 64 6 P+Al Anatomic 4 100 14 84 With limitations 0.70 4 EF 46 4 P+Al Satisfactory 4 100 14 82 Fully 0.72 5 Cemented spacer 64 3 P+Al Anatomic 3 100 16 90 With limitations 0.88 6 Cemented spacer 78 3 P+Al Anatomic 4 78 0 0 No 0.11 7 Cemented spacer 55 4 P+Al Anatomic 5 90 24 76 With limitations 0.76 8 Cemented spacer 26 5 P+Al Good 6 100 38 84 With limitations 0.80 9 Cemented spacer 29 4 P+Al Good 5 100 36 72 With limitations 0.74 10 Cemented spacer 84 3 Art+P+Al Satisfactory 2 96 0 64 With limitations 0.64 11 Cemented spacer 44 4 P+Al Anatomic 4 82 0 60 With limitations 0.77 12 Cemented spacer+EF 67 3 P+Al Good 3 100 28 62 Fully 0.78 13 Cemented spacer EF 72 3 P+Per Good 8 100 34 76 With limitations 0.81 14 Cemented spacer+EF 54 2 P+Al Anatomic 4 98 40 72 With limitations 0.79 15 Cemented spacer+EF 66 3 P+Per Good 5 98 42 68 Fully 0.63 16 Cemented spacer+EF 68 4 P+Al Anatomic 6 100 34 78 With limitations 0.77 17(Fig.1) Cast 71 2 P+Al Anatomic 2 100 34 100 Fully 1 18 Cast 64 4 P+Al Anatomic 3 100 32 100 Fully 1 19 Cast 66 3 P+Al Anatomic 3 100 36 96 Fully 0.92 NSSU, Non-Union Scoring System; QDASH, Quick Disabilities of the Arm, Shoulder and Hand; P, plate; Al, allograft; EF, external fixator; Art, arthrodesis; Per, peroneous autograft; ADL, activities of daily living

stability and bone stock was offered to achieve to proceed to the second step seems long in our union. As after the first stage of infection compo- cohort, yet preliminary stability was provided for nent was eliminated, the second stage was equi- using a cast or external fixator, and patients were valent to the treatment of aseptic non-unions at motivated enough to wait for optimal soft tissue our centre, which is used in other centres as well recovery to minimalize chances of failure. (7,12,13). The Masquelet technique, which uses Return to work is one of the factors we believe the same concept of debridement and membra- is highly important for these young patients who ne induction before final reconstruction without work with a median age of 37 years. In our group adding a bone strut, can also be used for septic all patients without pre-existing nerve injury could non-unions of the upper limb (20). The main ad- return to work. Of those returning to work, 39% vantage of our bone-strut-technique is that additi- needed adjustments. In previous literature, return onal stability and bone stock was provided using to normal activities of daily living within 90 days bone graft, which in our opinion provides more after the initial trauma was a predisposing factor biological impulses for fracture healing. for return to work (21). However, this 90-day peri- The bacteria causing the non-union in our cohort od had already passed for the patients visiting our were comparable to other similar cohorts with referral centre, and therefore, the rate of return-to- non-unions of the upper limb (7,20). The antibac- work in our cohort still seems satisfactory. terial treatment in our cohort consisted of local The functional and clinical results after two ye- control by debridement and removal of all seque- ars with a relatively small interquartile range are strae, followed by long (intravenous) antibiotic promising, and reflect the motivation of these yo- treatment. This approach is used generally to tre- ung patients to undergo the long trajectory after at infections after fractures and supported by in- an infection. The outcomes are comparable to si- ternational experts in order to treat infections and milar studies (7). The patient-reported outcomes to prevent chronic osteomyelitis (5,6). However, improved significantly after the whole treatment the skin and subcutaneous tissue have to be hea- to an acceptable level. Half of the patients had led completely before definitive reconstruction; initially an associated trauma, with almost all of otherwise, a secondary (wound) problem might those being a polytrauma. As the septic non-uni- occur and the treatment fails. Therefore, the time ons concerned the upper limb, the activities of

236 Meccariello et al. Bone-and-strut for septic non-unions

Figure 1. Female patient operated two years before presentation, with a plate fixation of a radial shaft fracture on the left side, with a fistula (A) because of a septic non-union (B) distal radius shaft fracture on the left side, with presence of a fistula (A) and a septic non-union (B). After debridement (C) and wide resection of the septic non-union (D-F) a cast was provided. Post-operative radio- graphs show complete resection of avital bone (G, H). After 2 months, osteosynthesis with the plate and bone-and strut technique was performed, using an allograft (I, J). After two years, excellent functional results were obtained (K, L) (Meccariello L, 2018) daily life were more compromised than mobility. In conclusion, the two-stage bone-and-strut tech- When radiographic signs of union (measured with nique proved to be a successful treatment of sep- radiographic union scale), and the patient related tic non-unions of the upper limb without recu- outcomes (measured with the QuickDASH) were rrence of infectious sequelae during a follow-up correlated, a high kappa value was seen. This me- period of two years. The patient-reported outco- ans that when patients were allowed to perform mes had increased to a satisfactory level after two more activities with their arm (based on consoli- years of follow-up and with a high percentage of dation on radiographical imaging) they also star- return-to-work. Therefore, we would recommend ted to use this arm more, resulting in better Qu- our two-stage bone-and-strut technique in these ickDASH scores. One patient who was not able cases of high-demand, young patients with septic to perform any activities because of nerve injury, non-unions of the upper limb. scored a low correlation because he did not func- FUNDING tionally improve, in contrast to the radiographic images. Therefore, patients’ related outcomes are No specific funding was received for this study. of high importance in our opinion and not only radiographic outcomes. TRANSPARENCY DECLARATION Conflict of interest: None to declare.

237 Medicinski Glasnik, Volume 18, Number 1, February 2021

REFERENCES 1. Bowen TR, Widmaier JC. Host classification predicts 12. Rollo G, Rotini R, Pichierri P, Giaracuni M, Stasi A, infection after open fracture. Clin Orthop Relat Res Macchiarola L, Bisaccia M, Meccariello L. Grafting 2005; (433):205–11. and fixation of proximal humeral aseptic non union: a 2. Allende C, Mangupli M, Bagliardelli J, Diaz P, prospective case series. Clin Cases Miner Bone Me- Allende BT. Infected nonunions of long bones of tab 2017; 14:298–304. the upper extremity: staged reconstruction using 13. Rollo G, Prkić A, Pichierri P, Eygendaal D, Bisaccia polymethylmethacrylate and bone graft impregnated M, Filipponi M, Giaracuni M, Hitov P, Tanovski K, with antibiotics. Chir Organi Mov 2009; 93:137–42. Meccariello L. Plate-and-bone-strut fixation of distal 3. Struijs PAA, Poolman RW, Bhandari M. Infected third humeral shaft aseptic non-unions: a consecutive nonunion of the long bones. J Orthop Trauma 2007; case series. J Clin Orthop Trauma 2019; 10:127–32. 21:507–11. 14. Ebied A, Elseedy A. Treatment of infected nonunion 4. Hanssen AD. Local antibiotic delivery vehicles in the of forearm bones by ring external fixator. Egypt Ort- treatment of musculoskeletal infection. Clin Orthop hop J 2018; 53:341–7. Relat Res 2005; (437):91–6. 15. Paley D, Catagni MA, Argnani F, Villa A, Battista Be- 5. Metsemakers WJ, Morgenstern M, Senneville E, nedetti G, Cattaneo R. Ilizarov treatment of tibial no- Borens O, Govaert GAM, Onsea J, Depypere M, nunions with bone loss. Clin Orthop Relat Res 1989; Richards RG, Trampuz A, Verhofstad MHJ, Kates 241:146–65. SL, Raschke M, Martin A McNally 13, Obremskey 16. Calori GM, Colombo M, Mazza EL, Mazzola S, Ma- WT, Fracture-Related Infection (FRI) group. General lagoli E, Marelli N, Corradi A. Validation of the Non- treatment principles for fracture-related infection: re- Union Scoring System in 300 long bone non-unions. commendations from an international expert group. Injury 2014; 45:S93–7. Arch Orthop Trauma Surg 2020; 140:1013–27. 17. Whelan DB, Bhandari M, Stephen D, Kreder H, Mc- 6. Metsemakers WJ, Kuehl R, Moriarty TF, Richards kee MD, Zdero R, Schemitsch EH. Development of RG, Verhofstad MHJ, Borens O, Kates S, Morgen- the radiographic union score for tibial fractures for stern M. Infection after fracture fixation: Current the assessment of tibial fracture healing after intrame- surgical and microbiological concepts. Injury 2018; dullary fixation. J Trauma 2010; 68:629–32. 49:511–22. 18. Rollo G, Rotini R, Eygendaal D, Pichierri P, Bisaccia 7. Perna F, Pilla F, Nanni M, Berti L, Lullini G, Traina M, Prkic A, Stasi A, Meccariello L. Effect of troc- F, Faldini C. Two-stage surgical treatment for sep- hleocapitellar index on adult patient-reported outco- tic non-union of the forearm. World J Orthop 2017; mes after noncomminuted intra-articular distal hume- 8:471–7. ral fractures. J Shoulder Elb Surg 2018; 27:1326–32. 8. Julka A, Ozer K. Infected nonunion of the upper 19. Landis JR, Koch GG. The measurement of observer extremity. J Hand Surg. 2013; 38:2244–6. agreement for categorical data. Biometrics 1977; 9. Moroni A, Caja VL, Sabato C, Rollo G, Zinghi G. 33:159. Composite bone grafting and plate fixation for the 20. Raven TF, Moghaddam A, Ermisch C, Westhauser F, treatment of nonunions of the forearm with segmental Heller R, Bruckner T, Schmidmaier G. Use of Masqu- bone loss: a report of eight cases. J Orthop Trauma elet technique in treatment of septic and atrophic 1995; 9:419–26. fracture nonunion. Injury 2019; 50:40–54. 10. Moroni A, Rollo G, Guzzardella M, Zinghi G. Sur- 21. Murgatroyd DF, Harris IA, Tran Y, Cameron ID. gical treatment of isolated forearm non-union with Predictors of return to work following motor vehicle segmental bone loss. Injury 1997; 28:497–504. related orthopaedic trauma. BMC Musculoskelet Dis- 11. Rollo G, Pichierri P, Marsilio A, Filipponi M, Bi- ord 2016; 17:171 saccia M, Meccariello L. The challenge of nonunion after osteosynthesis of the clavicle: Is it a biomecha- nical or infection problem? Clin Cases Miner Bone Metab 2017; 14:372–8.

238 ORIGINAL ARTICLE

Radiographic evaluation of the tunnel position in single and double bundle anterior cruciate ligament reconstruction

Michele Losco1, Francesco Giron1, Luca Giannini1, Pierlugi Cuomo2, Roberto Buzzi1, Stefano Giannotti3, Nicola Mondanelli3

1Traumatology and General Orthopaedics, Azienda Ospedaliera Universitaria Careggi, Florence, Italy, 2Royal National Orthopaedic Hospital, Stanmore, United Kingdom, 3Section of Orthopaedics, Department of Medicine, Surgery and Neurosciences, University of Siena, Siena, Italy

ABSTRACT

Aim To evaluate tunnel positioning on radiographs in single- bundle (SB) and double-bundle (DB) anterior cruciate ligament (ACL) reconstruction, to evaluate if measurement is accurate and reproducible.

Methods Radiographs of 30 SB and 30 DB ACL reconstruction were reviewed by two examiners who measured tunnel positio- ning with the quadrant method on the femur (a=depth, b=height) and the Amis and Jakob method on the tibia. Intra- and inter-ob- server reliability were evaluated with intra-class correlation co- efficient (ICC).

Corresponding author: Results A radiographic analysis was completed in all patients in a Nicola Mondanelli SB-group and in 27 in a DB-group (p>0.05). Intra-observer reli- ability was almost perfect on femoral (ICC: a=0.85, b=0.83) and Section of Orthopaedics. Department of tibial (ICC=0.87) side in the SB-group. In the DB-group, it was Medicine, Surgery and Neurosciences, almost perfect for tibial anteromedial (AM) and posterolateral University of Siena (PL) bundles (ICC: AM=0.84, PL=0.81) and for femoral PL bun- Viale Mario Bracci 16, 53100 Siena, Italy dle (ICC: a=0.83, b=0.82), and substantial for femoral AM bun- Phone: +39 0577 585 675; dle (ICC: a=0.78, b=0.74). Inter-observer reliability was almost Fax +39 0577 233 400; perfect on tibial (ICC=0.81) and femoral (ICC: a=0.81, b=0.87) side in the SB-group, and substantial on tibial (ICC: AM=0.71, E-mail: [email protected]; PL=0.77) and femoral (ICC: AM a=0.73, b=0.78; PL a=0.74, Losco Michele ORCID ID: https://orcid. b=0.76) side in the DB-group. Standard deviation (SD) was low org/0000-0003-4578-9079 (±9%) with respect to the centre of tunnel(s).

Conclusion The quadrant method and the Amis and Jakob method are accurate and reproducible measurement methods. Also, as SD Original submission: was low, an outside-in approach with a front-entry guide, which is free-hand positioned, can be postulated as a reliable method to 16 November 2020; locate the femoral tunnel in SB reconstruction and the AM bundle Revised submission: in DB reconstruction. 23 November 2020; Accepted: Key words: anatomic reconstruction, quadrant method, radio- graphic analysis, tunnel placement 24 November 2020 doi: 10.17392/1316-21

Med Glas (Zenica) 2021; 18(1):239-246

239 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION PATIENTS AND METHODS Anatomical anterior cruciate ligament (ACL) re- Patients and study design construction has been deemed necessary to obtain better clinical results (1–3). Correct positioning of Two groups of 30 patients each who underwent tunnels is pivotal, and femoral tunnel position gre- an arthroscopic assisted SB or DB ACL recon- atly affects tension and isometry of the graft (4,5). struction at Azienda Ospedaliero-Universitaria Different approaches to drill the femoral tunnels Careggi from 2015 to 2018 were retrospecti- have been proposed: transtibial, transportal thro- vely picked-up in a casual fashion from a pros- ugh the anteromedial (AM) or an accessory AM pectively collected database and included into portal, outside-in using a rear-entry guide through the study. Informed patient consent and Ethical a posterolateral (PL) incision or a front-entry guide Committee consent were obtained at the time of through the anterolateral (AL) portal. At our Insti- previous studies; no further consent was required tution, an outside-in approach using a front-entry for this study. guide was finally opted for; also, a prototype guide to drill the PL bundle in double bundle (DB) recon- Methods struction was developed and techniques and results ACL reconstruction was performed using an au- have been previously published (3,6). While the- tologous hamstring graft and a double incision re is no clear evidence in literature that supports outside-in approach in all cases (6). In SB-group, DB over single bundle (SB) technique with respect tunnels were drilled aiming to exit in the centre of to post-operative results (7–9), recent literature tibial and femoral ACL insertion areas. The tibial supports the concept that clinical outcomes of ACL tunnel was drilled referring to anatomic landmar- reconstruction surgery would be linked to correct ks described by Jackson and Gasser (12) using femoral tunnel positioning (10). Native ACL fe- the 65° Howell Tibial Guide (previously Arthro- moral insertion has been described (5,11), and tek, Ontario, CA; now ZimmerBiomet, Warsaw, cadaveric biomechanical and radiographic studies IN, USA). The femoral tunnel was drilled on on anatomical landmarks useful for femoral tunnel the lateral femoral condyle (LFC) viewing via positioning have been published (12–17). the AM portal using a front-entry guide inser- Radiologic data of tunnel position can be used for ted through the AL portal (Acufex Director Drill surgical purposes and for post-operative evaluati- Guide, Smith & Nephew, Andover, MA, USA). on, and three-dimensional (3D) computed tomo- In DB-group, the AM tunnels were drilled first. graphy (CT) analysis for determining tunnel posi- On the tibia, the bullet of the 65° Howell Tibi- tion is deemed as the gold standard although this al Guide was rotated to get a more AM position, technique is not convenient in terms of costs and then the PL guide-wire was positioned using the radiation exposure. Also, 3D magnetic resonance prototype rod-guide inserted into the AM tunnel imaging (MRI) has been proven to be as accurate that allowed to exit posterior and lateral to the as CT and more accurate than plain radiographs AM tunnel at a fixed distance of 8 mm. On the (18,19), but it is much more expensive. Never- femur, the AM guide-wire was inserted near the theless, measurements achieved by radiograph posterior cartilage below the over-the-top positi- analysis are reliable if compared with those obta- on with the above-mentioned front-entry guide; ined by CT-scan (20–23). Substantial evidence the PL tunnel was drilled 9 mm apart, distal and supports, as the most reproducible ad reliable met- shallow from the AM tunnel, about 5 mm from hods to identify tunnel position on sagittal radio- the cartilage border, using the prototype rod-gu- graphs, the “quadrant method” described by Ber- ide through a different hole. nard and Hertel (24) for the femur and the Amis Standard anterior-posterior (AP) and true lateral and Jakob’s method (13) for the tibia. radiographic views were taken at 1- and 2-year The aim of this study was to investigate the radi- follow-up (FU) in every patient, according to ographic positions of the tunnels in SB and DB our protocol (25). In order to achieve the best reconstruction using these two radiographic met- femoral condyles superimposition, lateral ra- hods, and to compare results with those reported diographs were taken with fluoroscopic image in literature. intensifier to find correct rotation. Femoral and

240 Losco et al. Tunnel position in SB and DB ACL

tibial intra-articular tunnel aperture positions Statistical analysis were measured, independently and twice with Reliability of measurements was evaluated 8-week interval, by two examiners on the 2-year by means of intra-class correlation coefficient FU lateral views. On the femur, the centres were (ICC). Test-retest reliability was determined measured according to the quadrant method (24). with intra-observer ICC which measures the The total sagittal diameter of the LFC along the correlation between results obtained by the same Blumensaat’s line (distance t) and the maximum observer on separate occasions, and with inter- intercondylar notch height (distance h), tangent observer ICC which measures the correlation to the most dorsal subchondral contour of the between results obtained by different examiners. LFC and perpendicular to the distance t were me- ICC ranged from 0 to 1. According to Landis and asured. Then the distance from the tunnel centres Koch guidelines (26), the degree of agreement to the distance h (distance a) and to the distance was considered to be excellent (ICC greater than t (distance b) were measured and expressed as 0.91), almost perfect (ICC between 0.9 and 0.81), percentage of distance t (depth, being 0% deep/ substantial (ICC between 0.61 and 0.80), mode- posterior and 100% shallow/anterior) and distan- rate (ICC between 0.41 and 0.60) or fair (ICC ce h (height, being 0% high/superior and 100% between 0.21 and 0.40). low/inferior) (Figure 1). On the tibia, the centres of tunnels were orthogonally projected onto the RESULTS maximum sagittal tibial diameter and then expre- ssed as percentage being 0% anterior and 100% A radiographic analysis was completed in all pa- posterior (Figure 2). tients in the SB group and in 27 in the DB group (p>0.05). None of the two examiners was able to define PL femoral aperture in one patient, AM femoral aperture in another patient and PL tibial aperture in the third patient (Table 1, 2). Table 1. Centre of intra-articular tunnel apertures in single bundle (SB) group according to the quadrant method (24) on the femur and to the Amis and Jakob’s method (13) on the tibia Variable Femur (mean±SD) (%) Tibia (mean±SD) (%) Figure 1. The quadrant method described by Bernard and Her- a b tel (24) for femoral tunnel(s) position. Depth is expressed as Observer 1 25±4 28±7 46±5 0% deep/posterior and 100% shallow/anterior. Height is ex- Observer 2 25±6 33±5 43±7 pressed as 0% high/superior and 100% low/inferior. Location Average 25±5 30.5±6 44.5±6 of intra-articular opening of the tunnel(s) was not an obstacle a, height; b, depth on the lateral femoral condyle for superimposition of the metallic interference screws (Gi- Table 2. Centre of intra-articular tunnel apertures in double annini L, 2019) bundle (DB) group according to the quadrant method (24) on AM, anteromedial bundle; PL, posterolateral bundle; h, maximum the femur and to the Amis and Jakob’s method (13) on the tibia intercondylar notch height; t, total sagittal diameter of the lateral Femur (mean±SD) (%) Tibia (mean±SD) (%) femoral condyle along the Blumensaat’s line; a, distance from Variable the femoral tunnel centres to the distance h; b, distance from the AM PL AM PL femoral tunnel aperture to the distance t a b a b Observer 1 24±4 26±9 35±4 42±8 43±6 53±3 Observer 2 22±6 22±7 36±6 47±6 38±5 50±5 Average 23±5 24±8 35.5±5 44.5±7 40.5±4 51.5±4 AM, anteromedial bundle; PL, posterolateral bundle; a, height; b, depth on the lateral femoral condyle Intra-observer reliability of the quadrant method was quoted almost perfect in the SB group (ICC: a=0.85, b=0.83), substantial for AM tunnel (ICC: a=0.78, b=0.74) and almost perfect for PL tunnel (ICC: a=0.83, b=0.82) in the DB group. Inter- observer reliability was almost perfect in the SB Figure 2. The method described by Amis and Jakob (13) for tibial tunnel(s) position. Length is expressed as 0% anterior group (ICC: a=0.81, b=0.87) and substantial in and 100% posterior (Giannini L, 2019) the DB group for both AM (ICC: a=0.73, b=0.78) AM, anteromedial bundle; PL, posterolateral bundle and PL tunnels (ICC: a=0.74, b=0.76).

241 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 3. Comparison between results of the present study (in vivo) versus previous in vitro studies on cadaveric knees, entire anterior cruciate ligament (ACL) footprints* Femur (mean±SD) (%) Tibia (mean±SD) (%) Comparison Method Entire ACL Notes Entire ACL a b Present study X-rays 25 ± 5 30.5 ± 6 44.5 ± 6 Cadaveric knees Bernard and Hertel. (13) X-rays 24.8 ± 2.2 28.5 ± 2.5 - X-rays 27.5 ± 3.2 26.9 ± 3.5 46.2 ± 2.8 Musahl et al. (27) No differences between X-rays and CT-scan CT-scan 26.6 ± 1.9 26.3 ± 2.4 45.4 ± 2.1 De Abreu-e-Silva et al. (28) CT-scan 35.3 ± 4.5 30 ± 1.6 40.5 ± 5.3 Guo et al. (29) X-rays 38.3 ± 2.7 43.1 ± 4.6 - Slightly different method *The quadrant method of Bernard and Hertel (13) as in Figure 1 was used for the femoral footprint and the Amis and Jakob’s methods (13) as in Figure 2 for the tibial footprint except where noted a, height; b, depth on the lateral femoral condyle; X-rays, radiographs; CT-scan, computed tomography scan

Intra-observer reliability of the Amis and Jakob The centre of the femoral tunnel in the SB method was quoted almost perfect in the SB group was located at 25±5% in deep-shallow group (ICC=0.87) and almost perfect in the DB and 30.5±6% in high-low direction, a position group (ICC: AM=0.84, PL=0.81). Inter-observer superimposable to those reported on cadaveric reliability was almost perfect in the SB group knees (24, 27–29). Bernard and Hertel found the (ICC=0.81) and substantial in the DB group for centre of ACL femoral insertion to be located at both tunnels (ICC: AM=0.71, PL=0.77). 24.8±2.2% of depth and 28.5±2.5% of height on Standard deviation (SD) was low (±9%) with res- the LFC (24). Musahl et al. (27) and de Abreu-e- pect to the centre of tunnel(s). Silva et al. (28) found similar results; differences between radiographs and CT-scan evaluations DISCUSSION were not statistically significant (27). On the other hand, Guo et al. found different data, but a slightly The main finding of this study in our opinion is different method of measurement was used (29). the reliability of femoral tunnel positioning with the outside-in technique using a guide which is The AM femoral tunnel in the DB group was free-hand positioned to locate the intra-articular located at 23±5% / 24±8% of depth / height of tunnel opening, referring to visible landmarks the LFC. Again, this position was consistent with (5,16). This technique was used to locate the fe- that reported in literature (14,17,21,23,30). Inte- moral tunnel in the SB group and the AM femoral restingly, Lee et al. did not find any difference in tunnel in the DB group. Comparison between in evaluated parameters between anatomic dissecti- vivo results of the present study and in vitro re- on, radiographs and CT-scan (23). sults of literature are reported in Table 3 (entire In this series, the femoral PL tunnel in the DB ACL) and Table 4 (separate AM and PL bundle group was positioned at 35.5±5% and 44.5±7% measurements). of depth and height of the LFC, respectively. This

Table 4. Comparison between results of the present study (in vivo) versus previous in vitro studies, double bundle anterior cruciate liga- ment footprints* Femur (mean±SD) (%) Tibia (mean±SD) (%) Comparison Method AM PL Notes AM PL a b a b Present study X-rays 23±5 24±8 35.5±5 44.5±7 40.5±4 51.5 ± 4 Cadaveric knees Colombet et al. (14) X-rays 26.4±2.6 25.3±4.2 32.3±3.9 47.6±6.5 36 52 Zantop et al. (17) X-rays 18.5 22.3 29.3 53.6 30 44 Stäubli and Rauschning’s technique (33) Iriuchishima et al. (21) X-rays 15±6 26±8 32 52 31 50 Stäubli and Rauschning’s technique (33) Pietrini et al. (30) X-rays 21.6±5.6 14.2±7.7 28.9±4 42.3±6 36.3 51 X-rays 33.5±4.7 27.6±5.4 38.3±4 55.1±7.1 36.3±5.6 43.4±5 CT-scan 34.2±4.3 26.3±5.8 38.7±4 53±5 36.7±3.8 42.2±4.2 no differences between X-rays, CT-scan Lee et al. (23) Anatomic dissection 33.9 ± 5.6 25.6±5.5 40.6±4.3 56.4±6.3 37.6±5.7 43.8±6.5 and anatomic dissection Mean 33.9 26.5 39.2 54.8 36.9 43.1 Doi et al. (31) X-rays - - - - 34.6 38.4 *The quadrant method of Bernard and Hertel (13) as in Figure 1 was used for the femoral footprint and the Amis and Jakob’s methods (13) as in Figure 2 for the tibial footprint except where noted. AM, anteromedial bundle; PL, posterolateral bundle; a, height, b, depth on the lateral femoral condyle; X-rays, radiographs; CT-scan, computed tomography scan

242 Losco et al. Tunnel position in SB and DB ACL

position is close to that found by different aut- while the distance on the tibial side could be extra- hors (14,21,23), and shallower than those found polated in 7 mm (11,34). With regard to the femo- by others (17,30). ral side, Zantop et al. found the distance between The centre of tibial tunnel in the SB group was bundles to be 8-10 mm (17), while Tashiro et al. located at 44.5±6% of the tibial plateau length. superimposed the anatomical information obtai- Musahl et al. found the same location for the entire ned by previous authors onto 3D CT-scan models ACL tibial footprint; differences between radio- and found that the distance between AM and PL graphs and CT-scan evaluation were not statisti- centres could be evaluated 10.2±0.6 mm in males cally significant (27).De Abreu-e-Silva et al. found and 9.4±0.5 mm in females (35). Our prototype it to be located at 40.5±5.3% in the AP direction, rod-guide was designed to locate the centre of the on 3D CT-scan with a reference consistent with the PL bundle at the fixed distance from the centre of radiographic method of Amis and Jakob) (28). the AM bundle of 9 mm on femur and 8 mm on the tibia. This distance seems therefore to be 1 mm In the present study, the centre of tibial AM tunnel excessive with respect to data from cadaveric stu- in the DB group was located at 40.5±4% of depth, dies, but it was chosen as a safe standard distance that is a more posterior position than reported by to secure a bony bridge of 2 mm between tunnels other authors (14,30,31). A 5% difference was with a 6-mm diameter, taking into account possi- found, corresponding to about 2.5 mm as abso- ble ovalization of the tunnel aperture as well (6). lute value in Colombet’s (14) and Doi’s samples (31); in our opinion this was due to the use the 65° The second finding of this study is the intra- and Howell guide that tends to locate the tibial tunnel inter-observer reliability of tunnel evaluation on more posteriorly to prevent roof impingement in radiographs using two simple methods of mea- extension Cuomo et al(32). Iriuchishima et al. surement. With respect to the quadrant method (21) and Zantop et al. (17) found a more anterior for the femur, intra-observer reliability was quo- location for the centre of the tibial AM footprint; ted almost perfect for the SB group and PL tunnel anyway, they determined the centre of the tunnels in the DB group, and substantial for AM tunnel according to the technique described by Stäubli in the DB group; inter-observer reliability was al- and Rausching (33) that uses a different reference most perfect for the SB group and substantial for to define the maximum tibial sagittal diameter. both AM and PL tunnels in the DB group. With respect to Amis and Jakob’s method for the tibi- In the DB group, the centre of PL bundle was al tunnel(s), intra-observer reliability was almost located at 51.5±4% of the tibial sagittal diameter, perfect either in the SB group and the DB group in line with the studies of Colombet et al. (14) for both tunnels, whereas inter-observer reliability and Pietrini et al. (30), while Doi et al. (31) found was almost perfect in the SB group and substantial it to be more anterior. This could be related to in the DB group for both tunnels. Colombet et al. the large variation in tibial insertion patterns of calculated the inter-observer error in millimetres AM and PL bundles (14,15,34). Also, differences but did not calculate the ICC (14). Doi et al. me- between all these studies can be due to the small asured twice the landmarks but did not evaluate number of knees analysed in every cadaveric stu- any intra- or inter-observer variability (31). Pietri- dy, to different insertion patterns of AM and PL ni et al. instead found an excellent reliability (ICC native bundles (11,14,15,34) and to anatomical ≥ 0.989 in all analyses) either for intra-observer differences which can be found in individuals of and inter-observer measurements (30). Anyway, various ethnicities (having been the studies per- their excellent reliability was due to the prepara- formed in different continents). tion of cadaveric knees (dissected free from soft Colombet et al. found that the distance between tissues except for ACL, menisci and collateral li- the centre of AM and PL bundles was 8.2±1.2 mm gaments), the use of 2-mm stainless steel spheres on femoral side and 8.4±0.6 mm on tibial side to label the centres of the bundles, the marking of (14), while Lee et al. found such distance to be AM and PL footprints with a radio-opaque bari- 6.4±1.2 mm and 6.2±1 mm for femoral and tibi- um sulphate emulsion. With such a preparation, al attachments, respectively (23). Edwards et al. intra- and inter-observer variability related only to found that the distance between the centre of AM generate reference lines and to measure distances. and PL bundles on femoral side was 8±1.3 mm,

243 Medicinski Glasnik, Volume 18, Number 1, February 2021

In the present study, each observer had to “find” only in cadaveric dissected knees (29), while it is the tunnel(s)’ centre on radiographs in vivo, to ge- not possible to get an adequate axial imaging in nerate reference axes and to measure distances; vivo. The Rosenberg view has been described as it was therefore a multiple-step measuring which useful to evaluate the height of the tunnel (22), may amplify errors. Nevertheless, the reliability and a modified clock evaluation on the same was graded “almost perfect” for the SB group view has been proposed (36), but such an ima- and at least “substantial” for the DB group. This ging of the distal femur is not actually mimicking may be due to the fact that tunnel apertures in the the arthroscopic view, it lacks further usefulness DB group were smaller and superimposed each and can create confusion with arthroscopic no- other, making them more difficult to be detected. menclature. As for the tibia, anatomical landmar- To bypass such a problem, Horie et al. proposed ks have been well known since the 1990s (12) a modified quadrant method technique (22). They and they are clearly evident intra-operatively to first calculated the position of femoral tunnels on locate the tunnel(s). Also, a CT-scan and/or MRI a Rosenberg view, then reported the lines over the study was not performed, which would have ad- lateral view respecting the calculated height ratios ded more precise data on the tunnel(s) position and then drawn the axes of the femoral tunnels up both on the tibia and the femur; anyway, radio- to the intersection with these horizontal lines to graph analysis seems to be reliable and reprodu- obtain the position of tunnel apertures relatively to cible when compared with 3D CT-scan (20–23). depth. They found this method to have excellent The second limitation is that current results could intra-observer reliability, almost perfect inter-ob- not be compared with data about ACL footprints server reliability for AM tunnel and substantial obtained on cadaveric specimens by same aut- inter-observer reliability for PL tunnel. Also, the hors (15,32), and only to the limited number of accuracy of the method was found to be almost mentioned studies because of different methods perfect comparing it to 3D CT-scan. In the pre- used to evaluate native ACL footprints and tunnel sent series, it was not possible to identify femoral position. In previous papers (15), the circle met- tunnel apertures on the lateral view in only 2 over hod (37) had been used to measure the position of 30 cases in DB-group. Moreover, the modified the tunnels on the femoral side, but the quadrant quadrant method proposed by Horie et al. (22) method (24) seems to be easier and more used requires a Rosenberg view and a careful reporting nowadays. In our opinion, this is currently and of the horizontal lines on the lateral view as a ratio. actually the main issue that needs to be addre- There are several limitations in this study. First, ssed, as different methods and modification of radiographic analysis was performed only on la- historical methods are being proposed, each of teral radiographs, even if AP radiographs were which has advantaged and disadvantages, and available, and a CT-scan and/or MRI study sho- even if they seem to be tantamount, they are not. uld also have been performed. Studying only The third limitation of this study is that a correla- the lateral view can be enough to evaluate the tion between the tunnel position and clinical re- tunnel(s) position on the femur, while it gives sults was not evaluated; it would be interesting to only depth on the tibia: for practical purposes, understand if a particular position would have led it seems sufficient to collect such data (31). As to a slack ACL (increased AP or rotational laxity). for the femoral side, there is a need to evaluate In conclusions, the quadrant method of Bernard tunnel(s) position on 2 axes (height, depth), and and Hertel for the femoral side and the Amis and this can be done on lateral radiographs, while the Jakob’s method for the tibial side are reliable, third axis (width) is obviously on the medial wall useful and easy methods for radiographic des- of the LFC. Nevertheless, methods to evaluate cription of the tunnel position. A universal con- femoral tunnel(s) position on AP radiographs sensus on radiographs views, methods of evalu- have been proposed. The clock method, which ation and nomenclature are deemed appropriate is used intra-operatively to evaluate where to put to improve opportunity of comparison between the tunnel(s), (5) is neither a pure axial nor a co- studies. Also, it can be postulated that anatomical ronal view of the notch, it can be evaluated on 3D landmarks are useful and sufficient to locate the CT-scan or 3D MRI or even radiographically but tunnel(s) on the tibial side, while on the femoral

244 Losco et al. Tunnel position in SB and DB ACL

side anatomical landmarks are less clearly evi- anterior cruciate ligament reconstructions at our dent and have to be integrated with data obtained Institution, and whose teachings always guide us by radiographic and cadaveric measurements. in clinical practice and scientific studies.

KNOWLEDGEMENT FUNDING We would like to thank Professor Paolo Aglietti No specific funding was received for this study. (26 September 1941 † 17 April 2013), our men- tor, who was the one who conceptualized the en- TRANSPARENCY DECLARATION tire program about double-bundle and anatomic Conflict of interest: None to declare. REFERENCES 1. Hamada M, Shino K, Horibe S, Mitsuoka T, Miya- 11. Edwards A, Bull AMJ, Amis AA. The attachments ma T, Shiozaki Y, Mae T. Single- versus bi-socket of the anteromedial and posterolateral fibre bundles anterior cruciate ligament reconstruction using au- of the anterior cruciate ligament: Part 2: Femoral togenous multiple-stranded hamstring tendons with attachment. Knee Surgery Sport Traumatol Arthrosc endoButton femoral fixation: a prospective study. Ar- 2008; 16:29–36. throscopy 2001;17:801–7. 12. Jackson DW, Gasser SI. Tibial tunnel placement in 2. Cha PS, Brucker PU, West R V, Zelle BA, Yagi M, ACL reconstruction. Arthroscopy 1994; 10:124–31. Kurosaka M, Fu FH. Arthroscopic double-bundle an- 13. Amis AA, Jakob RP. Anterior cruciate ligament graft terior cruciate ligament reconstruction: an anatomic positioning, tensioning and twisting. Knee Surgery approach. Arthroscopy 2005; 21:1275e.1-8. Sport Traumatol Arthrosc 1998; 6(Suppl.1):2–12. 3. Aglietti P, Giron F, Cuomo P, Losco M, Mondanelli N. 14. Colombet P, Robinson J, Christel P, Franceschi JP, Single-and double-incision double-bundle ACL recon- Djian P, Bellier G, Sbihi A. Morphology of anterior struction. Clin Orthop Relat Res 2007; 454:108–13. cruciate ligament attachments for anatomic recon- 4. Zavras TD, Race A, Amis AA. The effect of femoral struction: a cadaveric dissection and radiographic stu- attachment location on anterior cruciate ligament re- dy. Arthroscopy 2006; 22:984–92. construction: graft tension patterns and restoration of 15. Giron F, Cuomo P, Edwards A, Bull AMJ, Amis AA, normal anterior-posterior laxity patterns. Knee Surg Aglietti P. Double-bundle “anatomic” anterior cru- Sports Traumatol Arthrosc 2005;13:92–100. ciate ligament reconstruction: a cadaveric study of 5. Giron F, Cuomo P, Aglietti P, Bull AMJ, Amis AA. tunnel positioning with a transtibial technique. Ar- Femoral attachment of the anterior cruciate liga- throscopy 2007; 23:7–13. ment. Knee Surgery Sport Traumatol Arthrosc 2006; 16. Ferretti M, Ekdahl M, Shen W, Fu FH. Osseous lan- 14:250–6. dmarks of the femoral attachment of the anterior cru- 6. Aglietti P, Giron F, Losco M, Cuomo P, Ciardullo A, ciate ligament: an anatomic study. Arthroscopy 2007; Mondanelli N. Comparison between single- and dou- 23:1218–25. ble-bundle anterior cruciate ligament reconstruction: 17. Zantop T, Wellmann M, Fu FH, Petersen W. Tunnel a prospective, randomized, single-blinded clinical tri- positioning of anteromedial and posterolateral bun- al. Am J Sports Med 2010; 38:25–34. dles in anatomic anterior cruciate ligament recon- 7. Chen H, Tie K, Qi Y, Li B, Chen B, Chen L. Antero- struction: anatomic and radiographic findings. Am J medial versus transtibial technique in single-bundle Sports Med 2008; 36:65–72. autologous hamstring ACL reconstruction: a meta- 18. Drews BH, Merz C, Huth J, Gulkin D, Guelke J, Geb- analysis of prospective randomized controlled trials. hard F, Mauch F. Magnetic resonance imaging in eva- J Orthop Surg Res 2017; 12:1–10. luation of tunnel diameters prior to revision ACL re- 8. Tiamklang T, Sumanont S, Foocharoen T, Laopaiboon construction: a comparison to computed tomography. M. Double-bundle versus single-bundle reconstructi- Skeletal Radiol 2017; 46:1361–6. on for anterior cruciate ligament rupture in adults. 19. Ducouret E, Loriaut P, Boyer P, Perozziello A, Pesqu- Cochrane Database Syst Rev 2012; 11:CD008413. er L, Mounayer C, Dallaudiere B. Tunnel positioning 9. Mascarenhas R, Cvetanovich GL, Sayegh ET, Ver- assessment after anterior cruciate ligament recon- ma NN, Cole BJ, Bush-Joseph C, Bach BRJ. Does struction at 12 months: Comparison between 3D CT double-bundle anterior cruciate ligament reconstruc- and 3D MRI. A pilot study. Orthop Traumatol Surg tion improve postoperative knee stability compared Res 2017; 103:937–42. with single-bundle techniques? A systematic review 20. Lee SR, Jang HW, Lee DW, Nam SW, Ha JK, Kim of overlapping meta-analyses. Arthroscopy 2015; JG. Evaluation of femoral tunnel positioning using 31:1185–96. 3-dimensional computed tomography and radio- 10. Osti M, Krawinkel A, Ostermann M, Hoffelner T, graphs after single bundle anterior cruciate ligament Benedetto KP. Femoral and tibial graft tunnel pa- reconstruction with modified transtibial technique. rameters after transtibial, anteromedial portal, and Clin Orthop Surg 2013; 5:188–94. outside-in single-bundle anterior cruciate ligament 21. Iriuchishima T, Ingham SJM, Tajima G, Horaguchi T, reconstruction. Am J Sports Med 2015; 43:2250–8. Saito A, Tokuhashi Y, van Houten AH, Aerts MM, Fu FH. Evaluation of the tunnel placement in the anato- mical double-bundle ACL reconstruction: A cadaver study. Knee Surgery Sport Traumatol Arthrosc 2010; 18:1226–31.

245 Medicinski Glasnik, Volume 18, Number 1, February 2021 Losco et al. Tunnel position in SB and DB ACL

22. Horie M, Muneta T, Yamazaki J, Nakamura T, Koga 30. Pietrini SD, Ziegler CG, Anderson CJ, Wijdicks CA, H, Watanabe T, Sekiya I. A modified quadrant met- Westerhaus BD, Johansen S, Engebretsen L, LaPrade hod for describing the femoral tunnel aperture po- RF. Radiographic landmarks for tunnel positioning in sitions in ACL reconstruction using two-view plain double-bundle ACL reconstructions. Knee Surgery radiographs. Knee Surgery Sport Traumatol Arthrosc Sport Traumatol Arthrosc 2011; 19:792–800. 2015; 23:981–5. 31. Doi M, Takahashi M, Abe M, Suzuki D, Nagano A. 23. Lee JK, Lee S, Seong SC, Lee MC. Anatomy of the Lateral radiographic study of the tibial sagital inser- anterior cruciate ligament insertion sites: comparison tions of the anteromedial and posterolateral bundles of plain radiography and three-dimensional computed of human anterior cruciate ligament. Knee Surgery tomographic imaging to anatomic dissection. Knee Sport Traumatol Arthrosc 2009; 17:347–51. Surgery Sport Traumatol Arthrosc 2015; 23:2297–305. 32. Cuomo P, Edwards A, Giron F, Bull AMJ, Amis AA, 24. Bernard M, Hertel P, Hornung H, Cierpinski T. Fe- Aglietti P. Validation of the 65° Howell guide for an- moral insertion of the ACL. Radiographic quadrant terior cruciate ligament reconstruction. Arthroscopy method. Am J Knee Surg 1997; 10:12–4. 2006; 22:70–5. 25. Giron F, Aglietti P, Cuomo P, Mondanelli N, Ciardullo 33. Stäubli HU, Rauschning W. Tibial attachment area of A. Anterior cruciate ligament reconstruction with do- the anterior cruciate ligament in the extended knee uble-looped semitendinosus and gracilis tendon graft position - Anatomy and cryosections in vitro com- directly fixed to cortical bone: 5-Year results. Knee plemented by magnetic resonance arthrography in Surgery Sport Traumatol Arthrosc 2005; 13:81–91. vivo. Knee Surgery Sport Traumatol Arthrosc 1994; 26. Landis JR, Koch GG. The measurement of observer 2:138–46. agreement for categorical data. Biometrics 1977; 34. Edwards A, Bull AMJ, Amis AA. The attachments 33:159–74. of the anteromedial and posterolateral fibre bun- 27. Musahl V, Burkart A, Debski RE, Van Scyoc A, Fu dles of the anterior cruciate ligament - Part 1: Tibial FH, Woo SLY. Anterior cruciate ligament tunnel pla- attachment. Knee Surgery Sport Traumatol Arthrosc cement: Comparison of insertion site anatomy with 2007; 15:1414–21. the guidelines of a computer-assisted surgical system. 35. Tashiro Y, Okazaki K, Iwamoto Y. Evaluating the Arthroscopy 2003; 19:154–60. distance between the femoral tunnel centers in anato- 28. Abreu-e-Silva GM de, Oliveira MHGCN de, mic double-bundle anterior cruciate ligament recon- Maranhão GS, Deligne L de MC, Pfeilsticker RM, struction using a computer simulation. Open Access J Novais ENV, Nunes TA, Andrade MAP de. Three-di- Sports Med 2015; 6:219–24. mensional computed tomography evaluation of ante- 36. Yamazaki J, Muneta T, Koga H, Sekiya I, Ju YJ, Mo- rior cruciate ligament footprint for anatomic single- rito T, Yagishita K. Radiographic description of femo- bundle reconstruction. Knee Surgery Sport Traumatol ral tunnel placement expressed as intercondylar clock Arthrosc 2015; 23:770–6. time in double-bundle anterior cruciate ligament re- 29. Guo L, Yang L, Wang AM, Wang XY, Dai G. Ro- construction. Knee Surgery Sport Traumatol Arthrosc entgenographic measurement study for locating fe- 2011; 19:418–23. moral insertion site of anterior cruciate ligament: A 37. Zavras TD, Amis AA. Method for visualising and cadaveric study with X-Caliper. Int Orthop 2009; measuring the position of the femoral attachment of 33:133–7. the ACL and ACL grafts in experimental work. J Bi- omech 1998; 31:387–90.

246 ORIGINAL ARTICLE

Single use instruments for total knee arthroplasty

Michele Romeo1, Giuseppe Rovere2, Leonardo Stramazzo1, Francesco Liuzza2, Luigi Meccariello3, Giulio Maccauro2, Lawrence Camarda1

1 Department of Orthopaedic Surgery (DICHIRONS), University of Palermo, Palermo, 2Department of Orthopaedics and Traumatology, Fondazione Policlinico Universitario A. Gemelli IRCCS - Università Cattolica del Sacro Cuore, Rome, 3Department of Orthopaedics and Traumatology, Vito Fazzi Hospital, Lecce; Italy

ABSTRACT

Aim Total knee arthroplasty represents a procedure that is succe- ssfully performed to relieve functional limitation and pain in ad- vanced stages of osteoarthritis. In the next 20 years the number of these procedures will be increased about four times. Patient spe- cific instrumentation (PSI) has been introduced in the past years. The aim of this study was to evaluate whether SUI are more useful in clinical, organizational and economic terms.

Methods A database search about single use instrumentation (SUI) was conducted on PubMed and Google Scholar for the pe- riod 2010-2020 using the following key “total knee replacement”, “total knee arthroplasty”, “single use instruments”, and “disposa- Corresponding author: ble instruments”. The results of the selected studies were classifi- Leonardo Stramazzo ed according to clinical, economic and organizational criteria. Department of Orthopaedic Surgery (DICHIRONS), University of Palermo Results The main advantage of SUI has been reported to reduce costs, timely turnover of operating rooms, maximizing the opera- Via del Vespro, 90100, Palermo, Italy ting room utilization and patient throughput, improving the num- Phone: +393395493581; ber of outpatient total joint replacements. No difference has been Fax: +390916554115 found other than with regard to conventional instruments in terms Email: [email protected] of clinical outcome such as hip-knee-ankle angle and other radio- Michele Romeo ORCID: https://orcid. graphic parameters, Oxford Knee Score, while a decreased infec- org/0000-0001-5289-4892 tion rate has been demonstrated. Regarding the economic aspect, a reduction of direct and indirect reduction of costs has been shown for the cost of instruments reprocessing, tray sterilization, 90-day infection rate.

Conclusion The SUI can be an alternative to conventional instru- Original submission: ments, but there are still few studies in the literature regarding cli- 28 November 2020; nical outcomes. Revised submission: Keywords: joint replacement, prosthesis, osteoarthrosis 12 December 2020; Accepted: 18 December 2020 doi: 10.17392/1321-21

Med Glas (Zenica) 2021; 18(1):247-251

247 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION ds: total knee replacement, total knee arthro- plasty, single use instruments, and disposable Total knee arthroplasty (TKA) represents a proce- instruments. The results of the selected studies dure that is successfully performed to relieve func- were classified according to clinical, economic tional limitation and pain in advanced stages of and organizational criteria. Clinical criteria were osteoarthritis (1). In 2009, 686,000 total knee pro- focused on hospital clinical, functional and ra- stheses were performed in the USA alone; a recent diological data. For the organizational data, the study predicted that in 2040 there will be a 401% operating room times have been considered. increase in the number of procedures performed (1). Usually, the instruments used for knee repla- RESULTS AND DISCUSSION cement surgery are reusable and are sterilized and packaged (1). The number of trays used for each Clinical aspects traditional individual procedure is between 6 and 8: Concerning the use of SUI, controversy still exists this could increase the possibility of contamination concerning the use of this technology improving of the surgical instruments (2). In the past years the clinical outcomes after TKA (5-7). This depends instrumentation for knee replacement surgery has on the lack of mild and long-term results available undergone innovations with the introduction of the that describe clinical outcomes, cost- effectiveness patient specific instrumentation (PSI) (1). Using and revision rates (Table 1). Abane et al. (5) eva- specific MRI of CT knee scans, cutting masks spe- luated 210 performed TKAs using different types cific to the patient’s knee anatomy are produced, of instruments: conventional (CI), patient-specific and used as cutting jigs during knee replacement cutting guides (PSI) and single use and patient- spe- surgery (1). Several advantages have been advoca- cific cutting guides (SUI) groups. The use of a SUI ted using PSI technology, such as reduced surgical in TKA provided similar results to those obtained time, no violation of the intramedullary canal, de- with traditional PSI and CI: no difference was fo- creased blood loss and decreased in the instrumen- und in terms of clinical results, operative time, tation trays optimizing the operative room time number of unit transfusion and length of hospital (3). Recently, on the basis of PSI clinical results stay (5). The mean hip-knee-ankle angle was signi- and following a progressive improvement of the ficantly lower for the SUI group when compared to manufacturing process of cutting guides, Single the CI group, indicating an overall varus alignment Use Instrumentation (SUI) has been introduced of the lower limb with the SUI instrumentation. Si- and proposed as a method to increase the sterility milar findings were observed for femoral and tibial rate of the instruments (2), reducing post-operative components positioning. In addition, patient-speci- knee infection risk. It consists of plastic-disposable fic guides both traditional and single-use (PSI and instruments that faithfully replicate metallic instru- SUI) showed significant trend towards varus place- mentation used during knee replacement surgery: ment of the tibial component (5). Attard et al. (6), in two or three sterile packs which include femoral a randomized controlled trial, allocated the patients and tibial guides, rods, and jigs (2). The main ad- into four separate groups by block stratification. vantage of this technology has been reported to re- The four instrument groups were: conventional/ duce costs, the timely turnover of operating rooms, reusable (CVR), patient-specific/reusable (PSR), maximizing the operating room utilization and pa- conventional/single-use (CVS) and patient-speci- tient throughput, improving the number of outpati- fic/single-use (PSS) instrumentation. Clinically, ent total joint replacements (4). at 6 weeks post operatively, in terms of Oxford The aim of this article was to evaluate whether Knee Score, the best result was achieved by the SUI are more useful in clinical, organizational PSR group. Meanwhile at 1-year follow up the best and economic terms. score was achieved by the CVR group: this score MATERIALS AND METHODS was significantly greater than the one reported in the PSR group. The lowest average score at 1 year A database search about single use instrumenta- was reported in the CVS group, but not statistically tion (SUI) on PubMed and Google Scholar was different if compared with the CVR group (6). A conducted to look for articles in English for the recent prospective, non-randomized multi-centre period 2010-2020 using the following key wor- clinical study (7) was conducted on 2 separate in-

248 Romeo et al. Single Use Instruments for TKA

Table 1. Evidence on the use of single use instruments (SUI) for total knee arthroplasty (TKA) compared with other instruments Author (reference Aim of the study Outcomes Economic analysis Organizational aspects number) No significant differences in terms of clinical results, operative time, number of unit transfusion and length of hospital Operative time was not reduced in Comparison between CI, Abane et al. (5) stay; both patient-specific groups when PSI and SUI Hip-knee-ankle angle was significantly compared to CI N/A lower for the SUI group compared to the CI group SUI instrument took longer to set At 6 weeks post-operatively the best OKS The cost for surgery up the operating room than the CI result was achieved by PSR group; was cheaper in instrumentation; Comparison between At 1-year follow up the best OKS was Attard et al. (6) SUI procedures CI All variables recorded after the set- CVR, PSR, CVS, SUI achieved by the CVR group and lowest (-24,6%, £ 320 vs £ up of the instruments were quicker average score was reported in the CVS 424,12) with the SUI and were significantly group shorter with SUI No significant differences between SU Using SUI than CI the OR set up Comparison between 2 and CI in most radiographic parameters; time was decreased by 30%, while Bugbee et al. (7) CI and SUI Post-operative adverse events in 5 CI other times such as surgical, OR cle- subjects and in 3 SUI subjects N/A an down, and total OR were similar Instrument set-up time and instru- The time and ment clean-up time were decreased equipment cost Comparison of rate of using SUI; Lower infection rate seen in SUI group savings from using Siegel et al. (8) surgical site infection Central supply clean-up time was (0.2%) than in CI group (3%) SUI amounted to between CI and SUI decreased by 60 minutes using SUI; between $480 and There was no change in operative $600 per case time Time parameters were significantly Comparison of time shorter with the SUI: they calculated Mont et al. (14) parameters between CI N/A N/A a potential total reduction of 17.1 and SUI minutes per case Potential economic Comparison of periope- No significant differences in terms of ma- Decrease of the operating room turn- benefit is $1198 per rative complication and jor perioperative complications including around time with SUI; Goldberg et al. (15) TKA procedure, economic differences re-admission, infection, reoperation and Reduction of the logistical burden of comparing the SUI between: CI and SUI revision loaner instrumentation with SUI to the CI CI, conventional instrument; PSI, patient-specific cutting guides; SUI, single use and patient-specific cutting guides; CVR, conventional/reusable; PSR, patient-specific/reusable; CVS, conventional/single-use; PSS, patient-specific/single-use instrumentation; OKS, Oxford Knee Score; OR, operative room strument systems, one reusable instrument and the enhanced maintenance of sterility and decreased other SUI, both designed for implanting the Attune risk of contamination when using single use in- Knee System (De Puy Synthes Joint Reconstructi- struments (8). This result is in contrast with those on, Warsaw, USA). Seventy-five subjects comple- of Goldberg et al. (9), which did not find statisti- ted the study (41 SUI/34 reusable instrument). No cally significant differences in terms of major pe- significant difference was found between SUIs and rioperative complications including re-admissi- reusable instruments (RUI) in most radiographic on, infection, reoperation and revision, between parameters (distal femoral varus-valgus, proximal the single use instruments or traditional reusable tibial varus-valgus, tibial slope, or subjects within instrument groups.

3° of target). There were six post-operative adver- se events in five reusable instrumentation subjects Organizational aspects and three post-operative adverse events in the SUI By 2026, more than a half (51%) of all total joint group (7). replacements will occur in the outpatient setting Siegel et al. (8) compared the rate of surgical site (vs 49% inpatients) (10). Several studies have infection between two groups (SUI and Reusable focused on the turnover in operating room (OR) instrument). A total of five patients in the reusa- for optimizing the time thus to increase the num- ble instrument group (3%) underwent revision ber of cases a day (10). Bert et al. (4) in their pa- surgery for infection, whereas only 1 patient in per have concluded that the ability to reduce costs, the SUI cohort (0.2%) required a revision surgery timely turnover of operating rooms to maximize (p=0.006). They concluded that the decreased in- operating room utilization and patient throughput fection rate seen in the study is most likely due to could improve the number of outpatient total joint

249 Medicinski Glasnik, Volume 18, Number 1, February 2021

replacements. Cendan and Good (11) concluded sult of the smaller number of trays. The time sa- that with a reduction of turnover time from 15 to vings for rewrapping trays was shorter, for each 20 minutes 3 or 4 times a day, 1 more surgery could instrument case, the saving was between $75 and be performed. On the other hand, Dexter et al. (12) $330. On the other hand, the cost of disposable have highlighted that reducing the turnover time is cutting blocks is higher than conventional instru- generally only important when multiple operations ments. In addition, it could be necessary to open of short duration are anticipated. Furthermore, an multiple sets of instruments, for example in case excessive number of tray instruments may also of an intraoperative femoral sizing change (2). cause operative delays by the surgical technician Attard et al. (6) in their analysis found that the spending extra time setting up the instruments, fin- cost for surgery was cheaper in SUI procedu- ding the correct instrument on a cluttered tray, or res than in conventional/reusable procedures handing the surgeon an incorrect instrument due to (-24.6%; £ 320 vs £ 424.12). clutter (13). However, Attard et al. (6) found that A Goldberg study (15) was focused to investigate the SUI, used in conventional procedures, took lon- a range of potential costs savings for TKA pro- ger to set up the operating room than the conven- cedures performed with single use instruments. tional reusable instrumentation. Conversely, all va- Four variables related to TKA costs and logistics riables which were recorded after the set-up of the were considered in this study: turnover time, tray instruments were quicker with the SUI and were sterilization, tray management time, and 90-day statistically significantly shorter when the SUI was infection rates. They simulated 200 sites: in 95% used (6). Similarly, in their study Mont et al. (14) of cases, at least $500 per case and in 48% of observed that time parameters were significantly cases at least $1000 were saved (15). shorter with the SUI when compared to the con- ventional instrumentation for most of the operating Siegel et al. (8) have calculated a saving per room parameters evaluated (navigated and non-na- surgery, with single use instrumentation, when vigated cases). In the best scenario, if single use compared with traditional use in this way: OR instruments were used, they calculated a potential man-hours decreased by $55.50, central supply total reduction of 17.1 minutes per case: 9-minute man-hours decreased by $50.36, supply and ste- savings in instrument set-up time, a 1.2-minute sa- rile rewrapping costs decreased by $375.00 per vings in procedure time, and a 6.9-minute savings 5 traditional trays. The cost of the SUI set was in instrument clean-up time (14). quoted at $490. They concluded stating that the time and equipment cost savings from using sin- A similar result was found by another study (8): gle-use equipment for total knee arthroplasties in a single-use cohort, instrument set-up time was amounted to between $480 and $600 per case (8). decreased by 15 minutes and instrument clean-up time was decreased by 14 minutes (p<0.05). The The incidence of periprosthetic infections is aro- central supply clean-up time was decreased by 60 und 1-2%, but considering the increase in the minutes (p<0.05). There was no change in opera- number of cases per year, the number of revision tive time. Similar results were found by Bungbee surgeries is expected to grow (16). et al. (7): using the SUIs rather than reusable in- Periprosthetic infections remain a challenge as struments, the OR set up time was decreased by well as a problem in prosthetic surgery. A study 30%, while other times such as surgical, OR cle- showed that the cost of a periprosthetic infection an down, and total OR were similar. procedure is roughly $ 116,000, which is approxi- mately 5 times higher than the cost of a primary Economic analysis implant (17). Surgical site infections (SSIs) are An economic analysis has been studied by many the most common (25.2%) indication for revision authors (2,6,8,15). The main question about the total knee arthroplasty (TKA) and it is the most single use instruments is if there is a direct and common reason for revision surgery such as arthro- indirect reduction of costs of a case. tomy and prosthesis components removal (79.1%) (18). The SSIs impose a higher cost for the prolon- In a paper made by Bonutti et al. (2), the cost ged hospital stay or hospital readmission. A study of instruments reprocessing was estimated to be observed a correlation between SSIs and post-ste- lower by between $140 and $220 per set, as a re- rilization contamination of sets containing surgi-

250 Romeo et al. Single Use Instruments for TKA

cal instruments (19). Further, it was observed that times. On the other hand, there are still few studies the current method of checking and maintaining in the literature regarding clinical outcomes. sterility in the OR is inadequate (20). In addition, a study (21) has shown that even a wrap defect FUNDING of 1.1 mm could allow bacterial contamination. No specific funding was received for this study. In conclusion, single use instruments can be an al- ternative to conventional instruments. Many studi- TRANSPARENCY DECLARATION es agree in stating that there is an economic saving Conflict of interest: None to declare. and a reduction in the operating room turn over

REFERENCES 1. Singh JA, Yu S, Chen L, Cleveland JD. Rates of total 12. Dexter F, Abouleish AE, Epstein RH, Whitten CW, joint replacement in the united states: future projecti- Lubarsky DA. Use of operating room information ons to 2020-2040 using the National Inpatient Sam- system data to predict the impact of reducing tur- ple. J Rheumatol 2019; 46:1134-40. nover times on staffing costs. Anesth Analg 2003; 2. Bonutti P, Zywiel M, Johnson A, Mont M. The use of 97:1119-26. disposable cutting blocks and trials for primary total 13. Cichos KH, Hyde ZB, Mabry SE, Ghanem ES, knee arthroplasty. Tecn Knee Surg 2010; 9:249–55. Brabston EW, Hayes LW, McGwin G Jr, Ponce 3. Camarda L, D'Arienzo A, Morello S, Peri G, Valenti- BA. Optimization of orthopedic surgical instrument no B, D'Arienzo M. Patient-specific instrumentation trays: lean principles to reduce fixed operating room for total knee arthroplasty: a literature review. Mus- expenses. J Arthroplasty 2019; 34:2834-40. culoskelet Surg 2015; 99:11-8. 14. Mont MA, McElroy MJ, Johnson AJ, Pivec R. Sin- 4. Bert JM, Hooper J, Moen S. Outpatient total joint gle-Use Multicenter Trial Group Writing Group. arthroplasty. Curr Rev Musculoskelet Med 2017; Single-use instruments, cutting blocks, and trials 10:567–74. increase efficiency in the operating room during to- 5. Abane L Zaoui A, Anract P, Lefevre N, Herman S, tal knee arthroplasty: a prospective comparison of Hamadouche M. Can a single-use and patient-speci- navigated and non-navigated cases. J Arthroplasty fic instrumentation be reliably used in primary total 2013; 28:1135–40. knee arthroplasty? A multicenter controlled study. J 15. Goldberg TD, Maltry JA, Ahuja M, Inzana JA. Lo- Arthroplasty 2018; 33:2111-8. gistical and Economic Advantages of sterile-packed, 6. Attard A, Tawy GF, Simons M, Riches P, Rowe P, single-use instruments for total knee arthroplasty. J Biant LC. Health costs and efficiencies of patient- Arthroplasty 2019; 34:1876–83. specific and single-use instrumentation in total knee 16. Haddad FS, Ngu A, Negus JJ. Prosthetic joint infec- arthroplasty: a randomised controlled trial. BMJ tions and cost analysis? Adv Exp Med Biol 2017; Open Qual 2019: 8:e000493. 971:93-100. 7. Bugbee WD, Kolessar DJ, Davidson JS, Gibbon AJ, 17. Kapadia BH, McElroy MJ, Issa K, Johnson AJ, Bo- Lesko JP, Cosgrove KD. Single use instruments for zic KJ, Mont MA. The economic impact of peripro- implanting a contemporary total knee arthroplasty sthetic infections following total knee arthroplasty system are accurate, efficient, and safe. J Arthropla- at a specialized tertiary-care center. J Arthroplasty sty 2020; S0883-5403(20)30782-8. 2014; 29:929–32. 8. Siegel GW, Patel NN, Milshteyn MA, Buzas D, 18. Patel H, Khoury H, Girgenti D, Welner S, Yu H. Lombardo DJ, Morawa LG. Cost analysis and sur- Burden of surgical site infections associated with gical site infection rates in total knee arthroplasty arthroplasty and the contribution of Staphylococcus comparing traditional vs. single-use instrumentati- aureus. Surg Infect 2016; 17:78–88. on. J Arthroplasty 2015; 30:2271-4. 19. Dancer SJ, Stewart M, Coulombe C, Gregori A, Vir- 9. Goldberg T, Seaveyet R, Kuse K, Domyahn M, di M. Surgical site infections linked to contaminated Torres A. Value in single use instruments for total surgical instruments. J Hosp Infect 2012; 81:231–8. knee arthroplasty: patient outcomes and operating 20. Mobley KS, Jackson JB 3rd. A prospective analysis room efficiency. White paper; 2017. of clinical detection of defective wrapping by opera- 10. DeCook CA. Outpatient joint arthroplasty: transitio- ting room staff. Am J Infect Control 2018; 46:837–9. ning to the Ambulatory Surgery Center. J Arthropla- 21. Waked WR, Simpson AK, Miller CP, Magit DP, Gra- sty 2019; 34:S48-50. uer JN. Sterilization wrap inspections do not adequ- 11. Cendan JC, Good M. Interdisciplinary work flow ately evaluate instrument sterility. Clin Orthop Relat assessment and redesign decreases operating room Res 2007; 462. turnover time and allows for additional caseload Arch Surg 2006; 141:65e9.

251 ORIGINAL ARTICLE

Medial pivot vs posterior stabilized total knee arthroplasty designs: a gait analysis study

Nicola Bianchi1,2, Andrea Facchini3,4, Nicola Mondanelli3,4, Federico Sacchetti1,2, Roberta Ghezzi3,4, Marco Gesi5, Rodolfo Capanna1,2, Stefano Giannotti3,4

1Department of Orthopedic and Trauma Surgery, University of Pisa, Pisa, Italy; 2Department of Orthopedic and Trauma Surgery, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy; 3Section of Orthopedics and Traumatology, Department of Medicine Surgery and Neurosci- ences, University of Siena, Siena, Italy; 4Section of Orthopedics and Traumatology, Azienda Ospedaliera Universitaria Senese, Siena, Italy; 5Center for Rehabilitative Medicine “Sport and Anatomy”, University of Pisa, Pisa

ABSTRACT

Aim To compare a medial pivot (MP) total knee arthroplasty (TKA) with posterior stabilized (PS) TKA designs from a subjec- tive, clinical and biomechanical point of view, in a single-centre, single-surgeon, case-control non-randomized trial.

Methods Sixteen patients were randomly picked up from case se- ries into each group. Subjective outcome was assessed using the Forgotten Joint Score Questionnaire (FJSQ). Clinical evaluation included range of motion (ROM). All patients underwent gait analysis by a treadmill with force-measuring plaques and video- recording device; data were recorded for 30 seconds and included Corresponding author: cadence, step length, stance time and walking speed. A blinded Nicola Mondanelli qualitative analysis of the pattern of gait was defined as biphasic or Section of Orthopaedics. Department of non-biphasic. Descriptive statistics for the continuous study varia- Medicine, Surgery and Neurosciences, bles and statistical significance were calculated for all parameters with independent-samples t-test and χ2 test to analyse difference in University of Siena pattern of gait between groups. Viale Mario Bracci 16, 53100 Siena, Italy Phone: +39 0577 585675; Results Mean FJSQ in the MP group was 91.87 (CI 95%: 88.12- fax +39 0577 233400; 95.46) and 75.31 (CI 95%: 67.97-81.56) in the PS group (p=0.029). Mean post-operative ROM was 117° (CI 95%: 113°-122°) in the E-mail: [email protected] MP group and 112° (CI 95%: 108°-117°) in the PS group (p=0.14). Nicola Bianchi ORCID ID: 0000-0002- No statistical difference was found between groups regarding all 1313-8493 gait analysis parameters which have been recorded.

Conclusion MP TKA design showed better subjective results using the FJSQ, but it did not improve significantly clinical and Original submission: functional outcomes compared to PS TKA design, at a short-term follow-up. 09 November 2020; Revised submission: Key words: forgotten joint, mid-flexion instability, PROMs, su- 16 November 2020; bjective results, TKA Accepted: 23 November 2020 doi: 10.17392/1312-21

Med Glas (Zenica) 2021; 18(1):252-259

252 Bianchi et al. Gait analysis in MP vs PS TKA

INTRODUCTION out a control group (17,19–21,23). In literature there are few direct comparisons between MP and Total knee arthroplasty (TKA) is one of the most other designs (24,25). These reports showed some performed surgical procedures in orthopaedics. It is advantages of the MP design as a better range of the most effective operative treatment for end-stage motion (ROM) or better results at patient-reported knee osteoarthritis (OA), and it has been designed outcomes measurements (PROMs), but a recent to allow patients to regain an acceptable function of paper by Benjamin et al. showed no difference in the operated knee while treating pain-related symp- the in vivo kinematics and clinical results between toms (1,2). In France, more than 100.000 TKAs MP and PS TKAs (26). were implanted during 2017 only (3), and some authors predict that by 2030, the number of pri- The aim of this study was to investigate if in our mary TKA will raise by 600% (4). However, up to hands a MP design could lead to better in vivo 25% of the patients report unsatisfactory subjective kinematics and clinical outcomes compared to a outcomes after TKA, mostly a subset of younger PS design at short-term FU. population who are not satisfied with the inability PATIENTS AND METHODS to perform high level activities after surgery (5,6). Since 1974, when the total condylar knee pro- Patients and study design sthesis was firstly used, several prosthetic desi- gns have been introduced, among them the poste- This retrospective case-control, single-centre, sin- rior stabilized (PS) design, the cruciate retaining gle-surgeon, double-blinded, non-randomised trial (CR) design and the medial pivot (MP) design compared 16 patients who underwent TKA with a introduced in 1994. In the United States, in 2016, MP implant (MP group) (Evolution Medial Pivot; approximately 50% of TKAs were PS and 42% Microport, Shangai, CHN) and 16 patients opera- were CR in design (7). ted on of TKA with a PS (PS group) (Persona-PS; Zimmer, Warsaw, IN, USA) prosthesis. Patients One possible explanation of the unsatisfactory were picked up casually in both groups from the reports after TKAs might be found in the altered database of the Azienda Ospedaliera Universitaria biomechanics. The TKAs do not reproduce physi- Pisana and the Azienda Ospedaliera Universitaria ological knee biomechanics, in particular with PS Senese during the period 2015 – 2019 matching and CR designs: several studies have demonstrat- them for age and gender; minimum FU was set at ed paradoxical anterior movement of the femur in one year post-operatively for both groups. Seve- respect of the tibial plateau from 5° of extension rity of OA or type of deformity were not matched, to 90° of flexion with a phenomenon called mid- but all surgeries were performed by a single surge- flexion instability (7–11). Normal knee kinematics on (SG) for primary knee OA. Exclusion criteria studies have demonstrated that the medial condyle were rheumatoid arthritis and post-traumatic OA, has minimal to no rollback while the lateral con- comorbidities such as cognitive impairment and/ dyle can show more rollback movements (12–16). or neurological deficits that could alter gait, and a The rationale of the MP design is to better re- pre-existing contralateral TKA. produce the normal knee kinematics. The design is characterized by a ball and socket geometry, The two groups were comparable regarding a high congruence in the medial side between the age, gender, pre-operative alignment on the condyle and tibial insert, and the morphology of frontal plane and ROM (Table 1), and FU period the tibial insert which prevents from paradoxi- (mean 25.4 months and 23.4 months for the MP cal anterior translation of the femur on the tibial and PS group, respectively). Both patients and plateau. The MP design seems not to increase the researchers who performed gait analysis were risk of post-operative complications such as asep- blinded to the design of the implant. tic loosening, and it showed similar survival rates All patients gave their written consent to the tre- compared to PS and CR designs also at long-term atment and anonymous use of data and images follow up (FU) (17–22). In vitro, biomechanics of for research and academic purposes. At our In- MP prostheses have been proven to be similar to stitutions, no Ethical Committee nor Institutional that of the native knee joint. Some authors have re- Review Board approval are needed for retrospec- ported good clinical outcomes of MP TKAs with- tive studies.

253 Medicinski Glasnik, Volume 18, Number 1, February 2021

Methods lary alignment aiming to with an anatomical valgus angle between 5° and 7° and an external A medial parapatellar approach was used in all rotation of the femoral component of 3° for varus cases; all implants were cruciate-sacrificing, and knees and 5° for valgus knees, using a posterior the patella was not resurfaced. After joint expo- condylar referenced cutting jig. Tibial cuts were sure, the femur was prepared using intramedul- made using an extramedullary guide perpendicu- lar to the long axis of the tibia with a posterior slope of between 0° and 3°. After osteophytes re- moval, soft-tissue balancing in the frontal plane and flexion-extension gaps were assessed. All components were cemented. One drainage was then inserted and left in place for a maximum of two days post-operatively. On the first post-operative day, early passive ROM exercises began, and isometric contractions of the quadriceps were also advised. Weight-bearing and short walks were encouraged by the second post- operative day. By the fifth post-operative day, pa- tients were expected to walk with crutches, climb stairs and have a 90° of flexion of the knee. Pati- ents were evaluated at the outpatient clinic at 1, 2, 6 and 12 months post-operatively, and then annu- ally, with clinical and radiological FU. Subjective outcome was assessed using the For- gotten Joint Score Questionnaire (FJSQ) (27) at every FU. Pre- and post-operative clinical evalua- tion including ROM were recorded by an investi- gator blinded to the implant using a goniometer. Figure 1. Patient during gait analysis on Walker View 3.0 (Gesi All patients underwent gait analysis by a tread- M, 2019) mill with force measuring plaques and video-

Figure 2. Knee range of motion (ROM) during 30’ gait on the Walker view (gait analysis from second 15 to 30 is presented). A) Pa- tient with right total knee arthroplasty (TKA) presenting a biphasic pattern of gait; B) Patient with left TKA presenting a non-biphasic gait. Time (s) in the horizontal axis and knee flexion degrees (°) in the vertical axis. Red is left knee ROM, Green in right knee ROM

254 Bianchi et al. Gait analysis in MP vs PS TKA

recording device (Walker View 3.0, Tecnobody, As for gait analysis results, step length was 25.2 Dalmine, I) (Figure 1) at 2-years FU. Patients (CI95%: 20.7–31.7) cm in the MP group and 21.1 were told to walk at a comfortable speed. Data (CI95%: 19.2–22.7) cm in the PS group (p=0.26). about gait were taken for 30 seconds and inclu- Mean cadence was 0.68 (CI95%: 0.61–0.76) ded cadence, step length, stance time and wal- cycles/s and 0.62 (CI95%: 0.57–0.66) cycles/s king speed. A qualitative analysis of the pattern in the MP and PS group, respectively (p=0.11). of gait was also performed and we defined it as Mean stance time was 1.2 (CI95%: 1.0–1.2) s biphasic or non-biphasic (Figures 2A and 2B). in the MP group and 1.2 (CI95%: 1.1–1.3) s in All gait analyses were recorded by a specialized the PS group (p = 0.19). Walking speed was 1.24 investigator blinded to the implant design. m/s in the MP group and 1 m/s in the PS group (p=0.24). A biphasic pattern of gait was detected Statistical analysis in 6 out of 16 patients in the MP group and in 4 Descriptive statistics for the continuous study out of 16 patients in the control group; a ꭓ2 test variables was used. Statistical significance was did not detect any significant difference (p=0.58) calculated for all parameters with independent- (Table 2). samples t-test calculation and a χ2 test to analyse Table 2. Subjective, clinical and gait analysis results of two difference in pattern of gait between groups. Sta- groups of patients tistical significance was set as p<0.05, the con- Variable MP group PS group 91.87 75.31 FJSQ (points) fidence interval (CI) was set at 95%. (88.12 – 95.46) (67.97 – 81.56) Post-operative ROM (°) 118 112 RESULTS (mean; CI 95%) (113 – 122) (107 – 117) In the MP group there were eight males and eight Walking speed (m/s) 1.24 1.00 females, with a mean age of 72 (CI95%: 68 – Walking Cadence (cycle/s) 0.68 0.62 76) years, while in the PS group there were nine (mean; CI 95%) (0.61 – 0.76) (0.57 – 0.66) Step Length (cm) 25.2 21.1 males and seven females with a mean age of 71 (mean; CI 95%) (20.7 – 31.7) (19.2 – 22.7) (CI95%: 69 – 74) years. Pre-operative ROM was Stance Time (s) 1.2 1.2 105° (CI95%: 96° – 107°) and 106° (CI95%: 95° (mean; CI 95%) (1.0 – 1.2) (1.1 – 1.3) Biphasic Gait Pattern (No) YES: 6; YES: 4; – 109°) for the MP and PS groups, respectively. (YES/NO) NO: 10 NO: 12 Mean pre-operative mechanical axis was 4° of P, medial pivot; PS, posterior stabilized; FJSQ, Forgotten Joint Score varus (CI95%: 12° varus – 13° valgus) in the MP Questionnaire group and 6° of varus (CI95%: 13° varus – 4° DISCUSSION valgus) in the PS group (Table 1). During knee ROM, from 0° to 110° of flexion, the Table 1. Characteristics of two groups of patients medial condyle does not make any antero-poste- Characteristic MP group PS group rior translation while the lateral condyle usually Gender (No) 8 males / 8 females 9 males / 7 females translates 0–15 mm posteriorly (28). Stability Age (years) 72 (68 – 76) 71 (69 – 74) of the medial compartment derives from bone/ (mean; CI 95%) Pre-operative ROM (°) cartilage congruence between the medial femo- 105 (96 – 107) 106 (95 – 109) (mean; CI 95%) ral condyle and the medial tibial plateau: during Pre-operative 4 varus 6 varus ROM no rollback is observed; furthermore, the mechanical axis (°) (12 varus - 13 valgus) (13 varus – 4 valgus) (mean; CI 95%) medial meniscus has little motility and adds sta- MP, medial pivot; PS, posterior stabilized; ROM, range of motion bility to the compartment. For that reason, the Mean FJQS was 91.87 (CI95%: 88.12 – 95.46) in medial compartment of the knee has been defined the MP group and 75.31 (CI95%: 67.97 – 81.56) as a ball-in-socket articulation (16). Paradoxical in the PS group; with a two-tailed, independent anteposition of the femoral condyle to the tibial samples t-test the difference was statistically sig- plateau in a ROM between 0° and 90° of flexion nificant (p=0.029). Mean post-operative ROM has been reported after implantation of a CR-de- was 117° (CI95%: 113°–122°) and 112° (CI95%: sign TKA; such an altered kinematics was linked 108°–117°) in the MP and PS groups, respective- to a phenomenon called ‘mid-flexion instability’ ly (p=0.14). that was associated with sub-optimal outcomes following implantation of a CR implants. Also,

255 Medicinski Glasnik, Volume 18, Number 1, February 2021

the subgroup of patients with CR prostheses re- higher revision rates following implantation of ferred to have a sensation of a non-native knee a MP TKA compared to PS TKA for aseptic lo- joint after surgery, and this was related also to the osening or anterior knee pain (19). The present mid-flexion instability (29). study showed similar results compared to the li- The PS and MP design prosthesis have been terature regarding ROM improvements (36,37). developed to give a more stable knee motion In particular, no significant differences between during the full ROM. To reach that goal, these the post-operative ROM of MP or PS TKAs were designs were planned to eliminate the femoral found; however, a positive trend in favour of MP anteposition towards the tibial plateau (30). The TKA has been detected. Shakespeare et al. have PS design was set to give stability preventing the reported a mean post-operative ROM of 111° in anteposition of the femur by the contact of the the MP group compared to 109° in the PS gro- femoral cam with the post of the tibial insert. Se- up at 1-year FU (36). Another randomized trial veral authors have reported the mechanism to be reported higher post-operative ROM at 1- and effective in a ROM from 0° to more than 90° of 2-years FUs in the MP group compared to the PS flexion (30,31). However, when the knee passes group (24). Samy et al. showed a higher ROM in 90°of flexion, also PS TKAs could reproduce pa- the MP group compared to the PS group (122° vs radoxical movements (11). On the other hand, the 116°); however, again, that result was not statisti- highly congruent design of the medial compar- cally significant (25). On the other hand, another tment in MP TKAs has been developed to ma- paper reported worse outcomes in post-operative intain maximal stability during the entire ROM ROM in MP TKA compared to a mobile bearing (10). Thanks to the congruence between femoral design (38). component and the medial part of the polyethyle- The most relevant result of this study is statisti- ne insert, with a more pronounced anterior and cally significant better results in subjective out- posterior borders, femoral anteposition should be comes as measured by PROMs. In the last 20 avoided during the entire knee ROM. On the la- years of practice, patients’ expectations follow- teral side, a less pronounced congruence between ing a TKA have changed enormously: nowadays the femoral component and the tibial insert gives patients want to regain high levels of functional- the opportunity to have rollbacks and to better ity after TKA. In the presented study, to assess reproduce the native knee joint kinematics (31). the clinical outcomes, the authors preferred to The aim of the presented study was to compa- use the FJSQ, that has been tested in various re- re subjective, clinical and functional outcomes ports (25,39–43). Many authors preferred to as- of patients that underwent the implantation of a sess subjective outcomes using other scores, for MP TKA compared to a group of patients that example the WOMAC (44), the SF-36 (45), Knee underwent implantation of a PS TKA. Seve- Society Score (KSS) (46) and the Oxford Knee ral papers have reported favourable outcomes Score (47). Hossain et al. showed better clinical after implantation of a MP TKA, with satis- outcomes in MP patients group compared to PS factory results at a medium- and long-term FU using SF-36 score (24). Samy et al. detected a (18,19,21,32–34). Fan et al. showed significant significantly higher FJSQ in patients that under- improvements of ROM and scores to assess pa- went implantation of a MP TKA compared to a in-related symptoms at 5-years FU (35). Bordini control group that underwent implantation of a et al. reported good clinical outcomes following PS TKA (25). On the other hand, several authors the implantation of a MP TKA with a 96% sur- did not find any differences in the subjective vival to any failure of the implant at 5-years FU and clinical outcomes between the patients from (34). These authors hypothesized that the high MP or PS groups (36,37). In particular, Bae et congruence of the medial compartment could al. did not find any differences in the outcomes lead to less polyethylene wear and consequently detected by WOMAC and KSS. In the presented to lower rates of failure due to the subsequent study, a statistically significantly better FJSQ ‘debris’ osteolysis and aseptic mobilization. was detected in the MP group compared to the However, the Australian Orthopaedic Associati- PS group. Furthermore, an interesting clue was on National Joint Replacement Registry detected that the question with the most different reports

256 Bianchi et al. Gait analysis in MP vs PS TKA

between MP and PS group was the following: In the current study, no statistical difference was “Do you feel to have a TKA with a sensation found between groups regarding all gait analysis of an artificial knee when you stand up from a parameters which have been recorded. Also, Be- chair?”. During daily activities, the full load on njamin et al. did not show any differences in gait the patient knee acts not only in extension but analysis of spatial-temporal parameters between also when the knee is flexed (48). To stand up two groups of patients treated by TKA with PS from a chair, a high level of mid-flexion stability or MP design (26). As for the gait pattern (bipha- is required. Mid-flexion instability is defined as a sic or altered), we did not detect any differences dynamic antero-posterior instability of the knee between groups being 4 patients in the PS group during the motion between 0° and 90° of flex- and 6 in the MP group that showed a biphasic ion (9). When the knee is fixed at 0° or 90°, the pattern of gait (similar to a native joint). These mid-flexion instability cannot be detected; this results are in line with a report by Wilson et al; instability is not linked to a varus-valgus laxity they found that only 25% of patients treated with of the knee (9). Mid-flexion instability has been a PS TKA had regained a biphasic pattern of gait recognized as a major cause of revision. Several at 4-years FU (56). reports have demonstrated that the mechanism One of the major limitations of the current stu- underlying mid-flexion instability could be an el- dy is the lack of a pre-operative gait analysis. It evation of more than 4 mm of the articular joint would have been useful to compare pre-operative line conjoined with the anterior shift of the femo- gait analysis between the two groups. Another li- ral component to the tibial plateau (49–51). In a mitation is the length of the FU, with a collection review by Ramappa, it was highlighted that the of data at a short term. This could be an important mid-flexion instability was linked to a high joint element since the expected gain in function and line, to a multi-ray TKA design and to a laxity of ROM in MP design TKA could be reached at 2 the medial collateral ligament (52). The results of years post-operatively. the presented study, in particular the perception In conclusions, the results showed that patients of stability in the mid-flexion activities such as who underwent the implantation of a MP TKA standing up from a chair in the patients of MP showed better subjective results compared to group, are in favour of the MP design. patients who received a PS TKA. In particular, In gait analysis, several spatial-temporal parame- patients who received a MP design TKA reported ters can be used to study the ROM and the force a better mid-flexion stability and a better percep- applied on the inferior limb joints (53,54). When tion about the prosthesis towards a sensation of a the step length and walking speed increase, the more ‘natural’ knee joint. As for clinical and ki- knee ROM increases consequently, and when the nematics results, the MP design did not show any walking cadence increases, also the force peak in improvements in ROM and gait analysis compa- stride increases and consequently the knee joint red to the PS design at a short-term FU. momentum (54). A decrease in the length of stri- de is usually associated to a post-operative adap- FUNDING tive strategy to decrease an excessive loading No specific funding was received for this study. on the joint and it is associated with worse post- operative outcomes after TKA implantation (55). TRANSPARENCY DECLARATION Conflict of interest: None to declare. REFERENCES 1. Bourne RB, Chesworth BM, Davis AM, Mahomed 3. Vaillant T, Steelandt J, Cordonnier AL, Haghighat S, NN, Charron KDJ. Patient satisfaction after total knee Anract P, Paubel P, Duhamel C. Review of patient- arthroplasty: Who is satisfied and who is not? Clin specific instrumentation for total knee prosthesis. Ann Orthop Relat Res 2010; 468:57–63. Pharm Fr 2018; 76:228–34. 2. Hamilton DF, Howie CR, Burnett R, Simpson AHRW, 4. Kurtz S, Ong K, Lau E, Mowat F, Halpern M. Projec- Patton JT. Dealing with the predicted increase in de- tions of primary and revision hip and knee arthropla- mand for revision total knee arthroplasty: Challenges, sty in the United States from 2005 to 2030. J Bone risks and opportunities. Bone Jt J 2015; 97-B:723–8. Joint Surg Am 2007; 89:780–5.

257 Medicinski Glasnik, Volume 18, Number 1, February 2021

5. Tolk JJ, van der Steen MC, Janssen RPA, Reijman M. 22. Amin A, Al-Taiar A, Sanghrajka AP, Kang N, Scott Total knee arthroplasty: what to expect? A survey of G. The early radiological follow-up of a medial rota- the members of the Dutch Knee Society on long-term tional design of total knee arthroplasty. Knee 2008; recovery after total knee arthroplasty. J Knee Surg 15:222–6. 2017; 30:612–6. 23. Sabatini L, Risitano S, Parisi G, Tosto F, Indelli PF, 6. Noble PC, Gordon MJ, Weiss JM, Reddix RN, Con- Atzori F, Massè A. Medial pivot in total knee arthro- ditt MA, Mathis KB. Does total knee replacement plasty: literature review and our first experience. Clin restore normal knee function? Clin Orthop Relat Res Med Insights Arthritis Musculoskelet Disord 2018; 2005; (431):157–65. 11:1–4. 7. Vaishya R, Agarwal AK, Vijay V. Extensor mecha- 24. Hossain F, Patel S, Rhee S-J, Haddad FS. Knee ar- nism disruption after total knee arthroplasty: a case throplasty with a medially conforming ball-and-soc- series and review of literature. Cureus 2016; 8:e479. ket tibiofemoral articulation provides better function. 8. Komistek RD, Dennis DA, Mahfouz M. In vivo flu- Clin Orthop Relat Res 2011; 469:55–63. oroscopic analysis of the normal human knee. Clin 25. Samy DA, Wolfstadt JI, Vaidee I, Backstein DJ. A Orthop Relat Res 2003; (410):69–81. retrospective comparison of a medial pivot and poste- 9. Vince K. Mid-flexion instability after total knee- ar rior-stabilized total knee arthroplasty with respect to throplasty: woolly thinking or a real concern? Bone patient-reported and radiographic outcomes. J Arthro- Joint J 2016; 98-B(1 Suppl A):84–8. plasty 2018; 33:1379–83. 10. Schmidt R, Komistek RD, Blaha JD, Penenberg BL, 26. Benjamin B, Pietrzak JRT, Tahmassebi J, Haddad FS. Maloney WJ. Fluoroscopic analyses of cruciate-re- A functional comparison of medial pivot and condylar taining and medial pivot knee implants. Clin Orthop knee designs based on patient outcomes and para- Relat Res 2003; (410):139–47. meters of gait. Bone Joint J 2018; 100-B(1 Suppl 11. Dennis DA, Komistek RD, Mahfouz MR, Haas BD, A):76–82. Stiehl JB. Multicenter determination of in vivo kine- 27. Behrend H, Giesinger K, Giesinger JM, Kuster MS. matics after total knee arthroplasty. Clin Orthop Relat The “forgotten joint” as the ultimate goal in joint Res 2003; (416):37–57. arthroplasty: validation of a new patient-reported 12. Blaha JD, Mancinelli CA, Simons WH, Kish VL, outcome measure. J Arthroplasty 2012; 27:430-6. Thyagarajan G. Kinematics of the human knee using 28. Moonot P, Mu S, Railton GT, Field RE, Banks SA. an open chain cadaver model. Clin Orthop Relat Res Tibiofemoral kinematic analysis of knee flexion for 2003; (410):25–34. a medial pivot knee. Knee Surg Sports Traumatol Ar- 13. Freeman MAR, Pinskerova V. The movement of the throsc 2009; 17:927–34. knee studied by magnetic resonance imaging. Clin 29. Varadarajan KMM, Zumbrunn T, Rubash HE, Orthop Relat Res 2003; (410):35–43. Malchau H, Li G, Muratoglu OK. Cruciate retaining 14. Pinskerova V, Johal P, Nakagawa S, Sosna A, implant with biomimetic articular surface to reprodu- Williams A, Gedroyc W, Freeman MAR. Does the ce activity dependent kinematics of the normal knee. femur roll-back with flexion? J Bone Joint Surg Br J Arthroplasty 2015; 30):2149-53.e2. 2004; 86:925–31. 30. Blaha JD. The rationale for a total knee implant that 15. van Duren BH, Pandit H, Beard DJ, Zavatsky AB, confers anteroposterior stability throughout range of Gallagher JA, Thomas NP, Shakespeare DT, Murray motion. J Arthroplasty 2004; 19(4 Suppl 1):22–6. DW, Gill HS. How effective are added constraints 31. Wang H, Simpson KJ, Chamnongkich S, Kinsey T, in improving TKR kinematics? J Biomech 2007; Mahoney OM. A biomechanical comparison between 40(Suppl 1):S31–7. the single-axis and multi-axis total knee arthroplasty 16. Freeman MAR, Pinskerova V. The movement of the systems for the stand-to-sit movement. Clin Biomech normal tibio-femoral joint. J Biomech 2005; 38:197– (Bristol, Avon) 2005; 20:428–33. 208. 32. Barnes CL, Lincoln D, Wilson B, Bushmaier M. Knee 17. Mannan K, Scott G. The Medial Rotation total knee manipulation after total knee arthroplasty: compari- replacement: A clinical and radiological review at a son of two implant designs. J Surg Orthop Adv 2013; mean followup of six years. J Bone Jt Surg - Ser B 22:157–9. 2009; 91:750–6. 33. Chinzei N, Ishida K, Tsumura N, Matsumoto T, Ki- 18. Fitch DA, Sedacki K, Yang Y. Mid- to long-term tagawa A, Iguchi T, Nishida K, Akisue T, Kuroda outcomes of a medial-pivot system for primary total R, Kurosaka M. Satisfactory results at 8 years mean knee replacement: a systematic review and meta- follow-up after ADVANCE® medial-pivot total knee analysis. Bone Joint Res 2014; 3):297–304. arthroplasty. Knee 2014; 21:387–90. 19. Brinkman JM, Bubra PS, Walker P, Walsh WR, Bruce 34. Bordini B, Ancarani C, Fitch DA. Long-term survi- WJM. Midterm results using a medial pivot total knee vorship of a medial-pivot total knee system compared replacement compared with the Australian National with other cemented designs in an arthroplasty regi- Joint Replacement Registry data. ANZ J Surg 2014; stry. J Orthop Surg Res 2016; 11:44. 84:172–6. 35. Fan CY, Hsieh JTS, Hsieh MS, Shih YC, Lee CH. Pri- 20. Macheras GA, Galanakos SP, Lepetsos P, Anastaso- mitive results after medial-pivot knee arthroplasties. poulos PP, Papadakis SA. A long term clinical outco- A minimum 5-year follow-up study. J Arthroplasty me of the Medial Pivot Knee Arthroplasty System. 2010; 25:492–6. Knee 2017; 24:447–53. 36. Shakespeare D, Ledger M, Kinzel V. Flexion after 21. Karachalios T, Roidis N, Giotikas D, Bargiotas K, Va- total knee replacement. A comparison between the ritimidis S, Malizos KN. A mid-term clinical outcome Medial Pivot knee and a posterior stabilised implant. study of the Advance Medial Pivot knee arthroplasty. Knee 2006; 13:371–3. Knee 2009; 16:484–8.

258 Bianchi et al. Gait analysis in MP vs PS TKA

37. Bae DK, Cho S Do, Im SK, Song SJ. Comparison of 46. Insall JN, Dorr LD, Scott RD, Scott WN. Rationale of midterm clinical and radiographic results between the Knee Society clinical rating system. Clin Orthop total knee arthroplasties using medial pivot and po- Relat Res 1989; (248):13–4. sterior-stabilized prosthesis-a matched pair analysis. J 47. Dawson J, Fitzpatrick R, Murray D, Carr A. Questi- Arthroplasty 2016; 31:419–24. onnaire on the perceptions of patients about total knee 38. Kim YH, Yoon SH, Kim JS. Early outcome of TKA replacement. J Bone Joint Surg Br 1998; 80-B(1):63–9. with a medial pivot fixed-bearing prosthesis is worse 48. Kettelkamp DB, Johnson RJ, Smidt GL, Chao EY, than with a PFC mobile-bearing prosthesis. Clin Ort- Walker M. An electrogoniometric study of knee mo- hop Relat Res 2009; 467:493–503. tion in normal gait. J Bone Joint Surg Am 1970; 52- 39. Thienpont E, Opsomer G, Koninckx A, Houssiau F. A:775–90. Joint awareness in different types of knee arthroplasty 49. Martin JW, Whiteside LA. The influence of joint line evaluated with the Forgotten Joint score. J Arthropla- position on knee stability after condylar knee arthro- sty 2014; 29:48–51. plasty. Clin Orthop Relat Res 1990; (259):146–56. 40. Matsumoto M, Baba T, Homma Y, Kobayashi H, 50. Cross MB, Nam D, Plaskos C, Sherman SL, Lyman Ochi H, Yuasa T, Behrend H, Kaneko K. Validation S, Pearle AD, Mayman DJ. Recutting the distal femur study of the Forgotten Joint Score-12 as a universal to increase maximal knee extension during TKA cau- patient-reported outcome measure. Eur J Orthop ses coronal plane laxity in mid-flexion. Knee 2012; Surg Traumatol 2015; 25:1141–5. 19:875–9. 41. Hamilton DF, Loth FL, Giesinger JM, Giesinger K, 51. Clavé A, Le Henaff G, Roger T, Maisongrosse P, Ma- MacDonald DJ, Patton JT, Simpson AHRW, Howie bit C, Dubrana F. Joint line level in revision total knee CR. Validation of the English language Forgotten Jo- replacement: assessment and functional results with int Score-12 as an outcome measure for total hip and an average of seven years follow-up. Int Orthop 2016; knee arthroplasty in a British population. Bone Joint J 40:1655–62. 2017; 99-B(2):218–24. 52. Ramappa M. Midflexion instability in primary total 42. Thomsen MG, Latifi R, Kallemose T, Barfod KW, knee replacement : a review. SICOT J 2015; 1:24. Husted H, Troelsen A. Good validity and reliability of 53. Chiu M-C, Wang M-J. The effect of gait speed and the forgotten joint score in evaluating the outcome of gender on perceived exertion, muscle activity, joint total knee arthroplasty. Acta Orthop 2016; 87:280–5. motion of lower extremity, ground reaction force and 43. Schotanus MGM, Pilot P, Vos R, Kort NP. No diffe- heart rate during normal walking. Gait Posture 2007; rence in joint awareness after mobile- and fixed-be- 25:385–92. aring total knee arthroplasty: 3-year follow-up of a 54. Riley PO, DellaCroce U, Kerrigan DC. Effect of age randomized controlled trial. Eur J Orthop Surg Trau- on lower extremity joint moment contributions to gait matol 2017; 27:1151–5. speed. Gait Posture 2001; 14:264–70. 44. Bellamy N, Buchanan WW, Goldsmith CH, Campbell 55. Stan G, Orban H. Human gait and postural control J, Stitt LW. Validation study of WOMAC: a health after unilateral total knee arthroplasty. Maedica (Buc- status instrument for measuring clinically important har) 2014; 9:356–60. patient relevant outcomes to antirheumatic drug the- 56. Wilson SA, McCann PD, Gotlin RS, Ramakrishnan rapy in patients with osteoarthritis of the hip or knee. HK, Wootten ME, Insall JN. Comprehensive gait J Rheumatol 1988; 15:1833–40. analysis in posterior-stabilized knee arthroplasty. J 45. Weinberger M, Samsa GP, Hanlon JT, Schmader Arthroplasty 1996; 11:359–67. K, Doyle ME, Cowper PA, Uttech KM, Cohen HJ, Feussner JR. An evaluation of a brief health status measure in elderly veterans. J Am Geriatr Soc 1991; 39:691–4.

259 ORIGINAL ARTICLE

Allogenic platelet concentrates from umbilical cord blood for knee osteoarthritis: preliminary results

Vincenzo Caiaffa1, Francesco Ippolito1, Antonella Abate1, Vittorio Nappi1, Michele Santodirocco2, Domenico Visceglie3

1Orthopaedic and Traumatology Unit, Di Venere Hospital Bari, 2Apulia Cord Blood Bank Casa Sollievo della Sofferenza, San Giovanni Rotondo, 3Blood Transfusion Service, Di Venere Hospital Bari; Italy

ABSTRACT

Aim To investigate the role of cordonal blood platelet-rich plasma (PRP) intra-articular injections for treating the patients with knee osteoarthritis in terms of procedure safety and clinical outcomes.

Methods Twenty-five patients affected by knee osteoarthritis were enrolled and received one single intra-articular knee injection of umbilical cord PRP in a volume of 10 mL. A follow-up was inve- stigated at time 0, 4, 8, 12 weeks and 6 months, evaluating clinical parameters and functional performances.

Corresponding author: Results No serious adverse events were identified. The paired t- Antonella Abate test analysis showed a significant difference between baseline and Orthopaedic and Traumatology Unit, each follow-up times for all clinical scales (p<0.05), with a signi- Di Venere Hospital ficant improvement of clinical outcomes. Via Ospedale Di Venere 1, Conclusion Allogeneic PRP can generate reliable therapeutic 70012 Bari, Italy effect. The high content of tissue regenerative factors in cord blo- Phone/fax: 080 5015 462; od platelets makes cordonal blood one of the ideal sources of PRP. E-mail: [email protected] Key words: allogenic, cordonal, osteoarthritis, platelet rich Vincenzo Caiaffa ORCID ID: https://orcid. plasma org/0000-0002-8105-1285

Original submission: 05 December 2020; Revised submission: 09 December 2020; Accepted: 30 December 2020 doi: 10.17392/1330-21

Med Glas (Zenica) 2021; 18(1):260-266

260 Caiaffa al. PRP and knee osteoarthritis

INTRODUCTION osteoarthritic symptoms, potentially delaying the need for joint replacement surgery. PRP injecti- Osteoarthritis (OA) of the knee is a common pro- ons have shown to influence the entire joint envi- blem characterized by joint pain, swelling, stiffne- ronment, leading to a short-term clinical improve- ss and disability (1,2). The main problem associa- ment (17) with PRP injections being considered ted with OA is articular cartilage defect which has a safe procedure with more favourable outcomes limited capacity for repair. This imposes a major when compared to alternative treatments (18). social burden due to elevated healthcare cost and absence from work (3,4). Despite decades of re- However, autologous platelet-rich plasma (PRP) search, no true disease-modifying OA drugs are application is harassed by controversial outco- described, and clinical effects of pharmacological me, due to highly variable PRP quality among interventions remain of short duration. patients, being influenced by age, comorbidities, modality of preparation. Allogeneic PRP from Cells, scaffolds, and growth factors have conven- well-characterized donors can either generate tionally been considered as the “three elements” more consistent and reliable therapeutic effect of regenerative medicine (5). Both growth factors and avoid harvesting large quantities of blood, and mesenchymal stem cells (MSCs) were iden- an additional health burdens to patients. Howe- tified as a new option in the field of cartilage re- ver, the use of allogeneic PRP is generally less generation, and some authors have reported that investigated, especially for its immunogenicity in patients with knee OA treated with implantation such application. Allogenic PRP consist of a new and injection showed clinical improvement (6-9). aim in regenerative medicine; novel results are Growing attention has been paid to growth factors encouraging (19). because of their critical role in cytogenesis and The high content of tissue regenerative factors histogenesis. In recent years, platelet-rich plasma in cord blood platelets makes cordonal blood the (PRP) containing growth factors has attracted ideal source of allogenic PRP. A recent study attention as a biomaterial useful for regenerative proposed a standardized production of allogenic medicine. The PRP, which is a physiological bi- cryopreserved cord blood platelet concentrates omaterial (10) and contains various types of pla- (CBPC) suitable for later preparation of clinical- telet-derived growth factors, can be expected to grade cord blood platelet gel (20). exert the actions of multiple growth factors that are required for histogenesis (11) without the arti- The availability of the CBPC units previously ficial enhancement of a single growth factor (12). prepared and cryopreserved allowed us to con- duct a prospective study to investigate the role of The therapeutic use of platelet concentrates was umbilical cord (UC) - PRP injections for treating first described by Whitman in 1997 (13), although patients with knee osteoarthritis, in terms of pro- blood-derived fibrin glues were already used 30 cedure safety and clinical outcomes. years earlier to seal wounds and stimulate their he- aling (14). In 1998, platelet concentrates started to PATIENTS AND METHODS be known as platelet-rich plasma (PRP), generally defined as a volume of autologous plasma contai- Patients and study design ning a higher platelet count than peripheral blood (150,000–350,000 platelets/μL) (15). Thereafter, For the realization of the project, we had the multiple systems have been developed to concen- collaboration of the Transfusion Medicine Unit- trate platelets and remove erythrocytes (red blood Di Venere Regional Hospital and Puglia Cord cells) (RBCs) and, in some cases, also leukocytes Blood Bank. (white blood cells) (WBCs) (16). On the benealth of the 2011 public Cordonal Blo- Application of autologous PRP for cartilage rege- od (CB) banks research project standardized pro- neration and OA treatment, our field of interest, duction of cryopreserved CBPC from CB units has been getting more and more attention over not fulfilling the criteria for banking for haemato- the last decade. poietic transplant purposes, but otherwise poten- tially usable for other therapeutic applications, In knee OA, autologous PRP injections aim to was started on November 1st, 2013. Meanwhile, stimulate cartilage repair and offer relief to other the Italian National Institute of Health (Istitu-

261 Medicinski Glasnik, Volume 18, Number 1, February 2021

to Superiore di Sanità) and the Italian National Methods Health Council (Consiglio Superiore di Sanità), Cord blood units collected at public banks with determined that allogeneic cordonal blood platlet total nucleated cell counts <1.5×109, platelet co- concentrates (CBPC) and cordonal blood platlet unt >150×109/L and volume >50 mL, at first, gel (CBPG), in analogy to similar products obta- underwent soft centrifugation within 48 hours of ined from adult peripheral blood, belong to the collection. Then, platelet-rich plasma was centri- category of blood component. fuged at high speed to obtain a CBPC with target The CB units were collected after the mothers’ platelet concentration of 800–1,200×109/L, which informed consent and processed within 48 hours. was cryopreserved without cryoprotectant below Standard parameters regarding platelets concen- −40 °C. The cost of preparation was estimated at € tration and centrifugation cycles were applied. 160.92/CBPC. About 2 hours were needed for one The CBPC units were finally transferred into a technician to prepare four CBPCs (20). storage bag and cryopreserved without cryopro- One independent orthopaedic surgeon performed tectant in a mechanical freezer at a temperature a clinical evaluation as assessed by validate clini- below −40 °C in view of future clinical use of cal outcome scales: Visual Analogue scale (VAS) the CBPG, which requires thawing at 37 °C in a (22), Western Ontario and McMaster Universiti- water-bath before activation. es Arthritis Index (WOMAC) (23), Knee Injury The target population included individuals of and Osteoarthritis Outcome Score (KOOS) (24), 43-79 years of age recruited between December and International Knee Documentation Com- 2019 and April 2020. All patients signed an infor- mittee (IKDC) (25). med consent for the treatment. This clinical analysis was performed at baseline Patients meet the following criteria: symptomatic (T0), 4 weeks (T1), 8 weeks (T2), 12 weeks (T3) knee OA (daily pain for at least 3 months not res- and 24 weeks follow-up (T4). ponding to pain medication), grade I-III Kellgren The primary endpoint of our trial was the safety Lawrence radiographic changes (21) without acute of UC-PRP treatment, evaluating the number of meniscal rupture: describe grades. One radiologist treatment related adverse events. The secondary independently staged knee OA according to Kell- endpoint of the trial was the efficacy of the tre- gren-Lawrence system (21) using standard knee atment validated by clinical scales. X-ray imaging of standing anteroposterior and ho- rizontal lateral projection. Patients were excluded The visual analogue scale (VAS) (22) is a va- in case of condylar or tibial plateau bone marrow lidated, subjective measure for acute and chro- oedema on MRI, major axial deviation defined by nic pain. Scores are recorded by making a valgus (>10°) or varus (>5°) deformity of the in- handwritten mark on a 10-cm line that represents volved leg, recent use of intra-articular hyaluronic a continuum between “no pain” and “worst pain.” acid/steroids in the past 6 months, ipsilateral hip or The Western Ontario and McMaster Univer- ankle arthritis, previous malignancy. sities Osteoarthritis Index (WOMAC) (23) is a widely used, proprietary set of standardized que- All patients underwent blood type definition and stionnaires to evaluate the condition of patients compatibility (ABO-Rh). with osteoarthritis of the knee and hip, including Twenty-five patients were enrolled and received pain, stiffness, and physical functioning of the one single intra-articular knee injection of CBPG joints. The Knee Injury and Osteoarthritis Outco- containing a volume of 10 mL. me Score (KOOS) (24) is self-administered and All injections were performed by two orthopae- assesses five outcomes: pain, symptoms, activiti- dic surgeons with more than five-year experience es of daily living, sport and recreation function, in knee intra-articular injections in syringes of 10 and knee-related quality of life. mL using an anterolateral approach at the medial The International Knee Documentation Com- joint line with 90° of knee flexion. mittee (IKDC Questionnaire) (25) is a knee-spe- Patients were asked to avoid physical activity cific patient-reported outcome measure. It is con- for 72 hours after the procedure. Acetaminophen sidered to be one of the most reliable outcome was allowed in case of pain (3 g/day). reporting tools in its category.

262 Caiaffa al. PRP and knee osteoarthritis

Statistical analysis re 31.8 (range 13-40), KOOS score 59 (range 45- 80), IKDC score 59.32 (range 44-78). Statistical analysis was performed using paired-t test to investigate a statistical difference in terms of At the final 6-month follow-up (T4), the mean valu- painful symptoms and functional outcomes betwe- es were: VAS score 6.72 (range 4-8), WOMAC sco- en preoperative and postoperative times. The test re 37.24 (range 20-45), KOOS score 53.64 (range was performed with a confidence level of 5%; a 41-75), IKDC score 54.4 (range 41-74) (Table 2). p<0.05 was considered statistically significant. Table 2. Clinical outcomes at baseline (T0), 4 (T1), 8 (T2), 12 weeks (T3) and 6 months (T4) follow-up RESULTS scale Follow-up period (mean The sample consisted of 25 patients, 10 males ±SD) T0 T1 T2 T3 T4 (40%) and 15 females (60%). The average age VAS 7.36±1.73 3.24±1.42 3.16±1.07 3.64±1,11 6.72±1.1 womac 53.96±9.55 30.6±6.43 29.84±5.44 31.8±6.55 37.24±6.18 was 62.68±8.76 (range 43-79) years. In 10 (40%) koos 46.39±6.79 61.77±7.06 60.56±6.87 59±7.61 53.64±7.15 cases the right limb was involved. In 14 (56%) ikdc 39.79±6.94 60.93±7.85 60.88±7.24 59.32±8.26 54.4±8.00 cases there was radiographic evidence of joint VAS, Visual Analogue Scale; WOMAC, Western Ontario and McMaster Universities Osteoarthritis Index; KOOS, Knee Injury and change based on Kellgren–Lawrence grade II Osteoarthritis Outcome Score; IKDC, International Knee Documen- and in 11 (44%) cases grade III (Table 1). tation Committee The paired t-test analysis showed a significant Table 1. Demographic characteristics of 25 patients Variable difference between baseline and each follow-up Gender (No; %) times for all clinical scales (p<0.05), with a si- Males 10 (40) gnificant improvement of these clinical outcomes Females 15 (60) (Table 3). Average age (±SD) (years) 62.68 (±8.76) Average BMI (±SD) 27.84 (±3.04) Table 3. Statistical analysis of clinical outcome at baseline Kellgren-Lawrence grade (No; %) (T0), 4 (T1), 8 (T2) 12 weeks (T3) and 6 months (T4) follow-up I 0 Paired t test between two time points* Scale II 14 (56) T0-T1 T0-T2 T0-T3 T0-T4 III 11 (44) vas 3,65948E-13 2,9892E-11 3,12881E-11 0.029 Kellgren-Lawrence grade (average; SD) 2.44 (±0.51) womac 4,84524E-16 1,65277E-15 1,70372E-16 1,03557E-12 BMI, body mass index; koos 6,12018E-12 9,79963E-12 3,42026E-10 2,05478E-06 No serious adverse events were identified. The IKDC 1,09353E-10 4,09279E-11 5,17389E-10 9,66833E-08 *The paired t test was used to test the statistical differences between most common adverse event was acute and pain- two time points; Number E-n, in which E (exponent) multiplies the ful synovitis during for a mean of 4 days. Local preceding number by 10 to the nth power. VAS, Visual Analogue Scale; WOMAC, Western Ontario and oedema and difficulties in walking were -obser McMaster Universities Osteoarthritis Index; KOOS, Knee Injury and ved in two patients and completely resolved in Osteoarthritis Outcome Score; IKDC, International Knee Documen- 7 days. No one required hospitalization or ar- tation Committee; throcentesis. Thirteen patients assumed acetami- DISCUSSION nophen with partial complete regression of pain. One of the most interesting therapeutic choices The average values at baseline (T0) were: VAS in regenerative medicine for the gonarthrosis tre- score 7.36 (range 3-10), WOMAC score 53.96 atment is the use of Platelet Rich Plasma (PRP). (range 28-65), KOOS score 46.39 (range 32-64), The PRP is a concentrate of platelets and growth IKDC score 39.79 (range 25-51.33). factors (GFs) obtained by the centrifugation of At T1 (4 weeks) the mean values were: VAS sco- venous blood. It may be autologous or alloge- re 3.24 (range 1-7), WOMAC score 30.6 (ran- nic. Preparation methods are not standardized yet ge 12-40), KOOS score 61.77 (range 46-74.2), and regenerative mechanisms related to the bio- IKDC score 60.93 (range 46-72). molecular pathway are still the object of study. The average values at 8-week follow-ups (T2) In the last fifteen years, the PRP application has were: VAS score 3.16 (range 2-5), WOMAC sco- expanded to a wide range of clinical fields, inclu- re 29.84 (range 20-37), KOOS score 60.56 (range ding plastic surgery, dermatology, maxillofacial 48-78), IKDC score 60.88 (range 47-73). surgery, orthopaedics and others (26-30). At 12-week follow-ups (T3) the mean values PRP in vitro studies actually available state almost were: VAS score 3.64 (range 1-6), WOMAC sco- consistently that PRP stimulates the proliferation

263 Medicinski Glasnik, Volume 18, Number 1, February 2021

of the human cell. This observation is also the case telet gel and autologous cancellous bone) in nine regarding cell motility and exocytosis of several patients (36). They used random single-donor important regenerative extracellular ground sub- allogenic PCs (ABO and RhD matched, serologi- stances, for example, collagen type I and III, HA cally HIV, hepatitis B virus, hepatitis C virus and and so forth. Regarding the optimal platelet con- lues-negative, leukocyte depleted, and irradiated) centration for cell proliferation, the studies diverge from standard blood bank stocks. As in the case severely. An overall trait is seen - when the PRP study, screening for HLA antibodies class I, and concentration increases, the volume of culture me- human platelet antibodies was performed befo- dia (nutrition) decreases and a lower optimal con- re implantation and after 3 months, also without centration for cell proliferation is observed (31). detecting any sign of immunologic reactions. At In vivo studies are actually still limited and results 1 year after surgery, seven out of nine patients are not of unique interpretation. Variables such as treated achieved complete healing. type of PRP, proper timing, treatment periodicity, More recently, Bottegoni and colleagues (37) location and technique for injection would need to performed a prospective open-label, uncon- be selected to establish efficacy in each treatment trolled, single-centre, pilot study with 60 pati- and to compare different studies. However autolo- ents. Participating patients (aged 65–86 years) gous PRP is generally considered microbiologically suffered symptomatic early or moderate knee OA safe with regard to the risk of acquiring microbial (Ahlbäck grade I–III) and were affected by he- and viral transmissible infections. Moreover, au- matologic disorders, preventing autologous PRP tologous PRP has significant practical limitations treatment. Effectiveness, as measured with the which may prevent its clinical use in different cate- International Knee Documentation Committee gories of patients, for example elderly hypo-mobile (IKDC), knee injury and osteoarthritis outco- patients, chronic inflammatory diseases; repeated me score (KOOS) and EuroQol-visual analogue blood collections for multiple PRP applications scales (EQ-VAS), was varied. As noted in other may be difficult or clinically inappropriate. These trials, younger patients with lower degree of de- limitations prompted several groups to standardi- generation showed a better response. In addition, ze platelet gel as an allogeneic blood component they did not report any severe complications rela- obtained from healthy adult blood donors, to be ted to the allogenic nature of the PRP. routinely offered to clinicians for the treatment of The high content of tissue regenerative factors in patients suffering from different conditions, thus cord blood platelets makes this a great source of avoiding the inconvenience of autologous blood allogenic PRP. Moreover the widespread availa- collection (32-34). This approach also reduces po- bility of allogeneic cord blood units generously tential negative effects of pathological biological donated for hematopoietic transplant but unsuita- effectors possibly present in the patient’s blood in ble for this use solely because of low hematopoi- relation to his or her morbid conditions (35). etic stem cell content prompted different Italian At least three recent works have described the cordonal Bank to develop a national programme use of allogenic PRP clinically. Smrke et al. per- to standardise the production of allogeneic formed a case study in which a 50-year-old male cryopreserved cord blood platlet concentrates with type 2 diabetes suffering from a comminu- (CBPC) suitable for later preparation of clinical- ted fracture of the tibia and delayed union was grade cord blood platelet gel (38-43), which can treated with a graft composed of allogenic pla- be used in different fields including osteoarthritis. telet gel mixed with autologous cancellous bone. Our study can be considered a clinical applica- After 6 months the graft was incorporated, the tion of a multicentric Italian study performed to bone defect was fully bridged and full weight- standardize a clinical grade procedure for the pre- bearing capacity was achieved. No side effects paration of allogeneic PCs from umbilical cord were observed and no platelet or HLA class I blood (20). antibodies were detected (35). As a follow-up, a Our results were encouraging. No adverse events prospective clinical study was conducted by the were observed. Transitory pain resulted respon- same group to treat long bone non-unions using der to acetaminophen. VAS reduction at first the same type of allogenic product (allogenic pla- follow-up promoted quality of life and function

264 Caiaffa al. PRP and knee osteoarthritis

improvement, maintaining good clinical outco- OA remains a heterogenous and multifactori- mes all six months long. al pathology. It is now known that in OA pati- The limitation of our study was the short follow- ents who present the same symptoms, the un- up. Additionally, a cohort study with a control derlying mechanisms causing them might be group (for example, hyaluronic injection) could different. Different OA phenotypes are emerging provide more information regarding the real effi- that will most likely require different treatment cacy of the procedure. approaches. Therefore, PRP might be effective in a selected group of patients, and those need to be In conclusion, we strongly believe that PRP is identified to obtain a target effective treatment. effective for tissue regeneration through a com- posite of actions exerted by types of growth fac- FUNDING tors that are released from concentrated platelets. Autologous PB-PRP injection is actually consi- No specific funding was received for this study. dered a safe procedure. Allogenic PRP still needs TRANSPARENCY DECLARATION to be evaluated throughout more high-quality tri- als. In both cases PRP formulations need to be Conflict of interest: None to declare. standardized to allow comparison across studies.

REFERENCES 1. Lawrence RC, Felson DT, Helmick CG, Arnold LM, 9. Horie M, Choi H, Lee RH, Reger RL, Ylostalo J, Choi H, Deyo RA, Gabriel S, Hirsch R., Hochberg Muneta T, Sekiya I, Prockop DJ. Intraarticular injec- MC, Hunder GG, Jordan JM, Katz JN, Kremers HM, tion of human mesenchymal stem cells (MSCs) pro- Wolfe F. Estimates of the prevalence of arthritis and motes rat meniscal regeneration by being activated other rheumatic conditions in the United States. Part to express Indian hedgehog that enhances expressi- II. Arthritis Rheum 2008; 58:26e35. on of type II collagen. Osteoarthritis Cartilage 2012; 2. Schuster P, Schulz M, Mayer P, Schlumberger M, 20:1197–207. Immendoerfer M, Richter J. Open-wedge high tibial 10. Dohan Ehrenfest DM, Pinto NR, Pereda A, Jime- osteotomy and combined abrasion/microfracture in nez P, Corso MD, Kang BS, Nally M, Lanata N, severe medial osteoarthritis and varus malalignment: Wang HL, Quirynen M. The impact of the centri- 5-year results and arthroscopic findings after 2 years. fuge characteristics and centrifugation protocols on Arthroscopy 2015; 31:1279e88. the cells, growth factors, and fibrin architecture of a 3. Gore M, Tai KS, Sadosky A, Leslie D, Stacey BR. leukocyte- and platelet-rich fibrin (L-PRF) clot and Clinical comorbidities, treatment patterns, and direct membrane. Platelets 2018; 29:171-84. medical costs of patients with osteoarthritis in usual 11. Murphy MB, Blashki D, Buchanan RM, Yazdi IK, care: A retrospective claims database analysis. J Med Ferrari M, Simmons PJ, Tasciotti E. Adult and umbi- Econ 2011; 14:497–507. lical cord blood-derived platelet-rich plasma for me- 4. McKenna MT, Michaud CM, Murray CJ, Marks JS. senchymal stem cell proliferation, chemotaxis, and Assessing the burden of disease in the United States cryo-preservation. Biomaterials 2012; 33:5308-16. using disability-adjusted life years. Am J Prev Med 12. Mendonça-Caridad JJ, Juiz-Lopez P, Rubio-Rodri- 2005; 28:415–23. guez JP. Frontal sinus obliteration and craniofacial 5. Yamada Y, Ito K, Nakamura S, Ueda M, Nagasaka T. reconstruction with platelet rich plasma in a patient Promising cell-based therapy for bone regeneration with fibrous dysplasia. Int J Oral Maxillofac Surg using stem cells from deciduous teeth, dental pulp, 2006; 35:88-91. and bone marrow. Cell Transplant 2011; 20:1003-13. 13. Whitman DH, Berry RL and Green DM. Platelet 6. Liu Q, Niu J, Huang J, Ke Y, Tang X, Wu X, Li R, gel: an autologous alternative to fibrin glue with Li H, Zhi X, Wang K, Zhang Y, Lin J. Knee osteoar- applications in oral and maxillofacial surgery. J Oral thritis and all-cause mortality: The Wuchuan Osteo- Maxillofac Surg 1997; 55:1294–9. arthritis Study. Osteoarthr Cartil 2015; 23:1154–7. 14. Matras H. Effect of various fibrin preparations on re- 7. Ogura T, Mosier BA, Bryant T, Minas T. A 20-year implantation in the rat skin. Osterr Z Stomatol 1970; follow-up after first-generation autologous chondro- 67:338–59. cyte implantation. Am J Sport Med 2017; 45:2751– 15. Marx RE, Carlson ER, Eichstaedt RM, Schimmele 61. SR, Strauss JE, Georgeff K R. Platelet-rich plasma 8. Diekman BO, Wu CL, Louer CR, Furman BD, - Growth factor enhancement for bone grafts. Oral Huebner JL, Kraus VB, Olson SA, Guilak F. Intra- Surg Oral Med Oral Pathol Oral Radiol Endod 1998; articular delivery of purified mesenchymal stem 85:638–46. cells from C57BL/6 or MRL/MpJ super healer mice 16. Xie X, Zhang C and Tuan RS. Biology of platelet- prevents posttraumatic arthritis. Cell Transplant rich plasma and its clinical application in cartilage 2013; 22:1395–408. repair. Arthrit Res Ther 2014; 16:204.

265 Medicinski Glasnik, Volume 18, Number 1, February 2021

17. Filardo G, Di Matteo B, Di Martino A, Merli ML, 31. Bhanot S, Alex JC. Current applications of platelet Cenacchi A, Fornasari P, Marcacci M, Kon E. Plate- gels in facial plastic surgery. Facial Plast Surg 2002; let-rich plasma intra-articular knee injections show 18:27-33. no superiority versus viscosupplementation: a ran- 32. Straum OK. The optimal platelet concentration in domized controlled trial. Am J Sports Med 2012; platelet-rich plasma for proliferation of human cells 43:1575–82. in vitro—diversity, biases, and possible basic expe- 18. Laver L, Marom N, Dnyanesh L, Mei-Dan O, rimental principles for further research in the field: a Espregueira-Mendes J, Gobbi A. PRP for degenera- review. Peer J 2020; 8:e10303. tive cartilage disease: a systematic review of clinical 33. Jeong SH, Han SK, Kim WK. Treatment of diabetic studies. Cartilage 2017; 8:341–64. foot ulcers using a blood bank platelet concentrate. 19. Zhang Z-Y, Huang A-W, Fan JJ, Wei K, Jin Plast Reconstr Surg 2010; 125:944-52. D, Chen B, Li D, Bi L, Wang J, Pei G. The potential 34. Crovetti G, Martinelli G, Issi M, Barone M, Guizzar- use of allogeneic platelet-rich plasma for large bone di M, Campanati B, Moroni M, Carabelli A. Platelet defect treatment: immunogenicity and defect healing gel for healing cutaneous chronic wounds. Transfus efficacy. Cell Transplant 2013; 22:175-87. Apher Sci 2004; 30:145-51. 20. Rebulla P, Pupella S , Santodirocco M , Greppi N 35. Smrke D, Gubina B, Domanoviç D, Rozman P. Allo- , Villanova I , Buzzi M, De Fazio N , Grazzini G; geneic platelet gel with autologous cancellous bone Italian Cord Blood Platelet Gel Study Group. Multi- graft for the treatment of a large bone defect. Eur centre standardisation of a clinical grade procedure Surg Res 2007; 39:170-4. for the preparation of allogeneic platelet concentra- 36. Gubina B, Rožman P, Bišcević M, Dromanovic D, tes from umbilical cord blood. Blood Transfus 2016; Smrke D. The influence of allogeneic platelet gel on 14:73-9. the morphology of human long bones. Coll Antropol 21. Kellegren JH, Lawrence JS. Radiological asse- 2014; 38:865–70. ssment of osteo-arthrosis Ann Rheum Dis 2000; 37. Bottegoni C, Dei Giudici L, Salvemini S, Chiurazzi 16:494-502. E, Bencivenga R, Gigante A. Homologous platelet- 22. D. Gould et al. Visual Analogue Scale (VAS). J Clin rich plasma for the treatment of knee osteoarthritis Nurs2001; 10:697-706. in selected elderly patients: an open-label, uncon- 23. WOMAC Osteoarthritis Index http://www.upphar- trolled, pilot study. Ther Adv Musculoskelet Dis ma.it/wp-content/uploads/2019/03/scale-valutazio- 2016; 8:35–41. ne-osteoartrite-WOMAC.pdf. 38. Rosso L, Parazzi V, Damarco F, Righi I, Santabrogio 24. Roos EM, Roos HP, Lohmander LS, Ekdahl C, L, Rebulla P, Gatti S, Ferrero S, Nosotti M, Lazzari Beynnon BD: Knee Injury and Osteoarthritis Outco- L. Pleural tissue repair with cord blood platelet gel. me Score (KOOS)--development of a self-admi- Blood Transf 2014; 12 Suppl 1(Suppl 1) s235–42. nistered outcome measure. J Orthop Sports Phys 39. Tadini G, Pezzani L, Ghirardello S, Rebulla P, Es- Ther 1998, 28:88–96. posito S, Mosca F. Cord blood platelet gel treatment 25. Padua R, Bondi R, Ceccarelli E, Bondi L, Romanini of dystrophic recessive epidermolysis bullosa. BMJ E, Zanoli G, Campi S. Italian version of the Interna- Case Rep 2015; 2015:bcr2014207364. tional Knee Documentation Committee Subjective 40. Gelmetti A, Greppi N, Guez S, Grassi F, Rebulla P, Knee Form: cross-cultural adaptation and validation. Tadini G. Cord blood platelet gel for the treatment of Arthroscopy 2004; 20:819-23. inherited epidermolysis bullosa. Transfus Apher Sci 26. Petrungaro PS. Using platelet-rich plasma to acce- 2018; 57:370–3. lerate soft tissue maturation in esthetic periodontal 41. Piccin A, Rebulla P, Pupella S, Tagnin M, Marano G, surgery. Compend Contin Educ Dent 2001; 22:729- Di Pierro AM, Santodirocco M, Di Mauro L, Bequiri 32. L, Kob M, Primerano M, Casini M, Billio A, Eisen- 27. Robiony M, Polini F, Costa F, Politi M. Osteogenesis dle K, Fontanella F. Impressive tissues regeneration distraction and platelet-rich plasma for bone restora- of severe mucositis post stem cell transplantation tion of the severely atrophic mandible: preliminary using cord blood platelet gel. Transfusion 2017; results. J Oral Maxillofac Surg 2002; 60:630-5. 57:2220–4. 28. Anitua E. Plasma rich in growth factors: prelimi- 42. Volpe P, Marcuccio D, Stilo G, Alberti A, Foti G, nary results of use in the preparation of future sites Volpe A, Princi D, Surace R, Pucci G, Massara M. for implants. Int J Oral Maxillofac Implants 1999; Efficacy of cord blood platelet gel application for 14:529-35. enhancing diabetic foot ulcer healing after lower 29. Della Valle A, Sammartino G, Marenzi G, Tia M, Es- limb revascularization. Semin. Vasc. Surg 2017; pedito di Lauro A, Ferrari F, Lo Muzio L. Prevention 30:106–12. of postoperative bleeding in anticoagulated patients 43. Bisceglia G., Santodirocco M, Faienza A, Mastrodo- undergoing oral surgery: use of platelet-rich plasma nato N, Urbano F, Totaro A, Bazzocchi F, Di Ma- gel. J Oral Maxillofac Surg 2003; 61:1275-8. uroL. First endocavitary treatment with cord blood 30. Marx RE. Platelet-rich plasma: evidence to support platelet gel for perianal fistula. Regen Med 2020; its use. J Oral Maxillofac Surg 2004; 62:489-96. 15:1171-6.

266 ORIGINAL ARTICLE

Intra-operative local plus systemic tranexamic acid significantly decreases post-operative bleeding and the need for allogeneic blood transfusion in total knee arthroplasty

1,2 1,2 1,2 1,2 1,2 1,2 Lidia De Falco , Elisa Troiano , Martina Cesari , Pietro Aiuto , Giacomo Peri , Nicolò Nuvoli , Mat- tia Fortina1,2, Nicola Mondanelli1,2, Stefano Giannotti1,2

1Section of Orthopaedics and Traumatology, Department of Medicine Surgery and Neurosciences, University of Siena, 2Section of Orthopaedics and Traumatology, Azienda Ospedaliera Universitaria Senese; Siena, Italy

ABSTRACT

Aim To evaluate the efficacy of systemic plus local tranexamic acid (TXA) in reducing post-operative bleeding, haemoglobin loss and the need for allogeneic blood transfusion (ABT) in total knee arthroplasty (TKA).

Methods All patients undergoing TKA between January 2017 and September 2019 were retrospectively evaluated. Exclusion criteria were cardiovascular comorbidities, diabetes and the assumption of any anticoagulant/antiaggregant therapy in the pre-operative peri- od. All patients received the same prosthesis with the same surgi- cal technique and were operated on by the same surgeon. Twenty Corresponding author: patients were found (group A) that received intra-operative TXA Nicola Mondanelli (20 mg/kg intravenous 10 minutes before deflating tourniquet and Section of Orthopaedics, Department of 1g intra-articular after capsular suture). A control group of 26 pati- Medicine, Surgery and Neurosciences, ents not receiving TXA was matched for demographics (group B). University of Siena Results Two (10%) patients in group A and 16 (61.5%) in group B Viale Mario Bracci 16, 53100 Siena, Italy needed ABT in the post-operative period (p=0.0004). Each patient E-mail: [email protected] in group A received 2 red blood cells (RBCs) units, while in group Phone: +39 0577 585 675; B 2 patients received one RBCs unit and one patient 4 RBCs units, for a total of 4 and 32 RBCs units in group A and B, respectively Fax +39 0577 233 400; (p=0.0006). The minimum haemoglobin level was observed at 48 De Falco Lidia ORCID ID: https://orcid. hours post-operatively in both groups: mean decrease was 3.54 org/0000-0003-3756-9033 and 4.64 g/dL in group A and B, respectively (p=0.0126).

Conclusion The association of systemic and local TXA admini- stration seems to significantly reduce post-operative bleeding and the need for RBCs transfusions after TKA in patients not assuming Original submission: any anticoagulant / antiaggregant therapy and without cardiovas- 02 December 2020; cular and diabetic morbidities. Accepted: 11 December 2020 Key words: bleeding, haemoglobin, orthopaedic surgery, post- doi: 10.17392/1327-21 operative anaemia, red blood cells

Med Glas (Zenica) 2021; 18(1):267-272

267 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION intra-articular TXA administration in reducing post-operative bleeding, haemoglobin loss and Total knee arthroplasty (TKA) generally involves the need for ABT in TKA. significant post-operative anaemia, because of the continuous bleeding from the cut bone surfaces (the PATIENTS AND METHODS femur more than the tibia and the patella) not co- vered by the prosthetic elements, from the opened Patients and study design medullary canal (again, from the femur) and the dissected periarticular soft tissues (especially po- Medical records of all patients who underwent steriorly) (1). This may cause a reduction of about TKA between January 2017 and September 2019 20% in circulating blood volume resulting in major at Azienda Ospedaliera Universitaria Senese cardiovascular complications and increased mor- were retrospectively evaluated. Selection criteria tality. A great number of patients (between 10 and were primary osteoarthritis (OA) of the knee as 30%) require red blood cells (RBCs) allogeneic diagnosis, a single surgeon performing the pro- transfusion (2) that may be accompanied by im- cedure, same surgical access and technique and portant risks such as allergic reaction, anaphylactic same cemented prosthesis, surgery conducted shock, fever, infections (3). Also, allogeneic blood under spinal anaesthesia, non-resurfacing pa- transfusion (ABT) implies high costs, and ortho- telloplasty to address the patella, and systemic paedic surgery accounts for over 10% of all ABTs, IV and local intra-articular TXA administration 40% of which for joint replacement procedures at surgery. Exclusion criteria were cardiovascular (4,5). In order to reduce blood loss in TKA pati- comorbidities, diabetes, ongoing anticoagulant/ ents, various methods have been proposed: autolo- antiaggregant therapy, history of previous VTE gous transfusions (6), hypotensive anaesthesia (7), or any other condition suggesting a pharmacolo- drainage clamping (8), application of fibrin tissue gical prophylaxis other than our standard proto- adhesive (9), compression bandage and cryothe- col with low molecular weight heparin (Enoxa- rapy (10). Furthermore, systemic or local admini- parine 4000 IU subcutaneously every 24 hours, stration of tranexamic acid (TXA) is a well-known starting 12 hours after surgery and going on up to method to reduce bleeding and therefore the need regular crutch-free walking). for ABTs after surgery (11,12). The use of antifibri- Twenty patients were found (group A) that met nolytic agents is based on evidence that the surgical the selection criteria. A control group with same trauma, as well as promoting the formation of clots inclusion and exclusion criteria, except that by activating the intrinsic and extrinsic coagulation for the administration of TXA at surgery, was cascade, also leads to a concomitant activation of matched for age, gender and body mass index the plasminogen which induces a hyperfibrinolytic (BMI), and 26 patients were recruited (group B). state that accelerates clot’s degeneration, thus incre- All patients gave their written consent to the tre- asing bleeding from the surgical site (13). atment and anonymous use of data and images The TXA is a synthetic derivative of the amino acid for research and academic purposes. At our In- lysine that acts as a competitive inhibitor of the ac- stitutions, neither the Ethical Committee nor In- tivation of plasminogen that interferes with fibri- stitutional Review Board approval are needed for nolysis (14); its intravenous administration during retrospective studies. joint replacement reduces post-operative bleeding Methods (15). The theoretical risk of venous thromboembo- lism (VTE) associated with the use of TXA has not Surgery was performed with tourniquet at the been proven clinically in several trials (16), even root of the high, inflated at 300 mmHg of pre- with high dosages (17). In contrast, a tendency ssure after the preparation of the sterile field and towards a protective effect against pulmonary em- limb exsanguination by elevation and elastic bolism has been described, probably linked to the compression with an Esmark bandage. A medial reduced need for RBCs transfusion that is a throm- parapatellar approach was performed in all cases. bogenic intervention (16). The femur was prepared at first, with intrame- The aim of this study was to evaluate the efficacy dullary instrumentation. The entry point of the of systemic intravenous (IV) and local low-dose femoral rod guide was closed by an autologous

268 De Falco et al. Tranexamic acid in TKA

bone plug in all cases, to reduce post-operative Statistical analysis bleeding (18). The tibia was prepared with an Patients’ age, body mass index (BMI) and hae- extramedullary guide. moglobin values at surgery were assessed for The same cemented prosthesis was implanted normality (Anderson-Darling test) and compared in all cases (Evolution Medial Pivot; Microport, between groups using the Mann-Whitney U test. Shangai, China); the femoral component presents The χ2 test was used to compare gender distribu- an open intercondylar box which eventually does tion and the differences in the number of patients not stop residual bleeding from the femoral ca- requiring ABT. The Mann-Whitney U test was nal. A non-resurfacing patelloplasty was perfor- also used to compare fall rates of haemoglobin med in all cases (circumferential resection of the and the number of transfused RBCs units in the osteophytes plus patellar reshaping with resec- post-operative period. The level of significance tion of the cartilage layer), aiming to improve was set at p<0.05. the congruency between the native patella and the femoral trochlea (19). After cementation of RESULTS prosthetic components, tourniquet was released, Patients’ age at surgery was between 51 and 88 accurate haemostasis was performed, and an in- years, with an average of 72.8 years; there were tra-articular drainage was positioned. 17 males and 29 females. Group A consisted of Patients in group A intra-operatively received 20 patients (nine males, 11 females) aging betwe- TXA according to the proposed protocol: 20 mg/ en 51 and 87 (mean 72.5) years old. Group B was kg in 100 mL of saline solution IV around 10 mi- composed of 26 patients (eight males, 18 fema- nutes before release of the pneumatic tourniquet les) with a mean age of 73.1 (range 52 – 88) ye- (to allow the drug to reach the plasma peak pla- ars old. No significant differences were observed sma at the time of release) (15) plus intra-articular between groups for demographics (age, gender, application of TXA 1 g after suturing the capsule BMI) and haemoglobin values at surgery. (so to have a “topic” effect). Patients in group B Two (10%) patients in group A and 16 (61.5%) in did not receive TXA at all. Finally, a sterile wo- group B required ABT (p=0.0004). Each patient und dressing and a bulky compression dressing (so in group A received 2 units of RBCs, while in called modified Robert Jones bandages) (20) were group B, two patients received 1 unit of RBCs applied. No anti-bleeding adjuvant pharmacologi- each and one patient received 4 units, for a total cal actions were undertaken in the post-operative number of transfused RBCs units of 4 in group A period, and post-operative rehabilitation protocol and 32 in group B (p=0.0006). was the same in both groups. Open suction draina- ge was left in situ for 48 hours after surgery; inter- The decrease in haemoglobin values observed at mittent cryotherapy was used in the post-operative 48 hours after surgery represented the minimum period (20 minutes every 2 hours for the first 2 value recorded in the post-operative period for post-operative days and after physiotherapy sessi- both groups: in group A there was a decrease ran- ons afterwards, ter in die), and continuous passive ging from 6.4 to 0.3 g/dL with a mean of 3.54 g/dL, motion was initiated from the first post-operative in group B the mean decrease was 4.64 g/dL with a day (2 hours bis in die). range from 7.2 g/dL to 1.8 g/dL (p=0.0126). Haemoglobin was measured pre-operatively No major side effects or complications in the and at 1, 24, 48 and 72 hours after surgery. ABT immediate post-operative period regarding ana- criteria for elective orthopaedic surgery were set emia or ABTs were recorded. at our Institution, at haemoglobin level equal to DISCUSSION or less than 8 g/dL in healthy patients younger than 80 and haemoglobin level equal to or less The main finding of this study confirmed the than 9 g/dL in patients with cardiovascular co- efficacy of a combined systemic and local TXA morbidities (exclusion criterion in this study) or administration to reduce post-operative bleeding age equal to or over 80 years. and haemoglobin post-operative loss, and su- bsequent need for ABT, in TKA performed for primary knee OA in patients without cardiovas-

269 Medicinski Glasnik, Volume 18, Number 1, February 2021

cular comorbidities. Several studies already exist Table 1. Efficacy of tranexamic acid (TXA) against bleeding in that described the efficacy of TXA administration different studies* in joint replacement surgery with special regards Study Protocol of TXA administration Hiippala et al. (1995) IV 15 mg/kg after deflating tourniquet to TKAs (1,8,11,12,15,17, 21–25). Also, several Vs placebo (equal volume of 0.9% sodium (RCT double-blinded) meta-analyses have been conducted (16, 26-34), chloride solution) IV 15 mg/kg after deflating tourniquet + 10 and all studies agreed about the efficacy of TXA Hiippala et al. (1997) mg/kg x 2 (at 3- and 6-hours post-op) against bleeding and confirmed its safety, as well. Vs placebo (equal volume of normal saline (RCT double-blinded) Unfortunately, their results cannot be easily com- solution) IA (topic) 1.5 g at the end of surgery for 5 pared to each other as every single study presented Wong et al. (2010) minutes a different protocol of TXA administration with Vs IA (topic) 3 g at the end of surgery for 5 (RCT double-blinded) respect to timing, route and/or dosage (Table 1). minutes Vs placebo (100 mL of normal saline soluti- In the pioneering studies of Hiippala et al. TXA was administered IV towards the end of surgery, on) at the end of surgery for 5 minutes IV 1 g before inflating tourniquet + 1 g after Sepah et al. (2011) after deflating tourniquet, and afterwards also in deflating tourniquet the immediate post-operative hours, always in IV (RCT double-blinded) Vs nothing route (11,12). Sepah et al. also compared IV admi- Ishida et al. (2011) IA (injected) 2 g (RCT) Vs placebo (saline) nistration of 2 doses of TXA versus no administra- IA (injected) 1 g + carbazochrome sodium sul- Onodera et al. (2011) tion at all (1). Wong et al. found that “topic” TXA fonate hydrate 50 mg + amikacin sulfate 200 mg administration left in situ for 5 minutes at the end (RCT) Vs saline + amikacin sulfate 200 mg Seo et al. (2013) IV 1.5 g of surgery was beneficial with respect to placebo; (RCT) Vs IA (topic) 1.5 g at suture also, a slightly statistical difference between low- Vs saline both IV and IA IV 1 g pre-operatively + IA (injected) 3 g after dose (1.5 g) and high-dose (3 g) TXA was present Nielsen et al. (2016) capsular closure with regards to ABT (21). (RCT double-blinded) Vs IV 1 g pre-operatively + placebo (saline) Also, different pharmacological VTE prophylaxis Wang et al. (2018 IV 20 mg/kg before inflating tourniquet Vs IA (topic) 1 g before cementation + IA were administered, different prostheses were im- (RCT double-blinded) (injected) 1 g at suture planted, and different post-operative protocols Vs Oral 2 g 2 hours before surgery were used. Ishida et al. compared intra-articular Yuan et al. (2018) (in IV 20 mg/kg 1 hour before surgery + IA injection of 2 g of TXA versus saline solution left RTKA) (injected) 3 g at suture (RCT double-blinded) Vs IA (injected) 3 g at suture into the joint with a clamped drainage for 30 minu- Zhang et al. (2019) IV 20 mg/kg pre-operatively tes, but a miscellaneous cohort of prostheses were (RCT double-blinded) Vs IA (injected) 3 g after suture implanted, some with a closed intercondylar box Vs IV 20 mg/kg pre-operatively + IA (injec- ted) 3 g after suture (22). A great difference in the use of the draina- IV 20 mg/kg 10 minutes before deflating Present study ge was present, too: some authors did not use it at tourniquet + IA (injected) 1 g at suture (retrospective cohort all (21), some had the drainage clamped for some Vs nothing study) period (8,22), some had it open, as in the present * Different studies could not be compared with each other as study study (17). Lastly, some confusion exists in the designs are differed in TXA administration (dose, route, timing) terminology: which is the difference between “in- RCT, randomized clinical trial; IV, intravenous; Vs, versus; IA, intra- articular; RTKA, revision total knee arthroplasty tra-articular” and “topic” administration? These terms can be interpreted as mutual but they are topical administration of TXA showed the lowest intended in different meaning in different studies: relative risk ratio for ABT when compared to “pla- some authors injected the TXA through the drai- cebo”, to routine haemostasis and to other mode nage (8,22), others poured the TXA into the joint of administration; also, no additional benefits were before capsular closure (21,23), others injected the found with increasing dose; the 15 mg/kg IV dose TXA into the joint after capsular closure as in the together with 1 g topical dose seems to be sufficient present study (17,24,25,30) and others both soa- to significantly reduce the need for blood transfusi- ked the joint with TXA solution and injected the on, and no safety issue was underlined (32). same solution after capsular closure (15). In conclusion, systemic IV and local topic in- Nevertheless, all studies indicated that the admini- tra-articular administration of TXA is confirmed stration of TXA is beneficial. Xu et al., in a recent as a safe, inexpensive and effective method to meta-analysis, found that the systemic IV plus local reduce post-operative bleeding, drop in haemo-

270 De Falco et al. Tranexamic acid in TKA

globin values and the subsequent need for ABT FUNDING after primary cemented TKA in patients without No specific funding was received for this study. cardiovascular comorbidities. The routine use of TXA to reduce bleeding can be recommended in TRANSPARENCY DECLARATION joint replacement surgery. Routes of administra- Conflict of interest: None to declare. tion can be various, while systemic IV plus local topical seem to be the most effective.

REFERENCES 1. Sepah YJ, Umer M, Ahmad T, Nasim F, Umer Cha- 13. Oremus K. Tranexamic acid for the reduction of blo- udhry M, Umar M. Use of tranexamic acid is a cost- od loss in total knee arthroplasty. Ann Transl Med effective method in preventing blood loss during and 2015; 3:1-4. after total knee replacement. J Orthop Surg Res 2011; 14. Mannucci PM. Hemostatic drugs. N Engl J Med 6:22. 1998; 339:245-53. 2. Ponnusamy KE, Kim TJ, Khanuja HS. Perioperative 15. Wang D, Wang HY, Cao C, Li LL, Meng WK, Pei blood transfusions in orthopaedic surgery. J Bone Jo- FX, Li DH, Zhou ZK, Zeng WN. Tranexamic acid in int Surg Am 2014; 96-A:1836–44. primary total knee arthroplasty without tourniquet: a 3. Frisch NB, Wessell NM, Charters MA, Yu S, Jeffri- randomized, controlled trial of oral versus intraveno- es JJ, Silverton CD. Predictors and complications of us versus topical administration. Sci Rep 2018; 8:1-9. blood transfusion in total hip and knee arthroplasty. J 16. Alshryda S, Sarda P, Sukeik M, Nargol A, Blenkin- Arthroplasty 2014; 29(Suppl 9):189–92. sopp J, Mason JM. Tranexamic acid in total knee re- 4. Stanworth SJ, Cockburn HAC, Boralessa H, Contre- placement: a systematic review and meta-analysis. J ras M. Which groups of patients are transfused? A stu- Bone Joint Surg Br 2011; 93:1577-85. dy of red cell usage in London and southeast England. 17. Yuan X, Wang J, Wang Q, Zhang X. Synergistic Vox Sang 2002; 83:352-7. effects of intravenous and intra-articular tranexamic 5. Wells AW, Mounter PJ, Chapman CE, Stainsby D, acid on reducing hemoglobin loss in revision total Wallis JP. Where does blood go? Prospective observa- knee arthroplasty: a prospective, randomized, con- tional study of red cell transfusion in north England. trolled study. Transfusion 2018; 58:982-8. Br Med J 2002; 325:803-4. 18. Kumar N, Saleh J, Gardiner E, Devadoss VG, Howell 6. Parvizi J, Chaudhry S, Rasouli MR, Pulido L, Joshi FR. Plugging the intramedullary canal of the femur A, Herman JH, Rothman RH. Who needs autologo- in total knee arthroplasty: Reduction in postoperative us blood donation in joint replacement? J Knee Surg blood loss. J Arthroplasty 2000; 15:947-9. 2011; 24:25-31. 19. Cerciello S, Robin J, Lustig S, Maccauro G, Hey- 7. Juelsgaard P, Larsen UT, Sørensen JV, Madsen F, se TJ, Neyret P. The role of patelloplasty in total Søballe K. Hypotensive epidural anesthesia in total knee arthroplasty. Arch Orthop Trauma Surg 2016; knee replacement without tourniquet: Reduced blo- 136:1607–13. od loss and transfusion. Reg Anesth Pain Med 2001; 20. Brodell JD, Axon DL, Evarts CM. The Robert Jones 26:105-10. bandage. J Bone Joint Surg Br 1986; 68:776-9. 8. Onodera T, Majima T, Sawaguchi N, Kasahara Y, Is- 21. Wong J, Abrishami A, El Beheiry H, Mahomed NN, higaki T, Minami A. Risk of deep venous thrombosis Davey JR, Gandhi R, Syed KA, Hasan SMO, De Sil- in drain clamping with tranexamic acid and carbazoc- va Y, Chung F. Topical application of tranexamic acid hrome sodium sulfonate hydrate in total knee arthro- reduces postoperative blood loss in total knee arthro- plasty. J Arthroplasty 2012; 27:105–8. plasty: a randomized, controlled trial. J Bone Joint 9. Levy O, Martinowitz U, Oran A, Tauber C, Horo- Surg Am 2010; 92:2503-13. szowski H. The use of fibrin tissue adhesive to re- 22. Ishida K, Tsumura N, Kitagawa A, Hamamura S, duce blood loss and the need for blood transfusion Fukuda K, Dogaki Y, Kubo S, Matsumoto T, Mats- after total knee arthroplasty. A prospective, randomi- ushita T, Chin T, Iguchi T, Kurosaka M, Kuroda R. zed, multicenter study. J Bone Joint Surg Am 1999; Intra-articular injection of tranexamic acid reduces 81:1580–8. not only blood loss but also knee joint swelling af- 10. Gibbons C, Solan M, Ricketts D, Patterson M. ter total knee arthroplasty. Int Orthop (SICOT) 2011; Cryotherapy compared with Robert Jones bandage 35:1639-45. after total knee replacement: A prospective randomi- 23. Seo JG, Moon YW, Park SH, Kim SM, Ko KR. The zed trial. Int Orthop (SICOT) 2001; 25:250-2. comparative efficacies of intra-articular and IV tra- 11. Hiippala S, Strid L, Wennerstrand M, Arvela V, nexamic acid for reducing blood loss during total Mantyla S, Ylinen J, Niemela H. Tranexamic acid knee arthroplasty. Knee Surgery Sport Traumatol Ar- (Cyklokapron) reduces perioperative blood loss asso- throsc 2013; 21:1869–74. ciated with total knee arthroplasty. Br J Anaesth 1995; 24. Nielsen CS, Jans Ø, Ørsnes T, Foss NB, Troelsen A, 74:534-7. Husted H. Combined intra-articular and intravenous 12. Hiippala ST, Strid LJ, Wennerstrand MI, Arvela JV tranexamic acid reduces blood loss in total knee ar- V, Niemelä HM, Mäntylä SK, Kuisma RP, Ylinen JE. throplasty a randomized, double-blind, placebo-con- Tranexamic acid radically decreases blood loss and trolled trial. J Bone Joint Surg Am 2016; 98:835-41. transfusions associated with total knee arthroplasty. Anesth Analg 1997; 84:839-44.

271 Medicinski Glasnik, Volume 18, Number 1, February 2021

25. Zhang YM, Yang B, Sun XD, Zhang Z, Figueiredo 30. Yuan ZF, Yin H, Ma WP, Xing DL. The combined N. Combined intravenous and intra-articular tranexa- effect of administration of intravenous and topical mic acid administration in total knee arthroplasty for tranexamic acid on blood loss and transfusion rate in preventing blood loss and hyperfibrinolysis: A ran- total knee arthroplasty: combined tranexamic acid for domized controlled trial. Med (United States) 2019; TKA. Bone Joint Res 2016; 5:353-61. 98:1-8. 31. Fillingham YA, Ramkumar DB, Jevsevar DS, Yates 26. Yang Z-G, Chen W-P, Wu L-D. Effectiveness and sa- AJ, Shores P, Mullen K, Bini SA, Clarke HD, Sche- fety of tranexamic acid in reducing blood loss in total mitsch E, Johnson RL, Memtsoudis SG, Sayeed SA, knee arthroplasty: a meta-analysis. J Bone Joint Surg Sah AP, Della Valle CJ. The efficacy of tranexamic Am 2012; 94:1153-9. acid in total knee arthroplasty: a network meta- 27. Zhang H, Chen J, Chen F, Que W. The effect of tra- analysis. J Arthroplasty 2018; 33:3090-8. nexamic acid on blood loss and use of blood products 32. Xu S, Chen JY, Zheng Q, Lo NN, Chia SL, Tay KJD, in total knee arthroplasty: A meta-analysis. Knee Sur- Pang HN, Shi L, Chan ESY, Yeo SJ. The safest and gery Sport Traumatol Arthrosc 2012; 20:1742-52. most efficacious route of tranexamic acid administra- 28. Panteli M, Papakostidis C, Dahabreh Z, Giannoudis tion in total joint arthroplasty: A systemic review and PV. Topical tranexamic acid in total knee replace- network meta-analysis. Thromb Res 2019; 176:61-6. ment: A systematic review and meta-analysis. Knee 33. Sun Q Li J, Chen J, Zheng C, Liu C, Jia Y. Comparison 2013; 20:300-9. of intravenous, topical or combined routes of tranexa- 29. Tan J, Chen H, Liu Q, Chen C, Huang W. A meta- mic acid administration in patients undergoing total analysis of the effectiveness and safety of using tra- knee and hip arthroplasty: A meta-analysis of randomi- nexamic acid in primary unilateral total knee arthro- sed controlled trials. BMJ Open 2019; 9:1-14. plasty. J Surg Res 2013; 184:880-7. 34. Moskal JT, Capps SG. Intra-articular tranexamic acid in primary total knee arthroplasty: meta-analysis. J Knee Surg 2018; 31:56-67.

272 ORIGINAL ARTICLE

Radiographic and functional outcome of complex acetabular fractures: implications of open reduction in spinopelvic balance, gait and quality of life Vitaliano F. Muzii1, Giuseppe Rollo2, Guido Rocca3, Rocco Erasmo4, Gabriele Falzarano5, Francesco Liuzza6, Michele Bisaccia7, Giuseppe Pica5, Raffaele Franzese8, Luigi Meccariello5

1Department of Medicine, Surgery, and Neurosciences, Section of Neurosurgery, University of Siena, Siena, 2Department of Ortho- paedics and Traumatology, Vito Fazzi Hospital, Lecce, 3Department of Orthopaedics and Traumatology, Trauma Centre Pietro Cosma, Camposampiero, Padua, 4Department of Orthopaedics and Traumatology, Santo Spirito Hospital, Pescara, 5Department of Orthopaedics and Traumatology, AORN San Pio, Benevento, 6Department of Orthopaedics and Traumatology, A. Gemelli University Hospital, Catholic University, Rome, 7Department of Orthopaedics and Traumatology, Azienda Ospedaliera Santa Maria della Misericordia, Perugia, 8Orthopaedics and Traumatology Unit, Villa del Sole, Caserta; Italy

ABSTRACT

Aim To investigate the effects of surgical reduction of complex acetabular fractures on spine balance, postural stability and quality of life.

Methods Twenty-six patients with acetabular fractures surgically treated by open reduction and internal fixation were divided into two groups according to the amount of reduction. Group A consi- sted of 18 patients with satisfactory reduction (≤2 mm), and group Corresponding author: B of eight patients with incomplete reduction (>2 mm). Functional outcome was measured with Harris Hip Score (HHS), Oswestry Vitaliano Francesco Muzii Disability Index (ODI), and Short Form (12) Health Survey (SF- Department of Medicine, 12). Radiological parameters were assessed with standing whole Surgery, and Neurosciences, spine, pelvis and hip X-rays, including pelvic incidence (PI), pel- University of Siena. vic tilt (PT), sacral slope (SS), and sagittal vertical axis (SVA). V.le Mario Bracci 1, 53100 Siena, Italy Follow-up intervals were 1, 3, 6 and 12 months and annually the- E-mail: [email protected]; reafter. Gait analysis and baropodometry were performed after 24 months of operation. Phone: +39 0577 585252; Fax: +39 0577 586153; Results Mean HHS, ODI, and SF-12 was improved during the first ORCID ID: https://orcid.org/0000-0002- postoperative year in both groups. After two years average scores 2346-8728 kept improving for group A, but worsened for group B. Mean PI, PT, and SS increased in both groups during the first postoperative year, with further increase after two years only in group B. After two years, 16 (89%) patients in group A and four (50%) in group B had a balanced spine (SVA <50 mm). Gait analysis and baro- Original submission: podometry showed greater imbalance and overload for group B 22 October 2020; compared to group A. Revised submission: 26 October 2020; Conclusion In the long term, incomplete reduction of associated acetabular fractures may lead to poor outcome because of secon- Accepted: dary spinopelvic imbalance, with posture and gait impairment. 30 October 2020 doi: 10.17392/1300-21 Key words: acetabulum, fracture, gait analysis, open reduction, postural balance

Med Glas (Zenica) 2021; 18(1):273-279

273 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION Table 1. Patients’ demographics and characteristics Group A Group B Acetabular fractures are increasing, especially Characteristic* (fracture (fracture in the elderly population (1-3). However, even reduction ≤2mm) reduction >2mm) in the young, these fractures may have unfavou- Number of patients 18 8 rable outcomes from the quality of life and so- Mean age (range) (years) 32.3 (16-50) 30.7 (16-50) Male/female (ratio) 15/3 (5:1) 7/1 (7:1) cial health point of view (2,4). Open reduction No (%) of patients with internal fixation (ORIF) is the gold standard Employment of treatment for acetabular fractures in young Agriculture 8 (44.5) 3 (37.5) Industry 8 (44.5) 3 (37.5) adults, while prosthesis in the acute phase is Tertiary sector 2 (11) 2 (25.0) usually not considered in this age group (1,5,6). Type of accident However, even after surgical repair, such fractu- Car accident 14 (77.8) 4 (50) Motorbike accident 4 (22.20) 4 (50) res may lead to malalignment of the acetabulum Energy of trauma: with biomechanical compromise of the hip joint High 18 (100) 8 (100) (7,8). While the impact of misaligned acetabular Low - - Type of fracture (Letournel classification) fractures on the coxofemoral joint is well known “T” fracture 1 (5.5) 1 (12. 5) (9, 10-13), their influence on spine and spine ba- Posterior column+posterior 5 (28) 3 (37.5) lance is underestimated and not clearly explained wall in the literature (14). Transverse+posterior wall 8 (44.5) 2 (25) Anterior wall+hemitransverse 2 (11) 1 (12) The aim of our study was to evaluate retrospec- posterior fracture tively the impact of surgical reduction of com- Both columns 2 (11) 1 (12) Secondary THR 8 (44.5) 8 (100) plex acetabular fractures on sagittal balance, gait *No statistically significant difference was found between the groups analysis, foot loading, and overall quality of life, for all items (p>0.05), except for THR; THR, total hip replacement; with regard to the coxofemoral joint and the spi- ne, in young adult patients. reduction with residual diastasis >2 mm. Patients were treated according to the ethical standard of PATIENTS AND METHODS the Helsinki Declaration and were invited to read, Patients and study design understand and sign the informed consent form. The Azienda Sanitaria Locale Lecce/Italy Ethical From January 2016 to December 2018, 67 pati- Committee approved this research. ents with associated acetabular fractures accor- ding to Letournel classification (12) aged betwe- Methods en 16 and 50 (mean 31.5 years, 22 males and four Outcome measurements included functional and ra- females) were treated with ORIF at five Italian diographic parameters, comparing the two groups. institutions. Twenty-six patients were selected for the study after applying exclusion criteria: signi- Functional outcome was measured with the ficant associated head, chest, or abdomen injury, Harris Hip Score (HHS) (16), Oswestry Disabi- fractures of the lower limbs or spine, injury of the lity Index (ODI) (17), and The Short Form (12) pelvic ring, disease or axial deviation or previous Health Survey (SF-12). Spinopelvic parameters operation of the lower limbs, spinal deformities, (pelvic incidence – PI, pelvic tilt – PT, sacral slo- significant degenerative spine disease, disc her- pe - SS, sagittal vertical axis - SVA, and lumbar niation, spondylolysis or spondylolisthesis, avas- lordosis – LL) were assessed with standing who- cular necrosis of the femoral head throughout the le spine; pelvis and hips X-ray and X-ray of an follow-up (Table 1). affected hip carried out after surgery and during the follow-up. The patients were divided into two groups accor- ding to the degree of fracture reduction after Spinal balance is the result of a lordotic arran- ORIF, as assessed by postoperative three-pro- gement of the lumbar spine above a correctly jection Judet X-rays: group A consisted of 18 oriented pelvis, in such way that the centre of patients with anatomical reduction, i.e. residual gravity of the trunk is supported by the femoral fracture diastasis ≤2 mm (Figure 2A-C), and gro- heads, to maintain balance with minimal muscu- up B with eight patients who had unsatisfactory lar effort. Modifications of sagittal spinal curva-

274 Muzii et al. Outcome of acetabular fracture reduction

tures and changes in pelvic orientation are inter- Functional results connected, with PI index being the key anatomic The average HHS before the trauma was about parameter of sagittal balance regulation, and PT, 98/100 for both groups. At the time of the trauma, SS and LL changing depending on PI and spine both groups showed a severe mean functional alignment (14). The functional and radiographic loss without significant differences. At follow- tests were performed at 1, 3, 6 and 12 months, up, HHS improved until 1 year, with no signi- and annually thereafter. Static and dynamic ba- ficant differences between the groups, to about ropodometry with gait analysis were performed 82/100. However, a statistically significant diffe- at 24-month follow-up to assess loading on the rence occurred 24 months after the trauma, with injured limb. Dynamic pedography was perfor- mean HHS score of 88.6 points in group A, and med on a multifunctional platform on treadmills a worsening 74.6 in group B (p<0.05). During with Zebris system with video recording (FDM- the follow-up, five patients with severe arthrosis THM, GmbH. Munich, Germany). Patients were and three with hypotrophy of the gluteus, abdo- allowed unlimited number of barefoot walks. At minal and paravertebral muscles were found in least five accurate measurements for injured si- the group B; all patients of the group B showed a des were carried out in ten areas (heel, midfoot, significantly limited hip extension. Eight (44.5%) metatarsals 1-5, hallux, second toe, toes 3-5) for patients in the group A and eight (100%) in the pressure, loading, contact time during the roll- group B required secondary total hip replacement over process and force-time integral. Data were (THR) (p<0.05) (Figure 1). analysed and averaged, and gait axis was depic- ted. The average follow-up was 3.2 years (range Functional scores during 24 months of Follow Up 100 SF-12 2-4 years). 90 80 70 Statistical analysis HHS 60 Descriptive statistics were used to summarize the 50 40

characteristics of the study group and subgroups. Mean score 30 T-test was used to obtain continuous outcomes. 20 2 ODI The χ or Fisher exact tests were used to compare 10 0 categorical variables. Statistical significance was Basal 013612 24* defined with p<0.05. The correlation between Time (months) *p<0.05 functional outcome and X-ray was determined Group A Group B by the Cohen's k index. The correlation between Figure 1. The trend of functional scores (HHS, SF-12, and ODI): changing spinopelvic parameters was evaluated until 1-year follow-up scores were similar between the groups; with Pearson’s index. after 2 years there was a significant difference in favour of the group A; HHS, Harris Hip Score; SF, Short Form Health Survey; ODI, Oswestry Disability Index; RESULTS The average ODI before the trauma was 0% in There were no significant differences in demo- both groups. At the time of the trauma, patients graphic and patient’s characteristics between the of both groups had a moderate low back pain wit- groups. Surgery in both groups was performed hout significant difference. At follow-up until 1 between 5 and 7 days after the trauma. The mean year, ODI was similar in both groups A and B, time for anaesthesia and surgical procedures was with minimal to moderate disability. Statistically similar with an average of 160 min (range 120- significant difference appeared at 2-year follow- 180). During the surgery, no impact of the femo- up with ODI of 3.7% in the group A, and 21.7% ral head, no visible subchondral hematoma, and in the group B (p<0.05). After 2 years, all pati- no loss of articular cartilage of more than 25% ents in the group B and only two patients in the were found. The mean time to verticalization was group A were suffering from facet joint syndrome about nine days after the surgery, there were no of L3-L4 and L4-L5 (Figure 1). perioperative complications, and patients were The mean SF-12 score before injury was 100 po- able to flex their hips more than 90° and to sit ints in both groups, with a severe loss at the time down 21 days after surgery. of trauma. One month after trauma the mean SF-

275 Medicinski Glasnik, Volume 18, Number 1, February 2021

12 score was similar both in the groups A and Table 2. Summary of spinopelvic parameters during the B with 42.5 and 42.7 points, respectively, and follow-up kept improving until 1-year follow-up to about Parameter Pre-operativePost-operative 12 months 24 months Pelvic incidence (±SD) 89, without significant difference. Two years af- Group A 50.3° (±10.4) 52° (±10.6) 61.3° (±12.4) 61.9° (±12.7) ter trauma the SF-12 score continued to improve Group B 51.2° (±10.3) 56° (±10.2) 65.8° (±12.7) 74.8° (±12.8) in the group A to a mean score of 93.5, while it p <0.05 Pelvic tilt regressed to 78.6 in the group B, with statistically Group A - 17.4° (±10.2) 23.6° (±14.2) 23.7° (±12.2) significant difference (p<0.05) (Figure 1). Group B - 20.2° (±10.2) 25.8° (±12.6) 30.5° (±13.8) p <0.05 Radiological results Sacral slope (±SD) Group A - 33.4° (±10.2) 37.9° (±12.2) 37.9° (±12.1) Fracture reduction was assessed with postopera- Group B - 35.3° (±10.0) 40.6° (±12.2) 43.4° (±12.1) tive radiographs, with a mean residual diastasis p <0.05 Lumbar lordosis (±SD) of 0.9 mm (range 0-1.9 mm) in the group A, and Group A - - - 63.4° (±9.36) 2.7 mm (range 2-6.2 mm) in the group B. Group B - - - 51.6° (±10.4) p <0.05 All of the three pelvic parameters (PI, SS, and PT) SVA (≤50 mm) (No; %) showed an increase. Mean pre-operative PI was Group A - - - 16 (89) 50.3° and 51.2° in the group A and B, respective- Group B - - - 4 (50) p <0.05 ly (p>0.05); after surgery, average PI was 52° and SVA, sagittal vertical axis; 56° in the groups A and B, respectively (p>0.05), group B, and three patients with flat back syn- rising after 1 year to 61.3° and 65° in the groups, drome in the group A and five in the group B. In respectively (p>0.05); after 24 months, average the statistical analysis of the correlation betwe- PI was stable at 61.9° in the group A, while it fur- en functional and clinical results with Coen’s ther increased to 74.8° in the group B (p<0.05). k, HHS, ODI and SF-12 scores correlated with Mean postoperative SS was 33.4° in the group radiographic results both in the group A (mean A and 35.3° in the group B, rising after 1 year k=0.786) and B (k= 0.784) (p<0.05). to 37.9 and 40.6° in the groups, respectively (p>0.05); after 24 months, SS further increased Baropodometry in the group B only (43.4°) (p<0.05). Mean post- operative PT was 17.4° in the group A, and 20.2° At two-year follow-up, baropodometry revealed in the group B (p>0.05); after 12 months, mean greater static foot overload in the affected side in the PT was 23.6° and 25.8° in the groups, respective- group B compared to A (Figure 2, Figure 3) in the ly (p>0.05); after 24 months, mean PT was stable heel region (176±71 vs 146±53N; p<0.05), and un- at 23.7° in the group A, while it further increased der the fifth metatarsal (99±55 vs 67±44N; p<0.05) to 30.5° in the group B (p<0.05) (Table 2). (Figure 2E, Figure 3B). Also, there was higher for- ce-time integral on foot in the group B compared After 2 years, SVA ≤50 mm indicating a balan- to A (91±8 vs 64±17N·s; p<0.05). Dynamic pedo- ced spine, was found in 16 (89%) patients in the graphy revealed a severe impairment of walking group A (Figure 2D) and only in four (50%) of pattern in the group B: in the heel and first metatar- the group B (Figure 3A) (p<0.05). Moreover, the sal regions, there was greater loading (621±102 vs Pearson’s index showed moderate direct correla- 498±77N, and 181±81 vs 134±76N, respectively tion (ρ =0.56) between the increase of PI, PT XY (p<0.05) and higher force-time integral (177±82 vs and SVA after 2 years in the whole series. When 131±69N·s, and 98±67 vs 78±58N·s, respectively considering group B only, the direct correlation (p<0.05) in the group B compared to group A. ρ was strong ( XY =0.71). Also, there was moderate direct correlation when considering the evolution Overall, in all patients there was a deviation of of PI, PT and SVA over preoperative to 2-year the longitudinal axis towards the affected side ρ with functional overload on the affected foot, but follow-up period ( XY =0.62) (Table 2). these dysfunctions were significantly more pro- Overall, radiographic analysis showed that nor- minent in the group B. Moreover, gait analysis mal lordosis was maintained in 10 patients in the showed that such dysmetria was compensated group A and in one in the group B, five patients during ambulation in the group A (Figure 2F), with hyperlordosis in the group A and two in the

276 Muzii et al. Outcome of acetabular fracture reduction

while in the group B, the greater deviation of the longitudinal axis and the considerable functional overload were not compensated (Figure 3B-C).

DISCUSSION Associated acetabular fractures are the most common fractures in young adults, and also the most difficult to treat operatively, with a high risk of poor outcomes (9,15,18). The effects of misaligned acetabular fractures on coxofemoral joint are well known, with the amount of surgical reduction being crucial (7,8,9, 11-13, 19). Howe- ver, the impact of such fractures on spine balan- ce is less clear, and probably underestimated. In Figure 2. Illustrative case of the group A*. 39-year-old male fact, acetabular fractures and their repair, may sustaining car crash. A) 3D TC: right anterior wall and hemi- transverse anterior fractures; B) Postoperative radiograph alter spinopelvic relationships and lead to spine showing anatomical reduction of the fractures and fixation with imbalance, as well as foot load and gait distur- shaped plate and screws through ileo-inguinal approach. c bance depending on the quality of reduction (9). 6-month follow-up radiograph; C) 1-year follow-up three Judet’s projections radiographs show perfect healing; D) Lateral stand- In this retrospective observational study, we in- ing full spine X-rays after 24 (12) months of operation, depicting vestigated long-term effects of the amount of good sagittal balance (PI 48°, PT 4°, SS 43°, LL 55°, SVA -7 reduction of acetabular fractures on spine and mm); E) Static and F) dynamic baropodometry 24 months after the operation shows right overload compensated during walking hip function and overall outcome. Patients were (Meccariello L, 2017) retrospectively grouped and compared, with the Group A consisted of 18 patients with anatomical reduction, i.e. re- group A receiving anatomical reduction, and the sidual fracture diastasis ≤2 mm; ORIF, open reduction and internal fixation; PI, pelvic incidence; PT, pelvic tilt; SS, sacral slope; SVA, group B resulting in poor reduction. Both HHS sagittal vertical axis; and SF-12 scores improved during follow-up in both groups of patients until one year. At two-year follow-up, however, both scores worsened in gro- up B, while they kept improving in the group A, with significant differences. Consistently, we fo- und in the group B five patients with severe hip de- generation and three with gluteus hypotrophy, and all having very limited hip extension. A consistent body of literature showed that the quality of po- stoperative reduction correlated with HHS, posto- perative arthritis, and need for THR, underscoring the importance of anatomical reduction in achie- ving successful outcomes (3,5, 7-9, 11-13, 20,21). Among acetabular fracture outcomes, low back pain is very common and persistent over years Figure 3. Illustrative case of the group B*. Four years before, a (22). In our series, ODI was significantly worse in 45-year-old woman underwent ORIF of left both columns frac- the long term in patients with unsatisfactory fractu- tures. A) X-ray after plates removal, before hip replacement. re reduction, possibly because of greater changes Advanced spine degeneration and compensatory lumbar hy- polordosis (PI 40°, PT 9°, SS 31°, LL 46°, SVA 8 mm) with a in spinopelvic parameters. Our results showed a decompensation to the right: left arthritic hip, with protrusion of slight increase of mean PI in both groups during the the acetabular head, and right convex lumbar rotoscoliosis; B) first postoperative year. However, after two years, static and C) dynamic baropodometry shows a patent overload while mean PI remained substantially stable in the on the left foot (Meccariello L, 2017) Group B included eight patients who had unsatisfactory reduction group A, there was a further considerable increase with residual diastasis >2 mm; ORIF, open reduction and internal in the group B. We speculated that some bone re- fixation; PI, pelvic incidence; PT, pelvic tilt; SS, sacral slope; SVA, modelling occurred during months after ORIF, with sagittal vertical axis.

277 Medicinski Glasnik, Volume 18, Number 1, February 2021

axial load causing a change in acetabular alignment paper showed impairment of gait, muscle stren- with PI increase. The PI is a fundamental pelvic gth, and functional outcome in patients operated parameter, pivoting three-dimensional regulation for displaced acetabular fracture regardless of of spine sagittal balance (14). When considering the approach, suggesting that gait changes after spinopelvic parameters modification, PT, SS and injury and treatment are related to other factors lumbar lordosis are a function of PI, which is given (25). In our study, incomplete fracture reduction as an anatomical constant. However, in the present correlated with worse outcome in gait and foot study, PI has shown significant changes over time load analysis, indicating the amount of reduc- in the group B, thus inducing unusual modification tion as a crucial factor affecting gait function. of spinopelvic parameters. Expectedly, PI increase Incomplete recovery of hip muscles and loss of was paralleled by an increase of PT and SS, and circumferential resistance to load of repaired the equation PI=SS+PT was fulfilled in all patients acetabulum probably result in overload, and may during the entire follow-up. However, in the group represent a concurring factor of poor functio- B there was a relative greater increase of PT (+51% nal outcome (8,26). The restoration of hip joint on average) compared to SS (+23%). Functionally, centre (HJC) is another important biomechani- PT increase corresponds to pelvic retroversion, cal issue concerning gait function after ORIF. which in turn involves a decrease of SS with the A significant correlation was found between the decrease or loss of lumbar lordosis, resulting in restoration of HJC and the quality of reduction, sagittal imbalance, as defined by anterior displace- and misplacement of HJC may result in alteration ment of C7 plumb line with increase of SVA (23). of hip loading pattern (7,8). Therefore, HJC shi- On the other hand, as shown in healthy subjects, ft due to incomplete fracture reduction may be a with higher PI femoral heads are projected forward possible mechanism for poor gait outcome. relative to the sacrum (6,24). In our study, this may Main limitations of our study are the retrospective explain the need of flattening the back to repositi- design and limited number of the patients. Howe- on the C7 plumb line close to the femoral heads, ver, all patients in this consecutive, multicentric attempting a compensatory balance. Consistently, series matching inclusion criteria were included, after two years, we found 50% of cases with an im- and all underwent the same diagnostic protocol and balanced spine (SVA >50 mm) in the group B com- surgical treatment, thus limiting possible selection pared to only 11% in the group A. The correlation and treatment biases. Another limitation of the stu- found between the increase of PI, and PT and SVA dy is the lack of data about acetabular alignment increase, which becomes strong when considering and its correlation with PI, which could help to group B only, supports our hypothesis that during explain the underlying mechanism of spinopelvic the healing process of the fracture there might be a modifications after ORIF. Hopefully, our findings remodelling causing PI increase. While these chan- might trigger future, prospective investigation. ges seem to be minor and well tolerated after ana- In conclusion, associated acetabular fractures are tomical reduction, with poor reduction they may be challenging, and ORIF remains a mainstay of tre- greater and lead to significant modification of pel- atment, especially in young adults. According to vic parameters, and ultimately result in spinopelvic the literature, the results of our study underscore imbalance. In these patients, the greater increase of the importance of anatomical reduction to achie- PT compared to SS may represent an adaptation of ve favourable outcomes. Long term analysis hip joint in response to long term changes in aceta- showed a significant increase of pelvic incidence bular orientation (14). While changes in acetabular after incomplete fracture reduction, which corre- orientation after ORIF have been studied (7), this lates with spinopelvic imbalance and ultimate hip is, to our knowledge, the first study in which chan- and back disability. ges in PI have been shown. However, further inve- stigation correlating PI and acetabular orientation FUNDING is needed to draw definite conclusions. No specific funding was received for this study To the best of our knowledge, there are not pre- vious reports on the results of baropodometry in TRANSPARENCY DECLARATION. patients with acetabulum fracture. A previous Conflict of interest: None to declare.

278 Muzii et al. Outcome of acetabular fracture reduction

REFERENCES 1. Carta S, Falzarano G, Rollo G, Grubor P, Fortina M, 14. Lazennec JY, Brusson A, Rousseau MA. Hip-spine Meccariello L, Medici A, Riva A, Sampieri L, Ferra- relations and sagittal balance clinical consequences. ta P. Total hip arthroplasty vs osteosynthesis in acute Eur Spine J 2011; 20(suppl 5):686-98. complex acetabular fractures in the elderly: Evalua- 15. Letournel E, Judet R. Fractures of the Acetabulum. tion of surgical management and outcomes. J Acute 2nd ed. New York: Springer-Verlag, 1993. Dis 2017; 6:12-17. 16. Harris WH. Traumatic arthritis of the hip after dis- 2. Nusser M, Holstiege J, Kaluscha R, Tepohl L, Stuby location and acetabular fractures: treatment by mold F, Röderer G, Krischak G. Return to work after arthroplasty. An end-result study using a new met- fractures of the pelvis and the acetabulum. Z Orthop hod of result evaluation. J Bone Joint Surg Am 1969; Unfall 2015; 153:282-8. 51:737-55. 3. Rommens PM, Schwab R. Handrich K, Arand C, 17. Monticone M, Baiardi P, Ferrari S, Foti C, Mugnai Wagner D, Hofmann A. Open reduction and internal R, Pillastrini P, Vanti C, Zanoli G. Development of fixation of acetabular fractures in patients of old age. the Italian version of the Oswestry Disability Index Int Orthop 2020; 44:2123-30. (ODI-I): A cross-cultural adaptation, reliability, and 4. Cimerman M, Kristan A, Jug M, Tomaževič M. validity study. Spine 2009; 34:2090-5. Fractures of the acetabulum: from yesterday to to- 18. Dakin GJ, Eberhardt AW, Alonso JE, Stannard JP, morrow. Int Orthop 2020; Online ahead of print. Mann KA. Acetabular fracture patterns: associati- 5. Kelly J, Ladurner A, Rickman M. Surgical manage- ons with motor vehicle crash information. J Trauma ment of acetabular fractures – a contemporary litera- 1999; 47:1063-71. ture review. Injury 2020; 10:2267-77. 19. Mears DC, Velyvis JH, Chang CP. Displaced ace- 6. Boudissa M, Francony F, Kerschbaumer G, Ruatti tabular fractures managed operatively: indication of S, Milaire M, Merloz P, Tonetti J. Epidemiology and outcome. Clin Orthop Relat Res 2003; 407:173-86. treatment of acetabular fractures in a level-1 trauma 20. Tosounidis TH, Gudipati S, Panteli M, Kanakaris centre: Retrospective study of 414 patients over 10 NK, Giannoudis PV. The use of buttress plates in the years. Orthop Traumatol Surg Res 2017; 103:335-9. management of acetabular fractures with quadrila- 7. Shi HF, Xiong J, Chen YX, Wang JF, Wang YH. teral plate involvement: is it still a valid option? Int Radiographic analysis of the restoration of hip joint Orthop 2015; 39:2219-26. center following open reduction and internal fixation 21. Grubor P, Krupic F, Biscevic M, Grubor M. Contro- of acetabular fractures: a retrospective cohort study. versies in treatment of acetabular fracture. Med Arch BMC Musculoskelet Disord 2014; 15:277. 2015; 69:16-20. 8. Olson SA, Bay BK, Chapman MW, Sharkey NA. 22. Gerbershagen HJ, Dagtekin O, Isenberg J, Martens Biomechanical consequences of fracture and repair N, Ozgür E, Krep H, Sabatowski R, Petzke F. Chro- of the posterior wall of the acetabulum. J Bone Joint nic pain and disability after pelvic and acetabular Surg Am 1995; 77:1184–92. fractures-assessment with the Mainz Pain Staging 9. Matta JM. Fractures of the acetabulum: accuracy of System. J Trauma 2010; 69:128-36. reduction and clinical results in patients managed 23. Meccariello L, Cioffi S, Grubor P, Franzese R, Ci- operatively within three weeks after the injury. J offi R. Di Giacinto S. Flat back syndrome as post Bone Joint Surg Am 1998; 78:1632-45. traumatic or post scoliosis treatment disorder of the 10. Borg T, Hailer NP. Outcome 5 years after surgical spine. Med Inv 2013; 47:51-6. treatment of acetabular fractures: a prospective clini- 24. Boulay C, Bollini G, Legaye J, Tardieu C, Prat- cal and radiographic follow-up of 101 patients. Arch Pradal D, Chabrol B, Jouve JL, Duval-Beaupère G, Orthop Trauma Surg 2015;135: 227-33. Pélissier J. Pelvic incidence: a predictive factor for 11. Giannoudis PV, Grotz MR, Papakostidis C, Dinopo- three-dimensional acetabular orientation-a prelimi- ulos H. Operative treatment of displaced fractures of nary study. Anat Res Int 2014; 2014:594650. the acetabulum. A meta-analysis. J Bone Joint Surg 25. Engsberg JR, Steger-May K, Anglen JO, Borrelli JJr. Br 2005; 87:2-9. An analysis of gait changes and functional outcome 12. Tannast M, Najibi S, Matta JM. Two to 20-year sur- in patients surgically treated for displaced acetabular vivorship of the hip in 810 patients with operatively fractures. J Orthop Trauma 2009; 23:346-53. treated ace- tabular fractures. J Bone Joint Surg Am 26. Borrelli JJr, Goldfarb C, Ricci W, Wagner JM, En- 2012; 94:1559–67. gsberg JR. Functional outcome after isolated aceta- 13. Liebergall M, Mosheiff R, Low J, Goldvirt M, Ma- bular fractures. J Orthop Trauma 2002; 16:73-81. tan Y, Segal D. Acetabular fractures. Clinical outco- me of surgical treatment. Clin Orthop Relat Res 1999; 366:205–16.

279 ORIGINAL ARTICLE

Key factors influencing clinical and functional outcomes in extracapsular proximal femur fractures: the role of early weight- bearing - one-year follow-up cohort of 495 patients

Enrique Sanchez-Munoz, Beatriz Lozano-Hernanz, Daniel Vicente Velarde-Garrido, Leticia Alarma-Bar- cia, Victor Trivino Sanchez-Mayoral, Paula Romera-Olivera, Cristina Lopez Palacios

Orthopaedic Surgery Department, University Hospital Centre of Toledo, Toledo, Spain

ABSTRACT

Aim To establish a correlation between immediate post-surgical weight bearing in extracapsular hip fractures and final functional outcome as well as to study the correlation between immediate post-surgical weight bearing and morbidity and mortality during the first year.

Methods Retrospective observational cohort study including 495 consecutive patients ≥75 years old operated of extracapsu- lar proximal femur fracture. Medical records were reviewed and information of demographic data, radiological evolution, time to weight-bearing, mortality rate, medical and surgical complications and final ambulation status were recorded. Corresponding author: Enrique Sanchez-Munoz Results Patients’ mean age was 87 years; 378 (76.4%) were fema- Orthopaedic Surgery Department, les. One-year mortality rate was 21.2%. Immediate weight bearing University Hospital Centre of Toledo was associated with: decreased hospital stays (7.5 days vs. 9.2 days; p=0.001) and decreased medical complications (78.3% vs. Avenida de Barber 30, 82.3 %; p=0.02). Surgical complications prevalence was compara- ZIP 45004, Toledo, Spain ble (4.4% vs. 7.8 %; p=0.43) within the groups. Despite mortality Phone/fax: +34 925 269 200; rate was lower in patients with immediate weight bearing (21%) E-mail: [email protected] compared with delayed weight bearing (21.4%), the difference ORCID ID: https://orcid.org/0000-0001- was not statistically significant (p=0.9). 7437-6826 Conclusion Immediate weight bearing was associated with shorter hospital stay and fewer medical complications, improving functi- onal outcome. Also, no correlation was found between immediate weight bearing and increased surgical complications or mortality rate during first year after surgery. Original submission: Key words: hip injuries, osteoporotic fractures, recovery of func- 21 September 2020; tion, weight-bearing Revised submission: 11 November 2020; Accepted: 17 November 2020 doi: 10.17392/1276-21

Med Glas (Zenica) 2021; 18(1):280-286

280 Sanchez-Munoz et al. Hip fracture functional outcomes

INTRODUCTION gical complications, morbidity and mortality du- ring the first yearafter surgery. Hip fracture in an elderly patient constitutes a major public health issue with a high and rapidly rising PATIENTS AND METHODS incidence and associates high morbidity and mor- tality rates (1,2). Patients with hip fracture usually Patients and study design suffer functional worsening (2,3). Thus, hip fractu- re represents a substantial health-care burden (4). This retrospective observational cohort stu- dy included 495 patients with proximal femur Proximal femur fractures prevalence have risen fracture type 31.A1, 31.A2 and 31.A3 according significantly during last decades, with a predicted to Arbeitsgemeinschaft für Osteosynthesefragen/ increase of 66% by the year 2021 and an almost Orthopaedic Trauma Association (AO/OTA) cla- double-fold prevalence by 2051, due to the incre- ssification (18) operated at the Orthopaedic Sur- ase in life expectancy (2,5). The incidence of hip gery Department of the University Hospital Cen- fracture in Spain is over 500/100,000 persons per tre of Toledo (Spain) from 1 January 2016 to 31 year in people of 65 years and older (6). It has May 2018. minimum follow-up was 1 year. been estimated that the global cost of hip fractu- res by the year 2050 will rise up to 131.5 million Inclusion criteria were: age 75 years or older (this United States dollars (USD) per year (7). is the minimum age for patient admission into the orthogeriatric unit, a commonly used cutting po- Clinical guidelines of hip fracture encourage imme- int (10,19), fractures type 31.A1 (Figure 1), 31.A2 diate weight-bearing after surgery (8,9). Early (Figure 2) and 31.A3 according to AO/OTA cla- deambulation has a short-term impact, decreasing ssification (18) and minimum follow-up of one postoperative complications and shortening hospi- year. All surgeries where done using intramedu- tal stay (5,7). In the long-term, early deambulation llary fixation with proximal femur nail Gamma3 improves patient autonomy and reduces mortality (Stryker Osteosynthesis, Kiel, Germany) without rate (1,10). On the other hand, immediate weight- exception during the studied period. bearing does not correlate with increased failure neither of osteosynthesis or implant (11-13). The economic burden of delayed weight-bearing has been estimated to an increased cost of 8400 USD per patient and procedure (5). In contrast, a 2011 Cochrane (14) review pointed out the lack of good quality evidence data to establish the best strategy for hip fracture management after surgery. Even though current evidence favours early weight-be- aring after hip surgery, many studies that analyse medical practice and adherence to clinical guide- lines demonstrate irregular compliance with these recommendations (15-17). This is the case at our department, where a low adherence rate to the in- dication of early weight-bearing after extracapsu- lar proximal femur fracture has been noticed. The decision of early or delayed weight-bearing after surgery relies solely on the surgeon advice without established consensus criteria. This situation crea- Figure 1. Anteroposterior X-rays of a left hip pertrochanteric tes favourable conditions to develop a study that 31.A1 fracture (Complejo Hospitalario Universitario de Toledo, 2017) analyses the correlation between early deambula- tion and morbidity and mortality after hip surgery. Exclusion criteria were: fractures with diaphyse- al extension, periprosthetic or peri-implant The aim of the study was to investigate the corre- fractures, pathologic fractures, atypical fractures, lation of early weight-bearing after surgery with poor reduction or osteosynthesis and follow-up the following variables: functional outcome, sur- at another institution. Tip-apex distance, cortical

281 Medicinski Glasnik, Volume 18, Number 1, February 2021

not early weight-bearing, considering the type of fracture, patient’s features, intraoperative findings, surgical outcome and personal clinical experience. The study was approved by the Ethics Committee of the University Hospital Centre of Toledo. Methods Fracture type was assigned after independent eva- luation of anteroposterior and oblique hip x-ray view by two trainees orthopaedic surgeons. When there was discrepancy in the fracture classificati- on, a third senior surgeon (blinded for previous de- cision) was consulted, allocating the patient to the consensus of two of the three evaluators. No case of three-evaluator disagreement was recorded. After hospital discharge, patients were evalua- ted at consultation at one, three, six and twelve months after surgery. Its treating surgeon or one of the main investigators of this study carried out Figure 2. Anteroposterior X-rays of a left hip pertrochanteric 31. A2 fracture (Complejo Hospitalario Universitario de Toledo, 2017) a final evaluation including functional status one year after surgery. contact point of the lag screw at the lateral femo- ral cortex and screw position on the femoral head Functional independence was assessed by Barthel (Figure 3) were considered to evaluate quality of score (23) and cognitive function was assessed osteosynthesis (11, 20-22). Femoral neck-shaft by the Global Deterioration Scale (GDS) (24). angle and fragments displacement were conside- Both scales where recorded by geriatric speci- red to evaluate reduction quality (20). alist at the admission to the Orthogeriatric Unit and during the follow-up in outpatient consults. Weight-bearing in the first 48 hours was considered In Barthel scale, both numeric result and alloca- to be an exposition factor (early weight-bearing). tion into two groups (independent when less than This variable was recorded as dichotomic, alloca- 60 score) were recorded. The GDS scale divides ting patients to the early weight-bearing group or patients into two groups, with those rating 4 or the delayed weight-bearing group. In every pati- more being the group that need assistance. ent, it was the surgeon that decided to prescript or Comorbidities prior the hip fracture where me- asured using the American Society of Anaesthe- siology Scale (ASA) (25), registered by an ana- esthesiologist in the preoperative assessment of the patient. Objective outcomes were measured at one-year final evaluation. Principal outcome was - deam bulation status that intimately correlates with worsening of the functional status. Four catego- ries were defined to evaluate deambulation sta- tus: autonomous deambulation, need of cane or crutches, walker or not-ambulant. Pre-surgical Figure 3. Anteroposterior axial X-rays of a right femur 31.A1 residence (particular house, institutionalized), fracture after osteosynthesis. Adequate nail placement is characterized by an adequate tip-apex distance (less than length of hospital stays (in days), time from 10mm) (noted in both images), contact of the lateral extreme hospital admission to surgery (in days), medical of the lag screw with lateral femoral cortex (right image) and complications, surgical complications, hospital lag screw position on centre-centre quadrant on the femoral head (noted on both images) (Complejo Hospitalario Universita- readmission in the first month after surgery and rio de Toledo, 2017) one-year mortality rate were also recorded.

282 Sanchez-Munoz et al. Hip fracture functional outcomes

Statistical analysis Table 1. Demographic and functional features of early and delayed weight-bearing groups Quantitative variables were presented with me- No (%) of patients in the group dian and standard deviation, qualitative with Variable Delayed weight- Early weight-bea- p percentages. Weight-bearing and complications bearing (231) ring (264) were adjusted as dichotomous variables. An Gender Males 50 (21.6) 67 (25.4) 0.329 analysis was performed with 2 tests for qualita- Females 181 (78.4) 197 (74.6) tive variables and ANOVA for quantitative ones. Barthel scale (23) Independent or two-sided T-Student´s test or 100 24 (10.4) 44 (16.7) >60 -<100 119 (51.5) 130 (49.2) non-parametric Mann-Whitney test were used >40- <60 46 (19.9) 44 (16.7) 0.324 for continuous variables. Contingency table was >20 - <40 30 (13) 35 (13.3) used for dichotomus variables. The p<0.05 was <20 12 (5.2) 11 (4.2) GSD (24) considered statistically significant, with statisti- 1 128 (55.4) 147 (55.7) cal power of 80%. 2 21 (9.1) 27 (10.2) 3 21 (9.1) 13 (4.9) RESULTS 4 21 (9.1) 26 (9.8) 0.348 5 27 (11.7) 25 (9.5) A total of 495 patients met the inclusion criteria 6 11 (4.8) 22(8.3) and were included in the study; 378 (76.4%) were 7 2 (0.9) 4 (1.5) ASA (25) females and 117 (23.6%) males. Mean age was 87 I 5 (2.2) 10 (3.8) years (75-106): 86.3 and 87.4 years in the delayed II 112 (48.5) 113 (42.8) 0.433 and early weight-bearing group, respectively. II 106 (45.9) 128 (48.5) IV 8 (3.5) 13 (4.9) According to data published by the Spanish Sta- Fracture type (AO/OTA) tistical Office (Instituto Nacional de Estadística, 31.A1 53 (23.4) 115 (43.6) INE) (26), during the studied period (2014 to 31.A2 115 (50.7) 128 (48.5) 0.000 31.A3 59 (26) 21 (8.0) 2018) our hospital served an average populati- Deambulation status on of 478,134 people. People older than 74 ye- Autonomous 59 (25.5) 71 (26.9) Cane/Crutches 90 (39.0) 89 (33.7) ars represented 8.1% of the whole population. 0.209 Walker 69 (29.9) 96 (36.4) Among them, 58.7% were females and 41.3% Not-ambulant 13 (5.6) 8 (3.0) were males. In light of these data, the incidence Residence Particular house 171 (74.0) 184 (60.7) of extracapsular hip fracture in elderly populati- 0.286 Institutionalized 60 (26.0) 80 (30.3) on (75 years or older) in the studied population AO/OTA, Arbeitsgemeinschaft für Osteosynthesefragen/Orthopaedic was 567.5/100.000 persons per year. Trauma Association classification (18); Global Deterioration Scale (24); ASA, American Society of Scale (25) Two hundred sixty-four (53.3%) of 495 patients initiated deambulation in the first 48 hours after Early weight-bearing was associated with better fi- surgery and were allocated to the early weight-be- nal ambulatory status: 120 (51%) (CI: 0.57.0.7) pa- aring group, and the remaining patients, 231 were tients with early weight-bearing showed worsening allocated to the delayed weight-bearing group. of their pre-fracture ambulatory status, whereas 136 Both groups were comparable in terms of demo- (63%) (CI:0.57-0.7) patients with delayed weight- graphic characteristics, Barthel score, GDS, pre- bearing worsened their ambulatory status. Also, the surgical deambulation status and pre-surgical re- decrease of ambulatory status was only of one level sidence, thereby delayed weight-bearing was not in 78 (33.6%) patients with early-weight bearing. It associated with a worse functional status (Table1). was also noted that, independently of early or de- layed weight-bearing, 256 (57.1%) patients showed The most common fracture according to the AO/ worsening of at least one level of ambulatory sta- OTA classification (18) (reference) was 31.A2 in tus (Table 2). Early weight-bearing also associated 243 (49.5%) patients, followed by 31.A1 in 168 fewer time to hospital discharge (early deambulati- (34.2%) and 31.A3 in 80 (16.3%) patients. Accor- on 7.5 days (standard deviation, (SD) +/-4.7) vs. de- ding to the fracture type, in the early weight-bea- layed deambulation 9.2 days (SD+/- 6.6 (p=0.001) ring group only 21 (8%) had 31.A3 fracture, whe- and lesser global complications: early deambulati- reas 128 (48.5%) corresponded to 31.A2 fractures on in 206 (78.3%) vs. delayed deambulation in 190 and 115 (43.6%) to 31.A1 (Table 1). (82.3%) patients (p=0.02) (Table 2).

283 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 2. Time to surgical intervention and hospital stay, final DISCUSSION ambulatory status, medical and surgical complications and one-year mortality, and their relation to weight-bearing. Our results are similar to those found in the cu- Groups rrent literature. Patients with early deambulation p Variable Delayed Early weight- demonstrated less deambulation status deteriora- weight-bearing bearing tion (0.51 vs. 0.63), similarly to the studies by Time to surgical intervention 3.2 2.7 0.108 (days) (CI 95%) (CI: 2.8- 3.6) (CI:2.3-3.1) Petros et al. (2) and Barone et al (10). The rate of 9.22 7.50 Stay in hospital (days) (CI 95%) 0.001 complications was lower in early weight-bearing (CI: 8.5 – 10.0) (CI: 6.8 – 8.2) group (78.3%) than in delayed weight-bearing Ambulatory status (No; %) Same 80 (37.0) 112 (48.3) (82.3%), and also hospital stay (7.5 days in early One-level decrease 100 (46.3) 78 (33.6) weight-bearing vs. 9.2 days in delayed weight- 0.045 Two-level decrease 34 (15.7) 39 (16.8) bearing). Even though one-year mortality rate Three-level decrease 2 (0.9) 3 (1.3) Medical complications (No; %) was lower in early deambulation group (21%) Without 41 (17.6) 57 (21.6) than in delayed deambulation group (21.4%) this Minor 124 (53.7) 134 (50.8) 0.02 difference was not statistically significant. Major 66 (28.6) 73 (27.6) Surgical complications (No; %) Demographic characteristics of our population No 202 (92.2) 218 (95.6) are similar to others studies, with a slight increa- Cut-Out 8 (3.7) 4 (1.4) 0.25 se of the percentage of women compared to other Consolidation retard 6 (2.7) 5 (1.3) Pseudarthrosis 3 (1.4) 1 (0.4) series (7,27,28). The cutting point age that defi- Mortality (No; %) 49 (21.4) 53 (21.0) 0.90 nes elderly population is an issue when compa- ring different studies of proximal femur fractu- Mortality rate at one year was 21.2%. Despite res, because the range of age included differs in the fact that mortality rate was lower in the early many of them. In the studied group, mean age is weight-bearing group (21%) than in the delayed 87 years, with an ASA score of II or III in 94.5% weight-bearing group (21.4%) that difference of the patients, which characterizes our popula- was not statistically significant (p=0.904) (Ta- tion as an aged one with low baseline functional ble 2). One-year mortality rates were higher in status, similarly to other authors (10,28). males, 39 (out of 114; 34.2%), than in females, 63 (out of 368; 17.1%) (p=0.00). Patients older International clinical practice guidelines (8,9) than 89 years also presented an increased morta- encourage early weight-bearing because it had lity rate (p=0.01). Complications during hospital demonstrated a favorable short-term impact, de- stay correlated to the increased mortality rates at creasing postoperative complications and hos- one-year (24.6% vs. 6.5%; p=0.00), and to longer pital stay. In the long-term, early deambulation hospital stay (8.8 days vs. 6.3 days) (Table 3). associates with better functional outcome and reduced mortality rate, as many studies had de- Table 3. Correlation between one-year mortality and hospital monstrated (1,10,29). Despite all evidence that stay of 390 patients with and 92 patients without complications sustains early weight-bearing as a better option, Without With Variable p only 53% of the patients of the studied populati- complications complications on initiated early deambulation after hip surgery. No of patients 92 390 Mortality (overall 102; This may be related to the already reported ten- 6 (6.5) 96 (24.6) 0.000 21.2%) (No; %) dency to delay deambulation to avoid hardware Hospital stay (days) 6.3 8.8 0.000 or surgical complications (29) and reflecting that clinical guidelines are not always applied in daily No association was found between early practice as they should be (15-17). weight-bearing and an increase of surgical com- plications: early deambulation in 10 (out of 228; Current literature reports one-year mortality rate 4.4%) vs. delayed deambulation in 17 (out of after hip fracture between 26 to 33% (28,30). In 219; 7.8% (p=0.43) this cohort a 21.2% rate was found, similarly to that reported by Rosso et al. (31) with an 18.8% The rate of surgical complication was 6% (30 one-year mortality rate in a series of 1558 fractu- of 495 patients), with cut-out being the most res. In our patients, both male gender and age common, 2.5% (11/495), followed by pseudoar- over 89 years correlated to increased mortality at throsis 0.9% (4/495) (Table 2).

284 Sanchez-Munoz et al. Hip fracture functional outcomes

one-year, similarly to the report from Frost et al. Early weight-bearing associates with improved fi- (32) with increased mortality rate in males (odds nal ambulatory status, shorter hospital stay, fewer ratio 2.4) and patients over 90 years of age (odds short-term complications and does not increase ratio 8.7). mechanical complications. Overall, early deam- Intra-articular proximal migration of cephalic bulation proves to be an effective and safe thera- screw (cut-out) was the most usual implant-rela- peutic intervention. However, given the limitati- ted complication with an incidence of 2.5% (11 ons of this study, further prospective studies that out of 495 patients). This is a low rate compared evaluate cost-effectivity of early weight-bearing to other studies, where prevalence of cut-out ran- and the relation with patient’s quality of life are ges from 0 to 16% (12,33) without a correlation needed and, specially, studies that elucidate who between screw migration and early weight-be- are the patients that will benefit from delayed aring has been demonstrated. The lack of influ- weight-bearing, if there are any. ence of early weight-bearing with cut-out is also ACKNOWLEDGEMENTS supported in the study by Zuckerman et al. (34) demonstrating that forces through the hip were the The authors would like to thank Dr Barrero C, Dr same with deambulation and movements in bed. Blanco A, and Dr Araujo M, and all other mem- More recently Eberle et al. (35), based on a bio- bers of the Orthogeriatric Unit. Without them this mechanical model using Gamma3 nails (Stryker study would have not been possible. The authors Osteosynthesis, Kiel, Germany), found that “even would like to thank Dr Félix Sánchez Sánchez, in the absence of fracture healing the implants wo- Head of the Department of Orthopaedic Surgery, uld not fail during the first 100 days after surgery, for his support to the investigation and to all assuming 5000 cycles of walking per day” (35). other members of the Department. Koval et al. (13) also stated that early weight-bea- ring is safe and it is not associated with an increase FUNDING of mechanical complications. No specific funding was received for this study. This study has limitations in being a retrospec- TRANSPARENCY DECLARATION tive, observational cohort study. In addition, analysis of surgical complications could not be Conflict of interest: None to declare. performed due to insufficient sample size.

REFERENCES 1. Morri M, Forni C, Marchioni M, Bonetti E, Marseglia 6. Librero J, Peiró S, Leutscher E, Merlo J, Bernal-Del- F, Cotti A. Which factors are independent predictors gado E, Ridao M, Martínez-Lizaga N, Sanfélix-Gi- of early recovery of mobility in the older adults’ po- meno G. Timing of surgery for hip fracture and in- pulation after hip fracture? A cohort prognostic study. hospital mortality: a retrospective population-based Arch Orthop Trauma Surg 2018; 138:35–41. cohort study in the Spanish National Health System. 2. Petros RSB, Ferreira PEV, Petros RSB. Influence of BMC Health Serv Res. 2012; 12:15. proximal femur fractures in the autonomy and mor- 7. Sherrington C, Lord SR, Herbert RD. A randomized tality of elderly patients submitted to osteosynthesis controlled trial of weight-bearing versus non-weight- with cephalomedullary nail. Rev Bras Ortop 2017; bearing exercise for improving physical ability after 52(Suppl 1):57–62. usual care for hip fracture. Arch Phys Med Rehabil 3. Pareja Sierra T, Bartolomé Martín I, Rodríguez Solís 2004; 85:710–6. J, Bárcena Goitiandia L, Torralba González de Suso 8. Scottish Intercollegiate Guidelines Network. Preven- M, Morales Sanz MD, Calvo H. Predictive factors of tion and management of hip fracture in older people: hospital stay, mortality and functional recovery after a national clinical guideline. Scottish Intercollegiate surgery for hip fracture in elderly patients. Rev Esp Guidelines Network; 2002 https://pdf4pro.com/view/ Cir Ortop Traumatol 2017; 61:427–35. part-of-nhs-quality-improvement-scotland-67a27. 4. Adeyemi A, Delhougne G. Incidence and Econo- html (30 November 2018) mic Burden of intertrochanteric fracture: a medicare 9. Ftouh S, Morga A, Swift C, Guideline Development claims database analysis. JBJS Open Access 2019; Group. Management of hip fracture in adults: 4:e0045. summary of NICE. BMJ 2011; 21:d3304. 5. Wu J, Kurrle S, Cameron ID. Restricted weight be- 10. Barone A, Giusti A, Pizzonia M, Razzano M, Oliveri aring after hip fracture surgery in the elderly: Eco- M, Palummeri E, Pioli G. Factors associated with an nomic costs and health outcomes. J Eval Clin Pract immediate weight-bearing and early ambulation pro- 2009; 15:217–9. gram for older adults after hip fracture repair. Arch Phys Med Rehabil 2009; 90:1495–8.

285 Medicinski Glasnik, Volume 18, Number 1, February 2021

11. Hsueh KK, Fang CK, Chen CM, Su YP, Wu HF, Chiu 24. Reisberg B, Ferris SH, de Leon MJ, Crook T. The FY. Risk factors in cutout of sliding hip screw in inter- Global Deterioration Scale for assessment of pri- trochanteric fractures: An evaluation of 937 patients. mary degenerative dementia. Am J Psychiatry 1982; Int Orthop 2010; 34:1273–6. 139:1136–9. 12. Andruszkow H, Frink M, Frömke C, Matityahu A, 25. ASA House of Delegates, Executive Committee. ​ASA Zeckey C, Mommsen P, Suntardjo S, Krettek C, Hil- Physical Status Classification System | American So- debrand F. Tip apex distance, hip screw placement, and ciety of Anesthesiologists (ASA)2014 https://www. neck shaft angle as potential risk factors for cut-out asahq.org/standards-and-guidelines/asa-physical-sta- failure of hip screws after surgical treatment of inter- tus-classification-system (30 November 2018). trochanteric fractures. Int Orthop 2012; 36:2347–54. 26. Consejería de Sanidad. Catálogo de Hospitales y Alta 13. Koval KJ, Friend KD, Aharonoff GB, Zukerman JD. Tecnología | Gobierno de Castilla-La Mancha, 2020 Weight bearing after hip fracture: a prospective series (Regional Health Department. Hospitals and High of 596 geriatric hip fracture patients. J Orthop Trauma Technology Catalogue. Government of Castilla-La 1996; 10:526–30. Mancha) [In Spanish] https://www.castillalamancha. 14. Handoll HH, Sherrington C, Mak JC. Interventi- es/gobierno/sanidad/estructura/dgspoeis/actuaciones/ ons for improving mobility after hip fracture sur- catálogo-de-hospitales-y-alta-tecnología (7 Novem- gery in adults. Cochrane database Syst Rev 2011; ber 2020) (3):CD001704. 27. Mesa Ramos M. Tratamiento multidisciplinar de 15. Grimshaw JM, Russell IT. Effect of clinical guideli- las fracturas de cadera (Multidisciplinary treatment nes on medical practice: a systematic review of rigo- of hip fractures) [In Spanish] Madrid: Multimedica rous evaluations. Lancet 1993; 342:1317–22. Proyectos, 2009. 16. Carlson VR, Ong AC, Orozco FR, Hernandez VH, 28. Sanz-Reig J, Salvador Marín J, Pérez Alba JM, Lutz RW, Post ZD. Compliance with the AAOS Gu- Ferrández Martínez J, Orozco Beltrán D, Martínez idelines for treatment of osteoarthritis of the knee. J López JF. Factores de riesgo de mortalidad intra- Am Acad Orthop Surg 2018; 26:103–7. hospitalaria en la fractura proximal de fémur (Risk 17. Leigheb F, Vanhaecht K, Sermeus W, Lodewijckx C, factors for mortality during hospital stay in proximal Deneckere S, Boonen S, Faria Boto PA, Veloso Men- femur fracture) [In Spanish] Rev Esp Cir Ortop Trau- des R, Panella M. The effect of care pathways for hip matol 2017; 61:209–15. fractures: a systematic review. Calcif Tissue Int 2012; 29. Ariza-Vega P, Jiménez-Moleón JJ, Kristensen MT. 91:1–14. Non-weight-bearing status compromises the functio- 18. Muller ME, Koch P, Nazarian S, Schatzker J. The nal level up to 1 year after hip fracture surgery. Am J Comprehensive Classification of Fractures of Long Phys Med Rehabil 2014; 93:641–8. Bones. Berlin Heidelberg: Springer, 1990. 30. Fu MC, Boddapati V, Gausden EB, Samuel AM, Ru- 19. Pareja Sierra T, Bartolomé Martín I, Rodríguez Solís ssell LA, Lane JM. Surgery for a fracture of the hip J, Bárcena Goitiandia L, Torralba González de Suso within 24 hours of admission is independently asso- M, Morales Sanz MD, Hornilos Calvo M. Factores ciated with reduced short-term post-operative compli- determinantes de estancia hospitalaria, mortalidad y cations. Bone Joint J 2017; 99-B(9):1216–22. evolución funcional tras cirugía por fractura de cade- 31. Rosso F, Dettoni F, Bonasia DE, Olivero F, Mattei L, ra en el anciano (Decisive Factors for hospital stay, Bruzzone M, Marmotti A, Rossi R. Prognostic factors mortality and functional outcome after hip fracture in for mortality after hip fracture: Operation within 48 the elderly patient) [In Spanish] Rev Esp Cir Ortop hours is mandatory. Injury 2016; 47:S91–7. Traumatol 2017; 61:427–35. 32. Frost SA, Nguyen ND, Black DA, Eisman JA, 20. Baumgaertner MR, Curtin SL, Lindskog DM, Keggi Nguyen T V. Risk factors for in-hospital post-hip JM. The value of the tip-apex distance in predicting fracture mortality. Bone 2011; 49:553–8. failure of fixation of peritrochanteric fractures of the 33. Bojan AJ, Beimel C, Taglang G, Collin D, Ekholm hip. J Bone Joint Surg Am 1995; 77:1058–64. C, Jönsson A. Critical factors in cut-out complicati- 21. Herman A, Landau Y, Gutman G, Ougortsin V, on after gamma nail treatment of proximal femoral Chechick A, Shazar N. Radiological evaluation of fractures. BMC Musculoskelet Disord 2013; 14:1. intertrochanteric fracture fixation by the proximal fe- 34. Zuckerman JD, Koval KJ, Aharonoff GB, Skovron moral nail. Injury 2012; 43:856–63. ML. A functional recovery score for elderly hip 22. Abram SGF, Pollard TCB, Andrade AJMD. Inadequ- fracture patients: II. Validity and reliability. J Orthop ate “three-point” proximal fixation predicts failure of Trauma 2000; 14:26–30. the Gamma nail. Bone Jt J 2013; 95 B:825–30. 35. Eberle S, Gerber C, Von Oldenburg G, Hungerer S, 23. Baztán JJ, Pérez del Molino J, Alarcón T, San Cristo- Augat P. Type of hip fracture determines load share in bal E, Izquierdo G, Manzarbeitia J. Índice de Barthel: intramedullary osteosynthesis. Clin Orthop Relat Res Instrumento válido para la valoración funcional de 2009; 467:1972–80. pacientes con enfermedad cerebrovascular (Barthel Index: A valid instrument for functional assessment of patients with cerebral vascular disease) [In Spa- nish] Rev Esp Geriatr Gerontol 1993; 28:32–40.

286 ORIGINAL ARTICLE

Effectiveness of teriparatide combined with the Ilizarov technique in septic tibial non-union

Giuseppe Rollo1, Francesco Luceri2, Gabriele Falzarano3, Carlo Salomone4, Enrico Maria Bonura5, Dmitry Popkov6, Mario Ronga7, Giuseppe Pica3, Michele Bisaccia8, Valentina Russi1, Predrag Grubor9, Raffaele Franzese10, Giuseppe M. Peretti2,11, Luigi Meccariello3

1Department of Orthopaedics and Traumatology, Vito Fazzi Hospital, Lecce, 2Istituto di Ricovero e Cura a Carattere Scientifico IRCCS Ortopedico Galeazzi, Milan, 3Department of Orthopaedics and Traumatology, AORN SAN PIO “Gaetano Rummo Hospital”, Benevento, 4Malattie Infettive Osteo-Articolari MIOS, S. Maria di Misericordia Hospital, Savona, 5Department of Orthopaedics and Traumatology, Poliambulanza Foundation Hospital, Brescia; Italy, 6Russian Ilizarov Scientific Centre “Reconstructive Traumatology and Orthopaedics”, Kurgan, Russia, 7Department of Medicine and Health Sciences 'Vincenzo Tiberio' University of Molise, Campobasso, 8Division of Orthopaedics and Trauma Surgery, University of Perugia, “S. Maria della Misericordia” Hospital, Perugia; Italy, 9School of Medicine, University of Banja Luka, Bosnia and Herzegovina, 10Orthopaedics and Traumatology Unit, Villa del Sole Caserta, Caserta, 11Department of Biomedical Sciences for Health, University of Milan, Milan; Italy

ABSTRACT

Aim The septic non-union is a common compliance in bone hea- ling due to bone infection. Bone resection, associated with Ilizarov osteo-distraction technique, is commonly used in these cases. The aim of this study was to analyse clinical and radiological results of teriparatide in combination with the Ilizarov technique and to Corresponding author: compare this treatment with the standard treatment. Enrico Maria Bonura Methods Forty adult patients underwent surgery because of type Department of Orthopaedics and C of the Association for the Study and Application of Methods Traumatology, Poliambulanza Foundation of Ilizarov (ASAMI) classification non-union were enrolled. The Hospital, Via Leonida Bissolati, 57, 25124 patients were divided in two groups: those treated with Ilizarov technique (Norm group) and those treated with Ilizarov technique Brescia BS, Italy combined with teriparatide injection (Teri group). Surgical durati- Telefono: 030/3518716 on, complication rate, bone healing status, clinical and functional Phone: +39 3807777577; outcomes were assessed according to the A.S.A.M.I. classification E-mail: [email protected] in the mean follow-up of 12 months. The subjective quality of life Giuseppe Rollo ORCID ID: https://orcid. was assessed by the Short Form Survey (SF)-12. org/0000-0003-1920-1286 Results Teri group showed less time wearing Ilizarov's frame (p <0.05) than the Norm group and a statistical significance in the in- ter-rater reliability Cohen’s k (p>0.05) respect to Norm according the score between the bone healing and clinical outcome results. Original submission: There was no statistically significant difference between the two 24 September 2020; groups in other parameters that were assessed. Revised submission: Conclusion A benefit of teriparatide was found as adjuvant in the 12 October 2020; treatment of septic non-union. Accepted: 12 November 2020 Key words: bone regeneration, Ilizarov technique, teriparatide, doi: 10.17392/1280-21 tibia, limb salvage, osteomyelitis

Med Glas (Zenica) 2021; 18(1):287-292

287 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION and 2018. A total of 112 tibial non-unions were treated. Forty patients aged between 18 to 65 ye- Non-union is defined as a persistent failure of the ars, with the Type C of Association for the Study healing process of fractures, generally after 6-8 and Application of the Method of Ilizarov (ASA- months (1). This is often a delayed complication MI) non-union (9) were enrolled in the study. of fracture; the incidence of long bone non-uni- Other 72 patients were excluded according the ons has been estimated to 5-10% (1). Septic non- exclusion criteria: concomitant systemic disease union is defined as absence of the bone healing patients, acute infection, type A and B ASAMI of process and concomitant infection of the fracture bilateral tibial non-union. site for 6-8 months (2). Infected non-unions of tibia are a challenging clinical condition for both The patients were divided in two groups: 20 pati- orthopaedic surgeons and patients. In this com- ents treated with Ilizarov Technique (Norm group) plex scenario it is possible to find recalcitrant and those treated with Ilizarov technique combi- infection, complex deformities, sclerotic bone ned with teriparatide injection (Teri group). ends, large bone gaps, limb shortening, and joint All patients were assessed in a multidisciplinary stiffness (2). A correct bone and soft tissue surgi- clinic, comprising orthopaedic and plastic sur- cal debridement, associated with Ilizarov osteo- geons and infectologists. Antibiotic therapy was distraction technique, is commonly used in these stopped at least 14 days before the surgery to aid cases (3), as reported in war surgery (4,5). microbiologic diagnosis. Although the pathophysiology of non-union is lar- The Non-Union Scoring System (NUSS) (1) gely unknown, certain risk factors have been well was used to classify the non-union in retrospec- reported. Mechanical causes like low-grade fractu- tive mode1. re stability or repeated manipulations of a fracture The study was performed according to the crite- may delay the healing process. Systemic risk fac- ria set by the Helsinki Declaration, every patient tors like diabetes, smoking, osteoporosis, and estro- read, understood and signed a dedicated infor- genic deficiency reduce the chance of recovery (6). med consent. Recently the use of bone-regenerating adjuvant Clinical, surgical (postoperative complication drug and bioengineering therapy is of growing im- rate, surgical duration) and radiological outco- portance (6). One of these innovative therapeutic mes were retrospectively evaluated at a mini- possibilities is the use of teriparatide, a recombinant mum 12-month follow-up after the Ilizarov fra- human parathyroid hormone, that was proposed for me removal. the treatment of postmenopausal osteoporosis (7). Bone tissue and functional outcomes were evalu- Several studies reported the role of teriparatide ated according to the ASAMI classification, whi- on bone tissue (6-8). A randomized double-blind- le the Short Form 12 Health Survey (SF-12) was ed study tasted its beneficial effects on fracture used to assess the subjective quality of life after repair in in postmenopausal women with frac- surgery (9-10). tures of the distal radius; the authors noticed a Azienda Sanitaria Locale (ASL) Lecce/Italy Et- reduced time of fracture healing in a group of hical Committee approved this research. patients treated with 20 ηg/day of teriparatide compared with placebo (8). Methods The aim of this study was to analyse the role of Surgical technique. Firstly, the accurate evalua- teriparatide combined with the Ilizarov oestro- tion of fracture stability was performed: the non- genic distraction and to compare this therapeutic union was defined “stiff” if there was an angular option with the isolated Ilizarov technique. bending of less than 7° and/or axial movement PATIENTS AND METHODS of less than 5 mm. Adequate surgical debride- ment (Figure 1, Figure 2) until the healthy and Patients and study design bleeding bone tissue was evident. The medullary canal was reamed. Skeletally mature patients who enrolled in this Bone compression was performed in cases of retrospective study were operated between 2006 mobile non-unions with segmental bone loss af-

288 Rollo et al. Teriparatide and Ilizarov in septic tibial non-union

Figure 1. 45-year-old male with surgical wound complication at 4 months after proximal tibial fracture treated with straight plate and screws, in another hospital A,B) Cutaneous defect presentation; C,D) Radiographs preoperative; E) Bone resection; F) implant of modular external fixator; G) for healing the skin; H,I) implant of Ilizarov's frame; J,K) docking point; L) good clinical and radiographic results according to ASAMI classification; M) cutaneous defect healing (Falzarano G. 2014)

Figure 2. 42-year-old male. A, B) open fracture according Gustilo Anderson classification type IIIB of the distal leg extremity treated with external fixator frame; C) After 6 months there was Cierny Mader IV A: widespread bone marrow and cortical infection with multiple seizures on the bone periphery but free from systemic pathologies or severe local impairment; D, E) Bone resection; F,G,H) Implantation of the Iizarov’s frame; I, J) Proximal corticotomy after 4 weeks; K) X-ray of regenerated bone after 3 weeks of teriparatide injection; L) X-rays after removed of Ilizarov’s frame shows an excellent regeneration is an excellent result according to ASAMI clas- sification; M, N) Excellent clinical and aesthetic results (Meccariello L. 2016) ter excision. In cases of severe posttraumatic de- Postoperative protocol. In the postoperative formities, the fibular osteotomy was performed period, the bone transport technique was perfor- before the surgical reduction and compression med. Segment transport started at 7 days after of the fracture. In all patients, skin suture was surgery (1 mm/day). performed, either directly or using local or free The patients were treated with a standard postop- microvascular muscle flaps to restore a healthy erative antibiotic therapy (vancomycin and me- soft tissue envelope. ropenem) and then switched to a culture-specific

289 Medicinski Glasnik, Volume 18, Number 1, February 2021

antimicrobial therapy until the normalization of Table 1. Description of the Norm and Teri groups No (%) of patients in the the inflammatory markers in blood test. Knee and Characteristic group p ankle joints mobilization started on the second Description of Population Norm Teri day after surgery and early full-weight-bearing Number of patients 20 20 1.000 was encouraged. Radiographs were performed Average age (SD) (years) 39.55 (±4.10) 39.84 (±4.11) 0.678 2 weeks during the distraction period and month- Age range (years) 22-65 23-65 0.831 Gender (ratio M:F) 9 (18:2) 9 (18:2) 1.000 ly during the consolidation period. In the Teri Type of fracture 1.000 group, the subcutaneous injection of teriparatide Closed 5 (25.00) 5 (25.00) 20 μg/day for three months was applied, when Open 15 (75.00) 15 (75.00) Type of open fracture according Gustilo Anderson starting from the contact of the docking-site. I 1 (6.67) 1 (6.67) 1.000 The Ilizarov frame was removed when a solid II 3 (20.00) 3 (20.00) 1.000 IIIA 5 (33.33) 5 (33.33) 1.000 docking-site union and a minimum of three com- IIIB 5 (33.33) 5 (33.33) 1.000 plete cortices regeneration was evident at x-rays. IIIC 1 (6.67) 1 (6.67) 1.000 Average time (SD) from 3.54 3.87 Statistical analysis fracture to chronic infection 0.598 (±1.24; 2-8) (±1.26; 2-8) (range) (years)

Continuous variables were expressed as the Type of non-union according ASAMI classification mean±standard deviation (SD) as appropriate. C 20 (100) 20 (100) 1.000 The Shapiro-Wilk normality test was used to eva- Type Cierny Mader osteomyelitis’s classification luate the normal distribution of the sample. The Stage 3A 4 (20) 4 (20) 1.000 Stage 3B 6 (30) 5 (25) 0.637 t test was used to compare continuous parame- Stage 4A 5 (25) 5 (25) 1 ters. The Fisher exact test were used to compare Stage 4B 5 (25) 6 (30) 0.637 Average non-union scoring 69.32 69.72 Categorical variables (in the groups smaller than 0.053 System (SD; Range) (±3.40; 51-84) (±3.39; 51-84) 10 patients). A correlation between preoperative SD, standard deviation; ASAMI, Application of the Method of Ilizarov; radiographic indices and lowering effect in pa- The reliability and validity of the correlation between bone regenerate/ tellar height was calculated using the Pearson’s bone healing and X-ray was determined by the Cohen’s kappa (k) correlation. The study sample size was conside- 42-90) days. No statistically significant differen- red sufficient to evaluate a difference in post- to ces were reported in the complication rate. The pre-operative measurements greater than 0.5 SD mean treatment duration with Ilizarov frame was units with power >80%. 19.24 months (±10.34; range 9–32) in the Norm, The reliability and validity of the correlation while 16.24 months (±7.83; range 9–31) in the between bone regenerate/bone healing and X- Teri group (p<0.05) (Table 2). rays were determined by the Cohen’s kappa (k). The Teri group had a statistical significance in the Statistical significance was set at p <0.05. inter-rater reliability Cohen’s k (p>0.05) respect to the Norm according the score between the bone RESULTS healing and clinical outcome results (Table 2). The two study groups reported no statistically According to the ASAMI classification the significant differences in age, gender, mean sur- same results in both groups were found (Table gical time, bone resection, bone transport time, 3): excellent in eight (40%), good in six (30%), External Fixation Index (Table 1). moderate in six (30%) patients; no patients with The mean follow-up was 1.7 years (±0.6; range poor result. At 12-month follow-up the SF-12 in 1-5) in the Norm group and 1.6 years (±0.5; ran- the Norm group was 73.6 (range 62-90), and in ge 1-5) in the Teri group. The mean surgical time the Teri group, the SF-12 was 76.7 (range 64-90) was 230.2 (±28.8; range 164-284) minutes in the (p>0.05). At the moment of total weigh bearing Norm group and 230.6 (±28.7; range 162-283) in the SF-12 score in the Norm group was 66.3 the Teri group. The average bone resection was (range 40-84), while in the Teri group it was 66.4 9.2 (±3.75, range 5.2-15.3) cm in the Norm, whi- (range 40-84), p>0.05. At 12 months after Iliza- le 9.1 (±9.22 range 5.2-15.2) in the Teri group rov frame removal SF-12 was 82.9 (range 64-92) (p>0.05). The mean bone transport time was 79.4 in the Norm group, while it was 82.3 (range 64- (±8.34; range 44-92) days in the Norm group 92) in the Teri group (p>0.05). while in the Teri group it was 79.7 (±8.30; range

290 Rollo et al. Teriparatide and Ilizarov in septic tibial non-union

Table 2. Results and outcome in the Norm and Teri groups DISCUSSION Characteristic Norm group Teri group p The mean follow-up The main finding of the study was a benefit of after removal of the 1.7 1.6 0.05 teriparatide as adjuvant in the treatment of sep- Ilizarov’s apparatus (±0.6; 1-5) (±0.5; 1-5) (SD; range) (years) tic non-union. There was a difference in time to Average surgical time 230.2 230.6 0.05 remove Ilizarov frame between two groups and a (SD; range) (minutes) (±28.8 164 -284) (±28.71; 62 -283) Average bone resection 9.2 9.1±9.22 statistically significant correlation in Bone Rege- 0.05 in cm (SD; range) (cm) (±3.75; 5.2-15.3) (5.2-15.2) nerate-Bone Healing/X-rays. Bacteriological cause of infection (No; %) N/A In large patients’ cohorts, 4.9% of non-union rate Methicillin-resistant 5 (25) 5(25) Staphylococcus aureus (scaphoid 15.5%, tibia 14% and femur 13.9%) Staphylococcus aureus 2 (10) 2 (10) were reported (11). Therefore, all strategies that Pseudomonas spp. 3 (15) 3 (15) help to reduce healing time with faster resumption Enterobacter cloacae 2 (10) 2 (10) Escherichia coli 3 (10) 3 (10) of work and activities not only improve medical Klebsiella spp. 1 (5) 1 (5) outcome for the patient, they also reduce the finan- Enterococcus faecalis 1 (5) cial burden in fracture and non-union patients (11). Acinetobacter baumanii 1 (5) Streptococcus spp. 1 (5) 1 (5) Several studies investigating the effect of pa- Proteus mirabilis 1 (5) rathyroid hormone in accelerating bone formati- Morganella morganii 1 (5) Enterococcus sp. 1 (5) 1 (5) on in animal models found an increase of bone Average time of bone mineral density (BMD) by 24–33% and mecha- 79.4 79.7 transport took in days 0.05 (±8.34; 44-92) (±8.30; 42-90) nical stiffness and load to failure increased by (SD; range) Average External Fixa- over 50% (12). 0.97±0.47 0.94±0.53 tion Index (SD; range) 0.05 (0.44–1.76) (0.36–1.80) In humans, daily subcutaneous injection of teri- (months/centimetres) Type of complication paratide has been used to accelerate bone-healing Blood loss 1215±160.30 mL 1218±159.28 mL 0.0638 process. Patients treated with teriparatide healed Intra operative fracture 0 0 1.000 in 7.8 weeks, while the control group without te- Loosening of wires 2 (10) 2 (10) 1.000 or pins riparatide injection healed in 12.6 weeks and re- Limb shortening (from 6 (30) 6 (30) 1.000 ported worse functional outcomes (13). Another 1 to 2.9 cm) Local skin inflammation 6 (30) 6 (30) 1.000 study analysed 34 patients with osteoporotic dis- Docking point skin 3 (15) 3 (15) 1.000 tal radius fracture, treated with 0.20 mg teripara- retraction Retard to consolidation tide who healed in 7.4 weeks, whereas the control 4 (20) 4 (20) 1.000 of bone regenerate group healed in 9.1 weeks. Clinical scores were Average correlation k=0.815755 k=0.817252 between bone regenera- p<0.042 better in teriparatide patients and no difference ±0.104632 ±0.056518 te-bone healing/ X-rays in grip strength was reported (8). Teriparatide is Average time for remo- 19.24±10.34 16.24± 7.83 an anabolic drug that can help bone healing, but val of Ilizarov’s frame 0.036 (9–32). (range 9–31) (SD; range) (months) there is no consensus on the clinical indication. N/A, non-applicable Investigating 16 cases of septic tibial bone de- fects that underwent bone segment transport and Table 3. Outcome of patients in the Norm and Teri groups 8 weeks of treatment with daily subcutaneous 0.20 according to the application of the Method of Ilizarov (ASAMI) classification μg teriparatide injections followed by 8 weeks No (%) of pati- with no treatment, or 8 weeks with no treatment Outcome ents in the group p followed by 8 weeks with daily subcutaneous Norm Teri 0.20 μg teriparatide injection, the authors noticed Excellent: Union, no infection, deformity 8 (40) 8 (40) 1.000 that teriparatide during the consolidation phase <7°, limb length discrepancy (LLD) <2.5 cm Good: Union plus any two of the doubled the mineralization rate of the regenerate, following: absence of infection, deformity 6 (30) 6 (30) 1.000 compared to no treatment (14). Teriparatide use <7°, (LLD) <2.5 cm. should be limited to selected patients presenting Fair: Union plus any one of the following: severe forms of osteoporosis, presence or history absence of infection, deformity <7°, LLD 6 (30) 6 (30) 1.000 <2.5 cm. of multiple fractures, exposed high risk for subse- Poor: Nonunion/refracture/union plus infec- quent fractures, or patients with osteoporosis resis- 0 0 1.000 tion plus deformity >7° plus LLD >2.5 cm tant or intolerant to other specific therapies (15). LLD, Limb length discrepancy;

291 Medicinski Glasnik, Volume 18, Number 1, February 2021

Abuomira et al. compared Taylor spatial frame The limited number of enrolled patients and the trifocal and bifocal techniques for the treatment retrospective nature of the study were the most of seven segmental bone defects of the tibia; the important study limitations. docking site ossification phase was gradually sti- In conclusion, the use of teriparatide off-label mulated (16). The simple compression procedu- has a positive and additive effect when combi- re requires less invasive surgery and is probably ned with the Ilizarov technique. Positive effect less demanding and more cost-effective in short of teriparatide on fracture healing is well-docu- transports (10,16). mented, however, further studies are needed to In our study, teriparatide aimed to accelerate the confirm these promising hypotheses. bone healing and remove the Ilizarov frame fast- er. The 3-8 month teriparatide treatment (20 μg/ FUNNDING day) helps the non-unions consolidation in long No specific funding was received for this study. bone non-unions as well as in the animal mod- els (12, 17-20). The efficiency of this treatment TRANSPARENCY DECLARATION seems to be associated with patient comorbidities Conflict of interest: None to declare. comparing to the isolated Ilizarov technique (17).

REFERENCES 1. Calori GM, Colombo M, Mazza EL, Mazzola S, 11. Hak DJ. Editorial on "Epidemiology of fracture no- Malagoli E, Marelli N, Corradi A. Validation of the nunion in 18 human bones". Ann Transl Med 2017; Non-Union Scoring System in 300 long bone non- 5(Suppl 1):S19. unions. Injury 2014; 45(Suppl 6):S93-7. 12. Seebach C, Skripitz R, Andreassen TT, Aspenberg 2. Chaudhary MM (2017) Infected non-union of tibia. P. Intermittent parathyroid hormone (1-34) enhances Indian J Orthop 2017; 51:256-68. mechanical strength and density of new bone after 3. Gubin AV, Borzunov DY, Marchenkova LO, Malko- distraction osteogenesis in rats. J Orthop Res 2004; va TA, Smirnova IL. Contribution of G.A. Ilizarov 22:472–8. to bone reconstruction: historical achievements and 13. Peichl P, Holzer LA, Maier R, Holzer G. Parathyroid state of the art. Strategies Trauma Limb Reconstr hormone 1-84 accelerates fracture-healing in pubic 2016; 11:145–52. bones of elderly osteoporotic women. J Bone Joint 4. Grubor P, Falzarano G, Grubor M, Piscopo A, Fran- Surg Am 2011; 93:1583–7. zese R, Meccariello L. Treatment of the chronic war 14. Wagner F, Vach W, Augat P, Varady PA, Panzer S, tibial osteomyelitis, Gustilo type IIIB and Cierny- Keiser S, Eckardt H. Daily subcutaneous teriparati- Mader IIIB, using various methods. A Retrospective de injection increased bone mineral density of newly study. EMBJ 2014; 9:7-18. formed bone after tibia distraction osteogenesis, a 5. Grubor P, Milicevic S, Grubor M, Meccariello L randomized study. Injury 2019; 50:1478-82. (2015) Treatment of bone defects in war wounds: 15. Coppola C, Del Buono A, Maffulli N. Teriparatide in retrospective study. Med Arh 2015; 69:260-4. fracture non-unions. Transl Med UniSa 2014; 12:47- 6. Raghavan P, Christofides E. Role of teriparatide in 53. accelerating metatarsal stress fracture healing: a case 16. Abuomira IE, Sala F, Elbatrawy Y, Lovisetti G, Alati series and review of literature. Clin Med Insights S, Capitani D. Distraction osteogenesis for tibial no- Endocrinol Diabetes 2012; 5:39–45. nunion with bone loss using combined Ilizarov and 7. Andreassen TT, Ejersted C, Oxlund H. Intermittent Taylor spatial frames versus a conventional circu- parathyroid hormone (1–34) treatment increases lar frame. Strategies Trauma Limb Reconstr. 2016; callus formation and mechanical strength of healing 11:153–9. rat fractures. J Bone Miner Res 1999; 14:960–8. 17. Mancilla EE, Brodsky JL, Mehta S, Pignolo RJ, Le- 8. Aspenberg P, Genant HK, Johansson T, Nino vine MA. Teriparatide as a systemic treatment for AJ, See K, Krohn K, García-Hernández PA, Recknor lower extremity nonunion fractures: a case series. CP, Einhorn TA, Dalsky GP, Mitlak BH, Fierlinger Endocr Pract 2015; 21:136-42. A, Lakshmanan MC. Teriparatide for acceleration of 18. Nishitani K, Mietus Z, Beck CA, Ito H, Matsuda S, fracture repair in humans: a prospective, randomi- Awad HA, Ehrhart N, Schwarz EM. High dose te- zed, double-blind-study of 102 postmenopausal wo- riparatide (rPTH1-34) therapy increases callus volu- men with distal radial fractures. J Bone Miner Res me and enhances radiographic healing at 8-weeks in 2010; 25:404–14. a massive canine femoral allograft model. PLoS One 9. Yin P, Zhang Q, Mao Z, Li T, Zhang L, Tang P. The 2017; 12:e0185446. treatment of infected tibial non-union by bone tran- 19. Ciurlia E, Leali PT, Doria C (2017) Use of teriparati- sport using the Ilizarov external fixator and a syste- de off-label: our experience and review of literature. matic review of infected tibial non-union treated by Clin Cases Miner Bone Metab 2017; 14:28-34. Ilizarov methods. Acta Orthop Belg 2014; 80:426-35. 20. Pietrogrande L, Raimondo E. Teriparatide in the tre- 10. Iliopoulos E, Morrissey N, Cho S, Khaleel A. Outco- atment of non-unions: scientific and clinical eviden- mes of the Ilizarov frame use in elderly patients. J ces. Injury 2013; 44(Suppl 1):S54-7. Orthop Sci 2017; 22:783-6.

292 ORIGINAL ARTICLE

Rare and uncommon diseases of the hip: arthroscopic treatment

Christian Carulli1,2, Alberto Schiavo1,2, Alberto Rigon1,2, Wondi De Marchi1,2, Matteo Innocenti1,2, Luigi Meccariello2, Massimo Innocenti1,2

1Orthopaedic Clinic, Department of Health Sciences, University of Florence, 2Orthopaedic Traumatology Centre, Careggi University Hospital, Florence, 3Department of Orthopaedics and Traumatology, Vito Fazzi Hospital, Lecce, Italy

ABSTRACT

Aim Uncommon and rare hip diseases are sources of pain and functional limitation particularly in young patients. Some of the- se conditions may be nowadays treated by arthroscopy due to the expertise and technical tips that high-volume hip arthroscopies have achieved during the last decades ensuring a wider range of indications for such a procedure. The aim of this study was to eva- luate clinical results of arthroscopy in treating uncommon or rare diseases of the hip at a single Institution.

Methods Thirteen patients affected by several types of diseases were treated by a hip arthroscopy and retrospectively evaluated. All patients were operated by the same surgeon, instrumentation Corresponding author: and technique, but postoperative rehabilitative protocol was tai- lored on each patient and his disease. Each patient underwent a Christian Carulli specific imaging, consisting of dedicated x-rays and arthro-MRI. Orthopaedic Clinic, Modified Harris Hip score (mHHS) and Non-Arthritic Hip score Department of Health Sciences, (NAHS) were used before and after surgery to clinically assess the University of Florence outcome. Largo P. Palagi 1, 50139 Florence, Italy Results All patients reported satisfaction, pain relief, and a good Phone: +39 055 794 8200; functional recovery at the latest follow-up visit. Only one patient E-mail: [email protected] affected by chondromatosis reported a recurrence of synovitis and ORCID ID: http://orcid.org/ 0000-0002- needed a further arthroscopy 25 months after the index operati- 0845-7940 on. No complications were reported at the latest follow-up. The NAHS and mHHS showed good improvements and all patients referred high satisfaction.

Conclusion Hip arthroscopy performed by expert and high-volu- me surgeons may ensure good results in patients affected by un- Original submission: common and rare hip diseases. 05 October 2020; Key words: arthroscopy, hip, hip joint/pathology, hip joint/sur- Revised submission: gery 14 October 2020; Accepted: 17 October 2020 doi: 10.17392/1285-21

Med Glas (Zenica) 2021; 18(1):293-298

293 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION PATIENTS AND METHODS Hip arthroscopy is nowadays more diffused than a Patients and study design decade ago, even if it cannot be still considered a standard procedure: few surgeons in a limited num- Medical records of all patients undergoing a hip ber of centres per country reach a high volume of arthroscopy over a 7-year period (September procedures every year (1,2). Arthroscopy represents 2012 to October 2019) were retrospectively eva- the gold standard procedure for the treatment of fe- luated. Among these, 13 patients (7.3% of the to- moro-acetabular impingement (FAI), labral tears, tal hip scopes in the overall period) were conside- and conditions as early arthritis and snapping hip. red for the study because fulfilling the following During the last years several other diseases have inclusion criteria: patients of all ages undergoing been successfully treated by this technique (1). a hip arthroscopy for an uncommon or rare disea- Growing the confidence on this procedure, hip se, different from primary FAI, labral tears, early surgeons have proposed hip arthroscopy also for arthritis, and snapping hip. Exclusion criteria other uncommon or rare diseases, as a unique were: patients affected by FAI, labral tears, hip technique or support to mini-open approaches arthritis, snapping hip, and joint infection. and with encouraging outcomes (3–22). The Institutional Review Board accepted the pro- Chondromatosis is a proliferative benign disease posal of the study, and all patients were properly of synovium characterized by formation of osteo- informed before surgery about the treatment and chondral loose bodies, with an unknown patho- follow-up visits after discharge. genesis: classically treated by open surgery, it has The mean age of the selected patients at the time been recently treated by arthroscopy and associated of surgery was 32.2 (range 14-54) years. Ten pa- to good results (3, 4, 17, 18). Moreover, rheumatic tients were males and three were females (Table diseases, pigmented villo-nodular synovitis of the 1). Four patients were represented by traumatic hip and Ehlers-Danlos syndrome and hyperlaxity Table 1. Demographic data, diagnosis and surgical proce- have been recently introduced as potential indicati- dures during hip arthroscopy ons for hip arthroscopy with acceptable results and Patients’ Age Diagnosis Procedure early recovery after surgery (19–21). The confiden- No (years) Rheumatoid arthritis and Arthroscopic debridement ce of surgeons with such a technique leads also to 1 21 the proposal of arthroscopy for traumatic and post- symptomatic synovitis and synovectomy Hip sprain with partial Shrinkage by radiofrequen- 2 22 traumatic conditions such as Pipkin fractures, lo- tear of lig. teres and FAI cies and osteoplasty Femoral fracture and Osteochondral fragments ose intra-articular fragments after acetabular rim 3 38 fractures, ligamentum teres tears, and even slipped dislocation removal and microfractures Acetabular fracture and Loose bodies removal and 4 26 capital femoral head (5–10, 22) we conducted the dislocation synovectomy present prospective study to determine the value Rheumatoid arthritis and Arthroscopic debridement 5 28 of hip arthroscopy in the diagnosis and manage- symptomatic synovitis and synovectomy Loose bodies removal and Isolated ment of various causes of hip pain after traumatic 6 23 synovectomy (synovec- chondromatosis* conditions. The present study included a prospec- tomy*) Loose bodies removal and tive cohort of 17 patients with symptomatic post- 7 17 Isolated chondromatosis synovectomy traumatic hip pain. It was conducted between July Rheumatoid arthritis and Arthroscopic debridement 8 18 2013 and May 2018. The mean age was 22 (19–29.) symptomatic synovitis and synovectomy Bony fragments removal Additionally, an endoscopic approach for several Acetabular fracture and 9 18 and debridement & micro- dislocation conditions as iliopsoas tendonitis, bursitis and in- fractures Pigmented villo-nodular Loose bodies removal and ternal snapping hip has been proposed as a surgical 10 52 option (12–14, 23) operative reports and operative synovitis synovectomy Symptomatic chronic Partial bursectomy and 11 54 procedures. All patients received either labral debri- ileopsoas bursitis synovectomy Atypical rheumatic dement, labral repair, osteoplasty or a combination Arthroscopic debridement 12 28 disease and symptomatic of those procedures. A standardized rehabilitation and synovectomy synovitis protocol was used. Of 252 patients, 60 (24%). FAI secondary to Osteoplasty and screw 13 25 slipped capital femoral removal The aim of this study was to evaluate clinical re- epiphysis sults of arthroscopy performed for uncommon or * This female patient underwent a revision arthroscopy two years rare diseases of the hip at a single Institution. after the first hip scope due to synovitis recurrence without formation of chondromas; FAI, femoro-acetabular impingement;

294 Carulli et al. Rare and uncommon diseases of the hip

or post-traumatic lesions (fracture-dislocation, preoperative instrumental examination by x-rays hip sprain with partial rupture of ligamentum (standing true pelvis view, Dunn views at 45° and teres) (Figure 1) and four patients by rheuma- 90°, false profile views) and arthro-magnetic re- tologic diseases (rheumatoid arthritis, a specific sonance imaging (arthro-MRI) (15, 26). synovitis); two patients were with isolated hip All procedures were performed by a single sur- chondromatosis (Figure 2), one patient with pi- geon, in lateral position, with a dedicated leg gmented villo-nodular synovitis (PVNS), one traction, a short-term antibiotic regimen (with a patient with symptomatic chronic ileopsoas bur- preoperative first dose), and in general or loco- sitis, and one patient with complications after an regional anaesthesia depending on patient’s cha- epiphysiodesis by screw performed for slipped racteristics. capital femoral epiphysis (SCFE). All patients underwent a tailored postoperative rehabilitation protocol. Follow-up visits were planned at 1, 3, 6, and 12 months, then yearly; when indicated, a further imaging was requested during the follow-up period.

Statistical analysis Statistical analysis was performed by a sample size calculation based on a priori assumption of p=0.05 with a 95% confidence interval. The Student t-test was used to perform the scores’ analysis, testing each disease separately.

RESULTS All patients completed the minimum follow-up of 12 months. The mean follow-up was 52.4 (ran- Figure 1. Symptomatic isolated chondromatosis of a right hip ge 12-98) months. No intraoperative complicati- of a 17-year-old female patient (professional dancer). A, B) ons were recorded. A single case of early posto- Radiologic and MRI patterns and C) intraoperative aspect of perative complication was recorded; it consisted several chondromas in the joint space; D, E) within the hyper- plastic synovitis in the acetabulum; F, G) treatment consisted in a superficial infection of one of the arthrosco- in loose bodies removal and synovectomy by radiofrequencies pic portals, requiring a prolonged oral antibiotic (Azienda Ospedaliero Universitaria Careggi, 2018) therapy lasting for two weeks. Several associated lesions were found during ar- throscopy: FAI in nine (all managed by femoral osteoplasty), chondral lesions in 11 (eight on the acetabular side, managed by shaving and/or ra- diofrequencies), and loose bodies in six patients Figure 2. Left hip of a 22-year-old female athlete (professional (two in the chondromatosis patients, four in post- volley) undergoing a hip sprain. A) On the basis of a mild fem- traumatic conditions). oro-acetabular impingement (FAI), B) a partial rupture of the ligamentum teres was found at arthro-magnetic resonance im- The mean preoperative values of mHHS and aging (white arrow); C) during surgery, the ligament tear was NAHS were 42.6 (range 31-66) and 50.2 (range evident and associated to acute hemarthrosis: the treatment consisted in joint debridement, osteoplasty, and shrinkage of 41-68), respectively. The mean time of return to a the residual safe part ligament (Azienda Ospedaliero Universi- full physical activity was 4.8 months (range 3-7). taria Careggi, 2016) At follow-up, one single case of chondromatosis Methods required a revision arthroscopy about two years after the first operation, due to the recurrence of All patients were evaluated clinically by modified synovitis without loose bodies or chondromas. Harris Hip Score (mHHS) (24) and Non Arthritic Two years after surgery, mHHS and NAHS values Hip Score (NAHS) (25), before and after surgery significantly improved with a mean score of 84.5 at specific intervals. All patients underwent a

295 Medicinski Glasnik, Volume 18, Number 1, February 2021

(range 79-90) and 72.4 (range 69-80), respectively vitis, detected two years after the first operation, (p<0.05); at the time of the mean follow-up, all sco- needed a further arthroscopy for synovectomy. res remained substantially good (Table 2). At the Rheumatic diseases have been recently managed latest follow-up, all patients referred satisfaction by hip arthroscopy in the series of Zhou et al.: and a good health status, with no recurrence of their 27 patients (40 hips; 36 patients) affected by in- diseases and with an acceptable standard of life. flammatory arthritis (17 ankylosing spondylitis, Table 2. Pre- and post-operative score evaluation of thirteen 11 rheumatoid arthritis, and eight of psoriatic ar- patients thritis) undergoing arthroscopic debridement and Pa- mHHS* NAHS* tient Diagnosis synovectomy (mean follow-up of 67 months), No Pre-op Post-op Pre-op Post-op referred satisfaction regaining normal daily acti- Rheumatoid arthritis and 1 37 79 49 69 symptomatic synovitis vities (19). In our series, the four patients treated Hip sprain with partial tear of reported a significant improvement among all the 2 42 84 51 78 lig. teres and FAI clinical scores with no complications or further Femoral fracture and dis- 3 35 81 47 70 location surgeries at the final follow-up. Acetabular fracture and 4 33 84 45 76 Evaluation of arthroscopic treatment for PVNS dislocation of the hip of 13 patients (followed-up for a mi- Rheumatoid arthritis and 5 48 88 51 72 symptomatic synovitis nimum period of 2 years) showed good outcome 6 Isolated chondromatosis 66 90† 68 80* and minimal morbidity (20). Our findings are in 7 Isolated chondromatosis 40 84 53 78 line with this study. Rheumatoid Arthritis and 8 45 88 61 79 symptomatic synovitis Larson et al. presented the outcome in a series of Acetabular fracture and 9 56 90 67 80 12 patients (16 hips) affected by Ehlers-Danlos dislocation Pigmented villo-nodular syndrome and hyperlaxity: FAI was mostly asso- 10 56 88 57 78 synovitis ciated to severe capsular laxity, thus, osteoplasty Symptomatic chronic ileop- 11 51 82 41 72 for FAI and careful capsular plication were per- soas bursitis Atypical rheumatic disease formed with satisfactory results and no cases of 12 31 79 49 69 and symptomatic synovitis postoperative dislocations were recorded (21). In FAI secondary to slipped 13 37 81 51 70 our series of patients with FAI associated with se- capital femoral epiphysis *p< 0.05; †This female patient underwent a revision arthroscopy two vere capsular laxity all were treated with comple- years after the first hip scope due to synovitis recurrence without for- te capsular closure reporting satisfactory results mation of chondromas; mHHS, Modified Harris Hip score; NAHS, and no conversion to hip arthroplasty. Non-Arthritic Hip score; pre-op, pre-operative; post-op, post-operati- ve; FAI, femoro-acetabular impingement; Traumatic and post-traumatic conditions treated DISCUSSION by arthroscopy were reported recently by the analysis of 17 patients with symptomatic hip Chondromatosis was for first time arthroscopi- pain caused by different conditions (traumatic cally treated by Boyer with a study population labral tear, Pipkin fractures, and loose intra-ar- of 111 cases: at a mean follow-up of 78 months, ticular fragments) with a mean follow-up of 24 57% of patients referred no recurrence and sa- months, showing significant improvements of tisfaction, while almost 20% needed conversion mHHS (22). In our study performing loose body to hip arthroplasty (17) 120 patients underwent removal and synovectomy combined or not with arthroscopic management for primary synovial microfractures was associated with good post- chondromatosis of the hip. We report the outco- operative clinical outcome. Despite the presence me of 111 patients with a mean follow-up of 78.6 of initial early osteoarthritis in all patients, we months (12 to 196). Also Zini et al. (18) and Lee reported no subsequent surgical procedures at a et coll. (4) reported their series with 11 cases at minimum of 24-month follow-up. almost two years follow-up (1 failure, early con- Hip arthroscopy finds application also in -liga verted in arthroplasty) and 10 patients at 4-year mentum teres reconstruction caused by traumatic follow-up respectively, associated to acceptable tears as reported by O’Donnell et al. in a seri- outcomes. In our series, two patients with chon- es of nine patients using autologous semitendi- dromatosis were treated with good outcome, nosus tendons (five cases) and tibialis posterior even tough if in one case a recurrence of syno- allografts (four cases) with a minimum follow-up

296 Carulli et al. Rare and uncommon diseases of the hip

of 12 months; all patients appeared satisfied with Iliopsoas tendonitis, bursitis and internal hip mHHS and during the follow-up a single patient snapping may also cause hip pain especially underwent capsular tightening due to instability, among athletes (12–14, 23) operative reports and and another underwent debridement due to a operative procedures. All patients received either partial tear (5). We reported a single case of hip labral debridement, labral repair, osteoplasty or a sprain associated with partial tear of ligamentum combination of those procedures. A standardized teres and FAI in which we performed the oste- rehabilitation protocol was used. Of 252 patients, oplasty and a simple shrinkage by radiofrequ- 60 (24%). Perets et al. found statistically signifi- encies without augmentation; nevertheless, this cant improvement with mHHS and NAHS com- single patient referred significant improvement paring two patient groups (underwent/or not) ili- in all clinical scores at the final follow-up. opsoas fractional lengthening treated for FAI (60 Arthroscopy may be an option also for the tre- patients with a mean follow-up of 49.1 months atment of slipped capital femoral head (SCFE), post-operatively) and/or chondrolabral lesions in conjunction with acute treatment or as delayed (23). Satisfactory subjective outcome and mHHS surgery. In the latter cases, delaying the surgery were reported by Maldonado et al. in patients resulted in the worsening of the outcome (6–11) with painful snapping hip: revision arthroscopies EMBASE, and PubMed were searched and scre- were performed in 5.6% and conversion to THA ened in duplicate. Data such as patient demo- in 1.3% (14). Complete resolution of symptoms graphics, surgical technique, surgical outcomes was also reported in our case of symptomatic and complications were retrieved from eligible chronic ileopsoas bursitis treated by partial bur- studies. Results: Fifteen eligible level IV studi- sectomy and synovectomy. es were included in this review comprising 261 Independently from the type of uncommon dise- patients (266 hips). Chen et al. reported complete ase, it is clear from the literature that hip arthros- pain relief in 88%, mild residual pain in two pa- copy has shown encouraging outcomes, minimal tients (out of 34 patients with a mean follow-up invasiveness, and very low rates of complications of 22 months); two patients underwent subsequ- with respect to standard open surgery. ent open osteotomy due to residual extra rotation This study has several limitations. First of all, it is in flexion (7) 10 to 19 years. Among 19 patients a retrospective study, with a small patient sample, with SCFE (a mean follow-up of 40 months), and represented by a heterogeneous group of di- Wilye et al. found 14 with mild and five with seases. Moreover, an adequate statistical analysis moderate slips, and obtained an alpha angle im- was not feasible given the scarce numbers and provement in all patients, as well as all patients parameters to evaluate. However, it is hard to re- had pain relief and improvement at the clinical ach gross numbers of patients at a single institu- scores (10). Basheer et al. observed statistically tion due to rarity of these clinical issues, and the significant improvement of mHHS and NAHS literature witnesses such difficulty. postoperatively in a group of 18 patients with a Hip arthroscopy may be indicated for uncommon mean follow-up of 29 months (8)including osteo- and rare clinical conditions, ensuring good re- chondroplasty, for the sequelae of SCFE. Data sults and few complications, when performed by were prospectively collected on patients under- expert high-volume surgeons. Future experiences going arthroscopy of the hip for the sequelae of of other study groups will be useful to assess if SCFE between March 2007 and February 2013, outcomes and complications’ rate may be consi- including demographic data, radiological asse- dered actually acceptable. ssment of the deformity and other factors that may influence outcome, such as the presence of FUNDING established avascular necrosis. Patients comple- No specific funding was received for this study. ted the modified Harris hip score (mHHS. Our experience is limited to a single case presenting TRANSPARENCY DECLARATION with a FAI secondary to SCFE. After performing osteoplasty and screw removal the patient repor- Conflicts of interest: None to declare. ted good post-operative clinical outcome.

297 Medicinski Glasnik, Volume 18, Number 1, February 2021

REFERENCES 1. Wall PDH, Brown JS, Parsons N, Buchbinder R, 14. Maldonado DR, Krych AJ, Levy BA, Hartigan DE, Costa ML, Griffin D. Surgery for treating hip impin- Laseter JR, Domb BG. Does iliopsoas lengthening gement (femoroacetabular impingement). Cochrane adversely affect clinical outcomes after hip arthros- Database Syst Rev 2014 (9):CD010796. copy? A multicenter comparative study. Am J Sports 2. Pierannunzii L, Di Benedetto P, Carulli C, Fiorenti- Med 2018; 46:2624-31. no G, Munegato D, Panascì M, Potestio D, Randelli 15. Carulli C, Tonelli F, Melani T, Pietragalla M, De F, Della Rocca F, Rosolen V, Giangreco M, Santori Renzis AGD, Caracchini G, Innocenti M. Diagnostic N. Mid-term outcome after arthroscopic treatment accuracy of magnetic resonance arthrography in de- of femoroacetabular impingement: development of a tecting intra-articular pathology associated with fe- predictive score. HIP Int 2019; 29:303-9. moroacetabular impingement. Joints 2018; 6:104-9. 3. Rath E, Amar E, Doron R, Matsuda DK. Hip arthros- 16. Shifrin LZ, Reis ND. Arthroscopy of a dislocated hip copy for synovial chondromatosis: tips and tricks. Ar- replacement: a case report. Clin Orthop Relat Res throsc Tech 2014; 3:e709-12. 1980; (146):213-4. 4. Lee YK, Moon KH, Kim JW, Hwang JS, Ha YC, Koo 17. Boyer T, Dorfmann H. Arthroscopy in primary syno- KH. Remaining loose bodies after arthroscopic sur- vial chondromatosis of the hip. J Bone Joint Surg Br gery including extensive capsulectomy for synovial 2008; 90:314-8. chondromatosis of the hip. CiOS Clin Orthop Surg 18. Zini R, Longo UG, De Benedetto M, Loppini M, 2018; 10:393-7. Carraro A, Maffulli N, Denaro V. Arthroscopic ma- 5. O’Donnell J, Klaber I, Takla A. Ligamentum teres nagement of primary synovial chondromatosis of the reconstruction: indications, technique and minimum hip. Arthroscopy 2013; 29:420-6. 1-year results in nine patients. J Hip Preserv Surg 19. Zhou M, Li ZL, Wang Y, Liu YJ, Zhang SM, Fu J, 2020; 7:140-6. Wang ZG, Cai X, Wei M. Arthroscopic debridement 6. Oduwole KO, De Sa D, Kay J, Findakli F, Duong A, and synovium resection for inflammatory hip arthri- Simunovic N, Yi-Meng Y, Ayeni OR. Surgical tre- tis. Chin Med Sci J 2013; 28:39-43. atment of femoroacetabular impingement following 20. Byrd JWT, Jones KS, Maiers GP. Two to 10 years’ slipped capital femoral epiphysis: A systematic re- follow-up of arthroscopic management of pigmented view. Bone Joint Res 2017; 6:472-80. villonodular synovitis in the hip: a case series. Ar- 7. Chen A, Youderian A, Watkins S, Gourineni P. Ar- throscopy 2013; 29:1783-7. throscopic femoral neck osteoplasty in slipped capital 21. Larson CM, Stone RM, Grossi EF, Giveans MR, Cor- femoral epiphysis. Arthroscopy 2014; 30:1229-34. nelsen GD. Ehlers-Danlos syndrome: arthroscopic 8. Basheer SZ, Cooper AP, Maheshwari R, Balakumar management for extreme soft-tissue hip instability. B, Madan S. Arthroscopic treatment of femoroaceta- Arthroscopy 2015; 31:2287-94. bular impingement following slipped capital femoral 22. Alfikey A, El-Bakoury A, Karim MA, Farouk H, epiphysis. Bone Joint J 2016; 98-B:21-7. Kaddah MA, Abdelazeem AH. Role of arthroscopy 9. Allen MM, Rosenfeld SB. Treatment for post-slipped for the diagnosis and management of post-trauma- capital femoral epiphysis deformity. Orthop Clin tic hip pain: a prospective study. J Hip Preserv Surg North Am 2020; 5:37–53. 2019; 6:377-84. 10. Wylie JD, Beckmann JT, Maak TG, Aoki SK. Ar- 23. Perets I, Hartigan DE, Chaharbakhshi EO, Ashberg throscopic treatment of mild to moderate deformity L, Mu B, Domb BG. Clinical outcomes and return after slipped capital femoral epiphysis: Intra-opera- to sport in competitive athletes undergoing arthros- tive findings and functional outcomes. Arthroscopy copic iliopsoas fractional lengthening compared with 2015; 31:247-53. a matched control group without iliopsoas fractional 11. Tscholl PM, Zingg PO, Dora C, Frey E, Dierauer S, lengthening. Arthroscopy 2018; 34:456-63. Ramseier LE. Arthroscopic osteochondroplasty in 24. Harris WH. Traumatic arthritis of the hip after dis- patients with mild slipped capital femoral epiphysis location and acetabular fractures: treatment by mold after in situ fixation. J Child Orthop 2016; 10:25-30. arthroplasty. J Bone Jt Surg Am 1969; 51:737-55. 12. Adib F, Johnson AJ, Hennrikus WL, Nasreddine A, 25. Christensen CP, Althausen PL, Mittleman MA, Lee Kocher M, Yen Y-M. Iliopsoas tendonitis after hip J ann, McCarthy JC. The nonarthritic hip score: Re- arthroscopy: prevalence, risk factors and treatment liable and validated. Clin.Orthop Relat Res 2003; algorithm. J Hip Preserv Surg 2018; 5:362-9. (406):75-83 13. Gouveia K, Shah A, Kay J, Memon M, Simunovic 26. Aubry S, Bélanger D, Giguère C, Lavigne M. Ma- N, Cakic JN, Ranawat AS, Ayeni OR. Iliopsoas te- gnetic resonance arthrography of the hip: technique notomy during hip arthroscopy: a systematic review and spectrum of findings in younger patients. Insights of postoperative outcomes. Am J Sports Med 2020; Imaging 2010; 1:72-82. 363546520922551. 27. Newman JT, Saroki AJ, Philippon MJ. Hip arthrosco- py for the management of trauma: a literature review. J Hip Preserv Surg 2015; 2:242-8

298 ORIGINAL ARTICLE

A new prognostic pelvic injury outcome score Luigi Meccariello1, Cristina Razzano2, Cristina De Dominicis3, Juan Antonio Herrera-Molpeceres4, Franc- esco Liuzza5, Rocco Erasmo6, Guido Rocca7, Michele Bisaccia8, Enzo Pagliarulo9, Pietro Cirfeda10, David Gómez Garrido11, Giuseppe Pica1, Giuseppe Rollo12

1Department of Orthopaedics and Traumatology, AORN San Pio, Benevento, 2Health Direction Unit, Physiatric and Rehabilitation AIAS Centre, Lauria, 3Bachelor in Specialization School in Cognitive Neuropsychological Psychotherapy, Madre della Divina Providenza Reha- bilitation Centre, Arezzo; Italy, 4Department of Orthopaedics and Traumatology, University Hospital Virgen De La Salud, Toledo, Spain, 5Department of Orthopaedics and Traumatology, A. Gemelli University Hospital, Catholic University, Rome, 6Department of Orthopae- dics and Traumatology, Santo Spirito Hospital, Pescara, 7Department of Orthopaedics and Traumatology, Trauma Centre Pietro Cosma, Camposampiero, Padua, 8Department of Orthopaedics and Traumatology, Azienda Ospedaliera “Santa Maria della Misericordia”, Perugia, 9Department of Urology, Vito Fazzi Hospital, Lecce; Italy, 10Urology and Unit, Department of Emergency and , University Aldo Moro , Bari, Italy, 11QuironSalud Toledo Hospital and Solimat Hospital, Toledo, Spain, 12 Department of Orthopaedics and Traumatology, Vito Fazzi Hospital, Lecce, Italy

ABSTRACT

Aim To propose a new prognostic classification system for pelvic injuries based on a new detailed and all-encompassing evaluation of the injury pelvic outcome score and to check the prognostic value of this classification and evaluate its reliability and repro- ducibility.

Methods From January 2017 to June 2020 from 156 pelvic fractu- res treated at our hospitals, 98 patients with pelvic fractures were Corresponding author: recruited according to inclusion and exclusion criteria. All patients Juan Antonio Herrera-Molpeceres compiled three scores (New Score System, Majeed Score, SF-12) Department of Orthopaedics and sessions two times during the hospital stay to evaluate the endpo- Traumatology, University Hospital Virgen int before the trauma and two years after the trauma. All patients De La Salud carried out three tests independently. The evaluation of three sco- res included a pelvic and general complication after the surgery, Av. de Barber 30, 45004 Toledo, Spain the times needed to compile three score system. For reliability of Phone: +34 925 26 92 00; the new score systems we evaluated the inter-observer or intra- Fax: +34 925 26 92 00 observer agreement, the prediction strength of each score, and a E-mail: [email protected] prognostic value. Luigi Meccariello ORCID: http://orcid. Results A total of 98 patients were enrolled (74 were males and 24 org/0000-0002-3669-189X females) with mean age of 43.6 (±18.6) (range 16-75) years. Tau B Kendall value was 0.827 for the new score system, 0.673 for the Majeed score, 0.746 for SF-12, there was p<0.05 for the new score system. Original submission: 21 October 2020; Conclusion The new score system is prognostic, reliable, reprodu- cible and can become a useful instrument to adequately correlate Revised submission: the long-term outcomes of pelvic injury fractures. Also, it provides 10 November 2020; a better evaluation of pain, work, sexual possibilities and satisfac- Accepted: tion, balance-sitting-walking and psychological status. 14 November 2020 Key words: dysfunction, outcomes, pain, work doi: 10.17392/1298-21

Med Glas (Zenica) 2021; 18(1):299-308

299 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION The exclusion criteria were: haematological or oncological patients, acute or chronic infections, Pelvic injuries are rare and still represent a major age under 16 and over 75 years, no sexual acti- cause of death and disability in patients involved vity, no bone metabolism diseases, no rheuma- in high-energy trauma (1). These fractures are toid diseases, sexual or urological dysfunctions among the most complex injuries of the lower before the trauma, previous pelvic or lower limbs limb and their management is technically deman- vascular/nerve diseases. . ding (2). Surgical treatment of these fractures is challenging, and creates several difficulties, both All injuries were classified by seven pelvic surge- in restoration of the pelvic anatomy and sagittal ons; among them, four were senior surgeons with balance alignment or reduces pre surgery or post- more than 20 years of experience in orthopaedics surgery complications (2,3). trauma pelvic surgery, the other tree had more than 5 years of experience. Chronic posttraumatic pelvic dysfunctions after pelvic ring fracture impact negatively quality of life The polytrauma patients were classified according and mental health (4). The importance of the pre- to the Tile classification (7), the Injury Severity operative and post-operative subjective or objecti- Score (ISS) (3). Complete neurological examinati- ve score to evaluate the pelvic injuries outcomes on according to the American Spinal Injury Asso- has been demonstrated in several studies (1-4). ciation (ASIA) (8) was performed in all patients. Pelvic injury outcome is classified according to Patients were treated according to the ethical the Majeed score (5) prognostic classification standards of the Helsinki Declaration, and were systems, which are based only on five assessed invited to read, understand, and sign an informed and scored factors: pain, standing, sitting, sexual consent form. intercourse and work performance. The total sco- Azienda Sanitaria Locale (ASL) Lecce/Italy Et- re gave a clinical grade as excellent, good, fair hical Committee approved this research. or poor. The scoring system allows comparison The new score system is based on 6 labelling fac- between early and late results and also betwe- tors (Table 2), evaluated on common problems en various methods of treatment. This grading that patients suffered after pelvic injuries: pain, outcome of pelvic fractures is a non-validated work, sexual possibilities, sexual satisfaction, ba- self-developed pelvic fracture specific functional lance-sitting-walking, and psychological status. assessment instrument, and the disadvantage of Each labelling factor is assigned a maximum score the Majeed score is neurological impairments, whose sum has a minimum of 0 and a maximum which has relevant prognostic influence, not in- of 100 points: 100 points equals the patient’s state tegrated and measures exclusively the functional of health despite the fracture of the pelvis while 0 component of the long-term pelvic injury (6). equals the maximum degree of dysfunction. Each The aim of this study was to propose a new pro- labelling factor is composed of subcategories to gnostic classification system for the pelvic injuri- which it refers or a specific score or range. es based on a new detailed and all-encompassing evaluation of the injury pelvic outcome score, to Methods check the prognostic value of this classification, All patients underwent radiological exams: pel- and evaluate its reliability and reproducibility. vic X-rays in AP position and CT scan with re- constructions in 3D. PATIENTS AND METHODS The patients were treated by conservative or sur- Patients and study design gical treatment as previously described (1,2). To evaluate the overall outcome of our pati- From January 2017 to June 2020 out of 156 pa- ents, three scores were administered: new score tients’ pelvic fractures treated at the Department system, Majeed score (5) and the Short Form of Orthopaedics, Vito Fazzi Hospital, 98 patients (12) Health Survey (SF-12) (4). were recruited according inclusion criteria of the Tile classification (7); polytrauma, high energy All patients compiled the three score sessions two trauma, low energy trauma, age 16- 75 years, times during hospital stay with the help of medical sexual activity almost once a week. All patients staff to evaluate the endpoint before the trauma and joined at one follow-up year. two years after the trauma. The three scores, betwe-

300 Meccariello et al. Prognostic pelvic injury outcome score

en the endpoint before the trauma and the endpoint to compare prediction strength of each score. The trauma, were administered one week apart. partial Eta squared (η2) was used to measure pre- All patients carried out three tests independently diction strength. during the waiting time for the clinical and radi- To investigate the reliability of the new score ographic check-ups (other endpoints) at 1 month, systems the inter-observer agreement for the Tile 3 months, 6 months, 12 months from the trauma. classification using the weighted Kappa (K) sta- The chosen criteria to evaluate the three sco- tistics described by Fleiss was evaluated (9,10). res during clinical and radiological follow-up Three Kappa statistics were compared using the were: pelvic and general complication after the Wald test. To evaluate the reproducibility of the surgery, the time needed to compile the three new proposed classification system the intra-ob- score systems. The reliability of the new score server agreement was calculated using the Kappa system was evaluated by inter- or intra-observer statistics. The Wald test was performed according agreement, the prediction strength of each sco- to Shoukri et al. (11). Classification for K value re and prognostic value. Each labelling factor is was: less than 0.4 poor agreement, 0.4-0.6 mo- assigned a maximum score whose sum has a mi- derate agreement, 0.6-0.8 good agreement and nimum of 0 and a maximum of 100 points: 100 0.8-1 excellent agreement (12). The statistical points equals the patient’s state of health despi- significance was defined as p<0.05. te the fracture of the pelvis, while 0 equals the RESULTS maximum degree of dysfunction. Each labelling factor is composed of subcategories to which it A total of 98 patients were enrolled, 74 were refers or a specific score or range. males and 24 females. The mean age was 43.6 The evaluation end point was set at 1 year of (±18.6; range 16-75) years. The industry sector follow up. was more represented in previous work, in 31 (31.63%) patients. Traffic accidents and agricul- Statistical analysis tural accidents were the most frequent causes of injuries, in 32 (32.65%) and 26 (26.53) patients, Descriptive statistics were used to summarize the respectively (Table 1). characteristics of the study group and subgroups, including mean and standard deviation of all con- Table 1. Characteristics of 98 patients with pelvic injury tinuous variables. The t-test was used to compare Characteristic continuous outcome. The test or Fisher’s exact test Gender ratio (M:F) 3.08:1 (74:24) (in subgroups smaller than 10 patients) was used to Average age (SD) (years) 43.6 (±18.6) compare categorical variables. Pearson correlation No (%) of patients Age range (years) coefficient (r) was used to compare the predictive Overall 16-75 score of outcomes and quality of life. Mean age (and 16-35 28 (28.57) the range) of the patients were rounded at the closest 36-50 24 (24.49) 51-59 20 (20.41) year. The predictive score of outcome and quality of 60-65 12 (12.25) life and their ranges were approximated at the first 65-70 8 (8.16) decimal, while the Pearson correlation coefficient 70-75 6 (6.12) Occupation (n;%) was approximated at the second decimal (r). Agricultural activity 30 (30.61) The Kendall Tau-B correlation between the Tile Industrial sector 31 (31.63) Tertiary industry 26 (26.53) classification (7) at the last follow-up and the Unemployed 11 (11.23) new score, the Majeed score (5) and SF-12 was Type of accident used to assess the prognostic value of each type Fall from height 22 (22.45) of fracture according to the Tile classification (7). Traffic accident: 32 (32.65) Accident agriculture: 26 (26.53) A multivariate analysis using the multiple regre- Other accidents: 18 (18.37) ssion with backward Wald method was perfor- Type of fractures according Tile classification A 16 (16.33) med to detect: sex, age, associated lesions, type B1 29 (29.59) of surgery, etc. B2 18 (18.37) B3 8 (8.16) The general linear model (GLM), with the Tile C1 17 (17.35) classification (7) as a dependent variable and C2 6 (6.12) three scores as covariates, was finally performed C3 4 (4.08)

301 Medicinski Glasnik, Volume 18, Number 1, February 2021

The new score system is based on 6 labelling fac- that patients suffered after pelvic injuries: pain, tors (Table 2) evaluated on the common problems work, sexual possibilities, sexual satisfaction, ba- Table 2. The new pelvic outcome system score after pelvic injury Patients suffered after pelvic injuries Point Subcategory point Pain (10 points) Intense or continuous rest 0 Intense with slight activity 0-2 Tolerable, but limiting slight activity 3-4 With moderate activity, abolished by rest 5-6 Mild, intermittent, normal activity 7-9 Slight, occasional or no pain 10 Work (25 points) No regular work 0-3 Light work 4 Change of job 7 Same job, reduced performance 10 Same job, same performance 25 Sexual possibilities (10 points) Type of intercourse or no intercourse 0 Only oral sex 1-2 Oral sex or anal sex 3-4 Intercourse possible in uncomforted position 5-8 Intercourse possible in any position 0 Sexual satisfaction (25 points) (Inverse Modified Arizona Sexual Experiences Scale) How strong is your sexual drive 0-5 Are you sexually turned on? 0-5 Can you easily reach and maintain an erection? (for man only) 0-5 Does your vagina become moist during sex? (for female only) 0-5 How easily can you reach an orgasm? 0-5 Are you orgasms satisfying? 0-5 Balance-sitting-walking (20 points) 1. Bedridden or almost 0 2. Wheelchair 0 3. Cannot walk or almost 0-2 2 I cannot walk 0 I take less than 10 steps 1 I take more than 10 steps 2 4. Painful sitting 0-5 no pain 0 armchairs / chair with backrest reclining at 40 ° 1 armchairs / chair with backrest reclining at 60° 2 stool / high chair (65 ° - 70 °) 3 stool / low chair (40 ° -45 °) 4 armchair / chair with backrest around 90 ° 5 5. Standing & Walking Balance 0-5 no balance 0 balance in orthostatism and walking 1 balance in orthostatism and loss of balance when walking 2 loss of balance with imbalance on the operated limb 3 loss of balance with imbalance on the healthy limb 4 loss of balance both in orthostatism and when walking 5 6. Walking with aids 0-4 independent 0 supervised in an extra and home environment 1 with crutches in an extra and home environment 2 with rollator in an extra and home environment 3 addicted to 4 7. Limp walking without aids 0-4 no lameness 0 slight lameness (+ 200 mt) 1 moderate lameness (- 200 mt) 2 limb length discrepancy 3 severe lameness (- 100 mt) so it needs aids 4 8. Free 20 20 Psychological status (10 points) 1. Depression 0 2. Anxiety 1-2 cognitive symptoms (irritability, insomnia, difficulty concentrating, restlessness…) 1 somatic symptoms (tachycardia, choking sensation, excessive sweating, dizziness, stomach pain, diarrhoea, nausea, 2 chills, hot flashes, frequent urination, muscle twitching, tremors) 3. Heavy stress in life-chronic stress 3-4 endless worries, traumatic episodes in early childhood 3 suicidal thoughts 4 4. Middle stress in life-acute episodic stress 5-6 severe abuse, anger, tension 5 constant concern (migraine, hypertension, chest pain, heart disease) 6 5. Little stress in life-acute stress 7-9 emotional stress (anger, irritability, anxiety, depression) 7 muscle problems, stomach problems, liver and bowel problems 8 high blood pressure 9 6. Normal for age and general condition 10 10

302 Meccariello et al. Prognostic pelvic injury outcome score

lance-sitting-walking, and psychological status. the New Pelvic Score System expands the range Compared to the Majeeed Score and the SF-12, of scores available to the patient to characterize his

Table 3. Associated injures, type of fixation and complications for each pelvic fracture type according Tile classification (3) Groups according Tile classification Variable Non Surgery B1 B2 B3 C1 C2 C3 surgery Associated injures with pelvic trauma (No of patients) Cerebral concussion 9 3 16 12 8 16 6 4 Fat embolism 0 0 2 0 2 3 4 3 Hemopneumothorax 2 0 8 9 7 11 5 4 Liver injuries 1 1 3 5 6 5 4 3 Spleen injuries 2 1 4 6 4 5 4 3 Blow injuries 2 0 1 1 2 3 2 3 Tibial injuries 5 0 3 5 5 15 4 2 Femoral injuries 8 0 6 3 4 13 3 3 Rib fractures 2 3 22 8 8 12 6 4 Ankle and foot 0 0 6 4 0 19 5 2 Clavicle fractures 0 0 12 3 1 6 3 3 Proximal humerus 0 0 8 9 2 4 1 2 Humeral shaft 0 0 12 2 2 4 1 1 Elbow 0 0 2 4 1 5 1 2 Forearm 0 0 6 7 1 3 2 1 Wrist and hand 0 2 10 5 6 6 6 3 Spine fractures or sacral injuries 0 0 3 6 7 17 6 4 Lumbo-sacral nerve injuries 0 0 0 6 3 9 5 4 Cranial maxillofacial surgery 0 0 0 6 2 8 2 3 fractures Urogenital injuries 0 0 14 5 4 11 6 4 Average injury severity score 12 23 22 31 30 40 42 45 (±; range) (3) (±2.3; 1-15) (±6.8; 1-31) (±5.8; 1-33) (±8.3; 21-44) (±8.7; 21-42) (±4.2; 34-50) (±11.4; 32-55) (±12.6; 32-55) Type of pelvic fixation (No of patients) Rest in bed for 21 days 12 4 29 18 8 17 6 4 Anterior double plates none 0 23 16 6 17 6 4 anterior plates none 2 6 2 2 0 0 0 Posterior sacral plates none 0 0 5 4 6 0 0 Posterior sacral roars none 0 0 7 1 1 0 0 Spinopelvic none 0 0 6 3 10 6 4 Cannulated Screws none 2 0 0 0 0 0 0 X-ray reduction (No) Excellent 12 3 26 14 2 4 0 0 Very Good 0 1 3 4 2 6 1 1 Good 0 0 0 0 3 5 3 1 Fair 0 0 0 0 1 2 1 1 Bad 0 0 0 0 0 0 1 1 Complications (No of patients) Pelvic floor relapse 0 0 2 6 3 12 2 2 Bowel stoma 0 0 1 2 1 6 3 3 Impotence to erection 0 0 9 6 6 7 2 3 Urinary incontinence 0 0 3 3 2 4 1 1 Dyspareunia 0 0 4 4 5 5 4 1 Anal incontinence 0 0 0 1 2 2 1 1 Neurological bladder 0 0 0 0 1 2 1 1 Bladder prolapse 0 0 0 0 1 1 0 1 Sexual limitation 0 0 22 13 6 17 6 4 Reduced sexual interest 0 0 8 10 8 11 6 4 Less frequent orgasm 0 0 6 7 6 14 6 4 Sensitive crural Nerve injury 0 2 0 0 0 14 6 4 Other complications 0 0 10 12 9 12 6 4 New scoring System: 0.22 0.18 0.18 0.18 0.22 0.18 0.14 0.14 Partial Eta squared Majeed score: Partial 0.15 0.16 0.12 0.11 0.11 0.11 0.14 0.14 Eta squared SF-12: 0.19 0.16 0,14 0.14 0.15 0.15 0.13 0.14 Partial eta squared

303 Medicinski Glasnik, Volume 18, Number 1, February 2021

dysfunction or incapacity for work and daily life. re system, 14.2 minutes (±4.1; range 9-25) for the Compared to the Majeed score, the patient’s wor- Majeed score and 16.5 minutes (±6.8; range 9-27) king capacity is emphasized. The psychological for SF-12 (p=0.0536). At twelve months after the aspect not considered by the Majeed score instead surgery, the time needed for the patients to comple- of the same SF-12 is typified by 6 sub-categories te the test was: 12.5 minutes (±3.3; range 8-18) for that allows us to understand what the sequelae of the new score system, 15.2 minutes (±4.1; range dysfunctions and deficits really leave in the psycho- 9-21) for the Majeed score and 15.9 minutes (±5.5; logical state. The analysis of the injuries associated range 13-23) for SF-12 (p=0.0512) (Figure 1). with pelvic fractures and their outcomes showed that the largest subgroup was represented by type B1 fracture, according to the Tile classification, with 29 patients. The group with the most related trauma was represented by the C1 group with 175 associated injuries. The highest injury severity score was that of the C3 subgroup with 45 (± 12.6; range 32-55) points. In all surgical subgroups the anterior stabilization was performed mainly with 2 plates, while the posterior one with spinopelvic stabiliza- tion. We noticed that according to the difficulty of the fracture the anatomical reduction was reduced on radiographic control. Except in the non-surgery subgroup, in all other subgroups sexual dysfunc-

tions were the most common complications in all Figure 1. The overtime frequency trend time patients need subgroups and with any fixation method (Table 3). to compile the functional score (New Pelvic Score System): at the first month post-surgery there was no statistically sig- Time patients need to compile the functional nificant difference compared to other two scores, while after 6 score and 12 months there was a statistically positive difference in favour of the New Pelvic Score System in the compilation time The time needed for patients to complete the test Prognostic value before the trauma was: 23.4 minutes (±12.3; range 11-46) for new score system, 16.4 minutes (±5.4; The new score showed a higher ordinal correla- range 9-37) for the Majeed score and 18.5 minu- tion with the Tile classification (7) score than the tes (±6.7; range 13-35) for SF-12 (p=0.046). At the Majeed score. Tau B Kendall value was: 0.827 moment of trauma, the time needed for patients to for the new score system, 0. 673 for the Majeed complete the test was: 21.2 minutes (± 10.4; range score, 0.746 for SF-12 (p.<0.05). The result of 9-42) for the new score system, 15.9 minutes (±5.2; the analysis of factors other than the considered range 8-38) for the Majeed score and 16.3 minu- classification to predict outcomes of the Tile (7) tes (±6.4; range 13-34) for SF-12 p=0.044. There classification fractures showed that no other fac- was no statistical significance (p>0.05) between tors besides the type of fractures according the Tile the three scores at 1-month follow up after the sur- Table 4. Prognostic value and inter/intra-observer agreement gery: the time needed for patients to complete the of three different score systems test was 16.9 minutes (±12.3; range 8-34) for the Partial Eta Prognostic value score system p new score system, 16.4 minutes (±5.6; range 7-35) squared for the Majeed score and 18.5 minutes (±5.6; range New score system 0.18 0.0012 13-35) for SF-12 (p=0.0623). At the third month Majeed score 0.13 0.053 The Short Form (12) Health Survey (SF-12) 0.15 0.023 (p=0.0544) between the three scores, the time nee- K weighted ded for patients to complete the test was: 14.2 mi- value nutes (±4.7; range 8-28) for the new score system, Inter-observer agreement score system New score system 0.91 0.016 15.1 minutes (±5.8; range 8-30) for the Majeed Majeed score 0.73 0.064 score and 15.5 minutes (±3.9; range 13-24) for The Short Form (12) Health Survey (SF-12) 0.82 0.051 SF-12. At 6 months from the revision surgery, the Inter-observer agreement score system New score system 0.93 0.009 time needed for patients to complete the test was: Majeed score 0.79 0.056 13.8 minutes (±3.8; range 8-26) for the new sco- The Short Form (12) Health Survey (SF-12) 0.82 0.0503

304 Meccariello et al. Prognostic pelvic injury outcome score

classification influenced the SF-12 score, new sco- are pain (30%), return to work (20%), sitting re system, and Majeed score. A comparison of the disturbances (10%), sexual impairments (4%) prediction strength of each classification showed and walking ability (36%). The latter is subdivi- that the new score was the significant and had a ded into use of walking aids (12%), analysis of higher partial Eta squared (Table 4). unaided gait (12%), and walking distance (12%). The SF-12 is one of most commonly used valida- Inter-observer agreement ted outcome instruments and is used for subjec- Using the new score system, the average K tive self-assessment of mental health, physical weighted value among seven reviewers was and social aspects (4,12,13). It is a meaningful 0.91±0.083 showing significantly higher inter- measurement instrument for evaluating the overall observer agreement than the other two commonly quality of life. A potential disadvantage is that re- used, Majeed score classification and the SF-12, levant impairments of quality of life are captured, with 0.73±0.172 and 0.82±0.083, respectively but the relative individual importance of these li- (p=0.016) (Table 4). mitations is not sufficiently analysed (14). Scores range from 0 to 100, with higher scores indicating Intra-observer agreement a better health state (14). Eight different parts are Using the new score system, the average K analysed: physical functioning (PF), role limitati- weighted value showed a significantly higher ons due to physical health (RP), body pain (BP), intra-observer agreement than Majeed classifica- general health perceptions (GH), vitality (VT), so- tion and the SF-12, 0.93±0.056, 0.79±0.074 and cial functioning (SF), role limitations due to emo- 0.86±0.037, respectively (p=0.009). No differen- tional problems (RE), and general mental health ces were found between experienced and inexpe- (MH). Of these PF, RP, BP and GH are summa- rienced observers (Table 4). rized to measures of physical (PCS) and VT, SF, RE and MH to mental (MCS) health. SF-36, SF DISCUSSION 12- SF 8 Health Surveys measure the same eight health domains for adults aged 18 and older but An ideal pelvic outcome score system should the limit of adult age is a great limit to use in pelvic be simple, all inclusive, reliable and reproduci- fracture in young sexual adulthood (14). ble. Furthermore, a pelvic outcome score system classification should provide prognostic informa- The results of our study have shown that the new tion based on the outcomes of different fracture score system is simple, all-inclusive and has a patterns to help a surgeon to improve preope- high prognostic value; it is based on 6 labelling rative planning and treatments and predict the factors evaluated on the common problems that patient’s possible outcome. The dysfunctions patients suffered after pelvic injuries (pain, work, due to pelvic ring injuries are mainly due to the sexual possibilities, sexual satisfaction balance- fracturing mechanism as reported by Duramaz sitting-walking, and psychological status). et al. (15). Anteroposterior compression (APC) Chronic posttraumatic pelvic pain after pelvic is the most common cause of sexual dysfuncti- ring fractures impacts negatively on quality of on in both genders, independent of surgery, and life issues. Our study aimed to identify and qu- in addition, the most common cause of erection antify the problem more clearly. However, this disorder in males is vertical shear (VS) (15). Pati- unidimensional definition of chronic pain does ents with APC and VS injuries should be especi- not represent the biopsychosocial impact caused ally multidisciplinary evaluated at gynaecology, by chronic pain that can occur particularly after urology, and psychiatry departments (15). severe trauma like pelvic injury (16-17). In all pre- Currently tibial pilon fractures are classified vious studies on outcomes of pelvic ring fracture, according to the Majeed score (5). The Majeed unidimensional pain measures were used, such score is a non-validated self-developed pelvic as pain on sitting, standing, or strenuous activiti- fracture specific functional assessment instru- es (yes, no) (17): pain intensity, time since onset ment with a maximum of 100 points for patients of pain, or the SF-12 bodily pain subscale, which working before injury or 80 points for patients combines one item each of pain magnitude and in- not working before injury (32). The score items terference (17). The pain subcategory in our new

305 Medicinski Glasnik, Volume 18, Number 1, February 2021

score system is extremely easy and intuitive for have inverted the score on this scale, bringing it the patient because it traces the concept of pain from a range of 1 to 6 points to 0 to 5 where zero determination from 1 to 10 of the Visual Analogue is the pineal dysfunction and 5 is well-being. The Scale (VAS) compared to the Majeed score and Majeed score outlines only the sexual functional SF-12. Furthermore, the description of the type of capacity (6), while SF-12 only the psychological pain and the range of quantification of it allows an aspect of a possible dysfunction (24). excellent determination of the correct pain related There was no clear correlation between the fractu- only to the pelvis. re types treated and the weight-bearing protocols The return to work category was awarded the reported, or any apparent trend in the manage- highest score of 25 points in the new score ment over time (25). There was, however, a slight system. The ability to return to work is dictated trend in the management of type C fractures. Sci- by various factors besides fracture osteosynthe- entific literature papers reporting non-weight-be- sis, and the reduction of complications and aring protocols, 86% included type C fractures, dysfunctions is also related to associated injuri- the figure being 84% for partial weight-bearing es and their outcomes. According McMinn et al. protocols but only 33% for full weight-bearing. poor mental health outcomes are known to have The same figures for type B injuries were 56% a significant effect on recovery from an injury, for non- and full weight-bearing, and 68% for including more re-admissions and follow-up vi- partial weight-bearing protocols (25). sits, delayed return-to-work, and higher rates of Balance-sitting-walking is the fifth point of new disability. Indeed, relative to the number of pati- outcomes pelvic score. This category of the score ents who returned to work at 3 months, there was to which we have attributed 20 points summa- a significant increase at 12 months (18). rizes 5 points of the Majeed score (29). The 8 Pelvic fractures are usually a result of high energy subcategories allow to understand the patient’s trauma, and sexual dysfunction after a pelvic working capacity and his return to work based fracture is a frequent complication (18). Though on the ability of the movement and maintaining organic pathologies can be the reason for sexual balance. The SF-12, on the other hand, is a non- disorders, and psychological factors following specific test for this dimension of the pelvic inju- a trauma may also be linked to sexual problems ries problem. (18). Our third categories of the sexual possibili- Previous literature has suggested that traumatic ties are formed by a very intuitive score from 1 to pelvic injuries are predictive of lowered quality 10, which allows the subject to outline his sexual of life due to their association with dysfunction, ability after pelvic injuries. As for the pain scale, correlated injuries, and chronic pain (1-25). the descriptive sub-categories of the type of limi- Concurrent with this dysfunction, correlated inju- tation have a range for which the patient knows ries and chronic pain, participants also reported which score to attribute to his limitation. This worsening physical and mental health and quality category has been modulated to make males and of life in the year after their injuries (18, 26-28). females of any age respond. In fact, many studi- es demonstrated that differences in mood status The sixth and final evaluation category of the exist between older and younger males or fema- new score system is the one that evaluates the les following pelvic injuries and those injuries are patient’s psychological state. In the Majeed score associated with increased depression in older pati- this evaluation is absent (5), while in the SF-12 it ents. Assessment of mood status in both short and is well evaluated in general (18). long terms following fracture in the elderly seems The main limitation of this study is a low num- justified, with early detection and treatment likely ber of patients. Another problem is in scientific to result in improved outcomes (18-22). validation and cross-cultural adaptation of the The Arizona Sexual Experience Scale is the sim- patient’s lifestyle and cultural adaptation betwe- plest and most intuitive scale for evaluating pa- en Italian and Spanish culture. Additionally, the tient sexual dysfunctions (22,23). It is also most main limitations are the weakness of a longitudi- validated (22,23). To make it easier and more nal study applied on a great variability of surgical adaptable to the structure of our score system, we techniques. Other limits include various sexual

306 Meccariello et al. Prognostic pelvic injury outcome score

and daily patients’ activities before the trauma. psychological status of pelvic injuries; it can im- These limits probably resulted in some biases, prove preoperative planning and subsequently such as that of surgeons’ intra- and inter obser- rehabilitation and psychological support, which vational capacity. affect the quality of life more than other diseases. In conclusion, the new score system is progno- FUNDING stic, reliable, reproducible and can become a useful instrument to adequately correlate long- No specific funding was received for this study. term outcome of these fractures. In our opinion, the new proposed score system provides a better TRANSPARENCY DECLARATION evaluation of pain, work, sexual possibilities, Conflict of interest: None to declare. sexual satisfaction, balance-sitting-walking, and

REFERENCES

1. Falzarano G, Medici A, Carta S, Grubor P, Fortina 13. Müller FJ, Stosiek W, Zellner M, Neugebauer R, M, Meccariello L, Ferrata P. The orthopedic dama- Füchtmeier B. The anterior subcutaneous internal ge control in pelvic ring fractures: when and why-a fixator (ASIF) for unstable pelvic ring fractures: cli- multicenter experience of 10 years’ treatment. J of nical and radiological mid-term results. Int Orthop Acute Disease 2014; 3; 201-6. 2013; 37:2239-45. 2. Falzarano G, Rollo G, Bisaccia M, Pace V, Lan- 14. Huo T, Guo Y, Shenkman E, Muller K. Assessing the zetti RM, Garcia-Prieto E, Pichierri P, Meccariello reliability of the short form 12 (SF-12) health Sur- L. Percutaneous screws CT guided to fix sacroiliac vey in adults with mental health conditions: a report joint in tile C pelvic injury. Outcomes at 5 years of from the wellness incentive and navigation (WIN) follow-up. SICOT J 2018; 4:52. Study. Health Qual Life Outcomes 2018; 16:34. 3. Gokalp MA, Hekimoglu Y, Gozen A, Guner S, Asir- 15. Duramaz A, Ilter MH, Yıldız Ş, Edipoğlu E, İpek C, dizer M. Evaluation of severity score in patients with Bilgili MG. The relationship between injury mecha- lower limb and pelvic fractures injured in motor ve- nism and sexual dysfunction in surgically treated hicle front-impact. Collisions Med Sci Monit 2016; pelvic fractures. Eur J Trauma Emerg Surg 2020; 22:4692-8. 46:807-16. 4. Gerbershagen HJ, Dagtekin O, Isenberg J, Martens 16. Leserman J, Zolnoun D, Meltzer-Brody S, Lamvu N, Ozgür E, Krep H, Sabatowski R, Petzke F Chron- G, Steege JF. Identification of diagnostic subtypes ic pain and disability after pelvic and acetabular of chronic pelvic pain and how subtypes differ in he- fractures--assessment with the Mainz Pain Staging alth status and trauma history. Am J Obstet Gynecol System J Trauma 2010; 69:128-36. 2006; 195:554-60. 5. Majeed SA. Grading the outcome of pelvic fractures. 17. Steingrímsdóttir OA, Landmark T, Macfarlane GJ, J Bone Joint Surg Br 1989; 71:304-6. Nielsen CS. Defining chronic pain in epidemiologi- 6. Gänsslen A, Lindahl J. Evaluation Tools and Outco- cal studies: a systematic review and meta-analysis. mes After Osteosynthesis of Unstable Type B and C Pain 2017; 158:2092-107. Pelvic Ring Injuries. Acta Chir Orthop Traumatol 18. McMinn KR, Thomas EV, Martin KR, Khetan JN, Cech 2013; 80:305-20 McShan EE, Bennett MM, Solis J, Jones AL, Powers 7. Furey AJ, O’Toole RV, Nascone JW, Sciadini MF, MB, Warren AM. Psychological morbidity and func- Copeland CE, Turen C. Classification of pelvic tional impairment following traumatic pelvic injury. fractures: analysis of inter- and intra-observer varia- Injury 2020; 51:978-83. bility using the Young-Burgess and Tile classificati- 19. Williams LJ, Berk M, Henry MJ, Stuart AL, Brennan on systems. Orthopedics 2009; 32:401. SL, Jacka FN, Pasco JA. Depression following 8. Cohen J. A coefficient of agreement for nominal sca- fracture in women: a study of age-matched cohorts. les. Educ Psychol Meas 1960; 20:37–46. BMJ Open 2014; 4:e004226. 9. Fleiss JL. Measuring nominal scale agreement 20. Fanjalalaina Ralahy M, Parfaite Randriantsoa M, among many raters. Psychol Bull 1971; 76:378–82. Rakototiana A, Razafimahandry HJ. Incidence of 10. Shoukri MM, Colak D, Kaya N, Donner A. Compa- erectile dysfunction in pelvic ring injuries: Study of rison of two dependent within subject coefficients of 48 patients at the Antananarivo hospital, Madagas- variation to evaluate the reproducibility of measure- car. Orthop Traumatol Surg Res 2019; 105:885-8. ment devices. BMC Med Res Methodol 2008; 8: 24. 21. Laurent SM, Simons AD. Sexual dysfunction in 11. Landis JR, Koch GG. The measurement of observer depression and anxiety: conceptualizing sexual agreement for categorical data. Biometrics 1977; dysfunction as part of an internalizing dimension. 33:159–74 Clin Psychol Rev 2009; 29:573-85. 12. Shang K, Ke C, Fu YH, Han S, Wang PF, Zhang BF, 22. Copuroglu C, Yilmaz B, Yilmaz S, Ozcan M, Ciftde- Zhuang Y, Zhang K. Feasibility of anterior pelvic mir M, Copuroglu E. Sexual dysfunction of male, af- ring fixation alone for treating lateral compression ter pelvic fracture. Eur J Trauma Emerg Surg 2017; type 1 pelvic fractures with nondisplaced complete 43:59-63. sacral fractures: a retrospective study. PeerJ 2020; 8:e8743.

307 Medicinski Glasnik, Volume 18, Number 1, February 2021

23. Elnazer HY, Baldwin DS. Structured review of the 27. Muscatelli S, Spurr H, OʼHara NN, OʼHara LM, use of the Arizona sexual experiences scale in clini- Sprague SA, Slobogean GP. Prevalence of depre- cal settings. Hum Psychopharmacol 2020; 35:e2730. ssion and posttraumatic stress disorder after acute 24. Cannada LK, Barr J. Pelvic fractures in women orthopaedic trauma: a systematic review and meta- of childbearing age. Clin Orthop Relat Res 2010; analysis. J Orthop Trauma 2017; 31:47-55. 468:1781-9. 28. Goussous N, Sawyer MD, Wuersmer LA, Huebner 25. Rickman M, Link BC, Solomon LB. Patient weight- M, Osborn ML, Zielinski MD. Comparison of sexu- bearing after pelvic fracture surgery- A systematic al function and quality of life after pelvic trauma review of the literature: what is the modern eviden- with and without angioembolization. Burns Trauma ce base? Strategies Trauma Limb Reconstr 2019; 2015; 3: 21. 14:45-52. 26. Ter Kuile MM, Weijenborg PT, Spinhoven P. Sexu- al functioning in women with chronic pelvic pain: the role of anxiety and depression. J Sex Med 2010; 7:1901-10.

308 ORIGINAL ARTICLE

Navigated percutaneous screw fixation of the pelvis with O-arm 2: two years’ experience

Gianluca Ciolli1, Daniele Caviglia1, Carla Vitiello2, Salvatore Lucchesi3, Corrado Pinelli1, Domenico De Mauro1, Amarildo Smakaj1, Giuseppe Rovere1, Luigi Meccariello4, Lawrence Camarda5, Giulio Mac- cauro1, Francesco Liuzza1

1Department of Orthopaedics, A. Gemelli University Hospital Foundation IRCCS, Catholic University, Rome, 2Department of Radiology, Ospedali Riuniti - Area Vesuviana - ASL Napoli 3 sud, Napoli, 3Department of Radiology, A. Gemelli University Hospital Foundation IRCCS, Catholic University, Rome, 4Department of Orthopaedics and Traumatology, AORN San Pio, Benevento, 5 Department of Ortho- paedic Surgery, University of Palermo, Palermo; Italy

ABSTRACT

Aim To evaluate the case series of the patients operated with per- cutaneous fixation by the navigation system based on 3Dfluo- roscopic images, to assess the precision of a surgical implant and functional outcome of patients.

Methods A retrospective study of pelvic ring fractures in a 2-year period included those treated with the use of the O-Arm 2 in com- bination with the Stealth Station 8. Pelvic fractures were classifi- ed according to the Tile and the Young-Burgess classification. All Corresponding author: patients were examined before surgery, with X-rays and CT scans, Francesco Liuzza and three days after surgery with additional CT scan. The positio- Department of Orthopaedics, A. Gemelli ning of the screws was evaluated according to the Smith score, the outcome with the SF-36. University Hospital Foundation IRCCS, Rome, Results Among 24 patients 18 were with B and six with C type Italy Catholic University Rome, Italy fracture according to Tile, while eight were with APC, 10 LC, and Largo Agostino Gemelli 8, 00168 Roma six with VS type according to Young-Burgess classification. All Phone: 06 3015 4036; patients were treated in the supine position, except two. A total of 41 iliosacral or transsacral screws and five anterior pelvic ring Fax: 06 3015.5981; screws were implanted. The medium surgical time per screw was E-mail: 41 minutes. There was a perfect correspondence of screw scores [email protected] value from post-operative CT and intraoperative fluoroscopy. The Gianluca Ciolli ORCID ID: https://orcid. mean screw score value was 0.92. There were no cases of poor org/0000-0002-3653-4552 positioning. The median follow-up was 17.5 months. The patients were satisfied with their health condition on SF-36. Original submission: Conclusion The use of the O-arm guarantees great precision in the 02 December 2020; positioning of the screws and reduced surgical times with excellent Accepted: clinical results in patients. 08 December 2020 Key words: 3D-fluoroscopic navigation, fragility fracture of doi: 10.17392/1326-21 pelvis, iliosacral fixation, O-arm Stealth Station S8, pelvic ring fractures

Med Glas (Zenica) 2021; 18(1):309-315

309 Medicinski Glasnik, Volume 18, Number 1, February 2021

INTRODUCTION the SARS-CoV-2 pandemic began and our Hospi- tal became involved (10). Of the pelvic ring fractu- Fractures of the pelvic ring account for 3% of all res treated, we included only those treated with fractures with an incidence of about 20-40 cases the use of the O-Arm 2 system (Medtronic Inc., per 100,000 citizens (1-2). Pelvic ring fractures Louisville, Colorado, USA) in combination with have a bimodal distribution by age, with peaks in the Stealth Station 8 navigation system (Medtronic the 15-30- and 50-70-years age groups. In the yo- Navigation) without exclusion criteria. ung population, fractures mostly involve males, victims of high-energy trauma (road accidents Pelvic fractures were classified according to the or falls from great heights), while in the elderly Tile classification (11) based on the stability of osteoporotic population, females are prevalent the pelvic ring (type A: Stable fracture, type B: and following low-energy trauma (falls from the rotationally unstable and vertically stable, fractu- standing position or bed) (3). res, type C: rotationally and vertically unstable fractures), and the Young-Burgess classification Pelvic ring surgery is complex and requires deep (12) based on the direction of the forces causing knowledge of the anatomy of the pelvis and a long the injury (anterior posterior compression - APC, learning curve for the surgeon, as well as a hospital lateral compression - LC, vertical shear - VS). able to guarantee assistance to patients, often he- modynamically unstable. Open reduction and in- Methods ternal fixation of the pelvic ring requires extensive approaches and long operating times, which can All patients were examined before surgery with lead to various early and long-term complications pelvic specific X-ray views (anteroposterior, for the patient (4-5). Therefore, in recent years, inlet, outlet) and with thin-slice CT with multi- increasing attention has been directed towards mi- planar reconstructions. Three days after surgery, nimally invasive or percutaneous techniques that additional CT scan was obtained to assess the po- allow less blood loss, neurovascular risks, and a sition of the screws. The radiolucent carbon table lower rate of infections (6-7). In parallel with their was used to allow intra-operative radiological vi- increased use, the available surgical instrumentati- sualization without interference, and the position on has improved, with the introduction of naviga- taken by the patient on the operating bed during tion techniques using high-quality fluoroscopy or the surgery was reported. All patients were trea- CT with 3D reconstructions. Those technologies ted by the same surgeon and with the O-Arm 2 assist the surgeon in all phases of surgery, from system in combination with the Stealth Station the confirmation of the preoperative planning to navigation system S8. The O-Arm 2 System per- the evaluation of the reduction obtained, up to the forms an intraoperative 3D fluoroscopic scan of execution of the surgical procedure, reducing the the pelvis, which is visualized with the Stealth operating time and increasing the surgeon's con- Station Navigation System showing the position fidence (8-9). The aim of this study was to retros- of moving objects in three planes of space, with pectively evaluate the case series of the patients multiple imaging protocols (low or high dose) operated with percutaneous fixation guided by that allow the surgeon the flexibility to choosean the navigation system based on 3D fluoroscopic appropriate dose for the patient based on indivi- images (O-arm 2 Medtronic), judging the preci- dual clinical goals (Figures 1, 2). With this tech- sion of the performed surgical procedure and the nology, the surgeon can virtually navigate each functional outcome of the patients. screw intraoperatively with a viewfinder centring system, from the entry point to the different pla- PATIENTS AND METHODS nes and calculate, without any other measure, the length and diameter of the screw. Only after those Patients and study design virtual screw checks on the monitor, the surgeon can safely insert the k-wire, through a cannulated We conducted a retrospective study of all surgically trocar, and then the cannulated real screw into the treated pelvic ring fractures in the Department of bone (13). During the acquisition of imaging, the Orthopaedics, A. Gemelli University Hospital Fo- surgical team was outside the operating room. undation IRCCS Level II Trauma Centre, Rome, The operative objective was to obtain the anato- between February 2018 and February 2020, before

310 Ciolli et al. Pelvic fixation with O-arm

a "screw score" ranging from a minimum of 0 po- ints (in case of perfect positioning) up to a maxi- mum of 6 points (in case a revision is necessary) based on deviation from ideal screw position. In the Smith score, each screw was assigned a nu- merical score that was the sum of each perforation and angular grade. Perforations were graded as grade 0 - no perforation, grade 1 - perforation less Fig 1. A) Navigated percutaneous fixation with O-Arm2 and Stealth station Navigation System. Once the trackers have been than 2 mm, grade 2 - perforation between 2 and 4 positioned on the iliac crest to locate the bone target, a cannu- mm, and grade 3 - perforation more than 4 mm. lated trocar can be used for surgical navigation. B) The three- The angle of each screw relative to the respecti- dimensional mode allows to obtain a simultaneous and complete multiplanar examination on the screen. The 3-window configura- ve superior endplate was measured and assigned tion highlights the axial, sagittal and coronal planes related to a grade: grade 0 for an angle less than 5°, grade 2 each other by a "crosshair” centring system (Liuzza F, 2019) for an angle of 5°–10°, grade 3 for an angle of 11°– 15°, and grade 4 for an angle more than 15°. Each screw was assigned a numerical score that was the sum of each perforation and angular grade. Functional outcome was assessed with the use of the SF-36 (Short-Form 36 items health Survey). We compared the measurements obtained from post-operative CT and intraoperative fluoroscopy using O-arm. The SF health survey 36 (standard questionnaire test) was administered 6 months after surgery (17-18). Patients began physiotherapy, with passive and active-assisted mobilization starting from the se- Figure 2. A) 3D CT reconstruction: pelvic ring B1 injury accord- cond postoperative day, and began weight-bea- ing to Tile and APC 2 injury according to Young Burgess. It is possible to appreciate sacral dysmorphism (Bertolotti's Syn- ring in a personalized way based on the type of drome) with hemisacralization of L5 on the left side; B) antero- fracture and the type of patient. The functional posterior X-ray after damage control with supracetabular exter- evaluation was done with a periodic clinical and nal fixator; C-E) post-operative X-ray: AP, inlet and outlet view after reduction and fixation with anterior 3.5 sacroiliac plate and radiological follow-up at 1, 3, 6, and 12 months, two 7.3 trans-sacral screws; the pubic symphysis was fixed with then annually. a 3.5 dedicated plate and six screws (Liuzza F, 2019) RESULTS mical reduction, with the least possible invasive- ness, and to obtain a stable and strong fixation. Of 120 pelvic ring fractures treated surgically Axial traction on the lower limb or a large dis- between February 2018 and February 2020, we tractor or external fixator could be used. It is also considered only 24 (17 males and seven females) possible to obtain a solid and provisional fixation for which we used the O-Arm 2 system in combi- with the operating bed employing special frames, nation with the Stealth Station 8 navigation system like Matta or Starr Frame (14-15). (Table 1). The mean age was 48.9 years with a On the third postoperative day, patients un- maximum age of 89 and a minimum of 18. Accor- derwent radiographs and thin-slice CT with mul- ding to the Tile classification the patients were cla- tiple control reconstruction examinations, by a ssified as B1 (five cases), B2 (13 cases), and C1 (six team of musculoskeletal anatomy radiologists. cases), while according to the Young-Burgess cla- They evaluated accuracy of the positioning of the ssification there were eight APC, 10 LC, and 6 VS. implanted screws and compared the results of the All patients were treated in the supine position, three-dimensional reconstruction performed in except two prone. In one case, a patient was tre- the operating room with the postoperative CT. ated in two surgical stages, first supine and then The positioning of the screws was evaluated prone. In all cases of anterior and posterior fixati- according to the Smith score (16), which produces on, anterior surgical time was performed first and

311 Medicinski Glasnik, Volume 18, Number 1, February 2021

Table 1. Characteristics of 24 patients with fractures treated using the O-Arm 2 system in combination with the Stealth Station 8 navigation system Patient Gender Age Tile* Young-Burgess* Implants Position 1 M 33 61B2.3 APC III Symphysis plate, Iliosacral screw Supine 2 M 50 61B2.3 APC II Symphysis plate, Iliosacral screw Supine 3 M 28 61B2.3 APC II Symphysis plate, Iliosacral screw Supine 4 F 51 61B2.2 LC I Pubic ramus screw, Iliosacral screw Supine 5 M 52 61B1 LC I Iliosacral screw, InFix Supine 6 M 38 61B2.1 LC I Transsacral screw, Iliosacral screw, Symphysis plate Supine 7 F 79 61B2.1 LC I Pubic ramus screw, Iliosacral screw Supine 8 F 51 61B2.1 LC I Transsacral screw, InFix Supine 9 M 89 61B2.3 APC III Symphysis plate, Transsacral screw Supine 10 M 46 61B1 APC I Pubic ramus screw, Iliosacral screw, Transsacral screw Supine 11 M 58 61C1.3 VS Transsacral screw, Iliosacral screw, Neutralization plate, Suprapectineal plate Supine/Prone 12 F 35 61B2.1 LC I Iliosacral screw, Transsacral screw Prone 13 M 18 61B1 APC III Iliosacral screw, Transsacral screw Supine 14 M 69 61C1.3 VS Symphysis plate, RECON plate, Transsacral screw Supine Iliosacral screw, Transsacral screw, Lumbopelvic fixation with pedicle screws 15 F 30 61C1.3 VS Prone and rod 16 M 65 61C1.3 VS Symphysis plate, RECON plate, Transsacral screw Supine 17 M 44 61B2.1 LC I Transsacral screw, Iliosacral screw, Symphysis plate Supine 18 M 33 61B1 APC III Iliosacral screw, Transsacral screw Supine 19 M 50 61B2.1 LC I Transsacral screw, Symphysis plate Supine 20 M 48 61B1 APC III Transsacral screw Supine 21 F 35 61C1.3 VS Symphysis plate, Transsacral screw Supine 22 M 56 61B2.1 LC I Transsacral screw, Iliosacral screw, Symphysis plate Supine 23 F 67 61C1.3 VS Symphysis plate, RECON plate, Transsacral screw Supine 24 M 52 61B2.1 LC I Transsacral screw, Iliosacral screw, Symphysis plate Supine *classification M, male; F, female; then percutaneous posterior fixation with O-arm, two cases (Smith grade 1) with perforation less as we described previously (13). Anterior fixation than 2 mm. The angle of each screw relative to was performed open, with Pfannenstiel approach, the respective superior endplate in 13 cases was in 13 cases, while in five cases it was performed between 5° and 10° (Smith grade 1), and between percutaneously with O-arm. 5° and 10° (Smith grade 2) in three cases. The A total of 41 iliosacral or transsacral screws, other seven screws have an angulation less than three ramus pubic-screws, and two in-fix were 5° (Smith grade 0). In 3rd day post-operative CT, implanted. In 16 cases an iliosacral screw was the grade of perforation and angulation were com- placed for fixation of the posterior arc of the parable to those evaluated on intraoperative CT pelvis, six without any other posterior fixation. scans. No implant breakage was observed. Screw A transiliac screw was used in 18 cases, 10 with revision was not necessary. The radiation dose re- another iliosacral screw and eight without other ceived by the operative team outside the operating posterior fixation. To fix the anterior arc of the room, during the imaging acquisition, was consid- pelvis a symphysis plate in 13 cases was used, a ered null. There were no complications due to the retrograde screw in the pubic ramus in three ca- placement of the screws. ses, and an in-fix in two cases (Figure 2). The median follow-up was 17.5 (±6.7) months. Surgical time per screw of 41 minutes on average Nine of 24 (37.5%) patients were followed up at (±12.5) was observed. 2 years, two were followed up at 6 months, the others after almost one year. At the last follow- In all cases, there was a perfect correspondence of up, the patient was satisfied with his health con- the perforation and angle values; so there was a dition (SF-36: 120% for the physical component perfect correspondence of screw score values. The summary score, 85% for the mental component mean value was 0.92 (±0.76). A perfectly located summary score compared to the age and gender screw corresponds to a screw score of 0, obtained controlled German population (18). in seven patients. There were no cases of poor po- sitioning in any of the implanted screws, no screw DISCUSSION score more than 2 in all cases. The screws were found to be completely intraosseous without any Pelvic surgeons use traditional fluoroscopy for penetration in all cases (Smith grade 0) except in percutaneous and mini-open procedures. Traditi-

312 Ciolli et al. Pelvic fixation with O-arm

onal fluoroscopy uses specific projections (ante- merous recent works (32). The complication roposterior view, inlet view, outlet view, Judet's rate and malpositioning of the screws with this views and lumbosacral spine views) that allow technology vary from 0 to 15% against the 10- the surgeon to safely place the screws within the 20% of placement with traditional fluoroscopy, bony corridors of the pelvic ring (19,21). This which has an incidence of neurological damage imaging is the most popular among surgeons be- between 0.5 and 7.7% (32,33). Our study shows cause it is inexpensive and widely used; however, that navigated percutaneous screw fixation ofthe it presents some technical difficulties which may pelvis with O-arm 2 is a safe and reliable surgi- concern the positioning of the patient on the ope- cal technique, which produces an intraoperative rating table, in the case of severe thoracic injury image comparable to that obtained with the po- with concomitant sternal or rib fractures or pneu- stoperative CT scan; the functional outcome was mothorax that prevent the patient from assuming judged satisfactory for all patients. the prone position (19,20). Also, some characteri- The biggest problem observed in our study con- stics of the patient can cause interference such as cerns the sexual sphere and sexual dysfunctions obesity or intestinal gas, or urinary and abdominal of the patients following the trauma. viscera, which would deserve some preoperative The limitation of our study is that it is a retros- preparations that are not always possible (20,21). pective study, with a limited sample of patients Other disadvantages are the exposure of the sur- and no control group. However, it produces the gical team to radiation and the inability to obta- groundwork for targeted use of the O-Arm 2 in simultaneous images on different planes (22). in patients who require minimally invasive but To overcome these difficulties, new technologies mechanically stable surgery, particularly in diffi- have been introduced in recent years, capable of cult conditions such as in the presence of sacral helping the surgeon to increase the precision and dysmorphism, obesity, gas in the abdominal vis- safety of screw placement, with similar surgical ti- cera, or a case of supine positioning by the pati- mes (23,26). They also make it possible to naviga- ent. Kaiser et al. show that sacral dysmorphism te bony corridors otherwise difficult to synthesize, was found in 41% of the pelvis and described such as pubic branches (27). a sacral dysmorphism score that quantifies the A fundamental requirement for the fixation of the dysmorphism and can be used in preoperative pelvic ring is obtained an anatomical reduction, planning of iliosacral screw placement. They through a previous temporary reduction with K also add that all patients with a sacral dysmorp- wires, large distractor, or external fixators. The hism scored >70 do not have a safe corridor for only exception to achieving perfect anatomical percutaneous fixation of the first sacral vertebra reduction is fragility fractures of the pelvic ring, (34,35). In those cases, it is strongly recommen- which require a compromise between the least ded to place ileosacral or trans-sacral screws with possible invasiveness of the surgery and mecha- navigated procedures and we recommend the use nically stable fixation (28,30). of the O-arm 2 in combination with the stealth Rommens et al. explained that fragility fracture station navigation system S8. Furthermore, as de- of the pelvis requires patient-specific treatment, monstrated by a previous work of our group (36), where fixation rigidity is more important than it should be remembered that it is a procedure anatomical reduction and to achieve this, large that exposes the patient to a radiation dose com- exposures and long duration of surgery must be parable to traditional fluoroscopy and lower than avoided; preferred percutaneous or minimally that of a CT scan, while it eliminates the radiation invasive approaches (28,30). We hope that the- exposure by the surgical team that is outside the se patients, who require multidisciplinary tre- operating room during image acquisition. atment, will be able to have targeted anaesthesia In conclusion, surgical treatment of pelvic ring for the pelvic ring as those that already exists for fractures is complex and requires a long learning acetabular fractures (31). curve. The increased attention given to percuta- The increase in surgical precision in the positi- neous surgery in this type of fracture is justified oning of pelvic screws with the support of CT by the increase in fragility fracture of the pelvis navigated instruments has been reported in nu- and by the improvement of diagnostic imaging

313 Medicinski Glasnik, Volume 18, Number 1, February 2021

and surgical technology. The capacity to navigate team, and reduced surgical time. The results of with the support of 3D fluoroscopic or CT images this study confirm the excellent clinical results in turns into greater safety for the surgeon in the patients undergoing percutaneous navigation of placement of difficult percutaneous screws such the O-arm screws. as trans-iliac screws, which can guarantee grea- We hope that with the help of dedicated tools this ter mechanical stability to fixation. It also allows intervention can be even more implemented and for avoiding open approaches, long surgery time, enhanced. and other complications related to open reduction and internal fixation (ORIF). The use of the O- FUNDING arm in our experience guarantees us an instant No specific funding was received for this study. precision in the positioning of the screws, a ra- diation exposure similar to the fluoroscopic tech- TRANSPARENCY DECLARATION nique for the patient and absent for the surgical Conflict of interest: None to declare. REFERENCES 1. Buller LT, Best MJ, Quinnan SM. A nationwide 13. Liuzza F, Capasso L, Florio M, Mocini F, Masci G, analysis of pelvic ring fractures: incidence and tren- Cazzato G, Ciolli G, Silluzio N, Maccauro G. Tran- ds in treatment, length of stay, and mortality. Geriatr siliosacral fixation using the O-ARM2® and STE- Orthop Surg Rehabil 2016; 7:9–17. ALTHSTATION® navigation system. J Biol Regul 2. Giannoudis PV, Grotz MR, Tzioupis C, Dinopoulos Homeost Agents 2018; 32(Suppl. 1):163-71. H, Wells GE, Bouamra O, Lecky F. Prevalence of 14. Starr AJ, Walter JC, Harris RW, Reinert CM, Jones pelvic fractures, associated injuries and mortality: AL. Percutaneous screw fixation of fractures of the The United Kingdom perspective. J Trauma 2007; iliac wing and fracture-dislocations of the sacro-iliac 63:875–83. joint (OTA Types 61-B2.2 and 61-B2.3, or Young- 3. Pereira GJC, Damasceno ER, Dinhane DI, Bueno Burgess lateral compression type II pelvic fractures). FM, Leite JBR, Ancheschi BDC. Epidemiology of J Orthop Trauma 2002; 16:116-23. pelvic ring fractures and injuries. Rev Bras Ortop 15. Matta JM, Yerasimides JGJ. Table-skeletal fixation 2017; 52:260-9. as an adjunct to pelvic ring reduction. Orthop Trau- 4. Rommens PM. Is there a role for percutaneous ma 2007; 21:647-56. pelvic and acetabular reconstruction? Injury 2007; 16. Smith HE, Yuan PS, Sasso R, Papadopolous S, 38:463–77. Vaccaro AR. An evaluation of image-guided tech- 5. Barei DP, Bellabarba C, Mills WJ, Routt Jr ML. Per- nologies in the placement of percutaneous iliosacral cutaneous management of unstable pelvic ring dis- screws. Spine 2006; 31:234-8. ruptions. Injury 2001; 32:SA33-44. 17. Ware JE Jr, Sherbourne CD. The MOS 36-item 6. Routt Jr ML, Nork SE, Mills WJ. Percutaneous fixa- short-form health survey (SF-36). I. Conceptual fra- tion of pelvic ring disruptions. Clin Orthop 2000; mework and item selection. Med Care 1992; 30:473- 375:15-29. 83. 7. Routt ML Jr, Kregor PJ, Simonian PT, Mayo KA. 18. Bullinger M, Kirchberger I. SF-36 Questionnaire Early results of percutaneous iliosacral screws pla- for the Health Survey - Handbook for the German ced with the patient in the supine position. J Orthop Version of Questionnaire. Göttingen, Bern, Toronto, Trauma 1995; 9:207-14. Seattle: Hogrefe-Verlag, 1998. 8. Schep NW, Haverlag R, van Vugt AB. Computer- 19. Hinsche AF, Giannoudis PV, Smith RM. Fluorosco- assisted versus conventional surgery for insertion of pybased multiplanar image guidance for insertion 96 cannulated iliosacral screws in patients with po- of sacroiliac screws. Clin Orthop Relat Res 2002; stpartum pelvic pain. J Trauma 2004; 57:1299-302. 395:135–44. 9. Takao M, Hamada H, Sakai T, Sugano N. Factors 20. Routt MLC Jr, Gary JL, Kellam JF, Burgess AR. J. influencing the accuracy of iliosacral screw insertion Improved Intraoperative fluoroscopy for pelvic and using 3D fluoroscopic navigation. Arch Orthop Tra- acetabular surgery. Orthop Trauma 2019; (Suppl uma Surg 2019; 139:189-95. 2):S37-42. 10. De Mauro D , Rovere G , Smimmo A , Meschini 21. Liuzza F, Silluzio N, Florio M, El Ezzo O, Cazzato C, Mocini F , Maccauro G , Falez F , Liuzza F, Zira- G, Ciolli G, Perisano C, Maccauro G. Comparison nu A. COVID-19 pandemic: management of patients between posterior sacral plate stabilization versus affected by SARS-CoV-2 in Rome COVID Hospital minimally invasive transiliac-transsacral lag-screw 2 Trauma Centre and safety of our surgical team Int fixation in fractures of sacrum: a single-centre expe- Orthop 2020; 44:2487-91. rience. Int Orthop 2019; 43:177-85. 11. Tile M, Helfet D, Kellam J. Fractures of the Pel- 22. Zwingmann J, Konrad G, Mehlhorn AT, Südkamp vis and Acetabulum. Philadelphia, PA: Lippincott NP, Oberst M. Percutaneous iliosacral screw inser- Williams & Wilkins, 2003. tion: malpositioning and revision rate of screws with 12. Young JW, Burgess AR, Brumback RJ, Poka A. regards to application technique (navigated vs. con- Pelvic fractures: value of plain radiography in early ventional). J Trauma 2010; 69:1501–6. assessment and management. Radiology 1986; 160:445–51.

314 Ciolli et al. Pelvic fixation with O-arm

23. Peng KT, Huang KC, Chen MC, Li YY, Hsu RW. 30. Wagner D, Ossendorf C, Gruszka D, Hofmann A, Percutaneous placement of iliosacral screws for un- Rommens PM. Fragility fractures of the sacrum: stable pelvic ring injuries: comparison between one how to identify and when to treat surgically? Eur J and two C-arm fluoroscopic techniques. J Trauma Trauma Emerg Surg 2015; 41:349-62. 2006; 60:602–8. 31. Vergari A, Frassanito L, Tamburello E, Nestorini R, 24. Schep NW, Haverlag R, van Vugt AB: Computer- Sala FD, Lais G, Ciolli G, Liuzza F. Supra-inguinal assisted versus conventional surgery for insertion of fascia iliaca compartment block for postoperative 96 cannulated iliosacral screws in patients with po- analgesia after Acetabular fracture surgery. Injury stpartum pelvic pain. J Trauma 2004; 57:1299-302. 2020; 2:S0020-1383(20)30801-9. 25. Radetzki F, Wohlrab D, Goehre F, Wohlrab D, Goe- 32. Thakkar SC, Sirisreetreerux N, Carrino JA , Shafiq hre F, Noser H, Delank K S, Mendel T. Anatomical B, Hasenboehler EA. 2D versus 3D fluoroscopy- conditions of the posterior pelvic ring regarding bi- based navigation in posterior pelvic fixation: review segmental transverse sacroiliac screw fixation: a 3D of the literature on current technology. Int J Comput morphometric study of 125 pelvic CT datasets. Arch Assist Radiol Surg 2017; 12:69-76. Orthop Trauma Surg 2014; 134:1115–20. 33. Falzarano G, Rollo G, Bisaccia M, Pace V, Lan- 26. Theologis AA, Burch S, Pekmezci M. Placement zetti RM, Garcia-Prieto E, Pichierri P, Meccariello of iliosacral screws using 3D image-guided (O- L. Percutaneous screws CT guided to fix sacroiliac arm) technology and stealth navigation: compari- joint in tile C pelvic injury. Outcomes at 5 years of son with traditional fluoroscopy. Bone Joint J 2016; follow-up. SICOT J 2018; 4:52. 98–B:696–702. 34. Kaiser SP, Gardner MJ, Liu J, Routt ML Jr, Morshed 27. Mosheiff R, Liebergall M. Maneuvering the retro- S. Anatomic determinants of sacral dysmorphism grade medullary screw in pubic ramus fractures. J and implications for safe iliosacral screw placement. Orthop Trauma 2002; 16:594–6. J Bone Joint Surg Am 2014; 96:e120. 28. Rommens PM, Hofmann A. Comprehensive classi- 35. Miller AN, Routt ML Jr. Variations in sacral morp- fication of fragility fractures of the pelvic ring: Re- hology and implications for iliosacral screw fixation. commendations for surgical treatment. Injury 2013; J Am Acad Orthop Surg 2012 J; 20:8-16. 44:1733-44. 36. Michela F, Capasso L, Olivi A, Vitiello C, Leone A, 29. Rommens PM, Arand C, Hofmann A, Wagner D. Liuzza F. 3D - navigated percutaneous screw fixati- When and how to operate fragility fractures of the on of pelvic ring injuries - a pilot study. Injury 2020; pelvis? Indian J Orthop 2019; 53:128-37. 51 (Suppl 3):S28-33.

315 ORIGINAL ARTICLE

Comparing hand strength and quality life of locking plate versus intramedullary k wire for transverse midshaft metacarpal fractures Andrea Pasquino1, Alessandro Tomarchio2, Enio De Cruto1, Jacopo Conteduca1, Damiano Longo1, Valen- tina Russi1, Giuseppe Pica3, Luigi Meccariello3, Giuseppe Rollo1

1Department of Orthopaedics and Traumatology, Vito Fazzi Hospital, Lecce, Italy, 2Department of Orthopaedics and Traumatology, S. Croce e Carle Cuneo Hospital, Cuneo, Italy, 3Department of Orthopaedics and Traumatology, AORN San Pio, Benevento, Italy

ABSTRACT

Aim In the scientific literature there are no papers that clarify which method of surgical fixation in transverse metacarpal fractu- res has the best functional outcomes. The aim of this study was to compare the hand strength obtained using two different methods in the treatment of these fractures.

Methods A total of 52 patients who presented a transverse me- tacarpal fracture were enrolled. They were divided in two grou- ps: 26 patients treated with K-wire (IMN) and 26 patients treated with plate and screws (PW). The evaluation criteria were: fracture Corresponding author: healing time, performed force testing collected ultimate tensile Andrea Pasquino strength and grip, the Disability Arm Shoulder and Hand (DASH) Department of Orthopaedics and score, and the range of motion of the hand. Traumatology, Vito Fazzi Hospital Results In both groups obtained results were comparable in terms Piazzetta Filippo Muratore, of full hand function, healing and total range of motion and DASH. Block: A- Floor: V, Lecce, Italy Results in group K were slightly better than group PW in terms of Phone: +39 3474965988; strength and grip pain within 3 months from osteosynthesis. Fax: +39 0832661337; Conclusion Neither of the two techniques, either in the literatu- E-mail: [email protected] re or in biomechanical studies, shows to have superior functional ORCID: https://orcid.org/0000-0002- outcomes for fixation of transverse metacarpal fractures. Since the 7013-2442 K-wire is cheaper and has no intrinsic complications as compared with plating (such as scar and tendon irritation), fixation with the latter is preferable to the plate in the treatment of these fractures in non-expert hands. Original submission: Key words: finger, grip, hand, joint, pain, stiffness 29 October 2020; Revised submission: 10 November 2020; Accepted: 13 November 2020 doi: 10.17392/1310-21

Med Glas (Zenica) 2021; 18(1):316-321

316 Pasquino et al. K wire vs plate in metacarpal shaft fractures

INTRODUCTION while accidental fall was the mechanism of injury over a bimodal distribution of age groups less than Metacarpal fractures account for 6% of all fractures 9 or older than 50 years old. According to Nakas- in the adult population (1). De Jonge et al. perfor- hian et al. (3) a typical patient suffering from a med a retrospective analysis of 3,858 metacarpal metacarpal fracture is a young, sporty patient with fractures seen over a 23-year period in a Netherlan- a high demand for work on the part of the hands. ds institution, finding that men aged 10–29 years Many metacarpal fractures can be treated non-ope- showed the highest incidence of metacarpal fractu- ratively; however, some are treated most effecti- res (2.5%) (2). They found that bicycle accidents in vely with surgical stabilization. It was postulated particular accounted for the vast majority of meta- that plates (PW) (Figure 1) would have a signifi- carpal fractures across all demographic variables,

Figure 1. A 42-year-old male with transverse fracture of the right V metacarpus from fist. A, B) Pre-operative X-rays show the above fracture in detail; C-D) postoperative X-rays show the surgical treatment with plate and screws; E-F) control X-rays at 6 months; G) result in the grip test at 6 months (Pasquino A, 2018)

Figure 2. An 18-year-old male with transverse fracture of the right V metacarpus from bike fall. A, B) Pre-operative X-rays show the above fracture in detail; C-D) postoperative X-rays show the surgical treatment with plate and screws; E-F) control X-rays at 6 months; G) result in the grip test at 6 months (Pasquino A, 2017)

317 Medicinski Glasnik, Volume 18, Number 1, February 2021

cantly higher load to failure than crossed K-wires The final decision was made by the senior author and that intramedullary (Figure 2) metacarpal nails considering the patient’s preference. (IMNs) and XK-wires would have equivalent load Patients were treated according to the ethical to failure (4,5). standards of the Helsinki Declaration, and were The aim of this study was to compare the hand invited to read, understand and sign an informed strength obtained using two different methods in consent form. the treatment of these injuries. Azienda Sanitaria Locale (ASL) Lecce, Italy Et- hical Committee approved this research. PATIENTS AND METHODS Methods Patients and study design Both groups underwent the same rehabilitation The patient databases of the Vito Fazzi Hospital, S. protocol after 21 days of cast. Croce e Carle Cuneo Hospital and AORN San Pio. Stretching exercises included: wrist range of mo- Level I Trauma Centres were retrospectively eva- tion, flexion, extension, side to side. luated for the patients with a metacarpal fracture admitted during the period between January 2015 Wrist range of motion, flexion- gently bend your and January 2019. wrist forward, hold for 5 seconds, do 2 sets of 15); extension - gently bend your wrist backward, Patients with metacarpal shaft fracture were en- hold this position for 5 seconds, do 2 sets of 15; rolled after the application of the following exclu- side to side - gently move your wrist from side to sion criteria: multiple metacarpal fracture, meta- side - a handshake motion, hold for 5 seconds in carpal articular fracture, metacarpal metaphysis each direction, do 2 sets of 15. fractures, no previous upper limb fractures, no contralateral upper limb fracture, neurovascular Strengthening exercises included: opposition injuries, age under 14 years, coexisting haemato- stretch, wrist flexion, wrist extension, and finger logical or oncological disorders, metabolic bone spring. diseases, rheumatoid disorders, and a follow-up Opposition stretch: rest your hand on a table, palm period of less than 24 months. Images of the con- up, touch the tip of your thumb to the tip of your tralateral hand were not routinely obtained. little finger, hold this position for 6 seconds and All fractures were classified according the anato- then release, repeat 10 times; wrist flexion: hold mic X-ray’s classification (6-9). Fifty-two enrolled a can or hammer handle in your hand with your patients were divided into two groups: the plate and palm facing up, bend your wrist upward, slowly screws (PW) and the K-wires (IMN) groups (Table 1). lower the weight and return to the starting positi- The patients were informed in a clear and compre- on, do 2 sets of 15, gradually increase the weight hensive way of the type of treatment and other po- of the can or weight you are holding; wrist exten- ssible surgical and conservative alternatives. sion: hold a soup can or hammer handle in your hand with your palm facing down, slowly bend Table 1. Characteristics of the plate and screws (PW) and intramedullary metacarpal nail (IMN) patients your wrist up, slowly lower the weight down into Plate and Intramedullary meta- the starting position, do 2 sets of 15, gradually Variable screws (PW) carpal nail (IMN) increase the weight of the object you are holding; Number of patients 26 26 grip strengthening: squeeze a soft rubber ball and Age (mean±SD) (years) 33.62 (±18.64) 32.34 (±18.75) Gender (male/female) 23/3 24/2 hold the squeeze for 5 seconds, do 2 sets of 15; Mechanism of injury (No; %) finger spring: place a large rubber band around Fall from a bike 4 (15.38) 1 (3.85) the outside of your thumb and fingers, open your Traffic accident 6 (23.09) 5 (19.23) fingers to stretch the rubber band, do 2 sets of 15. Work accident 4 (15.38) 8 (30.77) Punch 12 (46.15) 12 (46.15) To assess bone healing on radiographs, the Non- Side of injury (Right/Left) 11/15 12/14 Union Scoring System (NUSS) was used (10). Metacarpal fracture site (No, %) I 1 (3.85) 1 (3.85) The functional evaluation of the two groups du- II 2 (7.69) 1 (3.85) ring the follow-up was quantified with the Di- III 5 (19.23) 6 (23.09) IV 8 (30.77) 8 (30.77) sability Arm Shoulder and Hand Score (Quick V 10 (38.46) 10 (38.46) DASH) (3-10). Other criteria were: the time NUSS Score (mean±SD) 12.6 (±8.33) 12.7 (±9.64) until complete bone healing, complications, re- NUSS, Non-union scoring system

318 Pasquino et al. K wire vs plate in metacarpal shaft fractures

operation rates, ability to return to full work, the range of motion of metacarpal, and performed force testing collected ultimate tensile strength and grip (average grip strength was calculated for 3 attempts in both the injured and healthy hand using a hand dynamometer). The minimum follow-up was designated as 12 months after sur- gery, and the functional evaluations were made at 1, 3, 6,12 and 24 months postoperatively.

Statistical analysis Continuous variables were expressed as the mean±standard deviation (SD) as appropriate. The Shapiro-Wilk normality test was used to eva- luate the normal distribution of the sample. The t Figure 3. The trend of hands grip. Until 3rd-month follow-up, test was used to compare continuous parameters. scores were statistically significant for IMN group (p<0.05); The study sample size was considered sufficient after the sixth month after surgery, there was no significant difference* to evaluate a difference in post- to pre-operative *IMN group, patients treated with K-wire; PW group, patients measurements greater than 0.5 SD units with a treated with plate and screws; power >80%. The trend of Quality life measured with Qu- The reliability and validity of the correlation ickDASH until 3rd-month follow-up scores was between bone regenerate/bone healing and x-rays statistically significant for IMN group between were determined by the Cohen’s kappa (k). Stati- the groups (p<0.05); however, after the sixth stical significance was set at p <0.05. month after surgery, there was no significant difference (p>0.05) (Figure 4). RESULTS The mean of follow-up was 26.54 (±1.12; range 24–56) months for IMN and 26.56 (±1.18; range 25–57) months for PW (p>0.05). The surgery lasted an average of 19.8 (±8.4; ran- ge 8-33) minutes in IMN, while 42.7 (±18.8; ran- ge 24-78) minutes for PW (p<0.05). In both groups, patients demonstrated wound he- aling within 25 days. During the follow up no complications in PW gro- up were detected. In only one case a non-union with malrotation of metacarpal bone was found. Figure 4. The trend of quality life measured with QuickDASH. The average time of bone healing was 96.5 (±6.7; Until 3rd-month follow-up, scores were statistically significant range 83-123) days after the surgery in IMN and for IMN group (p<0.05); after the sixth month after surgery, 93.8 (±12.6; range 65-128) days for PW (p>0.05). there was no significant difference* *IMN group, patients treated with K-wire; PW group, patients treat- The average range of motion (ROM) of meta- ed with plate and screws; DASH, disability arm shoulder and hand; carpal-phalanx joint was 254.2° (±8.9;248-260) The average correlation between osteosynthesis after the surgery in IMN while 256.1° (±9.4;249- and bone healing at the moment of X-ray callus 260) for PW (p>0.05). was absolutely correlated in IMN clinical results The trend of hands grip until 3rd-month follow- (k=0.8674±0.12) as in PW (k=0.8658±0.12) up scores was statistically significant for IMN (p>0.05) for surgery. group between groups (p<0.05); however, after the sixth month after surgery, there was no signi- DISCUSSION ficant difference (p>0.05) (Figure 3). Metacarpal fractures are common and can be sta- bilized in multiple ways (12). Unlike other meta-

319 Medicinski Glasnik, Volume 18, Number 1, February 2021

carpal fracture fixation methods, the biomechani- The biomechanical properties of absorbable cal stability of proximally-locked IMNs for MC implants are comparable with their metallic co- fractures has not previously been compared to unterparts, allowing them to achieve adequate other commonly utilized techniques. Our data su- fracture stabilization (12). ggest that plate-screw constructs were 11 and 15 Van Bussel et al. (13) reported the mid-term times more stable than XK-wires and IMNs, res- functional outcome using the validated patient- pectively. Although XK-wire constructs showed related outcome measurement tool (DASH) in a trend toward greater stability than proximally- 70% of the study population after 30 months on locked IMNs, these two techniques did not offer average; the results of these patients reported significant instability (4). outcome measures (PROMs) with a mean DASH Dreyfuss et al. reported that metacarpal shaft of 5 and of 7 showing that the functional outcome fractures plate fixation (statistically) is advanta- of this technique is excellent. Also, Van Busel et geous in several parameters as compared to pin al. reported the overall, the average grip strength fixation, including grip strength, digital range compared with the contralateral hand was 91.7% of motion, residual rotation, and DASH scores (n = 13, range: 68%-117%), with only one patient (11). Our results show that radiographic fracture having a persistent loss of >30% after 6 months reduction was achieved equally in both groups; (13); with regard to mobility, there was no signi- operative time was significantly longer for surgi- ficant difference in postoperative stiffness betwe- cal plate implantation as compared with pinning. en metallic and absorbable implants. A high number of our patients were found to have DASH scores in our study population were sli- residual rotational deformity in their fingers, es- ghtly higher than those in similar studies (11-16). pecially in fractures which were fixated by pins. Some patients with satisfactory range of motion Metallic plates allow rigid and stable fixation of still complained of residual pain and functio- metacarpal (MC) shaft fractures and adequately re- nal disability during daily activities. Two issues store length. Disadvantages include extensive pe- might explain these discrepancies. Average ra- riosteal dissection, that is less significant with the diographic union time was shorter in the IMN new generation locking plates, and the possible ne- group. The larger exposure required for plate cessity for future plate removal, reported in ~15% fixation over the bone, especially the stripping of of MC fractures in more recent studies (12-14). periosteum, seems to be at fault (11-16). The most important complications to consider In conclusion, none of the two techniques, either in when dealing with metallic implants are plate the literature or in biomechanical studies, shows to prominence, stiffness, tendon irritation and rup- have superior functional outcomes for fixation of ture, infection, bone atrophy, and osteoporosis transverse metacarpal fractures. Since the K-wire due to stress shielding. On the contrary, absor- is cheaper and has no intrinsic complications than bable implants incrementally transfer load to a plating (such as scar and tendon irritation), fixati- healing fracture thereby limiting stress shielding, on with the latter is preferable to the plate in the while promoting bone union (11-15). treatment of these fractures in non-expert hands. Physical properties of absorbable implants make FUNDING them useful for magnetic resonance imaging and for radiolucent, facilitating postoperative radiolo- No specific funding was received for this study. gical evaluation (12). In addition, they offer the theoretical advantage of circumventing the need TRANSPARENCY DECLARATION for a second operation for hardware extraction (12). Conflict of interest: None to declare.

REFERENCES 1. Court-Brown CM, Caesar B. Epidemiology of adult 3. Nakashian MN, Pointer L, Owens BD, Wolf JM. In- fractures: a review. Injury 2006; 37:691-7. cidence of metacarpal fractures in the US populati- 2. Jonge JJ, Kingma J, Van der Lei B, Klasen H J. on. Hand (N Y) 2012; 7:426-30. Fractures of the metacarpals. A retrospective analysis 4. Curtis BD, Fajolu O, Ruff ME, Litsky AS. Fixation of incidence and etiology and a review of the En- of metacarpal shaft fractures: biomechanical com- glish-language literature. Injury 1994; 25:365–9. parison of intramedullary nail crossed K-wires and plate-screw constructs. Orthop Surg 2015; 7:256-60.

320 Pasquino et al. K wire vs plate in metacarpal shaft fractures

5. Hiatt SV, Begonia MT, Thiagarajan G, Hutchison RL. 11. Dreyfuss D, Allon R, Izacson N, Hutt D. A compa- Biomechanical comparison of 2 methods of intrame- rison of locking plates and intramedullary pinning dullary K-wire fixation of transverse metacarpal shaft for fixation of metacarpal shaft fractures. Hand (NY) fractures. J Hand Surg Am 2015; 40:1586-90. 2019; 14:27-33. 6. Grandizio LC, Speeckaert A, Kozick Z, Klena JC. 12. Hazan J, Azzi AJ, Thibaudeau S. Surgical fixation Anatomic assessment of K-wire trajectory for tran- of metacarpal shaft fractures using absorbable im- sverse percutaneous fixation of small finger metacar- plants: a systematic review of the literature. Hand pal fractures: a cadaveric study. Hand (NY) 2018; (NY). 2019; 14:19-26. 13:86-9. 13. van Bussel EM, Houwert RM, Kootstra TJM, van 7. Romo-Rodríguez R, Arroyo-Berezowsky C. Mini- Heijl M, Van der Velde D, Wittich P, Keizer J. An- mal invasive osteosynthesis with cannulated screws tegrade intramedullary Kirschner-wire fixation of in metacarpal fractures. Acta Ortop Mex 2017; displaced metacarpal shaft fractures. Eur J Trauma 31:75-81. Emerg Surg 2019; 45:65-71. 8. Zhu H, Xu Z, Wei H, Zheng X. Locking plate alone 14. Beck CM, Horesh E, Taub PJ. Intramedullary screw versus in combination with two crossed kirschner fixation of metacarpal fractures results in excellent wires for fifth metacarpal neck fracture. Sci Rep functional outcomes: a literature review. Plast Re- 2017; 7:46109. constr Surg 2019; 143:1111-8. 9. Shanmugam R, Jian CYCCS, Haseeb A, Aik S. 15. Oh JR, Kim DS, Yeom JS, Kang SK, Kim YT. A Comparing biomechanical strength of unicortical comparative study of tensile strength of three opera- locking plate versus bicortical compression plate for tive fixation techniques for metacarpal shaft fractu- transverse midshaft metacarpal fracture. J Orthop res in adults: a cadaver study. Clin Orthop Surg Surg (Hong Kong) 2018; 26:2309499018802511. 2019; 11:120-5. 10. Calori GM, Colombo M, Mazza EL, Mazzola S, Malagoli E, Marelli N, Corradi A. Validation of the Non-Union Scoring System in 300 long bone non- unions. Injury 2014; 45 S93-7.

321 ORIGINAL ARTICLE

Minimally invasive sinus tarsi approach in Sanders II-III calcaneal fractures in high-demand patients

Paolo Ceccarini, Francesco Manfreda, Rosario Petruccelli, Giuseppe Talesa, Giuseppe Rinonapoli, Auro Caraffa

Department of Orthopaedics and Traumatology, SM Misericordia Hospital, University of Perugia, Italy

ABSTRACT

Aim To evaluate if the sinus tarsi approach treated with open re- duction and internal fixation (ORIF), without using plate fixation, provided good functional results in active adult population. The hypothesis was that the sinus tarsi approach with limited incision provided good results comparable to other approaches.

Methods A total of 78 patients (81 feet) surgically treated for arti- cular calcaneus fracture were reviewed according to inclusion cri- teria: Sanders fracture type II-III, minimum follow-up of 2 years, patients aged 18-65 years. Exclusion criteria were smokers, diabe- tics, non-collaborative patients and patients with Sanders fracture type I and IV. A mean follow-up was 52.6 months. Radiographic changes of the Bohler’s angle were reported. For the clinical eva- Corresponding author: luation, Visual Analogue Scale (VAS) for calcaneal fractures, Paolo Ceccarini American Orthopaedic Foot and Ankle Society (AOFAS) score Department of Orthopaedics and and Maryland Foot Score (MFS) were used. Traumatology, SM Misericordia Hospital, Results A statistically significant restitution of Böhler’s angle University of Perugia from preoperative to postoperative (13.5°-27°; p<.001) was found. Postal address of the institution Perugia, The AOFAS and MFS showed pain relief and good/excellent func- Italy tional activities at the final follow-up in 65 of 78 (83.3%) patients. Phone: +39075784049 In eight (out of 81; 10%) feet a superficial wound infection was E-mail: [email protected] observed. In three (3.8%) patients a subtalar arthrodesis was per- ORCID ID: https://orcid.org/0000-0003- formed. 3447-109X Conclusion The mini-invasive sinus tarsi approach for active adult population is a valid and reproducible technique with a low rate of major complications, but it is mandatory advice to patients regarding the expectation of the results. Original submission: Key words: foot and ankle trauma, hindfoot surgery, mini-invasi- 30 September 2020; ve surgery, outcome scoring Revised submission: 09 November 2020; Accepted: 19 November 2020 doi: 10.17392/1282-21

Med Glas (Zenica) 2021; 18(1):322-327

322 Ceccarini et al. Sinus tarsi approach in calcaneal fractures

INTRODUCTION After applied exclusion criteria 78 patients (81 feet) were eligible for an analysis. Calcaneal fractures present a significant challen- ge to orthopaedic surgeons. Calcaneal fractures All fractures were classified according to the CT are the most frequent tarsal fractures (approxi- Sanders classification system (4). The study only mately 60% of all tarsal fractures) and represent included Sanders II (48 feet) and Sanders III (33 2% of all adult fractures; in the most cases they cases feet) fractures. The average final follow-up involve young adults (1-4). ORIF is routinely was 52.6 (range 24-120) months. performed especially for displaced intra-articular The mean age of the patients was 44.8 (range 22- calcaneal fractures (5,6). 63) years. Of the 78 patients, 21 (27%) were fe- There are various approaches for calcaneal males and 57 (73%) were males; 41 had right feet fracture fixation in the literature (2-4). Surgical injury and 37 left one (three cases were bilateral). approach is one of the factors affecting outcomes This study was conducted in accordance with the of the surgical treatment (7,8). ethical guidelines of the Declaration of Helsin- The most commonly used approach is the exten- ki, and an informed consent was obtained from sile lateral approach described by Letournel (9). all patients. Data used in this retrospective study were recorded as part of a usual clinical evaluati- Soft tissue complications affect success of the on in the Orthopaedic and Traumatology Depar- surgery (7,8). Percutaneous techniques are deve- tment (Perugia, Italy), and consequently appro- loped in order to reduce the complications of the ved by a local review board. extensile approach but reduction of the articular surface could be difficult and may be considered Methods the main problem of percutaneous techniques (10,11). Soft tissue problems may accompany Radiographic modifications of the Bohler’s angle calcaneal fractures due to high-energy trauma. (pre-operative, immediately after surgery and at Particularly severe oedema may cause corruption final follow-up) were reported. For the clinical of the soft tissue, fracture blisters and compar- evaluation, Visual Analogue Scale (VAS) for cal- tment syndrome (12,13). caneal fractures (12 questions, 0 was the worst possible result and 10 was no pain), American The aim of this study was to evaluate if the si- Orthopaedic Foot and Ankle Society (AOFAS) nus tarsi approach fixed with k-wires or screws score (it includes both subjective and objective, provided good functional results in active adult or physician-assessed items, and is scored from 0 population. The hypothesis was that the sinus tar- to 100, with a higher score representing a better si approach with limited incision provided good outcome) for ankle/hindfoot and Maryland Foot results comparable to other approaches. Score (MFS) were used (in MFS 100 marks are PATIENTS AND METHODS possible with pain, function and movements each carrying 45, 50 and 5 marks respectively; less Patients and study design than 50 marks suggest failure, 50-74 fair, 75-89 good and 90-100 excellent results) (2, 14-17). Ninety-seven patients (109 feet) surgically trea- CT scans were performed to confirm Sanders cla- ted for articular calcaneus fracture in SM Miseri- ssification preoperatively. cordia Hospital - Orthopaedic and Traumatology Department (Perugia, Italy) in the period 2007- The radiographic evaluations included anterior- 2018 were retrospectively analysed. posterior (AP), lateral view of the calcaneus and Broden’s view in all patients. The Böhler’s angle Inclusion criteria were: Sanders fracture type II-III, was measured before surgery, one-day post-ope- minimum follow-up 2 years, patients aged 18-65 rative, and at the time of the final follow-up. years, workers and/or recreational sports, with a Tegner Activity Level >4 before injury. Exclusion Radiographic measurements of the calcaneus criteria were open fractures and polytrauma, chro- were also measured retrospectively, according nic smokers (more than 10 years), diabetics (with to Abdelgaid and Kikuchi, pre-operative, after 6 clinical manifestations), non-collaborative patients weeks and at the final follow-up (11,18). Osteo- and patients with Sanders fracture type I and IV. arthritic changes of the subtalar joint were evalu- All surgeries were performed by surgeons who had ated at the final follow-up using Paley and Hall considerable experience in foot and ankle surgery. scoring system (14).

323 Medicinski Glasnik, Volume 18, Number 1, February 2021

The functional and radiographic results were Post-operative. After surgery a cast below the evaluated by an orthopaedic surgeon who was knee without weight-bearing was placed for 6 blinded to the study and who was not involved in weeks. After this time hydro kinesitherapy and the surgical treatment. partial weight bearing was encouraged. Full In all patients, surgery was performed via sinus weight-bearing was possible after 8 weeks. Su- tarsi approach in lateral decubitus position and tures were removed approximately after 3 weeks the fragments are fixed either temporarily with and after a meticulous wound check. a Kirschner (K)-wire, or definitively with one or Statistical analysis more screws. No plate fixation was used in pati- ents (Figure 1). The nonparametric Wilcoxon test for paired data was used to compare scores before and after the sur- gery. Statistical significance for p <0.05 was used.

RESULTS A total of 78 (fifty-seven males and twenty-one females, with a mean age of 44.8 years) met the inclusion criteria. The average length of follow- up time was 52 (range 24-120) months. Preoperatively all patients were high demand workers and/or active in daily living (at least sport activities two times a week). All patients reported severe pain at the VAS dedi- cated for calcaneus fractures (in the pre-operati- ve, with an average of 1.6 (range 0-3). At the final follow-up the mean VAS for calcaneus fractures Figure 1. Surgical technique for reduction of calcaneal frac- was 7.58 (p<0.0001). tures with limited sinus tarsi approach. A) Sinus tarsi ap- According to the Sander’s CT classification for proach; B) after open reduction and internal fixation; C) axial view: direction of the screws and fracture reduction; D) lateral calcaneus intra-articular fractures, 48 (out of 81; view: Bohler’s angle reduction can be observed after Open 59%) fractures were classified as grade II and 33 Reduction and Internal Fixation (ORIF) (Ceccarini P, SM Miseri- (41%) as grade III. cordia Hospital, 2019) None of the patients improved his/her activity The average time from the trauma to surgery was level and the most of them, 80 (%) practiced non- 7.8 days (range 2-16 days) (Table 1). contact sports after trauma. Table 1. Characteristics of the study population For the clinical evaluation Maryland Foot Sco- No of patients/feet 78/81 re (MFS) and AOFAS score were used, repor- Age (years) 44.8 (22-63) ting a mean score of 85.28 and 86.34 at the final Gender (No, %) follow-up, respectively. A total of 65 (83.3%) of Female 21(27) Male 57 (73) 78 patients showed excellent/good result (range Side of injury (No, %) 80-100), seven (8.9%) fair (range 70-80) and six Right 41 (52) (7.6%) poor (score <70). Left 37 (44) Bilateral 3 (4) The patients with Sanders II and III fractures Time to surgery (days) 7.8 (range 2-16) reported an MFS of 88.1 and 82.46 respectively Sander’s classification (No, %) II 48 (61) (p>0.005) (Table 2, Figure 2). III 33 (39) Median Böhler’s angle in the pre-operative was Follow-up duration (months) 52.6 (24-120) Complications (No, %) 7.1° (range -11°-17°), on the day 1 after the sur- Deep infection / gery it was 21.5° (min. 6°, max. 34°), and at the fi- Wound dehiscence 8/81 (10) nal follow-up it was 20° (min. 2°, max. 33°), with Subtalar fusion 3/81 (4) mean improvement of 14° between pre-operative Sural nerve symptoms 6/81 (7) Non-union / and last follow-up (7°-20.1°) (p<0.001). No

324 Ceccarini et al. Sinus tarsi approach in calcaneal fractures

Table 2. Clinical and radiographic results of calcaneal fractures surgically treated Clinical results Pre-operative Post-operative (final follow-up) p VAS for calcaneus fractures 1.61 7.16 <0.0001 Maryland foot score Pain 38.57 Walked distance 8.85 Stability 3.71 Support 3.92 Limp 3.71 Shoes 8.86 Stairs 3.64 Terrain 2.42 Cosmesis 7.28 Motion 4.01 Total 85.28 Radiographic results Pre-operative 1 day after surgery After 6 weeks Final follow-up (52 months) P (post-operative) Böhler angle (°) 7.1 21.5 21.16 20 >0.05 Mean length (mm) 78.6 84.26 84.01 83.72 >0.05 Mean width (mm) 58.6 48.92 48.75 50.63 >0.05 Mean height (mm) 47.6 51.51 51.53 49.89 >0.05 Paley and Hall score for subtalar joint Grade 0 11 Grade 1 18 Grade 2 10 Grade 3 2 / Significant (p<0.001) improvement of VAS was observed after the surgery. No loss of correction of Bohler’s angle was observed at the final follow-up (p>0.05) VAS, visual analogue scale; NA, non-applicable; WB, weight-bearing radiographs

Figure 3. Changes in Bohler’s angle for Sanders II and III at the time of trauma, after surgery and at final follow-up

Figure 2. A) Maryland Foot Score (MFS) and B) American Or- thopaedic Foot and Ankle Society (AOFAS) for ankle-hindfoot score at the final follow-up (52.6 months) ficantly different from 6 weeks post-operatively statistical difference between post-operative re- to the final follow-up (p>0.05) (Table 2). sults was observed (p>0.05). The patients with Complications Sanders II fractures showed better results of the Bohler’s angle, however without statistical diffe- In eight of 81 (9.8%) fractures a superficial wo- rences (p>0.05) (Figure 3). und infection was observed, all healed with con- Radiographic outcomes of calcaneal measure- servative treatment. In three (3.8%) of 78 pati- ments (height, width and length) were not signi- ents a subtalar arthrodesis was performed due to

325 Medicinski Glasnik, Volume 18, Number 1, February 2021

post-traumatic subtalar arthritis 2 years after the tive comparative case series of 112 displaced surgery (Sanders III in all cases). In six (7.6%) intra-articular calcaneal fractures treated with cases, patients reported sural nerve symptoms minimally invasive technique and extensile la- in the first year; no patient required exploration teral approach, of 33 who were treated through or sural neuroma excision. One patient reported the sinus tarsi approach, malunion. There were no reported cases of oste- 6.6% required repeat surgery for infection omyelitis, deep infection and non-union. There and 94% of patients were satisfied versus 20% were no cases of displacement of hardware. and 84%, respectively, in the extensile lateral approach cohort. The study showed the mini- DISCUSSION mally invasive approach had a significantly lower The surgical treatment for the calcaneal fractures incidence of wound complications and secondary has been advocated for years; in general, open surgeries (23). In a retrospective cohort study Yeo reduction and internal fixation is indicated as a et al. compared 100 calcaneal fractures Sanders gold standard in the young and active population, II and III treated with sinus tarsi approach (40 but the debate is still open on the best surgical cases) or extensile lateral approach (60 fractures) treatment, with many surgical approaches propo- and found comparable clinical and radiographic sed in the last years, each with its advantages and results for both approaches, with a lower compli- disadvantages (3,6,19-21). cation rate for sinus tarsi approach (24). In 2011 Schepers et al. performed a systematic Our study showed that the articulation subtalar review on the sinus tarsi approach in intra-arti- joint can be restored without the use of plates and cular calcaneal fractures, examining eight case more invasive approaches. The infection rate and series of the last decade (2000-2010) with 271 major complications appear to be lower than cla- calcaneal fractures. The author concluded the ssic L-shaped approach. The prevalence of deep review asserting that the results of the sinus infection with this approach varies significantly tarsi approach compare similar or favourable to between different studies, suggesting that a larger the extended lateral approach, and in the pro- patient cohort would be required to better assess cess of tailoring the best treatment modality to the true incidence of this complication (23). The the right patient and the right fracture type the patients should therefore be explicitly advised sinus tarsi approach might be a valuable asset preoperatively of this risk. (6). Moreover, the author suggested to use in The clinical and radiographic results of our study all cases Sanders classification and more uni- in active population with high functional requests formity in the outcome scores. are very interesting and at midterm are comparable There are also other studies published after this and, in some cases, superior to other approaches systematic review in order to evaluate the results though a long-term outcome of subtalar arthrosis of the sinus tarsi approach. with this technique rests unidentified. In a prospective study Zhang et al. reported Limitations of this study include the retrospecti- similar clinical (with AOFAS score) and ra- ve study design, a relatively small patient cohort, diographic outcomes of two mini-invasive the absence of a control group and direct compa- approaches in 130 patients with Sanders II- rison with other approaches. A comparison radi- III intra-articular fractures of calcaneus (22). ographs of the contralateral calcaneum were not Kikuchi et al. reviewed 22 calcaneal fractu- performed and also weight bearing X-rays were res treated with ORIF with limited sinus tarsi not routinely done at the final follow up. approach and found a statistically significant To our knowledge this study reporting the results restoration of Böhler’s angle and calcaneal of the sinus tarsi approach in active adult popu- width; however, a clinical evaluation with de- lation could be an important reference for more dicated scores was not performed. The authors accurate studies for the treatment of the intra-ar- reported no cases of osteomyelitis, deep infec- ticular calcaneus fractures. tion, malunion or non-union and three out of In conclusion, the sinus tarsi approach for adult 22 cases of superficial wound infection (18). active population is a valid and reproducible Kline et al. found similar results in a retrospec-

326 Ceccarini et al. Sinus tarsi approach in calcaneal fractures

technique with a low rate of major complications, FUNDING: but is mandatory advice to patients regarding the No specific funding was received for this study. expectation of the result. TRANSPARENCY DECLARATION Conflict of interest: None to declare.

REFERENCES 1. Busel G, Mir HR, Merimee S, Patel R, Atassi O, 13. Myerson MS, Quill GE Jr. Late complications of De La Fuente G, Donohue D, Maxson B, Infante A, fractures of calcaneus. J Bone Jt Surg 1993; 75:331-41. Shah A, Watson D, Downes K, Sanders RW. Quality 14. Paley D, Hall H. Intra-articular fractures of the cal- of reduction of displaced intra-articular calcaneal caneus. A critical analysis of results and prognostic fractures using a sinus tarsi versus extensile lateral factors. J Bone Joint Surg Am 1993; 75:342–54. approach. J Orthop Trauma 2020; Sep 18. Online 15. Tegner Y, Lysolm J. Rating systems in the evaluati- ahead of print. on of knee ligament injuries. Clin Orthop Relat Res 2. Basile A. Subjective results after surgical treatment 1985; 198:43-9. for displaced intra-articular calcaneal fractures. J 16. Petruccelli R, Bisaccia M, Rinonapoli G, Rollo G, Foot Ankle Surg 2012; 51:182-6. Meccariello L, Falzarano G, Ceccarini P, Bisaccia 3. Buckley RE, Tough S. Displaced intra-articular O, Giaracuni M, Caraffa A. Tubular vs profile plate calcaneal fractures. J Am Acad Orthop Surg 2004; in peroneal or bimalleolar fractures: is there a real 12:172-8. difference in skin complication? A retrospective stu- 4. Bai L, Hou YL, Lin GH, Zhang X, Liu GQ, Yu B. Sinus dy in Three Level I Trauma Center. Med Arch 2017; tarsi approach (STA) versus extensile lateral approach 71265-9. (ELA) for treatment of closed displaced intra-articular 17. Kitaoka HB, Alexander IJ, Adelaar RS, Nunley JA, calcaneal fractures (DIACF): a meta-analysis. Orthop Myerson MS, Sanders M. Clinical rating systems for Traumatol Surg Res 2018; 104:239-44. the ankle-hindfoot, midfoot, hallux, and lesser toes. 5. Sanders R, Fortin P, DiPasquale T, Walling A. Ope- Foot Ankle Int 1994; 15:349 –53. rative treatment in 120 displaced intraarticular calca- 18. Kikuchi C, Charlton TP, Thordarson DB. Limited neal fractures. Results using a prognostic computed sinus tarsi approach for intra-articular calcaneus tomography scan classification. Clin Orthop 1993; fractures. Foot Ankle Int 2013; 34:1689-94. (290):87–95. 19. Abdelazeem A, Khedr A, Abousayed M, Seifeldin A, 6. Schepers T. The sinus tarsi approach in displaced in- Khaled S. Management of displaced intra-articular tra-articular calcaneal fractures: a systematic review. calcaneal fractures using the limited open sinus tarsi Int Orthop 2011; 35:697-703. approach and fixation by screws only technique. Int 7. Mehta CR, An VVG, Phan K, Sivakumar B, Ka- Orthop 2014; 38:601-6. nawati AJ, Suthersan M. Extensile lateral versus 20. Agren PH, Wretenberg P, Sayed-Noor AS. Operative sinus tarsi approach for displaced, intra-articular versus nonoperative treatment of displaced intra-ar- calcaneal fractures: a meta-analysis. J Orthop Surg ticular calcaneal fractures: a prospective, randomi- Res 2018; 13:243. zed, controlled multicenter trial. J Bone Joint Surg 8. Jiang N, Lin QR, Diao XC, Wu L, Yu B. Surgical Am 2013; 95:1351-7. versus nonsurgical treatment of displaced intra-arti- 21. Allmacher DH, Galles KS, Marsh JL. Intra-articu- cular calcaneal fracture: a meta-analysis of current lar calcaneal fractures treated nonoperatively and evidence base. Int Orthop 2012; 36:1615-22. followed sequentially for 2 decades. J Orthop Trau- 9. Letournel E. Open treatment of acute calcaneal ma 2006; 20:464-9. fractures. Clin Orthop 1993; 290:60–7. 22. Zhang T, Su Y, Chen W, Zhang Q, Wu Z, Zhang 10. Arastu M, Sheehan B, Buckley R. Minimally in- Y. Displaced intra-articular calcaneal fractures tre- vasive reduction and fixation of displaced calca- ated in a minimally invasive fashion: longitudinal neal fractures: surgical technique and radiographic approach versus sinus tarsi approach. J Bone Joint analysis. Int Orthop 2014; 38:539-45. Surg Am 2014; 96:302-9. 11. Abdelgaid SM. Closed reduction and percutaneous 23. Kline AJ, Anderson RB, Davis WH, Jones CP, Co- cannulated screws fixation of displaced intra-ar- hen BE. Minimally invasive technique versus an ticular calcaneus fractures. Foot Ankle Surg 2012; extensive lateral approach for intra-articular calca- 18:164–79. neal fractures. Foot Ankle Int 2013; 34:773–80. 12. Lim EV, Leung JP. Complications of intraarticular 24. Yeo J-H, Cho H-J, Lee K-B. Comparison of two calcaneal fractures. Clin Orthop Relat Res 2001; surgical approaches for displaced intra-articular cal- (391):7-16. caneal fractures: sinus tarsi versus extensile lateral approach. BMC Musculoskelet Disord 2015; 16:63.

327 ORIGINAL ARTICLE

Balance assessment after altering stimulation of the neurosensory system

Valentina Li Causi1,2, Alessandro Manelli3, Valentina Gianpaola Marini 3, Mario Cherubino4, Luigi Mec- cariello5, Michael Mazzacane6, Mario Ronga1

1 Orthopaedic and Trauma Operative Unit, Department of Biomedical and Dental Sciences and Morpho-Functional Imaging, University of Messina, University Hospital G. Martino, Messina, 2 "Vanico" Physical Medicine and Rehabilitation Centre and "Vittoria" Clinic of Intensive Rehabilitation, Castelvetrano (Trapani), 3Division of Rehabilitation Activities Management, Department of Primary Care, ASL1 Imperiese, Bussana di Sanremo (Imperia), 4Division of Plastic and Reconstructive Surgery, Department of Biotechnology and Life Sci- ences (DBSV), University of Insubria, Varese, 5Department of Orthopaedics and Traumatology, AORN San Pio, Benevento, 6Orthopaedic and Trauma Operative Unit, Department of Biotechnology and Life Sciences (DBSV), University of Insubria, Varese; Italy

ABSTRACT

Aim Posture requires fine integrative elaboration, performed by the central nervous system, of neurosensory information origina- ted from the visual, vestibular and spinal circuit. Many perturbing agents can influence this elaboration and then the postural stabi- lity. Several studies have evaluated only the effect of a single agent on the postural control. The study analysed the perturbing effect of several external agents on the different sensorial circuits in terms of postural balance loss in orthostatism.

Corresponding author: Methods The postural stability of 31 patients was evaluated with a static posturography platform in basal conditions and after exposure Mario Ronga to an external agent in the following order: stroboscopic light pro- Orthopaedic and Trauma Operative Unit, jecting, mechanical rotations on a swivel chair, feet desensitization Department of Biomedical and through ice, administration of an alcoholic drink at intervals which Dental Sciences and Morpho-Functional depended on the participant return to basic posturographic values. Imaging, University of Messina, Tests were performed with open eyes (OE), closed eyes (CE) and University Hospital G. Martino, reducing plantar perception through the use of a rubber pillow. Via Consolare Valeria, 1, Results The stroboscopic light altered the postural control. The 98124 Messina, Italy swivel chair disturbed only with CE. Ice and alcohol increased Phone: +39 90 2213041; the oscillation area. The alcohol test had a significant reduction E-mail: [email protected] in postural control with OE compared to CE. The rubber cushion increased the oscillation area in all OE tests and with CE in alcohol Valentina Li Causi ORCID ID: https://orcid. and ice tests. org/0000-0003-2305-5832 Conclusion The different agents did not trigger postural control Original submission: deficits in the same way. A cold environment with psychedelic li- 01 December 2020; ghts and the use of alcoholic beverages altered significantly the Revised submission: postural stability by influencing simultaneously all perceptions 12 December 2020; (visual, vestibular and somatosensory feedback). Accepted: Key words: central nervous system, postural balance, sensation 13 December 2020 disorders, vestibular, visual perception doi: 10.17392/1324-21

Med Glas (Zenica) 2021; 18(1):328-333

328 Li Causi et al. Perturbing agents influence posture

INTRODUCTION vious lower limbs fractures, lower limb sprains which occurred in 6 months prior to the test, pre- Posture requires a fine integrative elaboration, vious surgeries on lower limbs, muscular diseases, performed by the central nervous system (CNS), drugs intake that alter postural stability, migraine, of neurosensory information originated from the gastrointestinal diseases (celiac disease, malabsor- visual, vestibular and spinal circuit (1-4). The po- ption, peptic ulcer, etc.); pregnancy, nursing and stural stability is regulated by “eso” and “endo” every cause of basal stabilometric alteration. inputs. The first is defined as an external infor- mation that modifies the neurosensory response Thirty-one patients, 21 males and 10 females, met implied in standing posture control, the second these criteria; the average age was 26 years (range as an internal body component that responds to 18 – 39 years). A “Vertigo VSP 400N” (Vertigo external stress and provides the holding of the Static Platform) (Vertigo, Genova 2010) static body position in the space. Three “eso-input” posturography platform was used to evaluate the structures are known in the standing posture: eye, postural stability. Each patient was evaluated in vestibule and feet. Several studies have evaluated basal conditions and after exposure to every single the effect of a singular modifying agent on the external perturbing agent in the following order: postural control: alcohol (5), hypothermia (6), stroboscopic light projecting, mechanical rotations and alteration of foot perception (7) or just the on a swivel chair, feet desensitization through ice different agents (8). No studies in literature have and administration of an alcoholic drink at inter- tried to assess the diversity of response of the ne- vals which depended on the participant’s return to urosensory adaptation as the result of different basic posturographic values. external stimuli performing on the same subject. All the procedures described in the study and The aim of this study was to analyse the pertur- involving human subjects were implemented in bing effect of different external agents on diffe- accordance with the ethical standards established rent sensorial circuits in terms of postural balan- by the Helsinki Declaration of 1975 and subsequ- ce loss in orthostatism in a young and healthy ent amendments. An informed consent was obtai- population. Moreover, the strategy that the CNS ned from all patients included in the study. adopts to compensate external misleading sensi- The study did not require an approval of the Et- tive information while maintaining the upright hics Committee. static position was evaluated. The first hypothesis Methods to test was that every single external perturbing agent can modify significantly postural control. Posturography tests. The Test of Balance (ToB) The second hypothesis was that, according to calculates the percentages of the sensorial inte- different altering sources, there are no differen- ractions (visual, vestibular and somatosensory ces in the modification of the body balance. feedback) of postural balance using the postu- rography platform. The ToB correlates the data PATIENTS AND METHODS of area, length and angular speed, combining the data of test performed with open eyes (OE), Patients and study design closed eyes (CE), reducing plantar perception The study was performed from January 2019 to through the use of a rubber pillow. Normal ToB May 2019 at the Orthopaedic and Trauma Ope- values are (9): 21.49%-57.55% for view, 18.32%- rative Unit, Department of Biomedical and Den- 43.94% for vestibule, and 15.11%-48.65% for tal Sciences and Morpho-Functional Imaging, plantar proprioceptive perception. We also con- University of Messina, University Hospital G. sidered the area, length, and the ratio between Martino, Messina, Italy. Inclusion criteria were length and area (L/A) posturographic values for the patients aged between 18 and 40 without any the Status-kinesigram with OE and CE. L/A is neurological, ocular, vestibular, orthopaedic, me- the expression of the density of the Status-kinesi- tabolic or other pathological conditions that could gram, indirect and reverse index, as well as mo- influence postural stability, with “normal” body nitor of the proprioception (10). An upper limit mass index (BMI) (between 19 and 24kg/m2). of 200 mm2 of the test T0 area with OE was esta- Exclusion criteria were neurological diseases, pre- blished as an additional exclusion criterion (11).

329 Medicinski Glasnik, Volume 18, Number 1, February 2021

Tests execution. Patients fasted for at least 3 hours sturographic test has been performed 30 minutes and stood with bare feet on the platform. The room after the alcohol intake, when the alcohol reaches was illuminated by artificial light to reproduce the the blood concentration of 0.02% (15). same conditions of the external environment and isolated acoustically to prevent noise pollution. Statistical analysis The patients maintained a standing position with The results were expressed as mean, median and two centimetres between heels, feet 30° apart, standard deviation (SD). Statistical analysis of arms straight at their sides, looking straight ahead the difference in average was performed by using at a red target located at a distance of 1.5 meters the Student t test. The alpha value of 0.01 was (12). Posturography test was performed after al- considered. Therefore, the value of p<0.01 was teration of the visual component by projecting a rated as significant. strobe light, of the vestibular component through mechanical rotation of the participant, of the plan- RESULTS tar receptor component by inducing hypothermia The results of the OE tests showed an increased and further of the visual and vestibular compo- length of the stroboscopic effect (T0 207.9±58.5, nents by administrating alcohol. strobe 318.2±99.3; p<0.01) and an increa- Strobo light test. Using the program "Windows sed value of L/A (T0 1.7±0.6, strobe 2.3±0.8; Movie Maker", we created a video in which p<0.01); the swivel chair had not produced any black and white screens alternate for 3 times per significant changes, but a high standard deviati- second, and then we projected it on a wall facing on in the value area (T0 49.5, chair 801.1); the the participant, at a distance of 1.5 meters, throu- use of ice on feet showed an increased area (T0 gh a strobe light. 134.8±49.5, ice 188.5±107.5; p<0.01) and a len- Mechanical rotation test. Slow and mechanical gth (T0 207.9±58.5, ice 237.2±59.4; p<0.01); clockwise rotations were applied to the partici- the use of alcohol showed an increased area (T0 pant seated on a swivel chair without armrests. 134.8±49.5, alcohol 225.3±192.5; p<0.01) and Rotations were performed at a speed of about 10 a length (T0 207.9±58.5, alcohol 244.4±68.1; p seconds per revolution of 360°, for a total time <0.01) (Table 1). of 2 minutes (13). The CE test results did not show any significant Plantar receptor desensitization test. A round Table 1. Stabilometric data with open eyes container with a 40 cm diameter was filled with Average/Median (SD) p (Area, Length, Test crushed ice and used to determine feet hypother- Area (A) Length (L) L/A ratio L/A ratio) 134.8/130.0 207.9/195.1 1.7/1.6 mia, and an anaesthetic effect on mechanorecep- T0 (49.5) (58.5) (0.6) tors. The patients put their feet in the container 156.5/162.0 318.2/306.0 2.3/2.2 Strobo 0.1/<0.01/<0.01 for 20 minutes (14), interposing a sheet between (70.4) (99.3) (0.8) 303.2/145.0 222.6/213.0 1.5/1.5 feet and ice to prevent burns from ice. Swivel chair 0.3/0.3/0.2 (801.1) (81.7) (0.6) Alcohol administration test. A beverage with an 188.5/155.0 237.2/228.0 1.6/1.5 Ice 0.01/0.02/.07 alcohol content of 40° was used for the alcoho- (107.5) (59.4) (0.8) 226.3/167.0 244.4/238.0 1.5/1.4 lic administration test. The test contemplated a Alcohol 0.01/<0.01/0.2 (192.5) (68.1) (1.5) rapid intake of alcoholic substance. For dosing, the parameters were met based on the assumpti- statistical variation for the strobe light effect, on that 0.32g of ethanol per kilogram constituting for the swivel chair and for the ice. A significant patient’s weight is required in order for the test to value of L/A, in a statistically valid percentage be effective (15). reduction, resulted after the alcohol effect (T0 A digital breathalyser "Digital display alcohol 1.7±0.8, alcohol 1.2±0.5; p<0.01) (Table 2). breath tester" (HD TRADING sas, Vicenza, The results of the eso-input showed changes to the Italy) in accordance with the RoHs and CE mar- stroboscopic effect, indicating a reduction in the king, certificated by IMQ Milan, Italy (www. percentage of the visual component (27±10.1 vs hdtrading.eu, June 2012) has been used to test the 12.3±7.5), and an increase of the vestibular compo- alcohol content of the participants after intake, so nent (20.3±5.4 vs 24.5±6.7) and touch (52.8±10.5 that everybody would reach their limit. The po- vs 63.3±9.3) (p<0.01). The swivel chair induced

330 Li Causi et al. Perturbing agents influence posture

Table 2. Stabilometric data with closed eyes Table 5 completes the previous table in CE and Average/Median (SD) p (Area, Length, Test rubber pillow conditions. The results showed a va- Area (A) Length (L) L/A ratio L/A ratio) riation for the swivel chair effects (p<0.01) with 225.1/181.0 316.7/293.0 1.7/1.4 T0 (127.5) (121.1) (0.8) modest reduction of the area (T0 2416.4±1325.4, chair 1853.9±692.6) and length (T0 1419.1±448.7, Strobo NE NE NE chair 1190.2±340.1). Ice effect showed a reduction 188.6/145.0 322.4/289.0 1.9/2.0 Swivel chair 0.03/0.7/0.2 exclusively for the length (T0 1419.1±448.7; ice (97.0) (107.5) (0.7) 204.7/185.0 322.8/313.0 1.8/1.8 1152±283.6) (p<0.01). The alcohol effect showed Ice 0.3/0.7/0.4 (108.8) (105.9) (0.7) a reduction in the value L/A (T0 0.7±0.2; 0.5±0.2 532.6/285.0 384.0/346.0 1.2/1.2 Alcohol 0.03/0.02/<0.01 alcohol) (p<0.01) (Table 5). (732.0) (159.0) (0.5) NE, Not evaluable Table 5. Stabilometric data with rubber cushion and closes eyes an increase in the use of the vestibular component Average/Median (SD) p (Area, Len- Test (20.3±5.4 vs 24.7±7.8; p <0.01) (Table 3). Area (A) Length (L) L/A ratio gth, L/A ratio) 2416.4/2198.5 1419.1/1365.5 0.7/0.6 T0 Table 3. Stabilometric data of Test of Balance (1325.4) (448.7) (0.2) Average/Median (SD) p (Area, Length, Test Strobo NE NE NE Vista Vestibule Feet Length/Area ratio) 1853.9/1665.0 1190.2/135.5 0.7/0.7 27.0/29.0 20.3/20.0 52.8/52.0 Swivel chair <0.01/<0.01/0.8 T0 (692.6) (340.1) (0.2) (10.1) (5.4) (10.5) 2007,7/1860.0 1152.4/1154.0 0.6/0.6 12.3/11.0 24.5/25.0 63.3/63.0 Ice 0.07/<0.01/0.07 Strobo <0.01/<0.01/<0.01 (754.4) (283.6) (0.1) (7.5) (6.7) (9.3) 3162.3/2827.0 1320.8/1203.0 0.5/0.5 24.9/26.0 50.2/51.0 Alcohol 0.02/0.2/<0.01 Swivel chair 24.7/23.0(7.8) 0.2/0.01/0.2 (1848.7) (389.8) (0.2) (10.2) (12.1) 22.6/22.0 23.4/23.0 53.9/53.0 NE, Not evaluable Ice 0.03/0.02/0.6 (9.6) (4.9) (10.0) DISCUSSION 28.8/27.0 22.8/23.0 48.3/50.0 Alcohol 0.5/0.06/0.1 (11.6) (5.5) (12.3) The study evaluated the effects of different alte- The OE and rubber pillow values increased about ring agents on postural control. area (T0 460.4±205.4; strobe 895.7±333.7), length The results of stroboscopic light indicate an incre- (T0 508.8±131.1, strobe 846.2±230.4) and reduc- ase in the length not associated with an increase tion of L/A (T0 1.2±0.4, strobe 1.0±0.2) for the of the area, and an L/A ratio increased. Several stroboscopic effect (p<0.01); a reduction in length studies have shown, through visual stimuli in (T0 508.8±131.1, chair 836.8±104.7) and a slight motion, namely oscillating rooms or provocati- increase in an area (p<0.01), but not for the use on due to movies projected on the visual field, of the chair was observed (p >0.01); an incre- of saccadic or tracking of eye movements, that ase in area (T0 460.4±205.4, ice 604.0±338.7) the movement of the visual scene induces adapti- with decrease in length (T0 508.8±131.1, ice ve postural reactions, measured on the platform. 464.6±140.8) and L/A (T0 1.2±0.4; ice 0.9±0.4) However, in daily life the visual scene is not mo- for the use of ice (p<0.01); an increase in area (T0 ving and can be a spatial reference (16,17). The 460.4±205.4, alcohol 690.9±468.8) and reduction data obtained in our study indicate that the hol- of L/A (T0 1.2±0.4; alcohol 0.8±0.3) for the use of ding of the upright static position was not altered alcohol was also recorded (p<0.01) (Table 4). in amplitude of the area, which remains equal to the reference T0. However, the speed or the Table 4. Stabilometric data with rubber cushion and open eyes length of the path executed would be increased Average/Median (SD) p (Area, Length, Test and consequently the value L/A increases. These Area (A) Length (L) L/A ratio L/A ratio) 460.4/388.0 508.8/467.0 1.2/1.2 findings indicate a greater use of proprioception T0 (205.4) (131.1) (0.4) confirmed by reading the status-kinesigram as an 895.7/849.0 846.2/819.0 1.0/1.0 Strobo <0.01/<0.01/<0.01 increase in the density of the graph (10,12). The (333.7) (230.4) (0.2) 463.9/408.0 436.8/409.0 1.1/1.0 ToB shows that this disturbance greatly reduces Swivel chair 0.9/<0.01/0.02 (192.5) (104.7) (0.4) the visual eso-input, compensated in proportion 604.0/480.0 464.6/421.0 0.9/1.0 Ice <0.01/0.02/<0.01 by other systems (vestibular and foot tactile) (9). (338.7) (140.8) (0.4) 690.9/556.0 474.4/448.0 0.8/0.8 Data analysis of rotation on the swivel chair Alcohol <0.01/0.06/<0.01 (468.8) (140.4) (0.3) did not show any significant change in area and

331 Medicinski Glasnik, Volume 18, Number 1, February 2021

length values, but showed a very high standard coordination; >0.2% severe reduction of balance) deviation, index of extreme variability of results (15). The interesting finding which has emerged among the participants. The use of the chair with from the study is a clear reduction of postural con- closed eyes has the purpose to alter the vestibule, trol with OE than that of CE that is the opposite stressing the vestibulo-spinal reflex (13). Horak of what we would expect in normal conditions. et al. (13), in a clinical review in pathological This phenomenon has been detected by the group subjects before and after rehabilitation, showed of Palm through the use of a dynamic platform, that subjects with vestibular alterations were able in which it was concluded that even small amou- to maintain a stable upright static position with nts of alcohol in the blood induce negative effects open eyes. In contrast, a great vestibular com- on the use of the visual system in the maintenance pensation does not appear beneficial if there are of posture rather than on the vestibular or propri- problems in the vestibular-ocular reflex. In our oceptive system (15). Moreover, our data on the group of healthy subjects, we found that the vi- use of alcohol showed a high standard deviation, sual system has corrected the imbalance caused indicating a high variability in postural response by the vestibular alteration. The high standard in static station, and appears even if the participant deviation indicates a high variability inside the fasted. This finding has led to the conclusion that group. The ToB (9) did not indicate high value the increase of alcohol levels in blood reduces the changes as a percentage of eso-input despite a control that the view has in maintaining the balan- significant increase in the use of the vestibule. In- ce. The Test of Balance does not show a different ternal variables that could reduce this value and stimulation of eso-input in terms of percentage (9). help to explain the phenomenon (use of glasses, Rubber cushion analysis showed a constant varia- blind postural etc.) have not been found. tion of the area parameters in terms of increase in Plantar hypothermia results indicate that the incre- all stimulatory tests with OE. The length parame- ased values of area and length were not followed ters appear uniformly decreased and the L/A ratio by an increase in the L/A ratio. Studies engaging declined, especially in trials of plantar hypothermia young athletes have shown that those who practice and alcoholic administration. These results confir- sports at a competitive level are less susceptible to med the hypothesis that the instability of the plantar hypothermia than amateurs (14). The study groups support surface, in terms of drop in proprioceptive showed an increase in the value of area (14). Our plantar component, caused greater amplitude of os- study confirms this finding because the evaluated cillations with a slow recovery of posture. Careful population was nonprofessional. We can specula- analysis of data with OE without rubber cushion, in te that this difference is due to the development terms of area and length, revealed a change in the of a better postural balance control by subjects control of posture for all administered stimulations. that practice competitive sports. However, this In particular, there has been an increase in both the hypothesis needs to be confirmed with studies on length and the area values in plantar hypothermia a wider population. The simultaneous increase of and alcoholic administration tests. The cushion the length which confirms the value of L/A com- test has shown similar values to the alcohol test, pared to T0 has to be emphasized. The ToB does thus confirming the study of Palm, carried out on not show the sensitization of neurosensory circuits a dynamic platform (15). Evaluation with the same by a particular eso-input (9). parameters of rubber cushion tests with OE has Alcohol administration test showed similar re- reported an increase of the area especially for the sults to the hypothermia test with an increase in hypothermia and alcohol tests. This seems to su- the standard deviation as a wide diversity of par- ggest a new strategy implemented by the neuronal ticipants’ response. The alcoholic dose conditions system. The "floating sensation" of the rubber has the physical and mental state with a reduction of "awakened" the voluntary control to cope with a inhibition at blood concentrations over 0.02%. “new” and destabilizing surface of the soil. Only in the next stage, with the increase of the le- In conclusion, in literature there are no studies vel of ethanol in blood, it is possible to reveal a which have assessed on the same subjects the diver- beginning of impairment of posture and balance sity of response of neurosensory adaptation as a re- (>0.05% slow reactions; >0.1% reduced motor sult of different stimuli. The first hypothesis of the

332 Li Causi et al. Perturbing agents influence posture

present study (each single perturbing external agent setting against neurosensory input that control po- could significantly modify postural control) has sture such as in nightclubs (psychedelic lights and not been fully confirmed by ToB analysis (altering intake of alcoholic beverages) could significantly effects only for the strobe light and the swivel chair alter the maintenance of postural stability. This stu- tests). As for the second hypothesis (there are no dy should certainly be conducted on a larger po- differences when comparing the different altering pulation and increasing time of the single altering sources), it is clear that different tests do not trigger stimuli in order to assess the effects that some real- the deficit of postural control in the same way and world conditions may have on the nervous system. with the same features. Strobe light input and rota- tion of the body in space have influenced respecti- FUNDING vely the view (decrease compared to the vestibule No specific funding was received for this study. and touch) and the vestibule (increase compared to view and touch), while plantar hypothermia and TRANSPARENCY DECLARATION alcohol have increased the oscillation area. The- Conflicts of interest: None to declare refore, a particularly low temperature or stressful

REFERENCES 1. Collins JJ, De Luca CJ. The effects of visual input on 10. Storaci R, Manelli A, Schiavone N, Mangia L, Prigi- open-loop and closed-loop postural control mechani- one G, Sangiorgi S. Whiplash injury and oculomotor sms. Exp Brain Res 1995; 103:151-63. dysfunctions: clinical-posturographic correlations. 2. Hafström A, Fransson PA, Karlberg M, Ledin T, Ma- Eur Spine J 2006; 15:1811-6. gnusson M. Visual influence on postural control, with 11. Gagey PM, Bizzo G, Bonnier L, Gentaz R, Guillaume and without visual motion feedback. Acta Otolaryn- P, Marucchi C, Villeneuve P. Huit leçons de Posturo- gol 2002; 122:3927. logie (Eight lessons of Posturology) [French] 4th ed. 3. Krishnamoorthy V, Yang JF, Scholz JP. Joint coordina- Paris: Association Française de Posturologie, 1990. tion during quiet stance: effects of vision. Exp Brain 12. Gagey PM, Weber BG. Posturologia - Regolazione Res 2005; 164:1-17. e perturbazioni della stazione eretta (Posturology - 4. Horak FB. Role of the vestibular system in postural Regulation and perturbations of standing) [Italian] control. In: Herdman SJ (ed.) Vestibular Rehabilita- Rome: Marrapese, 2000. tion, 3rd ed. Philadelphia: Contemporary Perspectives 13. Horak FB. Postural compensation for vestibular loss in Rehabilitation, 2007: 32-53. and implications for rehabilitation. Postural compen- 5. Schmidt PM, Giordani AM, Rossi AG, Cóser PL. Ba- sation for vestibular loss and implications for rehabi- lance assessment in alcoholic subjects. Braz J Otor- litation. Restor Neurol Neurosci 2010; 28:57-68. hinolaryngol 2010; 76:148-55. 14. Paillard T, Bizid R, Dupui P. Do sensorial manipu- 6. Stal F, Fransson PA, Magnusson M, Karlberg M. lations affect subjects differently depending on their Effects of hypothermic anesthesia of the feet on vi- postural abilities? Br J Sports Med 2007; 41:435-8. bration-induced body sway and adaptation. J Vestib 15. Palm HG, Waitz O, Strobel J, Metrikat J, Hay B, Fri- Res 2003; 13:39-52. emert B. Effects of low-dose alcohol consumption on 7. Patel M, Fransson PA, Lush D, Gomez S. The effect postural control with a particular focus on the role of of foam surface properties on postural stability asse- the visual system. Motor Control 2010; 14:265-76. ssment while standing. Gait Posture 2008; 28:649-56. 16. Mergner T, Schweigart G, Maurer C, Blümle A. Hu- 8. Pyykkö I, Jäntti P, Aalto H. Postural control in elderly man postural responses to motion of real and virtual subjects. Age Ageing 1990; 19:215-21. visual environments under different support base con- 9. Bertora GO, Bergmann JM, Contarino D. Bipedestati- ditions. Exp Brain Res 2005; 167:535-56. on studied by posturography. Archives for Sensology 17. Schulmann DL, Godfrey B, Fisher AG. Effect of eye and Neurootology in Science and Practice – ASN – movements on dynamic equilibrium. Phys Ther 1987; Proceedings of the XXXIII Congress of the NES, Bad 67:1054-9. Kissingen/Germany, March, 2006. http//:www.neuro- otology.org (07 December 2020)

333