Official Publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 18, Number 2, August 2021

Official publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 18, Number 2, August 2021. 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 32 441 291; Printed by: Labirint d.o.o. Zenica. Zmaja od Bosne bb, 72000 Zenica 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: "Summer" (Author: Zdena Šarić Pisker) MEDICINSKI GLASNIK Official Publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 18, Number 2, August 2021 Free full-text online at: www.ljkzedo.com.ba, and www.doaj.org (DOAJ, Directory of Open Access Journals)

Review Holly Sacrament: the medicine of spiritual immortality or a vehicle for contamination? 334 Dimitrios Anyfantakis Dog bites and attacks on athletes: lack of effective prevention mechanisms 338 Muhamed Katica, Nadža Kapo, Nasreldin Hassan Ahmed, Anida Kapo-Gurda, Safet Kapo Original Regulation of the apothecary activity in Bosnia and Herzegovina over the Austro-Hungarian period 343 Vanda Marković-Peković article Fasting state requirements for blood sampling: a survey of patients in Cantonal Hospital Zenica, 352 Bosnia and Herzegovina Damira Kadić, Aida Avdagić-Ismić, Sabaheta Hasić, Ferhana Bošnjak Effect of atorvastatin on systolic and diastolic function in patients with heart failure with reduced 357 ejection fraction (HFrEF) Faris Zvizdić, Edin Begić, Mirza Dilić, Šekib Sokolović, Orhan Lepara Predictive factors for noninvasive mechanical ventilation failure among COVID-19 critically ill 362 patients - a retrospective cohort study Mirza Kovačević, Nermina Rizvanović, Adisa Šabanović Adilović Chest x-ray resolution after SARS-CoV-2 infection 370 Besim Prnjavorac, Aida Mujaković, Lejla Prnjavorac, Tamer Bego, Edin Jusufović, Edin Begić, Vildana Torlak-Arnaut, Mutapčić Meliha, Hasan Škiljo, Enes Hodžić, Emina Karahmet, Maja Malenica, Tanja Dujić, Jusuf Mehić, Nedžada Irejiz, Rifat Sejdinović, Anel Mahmutović, Ajdin Ibrahimović Clinical characteristics, comorbidities and mortality in critically ill mechanically ventilated patients 378 with Covid-19: a retrospective observational study Adisa Šabanović Adilović, Nermina Rizvanović, Mirza Kovačević, Harun Adilović Early predictors of severity and mortality in COVID-19 hospitalized patients 384 Sehveta Mustafić, Edin Jusufović, Fatima Hukić, Emir Trnačević, Anja Divković, Alma Trnačević

Mesenchymal stem cells under hypoxia condition inhibit peritoneal adhesion by suppressing the 398 prolonged release of interleukin-6 Adi Muradi Muhar, Agung Putra, Vito Mahendra Ekasaputra, Dewi Masyithah Darlan, Desiree Anggia Paramita, Iqbal Pahlevi Adeputra Nasution Association of rs211037 GABRG2 gene polymorphism with susceptibility to idiopathic generalized 404 epilepsy Marija Milanovska, Emilija Cvetkovska, Sasho Panov C-reactive protein and haemoglobin level in acute kidney injury among preterm newborns 410 Fiva Aprilia Kadi, Tetty Yuniati, Yunia Sribudiani, Dedi Rachmadi

A randomized, placebo-controlled trial of zinc supplementation during pregnancy for the prevention 415 of stunting: analysis of maternal serum zinc, cord blood osteocalcin and neonatal birth length Lili Rohmawati, Dina Keumala Sari, Makmur Sitepu, Kusnandi Rusmil

Male to female birth ratios over a 35-year period 421 Hrvoje Vraneš, Hrvojka Soljačić Vraneš, Ivka Djaković, Vesna Gall, Ana Meyra Potkonjak Retinal changes in febrile seizures in children: a retrospective analysis in Tuzla Canton, Bosnia and 427 Herzegovina Meliha Halilbašić, Amra Nadarević Vodenčarević, Anis Međedović, Amir Halilbašić, Almira Ćosićkić, Ajla Pidro

The role of aripiprazole in improvement of penile erection in schizophrenia patients with erectile 432 dysfunction Debby Handayati Harahap, Carla Raymondalexas Marchira, Eti Nurwening Solikhah, Dicky Moch Rizal Risk of surgical site infections after colorectal surgery and the most frequent pathogens isolated: a 438 prospective single-centre observational study George Panos, Francesk Mulita, Karolina Akinosoglou, Elias Liolis, Charalampos Kaplanis, Levan Tchabashvili, Michail Vailas, Ioannis Maroulis

Neurocysticercosis with symptomatic epilepsy manifestation 444 Nataliya Nekrasova, Olena Tovazhnyanska, Daryna Sushetska, Olena Markovska, Anton Shapkin, Rhea Singh, Yevhenija Soloviova, Dmytro Butov

Is preoperative hypoproteinemia associated with colorectal cancer stage and postoperative 450 complications? Amina Sofić, Ismar Rašić, Emsad Halilović, Alma Mujić, Denis Muslić

Comparison of early and delayed lumbar disc herniation surgery and the treatment outcome 456 Ermin Hadžić, Bruno Splavski, Goran Lakičević Single-centre experience of emergency hernia surgery during COVID-19 pandemic: a comparative 463 study of the operative activity and outcomes before and after the outbreak Adnan Malik, Mohamed Zohdy, Aftab Ahmad, Charalampos Seretis

Epidemiology of hospitalized patients with peripheral arterial disease in Bosnia and Herzegovina 468 Akif Mlačo, Nejra Mlačo, Mevludin Mekić, Alen Džubur

Anatomy of septocutaneous blood vessels of the anterior forearm 475 Darko Jović, Mirza Bišćević, Milan Milisavljevic, Zoran Aleksić, Milica Jakovljević, Nevena Tešović, Mićo Kremenović

Reamputation stumps below knee 479 Viktor I. Shevchuk, Yurii O. Bezsmertnyi, Yankai Jiang, Halyna V. Bezsmertna, Tetyana V. Dovgalyuk

Relapses of traumatic peroneal tendons subluxation already treated surgically: a new surgical 487 approach Alessandro Tomarchio, Luigi Meccariello, Dariush Ghargozloo, Andrea Pasquino, Enrico Leonardi

Factors related to anxiety among resident doctors assigned to emergency room during the COVID-19 493 pandemic: a multivariate study at Sumatera Utara Affiliated Teaching Hospital Elmeida Effendy, Ariwan Selian, Julius Martin Siagian

Knowledge, attitudes and practices during the second wave of COVID-19 outbreak: a cross-sectional 499 study from various perspectives Armin Šljivo, Sutanay Bhattacharyya, Ahmed Mulać, Arian Abdulkhaliq, Rexhep Sahatçiu

A measurement of irradiance of light-curing units in dental offices in three Croatian cities 505 Ante Dundić, Valentina Rajić Brzović, Gloria Vlajnić, Danijela Kalibović Govorko, Ivana Medvedec Mikić Assessment of mothers’ satisfaction with health care during childbirth in a tertiary-level maternity 510 ward Adriana Haller, Albert Haller, Dejan Tirić, Vajdana Tomić Mortality associated with seasonal changes in ambient temperature and humidity in Zenica-Doboj 516 Canton Suad Sivic

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Holly Sacrament: the medicine of spiritual immortality or a vehicle for contamination?

Dimitrios Anyfantakis

Primary Care Centre of Kissamos, Chania, Crete, Greece

ABSTRACT

The Holy Communion is the oldest Christian practice, through which a gathered group of people partake bread and wine with a shared spoon. The pandemic of COVID-19 produced unexpec- ted social instability and chaos. In Greece, a recent bishop’s death from COVID-19 re-awaked the issue of infection transmission by using a common chalice and spoon. This review evaluates the available medical literature on this topic from the beginning of the pandemic until today.

Key words: COVID-19, Eastern Orthodoxy, infections Corresponding author: Dimitrios Anyfantakis Primary Care Centre of Kissamos K. Hliaki 3, 73134Chania, Crete Phone: +306937473215; +30 282 234 0100; E-mail: [email protected] ORCID ID: 0000-0002-8932-9093

Original submission: 11 December 2020; Revised submission: 28 December 2020; Accepted: 26 January 2021 doi: 10.17392/1334-21

Med Glas (Zenica) 2021; 18(2):334-337

334 Anyfantakis D. Holly communion and infection

INTRODUCTION of existence and they are sanctified (4).The miracle of Jesus healing the bleeding woman when The ongoing pandemic triggered a severe eco- she touched the edge of his clothes described in nomic and social crisis worldwide. In Greece a the Gospels, illustrates this metaphor (Matthew three-week nationwide lockdown started on 7 9:20–22, Mark 5:25–34, Luke 8:43–48 (5). November in order to halt the transmission of the infection. Churches closed their doors and were HOLLY COMMUNION AND THE RISK OF INFECTION due to reopen before the Christmas period. Holy Communion is a core ritual of both Eastern Ort- Empirical information suggests that Christians hodox and Roman Catholic Christians. During who receive Holy Communion do not get ill more the Divine Liturgy the priest dips a spoon into a frequently compared to those who do not partici- chalice of wine and places it into the mouth of a pate in this religious ritual (1). The first literature parishioner (1). report on this subject appeared in 1894 (6). Since then, the only scientific data derive from a small Although no episode of disease attributable to the number of experimental studies (1,7). In 1988, shared communion cup has ever been documen- Kingston reported that the only available expe- ted (2), the death of Metropolitan Bishop Ioannis rimental studies until then tested the numbers of of Lagadas due to COVID-19 revived the debate the bacteria from the rim of the communion cup over the safety of receiving communion before (8). He also stated that a pathogen entering by the Christmas season. mouth can cause disease, under specific circum- This review evaluates available medical literatu- stances (8). Principal parameters that may favour re on this topic from the beginning of the pande- infection transmission are: concentration and sur- mic until today. vival time of a micro-organism in the chalice, in the cup, in the communion wine, and the saliva HISTORICAL ASPECTS OF THE HOLY COMMUNION (8). The majority of the investigations studied Before the eleventh century, Christians used to only the eventuality of bacterial and not viral receive Holy Communion in an entirely different contamination (8). Remarkably, the concentrati- way than the present practice in the Eastern Ort- on of bacterial pathogens isolated from the rim hodox Church (3). The worshipers extended their of the cup was found to be considerably low (8). hands and the priest placed a portion of the holy In alignment with this, research efforts by Hobbs bread (3). After consuming the bread, the commu- et al. found that the number of organisms deposi- nicants were offered wine by a common cup (3). ted on the rim of the chalice varied from person By the twelfth century lack of priests made the to person but was relatively small (9). No micro- administration of the bread and wine separately, organisms were found in wine (9). hard procedure (3). For this reason, the Church A noteworthy experimental study was conducted adopted the use of a shared spoon (3). The sa- by Burrows and Hemmens (10). They contami- cred bread and wine were mixed together in the nated the rim of the chalice as heavily as possible Communion cup and placed carefully into the with saliva. The authors stated that transfer of the mouths of the communicants (3). Historically the pathogen from a person to another did not occur tradition of spoon emerged during the 7th centu- (10). The bactericidal effect of wine on most pat- ry in Byzantium (3).Today, more than two billi- hogens was also remarked (8,9,11). Interestingly, on Christians worldwide consume in this way Serratia marcescens and Streptococcus pyogenes consecrated bread and wine every week or more died within minutes in wine, and this effect was frequently (3). Hierarchs, priests and deacons do amplified by the silver material of the cup (9,11). not receive Holy Communion from a spoon (3). A survey of 10-week duration by Loving and The sacramental bread and wine offered during Wolf among 681 volunteers, compared illness the mystery of Eucharist are the body and the rates among those who received Holy Communi- blood of Jesus Christ. The material objects (the on, those who simply reported church attendan- spoon and chalice) become part of the mystery ce, and those who stayed at home. No significant as they come into contact with Christ’s Body difference was found between the three populati- and Blood (4). Their nature is not changed, but, on groups of the survey (12). In the same directi- rather, they are transformed to a different mode on, Manangan et al. reported that the risk of tran-

335 Medicinski Glasnik, Volume 18, Number 2, August 2021

smission of a communicable disease through the Holy Communion in Greece (18). If this number Holy Communion is small (13). Until today, the- is compared to 130.000 recorded infected cases re is a lack of evidence regarding the transmissi- during this interval, there is a large discrepancy, bility of a viral infection through the chalice and showing a miracle of God in front of the Chris- the communion cup. Viral infections and SARS- tmas period (18). However, it cannot be excluded COV-2 are transmitted mainly through inhalation that religious gathering of the population, without of respiratory droplets rather than oral contact preserving the safety measures (wearing a mask (14). Infected people spread viral particles whe- and keeping distance) can be a vehicle of the tran- never they talk and breathe (14). Therefore, this smission of COVID-19 infection (19). investigation would be complex and would raise Greek health experts avoided commenting on unanswered issues (15). The practice of intincti- church practices (20). Athena Linou, Professor on, which consists of dipping the wafer of bre- of Epidemiology at the Medical School of EKPA ad into the chalice together with the consecrated and President of the Institute of Preventive, Envi- wine, was reported to be a promising strategy in ronmental and Occupational Medicine Prolepsis, lowering the risk of infection (15,16). reported that it has never been scientifically con- In a recent article, Spantideas et al. evaluated the risk firmed that the saliva of those who partake in the of severe acute respiratory syndrome coronavirus 2 Holy Communion is a vehicle for contamination (SARS-CoV-2) transmission by participating in the (20). She also stated that spiritual matters of the mystery of Holy Communion (7). The authors, see- Orthodox faith cannot be explained with logic (20). king an answer on this topic, underlined the lack of In conclusion, the transmission of any contagio- the existing evidence and limited knowledge about us disease by sharing utensils and contact with the bacterial or viral load in the communicants' sali- the chalice has never been scientifically proved. va, a crucial parameter for the transmission of viru- Adjustments on the way that the mystery of the ses such as the common cold, influenza and SARS- Holy Communion is traditionally served to faithful CoV-2(7). They emphasized the immune status of Christians, with measures such as disinfection of the worshipers, stating that immune-compromised the spoon and the use of individual cups for wine, patients should request alternate means of partaking are interventions of unknown benefit. Any sugge- in the Holy Communion (7). stion that the Holly Communion poses a danger The small risk of transmission of any pathogen for viral or bacterial respiratory infection is not by partaking in the Holy Communion may be evidence based and derives from the lack of faith. sustained by the argument that deacons after the For the human mind, it is not easy to understand dismissal and distribution of antidoron, consume how this illogical transformation of the bread and the remaining communion from the rim of the wine to the body and blood of Jesus Christ occurs. chalice at the end of the liturgy (1). I believe Holy Sacrament is a mystery that exceeds They should be the first infected persons from vi- the natural laws and cannot be fully explained ral lethal diseases in the past, such as tuberculosis with the human logic. It could be assumed that and Hansen’s disease. Severe infections among the worshipers who do not believe that the Son priests have never been reported (1). An intere- of God is truly present in the Holy Communion sting case is that of the Saint Anthimos of Chios, under the appearance of bread and wine are better who regularly communed patients with leper and to stay apart and not ask for it. Those who believe tuberculosis (17). Despite the suggestions not to in “the medicine of immortality” the “life-giving receive Holy Gifts from the same cup with all body and blood of Jesus Christ” they have the these sick people, he was never infected (17). right to participate in the Holy Eucharist without fearing of the coronavirus. HOLY SYNOD AND EXPERTS OPINION FUNDING The Greek Orthodox Church insists that it is impossible for any disease – including Covid-19 – to be No specific funding was received for this study. transmitted through Communion (18). Metropoli- tan Piraeus Serafim, argued that during the last 10 TRANSPARENCY DECLARATION months (from February 2020 to December 2020), Conflict of interest: None to declare. approximately 2,500,000 faithful people received

336 Anyfantakis D. Holly communion and infection

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1. Anyfantakis D. Holy communion and infection tran- 13. Manangan LP, Sehulster LM, Chiarello L, Simonds smission: a literature review. Cureus 2020; 12:e8741. DN, Jarvis WR. Risk of infectious disease transmi- 2. Gill ON. The hazard of infection from the shared ssion from a common communion cup. Am J Infect communion cup. J Infect 1988; 16:3-23. Control 1998; 26:538–39. 3. Taft R. Byzantine communion spoons: a review of 14. Jayaweera M, Perera H, Gunawardana B, Manatun- the evidence. DOP J1996; 50:209-38. ge J. Transmission of COVID-19 virus by droplets 4. Papageorgiou Fr. P. The chalice, the spoon, and and aerosols: a critical review on the unresolved our fear of death. 2020https://www.trisagion- dichotomy. Environ Res 2020; 188:109819. films.com/blog/2020/5/25/chalice (26 December 15. Vélez FJR. Spiritual care of the sick. Linacre Q 2020) 2017; 84:220–25. 5. Oke RO. Healing of the haemorrhaging woman as a 16. LaGrange Loving A. A controlled study on intinc- model for checkmating stigma of people living with tion: a safer alternative method for receiving Holy HIV. Verbum Eccles2017; 38:1-12. Communion. J Environ Health1995; 58:24–28. 6. Anders H.S. Prophylaxis in churches needed by the 17. Ekklisia. Agios Anthimos of Chios: He transmitted adoption of individual communion chalices or cups. the divine communion to the lepers and then ended Proc Philadelphia County Med Soc1894; XV:345– 2020 https://www.ekklisiaonline.gr/ekklisiaonline/ 52. agios-anthimos-chiou-metedide-ti-thia-kinonia-sto- 7. Spantideas N, Drosou E, Barsoum M, Bougea A. us-leprous-ke-katelye-meta/ (26 December 2020) COVID-19 and Holy Communion. Public Health 18. Ekklisia. Piraeus: 2,500,000 people have shared 2020; 187:134-35. in the last 10 months. 2020 https://www.ekklisia- 8. Kingston, D. Memorandum on the infections hazar- online.gr/nea/pireos-tous-telefteous-10-mines- ds of the common communion cup with especial re- kinonisan-2-500-000-atoma/?fbclid=IwAR1Ou ference to aids. Eur J Epidemiol 1988;4:164–70 pk7ddJ12B1u7ga2gJSXBNciCD5aP3ss_TgU- 9. Hobbs BC, Knowlden JA, White A. Experiments on tIPYSfnlrPwTPgHU0qc (26 December 2020) the communion cup. J Hyg Lond 1967; 65:37-48. 19. Quadri SA. COVID-19 and religious congregations: 10. Burrows W, Hemmens ES. Survival of bacteria Implications for spread of novel pathogens. Int J In- .on the silver communion cup. J Infect Dis 1943; fect Dis 2020;96:219-21. 73:180-90. 20. Orthodox times Epidemiology professor: No scienti- 11. Gregory KF, Carpenter JA, Bending GC. Infection fic study that proves COVID-19 transmission thro- hazards of the common communion cup. Can J Pu- ugh Holy Communion 2020 https://orthodoxtimes. blic Health 1967; 58: 305-10. com/epidemiology-professor-no-scientific-study- 12. Loving A, Wolf L. The effects of receiving Holy that-proves-covid-19-transmission-through-holy- Communion on health. J Environ Health 1997; communion/ (26 December 2020) 60:6–10.

337 REVIEW

Dog bites and attacks on athletes: lack of effective prevention mechanisms

Muhamed Katica1, Nadža Kapo2, Nasreldin Hassan Ahmed3, Anida Kapo-Gurda4, Safet Kapo5

1Department of Pathological Physiology, Veterinary Faculty, University of Sarajevo, 2Veterinary Faculty, University of Sarajevo, 3Emer- gency Medicine Clinic, Clinical Centre of the University of Sarajevo, 4Faculty of Educational Sciences, University of Sarajevo, 5Faculty of Sport and Physical Education of the University of Sarajevo; Sarajevo, Bosnia and Herzegovina

ABSTRACT

Athletes who train in public places in urban and rural areas are just as attacked and injured by dogs of known owners as they are by dogs with no owners, in a relatively equal proportion. The largest number of bites occurs in the summer, what makes up half of all bites, just when sports activity is most pronounced. Athletes who are most often exposed to potential attacks and bites are cyclists, long-distance athletes, marathon runners, recreational athletes, etc. both during training and competitions. Off-road cyclists Corresponding author: are at a significantly higher risk of dog attacks because cycling Muhamed Katica takes place off-road, that is, away from urban areas. Dog attacks Department of Pathological Physiology, can adversely affect the psycho-physical readiness of athletes. In Veterinary Faculty, University of Sarajevo Bosnia and Herzegovina there have been no cases of injuries to Zmaja od Bosne 90, 71000 Sarajevo, athletes recorded by competent medical institutions or umbrella sports associations. It is necessary to work on more efficient admi- Bosnia and Herzegovina nistration (registration and recording of attacks and bites of dogs). Phone: +387 33 729 155; It is of utmost importance to educate athletes on dog behaviour, the fax: +387 33 617 850; reasons for their aggressive behaviour and causal mechanisms of E-mail: [email protected] dog attacks as well as the first aid education, what can have a great ORCID ID: https://orcid.org/0000-0002- impact on reducing further complications. 8184-0065 Key words: aggression, animals, first aid, marathon running, sports

Original submission: 15 January 2021; Revised submission: 28 February 2021; Accepted: 18 March 2021 doi: 10.17392/1344-21

Med Glas (Zenica) 2021; 18(2):338-342

338 Katica et al. Dog bites and attacks on athletes

INTRODUCTION areas from aggressive dogs is high. In addition to the mentioned risks of the infection occurrence, A domesticated dog has successfully adapted for dog bites also cause psychological trauma (2). many purposes: as a pet, a running dog-sports dog, a dog in the service of military and police Some breeds of dogs (Bull Terrier, German formations, a hunting dog (1). There is also a shepherd, Cocker Spaniel, Pit Bull, Collie very large population of dogs, especially in un- Rottweiler, Doberman Pinscher and Siberian Hu- derdeveloped countries, without owners-guardi- sky) have been identified as more aggressive than ans, referred to as "stray dogs" (2,3). The loss of other breeds (13-15). However, all dogs can show human support in this animal population causes aggressive behaviour under certain circumstances a number of problems, from territorial status to (13). A number of experts believe that most bites food security. Existential threats activate mecha- can be prevented. Bite victims misinterpret the be- nisms for self-preservation and return to natural haviour of an anxious and/or fearful dog (16). patterns of behaviour resulting in a variety of ad- Intervention educational programs are practiced verse events (4). Uncontrolled movement of dogs in some European Union countries aimed at edu- on the roads causes traffic accidents; dogs usurp cating children and adults about the behaviour of social peace by barking and they often attack dogs, and about the signals that, for example, a people in packs, while their bites cause physical dog is frightened and can bite (17-20). However, and mental injuries. Such a population of dogs there is little research and evidence on whether has no veterinary supervision (2,5). and how this approach actually prevents bites (21). It has been observed that bites by dogs of known Athletes during daily training, as well as acti- owners are increasing in the UK and Chile and ve athletes, cyclists during competitions, are not pose a public health concern as they can result in exempt from the danger of dog attacks. Dog bites serious injuries or even death (6,7). In addition, occur throughout the year, but most bites occur in there may be negative implications for the well- the summer (22), accounting for half of all bites, being of the dog that has bitten; e.g. a dog owner just when sports activity is most pronounced (23). waiving custody, placement in dog shelters, eu- Cyclists, long-distance athletes, marathon thanasia, etc. (8-10). Despite numerous studies, it runners, recreational athletes, etc. are most often is difficult to prove why dogs bite, at what point exposed to attacks, both during training and du- of the event they do it and what profile of a per- ring competitions (24). son is particularly at risk (2). In general, there is a lack of reports on affected People are just as attacked and injured by dogs athletes both in Bosnia and Herzegovina (B&H) of known owners as they are by dogs with no and worldwide. No reports of this kind have been owners, in a relatively equal proportion (1,2,11). published in B&H and so far no cases of injuries Dogs of known owners attack people in public to athletes have been recorded by competent me- areas, most often due to irresponsible behaviour dical institutions. There is also no such informati- of their owners (1). However, dogs of known on from umbrella sports associations and the like. owners are to a greater or lesser extent under The aim of this paper was to point out the exposu- health and veterinary supervision and are most re of athletes, due to their specific work and mo- often vaccinated against rabies, which is not the vement, to dog bites and attacks, and the shortco- case with dogs without owners. Owned dogs eat mings of the injury record system in B&H, with adequate dog food, while stray ones most often the intention to progress towards better and more look for food among the waste, so it can be assu- practical measures to improve public safety. med that stray dogs have a higher quantitative and qualitative presence of microorganisms, pri- CONSEQUENCES OF BITE WOUNDS marily in the mouth (4). There is a huge number of microorganisms li- Various microorganisms are introduced into the ti- ving in the oral cavity of dogs, especially in the ssue of the bitten person by a dog bite, which can population of stray dogs. At the moment of the consequently lead to an infection. The situation is bite, together with saliva, the microorganisms especially serious if there is a risk of developing are pressed into the injured tissue, most often tetanus or rabies (12). The risk of bites in urban

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that of extremities, arms, legs, head or neck. A bitten by a dog or were hit by thrown objects large number of bacteria with high virulence can (30). There is a wide range of different profiles immediately cause infection on this occasion (4). of athletes who are potential targets of stray dogs The force produced by dog's teeth during a bite or dogs of known owners. The most vulnerable varies between breeds, from 310 kPa to almost are athletes, cyclists, marathon runners, and va- 31,790 kPa in specially trained dogs (25). Along rious profiles of recreational athletes, who train with the superficial wound, skin, muscles,- ten or compete in open public areas. dons, blood vessels and nerves can be damaged Off-road (mountain) cycling tends to increase as well (26) that can permanently or temporarily in popularity in different regions (31,32). This remove athletes from sport fields. sport involves coaching and competition, mostly In wounds of deeper bites there is a risk of con- in subrural and rural areas. Off-road cyclists are tamination by tetanus spores and consequently at a significantly higher risk of dog attacks, as by tetanus, especially if the wound is not treated cycling usually takes place off-road, away from properly (12, 23, 26). However, the greatest risk urban areas (32). In such environment there is no associated with dog bites is the risk of rabies in presence (or it is very reduced) of any audience the event of injury by a rabid animal (27). Talan or security services. Such environment can often et al. (1999) (28) investigated the bacterial micro- favour the attack of aggressive stray dogs on this flora of bite wounds in humans, and found the profile of athletes, whose population in this area following isolated aerobic bacteria: Pasteurella is often large. At a particular risk is the group sp.(50%), Streptococcus spp. (46%), Staphylo- of cyclist individuals who are notably separated coccus spp. (46%), Neisseria spp. (32%), and from the largest group of cyclists who compete Corinebacterium sp. (12%). with each other (32). A particularly aggravating circumstance for such CLINICAL MANIFESTATIONS OF BITE WOUNDS cyclists is that they do not notice the potential Skin lesions are the most common injury, and danger during exercise or competition, or are of- they are often accompanied by bleeding. Accor- ten physically exhausted, or because they have ding to the research conducted by Talan et al. headphones on they do not hear the barking of (1999) (28), out of 50 patients with bite wounds, dogs attacking them (32). Two case studies (32) 60% were with tooth impression only, 10% were described one cyclist attacked and bitten on the minor perforations with lacerations, and 30% knee by a stray dog, and in another one recreatio- were a combination of both. Hematomas around nal cyclist was attacked by a sheepdog. the bite wound are usually evident. The most Cyclists who recreationally practice this sport common is purulent infection without abscess come in various interactions with dogs, often formation (58%), accompanied nonpurulent wo- get various injuries directly or indirectly. It can unds with cellulitis, lymphangitis, or both (30%), be assumed that injuries are not rare in the dog- and abscesses (12%). Limited and painful move- bicycle interaction which resulted in, on average ments of fingers and joints occur if the wound is 3500 visits to the emergency department per year located on the extremity, which gravitates close in the period from 2006 to 2015 (33). More serio- to the region of or is directly situated on the joint. us injuries occurred when cyclists hit dogs that Swelling and redness in the area of the injury are had been curled up or when they tried to avoid not uncommon (4,6). Microorganisms isolated such a dog. The most serious injuries occurred from infected bite wounds are similar to those when a person rode a bicycle while simultaneou- isolated from the oral cavity of dogs (29). sly leading a dog on a leash (33).

ATHLETE INJURIES CAUSED BY DOG AT- PSYCHOLOGICAL TRAUMA IN BITTEN ATHLETES TACKS Dog bites can have an adverse effect in terms of Athletes during exercise, as well as during com- the appearance of feelings of intense fear, helple- petition, often encounter various adverse effects ssness or horror, which ultimately often leads to including various injuries. Ten percent of sur- many symptoms of post-traumatic stress disor- veyed professional and recreational athletes were der (PTSD) (34,35). Every athlete attacked by a

340 Katica et al. Dog bites and attacks on athletes

dog has presumably suffered a greater or lesser of Notari et al. (2020) (40) suggest that educati- intensity of fear. Ultimately, this has adversely onal programmes for owners of dogs are funda- resulted in the success achieved, or will nega- mental tools to reduce aggression risk factors and tively affect the psycho-physical readiness of a prevent aggression. In addition to prevention, ad- potential athlete in future competitions. We have vice can be provided on how the victim should not found any research in the available literature behave during the bite, to keep injuries to a mini- that has dealt with this issue. mum, how to deter the dog from further attacks, Most scientific reports describe the problem of and how to seek adequate help. the occurrence of fear in children, with negative In conclusion, dog bites are a serious public he- consequences after dog attacks. Numerous studies alth problem, which, among other things, has ne- indicate that this risk to children's health is unde- gative implications for the animals in question, restimated in paediatric care, as they are the most such as abandonment, confiscation or euthanasia. vulnerable group in this regard (36). The reason This paper provides an opportunity to shed light why PTSD occurs in some children is not known. on the circumstances of dog bites in order to pro- It is evident that this is closely related to the in- gress towards better and more practical measures tensity, character and aggressiveness of the animal to improve public safety, and especially for the attack (37). Attacked people can become victims interaction between athletes and dogs. of dog bites and suffer significant emotional tra- It is necessary to take a set of measures and acti- uma, reliving for years the memories of fear and vities for more efficient administration (registra- pain caused by an aggressive dog attack (36, 38). tion and recording of dog bites and attacks), and based on adequate data obtained by future scien- CAUSAL MECHANISMS OF DOG ATTACKS tific research, in the near future, organize various There are very complex factors associated with types of education to reduce unwanted cases. It is dog bites, victim behaviour and/or dog behaviour necessary to educate athletes regarding dogs and prior to the bite. It is evident that most scientific the reasons for their aggressive behaviour, and to reports elaborate on the problem of dog attacks educate them on providing necessary first aid on and bites solely on the basis of clinical and hos- the spot, after an injury by a dog, that can have a pital data (6). There are insufficient data in the great impact on reducing further complications. literature about the behavioural and legislative Tailored educational programs would help raise aspects of bites as well as the consequences for awareness, with the goal of reducing the frequ- dogs, which is of importance for improving our ency of this risk. In particular, in this case, the understanding of dog bites. risk related to professional and recreational athle- The causal mechanisms of the bite will be clarifi- tes would be reduced, just as that observed in a ed after a detailed analysis of factors surrounding broader context and to public health as a whole. the incident, i.e. the dog bite (2). Westgarth and FUNDING Watkins (2015) (21) and Chen et al. (2016) (39) prefer education in the first place on how to actu- No specific funding was received for this study. ally deal with dog bites, because it is an essential benefit that reduces the consequences of injuries. TRANSPARENCY DECLARATION In that sense, the results of a retrospective study Competing interests: None to declare. REFERENCES 1. Katica M, Gradascevic N, Hadzimusic N, Obradovic 3. Saleem SM, Jan SS, Khan SM. Qualitative analysis Z, Mujkanovic R, Mestric E, Coloman S, Dupovac of the perception of street dog bite victims and im- M. 2017. Widespread of stray dogs: methods for sol- plication for the prevention of dog bites at a teaching ving the problem in certain regions of Bosnia and hospital anti-rabies Clinic. J Family Med Prim Care Herzegovina. IJRG 2017; 5:414-22. 2020; 9:4118-26. 2. Katica M, Obradović Z, Ahmed NH, Mehmedika- 4. Katica M, Obradović Z, Ahmed NH, Dervišević E, Suljić E, Stanić Ž, Mohamed RSA, Dervišević E. Delibegović S. Dog bites and their treatment in Fe- Interdisciplinary aspects of possible negative effects deration of Bosnia and Herzegovina. Cyprus J Med of dogs on humans in Bosnia and Herzegovina. Med Sci 2019; 4:136-40. Glas (Zenica) 2020; 17:246-51.

341 5. Schalamon J, Ainoedhofer H, Singer G, Petnehazy implications for the prevention of dog bites. J Vet T, Mayr J, Kiss K, Höllwarth ME. Analysis of dog Behav Clin Appl Res 2015; 10:479-88. bites in children who are younger than 17 years. Pe- 22. Ali MI, Jamali S, Ashraf T, Ahmed N. Patterns and diatrics 2006; 117:e374-9. Outcomes of dog bite injuries presenting to emer- 6. Oxley JA, Westgarth C. Contexts and consequences gency department in a tertiary care hospital at Ka- of dog bite incidents. J Vet Behav 2018; 23:33-9. rachi. Pak J Med Sci 2021; 37 (In press). 7. Barrios CL, Bustos-López C, Pavletic C, Parra A, 23. Agarwal N, Reddajah VP. Epidemiology of dog bi- Vidal M, Bowen J, Fatjó J. Epidemiology of dog tes: a community-based study in India. Trop Doct bite incidents in Chile: factors related to the patterns 2004; 34:76-8. of human-dog relationship. Animals 2021; 1:96. 24. Reisner IR, Nance ML, Zeller JS, Houseknecht EM, 8. Alberghina D, Virga A, Buffa SP, Panzera M. Inciden- Kassam-Adams N, Wiebe DJ. Behavioural charac- ce and characteristics of hospitalizations after dog’s teristics associated with dog bites to children pre- bite injuries in Sicily (Italy) between 2012-2015. senting to an urban trauma center. Inj Prev 2011; Vet Ital 2017; 53:315-20. 17:348-53. 9. Casey RA, Loftus B, Bolster C, Richards 25. Morgan M, Palmer J. Dog bites. BMJ 2007; 7:413-7. GJ, Blackwel EJ. Human directed aggression in do- 26. Griego RD, Rosen T, Orengo IF, Wolf JE. Dog, cat mestic dogs (Canis familiaris): occurrence in diffe- and human bites: a review. J Am Acad Dermatol rent contexts and risk factors. Appl Anim Behav Sci 1995; 33:1019-29. 2014; 152:52-63. 27. Parize P, Docheux L, Bourhy H. Rabies. Rev Prat 10. Hunthausen W. Effects of aggressive behavior onca- 2019; 69:423-8. nine welfare. J Am Vet Med Ass 1997; 210:1134-6. 28. Talan DA, Citron FM, Abrahamian GJ, Moran E, 11. Vučinić M, Đorđević M, Radenković-Damnjanović Goldstein JC. Bacteriologic analysis of infected dog B, Janković LJ, Mirilović M. Bites to humans cau- and cat bites. N Engl J Med 1999; 340:85-92. sed by stray and owned dogs in Belgrade. Acta Vete- 29. Philipsen TEJ, Molderez C, Gys T. Cat and dog rinaria (Belgrade) 2008; 58:563-71. bites. What to do? Guidelines for the treatment of 12. Uzunović S, Skomorac M, Bašić F, Mijač-Musić I. cat and dog bites in humans. Acta Chir Belg 2006; Epidemiological features of human cases after bites/ 106:692-5. scratches from rabies-suspected animals in Zenica- 30. Koplan JP, Rothenberg RB, Jones EL. The natural Doboj Canton, Bosnia and Herzegovina. J Prev Med history of exercise: a 10-yr follow-up of a cohort of Public Health 2019; 52:170-8. runners. Med Sci Sports Exerc 1995; 27:1180-4. 13. Presutti RJ. Prevention and treatment of dog bites. 31. Jeys LM, Cribb G, Toms AD, Hay SM. Mountain Am Fam Physician 2001; 63:1567-72. biking injuries in rural England. Br J Sports Med 14. Özcan M, Öztürk N. 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Overall K, Love M. Dog bites to humans-demo- mon HK. Post-traumatic stress responses in chil- graphy, epidemiology, injury, and risk JAVMA dren: awareness and practice among a sample of 2001; 218:1923-34. pediatric emergency care providers. Pediatrics 2005; 17. Wilson F, Dwyer F, Bennet PC. Prevention of 115:1261-7. dog bites: evaluation of a brief educational inter- 36. Peters V, Sottiaux M, Appelboom J, Kahn A. Posttra- vention program for preschool children. J Comm umatic stress disorder after dog bites inchildren. J Psychol 2003; 3175–86. Pediatr 2004; 144:121-2. 18. Duperrex O, Blackhall K, Burri M, Jeannot E. Edu- 37. American Psychiatric Association: Diagnostic and cation of children and adolescents for the preventi- Statistical Manual of Mental Disorders, DSM-IV. on of dog bite injuries (Review). Cochrane DB Syst 4thed. Washington: APA, 1994. Rev 2009; 2:CD004726. 38. Westgarth C, Brooke M, Christley RM. How many 19. Davis AL, Schwebel DC, Morrongiello BA, Stewart people have been bitten by dogs? A cross-sectional J, Bell K. Dog bite risk: an assessment of child tem- survey of prevalence, incidence and factors associa- perament and child-dog interactions. Int J Environ ted with dog bites in a UK community. J Epidemiol Res Public Health 2012; 9:3002-13. Community Health 2018; 72:331-6. 20. Shen J, Rouse J, Godbole M, Wells HL, Boppana S, 39. Chen Y, Gao Y , Zhou L, Tan Y, Li L. A Comparative Schwebel DC. Systematic review: Interventions to study of dog- and cat-induced injury on incidence educate children about dog safety and prevent pe- and risk factors among children. Int J Environ Res diatric dog-bite injuries: A meta-analytic review. J Public Health 2016; 13:1079. Pediatr Psychol 2017; 42:779-91. 40. Notari L, Cannas S, Di Sotto YA, Palestrin C. A 21. Westgarth C, Watkins FA. 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342 ORIGINAL ARTICLE

Regulation of the apothecary activity in Bosnia and Herzegovina over the Austro-Hungarian period

Vanda Marković-Peković

Department of Social Pharmacy and Pharmaceutical Legislation, Faculty of Medicine, University of Banja Luka, Banja Luka, Bosnia and Herzegovina

ABSTRACT

Aim To present the regulations governing the operation of pharmacies in Bosnia and Herzegovina over the Austro-Hungarian rule (1878-1918).

Methods Qualitative secondary data analysis was used.

Results The Austro-Hungarian government had found poor population’s health, insufficient health facilities and qualified staff. For a long time, population was treated by old methods of medicine and pharmacy, and directed to folk doctors, healers and herbalists. As early as 1879, orders requesting mandatory possession of a university diploma to practice pharmacy and medicine, thus taking the Corresponding author: initial steps to combat quackery. The production and dispensing of Vanda Marković-Peković medicines became the exclusive competence of pharmacists. The University of Banja Luka, Law on Pharmacies adopted in 1907 comprehensively regulated Faculty of Medicine, Department of Social the apothecary activity. Pharmacy Gremium was founded, the first association of pharmacists with the task of protecting professional Pharmacy and Pharmaceutical Legislation interests. All types of quackery were explicitly forbidden to phar- Save Mrkalja 14, 78 000 Banja Luka, macy staff. Apothecary activity was regulated as a craft, not as a Republic of Srpska, Bosnia and Herzegovina health activity. During this period, pharmacy became a regulated Phone: +387 51 340 106; profession with educated and qualified personnel. The number of Fax: + 387 51 215 454; public pharmacies and qualified staff was growing. In 1878 only one Email: vanda.markovic-pekovic@med. graduated pharmacist was found, while in 1910 in 47 pharmacies there were 79 pharmacy staff. At the end of 1918, the masters of unibl.org pharmacy were the owners of 48 pharmacies, in 38 cities. ORCID ID: https://orcid.org/0000-0001- 8963-5720 Conclusion All enacted regulations contributed to the development and improvement of the apothecary activity over the ob- Original submission: served period, and laid the foundations for the future development 03 February 2021; of the profession. Revised submission: Key words: community pharmacies, history of pharmacy, legi- 18 February 2021; slation Accepted: 23 February 2021 doi: 10.17392/1355-21

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INTRODUCTION the work bans. They can be said to have been a transition from folk pharmacotherapy to modern Bosnia and Herzegovina (B&H) was under Austro- pharmacies, and as such were preferred by average Hungarian rule from 1878 to 1918. After the Berlin residents (1). Therefore, pharmacy owners com- congress, held in 1878, B&H fell under the Austro- plained of poor turnover, so they had to deal with Hungarian occupation, while the Sultan’s soverei- additional work in order to provide for normal life. gnty was formally retained and from the Austro- Pharmacist Anton Kluczenko, the pharmacy own- Hungarian annexation (1908) to the end of the First er in Livno, is an example of how pharmacy had World War (1918) it was under the full sovereignty struggled. He called himself a "photographer from of the Austro-Hungary Monarchy (Monarchy). Livno" because his pharmacy worked so poorly Over the Austro-Hungarian rule socio-political and that he also practiced photographic craft, (3) the social circumstances changed significantly, inclu- soda water production and the grocery store (6,7). ding health conditions in the country. Some other pharmacists were also engaged in ad- Little attention was paid to the population's health ditional jobs because accustoming of the people during the Ottoman rule, so the health and hygiene to be treated with medicines from pharmacies conditions of the population were very poor. The was slow (3). Pharmacy owners (in B&H cities) Austro-Hungarian government found a complete Eduard Rhein (Bihać), Stefan Variačić (Gradiška), absence of public hygiene, epidemics and numero- Theodor Heydušek (Bugojno), Gyulla Keller (Bo- us chronic infectious diseases were a regular occu- sanski Novi), Aleksander Sussmann (Derventa), rrence as a result of insufficient number of health Johann Stanislaus Niemczyk (Gračanica), Mi- facilities and qualified health personnel (1,2). The chael Hodža (Gradačac), Domenico de Mistura Turkish administration was deprived of an orga- (Jajce), Waclaw Babinski (Konjic), Rudolf Karl nized health service, everyone was allowed to Loebel (Maglaj), Wenzel Mikan (Mostar), Gavro practice medicine and prepare medications, with Peciković and Simon Zaloscer (Tuzla), Moritz the inherited and empirically acquired knowled- Kirtner (Županjac) also had the soda water produc- ge (3). The people were mostly focused on folk tion (6), Gustav Proche (Brčko) the liqueur factory doctors, self-taught healers, herbalists, and clergy and Karl Fürich (Foča) and Michael Finkelstein of all denominations. The new government found (Prnjavor) the grocery store (8). Despite all the only about five qualified doctors, but many Jewish difficulties, pharmacy in B&H began to develop doctors (hakims), Jewish and Muslim attars (tra- over the Austro-Hungarian period. As this was the ditional and herbal drug sellers), barbers, ranches, period when health conditions in B&H changed sorcerers, charms, who treated people and prepa- significantly and the modernization of medicine red medicines (3). Various medicines and spices and pharmacy began, a research was conducted in were sold for expensive money (4), and the tre- order to explore and present the apothecary activ- atment at that time can be more or less expressed ity for the period from 1878 until 1918. with “verbis, herbis et lapidibus” (5). The aim of this paper was to present the enacted The Austro-Hungarian administration immediately regulations governing the operation of pharmaci- approached the organization of the health service es over the Austro-Hungarian rule, by which the according to the canons that ruled in the Monarchy. pharmacy became a regulated profession with qu- This was not easy given the conservative habits of alified pharmacy staff providing a health service. the population, the limited financial resources needed to modernize the health service and the lack of MATERIALS AND METHODS qualified educated staff (1). Among many settlers in B&H there were qualified doctors and pharmaci- Materials and study design sts, who had taken treatment into their own hands. A retrospective and descriptive research was con- Since then, an increasing number of public phar- ducted at the Archives of the Republic of Srpska macies had started to open. Authorities sought to and the Museum of the Republic of Srpska du- combat quackery and banned such work (3). ring the period from November 2019 to August The people were treated for a long time according 2020 in order to present the regulations gover- to the old methods of folk medicine and pharmacy, ning the operation of pharmacies in Bosnia and and the attar shops survived for a long time despite

344 Marković-Peković V. Regulation of apothecary activity

Herzegovina over the Austro-Hungarian rule for the period from 1878 until 1918.

Methods The method of the qualitative secondary data analysis was applied.

The material available in its original form from Figure 1. An Order regulating the apothecary activity in Bos- the Archives of the Republic of Srpska and the nia and Herzegovina (Article 1, 1879) (9) (with permission of Museum of the Republic of Srpska, as well as the the Archives of the Republic of Srpska Banja Luka, Bosnia and Herzegovina) National and University Library of Bosnia and Herzegovina digital collections was used. Com- not meet this requirement had to be closed within pany registers, books (Administration Reports, three months of the publication of this Order. The Collections of Laws), address books and newspa- concession for the opening of the pharmacy was pers were used from these sources, as well as given by the Provincial Government, applying the other sources, as books, published papers in the principle that one pharmacy is needed per 10,000 journals and the Internet. The most important inhabitants. It was mostly received by pharma- enacted regulations that regulated the operation cists from the other parts of the Monarchy as of pharmacies over the observed period were pre- there were no locals. Preparation and dispensing sented chronologically. Firstly, an Austro-Hun- of medicines were allowed only to a master of garian Government’s order regulating the apothe- pharmacy or a pharmacy assistant with a tyroci- cary activity was presented as the first regulation nal exam. Medicines were allowed to be prepared enacted as early as in 1879, followed with an and dispensed only in accordance with the valid order on performing medical practice enacted in pharmacopoeia and the medicine tax. In order to the same year. The latter was briefly presented as determine the quality and availability of medici- it defined duties of other medical staff regarding nes, the inspection of pharmacies was conducted medicines preparation. The next regulation, the by the district doctor. In the same year, an Order Law on Pharmacies from 1907 regulated the pro- on performing medical practice was issued (10). fession in a more comprehensive way. Secondly, Only doctors, ranches, veterinarians, dentists and a Pharmacy Gremium established by the Law on midwives with an appropriate diploma obtained Pharmacies was presented as the first professio- in the Monarchy were allowed to practice me- nal organization. Thirdly, some other regulations dicine. Preparation and dispensing of medicines and prohibition orders pharmacies had to apply was the exclusive right of pharmacists, which were also briefly presented, as the introduction of the doctors and ranches could independently an adequate legislation was necessary to organize perform only with a special authorization. They a good health service. Finally, the changes in the received permission to run a home pharmacy number of pharmacy staff and pharmacies were from the Provincial Government only for a pla- shown, as the regulated profession in the country ce without a public pharmacy within two miles might also be a factor which enabled the increase of their residence place. Although this order did in professional staff through organized working not directly concern the pharmacies themselves, conditions in a certain social environment. it clearly defined the conditions for the preparation of medicines by other medical staff. The Au- RESULTS strian Pharmacopoeia was valid for all medical Regulations governing apothecary activity staff, starting with the 6th ed. from 1869, with an 1879 addendum (Figure 2) (11), the 7th ed. from Apothecary activity was regulated as early as 1890, the 8th ed. from 1906, also the last edition 1879 by the Order of the Provincial Government of "Pharmacopoeia Austriaca" in the Monarchy, (Figure 1) as a pharmacy craft (9). The pharmacy and the appendices from 1910 and 1916 (12,13). owner or provisor could only be a person with a doctor's degree in chemistry or a master's de- With the Law on Pharmacies adopted in 1907, gree in pharmacy obtained at an Austro-Hunga- after almost thirty years, the apothecary acti- rian university. All existing pharmacies that did vity was regulated more comprehensively and

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reliability in working in a pharmacy and at least five years of work in a pharmacy as an assistant. The competition for opening a new pharmacy was published in the official newspaper "Sarajev- ski list" (“Sarajevo Gazette”). A new concession was required if the pharmacy was legally or by inheritance transferred to another living person. It was not required in the case when a pharmacy was inherited by concession's owner widow or Figure 2. An Order determining the use of the Austrian Phar- children, or when the pharmacy heir was a pharmacopoeia (The 6th Ed., 1879) (11) (with permission of the Archives of the Republic of Srpska, Banja Luka, Bosnia and Her- macist, until he acquired the condition for an in- zegovina) dependent pharmacy operation. For example, af- the Pharmacy Gremium, a professional body of ter the death of Josef Oltvany from Derventa city masters of pharmacy, was established (Figure 3) (B&H), the pharmacy owner became the widow (14). The public pharmacy was considered as a Marie Oltvany (15), and after the death of Robert craft company, and a concession given by the Brammer from Banja Luka city (B&H) the owners Provincial Government was needed for opening became widow Jozefina and minor sons (16). One the pharmacy. The concession was personal and of them continued to run his father's pharmacy as tied only to a specific place where the doctor a pharmacist upon the same concession. performing practice had a permanent residence. The concession owner could exceptionally be When opening new pharmacies, attention had to allowed to operate a branch in a place with a be paid to the local need to enable the already temporary or occasional need for the medicines existing pharmacies in that place or the surroun- preparation and dispensing, like a seasonal phar- ding area to survive. macy in the spas. Such a pharmacy, operated during the bathing season in Ilidža (Sarajevo, B&H), was run by Stefan Dobóczky from 1898 to 1904 (6,7,17,18, 19-21), and Heinrich Schlezinger from 1908 to 1914 (8, 22-26), both pharmacy owners in Sarajevo. Doctors and veterinarians had the right to keep a home pharmacy if there was no public pharmacy in their place of residence, only until the pharmacy was opened. The Provincial Hospital, the Central Penitentiary in Zenica city (B&H) and the Sickness Foundation of the State Railways (B&H) were allowed to operate their own institutional pharmacies. Phar- macists Max Teich, a provisor, and Karl Anderle, an assistant, worked at the Provincial Hospital's pharmacy from 1898 to 1918 (6-8, 17-29). Max Teich was one of the extraordinary members of the Provincial Health Council in the period 1900- 1918 (7,8, 18-29). The Council gave an opinion Figure 3. The Law on Pharmacies in Bosnia and Herzegovina in all important health matters, including those (The first page, 1907) (14) (with permission of the Archives of related to the public pharmacies (30). the Republic of Srpska, Banja Luka, Bosnia and Herzegovina) Pharmacies were supervised by the Provincial The concession could only be granted to Bosni- Government through health authorities, and were an-Herzegovinian, Austrian or Hungarian citizens. inspected once a year. The pharmacy employed Those interested had to prove their personal abi- interns, with or without a tyrocinal exam, and lity, which meant having a master's or doctoral assistants, with a university diploma. Women degree in pharmacy from an Austrian or Hunga- could practice pharmacy under the same condi- rian university, full enjoyment of all civil rights,

346 Marković-Peković V. Regulation of apothecary activity

tions as men, but the permission for an indepen- The Pharmacy Gremium presidents were Edu- dent pharmacy operation had to be given by the ard Pleyel (in the period of 1910), Josef Patera Joint Ministry in Vienna, through the Provincial (1911-1913), Stevo Romčević (1919), vacant Government in Sarajevo. Pharmacy staff was not (1914-1918). The first deputies were Josef- Pa allowed to unfavourably comment the doctor and tera (1910), Eduard Pleyel (1911-1912) and He- his prescription, and had the obligation to keep inrich Schlesinger (1913-1914), all from Sara- professional secret. The pharmacist and his staff jevo. The second deputies were Martin Houška were forbidden any kind of quackery. (1910-1911) from Mostar, Tomo Mirković from Following the Law on Pharmacies, several im- Banja Luka (1912-1913), Ján Halla from Visoko plementing regulations were enacted. A detailed (1914), vacant (1916-1918). The treasurers were condition for operating the pharmacies, the ne- Heinrich Schlesinger (1910-1912) and Eduard cessary premises and equipment for medicines Pleyel (1913-1918), secretary Eustah Widen- preparation and dispensing were prescribed by hoffer (1910-1918) from Sarajevo and Gustav one (31). These premises included a dispensation Proche from Brčko (1918). Councillors without room, laboratory, medicine storage room, attic for a specific function were Gustav Proche (1910- storing herbal drugs, medicine cellar and inspec- 1917) and Theodor Heydušek from Bugojno tion room. The pharmacist was responsible for all (1910-1914) (22-29). medicines made or bought by him, and the manu- Other regulations that applied to pharmacies facturer for the specialties, cosmetics and other products sold in the original package. In additi- Some other regulations also applied to pharma- on to the pharmacopoeia and tax, the pharmacy cies in certain parts. Customs duties were paid had to have all the valid orders, gremial circulars on each imported commodity according to the and instructions, and professional journals. The tariff from the Law on General Customs Tariff pharmacist was allowed to dispense medicines in with customs price list (33). The customs duty emergencies when it was not possible to reach a on all medicines from the tariff class “Chemical doctor, which was not considered quackery. products, dyes, medicines and perfumes” was 24 forints, and for cosmetics 50 forints. All doctors Pharmacy Gremium and pharmacists were required to comply with The Pharmacy Gremium, with a headquarters in regulations relating to prescribing and prepa- Sarajevo (B&H), was established to represent ration of medicines at the expense of the state professional interests and assist the Provincial fund. Prescribing was limited to medicines nee- Government in relation to pharmacies. It consi- ded to treat or alleviate the disease sufficiently, sted of all public pharmacy owners and masters and to cheaper if more medicines were needed of pharmacy who worked in those pharmacies. (34). Pharmacists who wanted to procure tax-free The first session of the Gremium Assembly was brandy for pharmaceutical or scientific purposes held on January 29, 1908 (32). The governing had to have a special permit (35). The sale and body was the Board, consisted of a president, first purchase of poisons were subject to licensing and and second deputy, treasurer, secretary and two control, and was given exclusively to pharmaci- councillors without a specific function. They all sts and drugstore owners (36). had to be pharmacy owners, with the exception of The Provincial Government also issued orders the secretary, who had to be a graduated assistant. banning certain products from use, for example The president, who had to be from Sarajevo, re- Count Mattei's electro-homeopathic medicines presented the Gremium in public and managed (Figure 4) (37), all kinds of Brandt's Swiss pills the guild treasury. The members, confirmed by (38), Dr. Schiffman's asthma powder (39). These the Provincial Government, performed all functi- banns provide an insight into the various products ons free of charge. The Gremium revenues, con- sold here, though perhaps not all in the pharma- cession and membership fees, fines, gifts, inte- cies, but also the willingness of the authorities to rest, were used for administrative expenses, guild ban harmful products. From 1896, the import of library and to help poor pharmacists and widows saccharin, all other similar artificial sweeteners and children. and syrups containing saccharin or similar arti-

347 Medicinski Glasnik, Volume 18, Number 2, August 2021

Table 1. Pharmacy staff and number of pharmacies in the period from 1905 to 1918 Year Pharmacy staff 1905 1910 1914 1918 Owners 38 40 41 37 Tenants NA 3 4 4 Provisors 5 3 2 7 Graduated assistants 9 12 NA NA Interns 13 11 NA NA 1st year NA 6 NA NA 2nd year NA 4 NA NA 3rd year NA 1 NA NA Total 65 69 71 NA No. of pharmacies 44 47 47 48 No. of inhabitants per a pharmacy NA 41.261 40.384 NA NA, no information available

alth condition had significantly improved over time thanks to a well-organized network of health institutions and staff. In 1914, a total of 234 doctors, 141 midwives and 8 dentists worked in 17 hospitals (with 837 beds), 58 municipal dispensaries, and on average, there were 0.6 beds per inhabitant, 12,912 inhabitants per doctor and 16,222 per midwife. At Figure 4. A Circular on the import and sale ban of the count's the end of 1918, there were 48 operating pharma- Mattei electro-homeopathic medicine, 1886 (37) (with permis- cies distributed in 38 cities. In a forty-year period sion of the Archives of the Republic of Srpska, Banja Luka, Bos- 47 pharmacies were opened, and only a little over nia and Herzegovina) half of the urban settlements had a pharmacy (29). ficial sweeteners was banned (40). Pharmacists Women first appear in a report on pharmacy staff in and wholesalers of spice goods had to submit an 1909, one out of eight graduate assistants, and two application for import to the political area and out of six third year interns (44). The first modern register the shipment immediately upon receipt. European-style pharmacies were opened in Saraje- The Law introducing a monopoly on artificial vo, 1878, by Eduard Pleyel (3) and in Banja Luka, sweeteners (41) also applied to pharmacists. Dia- 1879, by Moritz Brammer (45). betics were allowed to pick up saccharin at the pharmacy upon a doctor's certificate, at the same DISCUSSION time withdrawing from the sugar supply (42). Du- The Austro-Hungarian government took the first ring the war period (1914-1918), orders banning steps in settling the difficult circumstances of the export of certain goods, including medicines earlier times at the very beginning of its rule by for humans, animals, medical instruments, vacci- organizing the health service according the laws nes, serums, and the like were issued. governed the Monarchy itself (1). This included Changes in the number of pharmacy staff and the enactment of the legislation for the regulati- pharmacies over this period on of the pharmacy and medicine practice on this territory. As early as in 1879, the orders stipulating Only one graduate pharmacist was found in 1878 the obligation of holding a university diploma to in B&H (30), and twenty years later there were practice pharmacy as well as to practice medicine forty masters of pharmacy who owned a pharmacy by other medical staff were enacted. This was a (6). At first, only one pharmacist worked in each novelty because during the Turkish rule there was pharmacy, but over time there were more staff, no such an obligation to have a formal education assistants and interns. Changes in the number of for preparation the medicines, and everyone was pharmacy staff and pharmacies were presented by allowed to practice mostly the inherited and em- Table 1, where an increase can be noticed (Table 1) pirically acquired knowledge (3). These were the (1,26,29,30,43). In 1910, the total pharmacies turno- initial steps in combating quackery, and raising the ver was 706,589 crowns and the number of doctor's quality of the health services and health educati- prescriptions was 43,576 (43). The population’s he- on of the population. Preparation and dispensing

348 Marković-Peković V. Regulation of apothecary activity

of medicines became the exclusive competence se this was the first organized association of phar- of pharmacists as formally educated health profe- macists with the task of representing professional ssionals, to which doctors and ranches were only interests, preserving and developing its community. exceptionally entitled by the approval of the Pro- Regulating the work of women in pharmacies was vincial Government (9,10). Such an approach can also a step forward, and in line with the modern be considered as good and rational, and probably practice at that time (48). Women were allowed to the only possible one when a very few graduated practice pharmacy, and until the end of this peri- pharmacists were available at the time. This excep- od, none owned a pharmacy. However, women tion, however, enabled the medicines availability graduate assistants and interns started to appear in to the population by a qualified health personnel the practice. All types of quackery were explicitly in places where there was no pharmacy. By these forbidden to pharmacy staff. During the entire pe- orders medicine and pharmacy practice were cle- riod the operation of pharmacies was regulated as a arly separated, which was not the case during the craft, rather than health activity, as it was, for exam- Turkish rule (3). The introduction of the obligation ple, in Croatia (49). Apothecary became a regulated to apply the medicine tax and pharmacopoeia was profession with educated and qualified personnel a major step forward, which guarantees the qua- who provided a service to the population. Intro- lity of the medicines and regulated prices. Prior duction of the necessary legislation by which the to the Austro-Hungarian rule, the population was operation of pharmacies was regulated, enabled the mostly treated by medicines and herbs prepared by arrival of a larger number of graduated pharmaci- self-taught healers, and there was a lot of quac- sts in the country, with the right and the obligations kery (1). The treatment procedures for various di- in the medicines production and dispensing they seases, prescriptions were passed from generation were entitled to by their education. The introduced to generation, and were later recorded by literate legislation, among other things, contributed to the people (46). These medicine recipes or a kind of increase in the number of pharmacies and qualified rulebooks were called “ljekaruše” by which the pharmacy staff, thus providing the necessary base people were mostly treated with, or with the me- for the organization of good pharmacy service in dical transcripts from other languages brought in towns and villages, and to the development and im- the country (Arabic manuscripts) (47). There were provement of the profession, health and social con- some written documents regarding treatment and ditions in the country during the Austro-Hungarian medicine preparations even then, providing the rule from 1878 to 1918. only possible quality and continuity level of the In conclusion, all enacted regulations had si- medicines use at that time. The introduction of the gnificantly changed the apothecary activity in mandatory use of pharmacopoeia and medicines B&H during the period from 1878 to 1918. They tax instantly raised medicines quality to the level contributed to the establishment, regulation and similar to the other parts of the Monarchy (13), development of modern apothecary activity and which could be provided only in the pharmacies professional service over the observed period, by the graduated pharmacists. These first regulati- and, thus, created the foundations for future de- ons were very valuable as they enabled graduated velopment of the profession. pharmacists to be the only quality, educated staff responsible for the medicine preparation, thus ra- FUNDING ising the quality of healthcare in the country and No specific funding was received for this study. started to combat quackery. Later the pharmacies were regulated in a more TRANSPARENCY DECLARATION comprehensive way, and the Pharmacy Gremium Conflicts of interest: None to declare. was established. This is extremely important becau-

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3. Đuričić A, Elazar S. Pregled istorije farmacije Bo- 14. Red za ljekarnice u Bosni i Hercegovini (Law on sne i Hercegovine (An overview of the history of Pharmacies in Bosnia and Herzegovina) [in Croati- pharmacy in Bosnia and Herzegovina) [in Serbian]. an]. Glasnik zakona i naredaba za Bosnu i Hercego- Sarajevo: Grafičar, 1958. vinu. Godina 1907. Sarajevo: Zemaljska štamparija, 4. Zajedničko ministarstvo financija. Izvještaj o upravi 1909: 254-73. Bosne i Hercegovine 1906 (Report on the admini- 15. Registar inokosnih firmi (Register of independent stration of Bosnia and Herzegovina 1906) [in Croa- companies) [in Croatian]. Okružni sud Banja Luka, tian]. Zagreb: Ministarstvo, 1906. p.78. 1883-1911. Ordinal No. 623. Banja Luka: Arhiv Re- 5. Zdravstvenost u Bosni i Hercegovini od 1878-1901. publike Srpske. (Health care in Bosnia and Herzegovina 1878-1901) 16. Registar firmi-društva (Register of socially-owned [in Croatian]. 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Wien: Aus der Kaiserlich-Königlichen Hof- und 30. Zajedničko ministarstvo financija. Izvještaj o upravi Staadsdruckerei, 1880: 112. Bosne i Hercegovine 1906 (Report on the Admini- 12. Okružnica od 23. decembra 1907., br. 206.884/I., stration of Bosnia and Herzegovina 1906) [in Croa- o provagjanju nekih odredaba dne 26. novembra tian]. Zagreb: Ministarstvo, 1906: 78-100. 1907. proglašenog Reda za ljekarnice u Bosni i Her- 31. Naredba Zemaljske vlade za Bosnu i Hercego- cegovini (Circular No. 11 of December 23, 1907, vinu od 4. aprila 1913., br. 144.302/I., kako da se No. 206.884/I., on the implementation of certain vode javne ljekarnice. (Order of the Provincial Go- provisions of the Law of Pharmacies in Bosnia and vernment for Bosnia and Herzegovina of 4 April Herzegovina proclaimed on November 26, 1907) 1913, No. 144.302/I., how to run public pharmacies) [in Croatian]. Glasnik zakona i naredaba za Bosnu [in Croatian]. Glasnik zakona i naredaba za Bosnu i Hercegovinu. Godina 1908. Sarajevo: Zemaljska i Hercegovinu. Godina 1913. Sarajevo: Zemaljska štamparija, 1909: 24-6. štamparija, 1913: 97-101. 13. Cletter K. The civil Pharmacopoeias of Austria. 32. Zajedničko ministarstvo financija. Izvještaj o upravi http://www.histpharm.org/ISHPWG%20Austria.pdf Bosne i Hercegovine 1908 (Report on the admini- (19 January 2021). stration of Bosnia and Herzegovina 1908) [in Croa- tian]. Zagreb: Ministarstvo; 1909: 23.

350 Marković-Peković V. Regulation of apothecary activity

33. Zakon od 25. maja 1882. tičući se sveobće carinske 39. Naredba zemaljske vlade za Bosnu i Hercegovinu tarife austro-ugarskog carinskog područja (Law of od 26. maja 1900., br 75.207/B.H., o zabrani uvo- 25 May 1882 concerning the universal customs tariff za praška protiv sipnje dra K. Šifmana (Order of the of the Austro-Hungarian customs territory) [in Cro- Provincial Government for Bosnia and Herzegovina atian]. Sbornik zakona i naredaba za Bosnu i Her- of 26 May 1893, No. 75.207/B.H., on the import ban cegovinu. Godina 1882. Sarajevo: Tisak Zemaljske of powder against asthma by Dr K. Šifman) [in Ger- tiskare, 1882: 341-78. man, Croatian]. Glasnik zakona i naredaba za Bosnu 34. Naredba zemaljske vlade za Bosnu i Hercegovinu od i Hercegovinu. Godina 1900. Sarajevo: Zemaljska 17. decembra 1885, broj 28211/I., kojom se izdaje štamparija, 1900: 256-57. pravilo ordinacije, po kom se svi ljekari i apotekari 40. Naredba zemaljske vlade za Bosnu i Hercegovinu vladati imaju, kad na račun zemaljskog erara ili jed- od 1. maja 1898., broj 61.638/II., o zabrani uvoza nog pod državnim nadzorom stojećeg fonda lijekove saharina i podobnih umjetnih slatkih materija, koje propisuju i pripravljaju (Order of the Provincial Go- pod drugim imenima u trgovinu dolaze, dalje šurupa, vernment for Bosnia and Herzegovina of 17 Decem- koji su s istim pomiješani (Order of the Provincial ber 1885, No. 28211/I, issuing the rule of the prac- Government for Bosnia and Herzegovina of 1 May tice, according to which all doctors and pharmacists 1898, No. 61.638/II, prohibiting the import of sacc- are governed, when at the expense of the terrestrial harin and other artificial sweet substances, as well era or one state fund under state supervision me- as syrups prepared therefrom) [in Croatian, Serbian]. dicines are prescribed and prepared) [in Croatian]. Glasnik zakona i naredaba za Bosnu i Hercegovinu. Zbornik zakona i naredaba za Bosnu i Hercegovi- Godina 1898. Sarajevo: Zemaljska štamparija, 1898: nu, Godina 1886. Sarajevo: Tisak Zemaljske tiskare, 72-3. 1886: 21-3. 41. Zakon od 18. jula 1917. kojim se uvodi monopol na 35. Naredba zemaljske vlade za Bosnu i Hercegovinu od umjetna sladila (Law of 18 July 1917 introducing 29. marta 1893, br. 29.427/II., o odredbama tičućim a monopoly on artificial sweeteners) [in Croatian]. se godišnje količine alkohola, koja se u slučaju pa- Glasnik zakona i naredaba za BiH. Godina 1918. Sa- ušaliranja smije dopustiti apotekarima da ju od po- rajevo: Zemaljska štamparija, 1919: 141-45. reza prosto upotrebljavaju u svrhe liječenja, kao i o 42. Saharin za diabetičare (Saccharin for diabetics) [in zabrani građenja nekih preparata od nedenaturirane, Croatian]. Sarajevski list 1918; No. 192, p. 2. od danka proste rakije (Order of the Provincial Go- 43. Zajedničko ministarstvo financija. Izvještaj o upravi vernment for Bosnia and Herzegovina of March 29, Bosne i Hercegovine 1911 (Report on the Admini- 1893, no. 29.427/II., on the provisions concerning stration of Bosnia and Herzegovina 1911) [in Croati- the annual amount of alcohol, which in the case of an]. Sarajevo: Ministarstvo, 1911: 27-32. a lump sum may allow pharmacists to simply use it 44. Zajedničko ministarstvo financija. Izvještaj o upravi for medical purposes, as well as on the prohibition of Bosne i Hercegovine 1909 (Report on the Admini- the preparation of some preparations of undenatured, stration of Bosnia and Herzegovina 1909) [in Croa- tax-exempt brandy) [in Croatian, Serbian]. Glasnik tian]. Sarajevo: Ministarstvo, 1910: 29-34. zakona i naredaba za Bosnu i Hercegovinu. Godina 45. Marković-Peković, V. Banjalučke javne apoteke 1890. Sarajevo, 1890: 167-70. u Austrougarskom periodu (Public Pharmacies in 36. Naredba zemaljske vlade za Bosnu i Hercegovinu od Banja Luka in the Austro-Hungarian Period). Gla- 24. maja 1899., br. 38.853/I, o prodaji i dobavljanju snik Udruženja arhivskih radnika Republike Srpske otrovnih tvoriva u Bosni i Hercegovini (Order of the 2011; 3:137-48. vanda.markovic-pekovic@med. Provincial Government for Bosnia and Herzegovi- unibl.org na of May 24, 1899, No. 38.853/I., on the sale and 46. Salihović H, Mašić I. Razvoj javnog zdravstva u supply of toxic substances in Bosnia and Herzegovi- BiH i Sarajevu (Development of Public Health in na) [in Croatian, Serbian]. Glasnik zakona i nareda- B&H and Sarajevo) [in Bosnian]. Med Arh 2006; ba za Bosnu i Hercegovinu. Godina 1899. Sarajevo: 60:333-6. Zemaljska štamparija, 1899: 130-35. 47. Mašić I. One hundred fifty years of organized- he 37. Okružnica zemaljske vlade za Bosnu i Hercegovi- alth care service in Bosnia and Herzegovina Med nu od 23. aprila 1886, broj 19978/II., obznanjuje da Arch 2018; 72:374–88. [email protected] je zabranjeno uvoziti elektro-homeopatičke ljeka- 48. Naredba kr. hrv.-slav.dalm. zemaljske vlade, odje- rije grofa Mattei-a (Circular of the Provincial Go- la za bogoštovlje i nastavu od 18. listopada 1901. vernment for Bosnia and Herzegovina of 23 April broj 16.074 u pogledu pripusta ženskinja k ljekar- 1886, No. 19978/II, declares that it is banned to ničkom zvanju u kraljevinama Hrvatskoj i Slavoniji import the electro-homeopathic medicines of Co- (Order of the Provincial Government, Department unt Mattei) [in German, Croatian]. Zbornik zakona of Worship and Instruction, dated 18 October 1901, i naredaba za Bosnu i Hercegovinu. Godina 1886. No. 16,074 regarding the admission of women to Sarajevo: Tisak zemaljske tiskare, 1886: 809. the medical profession in the kingdoms of Croatia 38. Naredba zemaljske vlade za Bosnu i Hercegovinu and Slavonia) [in Croatian]. Österreichische Na- od 21.jula 1894., br. 80.590/II., kojom se zabranjuje tionalbibliothek. ALEX Historischen Rechts- und uvažanje Brantovih švajcarskih pilula svake vrste Gesetzestexten. http://alex.onb.ac.at/cgi-content/ale (Order of the Provincial Government for Bosnia and x?aid=lks&datum=19010304&seite=00000643 (16 Herzegovina of July 21, 1894, No. 80.590/II., which February 2021) bans the import of Brant's Swiss pills of any kind) 49. Zakon o ljekarničtvu od 11. travnja 1894 (Pharmacy [in Croatian, Serbian]. Glasnik zakona i naredaba za Law of April 11, 1894) [in Croatian]. Liečnički Bosnu i Hercegovinu, Godina 1894. Sarajevo: Ze- viestnik 1894; 6, prilog. https://library.foi.hr/m8/ maljska štamparija, 1894: 214. S01101/1894/1894_00015.pdf (16 February 2021)

351 ORIGINAL ARTICLE

Fasting state requirements for blood sampling: a survey of patients in Cantonal Hospital Zenica, Bosnia and Herzegovina

Damira Kadić1, Aida Avdagić-Ismić1, Sabaheta Hasić2, Ferhana Bošnjak1

1Department of Medical Biochemistry and Immunology Diagnostics, Cantonal Hospital Zenica, Zenica, 2Department of Medical Bio- chemistry, School of Medicine, University of Sarajevo, Sarajevo; Bosnia and Herzegovina

ABSTRACT

Aim To explore patient’s awareness and appliance of the fasting state requirements for blood sampling.

Methods This observational survey was performed at the Depar- tment of Medical Biochemistry and Immunology Diagnostics, Cantonal Hospital Zenica, from June to July 2019. An anonymous questionnaire was conducted on 200 consecutive outpatients older than 18, who were admitted to the laboratory for routine blood testing. Corresponding author: Damira Kadić Results A total of 134 (67%) patients were informed that they needed to be at fasting to perform laboratory tests. Patients were Department of Medical Biochemistry and mostly informed by a requesting physician or a nurse, 68 (50.8 Immunology Diagnostics, %), and by other patients, members of the family and friends, 58 Cantonal Hospital Zenica (43.3%); only seven (5.2%) patients were informed in the labora- Crkvice 67, 72 000 Zenica, tory. A total of 75 (37.5%) patients arrived to the laboratory pro- Bosnia and Herzegovina perly prepared. Phone: + 387 32 447 257; Conclusion Most patients were not well informed about fasting E-mail: [email protected] state requirements for blood sampling and consequently they were ORCID ID: https://orcid.org/0000-0001- not adequately prepared for laboratory tests. Laboratory should 5860-9896 establish updated fasting recommendations available to patients and healthcare professionals, and conduct continuing education of patients and health care staff.

Key words: blood specimen collection, diagnostic errors, patient Original submission: safety, pre-analytical phase 27 January 2020; Revised submission: 11 February 2021; Accepted: 12 April 2021 doi: 10.17392/1347-21

Med Glas (Zenica) 2021; 18(2):352-356

352 Kadić et al. Fasting state requirements

INTRODUCTION dation about blood sampling for laboratory tests exists, but not on-line or on leaflets. Blood sam- Patient safety is one of the most important ples are taken in the morning, from 8-10 AM. challenges in healthcare. From the laboratory The previous evening, after 8 PM, patient should perspective, pre-analytical errors make the lar- not eat food and drink beverages (except water), gest contribution to the overall rate of diagno- or consume alcohol and cigarettes. stic and therapeutic errors (1,2). Pre-analytical phase is the main source of errors in labora- The aim of this study was to investigate patient’s tory testing procedures (3-5) and blood sample awareness and appliance of fasting state requi- collection for laboratory tests is a crucial pre- rements for blood sampling to gain insight into analytical activity. Errors occurring before this the compliance with EFLM WG-PA fasting repre-analytical step may impair sample quality commendations in Cantonal Hospital Zenica. and patient test results (6). These data will serve to all health professionals to understand the need for active clarification of Adequate preparation of the patient is the most fasting requirements to patients in order to pre- important step for ensuring the sample quality. vent spurious laboratory test results. Blood sampling in a non-fasted state, even a sugar-free gum chewing, may have an impact PATIENTS AND METHODS on the laboratory test results (7-10). Patients should be in the fasting state before blood sam- Patients and study design pling for most biochemical, haematological and coagulation analyses. Some biochemical, This observational survey was performed du- haematological and coagulation analytes are ring a 2-month period, from June to July 2019 changed directly, due to various metabolic and at the Department of Medical Biochemistry and hormonal mechanisms in non-fasting state. Immunology Diagnostics, Cantonal Hospital Ze- Other analytes are changed indirectly, due to nica (Zenica, Bosnia and Herzegovina), serving interference caused by lipemia, during the me- general and specialized clinical chemistry, hae- asurement of an analyte. Postprandial response matology, immunology and coagulation testing depends on a number of factors, such as fasting services. The laboratory is not accredited by the duration, eating behaviour, smoking, coffee and ISO 15189 standard. alcohol consumption. Therefore, it is of crucial An anonymous questionnaire (Tables 1-3) was importance to control as many of these factors conducted on a consecutive sample of 200 out- as possible in order to prevent spurious labora- patients older than 18 years, who were admitted tory test results (11-14). to the laboratory in the morning between 8-10 h Unfortunately, there is still grate heterogeneity AM for routine blood testing. All patients were in the fasting state definition in health care insti- informed about the survey and consented to par- tutions as well as in the scientific literature (12). ticipate in the survey. The standardization of fasting requirements, The research was done respecting ethical stan- among other preanalytical activities, is most im- dards of the Declaration of Helsinki. The study portant for mitigating the impact of pre-analyti- approval was obtained from the Ethics Com- cal variability (6). The European Federation of mittee of Cantonal Hospital Zenica. Clinical Chemistry and Laboratory Medicine Methods Working Group on Preanalytical Phase (EFLM WG-PA) recommends that fasting state require- Patients were interviewed by a specialist of the ments include 12 hours fasting state and water medical biochemistry. A 12-question survey con- ad libitum prior to blood sampling. Fasting in- tained data about patients’ demographics, degree structions also include abstinence from alcohol of education and the use of internet as a source a day before blood sampling, and abstinence of medical information, how often do they draw from coffee, tea and smoking intake in the mor- blood for laboratory tests, are they currently at ning before blood sampling (15). fasting, are they informed that they need to be In Cantonal Hospital Zenica, a written recommen- at fasting to perform laboratory tests, are they informed that consumption of food or beverages

353 Medicinski Glasnik, Volume 18, Number 2, August 2021

before laboratory tests affects their results; the vely (it affects only some test results). However, patients were asked to explain the term fasting when they were asked about the correct defini- state, what they consumed before blood sampling tion of the fasting state (at least 12 hours must in the case that they declared about non-fasting, pass from the last meal to blood sampling, it and if they consumed alcohol and cigarettes. A is allowed to drink water) 35 (17.5 %) patients questionnaire was developed by authors of this answered affirmatively. Almost half, 86 (43%) study in accordance with similar studies. patients believed that it was enough to take the last meal at any time on the day preceding the Statistical analysis blood sampling; 36 (18%) patients thought that Frequency of each answer given in the questi- they could consume light breakfast or coffee and onnaire was calculated. Data were shown in the tea before blood sampling (Table 2). table as absolute numbers and percentages. Table 2. Patients’ knowledge about the fasting state require- RESULTS ments Question No (%) of patients A total of the 200 outpatients enrolled in the stu- Are you informed that you need to be fasting to perform labora- dy (Table 1). When asked, 134 (67%) patients tory tests? NO 66 (33.0) reported that they were informed that fasting is YES (a way of information) 134 (67%) required to perform laboratory tests, of which 68 In laboratory verbally 6 (3.0) (50.8%) were informed by requesting an physi- In laboratory by written instruction 1 (0.5) By doctor 47 23.5) cian or nurse and 58 (43.3%) by others (other By nurse 21 (10.5) patients, members of the family, friends). Seven By internet 1 (0.5) (5.2%) patients were informed in the laboratory By other persons 58 (29.0) Does consuming food or beverages before laboratory tests affect and one (0.7%) from the Internet (Table 2), alt- your results? hough 101 (50. 5 %) patients stated that they NO 31 (15.5) used the Internet as the source of medical infor- YES 169 (84.5) for all tests 46 (23.0) mation (Table 1). only some tests 123 (61.5) What does the fasting state mean? Table 1. Characteristics of 200 outpatients From the last meal to blood sampling at least 35 (17.5) 12 hours must pass, it is allowed to drink water Characteristic No (%) of patients From the last meal to blood sampling at least 17 (8.5) Gender 12 must pass, it is not allowed to drink water Males 65 (32.5) From the last meal to blood sampling at least 11 (5.5) Females 135 (67.5) 10 hours must pass, it is allowed to drink water Age group From the last meal to blood sampling at least 10 2 (1.0) <25 years 16 (8.0) hours must pass, it is not allowed to drink water 25-49 years 43 (21.5) From the last meal to blood sampling at least 8 (4.0) 50-65 years 78 (39.0) 8 hours must pass, it is allowed to drink water >65 years 63 (31.5) From the last meal to blood sampling at least 8 5 (2.5) Degree of education hours must pass, it is not allowed to drink water Elementary school 49 (24.5) The last meal was taken the day before, the 86 (43.0) High school 113 (56.5) exact time does not matter College 31 (15.5) In the morning light breakfast can be con- Other 7 (3.5) 33 (16.5) sumed Use of internet as a source of medical information In the morning coffee or tea can be consumed 3 (1.5) YES 101 (50.5) NO 99 (49.5) How often do you draw blood for laboratory tests? Half of the patients, 102 (51%) stated that they First time 1 (0.5) were currently fasting. However, when they were Weekly 3 (1.5) Monthly 36 (18.0) asked what they had consumed before blood Half-yearly 61 (30.5) sampling, it turned out that 75 (37.5%) patients Yearly 29 (14.5) arrived to the laboratory properly prepared, i.e. Sporadically 70 (35.0) their last meal was 12 hours before blood sam- When they were asked if consuming food or pling; 80 (39%) patients consumed coffee or tea beverages before laboratory tests affected their and cigarettes in the morning, before blood sam- results, 123 (61.5 %) patients answered affirmati- pling (Table 3).

354 Kadić et al. Fasting state requirements

Table 3. Patient appliance to fasting state requirements were informed, 68.0% were informed by the he- Question No (%) of patients althcare professionals, and 11.9% knew that the 12- Are you currently fasting? hour fasting period was required. Although not all YES 102 (51.0) NO 43 (21.5) patients received information about fasting, 22% I do not know 55 (27.5) fasted for 12 hours (16). The results of the study of What did you consume before blood sampling? Sareen at al. (India) performed on 200 outpatients The last meal consumed 12 hours before 75 (37.5) The last meal consumed 10 hours before 6 (3.0) revealed a lack of knowledge and awareness about The last meal consumed 8 hours before 13 (6.5) the fasting requirements for blood glucose estima- Coffee or tea in the morning 45 (22.5) tion: 69% knew that 12-hour fasting was required Water 1 (0.5) for a test, their major source of instructions for la- Other 51 (25.5) I did not pay attention 9 (4.5) boratory testing was a requesting physician and nu- Did you consume alcohol? rse (73%). Very small amount of information was NO 188 (94.0) obtained from the laboratory (17). YES 12 (6.0) Within 24 hours before blood sampling 6 (3.0) The results of the study of Hepburn at al. (United More than 24 hours before blood sampling 6 (3.0) Kingdom and Republic of Ireland) performed on Did you consume cigarettes? NO 147 (73.5) 235 outpatients also revealed that majority of pa- YES 53 (26.5) tients were not informed or informed well about The day before blood sampling 18 (9.0) fasting state requirements; a total of 103 patients In the morning, before blood sampling 35 (17.5) did not receive any information about preparing DISCUSSION for blood sampling, none of the patients was in- To our knowledge, this is the first study evalua- formed about the need to fast for 12 hours (18). ting these pre-analytical requirements in any la- A crucial issue in the laboratory medicine is to boratory in Bosnia and Herzegovina. Our study assure the quality throughout the whole total te- showed that not all patients were informed about sting process (TTP) (19). Quality of the prea- fasting state requirements for blood sampling, nalytical phase as a part of TTP is the responsi- Although the majority of them were informed bility of the laboratory staff, even though many about that, they were still not well acquainted pre-analytical steps are performed by the non-la- with the correct definition of the fasting state. boratory staff (3). Although it is too late for pa- The major source of the instructions for labora- tients coming for the first time to be informed in tory testing were a requesting physician and nu- the laboratory, it would still be of great importance rse, then other people (other patients, members because our study showed that most of the patients of the family, friends). Unfortunately, very small come to the laboratory on a regular basis. Labora- amount of information was obtained from labo- tory staff should be more active in clarifying fa- ratory staff. The least amount of information was sting requirements to the patients. Despite the lack obtained from the Internet. of official written recommendations of national Our study also revealed that most patients were scientific communities, laboratories, even non- not adequately prepared for laboratory tests. But accredited, should have updated instructions for still, even though patients were not well enough the patient preparation for laboratory tests. informed about fasting requirements, they were Although we obtained results similar to other mostly aware of its relevance and the fact that studies, our study showed that only written re- consuming food or beverages before laboratory commendations inside our hospital are not enou- tests affected their results. This is probably the gh, and that we should provide those recommen- reason why twice as many patients then those dations in the form of leaflets as well as on-line. who knew proper definition of the fasting state Those should provide patients as well as physi- still came to the laboratory adequately prepared. cians and nurses with information for fasting Similar to our findings, López-Garrigós et al. (Spa- requirements, with clear and understandable in- in) found in their survey of 254 patients who atten- structions for the preparation of the patients for ded a blood sampling that substantial proportion laboratory tests. Since the instructions for labora- (27.6%) of patients did not receive any information tory testing are obtained by patients mainly from about fasting prior to the analysis; from those who physicians and nurses, laboratory professionals

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should be proactive in organizing educational se of EFLM in the absence of official written na- meetings for non-laboratory health professionals tional recommendations. And finally, laboratory to disseminate knowledge about the importance professionals should conduct continuous educa- of fasting requirements in preventing spurious tion of patients and healthcare staff in order to laboratory test results. reduce preanalytical errors. In conclusion, we detected that most patients FUNDING were not well informed about fasting state requirements for blood sampling and were not adequ- No specific funding was received for this study. ately prepared for laboratory tests. The study results point to the need for each laboratory to TRANSPARENCY DECLARATION establish its own updated, visible and available Competing interests: None to declare. fasting recommendations in accordance with tho-

REFERENCES 1. Cornes M, Ibarz M, Ivanov H, Grankvist K. Blood 11. Bajaña W, Aranda E, Arredondo ME, Brennan- sampling guidelines with focus on patient safety and Bourdon LM, Campelo MD, Espinoza E, Flores S, identification - a review. Diagnosis (Berl) 2019; Ochoa P, Vega V, Varela B, Lima-Oliveira G. Im- 6:33-7. pact of an Andean breakfast on biochemistry and 2. Cornes MP, Church S, van Dongen-Lases E, Gran- immunochemistry laboratory tests: an evaluation on kvist K, Guimarães JT, Ibarz M, Kovalevskaya S, behalf COLABIOCLI WG-PRE-LATAM. Biochem Kristensen GB, Lippi G, Nybo M, Sprongl L, Su- Med 2019; 29:020702. marac Z, Simundic AM; Working Group for Prea- 12. Koscielniak BK, Charchut A, Wójcik M, Sztefko K, nalytical Phase (WG-PRE) and European Federati- Tomasik PJ. Impact of fasting on complete blood on of Clinical Chemistry and Laboratory Medicine count assayed in capillary blood samples. Lab Med (EFLM). The role of European Federation of Cli- 2017; 48:357-61. nical Chemistry and Laboratory Medicine Working 13. Benozzi SF, Unger G, Campion A, Pennacchiotti Group for Preanalytical Phase in standardization and GL. Fasting conditions: influence of water intake harmonization of the preanalytical phase in Europe. on clinical chemistry analytes. Biochem Med 2018; Ann Clin Biochem 2016; 53:539-47. 28:010702. 3. Sonmez C, Yıldız U, Akkaya N, Taneli F. Preanalyti- 14. Arredondo ME, Aranda E, Astorga R, Brennan- cal phase errors: experience of a central laboratory. Bourdon LM, Campelo MD, Flores S, Medel C, Cureus 2020; 12:e7335. Manríquez I, Ochoa P, Varela B, Salinas CV, Lima- 4. Lippi G, von Meyer A , Cadamuro J, Simundic AM. Oliveira G. Breakfast can affect routine hematology Blood sample quality. Diagnosis (Berl) 2019; 6:25-31. and coagulation laboratory testing: an evaluation on 5. Lima-Oliveira G, Volanski W, Lippi G, Picheth G, behalf of COLABIOCLI WG-PRE-LATAM. TH Guidi GC. Pre-analytical phase management: a re- Open 2019; 3:e367-76. view of the procedures from patient preparation to 15. Grankvist K, Sigthorsson G, Kristensen GB, Pelan- laboratory analysis. Scand J Clin Lab Invest 2017; ti J, Nybo M. Status on fasting definition for blood 77:153-63. sampling in the Nordic countries - time for a har- 6. Giavarina D, Lippi G. Blood venous sample collec- monized definition. Scand J Clin Lab Invest 2018; tion: recommendations overview and a checklist to 78:591-4. improve quality. Clin Biochem 2017; 50:568-73. 16. López-Garrigós M, Flores E, Bourahel Y, Leiva-Sali- 7. Mrazek C, Lippi G, Keppel MH, Felder TK, Ober- nas P, Ortega-Lamaignere M, Puerta MJ. Patient pre- kofler H, Haschke-Becher E, Cadamuro J. Errors paration prior to the blood test: importance in qua- within the total laboratory testing process, from test lity of the results. J Healthc Qual Res 2020; 35:56-8. selection to medical decision-making - a review of 17. Sareen R, Kapil M, Gupta GN. Blood glucose fa- causes, consequences, surveillance and solutions. sting – harmonization of variables affecting pre Biochem Med 2020; 30:020502. analytical phase. J Fam Med Forecast 2019; 2:1014. 8. Stonys R, Banys V, Vitkus D, Lima-Oliveira G. Can 18. Hepburn S, Jankute M, Cornes MP, Rico Rios N, chewing gum be another source of preanalytical varia- Stretton A, Costelloe SJ. Survey of patient percep- bility in fasting outpatients? EJIFCC 2020; 31:28-45. tion of pre-analytical requirements for blood testing 9. Clinical Laboratory Standards Institute. Procedures in the UK and RoI. Ann Clin Biochem 2020; Online for collection of diagnostic blood specimens by ve- ahead of print. nipuncture. CLSI document GP41. CLSI: Wayne, 19. Lippi G, Baird GS, Banfi G, Bölenius K, Cadamu- PA; 2017. ro J, Church S, Cornes MP, Dacey A, Guillon A, 10. Simundic AM, Bolenius K, Cadamuro J, Church S, Hoffmann G, Nybo M, Premawardhana LD, Sa- Cornes MP, van Dongen-Lases EC, Eker P, Erdelja- linas M, Sandberg S, Slingerland R, Stankovic A, novic T, Grankvist K, Guimaraes JT, Hoke R, Ibarz Sverresdotter SM, Vermeersch P, Simundic AM. M, Ivanov A, Kovalevskaya S, Kristensen GBB, Li- Improving quality in the preanalytical phase throu- ma-Oliveira G, Lippi G, von Meyer A, Nybo M, Salle gh innovation, on behalf of the European Federati- B, Seipelt C, Sumarac Z, Vermeersch P. Joint EFLM- on for Clinical Chemistry and Laboratory Medicine COLABIOCLI Recommendation for venous blood (EFLM) Working Group for Preanalytical Phase sampling. Clin Chem Lab Med 2018; 56: 015-38. (WG-PRE). Clin Chem Lab Med 2017; 55:489-500.

356 ORIGINAL ARTICLE

Effect of atorvastatin on systolic and diastolic function in patients with heart failure with reduced ejection fraction (HFrEF)

Faris Zvizdić1, Edin Begić2, Mirza Dilić3, Šekib Sokolović1, Orhan Lepara4

1Department of Cardiology, Clinic for Heart, Blood Vessel and Rheumatic Diseases, Clinical Centre, University of Sarajevo, 2Department of Cardiology, General Hospital "Prim. Dr. Abdulah Nakaš", Sarajevo, 3School of Medicine, University of Sarajevo, 4Department of Physi- ology, School of Medicine, University of Sarajevo; Sarajevo, Bosnia and Herzegovina

ABSTRACT

Aim To investigate the benefit of high-dose lipophilic statin therapy on cardiac remodelling, function and progression of heart failure (HF) in patients with ischemic heart disease.

Methods A total of 80 patients with ischemic HF diagnosis were followed during 6 months, and they were divided in two groups. First group (n=40) was treated by high-dose lipophilic statin therapy (atorvastatin 40 mg) and conventional therapy for HF, while the second group (n=40) had no atorvastatin in the therapy.

Results In the beginning of study, from all of the observed parameters, only the ratio of flow rates in early and late diastole (E/A Corresponding author: ratio) differed between the test groups (p=0.007). After six months, Edin Begić a statistically significant increase in left ventricular end-diastolic Department of Cardiology, diameter (LVIDD) in patients who had not been treated with ator- General Hospital “Prim.dr Abdulah Nakaš“ vastatin was found. In the patients treated with atorvastatin, there was a significant reduction in basal right ventricle diameter in dia- Sarajevo, Bosnia and Herzegovina stole and systole (p<0.001 and p<0.001, respectively), and in tri- Kranjčevićeva 12, 71000 Sarajevo, cuspid annular plane systolic excursion (TAPSE) (p<0.001); there Bosnia and Herzegovina was a reduction in LVIDD (p<0.001), and an increase of ejection Phone: +387 33 285 100; fraction of the left ventricle according to Teicholtz and Simpson Fax: +387 33 285 370; (p<0.001 and p<0.001, respectively). Also, there was an increase E-mail: [email protected] of deceleration time of early diastolic velocity (DTE) (p<0.05) and a decrease of isovolumic relaxation time (IVRT) (p<0.001). ORCID ID: https://orcid.org/0000-0001- 6842-262X Conclusion The reduction in the right and left ventricle diameters was noted after the six-month atorvastatin therapy. Atorvastatin in the therapy resulted in increased EFLV and better systolic function Original submission: and should be a part of a therapeutic modality of HF. 16 April 2021; Key words: heart failure, hydroxymethylglutaryl-CoA reductase Revised submission: inhibitors, therapeutics 18 May 2021; Accepted: 26 May 2021 doi: 10.17392/1388-21

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INTRODUCTION tin, pravastatin) in relation to tissue selectivity (21). The question arises about the effect and a Heart failure (HF) is a clinical entity characte- choice of statins on the heart muscle in patients rized by structural and/or functional cardiac ab- diagnosed with HF. Atorvastatin, as a lipophilic normalities (1). Based on ejection fraction of statin, shows potential benefit in patients diagno- the left ventricle (EFLV) it is divided into HF sed with chronic HF (10), but still with a lot of with preserved ejection fraction (HFpEF), where questions in patients who have been verified with EFLV is ≥ 50%, HF with mid-range ejection frac- ischemic heart disease in addition to HF. tion (HFmrEF) with EFLV in the range of 40%- 49%, and as HF with reduced ejection fraction The aim of this study was to investigate the bene- (HFrEF) and EFLV <40% (1). fit of high-dose lipophilic statin therapy on cardiac remodelling, function and progression of HF The question of the use of statins in patients with in patients with ischemic HF. HF is raised, and their effect is already well known in the primary and secondary prevention of athe- PATIENTS AND METHODS rosclerotic cardiovascular disease (2,3). Statins in- hibited the activity of 3-hydroxy-3-methylglutaryl Patients and study design coenzyme A reductase, and are primarily used in the treatment of hyperlipidaemia (2). They have This prospective, clinical, and controlled study a lipid lowering effect, pleiotropic effects, impro- involved 80 patients, who received high-dose li- vement of endothelial function, anti-inflammatory pophilic statin therapy between January and June effect, immunomodulatory effect and anti-throm- 2020. According to New York Heart Association botic effect (2). It seems natural that the pleiotropic (NYHA) heart failure classification (10) all pati- effects of statins may play a useful role in patients ents belonged to class II, III, and IV. Criteria for with HF (2-4). It is known that in the patients with inclusion were verified HF of ischemic etiology, higher risk for a cardiovascular event, a benefit of ejection fraction of the left ventricle (EFLV) ≤ statins will be greater. In patients with HF with 40%, and due to the lipid profile, the patients diabetes and hypertension, statins will be more who did not need statin therapy. Exclusion crite- effective than in those who do not have these risk ria were: lipophilic statins therapy for more than factors (2-4). Statins have immuno-modulatory and 3 months before the start of the study, heart fa- anti-inflammatory effects, and they can induce an- ilure of non-ischemic origin, severe liver or kid- ti-atherosclerotic effects regardless of their antilipe- ney dysfunction, occurrence of statin side effects mic action (4). Statins have an effect on endothelial (increase in transaminases four times more than dysfunction, which may contribute to vascular re- reference range, muscle pain). modelling (5). The benefit of statin therapy in the Patients with ischemic heart failure were deter- process of atherosclerosis is well established (6-10). mined according to the previous clinical findings The use of statins is a part of routine premedica- of myocardial infarction or already by cardiac tion prior to primary percutaneous coronary in- catheterization, in the period of two years befo- tervention (pPCI) (11,12). High doses of statins re research, verified coronary artery disease with (atorvastatin 80 mg or rosuvastatin 40 mg), when stenosis over 50%. Patients were divided into administered prior to pPCI in patients with STE- two groups: the first group (n=40) was treated by MI are positive predictors of outcome, and they a high-dose lipophilic statin therapy (atorvasta- are associated with reduced occurrence of major tin 40 mg) and conventional therapy (angiotensin adverse cardiovascular events (MACE) (12-15) converting enzyme inhibitors or angiotensin II reducing infarct size by increasing myocardial receptor blockers, mineralocorticoid receptor an- microvascular perfusion (16-18). The statins si- tagonists, beta blockers) for HF, while the second gnificantly reduce the level of total cholesterol (n=40) had no atorvastatin in therapy. and LDL, and increase the level of high-density An informed consent was obtained from all pati- lipoproteins (HDL) (19,20). ents following an explanation of the purpose of Statins are divided into lipophilic (atorvastatin, the study. An ethical approval was obtained from simvastatin, lovastatin, fluvastatin, cerivastatin the Ethics Committee of Clinical Centre Univer- and pitavastatin) and hydrophilic (rosuvasta- sity of Sarajevo.

358 Zvizdić et al. Lipophilic statins in heart failure

Methods which an incorrect distribution was found. For comparative analysis of dependent numerical va- All the patients were undergoing transthoracic riables, a paired t-test, i.e. the corresponding non- echocardiographic examination (TTE) at the be- parametric test (Wilcoxon test) was used. In the ginning of the study and after 6 months. Two-di- analysis of the dependence between the category mensional TTE was performed at the beginning variables, χ2 exact test was used. Statistical level and 6 months after the inclusion of the high-dose of 95% (p<0.05) was taken as significant. lipophilic statin therapy. The left and right ven- tricles were displayed in a parasternal longitudinal RESULTS section (long axis view), a parasternal short section (short axis view) and in an apical four-chamber The average age of the patients with HF who section (four chamber view) to measure the ejecti- were treated with high-dose atorvastatin therapy on fraction. The EFLV was measured in two ways, was 69.07±8.53 years, while the patients with HF by Teicholz and by Simpson. The function of the who were not treated with atorvastatin therapy right ventricle (EFRV) was measured by the tri- were 71.81±7.73 years old (p=0.128). In the gro- cuspid annular plane systolic excursion (TAPSE). up of patients who were treated with atorvastatin, The analysis of trans mitral flows was performed 31 (77.5%) were males, while nine (22.5%) were in the apical section of four cavities by placing a females. In the group of patients who were not two-millimetre volume Doppler sample above treated with atorvastatin, 32 (80%) were males, the peaks of open mitral cusps during diastole. while eight (20%) were females. By the Doppler parameters of the mitral flow, the In the beginning of the study, from all of the obser- following was measured: flow rates in early and ved parameters, only the E/A ratio differed betwe- late diastole and their ratio (E, A, E/A, respecti- en the groups (p=0.007). After six months, there vely), deceleration time of early diastolic velocity was a statistically significant increase in the left (DTE) and isovolumic relaxation time (IVRT). ventricular end-diastolic diameter (LVIDD) in patients who are not treated with atorvastatin. Other Statistical analysis parameters did not differ significantly (Table 1). The Shapiro-Wilk test was used to test the si- In patients treated with atorvastatin, there was a significance of the difference in deviation from gnificant reduction in basal right ventricle diameter the normal distribution. For the comparative in both diastole and systole (p<0.001 and p<0.001, analysis of independent numerical variables, a t- respectively), in TAPSE (p<0.001), and a reducti- test was used for variables that met the conditions on in LVIDd (p<0.001), as well as an increase of for application, i.e. an appropriate non-parame- ejection fraction of the left ventricle according to tric test (Mann-Whitney U test) for variables in Teicholtz and Simpson (p<0.001 and p<0.001,

Table 1. Basal values of echocardiography parameters of patients with heart failure at the beginning and after 6 months without and with the atorvastatin 40 mg treatment Not treated (n=40) Treated (n=40) Parameter p p Basal After 6 months Basal After 6 months Basal right ventricle diameter (cm)* in diastole 3.55 (3.3-3.9) 3.55 (3.3-3.9) 0.166 3.7 (3.3-3.9) 3.4 (3.0-3.5) <0.001 in systole 3.1 (2.8-3.5) 3.05 (2.8-3.5) 0.166 3.35 (2.9-3.5) 2.9 (2.55-3.0) <0.001 Right ventricle function* TAPSE (mm) 13 (10.75-14) 13 (10-14) 0.286 12 (9.25-14) 16 (14.5-17.5) <0.001 Basal left ventricle diameter (cm) † LVIDD 5.97±0.49 6.01±0.49 0.002 6.02±0.62 5.60±0.61 <0.001 LVIDS 5.23±0.58 5.26±0.60 0.046 5.29±0.63 4.89±0.63 <0.001 Ejection fraction of left ventricle (%)* Teicholtz 31 (30-36.75) 31.5 (28-38.5) 0.730 34 (30-36.75) 40.0 (37-44.5) <0.001 Simpson 34 (30-40) 32 (30-40) 0.017 34 (30-38) 42 (38-47) <0.001 Other parameters E/A ratio* 0.6 (0.6-0.7) 0.6 (0.6-0.7) 1.0 0,73±0.12 0.73±0,12 1.0 DTE (ms)* 290 (280-310) 291 (280-310) 0.082 294 (290-310) 296 (286-310) 0.031 IVRT) (ms)† 113.32±9.22 113.77±9.34 0.261 116.60±7.04 116.39±7.74 <0.001 *median and interquartile range (25-75 percentile); †mean ± standard deviation (± SD); TAPSE, Tricuspid annular plane systolic excursion; LVIDD, left ventricular end-diastolic diameter; E/A ratio - flow rates in early and late diastole and their ratio (E, A, E / A); DTE, deceleration time of early diastolic velocity; IVRT, isovolumic relaxation time

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respectively). Also, there was an increase of DTE ≤35% taking atorvastatin increased significantly, (p<0.05) and decrease of IVRT (p<0.001) (Table 1). and the end-diastolic and end-systolic diameters were reduced (28). DISCUSSION Bielecka-Dabrowa et al. concluded that statin use The results of this study have proved the reduction has benefits regardless of the etiology of HF, and in the right and left ventricle diameters after a six- that lipophilic statins are more favourable in pati- month atorvastatin therapy in patients with EFLV ents with HF (29). The Myocardial Ischemia Re- ≤40%. Improving systolic and diastolic functi- duction with Aggressive Cholesterol Lowering on leads to a lower rate of re-hospitalization, as (MIRACLE) clinical trial showed that the use well as to the improvement in the quality of life. of atorvastatin was beneficial in mortality, - car Kjekshus et al. stated that the use of statins redu- diac arrest occurrence and re-hospitalizations in ces the risk of developing HF, as well as reducing the patients with unstable angina or non-Q acute the progression of coronary artery disease (22). myocardial infarction, who were treated with 80 Zhang et al. demonstrated an improvement in the mg of atorvastatin (18). left ventricular ejection fraction, reduction of the The use of high doses of lipophilic statins has left ventricular end-diastolic diameter, end-systo- benefits in the initial treatment of acute coronary lic diameter, and in brain natriuretic peptide (BNP syndrome, but also in the period after that, due to is also the New York Heart Association functional the effect on the heart muscle, especially in pati- class) (23), which was confirmed in this research. ents with reduced EFLV. This research imposes Liu et al. analysis of patients with respect to pres- the use of lipophilic statins in this type of patients. cribed statin (atorvastatin, simvastatin, or pitava- Their use should be preferred in patients with HF, statin) showed that the use of lipophilic statins in and potential side effects should not be the reason patients with HF reduced overall mortality and the not to include them in the therapy (pharmacokine- number of hospitalizations, while the use of lipo- tic interactions are particularly considered). philic statins alone increased the values of EFLV Despite the small number of patients included in (24). Also, the rates of hospitalization were lower this study, it can be a basis for further research in the patients with HF who were taking 80 mg of that would include a larger number of patients, atorvastatin, while no benefit was shown on the re- and examine the impact of comorbidities (the duction of the rate of HF onset (29). In an analysis presence of renal failure, liver disease, or diabe- of 110 patients with EFLV below 30%, Vrtovec tes mellitus) on the effect of statin therapy. et al. indicated that the mortality rate was lower in the patients using atorvastatin therapy during a In conclusion, atorvastatin in the therapy resulted one-year follow-up (25). in increased EFLV and better systolic function and should be a part of the therapeutic modality of HF. Hobbs et al. indicated that the use of lipophilic statins had an effect on the increase in EFLV, and FUNDING thus on patient outcome, during a five-year period (26). Wu et al. showed a significant improvement No specific funding was received for this study. in global strain imaging in patients undergoing TRANSPARENCY DECLARATION peritoneal dialysis in patients on atorvastatin therapy (27). It was shown that the EFLV valu- Conflicts of interest: None to declare. es of patients with nonischemic HF and EFLV of REFERENCES: 4. Swenne CA. Beyond lipid lowering: pleiotropic effects of statins in heart failure. Neth Heart J 2013; 1. Choi HM, Park MS, Youn JC. Update on heart fa- 21:406-7. ilure management and future directions. Korean J 5. Vogiatzi G, Oikonomou E, Siasos G, Tsalamandris Intern Med 2019; 3:11-43. S, Briasoulis A, Androulakis E, Latsios G, Papai- 2. Pinal-Fernandez I, Casal-Dominguez M, Mammen oannou S, Tsioufis K, Tousoulis D. Statins and in- AL. Statins: pros and cons. Med Clin (Barc) 2018; flammation in cardiovascular disease. Curr Pharm 150:398-402. Des 2017. 3. Correale M, Abruzzese S, Greco CA, Concilio M, 6. Wang L, Shi J, Zhang Y. Influences of simvastatin Biase MD, Brunetti ND. Pleiotropic effects of sta- on vascular endothelial function of patients with tin in therapy in heart failure: a review. Curr Vasc coronary heart disease complicated with congesti- Pharm 2014; 12:873-84. ve heart failure. Eur Rev Med Pharmacol Sci 2013; 17:1590-3.

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7. Oikonomou E, Siasos G, Zaromitidou M, Hatzis G, percutaneous coronary intervention: a collaborative Mourouzis K, Chrysohoou C, Zisimos K, Mazaris patient-level meta-analysis of 13 randomized studi- S, Tourikis P, Athanasiou D, Stefanadis C, Papava- es. Circulation 2011; 123:1622-32. ssiliou AG, Tousoulis D. Atorvastatin treatment im- 18. Schwartz GG, Olsson AG, Ezekowitz MD, Ganz P, proves endothelial function through endothelial pro- Oliver MF, Waters D, Zeiher A, Chaitman BR, Le- genitor cells mobilization in ischemic heart failure slie S, Stern T; Myocardial Ischemia Reduction with patients. Atherosclerosis 2015; 238:159-64. Aggressive Cholesterol Lowering (MIRACL) Study 8. Li J, Sun YM, Wang LF, Li ZQ, Pan W, Cao HY. Investigators. Effects of atorvastatin on early recu- Comparison of effects of simvastatin versus atorva- rrent ischemic events in acute coronary syndromes: statin on oxidative stress in patients with coronary The MIRACLE study: a randomized controlled tri- heart disease. Clin Cardiol 2010; 33:222-7. al. JAMA 2001; 285:1711-8. 9. Costa S, Reina-Couto M, Albino-Teixeira A, Sousa 19. Thompson PL, Meredith I, Amerena J, Campbell T. Statins and oxidative stress in chronic heart failu- TJ, Sloman JG, Harris PJ; Pravastatin in Acute Co- re. Rev Port Cardiol 2016; 35:41-57. ronary Treatment (PACT) Investigators. Effect of 10. Niazi M, Galehdar N, Jamshidi M, Mohammadi R, pravastatin compared with placebo initiated within Moayyedkazemi A. A Review of the role of statins in 24 hours of onset of acute myocardial infarction or heart failure treatment. Curr Clin Pharmacol 2020; unstable angina: the Pravastatin in Acute Coronary 15:30-7. Treatment (PACT) trial. Am Heart J 2004; 148:e2. 11. Zhang R, Ma S, Shanahan L, Munroe J, Horn S, 20. Bonsu KO, Reidpath DD, Kadirvelu A. Lipophilic Speedie S. Discovering and identifying New York statin versus rosuvastatin (hydrophilic) treatment for heart association classification from electronic- he heart failure: a meta-analysis and adjusted indirect alth records. BMC Med Inform Decis Mak 2018; comparison of randomised trials. Cardiovasc Drugs 18:48. Ther 2016; 30:177–88. 12. Selker HP, Udelson JE, Massaro JM, Ruthazer R, 21. Schachter M. Chemical, pharmacokinetic and phar- D'Agostino RB, Griffith JL, Sheehan PR, Desvigne- macodynamic properties of statins: an update. Fun- Nickens P, Rosenberg Y, Tian X, Vickery EM, Atkins dam Clin Pharmacol 2005; 19:117–25. JM, Aufderheide TP, Sayah AJ, Pirrallo RG, Levy 22. Kjekshus J, Pedersen TR, Olsson AG, Faergeman MK, Richards ME, Braude DA, Doyle DD, Frascone O, Pyörälä K. The effects of simvastatin on the inci- RJ, Kosiak DJ, Leaming JM, Van Gelder CM, Walter dence of heart failure in patients with coronary heart GP, Wayne MA, Woolard RH, Beshansky JR. One- disease. J Card Fail 1997; 3:249–54. year outcomes of out-of-hospital administration of 23. Zhang L, Zhang S, Jiang H, Sun A, Zou Y, Ge J. intravenous glucose, insulin, and potassium (GIK) Effects of statin treatment on cardiac function in in patients with suspected acute coronary syndromes patients with chronic heart failure: a meta-analysis (from the IMMEDIATE [Immediate Myocardial of randomized controlled trials. Clin Cardiol 2011; Metabolic Enhancement During Initial Assessment 34:117-23. and Treatment in Emergency Care] Trial). Am J Car- 24. Liu G, Zheng XX, Xu YL, Lu J, Hui RT, Huang XH. diol 2014; 113:1599–605. Effects of lipophilic statins for heart failure: a me- 13. Benjo AM, El-Hayek GE, Messerli F, DiNicolan- ta-analysis of 13 randomised controlled trials. Heart tonio JJ, Hong MK, Aziz EF, Herzog E, Tamis-Ho- Lung Circ 2014; 23:970-7. lland J. High dose statin loading prior to percutaneo- 25. Vrtovec B, Okrajsek R, Golicnik A, Ferjan M, Starc us coronary intervention decreases cardiovascular V, Schlegel TT, Radovancevic B. Atorvastatin the- events: a meta-analysis of randomized controlled rapy may reduce the incidence of sudden cardiac de- trials. Catheter Cardiovasc Interv 2015; 85:53-60. ath in patients with advanced chronic heart failure. J 14. Kim C, Choi D. Timing of high intensity statin for Card Fail 2008; 14:140-4. acute coronary syndrome: how earlier initiation ma- 26. Hobbs FD, Roalfe AK, Davis RC, Davies MK, Hare kes better? J Thorac Dis 2018; 10:S2149–52. R. Prognosis of all- cause heart failure and bor- 15. Navarese EP, Kowalewski M, Andreotti F, van Wely derline left ventricular systolic dysfunction: 5 year M, Camaro C, Kolodziejczak M, Gorny B, Wirianta mortality follow-up of the echocardiographic heart J, Kubica J, Kelm M, de Boer MJ, Suryapranata H. of Rengland 350 screening study (ECHOES). Eur Meta-analysis of time-related benefits of statin the- Heart J 2007; 28:1128–34. rapy in patients with acute coronary syndrome un- 27. Wu CK, Yeh CF, Chiang JY, Lin TT, Wu YF, Chiang dergoing percutaneous coronary intervention. Am J CK, Kao TW, Hung KY, Huang JW. Effects of ator- Cardiol 2014; 113:1753-64. vastatin treatment on left ventricular diastolic func- 16. Cannon CP, Braunwald E, McCabe CH, Rader DJ, tion in peritoneal dialysis patients-The ALEVENT Rouleau JL, Belder R, Joyal SV, Hill KA, Pfeffer clinical trial. J Clin Lipidol 2017; 11:657-66. MA, Skene AM; Pravastatin or Atorvastatin Eva- 28. Sola S, Mir MQS, Khan BV, Lerakis S, Tandon N. luation and Infection Therapy-Thrombolysis in Atorvastatin improves left ventricular systolic func- Myocardial Infarction 22 Investigators. Intensive tion and serum markers of inflammation in nonische- versus moderate lipid lowering with statins after mic heart failure. J Am Coll Cardiol 2006; 47:332-7. acute coronary syndromes. N Engl J Med 2004; 29. Bielecka-Dabrowa A, Bytyçi I, Von Haehling S, An- 350:1495-504. ker S, Jozwiak J, Rysz J, Hernandez AV, Bajraktari 17. Patti G, Cannon CP, Murphy SA, Mega S, Pasce- G, Mikhailidis DP, Banach M. Association of statin ri V, Briguori C, Colombo A, Yun KH, Jeong MH, use and clinical outcomes in heart failure patients: a Kim JS, Choi D, Bozbas H, Kinoshita M, Fukuda systematic review and meta-analysis. Lipids Health K, Jia XW, Hara H, Cay S, Di Sciascio G. Clinical Dis 2019; 18:188. benefit of statin pretreatment in patients undergoing

361 ORIGINAL ARTICLE

Predictive factors for noninvasive mechanical ventilation failure among COVID-19 critically ill patients - a retrospective cohort study

Mirza Kovačević, Nermina Rizvanović, Adisa Šabanović Adilović

Department of Anaesthesiology and Intensive Care Unit, Cantonal Hospital Zenica; Bosnia and Herzegovina

ABSTRACT

Aim To identify predictive factors related with noninvasive ventilation (NIV) failure that are not based on the patient's respiratory status or acid base gas analyses in COVID-19 critically ill patients, and to create a predictive model of NIV failure.

Methods A total of 73 COVID-19 critically ill patients who developed acute respiratory failure and underwent NIV were divided into two groups: Group 1, patients who required endotracheal intubation and invasive mechanical ventilation after NIV and Group 2, patients with successful weaning from NIV. Demographic data, clinical symptoms and signs, clinical index and scores, duration Corresponding author: indicators and laboratory data were analysed. Predictive factors Mirza Kovačević of NIV failure were assessed using univariate and multivariate regression analyses followed by the receiver operating characteristic Department of Anaesthesiology and (ROC) curve. Intensive Care Unit, Cantonal Hospital Zenica Results In the Group 1 (NIV failure) there were 54 (73.97%) pa- Crkvice 67, 72 000 Zenica, tients. Predictive factors for NIV failure were: the presence of dyspnoea on the day of admission at hospital (p<0.05; sensitivity Bosnia and Herzegovina 44.40%; specificity 84.20%), higher radiographic assessment of Phone: +387 32 447 000; lung oedema score (RALES) on the day of starting NIV (p<0.009; Fax: +387 32 226 576; sensitivity 70.40%; specificity 73.75%), higher length of NIV E-mail: [email protected] (p<0.014; sensitivity 48%; specificity 84.10%) and higher urea on ORCID ID: https://orcid.org/0000-0002- the day of starting NIV (p<0.004; sensitivity 70.44%; specificity 3492-4100 73.72%) Conclusion NIV treatment in COVID-19 critically ill patients has a high failure rate. In addition to respiratory parameters, dyspnoea, Original submission: higher RALES, higher length of NIV and increased urea value co- 06 April 2021; uld predict NIV failure. These factors should be considered in tre- Revised submission: atment decision making. 17 May 2021; Key words: acute respiratory failure, dyspnoea, RALES, urea Accepted: 25 May 2021 doi: 10.17392/1385-21

Med Glas (Zenica) 2021; 18(2):362-369

362 Kovačević at al. Noninvasive ventilation in COVID-19 ARDS

INTRODUCTION sensus among anaesthesiologists on acceptable predictors for NIV failure. COVID-19 virus is a new, predominantly respiratory virus, first recognized in China, in De- The aim of this study was to define predictive cember 2019 and has spread all over the world. factors for NIV failure and the necessity of en- Severe clinical condition with acute respiratory dotracheal intubation among COVID-19 criti- failure (ARF) caused by COVID-19 virus po- cally ill patients, regardless of the severity of ses a serious threat to citizens and healthcare hypoxemia, clinical respiratory variables, or systems or professionals (1,2). About 15-30% ventilation variables. We evaluated the predic- of patients with COVID-19 viral infection de- tive value of demographic parameters, clinical teriorate to acute respiratory distress syndrome signs and symptoms, clinical index and scores, (ARDS) within the first two days of hospital duration indicators, laboratory and radiological admission and require some type of respira- findings and created a corresponding model for tory support (3). Conventional oxygen therapy prediction of NIV failure. by face mask, high-flow nasal oxygen, NIV or PATIENTS AND METHODS invasive mechanical ventilation (IMV) are used in the treatment of hypoxemic ARF observed Patients and study design in COVID-19 viral infection. The IMV requires endotracheal intubation, which is associated This single centered retrospective cohort stu- with major medical complications, and which dy was conducted over the period of seven leads to significant medical costs. Indications months, between July 2020 and February 2021, for endotracheal intubation of patients with in the Department of Anaesthesiology and In- COVID-19 are strictly defined and based on the tensive Care Unit at the Cantonal Hospital in severity of hypoxemia, clinical respiratory va- Zenica, Bosnia and Herzegovina. During the riables, or ventilation variables. These indicati- observed period, 186 patients were admitted ons should be limited to: airway protection (dis- to the ICU with COVID-19 ARF. After obta- order of consciousness), severe decompensated ining the Ethical Committee’s approval and acidosis (pH <7.20-7.25), severe hypoxemia a written informed patient consent, 73 adult patients fulfilled the study criteria and were (PaO2 <50 mmHg or SaO2 <90% -92%), signs and symptoms of tissue hypoxia (4). included in the study. Inclusion criteria were The NIV is one of the first-line therapies in patients with a positive reverse transcription- order to avoid endotracheal intubation in pa- polymerase chain reaction of nasopharyngeal tients with ARDS (5). Limited data described swab samples for SARS-CoV-2, admitted to a high rate of NIV failure in a previously re- the ICU, presented with hypoxemic ARF and ported ARF caused by other types of coronavi- treated with NIV. Electronic data from the ICU rus infections, such as Middle East respiratory medical reports were used. syndrome corona virus (MERS-CoV) or se- The patients were divided into two groups: Gro- vere acute respiratory syndrome corona virus up 1 (54 patients, negative NIV outcome), pati- (SARS-CoV) (6). The patients with COVID-19 ents whose ICU treatment started with NIV but treated with NIV are admitted to intensive care required endotracheal intubation and invasive units (ICU) under the constant supervision of mechanical ventilation and Group 2 (19 patients, experienced medical staff, capable of endo- positive NIV outcome), patients whose ICU tre- tracheal intubation (7). atment started with NIV and finished successful Related factors that may impair ventilation and weaning from NIV. respiratory mechanics in NIV-treated patients One hundred and thirteen patients were excluded and contribute to endotracheal intubation have from the study due to non-fulfilment of the study not been precisely identified. Some studies have criteria. Exclusion criteria were: the patients tre- described risk factors for the requirement for ated with conventional oxygen therapy by face NIV support in critically ill COVID-19 patients mask, the patients treated with NIV less than 24 (8,9), but predictive factors for NIV failure are hours, the patients with severe ARDS who requ- not sufficiently investigated. There is no - con ired immediate endotracheal intubation, uncon-

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scious patients and other contraindications for Pharmaceutical strategy. All patients were trea- NIV such as facial abnormalities, fixed obstructi- ted according to the diagnosis and treatment pro- on of the upper airways and vomiting (Figure 1). tocol of the new coronavirus infection (12). The therapy included corticosteroids, anticoagulants, proton pump inhibitors, and vitamin supportive therapy. Antiviral medications, antibiotics and immunomodulatory therapy were administrated in consultation with an infectologist, according to clinical status and laboratory findings. Patients variables. The following variables were considered as possible predictors of NIV failure and involved in the regression analysis: demographic parameters, clinical symptoms and signs, clinical index and scores, duration indicators and laboratory data. Demographic parameters involved age and gender. Clinical symptoms and signs were recorded on the day of admission at hospital, marked with YES or NO and included: fever, cough, dyspnoea, chest pain, weakness, abdominal pain, diarrhoea, nausea, vomiting, headache, anosmia, myalgia, anorexia, heart rate, mean arterial pre- Fig.1. Flow diagram of the study protocol ssure (MAP) and temperature. Clinical index and scores were recorded on the Methods day of admission to the ICU: Charlson Comor- Ventilation strategy. After the admission to the bidity Index (CCI) (13), Simplified Acute Physi- ICU, the patients were selected for NIV by an ana- ology Score II (SAPS II) and Acute Physiology esthesiologist, according to the already established and Chronic Health Evaluation II (APACHE II) protocol at the ICU. Indications for the use of NIV (14). Radiographic Assessment of Lung Ede- were as follows: moderate to severe dyspnoea with ma Score (RALES) (15) was performed in two tachypnea (≥25 breaths/min), use of accessory following time: T1- on the day of admission to the ICU and T2- on the day of starting NIV. muscles, acid-base disturbance (pH <7.30; PaCO2

>45 mmHg; PaO2 <45 mmHg), SpO2 <85% with Evaluated duration indicators were measured in

FiO2 0.5 (10). Initial continuous positive airway days: length of symptoms to the day of hospitaliza- pressure (CPAP) was delivered to the patient using tion, length from admission day to starting of NIV,

an NIV mask with pressure values of 5-10 cmH2O. length of NIV and overall length of hospitalization. If hypoxemia (PaO <50 mmHg) or desaturation 2 Laboratory data consisted of blood count, bio- (SpO <80%) persisted after NIV administration, 2 chemistry and immunology data. Blood samples the positive end-expiratory pressure (PEEP) was for blood count (white blood cells, platelets, neu- increased for 1-2 cmH O, or inspiratory pressure 2 trophils, lymphocytes) and biochemistry parame- was increased for 2-3 cmH O to receive an inspi- 2 ters (urea, creatinine, sodium and potassium) were ratory volume of 6-8 mL/kg. In the case of further taken in three following time periods: T1- on the exacerbation, when patients met the criteria for en- day of admission at ICU, T2- on the day of starting dotracheal intubation (severe acidosis pH <7.25; NIV and T3- on the day of endotracheal intubation severe hypoxemia PaO <50 mmHg or impaired 2 for Group 1 or on the day of successful weaning consciousness), IMV was used as the main ven- from NIV for Group 2. Blood samples for analysis tilatory support. In contrast, successful respiratory of immunological parameters C-reactive protein support with NIV was based on improving gene- (CRP) and procalcitonin (PCT) were taken in two ral clinical condition of the patient, respiratory following time periods: T1- on the day of admissi- and heart rate, mental state and improving the gas on at ICU and T2- on the day of starting NIV. exchange index (11).

364 Kovačević at al. Noninvasive ventilation in COVID-19 ARDS

Statistical analysis (AUC), optimal cut-off, sensitivity and specificity were calculated. Categorical variables were presented as frequencies and percentages and analysed using χ2 RESULTS test. The Kolmogorov-Smirnov test was used to examine the normality of the distribution. Con- A total of 73 patients were included in the study. tinuous variables were presented as means and There were statistically significantly more males standard deviation (SD) and analysed using versus females, 49 (67.1%) and 24 (32.9%), respec- Student’s t test. All collected data were compa- tively (p<0.01). The mean age of the patients was red to identify the differences between the two 65.3 (±9.81) years. There were 54 patients in the groups. Statistical significance was considered as Group 1 (negative NIV outcome) and 19 patients in p< 0.05. Due to the dichotomous nature of the the Group 2 (positive NIV outcome). The NIV was dependent variable (NIV failure), the logistic applied with an overall success rate of 26%. regression method was used for further statisti- There were no statistically significant differences cal analysis. An univariate logistic regression in gender and age between the groups. The pre- analysis was used to calculate the independent sence of dyspnoea, anorexia and increased MAP association of each observed variable with NIV on the day of admission at hospital, higher RA- failure. Multivariate stepwise logistic regression LES on the day of starting NIV and higher length was used to estimate the predictive model of NIV of NIV showed a statistically significant predicti- failure. Adjusted odds ratios (OR) and 95% con- ve value for NIV failure (p<0.05). The CCI score fidence interval (CI) were calculated for potential was statistically significantly higher in the Gro- predictors. Predictive ability of variables in the up 1 than in the Group 2 (3.37% versus 1.68%; NIV failure model was evaluated by the receiver p<0.045), but a predictive value for NIV failure operating curves (ROC); area under the curve was not recorded (p=0.114) (Table 1). Table 1. Demographic data, clinical symptoms and sings, clinical index and scores and duration indicators according to the groups Patients group Univariate analysis Parameter p p Group 1 (n=54) Group 2 (n=19) OR 95% CI Age (years) (Mean±SD) 65.23 ±8.73 65.49 ±11.31 0.367 0.99 0.94-1.04 0.690 Male/Female (No, %) 36/18 (49.3/24.7) 13/6 (17.8/8.2) 0.889 1.08 0.35-3.32 0.889 Clinical symptoms and sings (YES/NO) No (%) Fever 42/12 (77.8/22.2) 16/3 (84.2/15.8) 0.551 1.52 0.16-2.63 0.553 Cough 36/18 (66.7/33.3) 16/3 (84.2/15.8) 0.146 0.37 0.09-1.45 0.157 Dyspnoea 30/24 (55.6/44.4) 3/16 (15.8/84.2) 0.026 0.23 0.06-0.89 0.034 Chest pain 6/48 (11.1/88.9) 17/2 (10.5/89.5) 0.944 1.06 0.19-5.77 0.944 Weakness 40/14 (74.1/25.9) 12/7 (63.2/36.8) 0.366 1.66 0.54-5.07 0.368 Abdominal pain 3/51 (5.6/94.4) 17/2 (10.5/89.5) 0.461 0.50 0.07-3.24 0.468 Diarrhoea 6/48 (13.0/87.0) 1/18 (5.3/94.7) 0.594 2.68 0.30-23.35 0.372 Nausea 7/47 (11.1/88.9) 3/16 (15.8/84/2) 0.355 0.66 0.14-2.9 0.596 Vomiting 5/49 (9.3/90.7) 1/18 (5.3/94.7) 0.585 1.83 0.20-16.80 0.590 Headache 2/52 (3.7/96.3) 1/18 (5.3/94.7) 0.768 0.69 0.05-8.90 0.769 Anosmia 10/44 (18.5/81.5) 1/18 (5.3/94.7) 0.165 4.09 0.48-34.33 0.194 Myalgia 14/40 (25.9/74.1) 17/2 (10.5/89.5) 0.163 2.97 0.60-14.53 0.178 Anorexia 21/33 (38.9/61.1) 2/17 (89.5/10.5) 0.022 5.40 1.13-1.09 0.034 Mean±SD HR (beat/min) 101.69±18.83 104.21 ±18.48 0.616 0.99 0.96-1.02 0.610 MAP (mmHg) 100.11±14.42 90.61 ±12.92 0.013 1.05 1.00-1.09 0.019 Temperature (°C) 37.14±0.93 37.25 ±1.09 0.66 0.88 0.51-1.51 0.655 Clinical index and scores (%) (Mean±SD) CCI 3.37±4.31 1.68±2.66 0.045 1.14 0.96-1.36 0.114 SAPS II 6.56±4.60 5.45±2.87 0.226 1.07 0.93-1.24 0.326 APACHE 22.9±9.15 20±6.87 0.264 1.04 0.97-1.11 0.265 RALES T1 30.85±10.10 26±11.70 0.88 1.04 0.99-1.09 0.093 T2 36.59±8.29 29.47±10.56 0.013 1.08 1.02-1.15 0.007 Duration indicators (days) (Mean±SD) LSH 6.39±3.03 7.47±2.72 0.173 0.88 0.74-1.05 0.178 LANIV 3.19±3.01 2.84±2.61 0.66 1.04 0.86-1.26 0.656 LNIV 3±1.48 4±1.76 0.019 0.69 0.50-0.95 0.026 LOH 9.63±5.1 11.89±5.23 0.102 0.92 0.83-1.01 0.113 Group 1, patients whose treatment started with noninvasive ventilation but required endotracheal intubation; Group 2, patients whose treatment finished successful weaning from noninvasive ventilation; OR, odds ratio; CI, confidence interval; SD, standard deviation; HR, heart rate; MAP, mean arterial pressure; CCI, Charlson Comorbidity Index; SAPS II, Simplified Acute Physiology Score II; APACHE II, Acute Physiology and Chronic Health Evaluation II; RALES, Radiological Asse- ssment Lung Edema Score; T1, on the day of admission to the ICU; T2, on the day of starting noninvasive ventilation; LSH, length of symptoms to the hospitalization, LANIV, length from admission to starting of noninvasive ventilation; LNIV, length of noninvasive ventilation; LOH, length of overall hospitalization

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Table 2. Laboratory data according to the groups Patients group Univariate analysis Parameter (reference) (Mean±SD) Time p p Group 1 (n=54) Group 2 (n=19) OR 95% CI T1 10.95±6.48 10.40±4.77 0.735 1.01 0.92-1.11 0.731 WBC (3.4-10x109/L) T2 13.40±7.32 13.68±5.89 0.882 0.99 0.92-1.07 0.880 T3 16.28±7.79 14.55±7.83 0.409 1.03 0.96-1.10 0.404 T1 232.24±97.93 227.36±83.6 0.847 1.00 0.99-1.00 0.845 Platelets (150-400x109/L) T2 252.90±109.58 244.57±82.66 0.764 1.00 0.99-1.00 0.760 T3 245.74±121.02 251.84±108.28 0.847 1.00 0.99-1.00 0.844 T1 1.52±2.56 2.00±3.47 0.530 0.94 0.79-1.12 0.530 Neutrophils (0.40-0.70 x109/L) T2 9.68±8.06 7.11±5.67 0.205 1.05 0.97-1.14 0.204 T3 9.79±8.16 7.01±3.02 0.199 1.19 0.94-1.11 0.258 T1 0.20±0.32 0.13±0.20 0.379 2.88 0.25-32.36 0.390 Lymphocytes (0.40-0.70 x109/L) T2 0.63±0.56 0.48±0.40 0.349 1.93 0.59-6.29 0.276 T3 0.52±0.45 0.47±0.37 0.426 1.04 0.51-6.10 0.291 T1 9.07±5.86 7.86±4.30 0.412 1.04 0.93-1.16 0.409 Urea (1.7-8.3 mmol/L) T2 14.84±9.13 9.77±3.87 0.038 6.65 2.05-21.06 0.002 T3 12.10±11.38 8.00±6.68 0.738 1.07 0.97-1.19 0.155 T1 96.92±80.21 80.52±49.05 0.406 1.00 0.99-1.01 0.419 Creatinine (44-84 mmol/L) T2 110.87±128.44 63.68±33.97 0.016 2.99 1.12-7.93 0.028 T3 140.40±158.03 87.47±75.37 0.007 3.05 1.33-6.99 0.008 T1 137.57±4.14 137.36±3.85 0.843 1.01 0.88-1.16 0.840 Sodium (136-146 mmol/L) T2 139.01±5.22 140.36±5.38 0.351 0.95 0.86-1.05 0.347 T3 141.35±7.03 139.10±5.32 0.209 1.05 0.96-1.15 0.209 T1 4.04±0.68 4.14±0.74 0.540 0.76 0.33-1.75 0.534 Potassium (3.6-5.4mmol/L) T2 4.14±0.79 4.11±0.68 0.883 1.05 0.52-2.11 0.881 T3 4.47±0.92 4.26±0.79 0.250 1.44 0.77-2.69 0.249 T1 83.03±29.32 83.15±15.29 0.990 1.03 0.98-1.02 0.986 CRP (0.5-10 g/L) T2 100.97±125.72 91.36±44.54 0.746 1.00 0.99-1.00 0.748 T1 0.56±1.01 3.60±12.39 0.074 1.33 0.51-1.33 0.439 PCT (0-0.046ng/mL) T2 2.64±4.17 3.47±7.75 0.563 1.06 0.88-1.06 0.562 Group 1, patients whose treatment started with noninvasive ventilation but required endotracheal intubation; Group 2, patients whose treatment finished successful weaning from noninvasive ventilation; OR, odds ratio; CI, confidence interval; SD, standard deviation;WBC, white blood cells; CRP, C-reactive protein; PCT, procalcitonin; T1, on the day of admission to the ICU; T2, on the day of starting noninvasive ventilation; T3, on the day of endotracheal intubation for Group 1 and on the day of successful weaning from noninvasive ventilation for Group 2

Higher mean value of urea and creatinine were Table 3. Multivariate analysis of the parameters indepen- recorded in the Group 1 compared with the Gro- dently associated with noninvasive ventilation failure up 2 (14.84 versus 9.77 and 110.87 versus 63.68, Multivariate analysis Parameter 95% CI respectively) on the day of starting NIV as well p OR Lower Upper as higher mean value of creatinine (140.40 ver- Dyspnoea 0.004 0.08 0.00 0.91 sus 87.47) on the day of starting IMV. Increased Anorexia 0.356 2.52 0.34 18.76 mean value of urea and creatinine on the day of MAP 0.067 1.48 0.98 2.28 RALES T2 0.001 1.18 1.06 1.30 starting NIV as well as increased mean value of LNIV 0.025 0.54 0.31 0.92 the creatinine on the day of starting IMV showed Urea T2 0.004 0.09 0.01 0.47 statistically significant predictive value for NIV Creatinine T2 0.597 0.61 0.10 3.77 Creatinine T3 0.698 1.46 0.21 10.07 failure (p<0.05) (Table 2). OR, odds ratio; CI, confidence interval; MAP, mean arterial pressure; After multivariate stepwise logistic regression RALES T2, Radiological Assessment Lung Edema Score on the day of starting noninvasive ventilation; LNIV, length of noninva- analysis of the parameters independently associa- sive ventilation; urea T2, mean value of urea on the day of starting ted with NIV failure, the presence of dyspnoea on noninvasive ventilation; creatinine T2, mean value of creatinine on the day of starting noninvasive ventilation; creatinine T3, mean value the day of admission to hospital (p<0.004), the of creatinine on the day of endotracheal intubation RALES on the day of starting NIV (p<0.001), the length of NIV (p<0.025) and the mean value of Table 4. The receiver operating characteristic (ROC) curve urea on the day of starting NIV (p<0.004) were data of predictive model for noninvasive ventilation failure SensitivitySpecificity Cut 95% CI included in the predictive model of NIV failure Parameters AUC p (Table 3). (%) (%) off Lower Upper Dyspnea 0.64 44.40 84.20 0.50 0.05 0.50 0.79 The most important predictive factor in the propo- RALES T2 0.70 70.40 73.75 31 0.009 0.56 0.84 sed model of NIV failure was increased mean va- LNIV 0.69 48.25 84.10 2.5 0.014 0.54 0.83 lue of urea on the day of starting NIV (sensitivity Urea T2 0.72 70.44 73.72 1.5 0.004 0.58 0.85 AUC, area under the curve; CI, confidence interval; RALES T2, 70.44%, specificity 73.72%; p<0.004) (Table 4). Radiological Assessment Lung Edema Score on the day of starting noninvasive ventilation; LNIV, length of noninvasive ventilation; urea T2, mean value of urea on the day of starting noninvasive ventilation

366 Kovačević at al. Noninvasive ventilation in COVID-19 ARDS

DISCUSSION by hypoxia. The predictive value of the MAP This single centered retrospective cohort study for NIV failure was not found. In the multicenter investigated predictive factors for NIV failure observational study, Liu et al. did not determine among COVID-19 respiratory critically ill pati- the statistical significance of MAP for predicting ents, that are not based on patient's respiratory NIV failure. The reported NIV failure rate was status or acid-base gas analyses. Demographic 81% versus 76% in our study (23). parameters, clinical signs and symptoms, clini- In our research, the mean CCI was 3.37 in the cal scores, coexisting illnesses, radiological and group with a negative NIV outcome, but the CCI laboratory findings were compared between pati- was not independently associated with NIV fa- ents who underwent IMV or NIV. The results of ilure. Other authors found CCI median 2 (1–3) in this study suggest that the presence of dyspnoea the NIV + IMV group (24). The RALES system on the day of admission at hospital, higher RA- is often used to quantify the progression of lung LES score on the day of starting NIV, higher len- involvement in patients with COVID-19 (25). gth of NIV and increased value of urea on the Our study showed the RALES on the day of star- day of starting NIV are included in the predictive ting NIV of 25-50% lung involvement (four po- model of NIV failure. ints) in the group with a negative NIV outcome; In the presented study, of a total of 74 patients it was strongly associated with NIV failure with a with NIV support, 54 patients required IMV. value of area under the curve or diagnostic accu- The NIV failure rate was 73.97%. Our results racy of tests of 0.70, a sensitivity 70.40% and a confirmed previous NIV failure rate data of 56- specificity 73.75%. The RALES on the day of 76% (16). These results could be explained by starting NIV is the second most significant factor a poorer response to NIV in patients with ARF in our predictive model of NIV failure. Burns et due to COVID-19 infection compared to patients al. concluded that the only statistical significan- with ARF due to community-acquired pneumo- ce for NIV success was lower level of the X-ray nia, a heterogeneity of the criteria for respiratory imaging score (26). support measures or leakage of objectivity in the The length of NIV of six days in our study clinical studies (17). showed a predictive value for negative NIV Dyspnoea has been reported in more than 50% of outcome; in the group with the positive NIV patients with COVID-19 (18) and a significantly outcome, the length of NIV was four days. Simi- higher incidence has been found in patients in lar results were reported by Mukhtara et al., the need of ICU care (19). In this study, the presence duration of successful NIV treatment was two to of dyspnoea was a predictive factor for NIV fa- five days (27). ilure, recorded in 55.6% patients with negative No statistically significant difference between the NIV outcome. Dyspnoea develops due to worse- groups in terms of white blood cells count, plate- ning of hypoxia, increased respiratory effort and lets, neutrophils, lymphocytes, CRP and procal- the use of accessory muscles and tachypnea (20). citonin was found in our study. Contrary, Guan et Malnourished patients have decreased immu- al. (1099 patients) recorded an increase of CRP in nity and bone marrow function, pancytopenia 91.1% and lymphocytopenia in 92.6% of patients and increased risk of severe morbidity (21). In requiring IMV (28). Opposite results could have the presented study, anorexia was more common been caused by a large difference in the number in the NIV failure group (38.9% patients) but of involved patients between the two studies. not showed a statistically significant value to be Urea and creatinine values did not differ stati- included in the predictive model. A severe form stically significantly in patients with COVID-19 of COVID-19 infection in patients with anorexia treated with a high-flow nasal cannula compared could be explained by disruption of the angioten- with NIV (29). The results of our study showed sin-converting enzyme 2 cell receptor function in mean urea and creatinine values higher in the the small intestine (22). group with NIV failure; creatinine value did not In our study hemodynamic instability, increa- show predictive significance for NIV outcome sed MAP and heart rate, recorded in the group although higher urea value on the day of starting with the negative NIV outcome was supported NIV proved to be the most significant factor in

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the predictive model of NIV failure. This result loped in this study showed that the presence of indicates accurate monitoring of the urea value in dyspnoea on the day of admission at hospital, the patients treated with NIV. higher RALES score on the day of starting NIV, There are some limitations of the study. The higher length of NIV and increased value of urea single centered, retrospective nature, without a on the day of starting NIV are strongly related control group and small number of patients could with NIV failure. In addition to respiratory pa- influence failure to achieve excellent prognostic rameters, this predictive model should be accura- accuracy of factors. A number of important labo- tely monitored and considered in making timely ratory data were not monitored due to collection therapeutic and diagnostic decisions. inconsistencies. For better insight into the pre- FUNDING dictors of NIV failure, future studies are needed, with more laboratory data (transaminases, immu- No specific funding was received for this study. nological and coagulation data). TRANSPARENCY DECLARATIONS In conclusion, the use of NIV remains a significant alternative to avoid IMV, during the CO- Competing interest: None to declare. VID-19 pandemic. The predictive model deve-

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369 ORIGINAL ARTICLE

Chest x-ray resolution after SARS-CoV-2 infection Besim Prnjavorac1-4, Aida Mujaković3,5, Lejla Prnjavorac1, Tamer Bego4, Edin Jusufović6,7, Edin Begić3,5, Vildana Torlak-Arnaut2,8, Meliha Mutapčić2,8, Hasan Škiljo1,2, Enes Hodžić1,2, Emina Karahmet4, Maja Malen- ica4, Tanja Dujić4, Jusuf Mehić1, Nedžada Irejiz1, Rifat Sejdinović1,2, Anel Mahmutović1, Ajdin Ibrahimović1

1General Hospital Tešanj, Tešanj, 2School of Medicine, University of Zenica, Zenica, 3Sarajevo School of Science and Technology, Sarajevo,4Faculty of Pharmacy, University of Sarajevo, Sarajevo, 5General Hospital “Prim.dr Abdulah Nakaš“ Sarajevo, 6School of Medicine, University of Tuzla, Tuzla , 7Health and Educational Medical Center for Pulmonary Diseases, Tuzla, 8Cantonal Hospital Zenica, Zenica; Bosnia and Herzegovina

ABSTRACT

Aim To analyse the resolution of chest X-ray findings in relation to laboratory parameters in patients infected with acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a two- month follow- up. Analysis of chest X-ray findings in the first few months after the disease is the main goal of our work.

Methods Out of the total of 343 patients chest X-ray findings were followed in 269 patients. Patients were divided into groups according to the severity of findings. D-dimer, inflammatory mar- Corresponding author: kers, blood cell count, neutrophil lymphocyte ratio (NLR) were Besim Prnjavorac analysed. Chest X-ray was analysed during the hospitalization Department of Pulmonology, on the day of admission, on the third, the seventh and the fourte- General Hospital Tešanj enth day (scoring method was used). After discharge chest X-ray Braće Pobrić 17, 74260 Tešanj, was performed in a two-week follow-up, then after one and two months, and after three months if necessary. Bosnia and Herzegovina Phone: +387 32 656 300; Results Incomplete chest X-ray resolution was identified in 24 Fax: 387 32 650 605; (39.34%) patients with severe, 27 (22.31 %) patients with mode- E-mail: [email protected] rate and in three (3.91%) patients with mild findings. Statistical significance was established in overall score by comparison betwe- ORCID ID: https://orcid.org/0000-0003- en all groups (p<0.001), and in the moderate compared to the mild 0331-065X group (p=0.0051). The difference of NLR in the severe compared to the moderate group was observed (p=0.0021) and in the severe group compared to the mild group (p=0.00013).

Original submission: Conclusion Chest X-ray findings persisted mostly in the severe group followed by the moderate and mild ones. Long-term follow- 05 May 2021; up is necessary for the appropriate treatment and prevention of Revised submission: fibrosis, and reduction of symptoms. 14 June 2021; Accepted: Key words: COVID-19, fibrosis, inflammation, lung 28 June 2021 doi: 10.17392/1391-21

Med Glas (Zenica) 2021; 18(2):370-377

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INTRODUCTION of inflammation? Which one of these parameters directs the inflammation towards type 1 (mostly Infection caused by acute respiratory syndrome exudative) or towards type 2 inflammation (with coronavirus 2 (SARS-CoV-2) is predominantly formation of granulomatous tissue)? What is the respiratory infection manifested most commonly role of cytokines in directing inflammation to a as interstitial pneumonia, but also other organs specific type (type 1 or type 2) (23,24). can be involved (1). Vascular complications are very common in SARS CoV-2 disease (CO- According to immunological patterns in patho- VID-19) (2). Due to pathological changes in the physiology of SARS CoV-2, type 1 inflammation blood vessels of the entire organism, COVID-19 is predominant (25,26). In late phases of the dise- can be presented as a disease involving several ase consolidations of lung parenchyma were seen. tissues and organ systems (3). The pathophysi- Condensation of lung tissue, seen on chest X-ray ology of disease can be analysed in two ways or CT scan, may be suspicious for lung fibrosis in – mostly as an analysis of organ involved in the the future. Occurrence of lung fibrosis is a irrever- disease, or analysis of tissue involved in pat- sible process, refractory to treatment. However, hogenesis of the disease. The second approach condensation of lung tissue, seen in radiologic seems to be more correct because of ubiquitous imaging, may be resolved by treatment (27). involvement of the vessels in the pathogenesis of Once started pulmonary fibrosis is a long-term pro- COVID-19 (4). The second fact to be analysed in cess, lasting for several months. Condensation of COVID-19 is a variant of the disease according the lung parenchyma seen in the acute phase of the to mostly impaired organ (5). According to re- disease does not imply fibrosis, but if left untreated, cent literature, lung variant presented as intersti- fibrosis may occur. Significant sequelae seen on tial pneumonia is present in approximately 85% chest X-rays, in the form of diffuse bright shadows, of cases (5). Involvement of other organs can be indicate the possibility of fibrosis occurrence (28). seen in other 15% of cases, with or without lung SARS CoV-2 infection has been present in the involvement at the same time (7,8). world for a relatively short period of time, about Pathogenesis of SARS CoV-2 pneumonia has 14 months. For fibrous processes in the lungs, this many patterns, different than other known pne- is not a long period of time, so the findings and umonias. In an earlier phase of the disease, type conclusions are subject to frequent changes (29). 1 of the inflammation is presented as implying Resolution of chest X-ray changes after SARS exudative patterns extensive exudation in intersti- CoV-2 infection is most often effective and ra- tial lung tissue, as well as in alveolar space (9,10). pid, but sometime it is significantly slow (30, 31). Substantial part of lung tissue is out of the function Chest CT scan is much more sensitive for dia- (11). In such a stage of the disease, the usage of gnosis of lung involvement in COVID-19 infec- mechanical ventilation (non-invasive and invasi- tion (32,33). Due to practical purposes repetitive ve) should be considered (12-14). In the immuno- chest CT scanning in post COVID-19 patients logical pattern, a decrease of absolute number of is inappropriate for follow up, chest X-ray with lymphocytes, as well as an increase in neutrophil temporally combination of chest CT scan is more to lymphocytes ratio (NLR) predominates (15,16). suitable (34,35). Moreover, NLR was significantly higher in SARS In recently published literature, we did not find CoV-2 patients, and this ratio was in correlation the topic regarding chest X-ray sequelae after with the severity of the disease (16-18). COVID-19 infection. However, in our everyday Chang revealed one of the first attempts for- qu clinical practice, we have observed many cases antification of chest X-ray sequelae (21). At the with prolonged sequelae detected on chest X- beginning of the pandemic and as it continues, nu- rays. A few scoring methods for X-ray analysis merous questions remain unanswered (22). Which were described in recent literature (19,36). one of the clinical signs, laboratory parameters The aim of this research was to find the most and/or chest X-ray findings are the most important appropriate method for chest X-ray analysis sui- for long time persistent sequelae after SARS CoV- table for comparison of changes on image findin- 2 infection still remains unknown. How the chan- gs during a follow-up period. ges in blood cell count can influence the behaviour

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PATIENTS AND METHODS method was based on an analysis of six fields of the lung (18). Patients and study design The scoring system is based on dividing chest x- Patients with SARS CoV-2 infection treated at ray image into six fields, all of which are analysed the Department of Pulmonology, Division for separately. Points belonging to each field were COVID-19 infection in General Hospital Tešanj, summarized as follows: no radiographically de- Bosnia and Herzegovina, were analysed. All tected changes 0 points; changes with only reti- 343 patients were divided in groups according cular interstitial changes 1 point; appearance of to adopted method of classification (38): “mild clear radiopacity in less than 50% of the analysed group” with limited symptoms of upper respira- field 2 points; a presence of radiopacity of more tory tract, cough, sore throat, myalgia, fatigue; than 50% of the analysed field 3 points (20). “moderate group” with productive cough, tempe- Statistical analysis rature >38 °C, prostration, chest X-ray signs of moderate, not severe pneumonia; “severe illness” Data were analysed using descriptive statistics, with prostration, drop of systolic blood pressure ANOVA and correlation test. The results of chest below 100 mmHg, extensive chest X-ray findin- X-ray scoring system in time-frame of follow up,

gs, decrease of oxygen saturation (spO2<92%). CRP, NLR and hematologic parameters in the Original classification includes additional two blood were analysed. Statistical significance was groups, asymptomatic and critical illness, but defined at the level of significance with p<0.05. these patients were not analysed in our study. RESULTS Methods Out of the total of 343 patients, 151 (44.04%) For all patients standard diagnostic protocol for were female and 192 (55.98%) male, 74 pati- SARS CoV-2 was performed. Laboratory para- ents had no signs of inflammation on chest X-ray meters including complete blood cell count with images and were excluded from further analysis. leukocyte formula, erythrocyte sedimentation Findings of chest X-ray images were followed rate, arterial blood gas analyses, d-dimer and blo- up in the time frame of 60 days for 269 patients, od sugar levels, C reactive protein (CRP), lactate 118 (43.87%) females and 151 (56.13%) males dehydrogenase (LDH), creatinine kinase (CK), (Table 1). aspartate aminotransferase (AST), alanine ami- Among the 269 patients in the group with mild notransferase (ALT), total and direct bilirubin, disease, there were 77 patients, of which 34 coagulation tests were performed. Real time PCR (44.16%) were females and 43 (55.84%) males. (RT PCR) for SARS CoV-2 infection was perfor- In the group with the moderate disease, there med for all patients (29). The neutrophil-lymphocyte ratio (NLR) was calculated in all patients. Chest X-ray was performed on the day of admission, on the third and seventh day of hospital stay and on discharge. Unscheduled chest X-rays were performed as it was needed. CT scan was performed in cases when the RT PCR SARS CoV-2 test was negative but suspicion of infection remained very high in order to confirm whether the infection is present or not, according to actual guidelines in use (34). The method of semi-quantification of chest X-ray findings for the analysis of acute inflammatory changes in the lungs in patients with RT PCR-po-

sitive COVID-19 tests used in this research, was Figure 1. Chest X-ray scoring system. The first patient: A) on the same as the methodology previously used in admission (score 4); B) after one month (score 7), C) after two Italy during SARS CoV-2 pandemic (20,37). The months (score 6) (Prnjavorac B, 2021)

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Table 1. Laboratory parameters according to the groups Table 2. Chest X-ray scoring according to severity of the Parameter Mean (± SD) (No of patients) in the group disease during two-month follow-up (reference value) Mild Moderate Severe Total Score Disease 43.77 62.89 65.26 58.91 classification At After 7 After 14 After 30 After 60 Age (18.93) (12.66) (13.52) (16.88) admission days days days days (82) (176) (85) (343) Severe 32.28 71.55 133.60 78.45 CRP 0 0 0 0 7 6 (51.32) (64.8) (114.81) (78.53) (0-8mg/L) 1-4 1 0 2 3 1 (67) (143) (74) (284) 5-6 2 0 7 10 8 58.74 55.17 49.83 54.60 pO2 7-10 2 17 14 12 10 (8.66) (10.35) (10.5) (10.48) (>70mmHg) (63) (137) (71) (271) 11-14 32 16 11 6 5 15-16 18 10 1 2 0 33.49 30.40 30.00 31.01 pCO2 (4.40) (6.54) (5.58) (5.99) 17-18 1 2 1 0 0 (35-45 mmHg) (63) (137) (71) (271) No data 5 16 25 21 31 91.57 88.94 83.83 88.04 Total 61 61 61 61 61 sO2 (3.56) (7.03) (10.82) (8.22) Moderate (>92 %) (27) (91) (42) (160) 0 0 1 9 58 47 151.20 134.08 134.46 138.24 1-4 5 3 12 9 3 Hb (12.16) (17.80) (18.51) (16.42) 5-6 1 23 41 19 17 (145 (g/L) (81) (176) (84) (341) 7-10 51 65 38 19 7 6.53 7.55 7.71 7.35 11-14 56 24 8 6 0 Le (2.63) (8.21) (4.47) (6.42) (6-10 x106L) 15-16 15 4 1 0 0 (82) (176) (86) (344) 17-18 0 0 0 0 0 27.01 19.66 14.23 20.22 Lymph No data 3 11 22 20 57 (11.78) (9.45) (8.56) (10.86) (25-30 (%) Total 131 131 131 131 131 (80) (157) (76) (313) Mild 7.13 7.29 6.40 7.08 Mid 0 0 21 48 44 31 (1.96) (4.26) (2.38) (3.39) (7-10 (%) 1-4 30 31 11 5 1 (80) (156) (76) (312) 5-6 25 10 7 2 2 65.75 73.18 79.38 72.78 Gran 7-10 18 4 4 1 0 (12.47) (10.53) (9.09) (11.76) (65-70%) (80) (156) (76) (312) 11-14 0 0 0 0 0 15-16 0 0 0 0 0 3.21 4.88 8.34 5.30 NLR (2.11) (3.03) (6.21) (4.29) 17-18 0 0 0 0 0 (3-5) (80) (157) (76) (313) No data 4 11 7 25 43 230.21 234.64 194.07 223.53 Total 77 77 77 77 77 Plt (76.69) (124.42) (73.17) (104.50) (150-300x109/L) were 131 patients, 63 (48.09%) and 68 (51.91%), (81) (174) (84) (339) 40.66 40.07 38.52 39.23 and in the group with severe illness, there were HTC (40-45%) (11.81) (3.78) (5.18) (4.22) 61 patients, 21 (34.42%) and 40 (65.58%) fema- (81) (174) (84) (339) les and males, respectively (Table 1). SD, standard deviation; CRP, C-reactive protein; pO2, partial pressure of oxygen; pCO2 partial pressure of pCO2; sO2, saturation Statistical significance was established in radio- of O2; Hb, haemoglobin; Le, leucocytes; Lymph, lymphocytes; logical analysis scoring of the chest X-ray findin- Mid, monocytes; Gran, granulocytes; Plt, platelets in blood; HTC, haematocrit; gs, by comparison between all groups (p <0.00) (Figures 1, 2). Significant negative correlation (p<0.001) was established between chest X-ray findings on the 7th day in severe and moderate group (Table 2). Among other followed parameters in the analysis of haemoglobin, haematocrit, total white blood cell count there was no statistical significance. Platelet count was different only between mild and severe (p=0.009) (Table 1). Neutrophil-lymphocyte ratio was different with statistical significance between mild and moderate group (p=0.0051), between mild and severe group (p=0.00013); the group with moderate ill- Figure 2. Chest X-ray scoring system. Second patient: A) on ness was different in comparison with the severe admission (score 5), B) after one month (score 13), C) after one p=0.0021 (Table 1). two months (score 6) (Prnjavorac B, 2021)

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The correlation analysis identified negative stati- of time, approximately 48 hours. Sometimes the stical significance between CRP level and chest highest progression was identified in the end of X-ray findings at admission and after seven days the second week after admission to the hospital.

(p<0.001). Partial pressure of oxygen (pO2) was Our patients were not admitted in the same phase significantly negatively correlated with CRP le- of the disease development. Moreover, most of vel (p<0.05). them were admitted in a later phase of the disease Residual findings were identified in chest X-ray when chest x-ray findings were widespread. images in some patients even after 60 days of Several studies have reported that NLR may follow-up (Table 2). differentiate between mild/moderate and severe/ In the group with mild disease in three (out of critical groups and probability of death in pati- 77; 3.91%) patients, chest X-ray resolution was ents with COVID-19 infection. not complete. In the group with moderate disease A systematic review and meta-analysis of Li et al. complete resolution was not identified in 27 (out (16) concluded that NLR had a good predictive of 121; 22.31%) patients; in the group with seve- value on disease severity and mortality in patients re disease incomplete resolution was identified in with COVID-19 infection. The use of NLR can 24 (out of 61; 39.34%) patients (Table 2). also help clinicians identify potentially severe cases early, which may reduce the overall mortality DISCUSSION of COVID-19. In terms of predicting the disease Late consequences of COVID- 19 infection have severity, the cut-off value in six studies covered been a topic of interest in recent literature. Accor- with meta-analysis (17) was higher than 4.5 (“high ding to the NICE guidelines used in the United cut-off value”); seven other studies used a lower Kingdom presence of symptoms related to CO- cut-off value. Similarly, ten studies that reported VID-19 disease “post COVID 19 syndrome” was the predictive value of NLR on mortality were di- defined as persistence of symptoms and signs 5 vided into “high cut-off value” (cut-off ≥ 6.5) and to 12 weeks after the disease, if it is not possible “low cut-off value” (< 6.5) subgroups (17). NLR to relate them as manifestations of other diseases is a marker of severe systemic inflammation and (34). “Long COVID” or post-COVID-19 syndro- next ongoing studies should consider the question me are still new clinical entities. Therefore, gu- of utility of it for the prediction of COVID-19 di- idelines for diagnosis and treatment are subject sease severity. We applied it on patients in our stu- to frequent changes (39). dy with the idea of determining the “cut-off” value for prediction of the disease severity. In our clini- Our prevalence of post COVID-19 sequelae ba- cal practice we have seen a correlation of NLR and sed on chest X-ray findings was substantially low severity of the disease with statistical significance as it is to be expected knowing that chest CT scan for mild and moderate cases and for severe cases is much more sensitive and rarely performed. as well. The mean cut-off value for severe cases In recently published guidelines chest CT scan in our study was 4.88 and 8.34 for moderate and is not routinely recommended for any case of severe cases, respectively. SARS CoV-2 patient (12). If the diagnosis is with Our findings in laboratory examinations overlap RT PCR test and chest X-ray findings undoubted- with worldwide findings in patients with- CO ly confirm, there is no need for performing chest VID-19 (15), identifying the absolute value of pe- CT scan. This radiologic procedure is reserved ripheral white blood cells as most often normal or for those patients with negative RT PCR SARS low, and lymphocyte count as decreased. However, CoV2 test. in severe cases with COVID-19, the lymphocytes Radiography findings peaked in the time-fra- count decreases progressively, while the neu- me 10-12 days after onset of the disease (40). trophils count gradually increases, identifying the According to the results of our study, worsening reason in excessive inflammation and/or immune of overall clinical status was in correlation to the suppression caused by SARS-CoV-2 (17). very quick progression in chest X-ray findings. As expected, in our patients, NLR was correlated Significant changes in the chest X-ray findings with CRP, that is, with the intensity of inflamma- were recorded in some cases in a short period tion. In the study of Yufei et al. among patients

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confirmed to have COVID-19, the NLR and CRP chest CT scan is capable of making better diffe- of the moderate group were lower than those of rentiation, but due to practical purpose in everyday severely ill patients (severe, critical and death clinical praxis CT scan is not available, and these groups), showing that the NLR, CRP, and lymp- findings in our study were avoided (47,48). hocyte percentages were independent risk factors Limitations of the study were a short period of for COVID-19 (18). follow-up, because the occurrence of fibrosis is It should be noted that according to our study a long process, and follow-up should be at least NLR was correlated with chest X-ray findings one year. on the 14th day after admission. This is another In conclusion, monitoring of changes in damaged confirmation of knowledge that the inflammation tissue is of great importance for the treatment de- is mostly intense in the period of 7-12 days after cision making in post COVID-19 period. Many the onset of the disease (41, 42). aspects involved in post-COVID lung patho- According to the results of our study, chest X-ray physiology should be considered, like dynamic imaging identified sequelae up to one year after changes of profibrotic interleukin (TGF-β) as the acute phase of disease. Chest X-ray imaging well as proinflammatory mediators. alone cannot accurately distinguish granulomatous changes, organized pneumonia and pulmonary FUNDING fibrosis (43-44). Recovery after SARS CoV-2 di- No specific funding was received for this study. sease is delayed in a very high percentage (45). Among 190 patients recruited for the study of TRANSPARENCY DECLARATION Sonnweber et al. at “zero date” (on discharge from Competing interests: None to declare. hospital) (46) as many as 77% chest CT scan ab- We would like to thank the Laboratory of Gene- normalities on the first visit and 63% on the se- ral Hospital Tešanj for performing the analyses. cond were found. For inflammation and fibrosis REFERENCE 1. Tong JY, Wong A, Zhu D, Fastenberg JH, Tham T. 6. Cummings MJ, Baldwin MR, Abrams D, Jacobson The Prevalence of olfactory and gustatory dysfunc- SD, Meyer BJ, Balough EM, Aaron JG, Claassen tion in COVID-19 patients: A systematic review and J, Rabbani LE, Hastie J, Hochman BR, Salazar- meta-analysis. Otolaryngol Head Neck Surg 2020; Schicchi J, Yip NH, Brodie D, O'Donnell MR. Epi- 163:3-11 demiology, clinical course, and outcomes of criti- 2. Ackermann M, Verleden SE, Kuehnel M, Haverich cally ill adults with COVID-19 in New York City: a A, Welte T, Laenger F, Vanstapel A, Werlein C, Stark prospective cohort study. Lancet 2020; 395:1763-70. H, Tzankov A, Li WW, Li VW, Mentzer SJ, Jonigk 7. Cui S, Chen S, Li X, Liu S, Wang F. Prevalence of D. Pulmonary vascular endothelialitis, thrombosis, venous thromboembolism in patients with severe and angiogenesis in Covid-19. N Engl J Med 2020; novel coronavirus pneumonia. J Thromb Haemost 383:120-8. 2020; 18:1421-4. 3. Lechien JR, Chiesa-Estomba CM, Place S, Van La- 8. Moriguchi T, Harii N, Goto J, Harada D, Sugawara ethem Y, Cabaraux P, Mat Q, Huet K, Plzak J, Ho- H, Takamino J, Ueno M, Sakata H, Kondo K, Myose roi M, Hans S, Rosaria Barillari M, Cammaroto G, N, Nakao A, Takeda M, Haro H, Inoue O, Suzuki- Fakhry N, Martiny D, Ayad T, Jouffe L, Hopkins C, Inoue K, Kubokawa K, Ogihara S, Sasaki T, Kino- Saussez S; COVID-19 Task Force of YO-IFOS. Cli- uchi H, Kojin H, Ito M, Onishi H, Shimizu T, Sasaki nical and epidemiological characteristics of 1,420 Y, Enomoto N, Ishihara H, Furuya S, Yamamoto T, European patients with mild-to-moderate coronavi- Shimada S. A first case of meningitis/encephalitis rus disease 2019. J Intern Med 2020; 288:335–44. associated with SARS-Coronavirus-2. Int J Infect 4. Helms J, Kremer S, Merdji H, Clere-Jehl R, Schenck Dis 2020; 94:55-8. M, Kummerlen C, Collange O, Boulay C, Fafi-Kre- 9. Tang N, Li D, Wang X, Sun Z. Abnormal coagulati- mer S, Ohana M, Anheim M, Meziani F. Neurologic on parameters are associated with poor prognosis in features in severe SARS-CoV-2 infection. N Engl J patients with novel coronavirus pneumonia. J Throm Med 2020; 382:2268-70. Haemost 2020; 18:844-7. 5. Batlle D, Soler MJ, Sparks MA, Hiremath S, South 10. Lee N, Hui D, Wu A, Chan P, Cameron P, Joynt GM, AM, Welling PA, Swaminathan S; COVID-19 and Ahuja A, Yung MY, Leung CB, To KF, Lui SF, Szeto ACE2 in cardiovascular, lung, and kidney working CC, Chung S, Sung JJ. A major outbreak of severe group. Acute kidney injury in COVID-19: emerging acute respiratory syndrome in Hong Kong. N Engl J evidence of a distinct pathophysiology. J Am Soc Med 2003; 348:1986–94. Nephrol 2020; 31:1380-3.

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37. Fang Y, Zhang H, Xie J, Lin M, Ying L, Pang P, Ji W. 43. Borghesi A, Zigliani A, Masciullo R, Golemi S, Ma- Sensitivity of Chest CT for COVID-19: Comparison culotti P, Farina D, Maroldi R. Radiographic severity to RT-PCR. Radiology 2020; 296:E115-7. index in COVID-19 pneumonia: relationship to age 38. Baj J, Karakuła-Juchnowicz H, Teresiński G, Bu- and sex in 783 Italian patients. Radiol Med 2020; szewicz G, Ciesielka M, Sitarz E, Forma A, Ka- 125:461–4. rakuła K, Flieger W, Portincasa P, Maciejewski R. 44. Wallace WAH, Fitch PM, Simpson JA, Howie SEM. COVID-19: Specific and non-specific clinical- ma Inflammation associated remodelling and fibrosis in nifestations and symptoms: The Current State of the lung: a process and an end point. Int J Exp Pathol Knowledge. J Clin Med 2020; 9:1753. 2007; 88:103–10. 39. Bramson RT, Griscom NT, Cleveland RH. Interpre- 45. Strieter RM. Pathogenesis and natural history of usu- tation of chest radiographs in infants with cough and al interstitial pneumonia: the whole story or the last fever. Radiology. 2005; 236: 22-9. chapter of a long novel. Chest 2005; 128:526S–32S. 40. Carfì A, Bernabei R, Landi F. Persistent symptoms 46. Sonnweber T, Sahanic S, Pizzini A, Luger A, in patients after acute COVID-19. JAMA 2020; Schwabl C, Sonnweber B, Kurz K, Koppelstätter S, 324:603-5. Haschka D, Petzer Verena, Boehm A, Aichner M, 41. Zeng Y, Fu J, Yu X, Huang Z, Yin X, Geng D, Zhang Tymoszuk Pjotr, Lener D, Theurl M, Lorsbach-Kö- J. Should computed tomography (CT) be used as a hler A, Amra Tancevski A. Cardiopulmonary reco- screening or follow-up tool for asymptomatic pati- very after COVID-19 – an observational prospective ents with SARS-CoV-2 infection? Quant Imaging multi-center trial. Eur Respir J 2020; (in press) Med Surg 2020; 10:1150–2. 47. Cool CD, Groshong SD, Rai PR, Henson PM, Scott 42. Kim HW, Capaccione KM, Li G, Luk L, Widemon SJ, Brown KK. Fibroblast foci are not discrete sites RS, Rahman O, Beylergil V, Mitchell R, D'Souza of lung injury or repair: the fibroblast reticulum. Am BM, Leb JS, Dumeer S, Bentley-Hibbert S, Liu M, J Respir Crit Care Med 2006; 174:654–8. Jambawalikar S, Austin JHM, Salvatore M. The 48. Venkataraman T, Frieman MB. The role of epider- role of initial chest X-ray in triaging patients with mal growth factor receptor (EGFR) signaling in suspected COVID-19 during the pandemic. Emerg SARS coronavirus-induced pulmonary fibrosis. An- Radiol 2020; 27:617–62. tiviral Res 2017; 143:142-50.

377 ORIGINAL ARTICLE

Clinical characteristics, comorbidities and mortality in critically ill mechanically ventilated patients with Covid-19: a retrospective observational study

Adisa Šabanović Adilović1, Nermina Rizvanović1, Mirza Kovačević1, Harun Adilović2

1Department of Anaesthesiology and Intensive Care Unit, 2Department of Internal Medicine; Cantonal Hospital Zenica, Bosnia and Herzegovina

ABSTRACT

Aim To analyse demographic data, clinical symptoms and signs, laboratory data and comorbidities in patients with COVID-19 pneumonia admitted to the intensive care unit (ICU), mechanically ventilated with fatal outcome.

Methods Medical records of 92 patients were retrospectively analysed. Demographic data, clinical symptoms and comorbidities were collected on the day of hospital admission. Clinical signs and laboratory data were collected on the day of hospital admission (T1), on the day of starting non-invasive ventilation (T2), and on the day of starting invasive ventilation (T3). Corresponding author: Results Average age of the patients was 60.05 years. Patients over Adisa Šabanović Adilović 50 years of age, 71 (77.1%) (p=0.000), and males, 62 (67.4%; Department of Anaesthesiology and p=0.001) were predominant. The most common patient symp- Intensive Care Unit, toms were exhaustion, myalgia, dyspnoea and cough. Hyperther- Cantonal Hospital Zenica mia was recorded on the day of hospital admission. Tachycardia, Crkvice 67, 72 000 Zenica, hyperglycaemia, hypoxemia were recorded at all observed study Bosnia i Herzegovina times. The most common comorbidity was hypertension arterialis with a very strong correlation with fatal outcome, followed by Phone: +387 32 447 000; diabetes mellitus and chronic heart disease that were moderately Fax: +387 32 226 576; correlated with fatal outcome. E-mail: [email protected] ORCID ID: https://orcid.org/0000-0002- Conclusion The treatment of COVID-19 patients in ICU with mechanical ventilation has a high failure rate. Demographic data, 1224-8877 clinical symptoms and signs as well as accompanying comorbidities can be a significant component in making decisions about diagnostic-therapeutic procedures. Original submission: 06 May 2021; Key words: comorbidity, fatal outcome, intensive care unit, inva- Revised submission: sive ventilation 28 May 2021; Accepted: 07 June 2021 doi: 10.17392/1394-21

Med Glas (Zenica) 2021; 18(2):378-383

378 Šabanović Adilović et al. COVID-19 patients with fatal outcome

INTRODUCTION PATIENTS AND METHODS In December 2019, acute respiratory disease, Patients and study design now known as the new pneumonia COVID-19, occurred in Wuhan, Hubei Province, China This observational, retrospective, cross-sectio- and quickly spread to other parts of the world nal study was conducted between July 2020 and (1,2). This ongoing global pandemic is caused February 2021 in the Department of Anaesthe- by severe acute respiratory syndrome corona- siology and Intensive Care Unit at the Cantonal virus 2 (SARS-CoV-2). From the first case in Hospital Zenica, Bosnia and Herzegovina. December 2019 in China to the pandemic in The study included 92 adult patients with positive March 2020 more than 120 million confirmed SARS-CoV 2 polymerase chain reaction (PCR) of cases and more than 2.8 million deaths have nasopharyngeal swabs. All observed patients were made this pandemic one of the deadliest in hi- invasively mechanically ventilated due to Co- story (3). The spectrum of clinical features of vid-19 ARDS. Patients with milder form of ARDS COVID-19 infection in the intensive care unit treated only with non-invasive ventilation (NIV) (ICU) varies from mild pneumonia to a criti- support measures were excluded from the study. cal condition with acute respiratory distress After admission at the ICU, the treatment of the syndrome (ARDS). Previous studies have des- patients followed internal institutional protocol cribed epidemiological characteristics, clinical made by the council consisting of an internist, an presentation, and outcomes of patients with infectologist and an anaesthesiologist. The drug COVID-19 pneumonia (4,5). therapy included corticosteroids, anticoagulants, Approximately one in ten patients with SARS- proton pump inhibitors, probiotics and vitamin CoV-2 becomes symptomatic (6). Symptoms of supportive therapy. Efforts were made to avoid COVID-19 are highly variable, ranging from intubation where feasible using NIV support me- asymptomatic, mild, or severe pneumonia–like asures and including prone positioning. The pati- symptoms (7). A large number of COVID-19 ents were selected for IMV by attending anaesthe- pneumonia leads to ARDS and it is usually deve- siologist according to the criteria (13). The IMV loped at day eight or nine after the symptom on- was initially managed with a lung protective venti- set (8). Reportedly, in most studies from Europe lator strategy targeting tidal volume of 6 mL/kg of and North America 10-20% of patients admitted ideal body weight, moderate positive end expira- to hospital were diagnosed with ARDS and were tory pressure (10-12 cmH2O) and plateau pressure treated with different forms of mechanical venti- <30 cmH2O. In patients with compliant lungs, ti- lation support according to the level of respiratory dal volumes were liberalized to 7-8 mL/kg of ideal failure, clinical condition and duration of illness body weight as long as plateau pressure remained (9,10). Mortality of patients with COVID19-pne- <30 cmH2O. Alternatively, some patients were umonia, especially those with the most severe switched to pressure control ventilation (14). form of ARDS when invasive mechanical venti- The Ethics Committee of the Cantonal Hospital lation (IMV) is required, is extremely high, and it Zenica approved this investigation. is up to 40.5% (11). A large number of patients hospitalized at the Methods ICU with COVID19 pneumonia have co- All data were collected from the ICU electronic morbidities that negatively affect the progno- medical report and included: demographic data, sis of the disease (12). In Bosnia and Herzego- comorbidities, clinical symptoms and signs, and vina there are no epidemiological data related to laboratory data. COVID-19 patients. Demographic data involved age and gender. The aim of our study was to analyse demo- Observed comorbidities were: diabetes mellitus, graphic data, clinical symptoms and signs, la- hypertension arterialis, chronic obstructive pul- boratory data and comorbidities in patients with monary disease (COPD), chronic heart disease, COVID-19 pneumonia admitted to the ICU and cerebrovascular disease (CVD) and malignant mechanically ventilated, with the fatal outcome. disease. Additionally, a correlation of the pre-

379 Medicinski Glasnik, Volume 18, Number 2, August 2021

valence of individual comorbidities with fatal RESULTS outcome was analysed. During eight months of the study period, 134 pa- Clinical symptoms were recorded on the day of tients were admitted to the ICU due to Covid-19 admission at hospital: cough, dyspnoea, chest ARDS and treated with various forms of respira- pain, exhaustion, abdominal pain, diarrhoea, na- tory support. The study sample consisted of 92 usea, vomiting, anorexia, headache, anosmia and patients requiring IMV and with a fatal outcome. myalgia. Clinical signs included temperature, The average age of the patients was 60.05 years heart rate, systolic and diastolic arterial pressure. (ranged between 26 and 75 years). Seventy one Laboratory data included blood glucose level, capi- (77.1%) patients were over 50 and 21 (22.9%) llary oxygen pressure and capillary carbon dioxide were under 50 years of age (p<0.000). There pressure. Blood samples were taken on the day of were 30 (32.6%) female and 62 (67.4%) male pa- hospital admission (T1), on the day of starting NIV tients (p<0.001). support measure (T2) and on the day of endotrache- The most common symptoms were exhaustion, al intubation and starting IMV (T3) (Figure 1). in 83 (90.2%), myalgia in 76 (82.6%), dyspnoea in 69 (75%) and cough in 62 (67.4%) patients. A lower prevalence of headache, anosmia, vomiting, nausea, diarrhoea, and abdominal pain was noted (Table 1). Table 1. Presentation of clinical symptoms in mechanically ventilated patients No (%) of patients with or without Symptom p (YES/ NO) symptoms 62 (67.4) / 30 (32.6) Cough 0.001 Dyspnoea 69 (75) / 23 (25) 0.000 Chest pain 46 (45.7) / 46(45.7) 0.404 Exhaustion 83 (90.2) / 9 (9.8) 0.000 Abdominal pain 9 (9.8) / 83 (90.2) 0.000 Diarrhoea 11 (11.96) / 81 (88.04) 0.000 Nausea 12 (13) / 80 (87) 0.000 Vomiting 15 (16.3) / 77 (83.7) 0.000 Anorexia 60 (67.4) / 32 (32.6) 0.004 Headache 27 (29.4) / 65 (70.6) 0.000 Anosmia 27 (29.4) / 65 (70.6) 0.000 Myalgia 76 (82.6) / 16 (17.4) 0.000 Mechanically ventilated patients with fatal outcome showed hyperthermia on the day of hospital admission, tachycardia, hyperglycaemia, and hypoxemia at all observed time periods, and hypocarbia on the day of hospital admission. In Figure 1. Flow chart of the study protocol terms of systolic and diastolic blood pressure, the Statistical analysis patients were normotensive at all observed study A descriptive analysis of the baseline and clini- time periods (Table 2). cal characteristics of the patients was performed. The most common comorbidity was hyperten- Categorical variables were presented as number sion, in 52 (56.5%) patients, followed by dia- and percentages and analysed using χ2 test. Con- betes mellitus, in 34 (37.0%) patients, while the tinuous variables were presented as median and history of malignant disease was the rarest co- interquartile range and analysed using Wilcoxon morbidity, in three (3.3%) patients. All observed rank test. A p value <0.05 was considered statisti- comorbidities showed a statistically significant cally significant. Pearson's correlation coefficient positive correlation with fatal outcome except hi- test was used to analyse the correlation of presen- story of malignant disease: hypertensio arterialis ted comorbidities with a fatal outcome. A positi- (r=0.735), diabetes mellitus (r=0.493), chronic ve correlation was considered as r>0.2: moderate heart disease (r=0.284), COPD (r=0.261), and 02.-0.5, very strong 0.5-0.8, excellent 0.8-1. malignant disease (r=0.118) (Table 3).

380 Šabanović Adilović et al. COVID-19 patients with fatal outcome

Table 2. Clinical signs and laboratory findings in mechani- vely analysed among 92 patients with COVID-19 cally ventilated patients pneumonia admitted to the ICU, mechanically Parameter Time period Median (IQR) Reference range ventilated and with fatal outcome. The average T1 37.3 (36.6-38.0) Temperature T2 36.6 (36.3-36.8) <37 age of 60 years was strongly associated with poor (°C) T3 36.5 (36.2-36.7) prognosis. On the other hand, results found in the T1 109 (90-120) Heart rate United States showed that the median age of 47 T2 102 (83-115) 60-90 (beat/minute) T3 85 (78-113) years was associated with deterioration of respi- T1 130 (120-146) ratory status of the patients (15). The number of Systolic pressure T2 124 (120-135) <140 (mmHg) males in our study was significantly higher than T3 120 (114-127) T1 80 (70-90) females. In the elderly population, COVID-19 Diastolic pressure T2 76 (69-76) <85 pneumonia predominates in females according to (mmHg) T3 71 (63.7-81.5) Guo et al. (16), as well as other studies (although T1 10.2 (8.6-12.4) Glucose T2 8.1 (6.5-8.6) 3.9-5.5 without statistical significance) (17,18). (mmol/L) T3 7.6 (6.5-8.6) Huan et al. first reported clinical features of the pati- T1 44.2 (36.2-51.6) pO2 ents with COVID-19 infection in the city of Wuhan T2 47.6 (40.5-54.1) 70-110 (mmHg) T3 45.2 (41.5-51.8) and found clinical manifestations (fever, cough, T1 33.9 (32.0-35.3) pCO2 dyspnoea, myalgia, and fatigue) were connected T2 35.1 (31.4-42.7) 32-42 (mmHg) to the younger age (19). In our study, the highest T3 36.5 (32.5-50.4) IQR, interquartile range; T1, on the day of hospital admission; T2, on prevalence of exhaustion, myalgia, dyspnoea, co- the day of starting noninvasive ventilation; T3, on the day of starting ugh, anorexia and chest pain was recorded in the invasive mechanical ventilation; pO2, capillary oxygen pressure; elderly; up to 90% of patients have more than one pCO2, capillary carbon dioxide pressure; Table 3. Prevalence of comorbidities and correlation with symptom, as it was previously reported (20). fatal outcome In our study, regarding clinical signs, hyperther- No (%) of patients Comorbidity mia was observed only on the day of hospital ad- with or without (YES/ p* r p† NO) comorbidity mission, indicating the following of the protocol related to antipyretic and anti-inflammatory dru- Diabetes mellitus 34 (37.0) / 58 (63.0) 0.000 0.493 0.000 Hypertensio arterialis 52 (56.5) / 40 (43.5) 0.000 0.735 0.000 gs during hospital treatment. COPD 13 (14.1) / 79 (85.9) 0.012 0.261 0.012 Hemodynamic instability in the form of tachycar- Chronic heart disease 15 (16.3) / 77 (83.7) 0.006 0.284 0.006 Cerebrovascular disease 11 (12.0) / 81 (88.0) 0.023 0.238 0.023 dia persisted at all observed time periods in our Malignant disease 3 (3.3) / 89 (96.7) 0.256 0.118 0.261 study as a compensatory response to the ongoing *χ2 test; †Pearson's correlation coefficient test; r, correlation coeffici- inflammatory process, hyperthermia, hypoxemia ent; COPD, chronic obstructive pulmonary disease; and consequent hypoperfusion in patients with Twenty four (26.1%) patients with one comor- COVID-19 pneumonia. Hemodynamic instabi- bidity and 24 (26.1%) with two comorbidities lity is supported by comorbidities of the observed had statistically significant correlation with fatal patients and the damage of the heart muscle due outcome; thirteen patients (14.1%) had three co- to COVID-19 infection (21). morbidities and only four (4.1%) had more than Despite therapeutic administration of insulin, three comorbidities (Table 4). hyperglycaemia was also maintained throughout Table 4. Number of present comorbidities and correlation with fatal outcome all three observed time periods in our mechanically ventilated patients with fatal outcome. This Number of No (%) of patients p* r p† comorbidities could be a sign of poorly regulated disease in pa- 1 24 (26.1) 0.000 0.383 0.000 tients with previously reported diabetes mellitus 2 24 (26.1) 0.000 0.383 0.000 (22). In patients without diabetes mellitus, hyper- 3 13 (14.1) 0.012 0.261 0.012 >3 4 (4.3) 0.188 0.137 0.191 glycaemia can be classified as a result of a strong *χ2 test; †Pearson's correlation coefficient test; r, correlation coefficient; stress response to the inflammatory process, new- onset diabetes, unrecognized pre-diabetes or direct DISCUSSION effect of the corona virus on the pancreas (23). In this observational, cross-sectional study, the de- We found a low level of capillary oxygen pressu- mographic data, clinical symptoms and signs, la- re at all three time periods, regardless of different boratory data and comorbidities were retrospecti- types of respiratory support administered. Severe

381 Medicinski Glasnik, Volume 18, Number 2, August 2021

hypoxemia, despite the application of mechanical outcome was moderate. Similarly, Phelps et al. ventilation, indicates a serious damage to the res- found that heart disease was presented in 22.1% piratory membrane due to COVID-19 infection, patients (32). consequent ARDS, and poor outcome (24,25). History of CVD was recorded in 12% patients Studies have shown a higher mortality rate in in our study with moderate correlation with fa- COVID-19 patients with pre-existing conditions tal outcome, which is in concordance with 11.9% compared to patients without comorbidities. The from Kummer et al. study (33). most common comorbidities are hypertension, In conclusion, the presented observational, cro- diabetes mellitus, cardiovascular diseases, arthri- ss-sectional study showed that patients with CO- tis, stroke and cancerous conditions (26,27). The VID-19 pneumonia admitted to the ICU, mecha- presented study confirmed a very strong correla- nically ventilated and with fatal outcome were on tion of hypertension with a poor clinical outco- average 60 years old, predominantly males. On me, and it was found in 56.5% patients, either as admission to the hospital, clinical presentation a single or in combination with other comorbi- included exhaustion, myalgia, dyspnoea, cough, dities. According to Du et al., hypertension was anorexia, chest pain, and hyperthermia, where- independently associated with increased risk of as tachycardia, hyperglycaemia, and hypoxemia mortality in 37% COVID-19 patients (28). existed until IMV administration. Concomitant In our study, diabetes mellitus was recorded in diseases strongly contributed to the fatal outco- 37.0% patients resulting in a moderate correla- me primarily in patients with previous hyperten- tion with fatal outcome similarly with 39.6% in sio arterialis, diabetes mellitus, or chronic heart Seiglie et al. study (29). disease. Close monitoring, prompt diagnostic History of COPD was revealed in 14.1% patients in and therapeutic procedures are necessary in these our research resulting in moderate correlation with patients from hospital admission to improve the fatal outcome. In contrast, Leung et al. has found clinical outcome. 33% of COPD patients (30). Patients with COPD FUNDING already have a disrupted anatomical-physiological component of the lung and increased vulnerability No specific funding was received for this study. to severe forms of COVID-19 infection (31). TRANSPARENCY DECLARATIONS Chronic heart disease was noted in 16.3% patients in our study and the correlation with fatal Competing interest: None to declare.

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383 ORIGINAL ARTICLE

Early predictors of severity and mortality in COVID-19 hospitalized patients

Sehveta Mustafić1,2, Edin Jusufović2,3, Fatima Hukić1,2, Emir Trnačević1,4, Anja Divković1,4, Alma Trnačević2,5

1Department of Laboratory Diagnostics, University Clinical Centre Tuzla, 2Faculty of Medicine, University in Tuzla, 3Department of Pulmonology, Public Educational and Health Centre, 4Pharmaceutical Faculty, University in Tuzla, 5Department of Infectious Diseases, University Clinical Centre Tuzla; Tuzla, Bosnia and Herzegovina

ABSTRACT

Aim To identify laboratory tests for early detection and the development of more severe illness and death in COVID-19 hospitalized patients.

Methods A prospective study was done on 66 hospitalized CO- VID-19 patients (males: 54.5%; mean age 70.1 ± 9.6 years) who were stratified into: moderate (n=36; 54.5%), severe (n=12; 18.2%), and critically ill (n=18; 27.3%). Besides clinical findings, a wide spectrum of laboratory parameters was monitored at admission and control during the first seven days of hospitalization and used to predict progression from non-severe to severe illness and Corresponding author: to predict the final outcome. Sehveta Mustafić Results Critically ill patients showed a higher control value of Department of Laboratory Diagnostics, white blood cell count, C-reactive protein, lactate dehydrogenase, University Clinical Centre Tuzla ferritin, but lower lymphocyte count and O2 saturation. Patients Prof. Ibre Pašića bb, 75 000 Tuzla, with fatal outcome (23; 34.85%) showed a higher control value of Bosnia and Herzegovina neutrophil, lactate dehydrogenase, ferritin, and lower lymphocyte

Phone: +387 35 303 516; and O2 saturation. Progression from moderate to severe or critical E-mail: [email protected] illness was predicted by increasing lactate dehydrogenase (95% CI 0.5803 to 0.8397;p=0.003729), increase in ferritin (95% CI ORCID ID: https://orcid.org/0000-0001- 0.5288 to 0.8221;p=0.03248), and by drop in O2 saturation (95% 6889-271X CI 0.5498 to 0.8179;p=0.01168). A fatal outcome was predicted by increase in ferritin (95% CI 0.5059 to 0.8195;p=0.04985),

as well as by drop in O2 saturation (95% CI 0.5916 to 0.8803; p=0.001861).

Conclusion Increase in ferritin, and drop in O saturation could be Original submission: 2 the most important prognostic parameters for the development of 28 January 2020; more severe clinical illness and death in COVID-19 hospitalized Revised submission: patients. 12 March 2021; Key words: ferritin, LDH, O saturation,SARS-CoV-2 Accepted: 2 01 April 2021 doi: 10.17392/1349-21

Med Glas (Zenica) 2021; 18(2):384-393

384 Mustafić et al.Predictors of severe COVID-19

INTRODUCTION Many patients deteriorate rapidly after a period of relatively mild symptoms, emphasizing the Coronavirus disease 2019 (COVID-19) is an need for early risk stratification (2,3). COVID-19 emerging infectious disease that has been decis associated with severe respiratory compromise lared a global public health emergency by the and mortality of up to 21% in hospitalized pati- World Health Organization (WHO) on February ents (14). So, it is necessary to urgently identify 28, 2020. reliable predictors of the disease severity and Although the majority of patients with CO- mortality. Comorbidities and laboratory markers VID-19 have a mild influenza-like illness or may have been proposed for risk stratification (15,16). be even asymptomatic, a small proportion of pati- There is mounting evidence that in critically ill ents develop severe pneumonia, acute respiratory patients, there are characteristics of hyperin- distress syndrome (ARDS), multi-organ failure, flammation, which consist of elevated serum and can even die (1-3). Pre-existing comorbidi- C-reactive protein (CRP), and hyperferritinemia ties such as medical histories of cardiovascular and many others. Laboratory biomarkers to fo- disease, chronic obstructive pulmonary disease recast the severity of COVID-19 are essential in (COPD), diabetes mellitus, obesity, immuno- a pandemic, because resource allocation must be compromised, renal disease, and liver disease are carefully planned, especially in the context of associated with severe disease and may have an respiratory support readiness (17). impact on the overall outcomes (1,4). Careful evaluation of laboratory indices at ba- It was shown already at the early stage of the pan- seline and during the disease course can assist demic that routine laboratory biomarkers could clinicians in formulating a tailored treatment provide some estimation. These laboratory tests approach and promptly provide intensive care to include haematological, haemostaseological, bi- those who are in greater need. ochemical and immunological parameters that The aim of this study was to identify laboratory are at least partially associated with a severe or tests for early detection and the development of mild course of the disease (5). Immune dysregu- more severe illness and death in COVID-19 pati- lation and prolonged inflammation might be the ents hospitalized at the University Clinical Hos- key drivers of the poor clinical outcomes in pa- pital Centre Tuzla. tients with COVID-19(6). Virus particles spread through the respiratory mucosa, initially using PATHIENTS AND METHODS the ACE2 receptor at ciliated bronchial epithelial cells, and infect other cells, induce a cytokine Patients and study design storm in the body, generate a series of immune responses, and cause changes in peripheral whi- This prospective study from the University Cli- te blood cells and immune cells such as lymp- nical Hospital Centre Tuzla (July to September hocytes (7,8). 2020) included 66 hospitalized patients aged 18 years or older with laboratory confirmed CO- There is consensus that in severe COVID-19 in- VID-19 infection, and pneumonia confirmed by fection, an exacerbated pulmonary and systemic chest X-ray or computerized tomography (CT), inflammatory response occurs, with increased with moderate, severe or critical degree of illne- serum levels of inflammatory markers, such as ss. COVID-19 infection was diagnosed accor- C-reactive protein (CRP), lactic dehydrogenase ding to the diagnosis and treatment of coronavi- (LDH), ferritin, D-dimer, and IL-6 (3,9,10), all of rus disease 2019 (COVID-19) recommended by which may result in the cytokine storm (10,11). the National Health Commission of China (China A hyperinflammatory environment has been a National Health Commission, 2020)(18). The la- hallmark of COVID19 infection and is thought boratory confirmed patient was defined as a posi- to be a key mediator of morbidity (12). Ferritin tive result on high throughput sequencing or real- is a key mediator of immune dysregulation, es- time polymerase-chain-reaction (RT-PCR) assay pecially under extreme hyperferritinemia, via di- of nasal and pharyngeal swab specimens. The rect immune-suppressive and pro-inflammatory illness severity of COVID-19 (mild, moderate, effects, contributing to the cytokine storm (13). severe and critical) was defined according to the

385 Medicinski Glasnik, Volume 18, Number 2, August 2021

Chinese management guideline for COVID-19 of carbon dioxide (cPCO2), capillary partial pre-

(version 7.0) (18). ssure of oxygen (cPO2), capillary blood oxygen

For intra-hospital determining progression of the saturation (cSaO2), white blood cell count (WBC), disease, the patients were divided in two groups: haematocrit (HCT), platelet count (PLT), medium non-severe (moderately ill) and severe (severely platelet volume (MPV), absolute value of neu- and critically ill). Patients with non-severe illne- trophil (NEU), neutrophil percentage (NEU%), ss were defined as COVID-19 confirmed patients absolute value of lymphocyte (LYM), lymphocyte with moderate clinical symptoms (cough, fe- percentage (LYM%), neutrophil to lymphocyte ver, respiratory symptoms, with imaging finding ratio (NLR), absolute value of monocyte (MON), of pneumonia, who needed hospital treatment). monocyte percentage (MON%), c-reactive protein Patients with “severe” illness were defined as (CRP), ferritin, lactate dehydrogenase (LDH), ala- COVID-19 confirmed patients with one of the nine aminotransferase (ALT), aspartate aminotran- following conditions: respiratory distress with res- sferase (AST), creatinine (Cr), blood urea nitrogen piratory rate (RR)>30/min, blood oxygen satura- (BUN), sodium, potassium, creatine kinase (CK), tion <93% at rest, arterial oxygen partial pressure creatine kinase isoenzyme-MB (CK-MB), high sensitive troponin I (hsTNI). The same laboratory (PaO2)/fraction of inspired O2 (FiO2) <300 mmHg, respiratory failure with mechanical ventilation, findings were also observed during the first week shock, or other extra pulmonary organ failures of their hospitalization and the worst findings were requiring intensive care unit (ICU) monitoring. observed (control value). The observed end-point was defined as recovery or Chest X-ray radiography or CT imaging was em- death recorded as "survivors" or "non-survivors". ployed to evaluate the ground-glass opacity and Survival time was defined as from illness onset to pulmonary infiltrate. death. The mortality group of non-survivors inclu- Anti-SARS-CoV-2 antibodies IgG were also ded patients who died during their hospital stay. tracked. IgG antibodies were measured between The patients with proven haematology disorders the 15th and 21st day of their hospitality stay with or with malignancy were excluded. SARS-CoV-2 IgG chemiluminescence test kit Patient’s clinical data were collected from the Me- (ABBOTT diagnostics, North Chicago, Illinois, dical Records, Laboratory Information System United States). The SARS-CoV-2 IgG assay is an (LIS) and Picture Archiving and Communicati- automated, two-step chemiluminescent micropar- on System (PACS). Final data were extracted 28 ticle immunoassay, designed for qualitative detec- days after finishing the observation. tion of immunoglobulin class G (IgG) antibodies The study was approved by the Institutional Et- to the nucleocapsid protein of SARS-CoV-2. The hics Committee of the University Clinical Hospi- resulting chemiluminescent reaction is measured tal Centre Tuzla (No 02-09/2-25/20). as a relative light unit (RLU). There is a direct relationship between the amount of IgG antibodies Methods to SARS-CoV-2 in the sample and the RLU de- A total of 50 indicators were collected from the tected by the system optics. This relationship is re- patients at hospital admission, including age, flected in the calculated index (S/C). The presence gender, pre-existing conditions (respiratory di- or absence of IgG antibodies to SARS-CoV-2 in sease, cardiac or vascular disease, diabetes, kid- the sample is determined by comparing the chemi- ney disease or other comorbidities), presenting luminescent RLU in the reaction to the calibrator symptoms (cough, dyspnoea, fatigue and wea- RLU. The Architect ci8200 system calculates the kness, symptoms from gastrointestinal tract) and calibrator mean chemiluminescent signal from 3 clinical findings (fever, blood pressure, medium calibrator replicates and stores results. The results arterial pressure-MAP, respiratory rate-RR, heart are reported by dividing the sample result by the beats, Glasgow coma score). Fever was defined stored calibrator results. The default results units as axillary temperature of 37.3°C or higher. for the SARS-CoV-2 IgG assay is index (S/C). The cut off value is 1.4 Index (S/C). Values < 1.4 Initial laboratory analysis at hospital admission of are interpretative (considered) as negative, and va- COVID-19 infection included: pH derived from lues ≥ 1.4 as positive results. capillary blood sample, capillary partial pressure

386 Mustafić et al.Predictors of severe COVID-19

Clinical and laboratory findings at admission and into two levels (0 for no and 1 for yes). The diffe- control laboratory findings were analysed and rence between the groups was considered signifi- compared between different stages of the disease cant when p<0.05. (moderate, severe, critical) and used to predict progression from “non-severely” to ”severely” ill RESULTS and to predict the final outcome in these patients. A total of 66 patients with the coronavirus disease 2019 were enrolled: 36 (54.5%) males and 24 Statistical analysis (45.5%) females, with mean age of 70.1±9.6 years Non-parametric and parametric methods are used were enrolled (the youngest patients was male, 41 to calculate statistical significance. The distribu- years old; the oldest patient was female, 89 years tion value was determined using Kolmogorov- old). Critically ill patients were younger (64.4±8.1 Smirnov and Shapiro-Wilk tests. Student's t-test, years) than moderately (71.5±10.1 years) and se- Mann-Whitney test, Fisher's test and χ2 test were verely ill patients (74.3±5.8 years). However, mo- used for calculating the difference between the gro- derately, severely and critically ill patients showed ups. ANOVA test was used to calculate the relative similar gender distribution (males: 52.8%, 41.7% difference distribution variance between variables. and 66.7%, respectively). On the other side, pati- The risk factors related to the development of se- ents with non-fatal and fatal outcome showed si- vere or critical clinical picture and mortality were milar age (69.9±9.9 vs70.4±9.2 years), and gender assessed using binary logistic regression analysis. distribution (males: 46.5 vs. 69.6%). Receiver operating characteristics (ROC) analysis According to the guidelines (18) all patients were was used to determine the optimum value of the stratified into: moderate (n=36; 54.5%), severe predictive score, and the Hanley and McNeil met- (n=12; 18.2%), and critically ill (n=18; 27.3%) hods were used to calculate the area under the cu- patients. Critically ill patients were younger, had rve. Qualitative variables were expressed as num- more comorbidities, treatment period was longer, bers and percentages, while quantitative variables and had more frequent fatal outcome than mode- were expressed as means and standard deviations rately or severely ill patients (Table 1). Cardiovas- and/or medians and interquartile ranges. Variables cular comorbidities were the most common in all included in the logistic models were categorized groups, followed by diabetes, and respiratory co- Table 1. Clinical characteristics of hospitalized COVID-19 patients with different clinical presentation according to severity of illness and outcome Severity of illness Outcome Clinical characteristic Moderate Severe Critical p Non-fatal Fatal (n=23; p (n=36; 54.5%) (n=12; 18.2%) (n=18; 27.3%) (n=43; 65.15%) 34.85%) Comorbidities (No; %) Diabetes mellitus 13 (36.1) 5 (41.7) 10 (55.6) 0.3944 16 (37.2) 12 (52.2) 0.2997 Respiratory comorbidities 7 (19.4) 2 (16.7) 2 (11.1) 0.7408 7 (16.3) 4 (17.4) 1.0000 Cardiovascular comorbidities 28 (77.8) 12 (100) 13 (72.2) 0.1472 34 (79.1) 19 (82.6) 1.0000 Renal comorbidities 6 (16.7) 0 (0.0) 2 (11.1) 0.3056 4 (9.3) 4 (17.4) 0.4346 Other comorbidities 14 (38.9) 8 (66.7) 16 (88.9) 0.0017 15 (34.9) 23 (100) <0.0001 p < 0.0001 < 0.0001 < 0.0001 - <0.0001 <0.0001 - Total (median [min, max]) 2 [1, 4] 2.5 [1, 4] 2.5 [1, 4] 0.0078 2 [0, 4] 3 [1, 4] 0.0019 Symptom (No; %) Cough 17 (47.2) 6 (50.0) 9 (50.0) 0.975 20 (46.5%) 12 (52.2%) 0.797 Breathing disorder 24 (66.7) 8 (66.7) 13 (72.2) 0.9111 29 (67.4%) 16 (69.6%) 1.0000 Gastrointestinal symptoms 13 (36.1) 6 (50.0) 9 (50.0) 0.5241 17 (39.5%) 11 (47.8%) 0.6045 Weakness and fatigue 30 (83.3) 10 (83.3) 18 (100) 0.1814 36 (83.7%) 22 (95.7%) 0.2444 p 0.0054 0.2231 0.0028 - <0.0001 0.0022 - Signs Fever (No; %) 23 (63.9) 8 (66.7) 13 (72.2) 0.8290 27 (62.8) 17 (73.9) 0.4211 SBD* (mmHg) (median [min, max]) 120 [80, 172] 120 [90, 150] 120 [86, 150] 0.8746 120 [86, 172] 120 [80, 150] 0.6766 DBP†(mmHg) (median [min, max]) 72.5 [60, 118] 70 [60, 86] 80 [50, 100] 0.5189 75.5 [50, 118] 70 [60, 90] 0.8151 MAP‡(mmHg) (median [min, max]) 95 [70, 145] 97.5 [75, 118] 101.25 [79.5, 200] 0.2071 95 [75, 145] 80 [60, 200] 0.5372 Pulse (number/minute) (median [min, max]) 84 [63, 134] 82 [76, 100] 81.5 [69, 113] 0.7759 83.5 [63, 105] 83 [69, 134] 0.9155 Glasgow coma score(median [min, max]) 15 [10, 15] 15 [10, 15] 15 [15, 15] 0.2213 15 [10, 15] 15 [10, 15] 0.8723 Days in hospital (median [min, max]) 15.5 [1,46] 18 [7,28] 24.5 [8,55] 0.0078 17 [7, 55] 19 [1, 34] 0.8526 Fatal outcome (No; %) 6 (16.7) 5 (41.7) 12 (66.7) 0.0012 - - - *Systolic blood pressure; †Diastolic blood pressure; ‡Mean arterial pressure

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morbidities (Table 1).The most common symptom Control values of AST, ALT, urea and creatinine in all groups was weakness and fatigue, followed were higher than initial values in patients with by breathing disorder, and cough (Table 1). fatal outcome only (Table 3; SDC). However, the Control values (at the first week of hospitalizati- patients with fatal outcome showed higher levels on) of lymphocytes, haematocrit, platelets, sodium of control neutrophils, neutrophils/lymphocytes ratio, LDH, ferritin, creatinine, creatine kinase, and O2 saturation were lower than initial values, but control values of leukocytes, neutrophils, ne- and both initial and control creatine kinase-MB, utrophils/lymphocytes ratio, monocytes, mean but lower levels of control monocytes (%) and platelet volume, CRP, AST, ALT, urea, creatinine, O2 saturation, as well as initial and control lymphocytes and initial neutrophils/lymphocytes ratio potassium and pCO2 were higher than initial values among all examined groups. Control values comparing to patients with non-fatal outcome. of pH and pO2 were lower than initial values, but Anti-SARS-CoV-2 IgG showed also similar dis- control values of LDH were higher than initial va- tribution between patients with fatal and non-fa- lues among severe and critically ill patients. Ferri- tal outcome (Table 3; SDC). tin was higher on control in critically ill patients, Binary logistic regression analysis showed that only. Creatine kinase was lower on control among the risk factors (significantly associated with moderately and critically ill patients, but creatine progression from moderately to severely or cri- kinase-MB was lower on control in moderately ill tically ill) were an increase in LDH and ferritin,

patients, only. When comparing specific groups, as well as a drop in O2 saturation. The risk fac- critically ill patients showed higher initial haema- tors significantly associated with mortality were

tocrit, creatine kinase-MB, and neutrophils/lymp- the drop of ferritin and O2 saturation (Table 4). hocytes ratio, as well as higher control mean pla- Progression from moderate to severe or critical telet volume, leukocytes, CRP, ferritin, but lower illness was predicted by increasing LDH (area

lymphocytes and O2 saturation than other two under the curve-AUC 0.71; 95% confidence in- groups. On the other hand, moderately ill patients terval - CI95% 0.5803 to 0.8397;p=0.003729),

showed lower control urea, creatinine, and pCO2 increase in ferritin (AUC 0.6755;CI95% 0.5288

than other two groups. Anti-SARS-CoV-2 IgG to 0.8221; p=0.03248), and by drop in O2 satu- showed similar distribution among moderately, ration (AUC 0.6838; CI95% 0.5498 to 0.8179; severe and critically ill patients (Table 2; SDC). p=0.01168). Patients with fatal outcome had more comorbiditi- In the prediction of progression to severe or cri- es than patients with non-fatal outcome. However, tical form of the disease for LDH increase values in both groups, with fatal and non-fatal outcome, of >18.5 and >54.5, sensitivity and specificity the most common were cardiovascular comorbidi- was 70.0% and 62.86%, as well as 63.33% and ties followed by diabetes, and then respiratory co- 74.29%, respectively. On the other hand, for ferri- morbidities. Also, in both groups the most common tin increase values of >433.5 and > 730.7, sensi- symptom was weakness and fatigue, followed by tivity and specificity was 53.57% and 69.57%, breathing disorder, and cough (Table 1). as well as 42.86% and 82.61%, respectively. At Control values of absolute number of lymphocyte, the same time, in the prediction of the disease progression to severe or critical form, sensitivity lymphocytes (%), sodium and O2 saturation were lower than initial values, but control values of and specificity were 53.33% and 79.41%, and absolute neutrophils, neutrophils/lymphocytes Table 4. Binary logistic regression analysis of factors related ratio, absolute and percentage of monocytes, to the development of progression from moderately to LDH, ferritin, and creatine kinase-MB were severely or critically ill and to fatal outcome higher than initial values among both groups of Variable 95% confidential interval p patients, with fatal and with non-fatal outcome. Lower Upper Progression to severely or critically ill Supplemental Digital Content (SDC) 1.Table 2. Laboratory Increase of LDH 2.194 67.224 0.005 findings between hospitalized COVID-19 patients with differ- Increase of ferritin 1.314 100.768 0.029 ent clinical presentation Drop in O2 saturation drop 3.431 102.119 0.0012 Supplemental Digital Content (SDC) 2. Table 3. Laboratory Fatal outcome findings between COVID-19 patients with non-fatal and fatal Increase of ferritin 1.004 26.111 0.022 outcome Drop in O2 saturation drop 1.05 24.601 0.041

388 Mustafić et al.Predictors of severe COVID-19

Figure 1. ROC curve analysis of increase in ferritin (A and B) and drop in O2 saturation (C and D) in patients with fatal outcome

56.67% and 64.71% for the drop in O2 saturation tients. This finding suggests that comorbidities of <-14.15 and <-11.00, respectively. associated with aging rather than advanced age Fatal outcome was predicted by increase in itself contribute to a worse prognosis. ferritin (AUC 0.6627; CI95% 0.5059 to 0.8195; However, crude case fatality ratio obtained by p=0.04985), as well as by drop in O2 satura- dividing the number of deaths by the number of tion (AUC 0.7359; CI95% 0.5916 to 0.8803; cases can be misleading (21). During a growing p=0.001861) (Figure 1). In the prediction of fatal epidemic, the final clinical outcome of most of the outcome, sensitivity and specificity was 57.14% reported cases is typically unknown (22). The in- and 66.67 %, and 52.38% and 83.33%, respec- hospital mortality of severe and critically ill pati- tively, for ferritin increase values of >433.5 and ents with COVID-19 could be up to almost 40%

>730.7, but for a values of drop in O2 saturation (23). The mortality rate in our study was 34.85%. of <-18.00 and <- 13.95 they were 52.17% and In our analysis those patients at highest risk for 87.8%, and 69.57% and 80.49%, respectively. severe disease and death included people over the age of 64 years and those with underlying con- DISCUSSION ditions. In Fei et al. (23) study in-hospital death One of the risk factors most strongly associated was higher in patients with diabetes or coronary with severe COVID-19 and death is advanced age. heart disease. The median age of patients in our study was 71 In contrast to many previous studies which relied years, compared to 76 years of Italian, Americans, on single data points, we estimated the association British and Spanish patients (19). Mandeep et al. between longitudinal measurements at admission (20) showed that out of the total of 8910 patients and control repeated measurements of laboratory 80.9% were below 60 years of age and 5.8% died markers and clinical outcomes. Several clinical in the hospital. Although the majority of patients and laboratory variables used in this study showed in our study were of older life age, there were no differences between different stages of the disease significant differences between survivors and non- already at admission but those were even more si- survivors. In our study in the critically ill group, gnificant during the hospital stay. the patients were younger, had more comorbidi- Among haematological parameters, lymphope- ties, were treated longer, and had more frequent nia is clearly associated with disease severity. fatal outcome than moderately or severely ill pa- The same as the other papers published previo-

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usly (26, 27), our data showed that the lymp- inflammatory biomarkers we monitored serum hocyte percentage descends with the disease level of CRP, LDH and ferritin. development, which indicates the direct result In our study, increased CRP value was associated of viral infection. We also found that both the with severe COVID-19, but it is not independent higher white blood cell count and absolute value of mortality. In the study Guan et al. (9) disease se- of neutrophil during first seven days of hospita- verity was associated with elevated CRP, and the lization were associated with the higher risk of primary composite endpoint (admission to an inten- severity (28). Although not significant, haema- sive care unit, use of mechanical ventilation, or de- tological abnormalities found in this study were ath) in their study was also associated with elevated more prominent among severe versus non-severe CRP. Also, Deng et al. (30) found that patients in cases. These results were consistent with three the death group exhibited significantly higher CRP other descriptive studies that were conducted in level on admission, and CRP levels remained high China (2,3,29). Huang et al. and Wang et al. (2,3) after the treatment in the non-survivors. highlighted an association between lymphopenia Elevated LDH indicates cell death and injury and and a need for ICU care, whereas Wu et al. (29) is associated with a poor host immune response, showed an association between lymphopenia and resulting in a higher susceptibility to severe vi- acute respiratory distress syndrome (ARDS) de- ral infections (32,33). Since LDH is present in velopment. It has also been reported that patients lung tissue (isozyme 3), patients with severe CO- with severe disease and fatal outcomes present VID-19 infections can be expected to release gre- with a decreased lymphocyte/white blood cell ra- ater amounts of LDH in the circulation (34). Early tio both on admission and during hospitalization data in COVID-19 patients have suggested signi- compared with those who survived (29). Con- ficant differences in LDH levels between patients trary to non-survivors, survivors demonstrated a and without severe disease (35). As the increasing nadir of lymphocytes count on day 7 from symp- experience with COVID-19 worldwide, numero- tom onset and subsequent restoration (23). The- us studies found that LDH was associated with refore, serial assessment of lymphocyte count the severity and poor outcomes of COVID-19 dynamics may be predictive of the patient outco- (32,34,36,37). In this study, we also proved that me. In our study baseline lymphocyte count was serum LDH was an independent prognostic fac- significantly higher in survivors than non-survi- tor for patients with COVID-19. vors; in survivors, whereas severe lymphopenia was also observed in non-survivors, but was not There are multiple publications showing that an independent predictor of mortality. higher ferritin levels, along with other pro-inflammatory markers are correlated with worse Coagulation disorders are relatively frequently en- outcomes and may even help predict these outco- countered among COVID-19 patients, especially mes (23,38-40). Particularly critically ill patients among those with severe disease (23,30). Altho- may exhibit an extreme increase in ferritin levels ugh a few studies showed a correlation between as an indication of a macrophage activation syn- thrombocytopenia and severity of the COVID-19 drome (41). This condition is most commonly disease, in our study there was no significant triggered by viral infections, which might lead to thrombocytopenia on admission but there was a a hypothesis of SARS-CoV-2 inducing this hyper slight fall-down during hospitalization in all exa- inflammatory syndrome (42). mined groups. A meta-analysis of nine studies has suggested that thrombocytopenia is significantly Our data showed that the common top predictors associated with the severity of the COVID-19 di- of progression from non-severe to severe disease sease, and a more sizeable drop in platelet counts were an increase in LDH and ferritin, and reduction in O saturation. At the same time, the best was noted especially in non-survivors (31). 2 predictors of mortality were an increase of ferri- Inflammatory biomarker characteristics of- pa tin levels and a drop in O saturation. The simi- tients with laboratory confirmed SARS-CoV-2 2 lar data were found in a retrospective analysis of infection have been described, and serum ferri- Ruan et al. clinical predictors of mortality in 150 tin seems to be relevant for assessing the dise- patients, higher ferritin concentrations correlated ase severity and outcome of patients (5). Of the with fatal disease development compared to pa-

390 Mustafić et al.Predictors of severe COVID-19

tients that were discharged (40). However, in our In our study there was no statistically significant study hyperferritinemia was observed in majority difference in antibody levels between severe and of patients on admission, as well as throughout non-severe patients nor between patients with fa- the clinical course, and increased with illness de- tal and non-fatal outcome. The number of patients terioration. Levels of serum ferritin were clearly was small in the present study, which might result elevated in non-survivors compared with survi- in less accurate results. More cases and time pe- vors on admission. The similar data were also riod tests are needed to verify these results. The found by Kappert et al. (5). similar data were found by Shang at al. (26).

We also found that a decrease of O2 saturation in In conclusion, timely analysis of the laboratory capillary blood sample is also strongly associated characteristics associated with COVID-19 can with severity of disease and mortality. Severe res- assist in the clinical prognosis. The increase in piratory failure and death caused by COVID-19 ferritin, increase in LDH and drop in O2 saturation may result from damaged alveoli and oedema could be the most important prognostic parame- formation, negatively affecting the lung’s abi- ters for the development of more severe clinical lity to oxygenate blood, as reflected in reduced illness and death in COVID-19 hospitalized pati- oxygen saturation (43). In studies by Han et al. ents. Early identification and adequate treatment and Erez et al. the common top predictors of ICU of COVID-19 patients at high risk for acute respi- admission and mortality were elevated LDH and ratory failure are paramount to avoid acute respi- ferritin, and reduced SpO2 (32,33). Xie et al. also ratory distress syndrome and end-organ damage, reported age, lymphocyte count, LDH and SpO2 and can probably reduce the death rate. to be independent predictors of mortality but a risk score was not developed (44). FUNDING It was previously reported that the serum antibody No specific funding was received for this study. in COVID-19 had potential diagnostic value and the higher titter of antibody was independently TRANSPARENCY DECLARATION associated with a worse clinical classification (45). Conflict of interests: None to declare REFERENCES 1. Nandy K, Salunke A, Pathak SK, Pandey A, Doctor 5. Kappert K, Jahić A, Tauber R. Assessment of serum C, Puj K, Sharma M, Jain A, Warikoo V. Corona- ferritin as a biomarker in COVID-19: bystander or virus disease (COVID-19): a systematic review and participant? Insights by comparison with other in- meta-analysis to evaluate the impact of various co- fectious and non-infectious diseases. Biomarkers morbidities on serious events. DiabetesMetabSyn- 2020;25:616-25. dr2020;14:1017-25. 6. Tahaghoghi-Hajghorbani S, Zafari P, Masoumi E, 2. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang Rajabinejad M, Jafari-Shakib R, Hasani B, Rafiei A. L, Fan G, Xu J, Gu X, Cheng Z, Yu T, Xia J, Wei Y, The role of dysregulated immune responses in CO- Wu W, Xie X, Yin W, Li H, Liu M, Xiao Y, Gao H, VID-19 pathogenesis. Virus Res 2020;290:198197. Guo L, Xie J, Wang G, Jiang R, Gao Z, Jin Q, Wang 7. Rodríguez-Morales AJ, MacGregor K, Kanagarajah J, Cao B. Clinical features of patients infected with S, Patel D, Schlagenhauf P. Going global - travel and 2019 novel coronavirus in Wuhan, China. Lancet the 2019 novel coronavirus. Travel Med Infect Dis 2020; 395:497-506. 2020;33:101578. 3. Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, 8. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang Liu L, Shan H, Lei CL, Hui DSC, Du B, Li LJ, Zeng W, Si HR, Zhu Y, Li B, Huang CL, Chen HD, Chen G, Yuen KY, Chen RC, Tang CL, Wang T, Chen PY, J, Luo Y, Guo H, Jiang RD, Liu MQ, Chen Y, Shen Xiang J, Li SY, Wang JL, Liang ZJ, Peng YX, Wei L, XR, Wang X, Zheng XS, Zhao K, Chen QJ, Deng F, Liu Y, Hu YH, Peng P, Wang JM, Liu JY, Chen Z, Li Liu LL, Yan B, Zhan FX, Wang YY, Xiao GF, Shi G, Zheng ZJ, Qiu SQ, Luo J, Ye CJ, Zhu SY, Zhong ZL. Addendum: a pneumonia outbreak associated NS; China Medical Treatment Expert Group for Co- with a new coronavirus of probable bat origin. Na- vid-19. Clinical characteristics of coronavirus disea- ture2020;588:6. se 2019 in China. N Engl J Med 2020; 382:1708-20. 9. Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, 4. Zhu N, Zhang D, Wang W, L, Yang B, Song J, Zhao Liu L, Shan H, Lei CL, Hui DSC, Du B, Li LJ, Zeng X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang G, Yuen KY, Chen RC, Tang CL, Wang T, Chen PY, D, Xu W, Wu i X G, Gao GF, Tan W; China Novel Xiang J, Li SY, Wang JL, Liang ZJ, Peng YX, Wei L, Coronavirus Investigating and Research Team. A Liu Y, Hu YH, Peng P, Wang JM, Liu JY, Chen Z, Li Novel Coronavirus from patients with pneumonia in G, Zheng ZJ, Qiu SQ, Luo J, Ye CJ, Zhu SY, Zhong China, 2019. N Engl J Med 2020;382:727-33. NS; China medical treatment expert group for Co- vid-19. Clinical characteristics of coronavirus disease 2019 in China. N Engl J Med 2020; 382:1708-20.

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10. Mehta P, McAuley DF, Brown M, Sanchez E, Tatter- 25. Li T, Lu H, Zhang W. Clinical observation and ma- sall RS, Manson JJ; HLH Across Speciality Colla- nagement of COVID-19 patients. Emerg Microbes boration, UK. COVID-19: consider cytokine storm Infect 2020; 9:687-90 syndromes and immunosuppression. Lancet 2020; 26. Dymond T. The Effects of viral infection on lymp- 395:1033-4. hocyte metabolism: a new perspective on disease 11. Hirano T, Murakami M. COVID-19: a new virus, but characterization. Viral Immunol 2018; 31:278-81. a familiar receptor and cytokine release syndrome. 27. Liu Z, Long W, Tu M, Chen S, Huang Y, Wang S, Immunity 2020; 52:731-3. Zhou W, Chen D, Zhou L, Wang M, Wu M, Hu- 12. Feld J, Tremblay D, Thibaud S, Kessler A, Nayma- ang Q, Xu H, Zeng W, Guo L. Lymphocyte subset gon L. 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Clinical characteristics of 113 S, Chao Y, Yang Z, Xu J, Zhou X, Chen D, Xiong deceased patients with coronavirus disease 2019: re- W, Xu L, Zhou F, Jiang J, Bai C, Zheng J, Song Y. trospective study. BMJ 2020; 368:1091. Risk factors associated with acute respiratory distre- 15. Pranata R, Huang I, Lukito AA, Raharjo SB. Ele- ss syndrome and death in patients with coronavirus vated N-terminal pro-brain natriuretic peptide is disease 2019 pneumonia in Wuhan, China. JAMA associated with increased mortality in patients with Intern Med 2020; 180:934-43. COVID-19: systematic review and meta-analysis. 30. Deng Y, Liu W, Liu K, Fang YY, Shang J, Zhou L, Postgrad Med J 2020; 96:387-91. Wang K, Leng F, Wei S, Chen L, Liu HG. Clinical 16. Huang I, Pranata R. Lymphopenia in severe corona- characteristics of fatal and recovered cases of coro- virus disease-2019 (COVID-19): systematic review navirus disease 2019 in Wuhan, China: a retrospecti- and meta-analysis. 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Erez A, Shental O, Tchebiner JZ, Laufer-Perl M, are-india-s-covid-19-patients-11586241562779. Wasserman A, Sella T, Guzner-Gur H. Diagnostic html(13 October 2021) and prognostic value of very high serum lactate 20. Mehra MR, Desai SS, Kuy S, Henry TD, Patel AN. dehydrogenase in admitted medical patients. Isr Med Cardiovascular disease, drug therapy, and mortality Assoc J 2014;16:439-43. in Covid-19. N Engl J Med 2020; 382:102 34. Henry BM, Aggarwal G, Wong J, Benoit S, Vikse J, 21. Lipsitch M, Donnelly CA, Fraser C, Blake IM, Cori Plebani M, Lippi G. Lactate dehydrogenase levels A, Dorigatti I, Ferguson NM, Garske T, Mills HL, predict coronavirus disease 2019 (COVID-19) se- Riley S, Van Kerkhove MD, Hernán MA. Potential verity and mortality: apooled analysis. Am J Emerg biases in estimating absolute and relative case-fa- Med 2020 Sep; 38:1722-6. tality risks during outbreaks. PLoSNegl Trop Dis 35. Henry BM, de Oliveira MHS, Benoit S, Plebani 2015; 9:0003846. M, Lippi G. 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Poggiali E, Zaino D, Immovilli P, Rovero L, Losi G, and risk factors for mortality of adult in patients with Dacrema A, Nuccetelli M, Vadacca GB, Guidetti D, COVID-19 in Wuhan, China: a retrospective cohort Vercelli A, Magnacavallo A, Bernardini S, Terracci- study. Lancet 2020; 395:1054-62. ano C. Lactate dehydrogenase and c-reactive protein 24. Velavan TP, Meyer CG. Mild versus severe CO- as predictors of respiratory failure in COVID-19 pa- VID-19: laboratory markers. Int J Infect Dis 2020; tients. ClinChimActa 2020; 509:135-8. 95:304-7.

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38. Giamarellos-Bourboulis EJ, Netea MG, Rovina N, 42. Thomas L, Thomas C. Detection of iron restriction Akinosoglou K, Antoniadou A, Antonakos N, Da- in anaemic and non-anaemic patients: New diagno- moraki G, Gkavogianni T, Adami ME, Katsaounou stic approaches. Eur J Haematol 2017; 99:262-8. P, Ntaganou M, Kyriakopoulou M, Dimopoulos G, 43. Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, Koutsodimitropoulos I, Velissaris D, Koufargyris P, Liu S, Zhao P, Liu H, Zhu L, Tai Y, Bai C, Gao T, Karageorgos A, Katrini K, Lekakis V, Lupse M, Kot- Song J, Xia P, Dong J, Zhao J, Wang FS. Patholo- saki A, Renieris G, Theodoulou D, Panou V, Kouka- gical findings of COVID-19 associated with acute ki E, Koulouris N, Gogos C, Koutsoukou A. Com- respiratory distress syndrome. Lancet Respir Med plex immune dysregulation in COVID-19 patients 2020; 8:420-2. with severe respiratory failure. Cell Host Microbe 44. Xie J, Wu W, Li S, Hu Y, Hu M, Li J, Yang Y, Huang 2020; 27:992-1000. T, Zheng K, Wang Y, Kang H, Huang Y, Jiang L, 39. Li Y, Hu Y, Yu J, Ma T. Retrospective analysis of Zhang W, Zhong M, Sang L, Zheng X, Pan C, Zheng laboratory testing in 54 patients with severe- or cri- R, Li X, Tong Z, Qiu H, Du B. Clinical characte- tical-type 2019 novel coronavirus pneumonia. Lab ristics and outcomes of critically ill patients with Invest 2020; 100:794-800. novel coronavirus infectious disease (COVID-19) in 40. Ruan Q, Yang K, Wang W, Jiang L, Song J. Clini- China: a retrospective multicenter study. Intensive cal predictors of mortality due to COVID-19 based Care Med 2020; 46:1863-72. on an analysis of data of 150 patients from Wuhan, 45. Jin Y, Wang M, Zuo Z, Fan C, Ye F, Cai Z, Wang Y, China. Intensive Care Med 2020; 46:846-8. Cui H, Pan K, Xu A. Diagnostic value and dynamic 41. Rosário C, Zandman-Goddard G, Meyron-Holtz variance of serum antibody in coronavirus disease EG, D'Cruz DP, Shoenfeld Y. The hyperferritinemic 2019. Int J Infect Dis 2020; 94:49-52. syndrome: macrophage activation syndrome, Still's disease, septic shock and catastrophic antiphospho- lipid syndrome. BMC Med 2013; 11:185.

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Table 2. Laboratory findings in hospitalized COVID-19 patients with different clinical presentations (continued) Laboratory finding Moderately ill Severely ill Critically ill p (reference value) (n=36; 54.5%) (n=12; 18.2%) (n=18; 27.3%) Initial 8.2±4.28 10.6±6.18 7.95±5.6 0.3912 Leukocytes x109/L Control 16.09±8.39 18.12±5.76 23.0±14.85 0.0494 (3.4-9.7 ) p 0.0309 0.0004 0.0002 - Initial 5.41±2.18 8.64±6.88 7.12±5.67 0.3592 Neutrophilsx109/L Control 12.75±8.67 16.06±7.64 15.1±5.27 0.263 (2.06-6.49 ) p 0.0122 0.0021 < 0.0001 - Initial 75.28±13.44 78.61±15.36 76.69±11.84 0.9235 Neutrophils % Control 81.77±10.55 88.85±5.6 89.13±4.52 0.6421 (44-72) p 0.0228 0.2263 0.1277 - Initial 1.13±0.55 1.15±0.56 0.96±0.37 0.6385 Lymphocytes x109/L Control 0.95±0.4 0.91±0.37 0.52±0.25 0.0038 (1.19-3.35) p 0.3652 0.6487 0.0429 - Initial 16.66±10.63 13.65±11.77 16.28±9.61 0.9905 Lymphocytes % Control 10.5±6.91 6.01±2.98 5.29±3.72 0.5307 (20-46) p 0.0165 0.0014 < 0.0001 - Initial 0.93±1.91 1.22±1.24 0.63±0.7 0.5935 Neutrophils/Lymphocytes ratio Control 17.36±13.82 20.84±5.88 30.66±14.84 0.0035 p < 0.0001 < 0.0001 < 0.0001 - Initial 0.45±0.17 0.48±0.33 0.38±0.21 0.4396 Monocytes x109/L Control 0.92±0.37 0.88±0.32 0.92±0.31 0.9308 (0.12-0.84) p < 0.0001 0.0079 < 0.0001 - Initial 7.0±3.26 4.78±2.18 5.75±2.94 0.0701 Monocytes % Control 10.27±3.31 9.66±4.31 8.62±3.39 0.2741 (2-12) p < 0.0001 0.0053 < 0.0001 - Initial 0.39±0.06 0.33±0.06 0.4±0.04 0.0048 Haematocrit L/L Control 0.34±0.06 0.28±0.07 0.27±0.07 0.0315 (M:0.415-0.530; F:0.356-0.470) p 0.0006 0.0009 < 0.0001 - Initial 203.17±91.91 233.75±157.45 218.28±88.42 0.6648 Plateletsx109/L Control 167.97±73.20 164.50±111.72 177.39±64.87 0.8883 (158-424) p 0.0003 0.0133 0.0065 - Initial 9.12±1.94 9.59±2.52 9.38±1.75 0.6909 Mean Platelet VolumefL Control 11.19±1.15 11.55±1.56 12.53±2.58 0.0231 (6.8-10.4) p 0.0294 0.001 < 0.0001 - Initial 56.44±88.46 25.67±11.93 48.28±35.48 0.4098 Aspartate Aminotransferase U/L Control 78.91±130.57 60.17±43.32 81.06±35.38 0.8278 (M:15-40; F:13-35) p 0.014 0.0185 0.0003 - Initial 63.06±95.48 16.25±7.12 54.39±55.67 0.1949 Alanine Aminotransferase U/L Control 97.26±132.67 63.42±61.77 108.22±71.5 0.5234 (M: 10-40; F:7-35) p 0.008 0.0194 0.0012 - Initial 12.19±11.69 12.0±6.49 11.06±8.75 0.5273 Urea mmol/L Control 14.03±12.24 27.6±13.21 27.65±21.04 0.0014 (2.1-7.1) p 0.0348 0.0022 0.0007 - Initial 156.08±159.94 145.08±65.94 137.0±79.44 0.8719 Creatinine µmol/L Control 164.03±169.12 342.42±104.32 296.11±242.96 0.006 (M:80-115; F:53-97) p 0.0073 < 0.0001 0.0068 - Initial 136.06±4.74 139.08±3.18 136.0±3.94 0.0927 Sodium mmol/L Control 134.29±4.10 135.58±4.08 134.0±4.04 0.5537 (136-145) p 0.0004 0.0018 0.0105 - Initial 4.45±1.14 3.78±1.0 4.08±0.77 0.7565 Potassium mmol/L Control 4.60±1.09 4.28±1.31 5.15±1.25 0.3585 (3.5-5.1) p 0.0183 0.0012 0.0008 - Initial 137.13±165.62 141.45±115.92 241.18±226.01 0.1239 Creatine kinase U/L Control 172.86±148.70 332.33±425.07 354.73±298.08 0.1052 (M:38-174; F:26-140) p 0.037 0.1698 0.0026 - Initial 20.10±8.31 18.55±4.25 28.47±8.47 0.001 Creatine kinase isoenzyme-MB U/L Control 25.91±9.78 36.78±38.67 38.6±21.83 0.1749 (<25) p 0.0005 0.2141 0.1013 - Initial 32.23±52.41 29.21±30.89 25.06±33.96 0.3351 Troponin pg/mL Control 73.91±116.81 10215.97±24879.6 1303.13±3326.53 0.3058 (M:0.0-34.2; F:0.0-15.6) p 0.3085 0.0853 0.1862 - Initial 94.23±83.28 107.36±61.43 103.41±70.35 0.5351 C reactive protein mg/L Control 167.85±91.37 206.81±108.02 223.86±68.74 0.0027 (<5.0) p 0.0056 0.0085 < 0.0001 - Initial 336.19±122.96 267.33±85.31 438.33±352.24 0.0781 LDH U/L Control 536.28±807.19 396.09±153.92 766.28±353.22 0.2647 (100-190) p 0.1624 0.0212 0.0002 - Initial 700.48±490.80 630.22±380.15 732.24±696.59 0.4716 Ferritin µg/L Control 1109.20±498.16 1186.5±1112.85 1841.86±1762.19 0.0012 (M:22-322; F:10-291) p 0.6346 0.6346 0.0009 - Initial 7.45±0.09 7.42±0.08 7.46±0.06 0.2216 pH Control 7.39±0.08 7.34±0.09 7.39±0.09 0.0682 (7.33-7.44) p 0.0879 0.0004 0.0013 -

394 Mustafić et al.Predictors of severe COVID-19

pCO2kPA Initial 3.81±0.81 3.97±0.59 3.6±0.77 0.1175 (4.69-6.42) Control 4.77±0.66 5.69±1.74 4.59±0.92 0.0397 p 0.0151 0.0006 0.0015 - Initial 7.78±1.42 7.4±1.57 7.27±1.21 0.9276 pO2kPA Control 6.23±1.02 6.09±1.5 5.21±1.02 0.806 (9.5-12.66) p 0.5454 0.0044 < 0.0001 - Initial 89.94±6.75 87.41±10.14 89.06±5.24 0.7671 O2 saturation % Control 82.14±7.59 72.72±12.42 70.54±13.18 0.0053 (85-90) p 0.0085 0.0007 < 0.0001 - Anti-SARS-CoV-2 IgG (S/C) - 6.56±2.17 5.71±2.47 7.47±1.36 0.0723 (≥ 1.4 positive) M, male; F, female

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Table 3. Laboratory findings in COVID-19 patients with non-fatal and fatal outcome (continued) Laboratory finding Non-fatal outcome Fatal outcome p (reference value) (n=43; 65.15%) (n=23; 34.85%) Initial 8.14±4.17 7.94±5.62 0.943 Leukocytes x109/L Control 14.31±6.79 22.91±11.63 0.2855 (3.4-9.7) p 0.3202 0.1754 - Initial 6.55±4.45 6.34±5.15 0.8644 Neutrophils x109/L Control 11.4±6.52 16.63±6.54 0.0029 (2.06-6.49) p 0.0001 < 0.0001 - Initial 76.57±12.95 76.69±12.0 0.4278 Neutrophils % Control 83.24±8.6 89.74±5.96 0.8331 (44-72) p 0.7396 0.2695 - Initial 1.03±0.49 0.99±0.55 < 0.0001 Lymphocytes x109/L Control 0.84±0.34 0.59±0.31 < 0.0001 (1.19-3.35) p 0.037 0.0039 - Initial 16.04±10.34 16.01±9.42 0.9905 Lymphocytes % Control 9.99±5.8 4.5±2.59 0.6263 (20-46) p 0.0012 < 0.0001 - Initial 0.97±1.8 0.78±0.94 < 0.0001 Neutrophils/Lymphocytes ratio Control 15.83±9.93 32.46±14.54 0.0286 p < 0.0001 < 0.0001 - Initial 0.45±0.22 0.4±0.21 0.3480 Monocytes x109/L Control 0.91±0.34 0.91±0.37 0.9962 (0.12-0.849 p < 0.0001 < 0.0001 - Initial 6.62±3.13 5.57±2.98 0.1919 Monocytes % Control 10.61±3.29 8.02±3.44 0.0039 (2-12) p < 0.0001 0.0132 - Initial 0.39±0.06 0.38±0.07 0.3644 Hematocrit L/L Control 0.33±0.06 0.3±0.06 0.0506 (M:0.415-0.530; F:0.356-0.470) p 0.4383 0.4873 - Platelets x109/L Initial 201.49±93.0 234.09±122.7 0.2302 (158-424) Control 166.88±72.99 175.57±88.67 0.6709 p 0.0583 0.0705 - Initial 9.29±1.83 8.83±2.32 0.7034 Mean Platelet VolumefL Control 11.28±1.37 12.01±2.08 0.9995 (6.8-10.4) p 0.343 0.3114 - Initial 50.28±83.46 45.52±23.86 0.7905 Aspartate Aminotransferase U/L Control 76.83±120.16 74.52±37.34 0.929 (M:15-40; F:13-35) p 0.2411 0.003 - Initial 57.16±90.96 42.87±43.48 0.4805 Alanine Aminotransferase U/L Control 96.22±124.23 90.04±69.75 0.8271 (M:10-40; F:7-35) p 0.1029 0.0086 - Initial 12.45±12.77 11.5±7.41 0.5019 Urea mmol/L Control 15.99±14.58 29.43±16.64 0.1588 (2.1-7.1) p 0.7496 0.0368 - Initial 153.56±148.55 140.13±75.55 0.6866 Creatinine µmol/L Control 194.39±169.57 306.35±224.54 0.0279 (M:80-115; F:53-97) p 0.2432 0.0016 - Initial 136.69±4.6 136.43±4.04 0.8241 Sodium mmol/L Control 134.68±4.28 134.04±4.06 0.5492 (136-145) p 0.0388 0.0516 - Initial 4.25±0.9 4.16±0.8 0.8454 Potassium mmol/L Control 4.59±0.86 4.92±1.06 0.3783 (3.5-5.1) p 0.5311 0.5438 - Initial 135.11±150.82 221.1±218.91 0.2022 Creatine kinase U/L Control 193.45±167.5 382.65±381.83 0.0217 (M:38-174; F:26-140) p 0.353 0.1380 - Initial 20.58±7.95 25.19±9.3 0.0494 Creatine kinase isoenzyme-MB U/L Control 25.3±8.26 43.94±32.31 0.0047 (<25) p 0.0197 0.0213 - Initial 28.27±55.15 44.0±57.59 0.2069 Troponin pg/mL Control 205.24±810.3 4146.±14042.8 0.4939 (M:0.0-34.2; F:0.0-15.6) p 0.2485 0.1854 - Initial 97.53±85.13 98.06±55.81 0.7241 C reactive protein mg/L Control 166.69±94.56 235.27±86.92 0.3278 (<5.0) p 0.0852 0.1252 -

396 Mustafić et al.Predictors of severe COVID-19

Initial 336.98±134.4 378.74±313.54 0.452 LDH U/L Control 430.63±184.59 823.78±956.75 0.0164 (100-190) p 0.0102 0.0397 - Initial 660.7±510.34 908.17±613.78 0.0945 Ferritin µg/L Control 1033.68±582.48 1858.63±1600.79 0.0124 (M:22-322; F:10-291) p 0.0058 0.0131 - Initial 7.45±0.1 7.45±0.07 0.5174 pH Control 7.39±0.09 7.36±0.09 0.7322 (7.33-7.44) p 0.4949 0.7446 - Initial 3.86±0.99 3.63±0.72 0.9592 pCO2kPA Control 5.12±1.18 4.96±1.17 0.8234 (4.69-6.42) p 0.282 0.5161 - Initial 7.93±1.59 7.6±2.53 0.5217 pO2kPA Control 6.17±1.07 5.78±2.87 0.7142 (9.5-12.66) p 0.9333 0.9732 - Initial 90.12±6.96 88.7±6.0 0.4085 O2 saturation % Control 81.37±9.03 70.24±11.32 < 0.0001 (85-90) p < 0.0001 < 0.0001 - Anti-SARS-CoV-2 IgG (S/C) (≥ 1.4 positive) - 6.69±2.0 6.6±2.32 0.8633 M, male; F, female

397 ORIGINAL ARTICLE

Mesenchymal stem cells under hypoxia condition inhibit peritoneal adhesion by suppressing the prolonged release of interleukin-6 Adi Muradi Muhar1, Agung Putra2-4, Vito Mahendra Ekasaputra5, Dewi Masyithah Darlan6, Desiree Ang- gia Paramita1, Iqbal Pahlevi Adeputra Nasution1

1Department of Surgery, Faculty of Medicine, Universitas Sumatera Utara, Medan, North Sumatera, 2Stem Cell and Cancer Research (SCCR), Medical Faculty, Universitas Islam Sultan Agung (Unissula), Semarang, Central Java, 3Department of Postgraduate Biomedical Science, Medical Faculty, Universitas Islam Sultan Agung (Unissula), Semarang, Central Java, 4Department of Pathological Anatomy, Medical Faculty, Universitas Islam Sultan Agung (Unissula), Semarang, Central Java, 5Department of Surgery, Medical Faculty, Univer- sitas Islam Sultan Agung (Unissula), Semarang, Central Java, 6Department of Parasitology, Faculty of Medicine, Universitas Sumatera Utara, Medan, North Sumatera; Indonesia

ABSTRACT

Aim To investigate the role of hypoxia-preconditioned mesenchymal stem cells (H-MSCs) in preventing peritoneal adhesion by regulating IL-6 at days 6 and 12.

Methods Twenty-four PAs rat model weighing 250 g to 300 g were randomly allocated into 4 groups: sham (Sh), control (C), Corresponding author: H-MSCs treatment group at dose 1.5 x 106 (T1) and 3 x 106 (T2). Agung Putra To induce H-MSCs, all MSCs population were incubated under o Sultan Agung Islamic University hypoxia state (5% O2), 5% CO2, and 37 C for 24 hours. Expre- ssion level of IL-6 was performed using ELISA. Morphological Kaligawe Raya Km. 4 Semarang Central appearance of adhesion was observed by visualizing the existence Java 50112, Indonesia of adhesion formation in intestinal. Phone: +62 8164251646; E-mail: [email protected] Results In this study we found that there was a trend of decrease of IL-6 level on day 6 following MSCs treatments. Interestingly, Adi Muradi Muhar ORCID ID: /orcid. there was a significant decrease of IL-6 level on day 12 in all tre- org/0000-0002-7727-1154 atment groups. Also, no adhesion occurred in T2 group.

Conclusions H-MSCs prevent PA development by suppressing the prolonged release of IL-6 at proliferation phase.

Key words: abdominal pain, abdominal surgery, cellular, inflammatory cytokines Original submission: 24 October 2020; Revised submission: 14 January 2021; Accepted: 04 June 2021 doi: 10.17392/1304-21

Med Glas (Zenica) 2021; 18(2):398-403

398 Muhar et al. Mesenchymal stem cells under hypoxia

INTRODUCTION tiation capacity into various specific mature cells such as adipocyte, osteocyte, chondrocyte and ne- Peritoneal adhesion (PA) is the fibrous adhesion urocyte (12). These immunomodulatory properti- that mostly occurs following abdominal surgery es of H-MSCs are potentially employed to control and prolonged intra-abdominal inflammation proinflammatory peritoneal by attenuating IL-6, (1). The incidence of PA post general abdominal which is associated with PA development. laparotomy ranges from 60 to 90% and reaches up to 97% post gynaecologic pelvic operation Peritoneal healing is initiated by the proliferation in which 10-20% of those may develop serious of new mesothelium in surrounding cellular ma- health problems, such as bowel obstruction that trix at the injury time up to 3 - 5 days. One major potentially increases mortality and morbidity step in PA formation is inadequate fibrinolytic ac- (2,3). The inflammation is the main actor in PA tivity to degrade the fibrin gel matrix containing formation due to the release of several cytoki- coagulation factors and inflammatory cells over nes, particularly IL-6 that have responsibility in the damaged peritoneal surface (13). IL-6 released maintaining the organization of fibrin gel matrix by inflammatory cells actively promotes the coa- leading to PA development (4). Delayed removal gulation cascade by increasing tissue factor and fi- of the fibrin gel matrix during remesothelializati- brinogen production without affecting fibrinolysis on process of wound peritoneal healing indicated (14). However, prolonged release of IL-6 may also inadequate activities of fibrinolytic in lysing the stimulate plasminogen activator PAI-1 and PAI-2 fibrin matrix associated with the continuous rele- to inhibit fibrinolytic activity leading to PA deve- ase of IL-6 (5-6). However, IL-6 also has a res- lopment (15). On the other hand, the enhancement ponsibility in promoting cellular proliferation du- of IL-6 at the early inflammatory phase may also ring the intestinal wound healing (7). Therefore, stimulate intestinal cell proliferation that is impor- to prevent PA formation, the control of inflamma- tant for wound peritoneal healing (16). The role of tion using immunosuppressive agents, including H-MSCs in preventing PAs has been explored by mesenchymal stem cells under hypoxia condition our previous study reporting that H-MSCs could (H-MSCs) is a crucial point to be explored. inhibit PA formation by accelerating the shift of inflammatory to proliferation phase through incre- The importance of the H-MSCs in the physio- asing IL-10 (17). Therefore, regulating IL-6 as one logy of multipotent cellular species is responsi- of the responsible factors in PA formation through ble for tissue organogenesis, including intestinal H-MSCs administration during peritoneal healing regeneration in PA. Accordingly, the initial stage remains to be elucidated. of repair at the wounds site, including PA occurs in a hypoxia milieu in which heterogeneous cell The aim of this study was to analyse the role of populations including MSCs are present. A pre- H-MSCs in suppressing the prolonged release of vious study reported that H-MSCs have a robust IL-6 during peritoneal adhesion prevention. ability in inhibiting PA formation than MSCs MATERIAL AND METHODS through accelerating the shift of inflammation to proliferation phase (8) leading to the acceleration Study design and animal model of injured tissues healing (9). The hypoxia precon- dition employed on MSCs in vitro could generate A total of 24 healthy 8-week-old male Wistar rats distinctive changes in stem cell characteristics and weighing 300 ± 30 gr (cv= 10%) were reared, influence the secretion of cytokines and growth acclimatized, and fed ad-libitum under 12 h light- factors. Moreover, mimicking the natural micro- dark photoperiod in battery cages at 24oC, 60% humidity. 12-h fasted rats were anesthetized in- environment by decreasing in vitro O2 tension on MSCs could also provide the optimum capability traperitoneally using ketamine and xylazine (90 of stem cell migration in vivo (10,11). H-MSCs as mg/kg and 10 mg/kg body weight, respectively). multipotent cells express the high level of speci- Briefly, 25 2 mm standardized injury area was fic markers such as CD29, CD44, CD73, CD90, applied to the right sidewall of ileum by scrub- CD105 and CD166 and lack of the expression of bing with a cytobrush (Gynobrush, Langenbrink, CD11b, CD14, CD34, CD45, or, CD79a or CD19, Emmendingen, Germany) until the punctuate red and HLA class II. H-MSCs also have the differen- colour visually appeared as an indication of ile-

399 Medicinski Glasnik, Volume 18, Number 2, August 2021

um trauma. The abdominal incision was sutured 10-7 mol/L/ 0.1 μM dexamethasone, 10 mmol/L with 3–0 polygelatin suture and the rats were re- β glycerophosphate, and 50 μmol/L ascorbate-2- ared in battery cages for 12 days. phosphate (Sigma-Aldrich, Louis St, MO) under

The study was approved by the Experimental 5% CO2, 37°C temperature, and 95% humidity Animal's Ethics Committee of the Medical Fa- until the calcium deposition was formed that could culty of Universitas Islam Sultan Agung (Unissu- be visualized after alizarin red staining (Sigma- la), Semarang, Indonesia. Aldrich Corp., St. Louis, MO, USA). H-MSCs preparation and administration. The Methods 6th passage RUC-MSCs were incubated in the MSCs isolation and culture. Mesenchymal stem hypoxic chamber under 5% O2, 37°C tempera- cells (MSCs) were isolated aseptically from the ture, and 95% humidity for 24 h, then collected 19-day pregnant female Wistar rats’ umbilical cord and diluted in 0.5 mL NaCl for the following ex- (RUC) by mincing into 1-2 mm-sized pieces and periment. The induced PAs rats were randomly carefully transferred into a T25 culture flask (Cor- divided into 4 groups: sham (S) received no heal- ning, Tewksbury, MA, USA). The culture medium ing agent, control (C) received NaCl injection contained DMEM (Gibco Invitrogen, NY, USA), around the abrasion site, T1 and T2 respectively 10% FBS (GibcoInvitrogen, NY, USA), 1% peni- received 1.5x106 and 3 x106 HMSCs injection cillin (100 U/mL) and 0.25% streptomycin (100 around the abrasion site. µg/mL) (GibcoInvitrogen, NY, USA) was added IL-6 level analysis. The blood samples from 6 and replaced every 3 days. The cultured umbilical and 12 days after the treatment were collected

cord (UC) was incubated under 37°C, 5% CO2 and from vena orbitalis and centrifuged at 3000×g 95% humidity condition until the cells reached for 10 minutes to obtain the serum. All samples 80% confluence (14 days). The 6th passaged were stored at –80°C until analysis. IL-6 level MSCs-like cells were used in this study. was analysed using an enzyme-linked immuno- MSCs characteristic. RUC-MSCs-like cells were sorbent assay (ELISA) plate reader (Bio-Rad, confirmed by analysing MSCs specific markers Berkeley, California, US) and IL-6 ELISA kit and the capability to differentiate into mature cells. (Wuhan Fine Biotech Co., Ltd, Wuhan, China). The 5th passage of RUC-MSCs-like was stained Macroscopic analysis. The induced PAs rat’s

with fluorescence-labelled specific MSCs - anti animal model was euthanized using CO2 inha- body including FITC mouse anti-rat CD45 (Clone lation in the 12th day after the treatment. The OX-1, 554877, BD Biosciences, CA, USA), PE adhesion formation was observed, categorized mouse anti-rat CD31 (Clone TLD-3A12, 555027, using Nair’s Adhesion Grade (18). Macroscopic BD Biosciences) PerCP mouse anti rat-CD90.1 grading was assessed by two general surgeons (Clone OX-7, 557266, BD Biosciences) and Alexa using the modification tool of Leach grade and flour 647 hamster anti-rat CD29 (Clone Ha2/5, Nair grade. The Nair’s grade was divided into 562153, BD Biosciences) and examined using a four grades according to the adhesion: grade 0 BD C6 Plus flow cytometer (BD Biosciences) and - no adhesion, grade 1 - only one adhesion band BD FACSDivaTM software (BD Biosciences). In between abrasion site and abdominal wall, grade accordance with the International Society of Cel- 2 - two adhesion bands between abrasion site and lular Therapy (ISCT) (12) the MSCs have speci- abdominal wall grade 3 - more than two adhesion fic marker profile, such as CD90 and CD29 and bands between abrasion site and abdominal wall, negative of Lineage marker (CD45/CD34/CD31/ and grade 4 - the highest grade is marked with CD11b/CD19/HLA-DR). adhesion of all viscera to the abdominal wall. MSCs osteogenic differentiation. The capability Statistical analysis of RUC-MSCs-like cells to differentiate into mature cell was confirmed using osteogenic differen- Descriptive data were expressed in mean±standard tiation. The RUC-MSCs-like cells at the 5th pas- error. For intergroup analysis, the Kruskal-Wallis sage were incubated in osteogenic differentiation variance analysis was used to analyse significant medium contained DMEM (Sigma-Aldrich, Louis differences among the groups. A p<0.05 was con- St, MO), 10% FBS (Gibco Invitrogen, NY, USA), sidered statistically significant.

400 Muhar et al. Mesenchymal stem cells under hypoxia

RESULTS The UC-MSCs expressed CD90.1 (53.8±0.92 %) and CD29 (97.1±0.87 %), and lacked the expression of CD31 (4.73±1.62 %) and CD45 (1.73±0.38 %) (Figure 1). Furthermore, MSCs showed fibroblast- like with spindle shape characteristic and became 80% confluent after 5-7 days culture (Figure 2A), and osteogenic differentiation analysis confirmed calcium deposition appearance following bright red colour after alizarin red staining (Figure 2B).

Figure 3. ELISA assay shows the significant increase of IL-6 level on T2 at day 6 (A), which became significantly decreasing following 12 days after the treatment (B (p<0.05)

12 following abdominal surgery. The Nair’s macroscopic adhesion grade was shown. There was Figure 1. Detection of UC-MSCs by flow cytometry demon- adhesion grade 4 in the control group, while T1 strates positive expression of CD 90.1, CD29, and negative showed adhesion grade 1. Moreover, there was expression of CD31 and CD45 no adhesion band in T2 (Grade 0) (Figure 4).

Figure 2. Morphology and differentiation of cultured mesenchymal stem cells. A) Umbilical cord candidate mesenchymal stem cells (MSCs) from in-vitro culture after 24h hypoxia; B) Osteogenic differentiation with alizarin red staining To determine the ability of MSCs in suppressing prolonged release of IL-6 associated with PA development, we assessed the level of IL-6 in proliferation and remodelling phases using ELISA on Figure 4. Peritoneal adhesion PAs were shown in the control group (A) (black arrow). There was grade 1 adhesion on T1 day 6 and 12. A trend of decrease of IL-6 level on (B), while no adhesion was shown in the T2 group (C) (red day 6 following MSCs treatments was found. In- arrow), which was similar to the sham group (D) (black arrow) terestingly, there was a significant decrease of IL-6 Table 1. IL-6 level after 6- and 12-days mesenchymal stem level on day 12 in all treatment groups (p<0.05), in cells (MSCs) treatment in peritoneal adhesion (Pas) rat model which T2 group showed the optimum decrease of IL-6 Level Group IL-6 level (6.82 ± 5.86 pg/mL) (Figure 3). Day 6 (pg/mL) Day 12 (pg/mL) To evaluate the PAs adhesion formation, a ne- Sham 58.36 ± 3.22 61.32 ± 2.78 Control 81.25 ± 4.84 84.46 ± 9.41 cropsy was performed by opening the abdominal T1 77.14 ± 5.85 70.48 ± 7.48 cavity through a reverse U-shape incision on day T2 75.56 ± 9.81 65.82 ± 5.86

401 Medicinski Glasnik, Volume 18, Number 2, August 2021

DISCUSSION that the peritoneal healing processes were not accomplished yet. Prolonged release of IL-6 may A study regarding the role of H-MSCs in pre- induce the activation of PAI1 and PAI2 as fibri- venting PA formation had previously reported a nolytic inhibitor triggering PA formation (15). result in effectively controlled adhesion through This finding is consistent with previous studies suppressing inflammation at the sites of surgical which reported that the delayed fibrin gel matrix (17,19) and modulating regulatory T cells (Treg) removal during the remesothelialization process (20). However, the role of H-MSCs in regulating of wound peritoneal healing potentially induces the inflammatory milieu-released IL-6 as the main PA development (25). In line with the increase initiator cytokines in PA formation has not been of IL-6 in the control group, the Nair’s macros- assessed. This is the first study that examines the copic of PA also obviously occurred in grade 3-4 H-MSC capabilities to control the release of IL-6 adhesion. Another study revealed that IL-6 is one during wound peritoneal healing particularly un- of the chemoattractant factors to mobilize the en- der the proliferation and remodelling phases. The dogenous MSCs to specific injury in addition to mechanisms of peritoneal healing differ from that leucocyte cells (27). The IL-6-induced endothelial of skin injury in which the new mesothelium deve- cells express E selectin that has an important role loping from islands of mesothelial cells proliferate in wound healing particularly for attracting stro- simultaneously into sheets of cells not from pe- mal cells including mesothelial cell and MSCs ripheries to the centre of injured areas as represen- (28). These findings suggested that H-MSC can ted by the reepithelialisation of skin healing (21). optimally control inflammation by suppressing Therefore, no time differentiation in peritoneal IL-6, however, the H-MSCs also simultaneously healing between the larger peritoneal wounds stimulate the proliferation of intestinal cell injury and small peritoneal wounds was due to all of the in an early inflammatory phase in the group trea- remesothelialization process covering injured pe- ted by H-MSCs. This indicated that H-MSCs have ritoneum within 5–6 days. Thus, to generate the robust ability to control the inflammatory milieu. PA animal model we scrubbed the right sidewall Furthermore, in terms of stimulating proliferation, of the ileum using a cytobrush to result in prolon- the existence of IL-6 in early inflammation was a ged inflammation that potentially triggers PA de- critical point due to the IL-6, it also has a role in velopment as described by a previous study (22). stimulating epithelial intestinal proliferation (26). In this study, the decrease of IL-6 level in the tre- This study has some limitations, in which the IL-6 atment groups indicates that H-MSC administra- in early inflammatory phase was not investigated. tion may robustly control the inflammation by Hence, we have no clear observation of the role decreasing pro-inflammatory cytokines involving of IL-6 as a stimulating factor for proliferation of the IL-6 levels. The competence of H-MSC in peritoneal injury. We did not analyse the PAI-1 and engrafting into the injury site and suppressing the PAI-2, as well as tPA as the critical molecule con- inflammatory milieu has been shown by several trolled by IL-6 associated with PA development. studies (23). Under controlled IL-6 level following Therefore, the association between IL-6 and PAI- H-MSC administration in those healing phases 1, PAI-2 and tPA still remains unclear. correlated with the optimum fibrinolysis activities in lysing the organization of a fibrin gel matrix le- In conclusion, our study showed H-MSCs could ading to inhibition of PA formation (24). The ac- attenuate the IL-6 level in PA through signifi- tivation of tissue plasminogen activator (tPA) and cantly decreasing IL-6 level on day 12. The rese- urokinase plasminogen activator in surgical sites arch was conducted based on the lack of research induce the plasmin to degrade fibrin matrix as the on the capability of MSCs as a potential immu- main initiator of PA formation (25). This finding is nomodulator in preventing PA development by in line with another study that reported the accumu- suppressing the prolonged release of IL-6 at a lation of H-MSCs in an injury site may accelerate proliferation phase. Therefore, this study co- the mesothelial proliferation that was necessary for uld provide beneficial knowledge for the - deve effective wound intestinal healing (17). lopment of PA patients. Our results showed an increasing trend of IL-6 ACKNOWLEDGEMENTS in the control group starting on day 6 and continuous increase significantly on day 12 indicating The authors gratefully acknowledge that this study is supported by Deputi Bidang Penguatan Riset

402 Muhar et al. Mesenchymal stem cells under hypoxia

dan Pengembangan, Kementrian Riset dan Tekno- FUNDING logi/Badan Riset Inovasi Nasional 2020, with the DRPM contract number : 11/AMD/E1/KP/ contract number 11/AMD/E1/KP/PTNBH/2020, PTNBH/2020, tanggal 11 May 2020. tanggal 11 May 2020. We would also like to thank the Stem Cell and Cancer Research Laboratory, TRANSPARENCY DECLARATION Medical Faculty, Universitas Islam Sultan Agung Semarang for all facilities to finish this research. Conflict of interests: None to declare.

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403 ORIGINAL ARTICLE

Association of rs211037 GABRG2 gene polymorphism with susceptibility to idiopathic generalized epilepsy

Marija Milanovska1,2, Emilija Cvetkovska1, Sasho Panov3

1University Clinic of Neurology, Saints Cyril and Methodius University, 2Neuromedica Hospital, 3Department of Molecular Biology and Genetics, Faculty of Sciences, Saints Cyril and Methodius University; Skopje, Republic of North Macedonia

ABSTRACT

Aim This case-control study aimed to determine a possible association of single nucleotide polymorphism rs211037 of the gamma-aminobutyric acid receptor subunit gamma-2 (GABRG2) gene with the susceptibility to idiopathic generalized epilepsy (IGE) in the Macedonian population.

Methods It enrolled 96 patients with clinically verified IGE and 51 healthy individuals without personal and family history of epilepsy or other neurological disorders as controls. A determination of the GABRG2 rs211037 polymorphism was performed using the Corresponding author: TaqMan-based genotyping assay. Emilija Cvetkovska University Clinic of Neurology Results A significant dominant association of the CC genotype (odds ratio - OR=2.100, 95% CI=1.018-4.332; p=0.043) and alle- Vodnjanska str. 17, MK-1000 Skopje, lic association of C allele (OR=1.902, CI=1.040-3.477; p=0.035) Republic of North Macedonia with susceptibility to IGE was found. Carriers of CC genotype Phone: +389 70 338 372; had approximately a 2-fold higher probability of developing IGE E-mail: [email protected] than the carriers of CT and TT genotypes. Carriers of the C allele Marija Milanovska ORCID ID: https:// had a 1.9-folds higher probability for IGE than the carriers of the orcid.org/ 0000-0003-1652-4661 T allele. Conclusion The polymorphism rs211037 of the GABRG2 gene increases the risk of the development of idiopathic generalized epilepsy in the Macedonian population.

Key words: case control studies, epilepsy, GABA receptors, single nucleotide polymorphism Original submission: 02 March 2021; Revised submission: 27 March 2021; Accepted: 25 April 2021 doi: 10.17392/1367-21

Med Glas (Zenica) 2021; 18(2):404-409

404 Milanovska et al. GABRG2 gene in idiopathic epilepsy

INTRODUCTION PATIENTS AND METHODS The gamma-aminobutyric acid type A (GABA ) A Patients and study design receptors are the major inhibitory neurotrans- mitter receptors in the mammalian brain and are Consecutive 96 patients aged ≥14 years with idi- crucial in controlling the activity of neuronal opathic generalized epilepsies (IGE) attending the networks (1,2). The GABAA receptor is a penta- Epilepsy Outpatient Clinic of a University Hospi- meric chloride ion channel and consists of two tal for Neurology in Skopje from March 1 2019 α, two β, and one γ subunits, encoded by the to February 28 2020 were enrolled. Patients (IGE gamma-aminobutyric acid type A receptor subgroup) were eligible for the study if they were unit alpha1 (GABRA1), gamma-aminobutyric meeting the criteria of the International League acid type A receptor subunit beta2 (GABRB2), Against Epilepsy (ILAE) guidelines for the classi- and gamma-aminobutyric acid type A receptor fication of seizures and epilepsy syndromes (16). subunit gamma2 (GABRG2) genes, respective- Specifically, we included patients with four well- ly, and with the most common subunit composi- established IGE syndromes: childhood absence tion being α1β2γ2 (1,3). Among epilepsy-asso- epilepsy (CAE), juvenile absence epilepsy (JAE), ciated mutations or variants in different GABAA juvenile myoclonic epilepsy (JME), and genera- subunits, a substantial amount is found in the lized tonic–clonic seizures alone (GTCS alone). GABRG2 gene and they are connected with a The diagnosis was made on clinical grounds, variety of seizures and epilepsy types from self- supported by the finding of generalized spike and limiting febrile seizures (FS) to drug-resistant wave or polyspike and wave pattern in EEG. epilepsies with comorbidities and epileptic en- We routinely performed 20-minute awake EEG cephalopathies (4-7). recording with standard activation procedures GABRG2 gene is located on the long arm of (hyperventilation and photostimulation). If the chromosome 5 (5q34). The rs211037 single initial EEG assessment was normal or inconclu- nucleotide polymorphism (588C>T or Asn196A- sive, we proceeded with a prolonged (2-hours) sn) in exon 5 on the GABRG2 gene results in a EEG, which was done in the morning after who- synonymous variant allele that may change its le night sleep-deprivation. Patients with normal expression affecting the transcription, mRNA sleep-deprived EEG were not included in the stu- stability, abnormal subunit folding, as well as dy, because of a lower degree of diagnostic cer- aberrant glycosylation and translation efficiency tainty for IGE. Patients with lesional MRI were of the GABRG2 receptor synthesis. Consequ- excluded from the study, too. The patients with ently, the receptor response to extrinsic envi- CAE were originally diagnosed and treated at ronmental signals may be altered by still un- Children’s hospital, and then because their seizu- known mechanisms (8). The results of the studies res persisted after the age of 14 years, they conti- that have examined exonic GABRG2 rs211037 nued their treatment at the Clinic for Neurology, locus have been inconsistent and the frequency where they were recruited for the study. of variants seems distinctive in different ethnical General information such as age, sex, prenatal groups (9-13). A few of them shed light on its po- and perinatal history, intellectual and motor de- ssible link with susceptibility to febrile seizures velopment as well as data regarding the history (FS) and idiopathic generalized epilepsy (IGE) of epilepsy i.e. age of onset and seizure types, specifically (14-15). family history, treatment, and seizure control was Given the reported variance in allele frequen- collected. cies and susceptibility to IGE between diverse Further, 51 age- and gender-matched healthy indi- populations, as well as lack of similar genetic viduals from the general population were recruited investigation in the Macedonian population, we as non-epilepsy controls. Subjects with personal performed a case-control study aimed to explore or family history of epilepsy or any other neurolo- the possible association of GABRG2 rs211037 gical disorders were excluded from the study. polymorphism with the susceptibility to IGE. The Ethics Committee of the Medical Faculty at the of Saints Cyril and Methodius University in

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Skopje approved this case-control study and all Table 1. Selected demographic and clinical characteristics participants signed informed written consents be- of the patients with idiopathic generalized epilepsy (IGE) and controls fore participating in the study. Characteristic IGE patients (n=96) Controls (n=51) Gender (No; %) Methods Males 47 (49 %) 27 (53 %) Females 49 (51 %) 24 (47 %) A venous blood sample (2-3 mL) was collected Age (mean ± SD) (range) (years) from each participant into an EDTA tube and Age overall 32.6 ± 13.6 (14-78) 39.7 ± 6.7 (28-53) the genomic DNA was extracted using Pure- Age at seizure onset 17.2 ± 12.5 (5-52) / Link Genomic DNA Mini Kit (Thermo Fisher SD, standard deviation Scientific) and the extracted DNAs were sto- The genotype and allele frequencies of the GA- red at -20°C. Determination of the GABRG2 BRG2 rs211037 polymorphism were determined rs211037 polymorphism was performed using in all participants (Table 2). The distributions of TaqMan fluorescence probes-based real-time analysed genotype frequencies were within the polymerase chain reaction amplification genoty- Hardy–Weinberg equilibrium (p>0.05) in both ping (Thermo Fisher Scientific, Thermo Fisher IGE patient and control groups. Scientific, Waltham, MA, USA) on a StepOne Table 2. Genotype frequencies in patients with idiopathic RT-PCR System (Applied Biosystems, Applied generalized epilepsy (IGE) patients and controls Biosystems, Foster, CA, USA) according to the GABRG2 rs211037 No (%) of subjects in the group manufacturer's instructions. The genotype calling polymorphism IGE patients Controls was performed based on the allele-specific -flu Genotype CC 72 (75.00) 30 (58.82) orescence by allelic discrimination utility of the CT 20 (20.83) 17 (33.33) StepOne software included in the system. TT 4 (4.17) 4 (7.84) Allele Statistical analysis C 164 (85.42) 77 (75.49) T 28 (14.58) 25 (24.51) Genotype and allele frequencies of the GA- The homozygous wild CC genotype was overre- BRG2 rs211037 polymorphism were determined presented among the IGE patients when compa- by allele counting. χ2 test or Fisher's exact test red against the control group. Genetic analysis (two-tailed, when the subgroups had less than 5 reveals a significant association of the samples) were used to estimate the significance CC genotype using the dominant model of genetic association with susceptibility to IGE. (OR=2.100, 95% CI=1.018-4.332; p=0.043) (Ta- The odds ratio (OR), as well as the corresponding ble 3). Carriers of CC genotype had approxima- 95% confidence interval (95% CI) were calcula- tely a two-fold higher probability for the deve- ted to evaluate polymorphism association with lopment of IGE than the carriers of heterozygous the probability-susceptibility to IGE. The asso- CT and homozygous TT genotypes. Similarly, ciation was considered significant when the p-va- a significant association of C allele with IGE lue was <0.05. Hardy-Weinberg equilibrium was susceptibility was found by the allelic model calculated by the χ2 test. Statistical and populati- (OR=1.902, 1.040-3.477; p=0.035) implying that on genetic analyses were performed by XLSTAT the carriers of C allele had 1.9 folds higher pre- 2016 and GenAlEx 6.5 software. disposition to IGE when compared to those with RESULTS a T allele. Table 3. Genotypes and allele distribution The IGE patients and control participants had Genetic model Genotype OR (95% CI) p matched gender and were of comparable age Dominant CC vs. CT + TT 2.100 (1.018 to 4.332) 0.043 Allelic C vs. T 1.902 (1.040 to 3.477) 0.035 structure. The JME was the most common syndrome presented in 52 (54%) patients (29 fe- DISCUSSION males and 23 males). Thirty-six (38%) patients fulfilled the criteria for GTCS alone (17 females This is the first study to evaluate the association and 19 males). Eight (8%) patients had absence of GABRG2 rs211037 polymorphism with the epilepsy (five with JAE and three with CAE). susceptibility to idiopathic generalized epilepsy in the population of patients in the Republic of

406 Milanovska et al. GABRG2 gene in idiopathic epilepsy

North Macedonia. We found that the presence of genotype/C allele) was found to be a risk factor C allele variant and CC genotype increased by for febrile seizures, focal seizures, and symp- the 2-fold probability of developing IGE in the tomatic epilepsy (SE) in Asians, particularly in Macedonian population. To our best knowledge, Chinese (12). On the other side, several studies there is only one prior study evaluating this ge- failed to present any evidence for association of netic association in Southeast Europe (15). Inte- GABRG2 rs211037 polymorphism with epilepsy restingly, in the former case-control study from susceptibility in general (10), as well as in diffe- Romania evaluating 114 children with IGE or FS, rent cohorts of temporal lobe epilepsy (TLE): GABRG2 Asn196Asn TT genotype polymorphi- familial TLE preceded by FS in the USA cohort sm was found to carry a 45 and 8 times higher risk (22), TLE with hippocampal sclerosis in the UK of developing IGE and recurrent FS, respectively and Ireland patients (18) and mesial TLE in Indi- (15). A higher frequency of the TT genotype and an population (23). Further, no association was T allele of the C588 T polymorphism of the GA- found between GABRG2 rs211037 polymorphi- BARG2 gene in patients than controls were also sm and susceptibility of Lennox-Gastaut syndro- found in the Egyptian cohort of children with me (19). IGE (17). In a similar cohort of all-encompassing The possible explanation for those divergent fin- IGE types in childhood population from Taiwan, dings might be that there is population-specific the relative risk of IGE in individuals with the variation implicated in influencing susceptibility GABRG2 rs211037-CC genotype was estima- to disease. Then, the sample size is a fundamen- ted to be 3.61 times greater compared with those tal determinant of the power to identify a causal with the GABRG2 rs211037-TT genotype (14) variant in genetic association studies, and large the γ2 subunit of GABA receptor (SNP211037; populations of patients with epilepsy are needed there was a higher frequency of the TT genotype to establish the role of common genetic variants and T allele of the C588 T polymorphism of the with small effect sizes. The combined results GABARG2 gene in patients than controls. On the of recent meta-analysis and expression quan- contrary, no positive association with IGE was titative trait loci analysis, which evaluated the established in a study examining the role of seve- role of GABRG2 in epilepsy, indicated that the ral GABRG2 gene single nucleotide polymorphi- GABRG2 C588T polymorphism was associated sms (including rs211037) in two separate cohorts with IGE risk under dominant and allelic models from England and Ireland (18). (24). Further polygenic risk scores that combine The results for distinct subgroups of IGE are the effect sizes of numerous variants into a single also inconsistent. Namely, CC genotype and the score can probably better stratify affected and he- C allele were found to be significantly overre- althy individuals (25). presented in the patients with JME in a study A substantial number of antiseizure medications from India (9), while there was a lack of asso- (ASM) target the GABAA receptor, for the ben- ciation between rs211037 of the GABRG2 gene zodiazepine and barbiturates; it is the primary and JME in another Indian study (19) as well or only known mechanism of antiseizure action, as Brazilian population (13). In the two studies while for topiramate, felbamate, and stiripentol, regarding CAE exclusively, allele and genoty- GABAA receptor modulation is one of several po- pe frequencies of the rs211037 of the GABRG2 ssible antiseizure mechanisms (26). Few studies gene polymorphisms did not differ significantly examined the possible association of GABRG2 between CAE patients and healthy controls in rs211037 polymorphism and drug-resistance in the German and Korean population, respectively patients with epilepsy (9,10,17). While in child- (20,21). Of note, more than half of the patients in hood cohort with IGE from Egypt, there was a our cohort were diagnosed with JME, while only substantial increase of the T allele among drug- a few with CAE. However, we did not evaluate resistant patients compared with those respon- subsyndromes separately because the relatively ding to ASM, i.e. children with the C allele were small number of participants in each group is four times more likely to be responsive ASM inadequate for statistical analyses. than non-C-allele- carriers (17) no correlation Apart from IGE, GABRG2 rs211037 (CC was found in other studies (9-10). In the Wang

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et al. meta-analysis it was found that the protein of 14 and only an insignificant number of patients encoded by the GABRG2 gene interacted with continue to have seizures after this age. Since 61 drugs, some of which are currently approved lack of a sufficient number of participants in each ASM, and others that have not yet been approved group would prevent accurate statistical evaluati- for epilepsy treatment but may have antiseizure on and conclusions, we did not break them down potential, and therefore, GABRG2 might be a po- into subcategories and perform analysis for the tential therapeutic target for epilepsy (24). whole group of IGE. Although only a relatively small number of pat- In conclusion, the results from our study have hogenic mutations involve genes ‘actionable’ established the association of polymorphism with current therapeutic tools (27), the therape- rs211037 of the GABRG2 gene with the suscep- utic strategy of the future might involve, among tibility to idiopathic generalized epilepsy in the

others, enhancement of the wild-type GABAA re- Macedonian population. ceptor channel function (28). FUNDING The main limitation of the study is the small number of patients with CAE in our cohort. The No specific funding was received for this study. reason for underrepresentation is that recruitment process took place at teenage and adult epilepsy TRANSPARENCY DECLARATION clinic (where patients older than 14 years are tre- Conflicts of interest: None to declare. ated), while CAE is usually resolved by the age

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409 ORIGINAL ARTICLE

C-reactive protein and haemoglobin level in acute kidney injury among preterm newborns

Fiva Aprilia Kadi, Tetty Yuniati, Yunia Sribudiani, Dedi Rachmadi

Department of Child Health, Universitas Padjadjaran Medical School/Dr Hasan Sadikin General Hospital, Bandung, West Java, Indonesia.

ABSTRACT

Aim To explore the possibility of C-reactive protein (CRP) and haemoglobin (Hb) in prediction and risk assessment of acute kidney injury (AKI) among preterm newborns. This is believed to be closely related to the incidences of AKI, and could be the most affordable in early detection of AKI.

Methods A case control study was carried out at Dr Hasan Sadi- kin Hospital in Bandung with a total of 112 preterms divided into two groups: with and without AKI based on the neonatal KDIGO Corresponding author: (Kidney Disease: Improving Global Outcomes). CRP and creati- Fiva Aprilia Kadi nine serum were measured within 6 hours and at 72-96 hours after birth. The routine blood count included haemoglobin, haematocrit, Department of Child Health, Universitas leucocyte, and thrombocyte in the first 24 hours of life. Padjadjaran Medical School/ Dr Hasan Sadikin General Hospital. Results CRP increase was the most influential factor for AKI with Jalan Pasteur no. 38 Bandung 40161, sensitivity of 80.6% and specificity of 60.2%. An increase in CRP >0.04 had an aOR (95% CI) of 5.64 (1.89–16.84). Haemoglobin West Java, Indonesia. <14.5 g/dL had slightly increased aOR (95% CI) of 1.65 (1.05- Phone +62 8112222908 8.63) E-mail: [email protected] ORCID ID: https://orcid.org/0000-0001- Conclusion CRP increases >0.04 and level Hb <14.5 g/dL showed acceptable as an early warning for AKI in preterm newborns. 5789-6010 Key words: AKI, anaemia, CRP, neonates, renal injury

Original submission: 05 March 2021; Revised submission: 23 April 2021; Accepted: 05 May 2021 doi: 10.17392/1371-21

Med Glas (Zenica) 2021; 18(2):410-414

410 Kadi et al. Anaemia and CRP preterm AKI

INTRODUCTION as early as possible, preferably with the simplest, easiest and most affordable examination (1–5). In Acute kidney injury (AKI) among neonates is this study an increased CRP and low haemoglobin a common problem in Neonatal Intensive Care were expected to be early acceptable predictors Unit (NICU) (1,2). Two studies reported its inci- of AKI. The risk of AKI will affect the choice of dence between 2.4% up to a staggering figure of antibiotics, because until now the macrolide group 56% (3,4). Mortality rate reached 50% or more with nephrotoxic effects is still empiric therapy. (1–6). The reported short-term findings were proteinuria and glomerulosclerosis, whereas with The aim of this study was to find a new predictor delayed or inadequate management its long-term marker for neonatal preterm AKI that is afforda- effect could be hypertension and chronic renal ble in a country with low health resources such as failure (3,5,7,8). CRP and haemoglobin. Early detection of AKI among neonates is im- PATIENTS AND METHODS portant to prevent these short and or long-term effects as well as mortality. Unfortunately no Patients and study design agreement has been reached on its best screening method, especially as some of the suggested This case control study was conducted at the Ha- markers were less accurate or impractical or not san Sadikin Hospital Bandung, Indonesia betwe- 24/7 readily unavailable in less developed cen- en the March and November 2020. All preterm tres (1–5). The use of elevated/raised serum cre- infants with 30-36 weeks gestational age were atinine (sCr) within 48-72 hours among neonates enrolled. Neonates born to prolonged prematu- had been reported to be affected by maternal cre- re rupture of membrane (PPROM) more than 18 atinine level (1–9). The latest diagnostic criteria hours, having 5-minute Apgar score <7 and/or for AKI as proposed by the Acute Kidney Injury showed respiratory distress (defined by Downe’s Working Group of KDIGO (Kidney Disease: Im- score >4 within the first 24 hours) were -exclu proving Global Outcomes) are based on absolute ded. The sample size formula was used to test the increase of sCr, at least 0.3 mg/dL (26.5 μmol/L) hypothesis of two proportions difference, with a within 48 hours or by a 50% increase in sCr from minimum of 52 subjects required per group. a baseline within 7 days, or a urine volume of less Written informed consents were obtained from than 0.5 mL/kg/h for at least 6 hours (7–10). all parents of the preterm newborns who were Acute kidney injury is a complex disorder with a enrolled in this study. wide variety of etiologies and corresponding risk The Ethics Committee of Padjadjaran University factors, the main risk factors are prematurity, sep- approved the study. sis, asphyxia and respiratory distress (11,12). It is Methods known that preterm delivery (PTD) is preceded by inflammation and or an infection (13). In addi- Apart from the routine examination of haemo- tion to systemic inflammation which promotes globin, haematocrit, leucocyte and thrombocyte tubular injury in a newborn, it also increases the count in 24 hours and examinations of CRP and risk of renal ischemia/reperfusion injury (11,12). sCr were added during an admission (within Prematurity comes with immature organs, inclu- 6 hours of life) and in 72-96 hours. Routine ding tubular immaturity. In such condition less haematology examination was done (Sysmex production to insufficient level of erythropoietin America). CRP and sCr were performed by (EPO) may result in a lower haemoglobin level. A Siemens USA dimension EXL 200 Integrated previous study had shown that tubular immaturity Chemistry System. Acute kidney injury diag- caused insufficient EPO production, which further nostic criteria in this study followed the consen- explained the presence of anaemia (14,15). As the sus by the Working Group of KDIGO, which is diagnosis of AKI in preterm infants is very difficult, an absolute increase in sCr, at least 0.3 mg/dL especially during the 1st until 3rd postnatal days of (26.5 μmol/L) within 48 hours, which were re- life, they are the most vulnerable to develop AKI. corded between hospital admission and the first In order to reduce AKI-related morbidity and mor- 72 hours, or a urine volume of less than 0.5 mL/ tality of prematurity we need to be able to detect it kg/h for at least 6 hours (7,10,11).

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Statistical analysis Table 2. Laboratory parameters characteristics of 112 premature newborns with and without acute kidney injury (AKI) The initial set of potential variables associated with Parameter AKI AKI was found during univariate analysis, which YES (n = 56) NO (n = 56) p Haemoglobin (g/dL) 0.03 was significantly different between the two groups Mean (SD) 13.57 (2.86) 17.60 (2.19) (p<0.05). A cut-off value for increased CRP level Median 14,37 18,2 Range 8,00 – 18,97 12,00 – 22,7 was set based on receiver operating curve (ROC), Leucocytes (mm3) 0.78 11.70 (1.93 – 11.99 (3,83 – sensitivity and specificity, while the cut-off value Mean (SD) 27.6) 31.15) of haemoglobin <14.5 g/dL was based on a pre- Median 10.77 11.26 Range 3.00 – 27,60 3.83 – 31.15 vious study (15). The identification of independent Haematocrit (%) 0.81 predictors of AKI in the whole population was Mean (SD) 51.3 (8.19) 51.9 (6.10) Median 50.50 51.95 assessed by a logistic regression analysis, which Range 23.2 – 71.3 33.4 – 64.9 included all variables with p<0.25 during bivariate Thrombocyte (mm3) 0.09 Range (SD) 125.78 (70.90) 259.38 (135.86) analysis. Results are presented as odds ratio (OR) Median 150 244,50 with 95% confidence intervals (CI). Range 69 – 390 151 – 460 CRP 1 (mg/dL) 0.01 Median (SD) 0.36 (2.81) 0.20 (0.59) RESULTS Median 0.3 0.15 Range 0.09 – 5.00 0.04 – 4.02 A total of 112 preterm neonates were enrolled in CRP 2 (mg/dL) 0.02 Mean (SD) 1.98 (4.79) 0.58(0.95) this study, 56 with AKI and 56 without AKI (no Median 0.4 0.2 AKI). The baseline clinical characteristics of the Range 0.07 – 30.71 0.03 – 5.03 patients with and without AKI are shown in Table 1, which shows no significant differences in birth weight, gestational age and sex.

Table 1. Characteristics of 112 premature newborns with and without acute kidney injury (AKI) Acute kidney injury Variable p YES (n = 56) NO (n = 56) Gender (No; %) 0.85 Male 27 (48.2) 26 (44.8) Female 29 (51.8) 27 (47.4) Gestational age (weeks) 0.82 Mean (SD) 32.3 (1.4) 32.3 (1.4) Median 32 32 Range 30 – 36 30 – 36 Birth weight (g) 0.97 Figure 1. Receiver-operating curve (ROC):n increase of C- Mean (SD) 1665.8 (309.4) 1639 (324.7) reactive protein (CRP) in preterm newborns with acute kidney Median 1630 1645 injury (AKI) Range 1000 – 2210 1100 – 2320 The cut-off points of CRP increase >0.04, haemoglobin <14.5 g/dL and platelet count <150,000/ Leucocytes, haematocrit and thrombocyte counts mm3 were included in the logistic regression cal- between the groups were also comparable (Table culation (16). 2), leaving that preterms with AKI had lower me- The logistic regression analysis showed that the dian haemoglobin value (14.37 vs. 18.2; p<0,05). increase of CRP (>0.04) and Hb level of <14.5 g/ The two median values of CRP in preterm neo- dL were the factors associated with preterm AKI nates with AKI were higher than those without (Table 3). AKI (consecutive comparison results 0.3 vs. Table 3. Factors associated with acute kidney injury* 0.15 (p=0.013) measured within 6 hours and 0.4 Variable Coeff-B SE (B) p ORadj (CI 95%) vs. 0.2 (p=0.016) measured within 72-96 hours Increasing CRP 1.729 0.558 0.002 5.64 (1.89 – 16.84) of life. Setting for the largest area under the cur- (>0.04) Hb 1.437 0.823 0.041 1.65 (1.05 – 8.63) ve, which was 67.2%, the cut-off point for CRP (<14.5 g/dL) Thrombocyte increase was >0.04. The sensitivity and specifi- 0.542 1.450 0.728 0.78 (0.20 – 3.09) (<150.000/mm3) city of CRP increase of >0.04 were 80.6% and *Based on Multiple Logistic Regression Analysis R2 (Nagelkerke) 60.2%, respectively (Figure 1). = 39%; ORadj (CI 95%), Odds ratio adjusted and confident interval 95%, accuracy = 75.0%;

412 Kadi et al. Anaemia and CRP preterm AKI

DISCUSSION by exacerbating local inflammation, impairing the proliferation of damaged tubular epithelial Recent studies already showed a few poten- cells (11,19,22). tial biomarkers in AKI are urinary such as neutrophil gelatinase- associated lipocalin The tubular maturation is required for initiati- (uNGAL), interleukin-18 (uIL-18), netrin-1 on EPO production. A human study showed the (uNTN-1), and sodium hydrogen exchanger level of Hb and Haematocrit (Ht) correlated isoform 3 (uNHE3) (17,18), but all the biomar- with tubular function (15). The previous study kers are expensive and not always available in showed anaemia in preterm newborns if Hb less limited hospitals. Our study intends to identify than 14.5 g/dL (15). Our study showed that the a usual and simple predictor to prevent AKI in median Hb in preterm AKI was less than in pre- preterm newborns. Prematurity by itself is an terms with no AKI (14,37 vs 18,2), with stati- independent risk factor for AKI as the result of stical logistic regression showing that the level an incomplete nephrogenesis, immature vaso- Hb <14.5 g/dL is one of the risk factors of AKI regulation with high renal vascular resistance, in preterm newborns with OR= 1.65 with 95% high plasma renin activity, low GFR and decre- confident interval and accuracy of 75%. This ased inter-cortical perfusion (1). study showed that PTD causing tubular immaturity could be a risk factor of AKI in preterm Mechanisms of CRP in acute AKI were shown newborns and further important for anaemia due by Tang et. Al. (19). CRP through its receptors to EPO dysfunction. promotes AKI by activating its downstream pathways including nuclear factor (NF)-κB and This study showed that birth weight, gestational transforming growth factor (TGF)-β to cause age and sex were not statistically different betwe- renal inflammation and macrophage activation. en preterm newborns with AKI and without AKI. A previous study by Nickavar (20) showed that This is the different result from a previous study CRP could be used as a predictive factor for AKI by Nickavar and Ghobrial (20) that showed that in neonate septicaemia with 83% sensitivity and neonatal AKI was influenced by gestational age 60% specificity. In this study we have exclud- and birth weight - the lower gestational age and ed the risk of infection in preterm infants such birth weight, the higher the risk of AKI (12,20). as preterm premature rupture of membranes In our study this difference is caused by done ho- (PPROM), maternal leukocytosis and maternal mogeneity of subjects before enrolment. fever, to determine preterm itself on inflamma- Difficulties in serum creatinine interpretation tion. This study has shown that the increase of make it more difficult to achieve a consensus CRP >0.04 with the AUC of-CRP 0.67 could be regarding AKI definition in newborns. Becau- used as a biomarker for early detection of pre- se of all these difficulties in diagnosing AKI in term AKI with sensitivity 80.6% and specificity newborns, new biomarkers are expected to be of 60.2%. This result is strengthened with the data greater importance in AKI approach in high-risk from logistic regression analysis which show neonatal populations such as preterm newborns, that the increase of CRP>0.04 in preterm new- but we need a biomarker that is easier to obtain in borns has 5.64 times higher risk to develop AKI limited hospitals (20,21). with 95% confident interval and accuracy of In conclusion, CRP and Hb estimation do 75%. A previous study by Cosentino (21) in pa- have a role in the diagnosis of AKI in preterm tients with acute myocardial infarction showed newborns, but the test is not specific enough to that the AUC of-CRP could be a prediction of be relied upon as the only indicator. The sen- AKI (0.69; p<0.0001). There has been no study sitivity is good enough, specificity not high of preterm infants with AKI yet (21). enough, positive and negative predictive values Growing attention has been focused on CRP, a were not calculated in this study. Considering simply detectable inflammation biomarker, as high morbidity and mortality associated with a possible predictor of AKI and it has been re- neonatal AKI, CRP and haemoglobin should be cently recognized that CRP actively contribu- considered to be one of the predictors for neo- tes in the pathogenesis and progression of AKI, natal AKI.

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ACKNOWLEDGEMENT FUNDING The authors thank to Abdurachman Sukadi, Sjarief An Academic Leadership Grant (ALG) from Hidajat and Aris Primadi of Paediatric Neonato- Padjadjaran University (Grant number 855/ logy Division for their support and contribution UN6.3.1/PL/2017) supported the work. to this research, as well as to the staff of medical TRANSPARENCY DECLARATION records for their assistance with this study. Conflict of interest: None to declare.

REFERENCES 1. Viswanathan S, Mhanna MJ. Acute Kidney Injury in 13. Bullen B, Jones NM, Holzman CB, Tian Y, Sena- Premature Infants. J Clin Pediatr 2013; 1. gore PK, Thorsen P, Skogtrands K, Hougaard DM, 2. Luyckx VA. Preterm birth and its impact on renal Sikorskii A. C-reactive protein and preterm delivery. health. Semin Nephrol 2017; 37:311–9. Reprod Sci 2013; 20:715–22. 3. Bruel A, Rozé J-C, Quere M-P, Flamant C, Boi- 14. Asada N. Tubular immaturity causes erythropoietin- vin M, Roussey-Kesler G, Allain-Launay E. Renal deficiency anemia of prematurity in preterm neona- outcome in children born preterm with neonatal acu- tes. Sci Rep 2018; 8:4448. te renal failure: IRENEO—a prospective controlled 15. Kates EH, Kate J. Anemia and polycythemia in the study. Pediatr Nephrol 2016; 31:2365–73. newborn. Pediatr Rev 2007; 28:33-4. 4. Stojanovic V, Barisie N, Milanovic B, Doronjski A. 16. Siller L, Slambrouck CV, Lapping-carr G. Neonatal Acute kidney injury in preterm infants admitted to a thrombocytopenia: etiology and diagnosis. Pediatr neonatal intensive care unit. Pediatr Nephrol 2014; Ann 2015; 44:175–80. 29:2213–20. 17. Oncel MY, Canpolat FE, Arayici S, Dizdar EA, Uras 5. Black MJ, Sutherland MR, Gubhaju L. Effects of N, Oguz SS. Urinary markers of acute kidney injury Preterm Birth on the Kidney. In: Sahay M. Editor. in newborns with perinatal asphyxia. Renal Failure Basic Nephrology and Acute Kidney Injury. 1st ed. 2016; 38:882–8. IntechOpen, London, UK: IAD Press 2011; 61–88. 18. Libório AB, Branco KMPC, Torres De Melo Be- 6. Momtaz H. Sabzehei M, Rasuli B, Torabian S, zerra C. Acute kidney injury in neonates: From urine The main etiologies of acute Kidney injury in the output to new biomarkers. Biomed Res Int 2014; newborns hospitalized in the neonatal intensive care 2014:601568. unit. J Clin Neonatol 2014; 3: 99. 19. Tang Y, Kwong-Mak S, Xu AP, Yao Lan -H. Role of 7. Makris K, Spanou L. Acute kidney injury: definition, C‐reactive protein in the pathogenesis of acute kid- pathophysiology and clinical phenotypes. Clin Bio- ney injury. Nephrology (Carlton) 2018); 23(Suppl chem Rev 2016; 37:85-97. 4):50-52. 8. Ottonello G, Dessì A, Neroni P, Trudu ME, Manus 20. Nickavar A, Khosravi N, Mazouri A. Predictive Fac- D, Fanos V. Acute kidney injury in neonatal age. J tors for Acute Renal Failure in Neonates with Septi- Pediatr Neonatal Individ Med 2014; 3:2–5. cemia. Arch Pediatr Infect Dis 2017; 5:e61627. 9. Durkan AM, Alexander RT. Acute kidney injury 21. Cosentino N, Genovese S, Campodonico J, Bo- post neonatal asphyxia. J Pediatr 2011; 158(Suppl nomi A, Lucci C, Milazzo V, Moltrasio M, Biondi 2):29–33. ML, Riggio D, Veglia F, Ceriani R, Celentano K, 10. KDIGO. Kidney Disease Improving Global Outco- Metrio MD, Rubino M, Bartorelli AL, Marenzi G. mes (KDIGO) Clinical Practice Guideline for Acute High-sensitivity c-reactive protein and acute kidney Kidney Injury. J Int Soc Nephrol 2012; 21. injury in patients with acute myocardial infarction: 11. Marenzy G, Consentino N, Bartorelli A. Acute kid- a prospective observational study. J Clin Med 2019; ney injury in patients with acute coronary syndro- 8:2192. mes. Heart 2015; 101:1778–85. 22. Pageus M, McCrory M, Zarjou A, Szalai A. C-reactive 12. Ghobrial EE, Elhouchi SZ, Eltatawy SS, Beshara protein exacerbates renal ischemia reperfusion injury LO. Risk factors associated with acute kidney injury (P4021). J Immunol 2013: 190 (Suppl):131–11. in newborn. Saudi J Kidney Dis Transpl 2018; 23. Stritzke A, Thomas S, Amin H, Fusch C, Abhay L. 29:81–7. Renal consequences of preterm birth. Mol Cel Pe- diatr 2017; 4:1–9.

414 ORIGINAL ARTICLE

A randomized, placebo-controlled trial of zinc supplementation during pregnancy for the prevention of stunting: analysis of maternal serum zinc, cord blood osteocalcin and neonatal birth length

Lili Rohmawati1, Dina Keumala Sari2, Makmur Sitepu3, Kusnandi Rusmil4

1Department of Paediatrics, 2Department of Nutrition, 3Department of Obstetrics and Gynaecology; School of Medicine, Universitas Sumatera Utara, Medan, 4Department of Paediatrics, School of Medicine, Universitas Padjadjaran, Bandung; Indonesia

ABSTRACT

Aim To investigate the influence of zinc supplementation on pregnant women for the prevention of stunting through an analysis of maternal serum zinc, cord blood osteocalcin and neonatal birth length.

Methods This study was conducted with pre-test/post-test control groups and double-blind randomization. Patients were pregnant mothers in second or third trimester and with their newborns who met the inclusion criteria. A total of 71 pregnant mothers and their newborns completed this study. They were divided into two groups of 35 and 36 patients, the supplementation (20 mg/day) and pla- Corresponding author: cebo groups, respectively for 12 weeks. The parameters assessed Lili Rohmawati were maternal serum zinc levels, cord blood osteocalcin and birth Department of Paediatrics, length measurements. School of Medicine, Results The mean maternal serum zinc level was 54.6±8.7 µg/dL Universitas Sumatera Utara from 71 patients. The mean maternal serum zinc levels after zinc Jl. dr. Mansyur 5, Medan, Indonesia supplementation were significantly higher than those of the place- Phone: +62 813 5618 5367; bo group: 55.1±9.9 to 59.1±8.6) μg/dL (p=0.017) and 54.2±7.5 to 50±8.6 μg/dL (p=0.001), respectively. The comparison of mean E-mail: [email protected] cord blood osteocalcin levels and median neonatal birth lengths in ORCID ID: https://orcid.org/0000-0002- the supplementation group was higher than in the placebo group: 1134-7689 131.8±35.3 vs 90.6±35.4 ng/ml (p=0.001) and 49.3 (46.5-51.3) vs 48.3 (46-50.8) cm (p=0.004), respectively. Maternal serum zinc levels after zinc supplementation had a positive significant correlation with cord blood osteocalcin and neonatal birth length: r=0.434 (p=0.001) and r=0.597 (p=0.001), respectively.

Original submission: Conclusion There was a significant correlation of maternal serum 31 August 2020; zinc with cord blood osteocalcin and neonatal birth length after Revised submission: zinc supplementation. 27 October 2020; Key words: bone growth, micronutrients, pregnant, pre-post test Accepted: design 24 January 2021 doi: 10.17392/1267-21

Med Glas (Zenica) 2021; 18(2):415-420

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INTRODUCTION PATIENTS AND METHODS Stunting is a condition that occurs due to chro- Patients and study design nic malnutrition, especially in the first 1000 days of life (1,2). Low linear birth length measures This was an experimental study with pre-test/ generally signal nutritional deficiency due to a post-test control groups, double-blind with ran- lack of energy and protein experienced over time domization block technique. This study was (3). The prevalence of stunting in Indonesia was conducted from March to December 2019 in the 30.8%, with 22.7% of all neonatal birth lengths at Obstetrics and Neonatology Unit of Universitas less than 48 cm (4). Sumatera Utara Hospital, Malahayati Islamic Malnutrition in both macro- and micronutrients in Hospital and Royal Maternity Hospital in Me- pregnant women causes an inadequate nutritional dan, Indonesia. Patients were pregnant mothers reserve for the physiological needs of the foetus du- with their newborns who met the inclusion crite- ring pregnancy, which results in growth and deve- ria: healthy pregnant mothers in their second or lopmental disorders, increased morbidity and mor- third trimester, 20-35 years old, with a height of tality, and may interfere with long-term intellectual >150 cm and no indication of cons following this capacity and academic proficiency (1,3,5). Zinc is study, as determined by obstetricians and gyna- one of the essential micronutrients, and a lack of ecology specialists. Births involving twins, con- zinc intake in small quantities can cause dysfunc- genital abnormalities, prematurity (gestation age tion in various systems and restriction of physical <37 weeks) or stillbirth were excluded. growth (6). Certain studies have found that low As the pregnant mothers got admitted in the Ob- maternal zinc levels are associated with foetal de- stetrics Unit, every consecutive patient was asse- ath, congenital malformations, intrauterine growth ssed for inclusion and exclusion criteria and was restriction (IUGR), prematurity, low birth weight randomized by a blocked randomization method. infants (LBW) and disorders of childbirth (7,8). One hundred and four patients were assessed for Zinc interacts with the development at varying de- this study, 82 were found to be eligible and were grees of synthesis, secretions, and hormonal acti- randomized, and assigned to two groups, with 41 ons that play an important role for linear growth patients in each group. One group administered (6), and the formation of foetal bones (9,10). zinc supplementation 20 mg/day and the other Zinc deficiency in pregnant women is suspected to group administered placebo, in the morning after be the cause of high birth rates with short birth len- meals for 12 weeks. Zinc tablets (zinc sulphate) gths (9). More specific research into the influence of and placebo were each inserted into a capsule of zinc on bone growth requires a marker that can be the same shape, colour and taste. All patients were measured through the examination of biomarkers also given iron and folic acid tablets in accordance in the blood or other tissues (11). Osteocalcin exa- with the Indonesian Government program. mination can be done in a clinical laboratory with An informed consent was given by all mothers sampling of the cord blood of newborns. Cord blo- following the provision of sufficient information od osteocalcin levels are rated almost equal to the prior to the study. newborn’s blood levels (11). This examination can This study was approved by the Health Research provide an overview of how much bone growth is Ethical Committee, School of Medicine, Univer- influenced by zinc or other micronutrients. To date, sitas Sumatera Utara (No.179/TGL/FK USU- there have been no studies linking the influence of RSUP HAM/2019). zinc supplementation in pregnant women on the Trial registration: Clinicaltrials.gov identifier: prevention of newborn stunting through analysis of NCT04559152. osteocalcin levels as markers of bone growth in the foetus and the newborn. Methods The aim of this study was to investigate the in- The parameters assessed in this study were ma- fluence of zinc supplementation on pregnancy in ternal serum zinc levels, cord blood osteocalcin the prevention of stunting through an analysis of and birth length. Maternal serum zinc levels were maternal serum zinc, cord blood osteocalcin and measured twice with 6 mL of vein blood each neonatal birth length. time, both during initial antenatal care and af-

416 Rohmawati et al. Zinc supplementation and osteocalcin

ter 12-weeks of supplementation. Samples were (21.1%) were excluded. Eighty-two pregnant subsequently centrifuged for 15 minutes at 3000 mothers with their newborns who met the inclu- rpm. Specimens were processed by the inducti- sion criteria were enrolled during the study, of vely coupled plasma-mass spectrometry (ICP- which 11 were excluded for the following rea- MS) method using Agilent 7700 analyser (Santa sons: seven dropped contact, two stopped ta- Clara, USA, 2014). Normal serum zinc levels king zinc supplementation, and two cord blood were defined based on a cut-off value of ≥56 µg/ samples were useless. Therefore, 71 pregnant dL in accordance with the Second National He- mothers and their newborns completed this stu- alth and Nutrition Examination Survey data from dy; they were divided into two groups of 35 and 1976-1980 (NHANES II) (12). 36 patients, the supplementation and placebo Cord blood osteocalcin levels were measured groups, respectively (Figure 1). with 6 mL cord blood samples prior to delivery. The serum was separated by centrifugation (15 minutes at 1000 rpm) and was frozen at −70 oC for subsequent analysis of osteocalcin. Osteo- calcin was measured using biotin and ruthenium specific monoclonal antibodies against N-Mid osteocalcin (N-Mid Osteocalcin, Roche Diagno- stics, Mannheim, Germany) by the electrochemi- luminescence immunoassay (ECLIA) on an automated Cobas e601 analyzer (Roche Diagnostics, Mannheim, Germany). Sample collection, tran- sportation, separation, storage, and analysis were performed according to Prodia clinical laboratory standards, Indonesia (ISO 9001 and ISO 15189). Neonatal birth length was performed with a SECA 232 digital baby scale (Hamburg, Ger- many) for length with an accuracy of 0.1 cm, independently duplicate by two trained persons for Figure 1. Flow diagram of study patient all births. Maternal zinc intake was calculated in Maternal serum zinc levels during pregnancy accordance with the NutriSurvey 2007 Indonesi- were of 54.6±8.7 µg/dL (range 40-74 µg/dL), and an version (13). the prevalence of levels <56 µg/dL was high, 46 Statistical analysis (64%) mothers. The mean cord blood osteocalcin levels of the supplementation and placebo grou- A Saphiro-Wilk’s test was performed to determi- ps were 131.8±35.3 ng/dL and 90.6±35.4 ng/dL, ne the normality of data spread. Descriptive data respectively (Table 1). were expressed as mean±SD and percentage (%); The supplementation group experienced a signif- and median (min-max) was for non-normal dis- icant increase of the mean maternal serum zinc tribution data. Bivariate analysis was performed level during pregnancy before and after 12 weeks with unpaired t-test, paired t-test and χ2 test; non- (p=0.017), while the placebo group showed a normal distribution was analysed with the Mann- significant decrease (p=0.001). Using a statisti- Whitney U-test. Correlation was used to measure cal unpaired t-test, mean maternal serum zinc two variables with Pearson correlation coefficients level between both groups after 12-weeks of zinc (r) and Spearman’s for nonparametric correlation. supplementation showed significant differences Statistical significance was considered at a p-value (p=0.001). All values of maternal serum zinc lev- <0.05 with a 95% confidence interval. el in both groups were below normal value (<56 RESULTS μg/dL) (Table 2). Of the 104 pregnant women assessed during the The relationship between maternal serum zinc le- period between March and December 2019, 22 vel and cord blood osteocalcin after 12-weeks of

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Table 1. Characteristics of 71 pregnant women involved in Table 3. Correlation between maternal serum zinc level after the study 12 weeks with cord blood osteocalcin and neonatal birth length Groups Variable r p Supple- Cord blood osteocalcin 0.434* 0.001 Characteristic Placebo Total p mentation Neonatal birth length 0.597† 0.001 (n=36) (n=71) (n=35) *Pearson correlation test; †Spearman’s rank correlation test Maternal age (years) 28.8±3.6 27.9±3.4 0.265* (Mean±SD) Pregnancy period (weeks) DISCUSSION 22.3±3.3 22.2±2.7 0.930* (Mean±SD) Maternal weight (kg) The patients in this study were pregnant mothers 64.2±11.7 66.0±7.2 0.447* (Mean±SD) because stunting prevention can start at concep- Maternal height (cm) 158.4±6.9 157.3±3.7 0.410* tion (1). The results of the presented study showed (Mean±SD) mean serum zinc level in pregnant women of Maternal BMI (kg/m2) 25.9±4.8 26.6±3.1 0.442* (Mean±SD) 54.6±8.7 μg/dL was under normal value in ac- Gravida (No, %) cordance with the recommendation of NHANES Primigravida 17 (48.6) 17 (47.2) 1.000† II (12). The proportion of pregnant women with Multigravida 18 (51.4) 19 (52.8) Maternal occupation (No, %) serum zinc level below normal was quite high Worker 20 (57.1) 21 (58.3) 1.000† (64%). Seriana et al. encountered low serum zinc Non-worker 15 (42.9) 15 (41.7) level during pregnancy (36.0±18.3 μg/dL) (14). Family economic status (No, %) Low 1 (2.9) 4 (11.1) In a similar study from Ethiopia, Mekonnen et Average 16 (45.7) 19 (52.8) 0.225† al. reported mean serum zinc level in pregnant High 18 (51.4) 13 (36.1) women of 58.7 μg/dL, with a proportion of zinc Maternal education (No, %) High school 5 (14.3) 15 (41.7) deficiency lower than 55.3% in this study (15). University, not graduate 2 (5.7) 7 (19.4) 0.002§ The average maternal zinc intake of only University, graduate 28 (80) 14 (38.9) 5.77±1.09 mg/day was found in our study, which Maternal zinc intake (mg/day) 6.0±1.3 5.5±0.8 0.061* (Mean±SD) is in accordance with the calculation of the Indo- Maternal serum zinc (µg/dL) 55.1±9.9 54.2±7.5 54.6±8.7 0.082* nesian version of NutriSurvey 2007 (13). This per- (Mean±SD) centage is low compared to the average needs of Range 40-74 Value <56 µg/dL No (%) 22 (62.9) 24 (66.7) 46 (64) pregnant women (13). A study in Bandung, West 0.930† Value ≥56 µg/dL, No (%) 13 (37.1) 12 (33.3) 25 (35.2) Java, Indonesia reported an average maternal Gestational age (weeks) 38.8±0.4 38.5±0.8 0.061* zinc intake of 5.1 mg/day (16). Gala et al. repor- (Mean±SD) Cord blood osteocalcin (ng/ ted lower levels than those found in this research 131.8±35.3 90.6±35.4 0.001* dL) (Mean±SD) (4.2±1.2 mg/day) (17). Some of the factors that Neonatal birth length (cm) 49.3; 48.3; 0.004‡ lead to the prevalence of maternal zinc deficiency (Median; Min.-max.) 46.5-51.3 46-50.8 *Unpaired t-test; †Chi Square test; ‡Mann-Whiney U-test; §Signifi- are low intake of animal protein sources and plant cant difference (p<0.05); BMI, Body mass index; based diets with high phytate contents, especially in developing countries. Animal proteins are the zinc supplementation was positive, moderate and best dietary source of zinc, as they contain amino significant (r=0.434; p=0.001). The Spearman acids that increase zinc absorption (3,18,19). rank correlation test showed that the relationship The need of zinc intake increases during pre- between maternal serum zinc level and the neo- gnancy due to the increasing need for foetus ossi- natal birth length was positive, strong and signi- fication causing a decrease in maternal serum zinc ficant (r=0.597; p=0.001) (Table 3). level (12). Choi et al. reported the serum zinc level of pregnant women decreased significantly throu- Table 2. Mean maternal serum zinc level before and after ghout the first, second and third trimesters of pre- zinc supplementation or placebo gnancy (20). This study found that maternal serum Maternal serum zinc level zinc level significantly decreased in the placebo Groups (Mean±SD) (µg/dL) p group, and increased significantly in the supple- Before After Supplementation 55.1±9.9 59.1±8.6 0.017† mentation group. According to WHO, pregnant 0.001* Placebo 54.2±7.5 50.0±8.6 0.001† women need at least 11 mg of zinc intake per day. *Unpaired t-test; †Paired t-test. In pregnant women with a low bioavailability, a zinc intake of 20 mg/day is required (21,22).

418 Rohmawati et al. Zinc supplementation and osteocalcin

Several studies reported the benefits of zinc Nearly none of the patients of this study compla- supplementation in pregnant women and birth ined of side effects. Two pregnant mothers repor- outcomes (23-25). This study found that neona- ted nausea after taking a zinc tablet, but continued tal birth length was higher in supplementation supplementation and reported no complaints af- group than placebo. In agreement with this stu- terwards. Nausea could also be attributed to the dy, Merialdi et al. reported a significant increase ongoing state of pregnancy. The limitations of in the foetal femur diaphysis length in pregnant this study include the use of telephone commu- mothers who were given 25 mg zinc supplemen- nication to monitor patients taking zinc tablets; it tation concurrently with 60 mg Fe and 250 g fo- could be possible that the patients forgot to take lic acid (26). Prawirohartono et al. reported that their zinc tablets, and some of them failed to be Zn supplementation during pregnancy improves contacted again. Also, this study did not control birth length after adjusting for maternal height the dietary intake of patients, and did not analyse and pre-pregnancy weight (27). foods containing zinc inhibitors that could affect Therefore, the examination of osteocalcin is a good the absorption of zinc in digestion. parameter for determining the metabolism of bone In conclusion, this study found a high prevalence formation (28,29).This study found that the average of zinc deficiency in pregnant women. There was cord blood osteocalcin level in the supplementation a positive correlation between maternal serum group was significantly higher than in the placebo zinc levels with cord blood osteocalcin levels as group, and they were higher than in previous rese- bone growth markers and neonatal birth length arch studies (30,31). Examination of osteocalcin in after 12 weeks of zinc supplementation. As a re- other research is taken from the serum, while this commendation, pregnant women require regular study took it from umbilical cord blood. There has examination of maternal serum zinc and admini- been no standard reference value for umbilical cord stration of 20 mg/day zinc prophylaxis dose du- blood osteocalcin levels. A study in Korea reported ring pregnancy. that the concentration of osteocalcin in serum was higher at puberty period. This value indicated that ACKNOWLEDGEMENTS osteocalcin greatly increased during growth spurt We thank the Universitas Sumatera Utara Hos- (31). High levels of umbilical cord blood osteocal- pital, Malahayati Islamic Hospital, Royal Mater- cin in this study are thought to be due to the rapid nity Hospital and Prodia Clinical Laboratory for growth rate during the foetal period. technical support during the conduction of this The effect of zinc supplementation of pregnant study. women on foetal bone growth caused by zinc stimulating osteoblast production and inhibiting FUNDING osteoclast activity (26). This study showed a po- This study was funded by the Ministry of Re- sitive and significant correlation between mater- search and Technology and Higher Education, nal serum zinc and cord blood osteocalcin levels Republic of Indonesia under the DRPM research as well as neonatal birth length after 12 weeks of grant of the year 2019 (No.9/UN5.2.3.1/PPM/ zinc supplementation. This relationship indicates KP-DRPM/2019). that maternal zinc level during pregnancy is one of the factors that can affect foetal growth. TRANSPARENCY DECLARATION Excess zinc can manifest itself through nausea, Competing interests: None to declare. vomiting, abdominal cramps, and diarrhoea (19).

REFERENCES

1. World Health Organization. Reducing stunting in 3. Mousa A, Naqash A, Lim S. Macronutrient and children: equity considerations for achieving the micronutrient intake during pregnancy: an overview global nutrition targets 2025. Geneva: World Health of recent evidence. Nutrients 2019; 11:443. Organization, 2018. 4. Kemenkes RI. Riset kesehatan dasar (Riskesdas) 2. Ashworth A. Nutrition, food security and health. In: 2018. (Basic health research 2018) [in Indonesian]. Kliegman RM, Stanton BF, St. Geme-III JW, Schor Jakarta: Kementerian Kesehatan Republik Indone- NF, Behrman RE, eds. Nelson Textbook of Pedia- sia, 2018. trics. 20th ed. Philadelphia: Elsevier, 2016:295-306.

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5. Beluska-Turkan K, Korczak R, Hartell B, Moskal K, 18. Wilson RL, Grieger JA, Bianco-Miotto T, Roberts Maukonen J, Alexander DE, Salem N, Harkness L, CT. Association between maternal zinc status, di- Ayad W, Szaro J, Zhang K, Siriwardhana N. Nutriti- etary zinc intake and pregnancy complications: a onal gaps and supplementation in the first 1000 days. systematic review. Nutrients 2016; 8:641. Nutrients 2019; 11:2891. 19. Grzeszczak K, Kwiatkowski S, Kosik-Bogacka D. 6. Terrin G, Canani RB, Di Chiara M, Pietravalle A, The role of Fe, Zn, and Cu in pregnancy. Biomole- Aleandri V, Conte F, De Curtis M. Zinc in early life: cules 2020; 10:1176. a key element in the fetus and preterm neonate. Nu- 20. Choi R, Sun J, Yoo H, Kim S, Cho YY, Kim HJ, Kim trients 2015; 7:10427-46. SW, Chung JH, Oh SY, Lee SY. A prospective study 7. Wang H, Hu YF, Hao JH, Chen YH, Su PY, Wang Y, of serum trace elements in healthy Korean pregnant Yu Z, Fu L, Xu YY, Zhang C, Tao FB, Xu DX. Ma- women. Nutrients 2016; 8:749. ternal zinc deficiency during pregnancy elevates the 21. Gibson RS, King JC, Lowe N. A review of die- risks of fetal growth restriction: a population-based tary zinc recommendations. Food Nutr Bull 2016; birth cohort study. Sci rep 2015; 5:11262. 37:443-60. 8. King JC, Brown KH, Gibson RS, Krebs NF, Lowe 22. Nasiadek M, Stragierowicz J, Klimczak M, Kila- NM, Siekmann JH. Biomarkers of nutrition for nowicz A. The role of zinc in selected female repro- development (BOND): zinc review. J Nutr 2016; ductive system disorders. Nutrients 2020; 12:2464. 146:858-85. 23. Özgan Çelikel Ö, Doğan Ö, Aksoy N. A multilateral 9. Suzuki T, Katsumata S, Matsuzaki H, Suzuki K. investigation of the effects of zinc level on pregnan- Dietary zinc deficiency induces oxidative stress and cy. J Clin Lab Anal 2018; 32:e22398. promotes tumor necrosis factor-α- and interleukin- 24. Mesdaghinia E, Naderi F, Bahmani F, Chamani M, 1β-induced RANKL expression in rat bone. J Clin Ghaderi A, Asemi Z. The effects of zinc supplemen- Biochem Nutr 2016; 58:122-9. tation on clinical response and metabolic profiles in 10. Rocha E, de Brito N, Dantas M, Silva A, Almeida pregnant women at risk for intrauterine growth re- M, Brandão-Neto J. Effect of zinc supplementation striction: a randomized, double-blind, placebo-con- on GH, IGF1, IGFBP3, OCN, and ALP in non-zinc- trolled trial. J Matern Fetal Neonatal Med 2019; 1-7. deficient children. J Am Coll Nutr 2015; 34:1-10. 25. Oh C, Keats EC, Bhutta ZA. Birth, child health and 11. Panero C, Cecchettin M, Mainard G, Sorice V, Gra- development outcomes in low- and middle-income nelli A, Tarquini B. Osteocalcin in maternal, ne- countries: a systematic review and meta-analysis. onatal, and cord blood. In: Berger H, ed. Vitamins Nutrients 2020; 12:491. and Minerals in Pregnancy and Lactation, Nestle 26. Merialdi M, Caulfield LE, Zavaleta N, Figueroa A, Nutrition Workshop Series. New York: Raven Press, Costigan KA, Dominici F. Randomized controlled 1988:145. trial of prenatal zinc supplementation and fetal bone 12. Hotz C, Peerson JM, Brown KH. Suggested lower growth. Am J Clin Nutr 2004; 79:826-30. cutoffs of serum zinc concentrations for assessing 27. Prawirohartono EP, Nyström L, Nurdiati DS, Haki- zinc status: reanalysis of the second national health mi M, Lind T. The impact of prenatal vitamin A and and nutrition examination survey data (1976-1980). zinc supplementation on birth size and neonatal sur- Am J Clin Nutr 2003; 78:756-64. vival - a double-blind, randomized controlled trial in 13. NutriSurvey. Nutrition surveys and calculations. a rural area of Indonesia. Int J Vitam Nutr Res 2013; 2007. http://www.nutrisurvey.de (05 July 2020) 83:14-25. 14. Seriana I, Yusrawati, Lubis G. Serum zinc level at 28. Gundberg CM. Biochemical markers of bone forma- term pregnancy and newborn anthropometry. Indo- tion. Clin Lab Med 2000; 20:489-501. nes J Obstet Gynecol 2015; 3:190-5. 29. Han Y, Xu G, Zhang J, Yan M, Li X, Ma B, Jun L, 15. Mekonnen A, Terefe W, Belachew AB, Adhanu AK, Wang SJ, Tan J. Leptin induces osteocalcin expre- Gezae KE. Prevalence and associated factors of zinc ssion in ATDC5 cells through activation of the deficiency among pregnant women attending ante- MAPK-ERK1/2 signaling pathway. Oncotarget natal care at Gambella hospital, Gambella, Ethiopia 2016; 7:64021-9. 2018. AJLS 2019; 5:91-9. 30. Bayer M. Reference values of osteocalcin and pro- 16. Rayahu S, Gumilang L, Astuti S, Nirmala SA, Judia- collagen type I N-propeptide plasma levels in a he- tiani R. Survei asupan asam folat dan seng pada ibu althy Central European population aged 0–18 years. hamil di Jawa Barat (Survey of folic acid and zinc Osteoporos Int 2014; 25:729–36. intake in pregnant women in West Java) [in Indone- 31. Choi JS, Park I, Lee SJ, Ju HJ, Lee H, Kim J. serum sian]. J Kesehat Vokasional 2019; 4:162-8. procollagen type I N-terminal propeptide and osteo- 17. Gala UM, Godhia ML, Nandanwar YS. Effect of calcin levels in Korean children and adolescents. maternal nutritional status on birth outcome. Int J Yonsei Med J 2019; 60:1174-80. Adv Nutr Health Sci 2016; 4:226-33.

420 ORIGINAL ARTICLE

Male to female birth ratios over a 35-year period

Hrvoje Vraneš1, Hrvojka Soljačić Vraneš2, Ivka Djaković 2, Vesna Gall2, Ana Meyra Potkonjak2

1School of Medicine, University of Zagreb, 2Department of Gynaecology and Obstetrics, Sestre milosrdnice University Hospital Centre; Zagreb, Croatia

ABSTRACT

Aim Along with changes to the human physique recorded over the past decades in certain countries, there are also changes concerning the male-female birth ratio. The aim of this study was to establish the movement of male-female birth ratios and factors affecting the ratio.

Methods This retrospective study was conducted in Zagreb, Croa- tia, in the period from 1985 to 2019 on a sample of 3804 newborns. Results In the 35-year period the ratio of boys and girls at birth did not change significantly. Girls had lower birth weight, and boys had higher birth length. In the war period (1992-1994), a mild Corresponding author: increase in the ratio of boys was noted, but not statistically signifi- Hrvoje Vraneš cant. Father’s age in the last period examined (2007-2009) showed School of Medicine, University of Zagreb to be a statistically significant predictor of the child’s gender. Na- Šalata 2, 10000 Zagreb, Croatia mely, the descendants of younger fathers were statistically significantly more frequently girls, while the descendants of older fathers Phone: +385 91 2507 992; were more frequently boys. Married mothers had higher percen- @ E-mail: hrvoje.vranes gmail.com tage of male births (51.5%), and a lower percentage (47.1%) by ORCID ID: https://orcid.org/0000-0003- unmarried mothers. 3544-8385 Conclusion The changes in birth ratios are particularly pronounced in different age and socioeconomic groups of parents nowadays when the growth of living standards is accompanied by significant changes of the human physique.

Key words: Croatia, gender, human body, newborn, parturition

Original submission: 21 September 2020; Revised submission: 08 December 2020; Accepted: 23 April 2021 doi: 10.17392/1275-21

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INTRODUCTION and father is greater. In women who live in urban areas and who are exposed to higher stress level Changes of the human physique are evident from the release of corticotrophin is elevated, which the very first pictures and recordings left by our encourages the release of androgens from the predecessors to this very day. The changes are mother’s adrenal gland, which might favour the particularly pronounced nowadays where the conception of a male child (12). In cases of long growth of living standards is accompanied by si- economic crises, it was also observed that there is gnificant changes of the human physique, such as a heightened ratio of male-female newborns (13). changes in height, weight and body mass index Certain racial differences were also observed in (1-3). Changes in the diet are also present, frequ- California in the period from 1960 to 1996, whe- ently inadequate (4), and there are certain authors re it was observed that there was a statistically who believe that the adoption of good nutritional significant drop in the ratio of births of male habits and motor skills should begin in childhood children in white men, where the drop was not (5) in order to reduce the trend of weight-gain. noted amongst black men, Japanese and Native In view of the losses of fertilised ova and early Americans, while in the case of Chinese an incre- pregnancy abortions, it is difficult to establish ase in male newborns was noted (14). A similar the exact ratio of conceived “male” and “female” trend was recorded in Canada (15). The ratio of zygotes. That is the reason why as a rule it is the male newborns rises during the periods of war, relationship between male and female newborns probably due to the stress mechanism, and in the that is described, which is mostly 100 female period after the war the ratio of female newborns newborns to 105-110 male newborns (6). rises (16). The ratio of male newborns depends Gender differentiation passively progresses towar- also on the number of foetuses. In a research con- ds the female phenotype, in other words, the pre- ducted on thirty-one million single-foetus births sence of functional testicle is essential for sex in America, there were 51.6% of boys, but in the differentiation towards male gender (7). These case of twins the ratio dropped to 50.9%, in the processes are determined by a complex network case of triplets to 49.5%, and in the case of qua- of genes and its expression (7). Recent advances druplets to 46.5% (17). In the case of monocho- in techniques and genome-wide epigenetic studies rionic-monoamniotic twins, 70% are female, and suggest that epigenetic mechanisms could play a in the case of Siamese twins 75% are female (18). role in gonadal sex determination (7). We have decided to conduct this research to find Over the past several decades, it was recorded out how the ratio of male and female newborns that there were less male newborns in developed has changed in recent Croatian history. Currently countries. As of 1970, the proportion of male chil- there are studies in the literature on the birth ratio, dren in Canada dropped by 2.2 boys per 1,000 but there are no studies for such a long period. newborns, and in the Atlantic region by 5.6 per The aim of this study was to establish the movement

1,000 newborns (8). A similar situation exists in of male-female birth ratio and factors affecting the Europe where, in the period from 1950 to 1996, it ratio. The obtained data can be used by anthropo- was observed that less male newborns were born in logists, demographers, historians and knowledge 11 countries, that there was a similar ratio of male- about the sex ratio in the population could later be female newborns in 8 countries, and that the ratio interesting to experts in the fields of medicine, mar- of male newborns increased only in 4 countries (9). keting, public relations and jobs employers. Recent inequality between male and female population in some countries, leading to the predomi- EXAMINEES AND METHODS nance of males, is thought to be affected by cul- tural differences, discrimination, nation’s policies Examinees and study design and widespread violence against women. (10) This study included 3804 randomly selected Certain authors established that the ratio of male mothers, their partners and newborns. Women newborns may positively depend on the age of the included in the study gave birth in the Sestre father (11), and that the ratio of male newborns milosrdnice University Hospital Centre over is greater if the age difference between mother a period of 35 years in four specific periods of

422 Vraneš et al. Birth ratios over a 35-year period

time: 1985-1986 (550 mothers), 1992-1994 (564 The ratio of boys and girls was significantly mothers), 2000-2002 (570 mothers) and 2007- different in the view of the weight of the babies in 2009 (730 mothers), 2018-2019 (3804 mothers). all four periods. In all four periods there was the same tendency that girls were born with a lower Methods weight (Figure 2). The data were collected by a random selection of the medical history of healthy mothers who had given vaginal birth to full-term babies in single pregnancies. Women and their partners were divided into age groups, and in groups based on their place of living (rural-urban) and degree of education (university or post-secondary school qualifications, secondary school qualifications and low professional qualifications). The mothers’ marital status was recorded, as well as and their height, weight, body mass index (BMI), weight-gain in pregnancy, parity. The mass, weight and gender of newborns was also registered. Figure 2. Overall newborns’ gender in terms of birth weight for all periods Statistical analysis Boys had significantly longer birth length than In terms of the statistical processing of the results, girls from 2000-2002 onwards, but the length of to test the significance of differences between se- newborns did not change significantly over all veral groups of results, a nonparametric χ2 test four periods examined (Figure 3). was used when the results were expressed in frequencies. In the case of testing the significance of differences between the arithmetic means of several groups of results, the parametric test of one-way or two-way analysis of variance was used. After the analysis of variance, further post hoc tests were done, if necessary, such as Tukey’s HSD and Scheffe’s tests to establish between which groups of results there was a significant difference (p<0.05). Figure 3. Length (cm) of girls and boys at birth by time period RESULTS The ratio of babies of a particular length showed There were no significant differences between significant difference in view of the gender of the the ratio of boys and girls in terms of different baby in all four periods (Figure 4). periods of time (Figure1).

Figure 4. Ratio (%) of newborns of a specific length (cm) in Figure 1. Ratio of boys and girls (%) by time period view of the baby’s gender for all periods together

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The ratio of boys and girls did not differ signifi- conditions, or is it the consequence of changes in cantly in view of the age of the mother, in overall the atmosphere and greater contamination of the or in a specific period. environment with chemicals, which might even- The ratio of boys and girls was significantly tually lead to an impact on male foetuses, is still different in terms of the age of the father only in not completely clear (9,21). So far, there is no the period 2007-2009, where it proved that in the convincing evidence of the association between group of younger fathers there was a greater ratio the impact of the coital rates around the time of of girls, and in the group of older fathers a greater conception and offspring sex ratio (22). ratio of boys (Figure 5). In the war period (1992-1994), a mild increase in the ratio of boys was noted in our research, but not statistically significant. During some wars, such as World War One and World War two, and the war in Bosnia (16,23,24), authors noted an increase in the ratio of male births. However, such a tendency is not evident in all wars. For example, during the war between Iraq and Iran, the percentage of births of female children rose (24). Mother’s age, based on research carried out over a period of 35 years, is not one of the more signifi- Figure 5. Newborn’s gender in view of father’s age, for the cant predictors of the newborn’s gender. Similar re- period 2007-2009 sults were also published by other researchers after The ratio of boys and girls did not differ signi- research (25). Nonetheless, other authors (26) pu- ficantly in terms of the age difference between blished opposite results to the ones mentioned, that fathers, degree of education of the mothers and is, they found a correlation between mother’s age fathers, the place of living, the marital status of and the higher ratio of births of female children. the mother in any of the four periods. Tendency As opposed to mother’s age, father’s age in the was noted of somewhat more frequent incidence last period examined (2007-2009) showed to be a of boys in the case of married mothers, and girls statistically significant predictor of the child’s gen- in the case of unmarried mothers. der. Namely, the descendants of younger fathers are statistically significantly more frequently girls, DISCUSSION while the descendants of older fathers are more Similar weight-gain was noted among pregnant frequently boys, which according to some resear- women carrying boys and those carrying girls. ch results in statistically significant somewhat gre- Although certain authors show that mothers’ ater ratio of births of male children (25). weight before pregnancy may influence the gen- Professional qualifications of the mother are not der of the child (19), there are no claims that wo- one of the statistically significant predictors of the uld show a statistically significant difference of child’s gender. Still, we can observe that women the birth of girls or boys depending on weight- having low professional qualifications are - so gain during mother’s pregnancy. mewhat more likely to have girls, and those with The ratio of boys and girls in our study did not secondary school qualifications boys. In terms of change significantly in different periods.- The women with university qualifications, the -per se results could be attributed to the fact that the centage of boys and girls is similar. According study was conducted on a sample of healthy wo- to some research (25), parents having better so- men with uncomplicated pregnancies. Authors in cioeconomic status are more likely to have sons. the United States of America and the developed Mothers from urban areas and mothers from rural countries of Western Europe record a mild drop areas give birth in a similar ratio to male and fema- in the birth of male children (9,20). Whether the le babies. Certain authors, however, presume that decrease in the ratio of male births in developed in urban areas, the lifestyle, which is frequently countries a consequence of sociodemographic more stressful than in the country, might have an development or better socioeconomic and health impact on the elevated release of gonadotropin,

424 Vraneš et al. Birth ratios over a 35-year period

which stimulates the release of androgens in the compare these ratios with certain stated factors, adrenal gland of the mother, resulting in the more what is a novelty and has not been shown so far. likely conception of a male child (12). Other aut- In conclusion, the changes in birth ratios are hors found that there is a mild decrease in the ratio particularly pronounced in different age and so- of male births in large urban centres (27). cioeconomic groups of parents nowadays when There is a higher percentage of male births the growth of living standards is accompanied (51.5%) by married mothers, and a lower percen- by significant changes of the human physique. tage (47.1%) by unmarried mothers. The diffe- Further research could study the differences in rence, however, did not prove to be statistically the newborns’ gender in countries surrounding significant, but might arise from a well-known Croatia and check whether they follow the same fact that a slightly higher percentage of boys are trends. born in better socioeconomic circumstances, and unmarried women still mostly have poorer so- FUNDING cioeconomic status than married women. No specific funding was received for this study. These results show a long-term detailed picture of changes in the ratio of male and female birth TRANSPARENCY DECLARATION ratios in Croatia during the time period and also Conflicts of interest: None to declare.

REFERENCES: 1. Vranes HS, Gall V, Jukić M, Vranes Z. Secular chan- 13. Venero Fernández SJ, Medina RS, Britton J, Fogarty ges in growth and obesity in perinatal population. AW. The association between living through a pro- Coll Antropol 2012; 36:549-54. longed economic depression and the male:female 2. Juresa V, Musil V, Tiljak MK. Growth charts for birth ratio--a longitudinal study from Cuba, 1960- Croatian school children and secular trends in past 2008. Am J Epidemiol 2011; 174:1327-31. twenty years. Coll Antropol 2012; 36(Suppl 1):47- 14. Smith D, Von Behren J. Trends in the sex ratio of Ca- 57. lifornia births, 1960-1996. J Epidemiol Community 3. Toselli S, Ventrella AR, Brasili P. Prevalence and Health 2005; 59:1047-53. tracking of weight disorders in Italian primary scho- 15. Ray JG, Henry DA, Urquia ML. Sex ratios among ol students: a three-year follow-up. Coll Antropol Canadian liveborn infants of mothers from different 2012; 36:63-7. countries. CMAJ 2012; 184:E492-6. 4. Mikulan R, Piko BE. High school students’ body 16. Jongbloet PH, Zielhuis GA, Groenewoud HM, Pa- weight control: differences between athletes and sker-De Jong PC. The secular trends in male:female non-athletes. Coll Antropol 2012; 36:79-86. ratio at birth in postwar industrialized countries. 5. Parízková J. The role of motor and nutritional indi- Environ Health Perspect 2001; 109:749-52. viduality in childhood obesity. Coll Antropol 2012; 17. Strandskov HH, Siemens GJ. An analysis of the sex 36:23-9. ratios among single and plural births in the total, the 6. World population prospects: the 2017 revision. Ge- white and the colored United States populations. Am neva: United Nations, Department of Economic and J Phys Anthropol 1946; 4:491-501. Social Affairs, Population Division; 2017 https:// po- 18. Machin GA. Some causes of genotypic and phe- pulation.un.org/wpp/ (18 November 2020). notypic discordance in monozygotic twin pairs. Am 7. Stévant I, Nef S. Genetic control of gonadal sex de- J Med Genet 1996; 61:216-28. termination and development. Trends Genet 2019; 19. Villamor E, Sparén P, Cnattingius S. Interpregnancy 35:346-58. weight gain and the male-to-female sex ratio of the 8. Allan BB, Brant R, Seidel JE, Jarrell JF. Declining second pregnancy: a population-based cohort study. sex ratios in Canada. CMAJ 1997; 156:37-41. Fertil Steril 2008; 89:1240-4. 9. Martuzzi M, Di Tanno ND, Bertollini R. Declining 20. Davis DL, Gottlieb MB, Stampnitzky JR. Reduced trends of male proportion at birth in Europe. Arch ratio of male to female births in several industrial Environ Health 2001; 56:358-64. countries: a sentinel health indicator? JAMA 1998; 10. Grech V. Evidence of socio-economic stress and fe- 279:1018-23. male foeticide in racial disparities in the gender ra- 21. Jargin S. Male to female ratio at birth: the role of tio at birth in the United States (1995–2014). Early background radiation vs. other factors. J Environ Hum Dev 2017; 106:63-65. Stud 2018; 4.1: 4. 11. Nicolich MJ, Huebner WW, Schnatter AR. Influence 22. James WH., Grech V. Offspring sex ratio: Coital of parental and biological factors on the male birth rates and other potential causal mechanisms. Early fraction in the United States: an analysis of birth Human Development 2018; 116: 24-27. certificate data from 1964 through 1988. Fertil Steril 23. Polasek O. Did the 1991-1995 wars in the former 2000; 73:487-92. Yugoslavia affect sex ratio at birth? Eur J Epidemiol 12. James WH. Hormonal control of sex ratio. J Theor 2006; 21:61-4. Biol 1986; 118:427-41.

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24. James WH. The variations of human sex ratio at 26. Gutiérrez-Adán A, Pintado B, de la Fuente J. Demo- birth during and after wars, and their potential expla- graphic and behavioral determinants of the reduction nations. J Theor Biol 2009; 257:116-23. of male-to-female birth ratio in Spain from 1981 to 25. Chahnazarian A. Determinants of the sex ratio at 1997. Hum Biol 2000; 72:891-8. birth: review of recent literature. Soc Biol 1988; 27. Figà-Talamanca I, Carbone P, Lauria L, Spinelli A, 35:214-35. Ulizzi L. Environmental factors and the proportion of males at birth in Italy. Arch Environ Health 2003; 58:119-24.

426 ORIGINAL ARTICLE

Retinal changes in febrile seizures in children: a retrospective analysis in Tuzla Canton, Bosnia and Herzegovina

Meliha Halilbašić1, Amra Nadarević Vodenčarević1, Anis Međedović1, Amir Halilbašić2, Almira Ćosićkić2, Ajla Pidro3

1Eye Clinic, 2Clinic for Children’s Diseases; University Clinical Center Tuzla, Tuzla, 3Eye Clinic Dr. Ismail, Sarajevo; Bosnia and Herzegovina

ABSTRACT

Aim To evaluate the possibility of retinal haemorrhages or any other retinal pathology caused by febrile seizures alone in children aged between 2 months and 15 years.

Methods Children aged between 2 months and 15 years admitted to the hospital following seizures were examined within 48 hours of admission. The seizures were classified by a paediatric neurologist and a detailed ocular examination, including indirect ophthalmoscopy, was performed by an ophthalmologist.

Results In the period between May 2019 and May 2020 a total number of 106 children were examined. There were 66 (62.3%) Corresponding author: male and 40 (37.7%) female children. The youngest patient was Meliha Halilbašić 2 months old and the oldest patient was 15 years old. None of Eye Clinic, University Clinical Centre Tuzla the children was found to have retinal haemorrhages or any other Prof. I. Pašića b.b, 75000 Tuzla, retinal pathology. Bosnia and Herzegovina Conclusion Retinal haemorrhages or any other acute retinal fin- Phone: +387 35 303 230; dings in children with febrile seizures are very rare, but we cannot Fax: +387 35 250 474; rule out its occurrence. The finding of retinal haemorrhages in a E-mail: [email protected] child admitted with a history of seizure should trigger a detailed search for other causes of those haemorrhages, especially shaken ORCID ID: http://orcid.org/0000-0002- baby syndrome. Due to the lack of any manifestations on the retina 4702-817X after febrile seizure, maybe it is time that the current protocol and guidelines, considering obligatory fundus examination, should be re-examined.

Key words: convulsions, fundus haemorrhage, ophthalmoscopy, retinal pathology Original submission: 02 March 2021; Revised submission: 04 May 2021; Accepted: 21 May 2021 doi: 10.17392/1366-21

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INTRODUCTION PATIENTS AND METHODS A febrile seizure (FS) is a seizure occurring in Patients and study design childhood, after one month of age-associated with a febrile illness and not caused by an infec- Tuzla Canton is an administrative unit of the tion of the central nervous system (1). They are Federation of Bosnia and Herzegovina, in the the most common neurologic disorder, and are Northeast part of Bosnia and Herzegovina. It is seen in 2-5% of children under the age of five in composed of 13 municipalities with the populati- Western Europe and the United States, and even on of 445.028 inhabitants (18). The administrati- more often, up to 10% in Japanese and Indian ve centre is Tuzla. children (2,3). The etiology of FS is multifacto- A retrospective cross-sectional observational rial, with genetic predisposition and viral illness study was conducted with an analysis of the as the most common factors (4). Many studies medical records of all children aged between 2 have investigated the seasonal distribution of FS months and 15 years presenting to the Clinic for and agreed that winter is the peak season for the Children’s Diseases, Emergency Department of onset of FS, but some studies found an increased the University Clinical Centre (UCC) Tuzla with number of FS also during the summer (5,6). The a diagnosis of FS between May 2019 and May FS is the most common reason for admission to 2020. Children with a history of trauma, suspec- the paediatric emergency department (7). Clini- ted child abuse or cardiopulmonary resuscitation cal evaluation of a child with FS includes a full were excluded. Children who were hospitalized paediatric examination and detailed neurological due to febrile seizures but live outside of Tuzla examination and should focus on identifying the Canton and children who were screened for reti- infection causing the fever (8). Paediatric litera- nopathy of prematurity and had the active disease ture recommends ocular examination of a child were also excluded from the study. with seizures to look for papilledema, retinal An approval from the Ethics Committee Board of haemorrhages, chorioretinitis, retinal coloboma, the University Clinic Centre Tuzla was obtained macular changes, as well as retinal phacomas to conduct this study. to guide a paediatrician on possible underlying systemic condition (9). Methods It is also recommended that an ocular fundus exa- Demographic data, age at presentation, deve- mination should be performed to look for signs lopmental history (developmental milestones of increased intracranial pressure (ICP) (10). The appropriate for the age reached on time), past ICP does not cause retinal haemorrhages (RH) medical and family history regarding allergies, in children except haemorrhages associated with intracranial infections, high fevers, previous se- papilledema (11-13). If RH are found they can be izures, head injuries, family history of febrile se- due to an injury, a variety of systemic diseases izures, and season when FS occurred were recor- (14,15), vomiting or coughing, and after resus- ded. All patients were examined by a paediatric citation. The likelihood of developing RH after neurologist who conducted detailed physical and seizures is less than 1% with a low prevalence neurological examinations and classified seizu- (16). However, in recent guidelines for febrile se- res. Only children with febrile seizures without izures, there are no recommendations for manda- evidence of intracranial infection or history of tory ocular fundus examination (17,18). nonfebrile seizures were included in this study. In Bosnia and Herzegovina, the frequency of retinal According to the age, the patients were divided haemorrhage or any fundus abnormalities in chil- into the following groups: 2-6 months, 6-12 dren with febrile seizures is not well established. months, 1-3 years, 3-5 years, 5-10 years, and The aim of this study was to establish the frequ- children 10-15 years old. It was noted if the child ency of retinal haemorrhages or any pathologi- required cardiopulmonary resuscitation. Previo- cal findings on the retina after febrile seizures in us eye examinations before the admission for FS children aged between 2 months and 15 years in were noted as well. The policy of the Children's the University Clinical Centre Tuzla. Hospital (UCC Tuzla) is to perform the manda-

428 Halilbašić et al. Retina and febrile seizures in children

tory fundus examination on all seizing children as a part of the routine physical examination. Ocular fundus was examined by an ophthalmologist with indirect ophthalmoscopy, without indentation, within 48 hours of the patient's admission to the Hospital. The pupils were dilated with tropicamide 0.5% eye drops before the examination. The examination was done by indirect ophthalmoscope (Heine, Gilching, Germany) Figure 1. Geographic distribution of children with febrile seizures in Tuzla Canton per 13 municipalities and using a 20D Volk lens. Findings on fundus were in detail written down in patient's records. Febrile seizures occurred throughout the year, but the largest number of children with FS was Statistical analysis recorded in the winter and autumn, 39 (36.8%) and 27 (25.5%), respectively (Table 1). Relevant data were presented as frequencies, means, and standard deviation (SD). Statistical No retinal haemorrhages were found in any of analysis was undertaken using Hanley’s rule of the children. Using Hanley’s rule of three, with three (19,20). If none of the “n” patients showed an upper limit of 95%, the chance of retinal hae- the event in question, the chance of this event morrhages occurring as a result of FS alone was (with 95% confidence limit; 0/n) is at most 3 in at the most 3 in 100 (2.8%). n (that is 3/n). We found one child with subatrophic optic disc (diagnosed before FS), while nine children had RESULTS fundus examination described as pale optic disc In the period from May 2019 till May 2020, a to- without signs of subatrophy or atrophy, or any tal of 106 children were examined. There were 66 other retinal changes. (62.3%) male and 40 (37.7%) female children. The None of the children with FS required cardiopul- youngest patient was 2 months old and the oldest monary resuscitation. patient was 15 years old. Most patients were in the Out of the total number, six (5.66%) children had age group 1-3 years (49.1%) (Table 1). an eye examination before FS due to eye deviati-

Table 1. Gender and age groups of the examined children on, of which esotropia was found in five children, with febrile seizure (FS) and seasonal variations while exophoria was found in only one child. Characteristic No (%) of children Gender DISCUSSION Male 66 (62.3) Female 40 (37.7) In our study, FS was more often present in boys Age and the age group 1-3 years, similar to the reports 2-6 months 7 (6.6) of other studies (1,21,22). Seasonal variations are 6-12 months 9 (8.5) 1-3 years 52 (49.1) known as well, with most cases occurring during 3-5 years 13 (12.2) winter, as we have found in our study (5,23,24). 5-10 years 20 (18.9) 10-15 years 5 (4.7) Current paediatric guidelines recommend that Season ocular fundus examination should be performed to Spring 22 (20.8) look for increased intracranial pressure (ICP) for Summer 18 (16.9) every child admitted with FS (1). Increased ICP Autumn 27 (25.5) Winter 39 (36.8) is a theory frequently proffered in legal settings as an alternative cause of severe RHs (12). Unli- There were 95 (89.6%) children born on time and ke neonatal RH, that are benign and are related to 11 children (10.4%) were preterm babies. A posi- birth trauma, that one found from infancy later in tive family history of FS in first-degree relatives life can be a sign of injury and variety of ocular was found in seven (6.6%) cases. Children with FS or systemic diseases (14). One of the mechanisms were admitted to UCC Tuzla from all 13 municipa- that can also result in RH is a sudden rise in intrat- lities of Tuzla Canton. Most of the children with FS horacic pressure (Valsava type manoeuver) that were from Tuzla city, 33 (31.1%) (Figure 1). can happen during seizures (26).

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The prevalence of retinal haemorrhage after seizu- 2 months to 15 years seizures alone were unlikely res is still unclear, and there are only a few studies a cause of retinal haemorrhages or any other acu- available in the literature (15,25,26). In our study, te retinal findings, although the possibility cannot none of the 106 children were found to have reti- be completely ruled out. nal haemorrhages or any other acute retinal finding The study conducted in 2008 in Tuzla Canton, Bo- within 48 hours of their admission following FS. snia and Herzegovina, examined clinical characte- In a similar study done by Sandramouli et al. on 32 ristics at onset of the first FS, but authors have not children (aged 4 months to 14 years) with seizures, analysed the connection between retinal findings none developed retinal haemorrhage (25). Tyagi et and febrile seizures (28). Taking into considerati- al. in a study on 32 infants with FS, found no cases on that until the present day, the available studies of retinal haemorrhage (26). Our study included show none, except one case, of the retinal hae- 106 children with a wider age group, and have morrhage associated with the febrile seizure, we found no retinal haemorrhage or any other retinal recommend that the current protocol and guideli- changes in children with FS. The likelihood to find nes be re-examined in children with FS. some retinal changes in our study is 0.0283 (less In conclusion, retinal haemorrhages or any other than 3/100). In a study on 153 children aged 2-24 acute retinal findings in children with febrile- se months with FS Mei Zahav et al. found unilateral izures are very rare, our study found none, but we retinal haemorrhages in only one 8-month-old girl, cannot rule out its occurrence. Finding a retinal with the prevalence of retinal haemorrhage secon- haemorrhage in a child after a febrile seizure episo- dary to seizures of 0.0065 (15). In two studies (25, de should therefore trigger an extensive search for 26) from the same Institution the upper limit of other causes, such as non-accidental injuries inclu- 95% confidence interval of retinal haemorrhages ding child abuse. Due to the lack of any manifesta- following seizures in children under the age of 14 tions on the retina after FS, maybe it is time that years was less than 5/100, which is insignificantly current protocol and guidelines be re-examined. higher than our finding. In a similar study on 34 children aged 3 months to 9 years, Guo et al. fo- FUNDING und prevalence of retinal haemorrhages in chil- No specific funding was received for this study. dren with FS lower than 10% (27). The limitation of all these studies is a relatively small number of TRANSPARENCY DECLARATION children in the study group. Conflict of interest: None to declare. A statistical analysis of our study, which included larger study group, showed that in children aged REFERENCES 1. Leung AK, Hon KL, Leung TN. Febrile seizures: an 8. Laino D, Mencaroni E, Esposito S. Management of overview. Drugs Context 2018; 7:212536. pediatric febrile seizures. Int J Environ Res Public 2. Patel N, Ram D, Swiderska N, Mewasingh LD, Health 2018; 15:2232. Newton RW, Offringa M. Febrile seizures. BMJ 9. Kliegman R, Geme J. Nelson Textbook of Pedia- 2015; 351:h4240. trics. 21th ed. Philadelphia: Elsevier, 2020. 3. Paul SP, Seymour M, Flower D, Rogers E. Febrile 10. Binenbaum G, Rogers DL, Forbes BJ. Patterns of convulsions in children. Nurs Child Young People retinal hemorrhage associated with increased intra- 2015; 27:14–15. cranial pressure in children. Pediatrics 2013; 132: 4. Smith DK, Sadler KP, Benedum M. Febrile Se- e430-e434. izures: Risks, evaluation, and prognosis. Am Fam 11. Shiau T, Levin AV. Retinal hemorrhages in children: Physician. 2019; 99:445-50. the role of intracranial pressure. Arch Pediatr Ado- 5. Sharafi R, Hassanzadeh RA, Aminzadeh V. Circadi- lesc Med 2012; 166:623–8. an rhythm and the seasonal variation in childhood 12. Shi A, Kulkarni A, Feldman KW. Retinal findings febrile seizure. Iran J Child Neurol 2017; 11:27-30. in young children with increased intracranial pre- 6. Han DH, Kim SY, Lee NM, Yi DY, Yun SW, Lim IS, ssure from nontraumatic causes. Pediatrics 2019; Chae SA. Seasonal distribution of febrile seizure and 143:e20181182. the relationship with respiratory and enteric viruses 13. Gayle MO, Kissoon N, Hered RW, Harwood-Nuss. in Korean children based on nationwide registry Retinal hemorrhage in the young child: a review of data. Seizure 2019; 73:9-13. etiology, predisposed conditions, and clinical impli- 7. Shankar P, Mahamud S. Clinical, epidemiological cations. J Emerg Med 1995; 13:233–9. and laboratory characteristics of children with febri- 14. Kaur B, Taylor D. Fundus hemorrhages in infancy. le seizures.Int J Contemp Pediatr 2020; 7:1598-605. Surv Ophthalmol 1992; 37:1–17.

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15. Mei-Zahav M, Uziel Y, Raz J, Ginot N, Wolach B, 22. Dreier JW, Li J, Sun Y, Christensen J. Evaluation of Fainmesser P. Convulsions and retinal hemorrha- long-term risk of epilepsy, psychiatric disorders, and ge: should we look further? Arch Dis Child 2002; mortality among children with recurrent febrile se- 86:334-5. izures: a national cohort study in Denmark. JAMA 16. Royal Cornwall Hospitals NHS Trust. Febrile Con- Pediatr 2019; 173:1164-70. vulsions Clinical Guideline V4.0 https://doclibrary- 23. Mikkonen K, Uhari M, Pokka T, Rantala H. Diurnal rcht.cornwall.nhs.uk/GET/d10290030 (03 April and seasonal occurrence of febrile seizures. Pediatr 2021) Neurol 2015; 52:424–427. 17. Alexander D. The Febrile Seizure http://brownem- 24. Millichap JJ, Millichap JG. Diurnal and seasonal blog.com/blog-1/2020/7/31/the-febrile-seizure (03 occurrence of febrile seizures. Pediatr Neurol Briefs April 2021) 2015; 29:29. 18. Agency for Statistics of Bosnia and Herzegovina. 25. Sandramouli S, Robinson R, Tsaloumas M. Retinal Census of population, households and dwellings in hemorrhages and convulsions. Archives of Disease Bosnia and Herzegovina, 2013. http://www.statisti- in Childhood 1997; 76:449-451. ka.ba/?lang=en (19 September 2020) 26. Tyagi AK, Scotcher S, Kozeis N, Willshaw HE. Can 19. Eypasch E, Lefering R, Kum CK, Troidl H. Proba- convulsions alone cause retinal hemorrhages in in- bility of adverse events that have not yet occurred: a fants? Br J Ophthalmol 1998; 82: 659-660. statistical reminder. BMJ 1995; 311:619-20. 27. Guo H, Lan Y, Wang M, Xiao J, Hu Y, Xia Z. Ma- 20. Hanley JA, Lippman-Hand A. If nothing goes nifestations of ocular fundus in children with febri- wrong, is everything all right? Interpreting zero nu- le seizures. J Pediatr Ophthalmol Strabismus 2011; merators. JAMA 1983; 249:1743-5. 48:182-186. 21. Choi YJ, Jung JY, Kim JH, Kwon H, Park JW, Kwak 28. Mustafić N, Tahirović H, Trnovcević J, Kapidzić YH, Kim DK, Lee JH. Febrile seizures: Are they A. Klinicke karakteristike prvih febrilnih konvul- truly benign? Longitudinal analysis of risk factors zija (Clinical characteristics at onset of first febrile and future risk of afebrile epileptic seizure based on convulsions) [in Croatian]. Acta Med Croatica 2008; the national sample cohort in South Korea, 2002- 62:511-515. 2013. Seizure 2019; 64:77-83.

431 ORIGINAL ARTICLE

The role of aripiprazole in improvement of penile erection in schizophrenia patients with erectile dysfunction

Debby Handayati Harahap1, Carla Raymondalexas Marchira2, Eti Nurwening Solikhah3, Dicky Moch Rizal4

1Doctoral Program, 2Department of Psychiatry, 3Department of Pharmacology, 4Department of Physiology; Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia

ABSTRACT

Aim To provide evidence regarding the effectiveness of aripiprazole in improving penile erection with a therapeutic strategy of add-on or switching therapy in patients with schizophrenia.

Methods. PubMed, Cochrane, Clinical Key, ProQuest, EBSCO- host, and ScienceDirect were searched for any design study that evaluated aripiprazole only or versus control (placebo or other antipsychotic) for erectile dysfunction in patients with schizophrenia. Three studies were identified and analysed from 295 initial articles. Data were then extracted from the studies and summarized descriptively. Corresponding author: Carla Raymondalexas Marchira Results Two hundred ninety-five articles were screened, and three Department of Psychiatry, studies were identified and eventually selected. After the add-on or Faculty of Medicine, Public Health and switching antipsychotic therapy to aripiprazole, the prevalence of erectile dysfunction and the score of erectile dysfunction or penile Nursing, Universitas Gadjah Mada erection assessed by Nagoya and Sexual Function Questionnaire Jl Kesehatan No 1, Sendowo, Sleman, (NSFQ) and Arizona Sexual Experience Scale (ASEX) decreased. Special Region of Yogyakarta, Indonesia, 55281 Conclusion Aripiprazole was effective to improve penile erectile Phone: +62 878 3716 0809; function in patients with schizophrenia. The therapeutic strategy is adjunctive treatment or switching therapy to aripiprazole. E-mail: [email protected] Debby Handayati Harahap ORCID ID: Key words: penile erection, prevalence, schizophrenia, sexual be- https://orcid.org/0000-0001-8467-1570 haviour

Original submission: 18 February 2021; Revised submission: 12 March 2021; Accepted: 23 March 2021 doi: 10.17392/1360-21

Med Glas (Zenica) 2021; 18(2):432-437

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INTRODUCTION mines the pharmacological profile and aripiprazole side effects (11). Aripiprazole is known to Schizophrenia is a severe psychiatric disorder have a low risk of extrapyramidal side effects and that has a profound effect on individuals and decrease the risk of sexual dysfunction, including society in the form of complex, heterogeneous erectile dysfunction (11). behavioural and cognitive syndromes, which ori- ginate from brain development disorders caused Treatment strategies for schizophrenic pati- by genetic, environmental, or both factors (1). ents with sexual dysfunction have been propo- Schizophrenia has the most prominent symptoms sed. Lowering the dose of antipsychotic drugs, including delusions and hallucinations, which are adjunctive treatment with dopamine agonists, and also called psychotic symptoms. The symptoms switching to prolactin-sparing drugs are a useful are loss of contact with reality and also experi- option (5). The ability of aripiprazole to reduce the encing negative symptoms that occur specifically incidence of erectile dysfunction can be seen in such as impaired motivation, decreased speech, several studies (7,12,13). Erectile dysfunction was and withdrawal from the social environment (2). significantly reduced 12 weeks after switching The incidence of schizophrenia in industrialized therapy to aripiprazole (12). Decreased erecti- countries is 10-70 new cases per 100,000 popu- le dysfunction also occurred from 8 to 26 weeks lation per year (1,3). Based on Indonesian Ba- after the treatment with aripiprazole compared to sic Health Research data, there is an increase in other antipsychotics based on total Arizona Sexual schizophrenia incidence in Indonesia, which was Experience Scale (ASEX) scores (13). Also, the 7 per 1000 adults in 2018, compared to 1.7 per prevalence of erectile dysfunction is less frequent 1000 adults in 2013 (4). in patients treated with aripiprazole (15.38%) than with haloperidol (45.83%) (14). Typical antipsychotic drugs have a greater level of affinity, risk of extrapyramidal side effects, This research aimed to review the effectivene- and hyperprolactinemia (5). Besides, one of the ss of the therapeutic strategy aripiprazole as an common side effects is sexual dysfunction, with adjunctive or switching therapy for patients with prevalence of 45-80% of male patients (6). Sexu- schizophrenia focusing on a comparison of evi- al dysfunction includes a reduction in desire or dence regarding the effectiveness of aripiprazole libido, reduced arousal, decreased frequency of in improving penile erectile function. sexual intercourse or inability to reach orgasm, MATERIALS AND METHODS retrograde ejaculation, and erectile dysfunction (7). Atypical antipsychotic drugs have a greater Materials and study design affinity for serotonin receptors than dopamine receptors. Most atypical antipsychotic drugs cause A comprehensive search was performed in July side effects such as weight gain and fat metaboli- 2020 in which we searched the Wiley Online sm disruption (8). Library, PubMed, Cochrane, ProQuest, and Sci- enceDirect databases, using keywords related to Antipsychotic drugs have the potential to cau- schizophrenia, aripiprazole, and erectile dysfunc- se hyperprolactinemia because the inhibition of tion without language restrictions. The following dopamine release effectively removes negative keywords were used in searches of all the data- feedback loops for prolactin secretion from the bases: "Schizophrenia" and "Aripiprazole" and anterior pituitary gland. Increased serum prolac- "Erectile Dysfunction." tin levels have shown to have profound effects on reproductive health and sexual function, inclu- This study reviews evidence from any design ding hypogonadism, decreased libido in both study with open-label treatment published in genders, amenorrhoea and infertility in women the period between 2005 and 2020 to assess the and low sperm counts and reduced muscle mass effect of aripiprazole on erectile dysfunction of in males (9). The prevalence of sexual dysfuncti- schizophrenic patients. The patients reviewed on was up to 50% for first-generation antipsycho- were male patients younger than 65 years. Race tic drugs groups (10). However, aripiprazole is and duration of follow-up were not considered. the only antipsychotic with partial agonist acti- The criteria for inclusion and exclusion were de- vity against dopamine D2; this difference deter- termined before the search. We included studies

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with add-on therapy or switching to aripiprazo- antipsychotic treatments), side effects of erectile with or without other control or antipsycho- le dysfunction, and methods used to analyse the tic groups. Studies with relevant titles are then results. The primary outcome assessed was the collected and filtered. Studies found in more than efficacy of aripiprazole treatment, classified as one database were removed. Full-paper manus- a decreased percentage of erectile dysfunction, cripts were then studied, and manuscripts that compared with baseline. were irrelevant to the theme are excluded. Three studies were included in a systematic review. RESULTS The search on the database was resulted in 295 Methods initial articles (93 articles from Wiley Online This systematic review was written based on the Library, 42 from PubMed database, four from Preferred Reporting Items for Systematic Re- Cochrane Online Library, 108 from ProQuest views and Meta-Analyses (PRISMA) guidelines and 48 articles from ScienceDirect). Because of for reporting the events evaluated by interventi- the irrelevant titles, 279 articles were excluded, ons and health care behaviours (15). Population, and nine articles were removed because of du- intervention, control, and outcome (PICO) que- plicate titles. After the discussion among the aut- stions (16) used in this systematic review were: hors, another four articles were excluded because P (population): schizophrenia patient, I (inter- of several reasons (among them, three were not vention): the use of aripiprazole, C (comparison/ found and attempts to contact the authors were control): without or with other antipsychotic or unsuccessful). The title and the abstract of the placebo and O (outcome): erectile dysfunction. articles were reviewed, and according to the results, only three fitted the eligibility criteria sta- Statistical analysis ted in this systematic review (Figure 1). Relevant information was extracted from selec- All three selected studies were conducted in Ko- ted studies. Relevant information included study rea (18), India (19) and Japan (17). There were 34 types, patient characteristics, intervention regi- schizophrenic patients with sexual dysfunction in mens, comparative regimens (placebo or other these studies. Two studies were on add-on therapy

Figure 1. Diagram flow of the article’s selection

434 Harahap et.al. Aripiprazole in erectile dysfunction

Table 1. Summary of data description from the included studies Length of Study Subject criteria and study design Intervention Outcome follow up Jeong et al. 2012 (Korea) (18) Subject criteria: Switching to aripiprazole 12 weeks Penile erection improved Taking a single second-generation oral antipsychotic (espe- on sexual dysfunction of 10 significantly over the study cially risperidone, olanzapine, or amisulpride), complaining male schizophrenia patients, period (score: baseline = of significant sexual dysfunction since taking antipsychotic who had been treated with 3.8±1.14, week 6 = 3.1±0.99, medication and patients aged 20–55 years. atypical antipsychotics, ris- week 12 =3.3±1.16; w2=9.33; Study design: peridone (n =6), amisulpride p =0.009). Prospective, open-label study without control group (n=3), and olanzapine (n=1)

Raghuthaman et al. 2015 (India) (19) Subject criteria: Adjunctive treatment 8 weeks Aripiprazole improved erectile Aged 15–45 years, patients were being prescribed a stable with 10 mg aripiprazole dysfunction in five out of six dose of risperidone for at least 12 weeks. on sexual side-effects in patients. In contrast, one additi- Study design: patients with schizophrenia onal patient developed erectile A double-blind, placebo-controlled study symptomatically maintained dysfunction in the placebo on risperidone group at follow-up (p=0.01) Fujioi et al. 2017 (Japan) (17) Subject criteria: Adjunctive treatment with 24 weeks Erectile dysfunction improved 9 males were 65 years of age or younger, hyperprolactinemia aripiprazole on sexual by week 24 (mean = 2.6±1.1 and a score of 3 or higher on any of the NSFQ items side-effects in patients with vs 2.0±1.1; p=0.049) Study design: schizophrenia Open-label and naturalistic design without a control group with aripiprazole, and one study was on switching at baseline to 3.1±0.99 at week 6, and continued to therapy to aripiprazole. Erectile dysfunction was improve at week 12 to 3.3±1.16 (p=0.009). assessed with the Nagoya and Sexual Function Raghuthaman et al. (19) enrolled fifteen male pa- Questionnaire (NSFQ) (17) and Arizona Sexual tients who had been prescribed a stable dose of Experience Scale (ASEX) (18,19). risperidone for at least 12 weeks, nine patients Erectile dysfunction or penile erection was one received adjunctive treatment with aripiprazole, of several items assessed from the two questi- and six patients received placebo. In the aripi- onnaires (NSFQ and ASEX). The NSFQ is a self- prazole group, six (66.7%) males had erectile administered sexual function scale that consists dysfunction at baseline, only one (11.1%) still of 7 items, which for males include pulsating had erectile dysfunction after eight weeks. In sensation in the breast/mammary area, galactorr- contrast, in the placebo group four (66.7%) males hea, interest in women, sexual interest, sexual had erectile dysfunction at baseline, which incre- self-confidence, erectile dysfunction and ejacula- ased to five (83.3%) males after eight weeks. The tory dysfunction (20), while the Arizona Sexu- difference in the proportion of men with erectile al Experience Scale (ASEX) consists of 5 items dysfunction at follow-up between the two groups comprising strength of sex drive, ease of sexual was statistically significant (p=0.01). arousal, penile erection, ability to reach orgasm, Fujioi et al. (17) reported a significant impro- and satisfaction with orgasm (21). vement of erectile dysfunction in nine male Two open-label studies without a control group schizophrenia patients after adjunctive treatment and one double-blind study with placebo-con- with aripiprazole. The score of erectile dysfunc- trolled were conducted in 2012, 2015, and 2017. tions improved from mean value of 2.6±1.1 at The patient's age ranged between 15 - 65 years. baseline to 2.0±1.1 at week 24 (mean differen- One of the open-label studies without the control ce=0.6 with 95% CI=0.003–1.2; p=0.049). group used the NSFQ to assess erectile dysfunction, and the other used the ASEX. Various lengths DISCUSSION of follow up were from 8 weeks (19), 12 weeks Based on three studies that have been analysed, (18), and 24 weeks (17) (Table 1). two studies were related to add-on therapy, and the Jeong et al. (18) found that the score of penile remaining one was related to switching therapy to erection in 10 male schizophrenia patients after aripiprazole. Erectile dysfunction or penile erecti- switching to aripiprazole improved significantly on was one of several items assessed from the two over the study period from a mean value of 3.8±1.14 questionnaires (NSFQ and ASEX). A significant

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improvement of erectile dysfunction was found in incidence of erectile dysfunction and the score of add-on therapy with aripiprazole at week 24 (17) erectile dysfunction or penile erection decrease. and week 8 (19) after adjunctive therapy. It can Despite the evidence of overall improvement of be concluded that adjunctive aripiprazole reduces erectile dysfunction after adjunctive or switch prolactin levels in schizophrenic patients treated therapy to aripiprazole, there were several limi- with risperidone and may be a potential treatment tations on these studies (17,18,19). The results for hyperprolactinemia following treatment with of Jeong's et al. study is based on a small sample, second-generation antipsychotics. and this will result in possible type II error/ fal- Switching therapy with aripiprazole could be se negative (26). Also, this study was unable to considered in patients with schizophrenia. The show whether improvements in sexual function study in Korea (18) found improvement in peni- persisted after 12 weeks. Fujioi's study included le erection after switching to aripiprazole. It can only participants in the aripiprazole group wit- be determined that sexual dysfunction in patients hout a control group. The statistical power for with schizophrenia in this study appeared to im- some assessment items is underpowered because prove after switching to aripiprazole from other of the small sample size. All patients had varied atypical antipsychotics. sexual functions; the therapeutic environment (in Erection is a neurovascular condition that is in- and out of the hospital) can also affect the results. fluenced by hormones, consists of arterial dilata- For further research, the sample size should be tion, relaxation of the trabecular smooth muscles, more extensive, and long-term research is re- and activation of the corporal veno-occlusive commended to ensure whether improvements in mechanism (22). The National Institutes of He- sexual function persisted overtime. alth (NIH) Consensus Development Conference This study's limitation was that a small number on Impotence defines erectile dysfunction as the of studies proved the effectiveness of aripiprazo- inability to achieve or maintain an erection suffi- le in improving erectile function in patients with cient for satisfactory sexual performance (23). schizophrenia, because only three studies fitted Blockade of dopamine receptors in the tuberoin- the eligibility criteria. fundibular pathway results in decreased dopami- In conclusion, dopamine blockade, as an antip- ne tone. Besides, inhibition of dopamine release sychotic mechanism of action in the tuberoinfun- effectively removes the negative feedback loop dibular pathway, can cause hyperprolactinemia. for prolactin secretion from the anterior pituitary Addition or switching of therapy to aripiprazole gland, thereby increasing the secretion of pro- decreases the incidence of erectile dysfunction lactin. Elevated serum prolactin levels have pro- and the score of erectile dysfunction or penile found effects on reproductive health and sexual erection, thereby improving erectile function. function, including erectile dysfunction (24, 25). ACKNOWLEDGEMENT Jeong et al. (18) reported that by switching therapy to aripiprazole, penile erection improved signifi- The authors would express their gratitude to cantly after 12 weeks. This is also the case with the Medical Research Unit of Faculty of Medicine, adjunctive therapy with aripiprazole, Raghutha- Health Science and Nursing, Universitas Gadjah man et al. (19) reported adjunctive therapy with Mada, Yogyakarta, Indonesia aripiprazole improved erectile dysfunction in five out of six patients after eight weeks of treatment. FUNDING Besides, Fujioi et al. (17) reported an improve- No specific funding was received for this study. ment of erectile dysfunction after 24 weeks of adjunctive treatment with aripiprazole. After the TRANSPARENCY DECLARATION addition or switching therapy to aripiprazole, the Conflict of interest: None to declare.

436 Harahap et.al. Aripiprazole in erectile dysfunction

REFERENCES 1. Owen MJ, Sawa A, Mortensen PB. Schizophrenia. 15. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Lancet 2016; 388: 86–97. Gøtzsche PC, Ioannidis JPA. The PRISMA statement 2. Fatani BZ, Aldawod RA, Alhawaj FA. Schizophre- for reporting systematic reviews and meta-analyses nia: etiology, pathophysiology, and management : a of studies that evaluate healthcare interventions: review. Egypt J Hosp Med 2017; 69:2640–46. explanation and elaboration. BMJ 2009; 339: 56-62. 3. Meyer N, MacCabe, JH. Schizophrenia. Med (Uni- 16. Byron C Wallace, Joel Kuiper, Aakash Sharma, ted Kingdom) 2016; 44:649-53. Mingxi Zhu IJM. Extracting PICO Sentences from 4. Indonesian Ministry of Health. Hasil utama riset ke- Clinical Trial Reports using Supervised Distant Su- sehatan dasar (RISKESDAS) (Main result of basic pervision. J Mach Learn Res 2016; 17:90-8. health data) [In Indonesian]. 2018; 44. 17. Fujioi J, Iwamoto K, Banno M, Kikuchi T, Aleksic B, 5. Miyamoto BE, Galecki M, Francois D. Guideli- Ozaki N. Effect of adjunctive aripiprazole on sexual nes for antipsychotic-induced hyperprolactinemia. dysfunction in schizophrenia: a preliminary open-la- Psychiatr Ann 2015; 45:266–72. bel study. Pharmacopsychiatry 2017; 50:74–8. 6. Yeon W, Yooseok K, Jun H. Antipsychotic induced 18. Jeong HG, Lee MS, Lee HY, Ko YH, Han C, Joe sexual dysfunction and its management. World J SH. Changes in sexual function and gonadal axis Mens Health 2012; 30:153-59. hormones after switching to aripiprazole in male 7. Anthony J, Rany S. Psychotropics and sexual schizophrenia patients: a prospective pilot study. Int dysfunction. Cent European J Urol 2013; 66:466-71. Clin Psychopharmacol 2012; 27:177–83. 8. Chisholm-Burns MA, Wells BG, Schwinghammer 19. Raghuthaman G, Venkateswaran R, Krishnadas TL, Malone PM, Kolesar JM, DiPiro JT. Pharma- R. Adjunctive aripiprazole in risperidone-indu- cotherapy: Principles & Practice. 4th ed. New York: ced hyperprolactinemia: a double-blind, rando- McGraw-Hill Education, 2016. mized, placebo-controlled trial. Br J Psych Open 9. Hanssens L, L’Italien G, Loze JY, Marcus RN, 2015;1:172–7. Pans M, Kerselaers W. The effect of antipsychotic 20. Kikuchi T, Iwamoto K, Sasada K, Aleksis B, Yoshi- medication on sexual function and serum prolactin da K, Ozaki N. Reliability and validity of a new levels in community-treated schizophrenic patients: sexual function questionnaire (Nagoya Sexual results from the Schizophrenia Trial of Aripiprazo- Function Questionnaire) for schizophrenic patients le (STAR) study (NCT00237913). BMC Psychiatry taking antipsychotics. Hum Psychopharmacol 2011; 2008; 8:1–11. 26:300–6. 10. Mahmoud A, Hayhurst KA, Drake RJ, Lewis SW. 21. McGahuey CA, Gelenberg AJ, Laukes CA, Moreno Second generation antipsychotics improve sexual FA, Delgado PL, McKnight KM. The Arizona Sexu- dysfunction in schizophrenia: a randomized con- al Experience Scale (ASEX): reliability and validity. trolled trial. Schizophr Res Treatment 2011; 596898. J Sex Marital Ther 2000; 26:25–38. 11. Tuplin EW, Holahan MR. Aripiprazole, a drug that 22. Tsertsvadze A, Fink HA, Yazdi F, Macdonald R, Be- displays partial agonism and functional selectivity. lla AJ. Clinical guidelines annals of internal medici- Curr Neuropharmacol 2017; 15:1192-207. ne oral phosphodiesterase-5 inhibitors and hormonal 12. Mir A, Shivakumar K, Williamson RJ, McAlli- treatments. Ann Intern Med 2009; 151:650–61. ster V, O’Keane V, Aitchison KJ. Change in sexual 23. Kevan W, Ian M. Erectile dysfunction. In: Richard dysfunction with aripiprazole: a switching or add-on B, R. Taylor S, ed. Handbook of Sexual Dysfuncti- study. J Psychopharmacol 2008; 22:244-53. on. New York: Taylor & Francis Ltd, 2005; 155–91. 13. Hanssens L, L’Italien G, Loze JY, Marcus RN, 24. Peter F, Timothy GD. Prolactin and dopamine: what Pans M, Kerselaers W. The effect of antipsychotic is the connection? a review article. J Psychopharma- medication on sexual function and serum prolactin col 2008; 2:12-9. levels in community-treated schizophrenic patients: 25. Saleem M, Martin H, Coates P. Prolactin biology results from the Schizophrenia Trial of Aripiprazo- and laboratory measurement: an update on physi- le (STAR) study (NCT00237913). BMC Psychiatry ology and current analytical issues. Clin Biochem 2008; 22:8:95. Rev 2018; 39:3-16. 14. Khan A, Nawaz H, Nazneen Z, Yousafzai A. Anti- 26. Banerjee A, Chitnis UB, Jadhav SL, Bhawalkar JS, psychotics induced sexual dysfunction. Pakistan J Chaudhury S. Hypothesis testing, type I and type II Physiol 2017; 13:3-7. errors. Ind Psychiatry J 2009; 18:127-31.

437 ORIGINAL ARTICLE

Risk of surgical site infections after colorectal surgery and the most frequent pathogens isolated: a prospective single-centre observational study

George Panos1, Francesk Mulita2, Karolina Akinosoglou1, Elias Liolis3, Charalampos Kaplanis2, Levan Tchabashvili2, Michail Vailas2, Ioannis Maroulis2

1Department of Internal Medicine and Infectious Diseases, 2Department of General Surgery, 3Department of Internal Medicine, Division of Oncology; University General Hospital, Patras, Greece

ABSTRACT

Aim To identify risk factors for developing surgical site infections (SSIs) based on a prospective study of patients undergoing colorectal surgery.

Methods Between November 2019 and January 2021, 133 patients underwent elective operation for colorectal cancer in our institution. The following variables were recorded for each patient: age, gender, body mass index (BMI), American Society of Ane- sthesiologists Classification (ASA class), duration of surgery, wound classification, skin preparation regimens, surgical approach, comorbidities (hypertension, diabetes, cardiovascular disease, Corresponding author: respiratory disease, chronic steroid use), and pathogens responsi- Francesk Mulita ble for surgical site infection. Univariate analysis was performed using χ2 tests for categorical variables. Department of Surgery, General University Hospital Results A total of 65 males and 68 females were enrolled. Posto- Rio 265 04, Patras, Greece perative SSI was diagnosed in 29 (21.8%) cases. Fifty five patients Phone: +30 6982785 142; were >70 years old, and SSIs were significantly more frequent in this group (p=0.033). There were 92 patients with BMI <30kg/m2 +30 2610 455 541; and 87 with ASA class ≤2; SSIs occurred significantly less frequ- E-mail: [email protected] ently in these patients (p=0.021 and p=0.028, respectively). Open ORCID ID: https://orcid.org/0000-0001- surgery was performed in 113 patients; 35 (out of 113; 31%) wo- 7198-2628 und infections were classified as contaminated or dirty, and SSI occurred more often in these two groups (p=0.048 and p=0.037, respectively). Nineteen patients had diabetes and 36 used steroids continuously; SSI was significantly more frequent in these patients (p=0.021 and p=0.049, respectively).

Original submission: Conclusion Following colorectal cancer procedures SSIs were 27 January 2021; significantly more common among patients over 70 years old, 2 Revised submission: BMI≥30kg/m , ASA score>2, with diabetes and chronic steroid use, 05 February 2021; undergoing open, dirty or contaminated surgery. Escherichia coli and Enterococcus spp. were the two most common pathogens isolated. Accepted: 18 March 2021 Key words: colorectal cancer, microorganism, risk factor, wound doi: 10.17392/1348-21 infection

Med Glas (Zenica) 2021; 18(2):438-443

438 Panos et al. SSIs after colorectal surgery

INTRODUCTION gan, evidence of abscess formation involving the organ/space, purulent drainage from a drain that Surgical site infection (SSI) is one of the most is placed into the organ/space, diagnosis of SSI- frequent healthcare-associated infections (HAI) O by the attending physician or surgeon (7,8). among surgical patients (1). According to recent data from the National Nosocomial Infection According to the literature, Escherichia coli (E. System (NNIS) of the US Centres for Disease coli), Pseudomonas aeruginosa (P. aeruginosa), Control and Prevention (CDC), the prevalence and gram-positive cocci (especially Enterococcus) of SSI ranges from 2.4% to 21.6% in patients are the most frequent isolated pathogens from pa- undergoing colorectal surgery (2). The SSI is the tients with SSI after colorectal surgery. However, most common postoperative complication after differences in the pathogens causes may exist de- colorectal surgery, causing pain and suffering to pending on the type of the colorectal surgery (left- patients (3). In addition, this complication has sided vs right-sided operations) (9,10). been associated with negative economic impact, The aim of this study was to identify the risk fac- increased morbidity, extended postoperative tors for developing SSI based on a prospective hospital stay, readmission, and death (4,5). The analysis of patients undergoing colorectal sur- process of SSI after colorectal surgery is very gery in a single centre between November 2019 complex and involves many factors, such as pa- and January 2021. tient-related factors (age, comorbidities, nutritional status), intra-operative factors (urgent vs PATIENTS AND METHODS elective surgery, open vs laparoscopic method, Patients and study design type of operating field, duration of surgery), and postoperative wound management (6). This prospective trial was conducted at the Ge- The Centres for Diseases Control and Prevention neral University Hospital of Patras in Greece, an (CDC) defined infection of a surgical site as: su- 800-bed tertiary Hospital in South-Western Gree- perficial incisional surgical site infection (SSI-S), ce that covers the population of approximately deep incisional surgical site infection (SSI-D), 1.5 million people, between November 2019 and and organ/space surgical site infection (SSI-O). January 2021, and included 133 patients under- In SSI-S only the skin and subcutaneous tissues going elective operation for colorectal cancer. are involved. They develop within 30 days from Inclusion criteria were patients older than 18 ye- surgery and the diagnostic criteria include the ars, preoperative hospital stay less than 48 hours, presence of at least one symptom of infection elective surgery, and diagnosis of colorectal can- (reddening, swelling, elevated skin temperatu- cer that was scheduled to be treated by elective re, or tenderness), isolated pathogen of material colorectal surgery. Exclusion criteria were patients collected after surgical opening of the incision younger than 18 years old, preoperative hospital site, purulent drainage from the surgical incisi- stay more than 48 hours, emergency surgery, pla- on, diagnosis of SSI-S by the attending physician cement of ileostomies or colostomies, and other or surgeon. One of these criteria has to be met. non-malignant colorectal diseases (infectious In SSI-D deeper tissues are involved, including bowel disease or diverticulitis). The patients gave fascial and muscle layers. Patients with SSI-D their informed written consent for the study. The have at least one of the following criteria: wo- day prior to surgery patients underwent preparati- und dehiscence, purulent drainage from the deep on of large bowel as well as antibiotic prophylaxis surgical incision but not from the organ com- based on standards established at our Institution: ponent of the surgical site, evidence of abscess 2nd generation cephalosporin and metronidazole formation, diagnosis of SSI-D by the attending administered 30 minutes before the first skin inci- physician or surgeon. SSI-O involves any part of sion, and two doses of each antibiotic administe- the anatomy in organs and spaces other than the red again after four hours of operation. incision, which was opened and manipulated du- All operations were conducted by the same group ring the surgical procedure. Patients with SSI-O of surgeons and anaesthesiologists. Patients recei- have at least one of following criteria: pathogens ved either a poviodone-iodine antisepsis regimen, isolated from a culture of fluid or tissue in the or- or chlorhexidine-alcohol skin preparation regimen.

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The day before the surgery, all patients provided Table 1. Postoperative surgical site infections (SSI) in 133 their written informed consent. patients undergoing colorectal surgery Patient groups No (%) of patients An ethical approval was obtained from the Ethics Non-SSI 104 Committee of the General University Hospital of SSI (according to classification) 29 (21.8) Patras (No 5461/40626-11/11/2019). Superficial (SSI-S) 20 (15.0) Deep incisional (SSI-D) 8 (6.0) Methods Organ/space (SSI-O) 1 (0.8) After patient’s extubating in the operating room, The univariate analysis showed no differen- surgical information was recorded (surgery time, ce with regard to SSI prevalence depending on intra-operative complications). Following surgery, patient gender, skin preparation, duration of the patients were transferred to the surgical ward. The procedure, and comorbidities (hypertension, car- patient follow-up and the surveillance of the surgi- diovascular and respiratory disease). The SSIs cal wound, as well as data collection and analysis were significantly more frequent in the group of were carried out by our team of surgeons from the patients >70 years old (55; 41.35%) comparing day of the surgery until hospital discharge. to that one of <70 (30.91% vs 15.38%; p=0.033). The following variables were recorded for each There were 92 (69.17%) patients with BMI patient: age (≤70 or >70 years), gender (male/ <30kg/m2 and 87 (65.41%) with ASA class ≤2 female), body mass index (BMI <30 or ≥30), in which SSI occurred significantly less frequ- American Society of Anesthesiologists Classifi- ently: 16.3% vs 34.15% (p=0.021) and 16.09% cation (ASA; ≤2 - healthy patients or with a mild vs 32.61% (p=0.028), respectively. systemic disease, or >2 - patients with at least An open approach was used in 113 (84.96%) one severe systemic disease) (11), duration of procedures, of which 35 (26.32%) wounds were surgery (≤240 minutes or >240 minutes), wound classified as contaminated or dirty. SSI occurred classification (clean/clean contaminated, or con- more often in these patients comparing to the pa- taminated/dirty), skin preparation regimens (po- tients with laparoscopic approach and the pati- viodone-iodine antisepsis or chlorhexidine-alco- ents with clean wounds (24.78% vs 5%; p=0.048 hol), surgical approach (open or laparoscopic), and 34.29 vs 17.35; p=0.037, respectively). comorbidities (hypertension, diabetes, cardiovas- Nineteen (out of 133; 14.29%) patients were fo- cular disease, respiratory disease, and continued und to have had diabetes and 36 (27.07%) used steroid use), and pathogens responsible for SSI. steroids; SSI was significantly more frequent in The criteria used to define SSI were those esta- these two groups of patients comparing to the pa- blished by the Centers for Diseases Control and tients who had not had diabetes or used steroids Prevention (CDC) (7). (42.11% vs 18.42%; p=0.021 and 33.33 vs 17.35; p=0.049, respectively) (Table 2). Statistical analysis Six different microbial pathogens were detected 2 A univariate analysis was performed using χ test from 25 SSIs of patients who underwent sur- for categorical variables to compare results betwe- gery for colorectal cancer: Escherichia coli in 11 en the groups (SSI group vs non-SSI group). A p< (44%) and Enterococcus spp. in six (24%) cases 0.05 was considered statistically significant. were the two most common pathogens (Table 3). RESULTS Table 3. Prevalence of microbial pathogens detected after From November 2019 to January 2021 a total of colorectal surgery 133 patients with colorectal cancer, including 65 Pathogen No (%) of patients Escherichia coli 11 (44.00) males and 68 females, met the inclusion criteria Enterococcus spp. 6 (24.00) for this prospective study. Postoperative SSI was Klebsiella pneumonia 3 (12.00) diagnosed in 29 (21.8%) patients. In 20 (15%) Pseudomonas aeruginosa 3 (12.00) Staphylococcus aureus 1 (4.00) patients diagnosed with SSI the infection was su- Candida albicans 1 (4.00) perficial, in eight (6%) deep, and in one patient (0.8%) infection involving an organ space was found (Table1).

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Table 2. Univariate analysis of demographic and clinical characteristics of the patients with and without surgical site infection (SSI) No (%) of patients in the group Variable Total (n=133) SSI (n=29) non-SSI (n=104) SSI % p Age ≤ 70 78 (58.65) 12 (41.38) 66 (63.46) 15.38 0.033 >70 55 (41.35) 17 (58.62) 38 (36.54) 30.91 Gender Male 65 (48.87) 15 (51.72) 50 (48.08) 23.08 0.448 Female 68 (51.13) 14 (48.28) 54 (51.92) 20.59 BMI (kg/m2) < 30 92 (69.17) 15 (51.72) 77 (74.04) 16.30 0.021 ≥ 30 41 (30.83) 14 (48.28) 27 (25.96) 34.15 ASA class ≤ 2 87 (65.41) 14 (48.28) 73 (70.19) 16.09 0.028 > 2 46 (34.59) 15 (51.72) 31 (29.81) 32.61 Duration of surgery > 240 minutes 25 (18.80) 7 (24.14) 18 (17.31) 28.00 0.405 ≤ 240 minutes 108 (81.20) 22 (75.86) 86 (82.69) 20.37 Wound classification Clean or clean contaminated 98 (73.68) 17 (58.62) 81 (77.88) 17.35 0.037 Contaminated or dirty 35 (26.32) 12 (41.38) 23 (22.12) 34.29 Skin preparation Poviodone-iodine 74 (55.64) 16 (55.17) 58 (55.77) 21.62 0.954 Chlorhexidine-alcohol 59 (44.36) 13 (44.83) 46 (44.23) 22.03 Surgical approach Open 113 (84.96) 28 (96.55) 85 (81.73) 24.78 0.048 Laparoscopic 20 (15.04) 1 (3.45) 19 (18.27) 5 Comorbidities Hypertension Yes 71 (53.38) 19 (65.52) 52 (50.00) 26.39 0.139 No 62 (46.62) 10 (34.48) 52 (50.00) 16.13 Diabetes Yes 19 (14.29) 8 (27.59) 11 (10.58) 42.11 0.021 No 114 (85.71) 21 (72.41) 93 (89.42) 18.42 Cardiovascular disease Yes 17 (12.78) 6 (20.69) 11 (10.58) 35.29 0.149 No 116 (87.22) 23 (79.31) 93 (89.42) 19.83 Respiratory disease Yes 28 (21.05) 8 (27.59) 20 (19.23) 28.57 0.329 No 105 (78.95) 21 (72.41) 84 (80.77) 20.00 Chronic steroid use Yes 36 (27.07) 12 (41.38) 24 (23.08) 33.33 0.049 No 97 (72.93) 17 (58.62) 80 (76.92) 17.53

DISCUSSION these reasons, reduction of SSI rates in patients undergoing colorectal surgeries remains an obser- Surgery of colorectal cancer has been associated vable priority for surgical quality improvement. with high risk of postoperative complications. The In addition, the incidence of SSI after colorectal SSIs are one of the most common postoperative operations ranges from 3% to 30 % (4,15). Howe- complication following colorectal procedures with ver, there are not many trials studying the risk of many negative consequences for patients, such as SSIs especially for oncologic operations. There is extended hospital stay, morbidity, readmission, a study in the literature finding SSI rate of 25% in and death (12,6). Because of the increase in the which more than 600 patients underwent elective SSIs incidence after elective and urgent admissi- surgery for colorectal cancer (16). In our study, the ons, there is an increase in the cost of care at disc- prevalence of SSIs was 21.8% and the majority of harge from hospital per patient (13). The additio- them were superficial (15%). nal average cost is estimated to be 20.000 dollars The outcomes of this prospective study suggest per infection (14). According to recent data from that there was no statistically significant diffe- CDC (14), SSIs represent 22% of all healthcare– rence in the presence of SSI among patients who associated infections (HAIs), and approximately received a proviodone-iodine antisepsis regimen 15% are associated with colorectal operations. For versus a chlorhexidine-alcohol skin preparation

441 Medicinski Glasnik, Volume 18, Number 2, August 2021

regimen. According to the literature, more than colorectal surgery and is associated with a redu- 70% of all SSIs arise from the micobieme of the ced risk of SSIs (10). patients, and the perioperative failure to control Six different microbial pathogens were detected their microbieme has as a result the occurrence from 25 SSIs after surgery for colorectal cancer of infections (17). In a study by Darouiche et al. in the present study, mostly Escherichia coli and 849 patients undergoing clean-contaminated sur- Enterococcus spp. This result is consistent with gery were randomized to receive either provio- the Chinese SSI surveillance study reported by Du done-iodine scrub, or chlorhexidine-alcohol. The et al., in which Escherichia coli and Enterococcus chlorhexidine-alcohol skin preparation regimen spp. were the two most common pathogens in pa- was superior for preventing SSI (18). tients undergoing colorectal surgery (10). It is worth mentioning that the major findings of One limitation of this study that should be con- this prospective study were that age >70 years, sidered is that we did not record data of patients 2 BMI ≥30kg/m , ASA score >2, dirty/contamina- undergoing urgent surgeries as well as cases with ted surgery, open surgery, as well as comorbidities an intestinal stoma (ileostomy or colostomy). (diabetes and chronic steroid use) were associated According to the literature, the placement of an with significantly higher incidence of SSIs. intestinal stoma is considered to be a significant Like the study of Kamboj et al., our study demon- risk factor for SSI (12,20). Furthermore, the de- strated that the prevalence of SSIs significantly velopment of SSI is much more frequent among differed according to the patients’ BMI, ASA patients undergoing urgent surgeries (6,12). A re- score, wound classification, surgical approach, trospective analysis of 310 patients with colorectal and comorbidities (diabetes and chronic steroid cancer conducted by Bayar et al. showed that SSI use) (4). However, in our study SSIs prevalen- was significantly more frequent among patients ce was affected by patient’s age, like the study undergoing urgent colorectal procedure (26.7% vs of Banaszkiewicz et al., whereas Kamboj et al. 10.9%) (21). Another limitation of our study is the did not find significant difference (4,6). This can small number of participants from a single centre. be explained by the difference in age-groups that In conclusion, our study indicates the need for a each study used. Specifically, we compared pati- prospective randomized controlled trial having a ents below or over 70 years old, whereas Kamboj larger number of participants. et al. and Banaszkiewicz et al. made comparison between patients below or over 65 and 75 years FUNDING of age, respectively. No specific funding was received for this study. We found that the open procedure was a risk factor for the appearance of SSIs, as it is demon- TRANSPARENCY DECLARATION strated in many studies (1,19). Laparoscopic Conflict of interests: None to declare. approach is an independent protective factor after

REFERENCES 1. Pedroso-Fernandez Y, Aguirre-Jaime A, Ramos MJ, 4. Kamboj M, Childers T, Sugalski J, Antonelli D, Hernández M, Cuervo M, Bravo A, Carrillo A. Pre- Bingener-Casey J, Cannon J, Cluff K, Davis KA, diction of surgical site infection after colorectal sur- Dellinger EP, Dowdy SC, Duncan K, Fedderson J, gery. Am J Infect Control 2016; 44:450-4. Glasgow R, Hall B, Hirsch M, Hutter M, Kimbro 2. Hou TY, Gan HQ, Zhou JF, Gong YJ, Li LY, Zhang L, Kuvshinoff B, Makary M, Morris M, Nehring S, XQ, Meng Y, Chen JR, Liu WJ, Ye L, Wang XX, Ramamoorthy S, Scott R, Sovel M, Strong V, Web- Zhao YH, Zhang Y. Incidence of and risk factors for ster A, Wick E, Aguilar JG, Carlson R, Sepkowitz surgical site infection after colorectal surgery: A mul- K. Risk of Surgical Site Infection (SSI) following tiple-center prospective study of 3,663 consecutive Colorectal Resection Is Higher in Patients With Dis- patients in China. Int J Infect Dis 2020; 96:676-81. seminated Cancer: An NCCN Member Cohort Stu- 3. GlobalSurg Collaborative. Surgical site infection dy. Infect Control Hosp Epidemiol 2018; 39:555-62. after gastrointestinal surgery in high-income, midd- 5. Turner MC, Migaly J. Surgical site infection: the cli- le-income, and low-income countries: a prospective, nical and economic impact. Clin Colon Rectal Surg international, multicentre cohort study. Lancet Infect 2019; 32:157-65. Dis 2018; 18:516-25.

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6. Banaszkiewicz Z, Cierzniakowska K, Tojek K, 14. Sanger PC, van Ramshorst GH, Mercan E, Huang S, Kozłowska E, Jawień A. Surgical site infection Hartzler AL, Armstrong CA, Lordon RJ, Lober WB, among patients after colorectal cancer surgery. Pol Evans HL. A prognostic model of surgical site in- Przegl Chir 2017; 89:9-15. fection using daily clinical wound assessment. J Am 7. Young PY, Khadaroo RG. Surgical site infections. Coll Surg 2016; 223:259-270.e2. Surg Clin North Am 2014; 94:1245-64. 15. Ju MH, Ko CY, Hall BL, Bosk CL, Bilimoria KY, 8. Hedrick TL, Sawyer RG, Hennessy SA, Turrentine Wick EC. A comparison of 2 surgical site infection FE, Friel CM. Can we define surgical site infection monitoring systems. JAMA Surg 2015; 150:51-7. accurately in colorectal surgery? Surg Infect (Lar- 16. Serra-Aracil X, García-Domingo MI, Parés D, Es- chmt) 2014; 15:372-6. pin-Basany E, Biondo S, Guirao X, Orrego C, Sitges- 9. Pochhammer J, Köhler J, Schäffer M. Colorectal Serra A. Surgical site infection in elective operations surgical site infections and their causative patho- for colorectal cancer after the application of preventi- gens: differences between left- and right-side resec- ve measures. Arch Surg 2011; 146:606-12. tions. Surg Infect (Larchmt) 2019; 20:62-70. 17. Wenzel RP. Surgical site infections and the microbi- 10. Du M, Liu B, Li M, Cao J, Liu D, Wang Z, Wang ome: An updated perspective. Infect Control Hosp Q, Xiao P, Zhang X, Gao Y, Zeng H, Yang J, Xu X, Epidemiol 2019; 40:590-6. Huang Y, Zhang Q, Zhang B, Chen W, Shi J, Fan S, 18. Darouiche RO, Wall MJ Jr, Itani KM, Otterson MF, Zhang F, Yang J, Yang H, Ding Z, Li H, Xiao S, Ran Webb AL, Carrick MM, Miller HJ, Awad SS, Crosby S, Zhai H, Wang F, Xing Y, Suo J, Liu Y. Multicen- CT, Mosier MC, Alsharif A, Berger DH. Chlorhexi- ter surveillance study of surgical site infection and dine-alcohol versus povidone-iodine for surgical-si- its risk factors in radical resection of colon or rectal te antisepsis. N Engl J Med 2010; 362:18-26. carcinoma. BMC Infect Dis 2019; 19:411. 19. Aimaq R, Akopian G, Kaufman HS. Surgical site in- 11. Mulita F, Karpetas G, Liolis E, Vailas M, Tchabashvi- fection rates in laparoscopic versus open colorectal li L, Maroulis I. Comparison of analgesic efficacy of surgery. Am Surg 2011; 77:1290-4. acetaminophen monotherapy versus acetaminophen 20. Blumetti J, Luu M, Sarosi G, Hartless K, McFarlin J, combinations with either pethidine or parecoxib in Parker B, Dineen S, Huerta S, Asolati M, Varela E, patients undergoing laparoscopic cholecystectomy: Anthony T. Surgical site infections after colorectal a randomized prospective study. Med Glas (Zenica) surgery: do risk factors vary depending on the type 2021; 18: (1) [online ahead of print]. of infection considered? Surgery 2007; 142:704-11. 12. Morikane K, Honda H, Yamagishi T, Suzuki S, 21. Bayar B, Yılmaz KB, Akıncı M, Şahin A, Kulaçoğlu Aminaka M. Factors associated with surgical site in- H. An evaluation of treatment results of emergency fection in colorectal surgery: the Japan nosocomial versus elective surgery in colorectal cancer patients. infections surveillance. Infect Control Hosp Epide- Ulus Cerrahi Derg 2015; 32:11-7. miol 2014; 35:660-6. 13. Gantz O, Zagadailov P, Merchant AM. The cost of surgical site infections after colorectal surgery in the United States from 2001 to 2012: a longitudinal analysis. Am Surg 2019; 85:142-9.

443 ORIGINAL ARTICLE

Neurocysticercosis with symptomatic epilepsy manifestation Nataliya Nekrasova1, Olena Tovazhnyanska1, Daryna Sushetska1, Olena Markovska1, Anton Shapkin2, Rhea Singh1, Yevhenija Soloviova1, Dmytro Butov3

1Department of Neurology No2, 2Department of Pathomorphology, 3Department of Phthisiology and Pulmonology; Kharkiv National Medical University, Kharkiv, Ukraine

ABSTRACT

Aim To present a unique case of a 22-year-old male patient with symptomatic epilepsy manifestation on a background of neurocysticercosis (NCC).

Methods An Indian student in Kharkiv, who lived in rural parts in India, presented with sudden episodes of seizure followed by severe headaches. Laboratory analyses and neurological status (MRI) were performed.

Results Neurological status of the patient revealed nystagmus and difficulty in performing co-ordination tests. General analysis of blood showed raised eosinophil count to 8%. The MRI showed a few small conglomerating peripherally enhancing thick-walled in- Corresponding author: fective granulomas in left frontal lobe with extensive surrounding Nataliya Nekrasova oedema in the left fronto-parietal lobe. The patient was treated Department of Neurology No2, with albendazol, levipil, methylprednisolone and pantoprazole. Kharkiv National Medical University Clinical symptoms and subsequent MRI showed improvement. Kharkiv Nauky Avenue 4, 61022, Conclusion Neurocysticercosis is often misdiagnosed in the early Kharkiv, Ukraine stages, which leads to adverse outcomes. Although seizures are the Phone: +380 50 615 45 80; most common clinical manifestation, it is a symptom that is not E-mail: [email protected] found in majority of the patients. The NCC of adult onset accom- ORCID ID: https://orcid.org/0000-0002- panying epileptic seizures is not well studied and a link between the helminthic invasion, epilepsy and psychiatric conditions needs 0900-4441 to be established. This disease is potentially eradicable with well- planned eradication programs targeting all stages of Taenia solium life cycle.

Key words: granuloma, frontal lobe, seizures, Taenia solium Original submission: 03 March 2021; Revised submission: 22 April 2021; Accepted: 18 May 2021 doi: 10.17392/1368-21

Med Glas (Zenica) 2021; 18(2):444-449

444 Nekrasova et al. Neurocysticercosis and epilepsy manifestation

INTRODUCTION are considered to be active cysts undergoing degeneration (colloidal cysts). Cysts degeneration Originally recognized as a swine disease in ancient usually happens fast within 6 to 12 months af- Greece, neurocysticercosis (NCC) is now consi- ter initial presentation. As a result, the rates of dered the most common helminthic disease of the seizure-recurrence increase during this period central nervous system in humans (1). Conditions (because of the conversion from vesicular cysts like a warm climate, severe poverty, illiteracy and to colloidal cysts). Epileptogenesis in patients unhygienic living conditions, all favour the tran- with NCC can be attributed to several factors: smission of this disease in most of the developing inflammation, gliosis, genetics, and predilection world, which makes it endemic to these regions for the cysts to travel to the frontal and temporal (2). Furthermore, according to various population- lobes (1). Furthermore, the response of the host based studies, the main reason for the higher pre- to degenerating cysts cannot be disregarded in valence of people with epilepsy in rural villages the process of epileptogenesis. of endemic countries is NCC in comparison to the prevalence of epilepsy in developed countries due A magnetic resonance imaging (MRI) scan is of- to other reasons (1). It has been called a "hidden ten needed for increased diagnostic sensitivity and epidemic" (3) and "arguably the most common pa- accuracy (11). Diagnosis may be confirmed by rasitic disease of the human nervous system"(4). detection of antibodies against cysticerci in cere- Cysticercosis in the United States, commonly brospinal fluid (CSF) or serum through ELISA or presenting in the form of neurocysticercosis, has immunoblotting techniques (2,12). Albendazole been classified as a "neglected tropical disease" (13,14) and praziquantel are the principal anti-pa- (5,6), commonly affecting the poor and home- rasitic drugs used to treat NCC, as they reduce the less, particularly those without access to a clean number of cysts and frequency of seizures. environment or those with ill-habit of inadequate Here we present a case of neurocystircerosis in a hand-washing and eating with such unclean hands. young adult with manifestation of symptomatic The NCC is a preventable parasitic infection cau- epilepsy and behavioural changes. sed by larval cysts of the pork tapeworm (Taenia PATIENT AND METHODS solium). The most important risk factor for acquiring cysticercos is being in close proximity to a Patient and study design tapeworm carrier. Larval cysts of the tapeworm can find their way to the brain and lead to epilepsy A 22-year-old male patient from India was hospi- (7). The NCC is acquired through consumption talized in the neurological department of Kharkiv of food or water contaminated with faeces of a city Students Hospital in November 2019 with carrier of T. solium tapeworm (faecal–oral rou- complaints of convulsion attacks with impaired te of transmission). Common symptoms of NCC consciousness occurring at his hostel in Kharkiv include seizures, headaches, blindness, meningi- and at the University. tis and dementia (8). Infected humans pass out The first episode of seizure occurred one day pri- eggs or gravid proglottids through their faeces, or to hospitalization. According to the roomma- which can be ingested by pig living in an area of tes, the patient fell suddenly in the evening while poor hygiene. The eggs further develop and com- walking with his head turned to the right and con- plete the complex life cycle of the tapeworm (9). vulsions in both his hands and feet, which lasted This disease often goes un- or misdiagnosed. for approximately 1 minute. The seizure was not Even when diagnosed, there is a huge gap in a preceded by an aura. The patient remained un- treatment. Generally, patients with NCC present conscious for 5-7 minutes after the seizure. After with either tonic-clonic seizures or partial-onset that, the patient complained of a severe headac- seizures. Usually, most of the patients have an he, but he had no confusion. A similar attack was active cyst—either vesicular or colloidal, at the repeated the next day during school hours, which time of the first seizure. New-onset seizures are was when the patient was examined by a physi- commonly associated with active cysts. Chro- cian and was sent to the neurological department nic epilepsy is usually associated with calcified Kharkiv Regional Hospital with a preliminary di- granulomas (10). The most epileptogenic cysts agnosis: Idiopathic epilepsy.

445 Medicinski Glasnik, Volume 18, Number 2, August 2021

From the anamnesis, the patient reported no na- General analysis of blood showed: red blood cells usea or vomiting. However, he reported having (RBC) 4.8x1012/L, haemoglobin 152g/L, mean experienced episodes of mild-moderate headac- corpuscular haemoglobin (MCH) 0.9, white blo- he of a waxing and waning character for the past od cells (WBC) 7.5x109/L, eosinophils 8.0%, year. He also complained of occasional tingling stab neutrophils 4%, segmental neutrophils 57%, feeling in his right lower limb, which had been lymphocytes 24%, monocytes 5% and erythro- progressively increasing in duration (usually la- cyte sedimentation rate (ESR) 20 mm/h. sting for 5-15 minutes) for the last 6 months. The An electroencephalograph (EEG) showed high patient also reported having difficulty in sociali- voltage activity of delta waves. Such abnormalities zing and increasing anger management issues for were detected because of the presence of 2.5-3-Hz- the past two years and that this was different from wave formations in left fronto-parietal distribution. his personality as a child. However, the patient The MRI (Figure 1) was conducted on 26 Novem- had never been diagnosed with any psychiatric ber 2019, which showed a cortical-subcortical in- disorder (as he did not find them concerning). homogeneous structure in the lateral parts of the The patient lived with his family in India in a left frontal lobe with volume formation, irregular small town before arriving in Kharkiv in 2018 shape, without clear outlines of size 24x26x25mm for studies. His living conditions were “normal”. with a pronounced perifocal oedema and mass The family claimed to be vegetarians and did not effect with compression of the left lateral ventricle consume meat of any kind. He had one younger and shift of midline structures to the right by 3mm. brother, who never had any medical conditions. The maxillary sinuses were swollen with a thic- Both his parents suffered from hypertension. In kening of the mucosa by 4-5 mm, along with the addition, the mother suffered from migraine. The presence of cysts on the right of size 25x17mm. patient did not smoke or drink alcohol.

Methods Biochemical laboratory analyses, neurological status with MRI was performed.

RESULTS On examination the patient had no fever (temperature was 37°C), heart rate of 85/minute, respiratory rate of 19/minute, oxygen saturation of 97%, and blood pressure of 130/89 mm of Hg. Figure 1. Magnetic resonance imaging (MRI) of the brain at admission at Kharkiv city Students Hospital on 26 November His skin was moist with good elasticity. He had 2019. A, B) On both slides in frontal section at different degrees no significant cardiac, respiratory, gastrointesti- of depth the lateral part of the left frontal lobe is depicted with nal or urinary symptoms. the presence of cortical-subcortical inhomogeneous structure with volume formation, irregular shape, without clear outlines In the neurological status: consciousness was clear, of 24x26x25 mm size with a pronounced perifocal oedema and orientation of space and time was present. Pupils mass effect with compression of the left lateral ventricle and and palpebral fissures were identical on both sides, shift of midline structures to the right by 3mm left and right. Convergence was saved. Horizontal Based on the epidemiological anamnesis, clini- nystagmus to the left was present. The face was cal and MRI research, the patient was diagno- symmetrical with the tongue in the midline. Deep sed with neurocysticerosis with a primary lesion tendon reflexes were quickened but symmetrical, of the left frontal lobe: symptomatic epilepsy, left and right. The sensitivity of the face and limbs cephalgic and asthenic syndrome. was saved. Pathological reflexes and meningeal si- The patient was prescribed (orally) carbamazepin gns were not determined. In the Romberg position, 200 mg 2 times a day for 6 months and albenda- the patient was stable. However, the patient had zole 400 mg 2 times a day for 3 weeks according trouble in performing coordination tests. to the local guidelines of Ukraine. The diagnosis on admission: Focal motor seizu- On 28 November 2019, the patient self-discharged res, idiopathic epilepsy. due to the desire to continue the treatment at home.

446 Nekrasova et al. Neurocysticercosis and epilepsy manifestation

On 2 December 2019, the patient referred to For- tis Hospital Limited in Mumbai, India, where anti-cysticercus antibody test was done, which came out to be positive and another brain MRI with contrast (Figure 2) was performed showing a few small conglomerating peripherally enhancing thick-walled infective granuloma sin left frontal lobe with extensive surrounding oedema in the left fronto-parietal lobe. These showed central T2 hyper intense signal intensity with Figure 3. The MRI of brain with contrast done in Fortis Hospi- peripheral T2 hypointensity. The conglomerate tal Limited in Mumbai, India on 06 January 2020. A, B) On both lesion measured approximately 20x12 mm along slides in frontal section at different degrees of depth left frontal lobe with persistent small well-defined heterogeneously enhanc- axial the dimension. Minimal shift of falx to the ing conglomerate lesion (measured 9x10 mm in axial and 15.5 right is noted. Findings likely - neurocysticerosis. mm in cranio-caudal dimension) with surrounding mild perilesional oedema with minimal blooming in gradient is depicted mild perilesional oedema, which appeared hype- rintense in T2-weighted images and isointense in T1W images. Minimal blooming was noted in gradient recalled echo (GRE) images. No areas of restricted diffusion in diffusion-weighted imaging (DWI) were seen. The conglomerate lesion measured approximately 9x10 mm in axial dimensions (previously measuring 20x12mm) and 15.5 mm in cranio-caudal dimension. On 31 January 2020, with consultation at neurology department №2, Kharkiv, some conclusions about the patient’s health were made: the patient's condition had improved, the dynamics of com- Figure 2. The MRI of brain with contrast in Fortis Hospital plaints (the absence of seizures, reducing the in- Limited in Mumbai, India on 02 December 2019. A) In frontal section persistent small well-defined heterogeneously tensity and frequency of headaches) and MRI enhancing conglomerate lesion involving left frontal lobe with picture were positive and the neurologic status surrounding mild perilesional oedema with minimal blooming was without any remarkable features. The patient in gradient is revealed. B) C) D) In cranio-caudal dimension at different degrees of depth the conglomerate lesion measured was recommended to be continually monitored by 9x10 mm in axial dimensions and 15.5mm in cranio-caudal neurologist, infectious diseases doctor and repeat dimension is depicted MRI in 6 months. Oral treatment was prescribed: On 4 December 2019, the patient was examined levipil 500 mg 2 times daily for 2-3 years, carba- by a neurosurgeon at Fortis Hospital Limited in mazepin 200 mg 2 times a day for 2 years. Mumbai, India and received recommendation for The patient was also advised to consider psychothe- a treatment (orally) according to the Indian Guide- rapy regarding the changes in his personality. lines: levipil 500 mg 2 times a day for 2-3 years, The patient reported decrease in the frequency albendazole 400 mg 2 times a day for 3 weeks, of headaches and the duration of tingling in the methylprednisolone 4 mg three times a day, with extremities after 4 months of treatment. Although a gradual reduction of the dose for 15 days, panta- the patient did not get an MRI done, clinically, he prazol 40 mg 2 times a day for 15 days, and carba- looked better. mazepin 200 mg 2 times a day for 2 years. On 6 January 2020, the patient referred again to DISCUSSION the Fortis Hospital Limited, where brain MRI with We present a case of NCC with epileptic syndrome contrast (Figure 3) revealed persistent small well- and psycho-behavioural changes. Although accor- defined heterogeneously enhancing conglomerate ding to WHO data, infection of the central nervous lesion involving left frontal lobe with surrounding system with Taenia solium is one of the leading

447 Medicinski Glasnik, Volume 18, Number 2, August 2021

causes of epilepsy, in our country such diseases as Patients with mental retardation were found to neurocystecyrcosis are rare and in the differential carry an increased risk of cysticercosis as compa- diagnosis of the causes of epileptic syndrome are red with patients with other psychiatric disorders not in the first place. It should be borne in mind (6). Although, our patient had no mental retarda- that today the population migration is very acti- tion, he had a history of poor social skills and ve and the endemicity of the spread of diseases is anger management issues. It has been estimated losing its relevance. Describing this clinical case, that a large proportion of psychiatric inpatients we want to pay the attention of doctors to this pro- with positive cysticercosis serology, despite blem, so that in the future they treat such patients carrying no adult Taenia spp. in their stool and no with greater caution. Another aspect of this matter CNS imaging, have high prevalence of NCC (6). is its treatment: various randomized studies eva- We presented a novel case of the young student luating the clinical benefits of the treatment have who, despite living in “normal” conditions and yielded conflicting data whereby some studies in- being vegetarian, developed neurocysticercosis dicate a benefit of the treatment and others suggest with symptomatic epilepsy and behavioural chan- that the treatment shows no difference in patients’ ges. We can only assume that the patient consu- condition (12). Our patient however, responded med contaminated vegetables or fruits growing well to the treatment. in unhygienic conditions and got infected with Cysticerci may be located in brain parenchyma, tapeworm, which was dormant in his body for qu- subarachnoid space, ventricular system, or spinal ite some time. Therefore, further efforts should be cord, causing pathological changes that are res- made to eradicate the disease via control program ponsible for the pleomorphism of neurocysticer- implementation against all interrelated steps in the cosis (1). In our patient, the disease was located in life cycle of T. solium, including human carriers of the left frontal lobe, which could explain the be- the adult tapeworm, infected pigs and eggs in the havioural changes. Seizures are the most common environment. Since these targets represent interre- clinical manifestation, but many patients present lated steps in the life cycle of T. solium, inadequate with focal deficits, intracranial hypertension, or coverage of even one of them may result in a rebo- cognitive decline (15). According to a study con- und prevalence of taeniosis or cysticercosis after ducted in India only 8% of the patients with anti- the program has been thought to be completed. cystercus antibodies had a history of seizures (16). Despite a lot of literature information about this Interpretation of clinical data, neuroimaging studi- syndrome and its many symptoms including seizu- es and immunological test help in accurate diagno- re-recurrence, it is often misdiagnosed, attention to sis of neurocysticercosis. which needs to be paid. The NCC of an adult onset, Another aspect of this matter is its treatment: va- accompanying with symptomatic epilepsy and be- rious randomized studies evaluating the clinical havioural changes is not well studied. Further rese- benefits of the treatment have yielded conflicting arch in this field needs to be carried out. data whereby some studies indicate a benefit of the treatment, and others suggest that the tre- ACKNOWLEDGMENTS atment shows no difference in patients’ condition We acknowledge the wholehearted support of all (12). Our patient, however, responded well to the clinicians, nurses, and lab staff who contributed treatment. The introduction of cysticidal drugs to this research and made this study possible. Our have changed the prognosis of most patients with sincere gratitude is expressed to the experts in the neurocysticercosis. These drugs have shown to tuberculosis field who kindly shared their opini- reduce the burden of infection in the brain and to ons and suggestions with us. improve the clinical course of the disease in most patients. The therapy for NCC, formerly restric- FUNDING ted to palliative measures, has advanced with the No specific funding was received for this study. advent of two drugs considered to be effective: praziquantel (PZQ) and albendazole (ALB) (17). TRANSPARENCY DECLARATION Even though there are treatment options, the tre- Competing interests: None to declare. atment gap in India is more than 90% (13).

448 Nekrasova et al. Neurocysticercosis and epilepsy manifestation

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1. Del Brutto OH. Neurocysticercosis: a review. Sci 12. Ahmad R, Khan T, Ahmad, B, Misra A, Balapure World J 2012; 159821. A. Neurocysticercosis: a review on status in India, 2. Murray P, Rosenthal K, Pfaller M. Medical Micro- management, and current therapeutic interventi- biology. 7th ed. Philadelphia, PA, USA: Elsevier Sa- ons. Parasitol Res 2017; 116:21–33. unders, 2013: p. 809. 13. Kaur S, Singhi P, and Khandelwal N. Combination 3. Ahmad FU, Sharma BS. Treatment of intramedu- therapy with albendazole and praziquantel versus llary spinal cysticercosis: report of 2 cases and re- albendazole alone in children with seizures and sin- view of literature. Surg Neurol 2007; 67:74–7. gle lesion neurocysticercosis: a randomized place- 4. White AC, Neurocysticercosis: A major cause of ne- bo-controlled double-blind trial. Pediatr Infect Dis urological disease worldwide. Clin. Infect. Dis 1997; 2009; 28:403–6. 24:101–31. 14. Nash TE, Pretell EJ, Lescano AG. Perilesional brain 5. Hotez PJ. Neglected parasitic infections and poverty oedema and seizure activity in patients with calcified in the United States. PLoS Negl Trop Dis 2014; 8: neurocysticercosis: a prospective cohort and nested e3012. case-control study. Lancet Neurol 2008; 7:1099– 6. Meza NW, Rossi NE, Galeazzi TN. Cysticercosis 105. in chronic psychiatric inpatients from a Venezuelan 15. Khurana S, Aggarwal A, Malla N. Prevalence of an- community. Am J Trop Med Hyg 2005; 73:504–9. ti-cysticercus antibodies in slum, rural and urban po- 7. Del Brutto, Neurocysticercosis. Semin Neurol 2005; pulations in and around Union territory, Chandigarh. 25:243–51. Indian J Pathol Microbiol 2006; 49:51-3. 8. World Health Organisation. 10 facts about neurocy- 16. Carpio A, Kelvin EA, Bagiella E. Effects of alben- sticercosis. https://www.who.int/features/factfiles/ dazole treatment on neurocysticercosis: a randomi- neurocysticercosis/en/ (10 January 2021) sed controlled trial. J. Neurol Neurosurg Psychiatry 9. DeGiorgio CM, Medina MT, Durón R, Zee C, Es- 2008; 79:1050-5. cueta SP. Neurocysticercosis. Epilepsy Curr 2004; 17. Keilbach NM, De Aluja AS, Sarti-Gutierrez E. A 4:107-11. programme to control taeniasis-cysticercosis (T. so- 10. Burneo JG, Plener I, Garcia HH. Neurocysticercosis lium): experiences in a Mexican village. Acta Leiden in a patient in Canada. CMAJ 2009; 180:639-42. 1989; 57:181-9. 11. Fleury A, Hernández M, Avila M. Detection of HP10 antigen in serum for diagnosis and follow-up of su- barachnoidal and intraventricular human neurocysticercosis. J Neurol Neurosurg Psychiatry 2007; 78:970-4.

449 ORIGINAL ARTICLE

Is preoperative hypoproteinemia associated with colorectal cancer stage and postoperative complications?

Amina Sofić1, Ismar Rašić2, Emsad Halilović3, Alma Mujić4, Denis Muslić1

1Clinic for Anaesthesiology and Intensive Care Medicine, Klinikum Kulmbach, Kulmbach, Germany, 2Department of Surgery, General Hos- pital “Prim. dr. Abdulah Nakaš”, 3Clinic for General and Abdominal Surgery, Clinical Centre of the University of Sarajevo; Sarajevo, Bosnia and Herzegovina, 4Department of Anaesthesiology, Reanimatology and Intensive Care, Hospital Travnik, Travnik, Bosnia and Herzegovina

ABSTRACT

Aim To investigate the relationship between preoperative level of serum albumin in patients with colorectal cancer (CRC), stage of CRC and postoperative complications.

Methods This cross-sectional retrospective study was conducted at the Clinic for General and Abdominal Surgery of the University Clinical Centre Sarajevo (UCCS). A total of 107 patients surgically treated for CRC in the period between 2013 and 2018 were enrolled in this study and divided into two groups: with hypoalbuminemia (group A) and without hypoalbuminemia (group B). Corresponding author: Results The average level of albumin in group A was 29 (25-32) Amina Sofić g/L versus 39 (37-41) g/L in group B (p<0.05). The average len- Clinic for Anaesthesiology and Intensive gth of hospital stay in group A was 18 (13-25) days, and in group Care Medicine, Klinikum Kulmbach B 14.5 (12-21) days. Patients with hypoalbuminemia (group A) Albert-Schweitzer-Strasse 10, had wound dehiscence more often and more re-interventions com- 95326 Kulmbach, Germany pared to group B (p<0.05). Binary logistic regression found that Phone: +49 9221 98 7436; serum protein, albumin and globulin levels were not statistically significant in the prediction of CRC stadium or postoperative com- Fax: +49 9221 98 5094; plications (p>0.05). E-mail: [email protected] ORCID: https://orcid.org/0000-0001- Conclusion Study results show that preoperatively measured 5191-8485 levels of serum albumin are not associated with the stage of colorectal cancer and cannot serve as predictors for postoperative complications.

Key words: colorectal neoplasms, complications, postoperative Original submission: period, serum 02 February 2021; Revised submission: 29 March 2021; Accepted: 24 May 2021 doi: 10.17392/1353-21

Med Glas (Zenica) 2021; 18(2):450-455

450 Sofić at al.Preoperative hypoproteinemia, colon cancer, and postoperative complications

INTRODUCTION postoperatively due to a physiological response to trauma and surgical stress at a level of about Colorectal cancer (CRC) is one of the most 33%, and Chinese authors suggest that a decline commonly diagnosed cancers in both genders of more than 15% two days after surgery could be and the most common gastrointestinal neopla- used to identify patients with a high probability sm in the world. The reduction of incidence and of developing postoperative complications and mortality rates over the last few decades has been having a poor outcome (11). associated with the reduction in the incidence of risk factors, the emergence of screening pro- Albumin is considered a controversial marker of grams, and improved therapeutic protocols (1). nutritional status (12). Some studies have pro- Patients who have surgery on colon and rectum ven that preoperative serum albumin is a good often have postoperative complications and thus predictor of mortality and morbidity after CRC an increased risk of mortality, poor oncological surgery (6,13), as well as a good predictor of outcomes, other complications and deterioration surgical outcome after emergency abdominal of life quality. The potential risks of colorectal surgery (14). However, there is no clear relati- surgery are the same as with any other abdominal onship of serum proteins to CRC stages and their surgery, and complications most often occur du- prognostic value. Similar studies on this subject ring hospitalization (2). have not been conducted in our country and the Balkan regions so far. Albumin has been found to have a protective effect on many biological processes, so low albumin le- The aim of this study was to determine whether vels are considered markers of disease and malnu- hypoproteinemia and hypoalbuminemia are asso- trition (3-5). Hypoalbuminemia is associated with ciated with the stage of colorectal cancer and higher mortality and morbidity of hospitalized pa- whether low level of serum albumin can have an tients, patients with a neoplasm and aging popula- effect on the development and severity of posto- tion. Truong et al. have found an increasing trend perative complications in surgically treated pati- in both postoperative mortality rate and postope- ents with colorectal cancer. rative complications in patients with preoperative PATIENTS AND METHODS low levels of serum albumin (6). Lai et al. found that preoperative hypoalbuminemia Patients and study design is a predictor of poor surgical outcome and a factor In this cross-sectional retrospective study 107 pa- of poor prognosis in long-term survival of patients tients of both genders, older than 18 years, who with colon cancer after curative surgery (7). In an were surgically treated for clinically, radiologi- epidemiological review study related to gastrointe- cally, colonoscopically and histopathologically stinal and lung cancers, female genital tract cancers, confirmed CRC, in the period from September and studies on various cancers, it was concluded 2013 to January 2018 at the Clinic for Gene- that the level of pre-therapy serum albumin may ral and Abdominal Surgery, University Clinical be a useful prognostic factor (8). Study results by Centre Sarajevo (UCCS ) were included. Gibbs et al. indicate that decreased concentrations of albumin were a predictor of mortality and morbi- All patients had a preoperative analysis of serum dity for surgical patients, as well as a good predictor proteins, and possible postoperative complicati- for certain postoperative morbidities such as sep- ons were recorded. Patients with a neoplasm of sis and infection; serum albumin is not used often different organs were excluded from the study. enough in preoperative diagnostics as it represents Patients were divided into two groups: the pati- a good and relatively inexpensive indicator of nega- ents with preoperative hypoproteinemia (group tive outcomes (9). Preoperative hypoalbuminemia A; 75 patients), and the patients with preoperati- represents an independent risk factor for the deve- ve reference values of serum proteins (group B; lopment of postoperative surgical wound infection 32 patients). after gastrointestinal surgery, and that the infections The research was accepted and approved by the were deeper with longer hospital stay (10). Department of Science and Education of the Clini- Most studies examine preoperative effects of cal Centre of the University of Sarajevo and com- low albumin; however, albumin tends to decline pleted in compliance with the Helsinki Declaration.

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Methods RESULTS An analysis of preoperative serum total protein The age of the patients of both groups did not and albumin levels was determined at the Clini- differ statistically significantly: 71 (62-76) vs. cal Chemistry and Biochemistry Clinic at UCCS 68 (62-75.8) years (p=0.552). There was also no on a Dimension®Clinical Chemistry System statistically significant difference in the gender (Siemens, Germany) apparatus using Flex® re- distribution of the patients in both groups: 36 agents from Siemens Healthcare Diagnostics (48%) vs. 13 (40.6%) males, and 39 (52%) vs. 19 (Newark, DE, USA). Referent values for total (59.4%) females, respectively (p=0.483). proteins were 62.0–82.0 g/L, and the referent va- Stage III CRC was the most common in both gro- lues for albumins were 35.0–50.0 g/L. All values ups: in 37 (49.3%) patients of group A and 20 below the set referent values were considered as (62.5%) of group B. The most common localiza- hypoproteinemia and hypoalbuminemia. tion of cancer in both groups was in the rectum The principle of oncological surgery with “en and sigmoid area: 32 (42.7%) in group A, 25 bloc” organ resection and associated lymphatic (78.1%) in group B. and vascular arcade was followed during opera- Preoperative hypoproteinemia was verified in 75 tive treatment. Pathohistological analysis of the (70%) patients with CRC (Figure 1). biopsy material was performed by a pathologist as part of the standard diagnostic procedure, while microbiological tests were performed by the Hospital’s microbiological laboratory. TNM classification of the American Committee for cancer of 2010 was used for classification of colorectal cancer stages (15): the stage of colorectal cancer was marked from I to IV. The values of serum proteins were observed in relation to the stage of the disease. The occurrence of early postoperative complications such as surgical wound dehiscence, anasto- motic dehiscence, abscess development, bactere- Figure 1. Presence of hypoproteinemia (lighter coloured) in mia, ileus, and the need for early reintervention individual stages of colorectal cancer (CRC) Statistically significant differences in the mean va- were analysed in monitored patients. lues of total serum proteins (p=0.276), serum al- Statistical analysis bumins (p=0.739) or serum globulins (p=0.081) in Kolmogorov-Smirnov test or Shapiro-Wilk test relation to CRC stage were not confirmed in the was used to examine normal distribution of data. patients with hypoproteinemia (group A) (Table 1). Variables with normal distribution were presented Table 1. Comparison of differences in the values of serum as mean±standard deviation (SD) and compared proteins according to the stage of colorectal cancer (CRC) in patients with hypoproteinemia by the parametric tests (t-test, ANOVA). Variables Variable CRC stage No of patients Mean±SD p not displaying normal distribution were presented I 3 63.00±8.30 Total proteins II 27 60.85±9.21 as median and interquartile range and compared 0.276 (g/L) III 37 62.29±7.86 by nonparametric tests (Mann-Whitney U test, IV 8 59.00±4,24 Kruskal-Wallis test). The ANOVA multiple com- I 3 25.50±2.12 II 27 28.56±4.78 Albumins (g/L) 0.739 parison test was used to examine differences in III 37 27.47±5.76 serum protein values with respect to TNM classi- IV 8 27.50±4.95 I 3 27.50±7.78 fication and stage of colorectal cancer. Nominal II 27 32.61±5.96 Globulins (g/L) 0.081 and ordinal variables in the study were analysed III 37 36.76±8.40 IV 8 31.50±0.71 using the χ2 test. Regression analysis examined The frequency of postoperative complications the predictor value of serum proteins in assessing (infection, anastomosis dehiscence, abscess and the stage of colorectal cancer and the occurrence ileus) did not statistically significantly differ of postoperative complications. Accepted statisti- between the observed groups. Wound dehiscence cal significance was at the level of p<0.05. occurred statistically more frequently in group A

452 Sofić at al.Preoperative hypoproteinemia, colon cancer, and postoperative complications

Table 2. Postoperative complications of patients with or cancer patients, and on mortality and morbidity without hypoproteinemia of surgical patients (16,17). In this regard the si- No (%) of patients gnificance of serum albumin values has also been Postoperative With hypoprote- Without hypo- p complication inemia proteinemia investigated (18,19). (n=75) (n=32) In our study we used preoperative serum levels Infection 13 (17.3) 7 (21.9) 0.587 of total proteins, albumins and globulins, as it is Surgical wound dehiscence 10 (13.3) 0 0.024 Anastomosis dehiscence 7 (9.3) 2 (6.3) 0.460 known that there is a relationship between nutri- Abscess 4 (5.3) 0 0.236 tional status and albumin levels in patients with Ileus 3 (4.0) 0 0.340 CRC (20,21); these patients, due to advanced di- Early reintervention 17 (22.7) 2 (6.3) 0.033 sease, are often in malnutrition, which results in Reinterventions 0.038 Without 58 (77.3) 30 (93.8) poor oral food intake, intestinal obstruction, fistula One 13 (17.3) 2 (6.3) formation, poor absorption capacity and large vo- Two 4 (5.3) 0 lume loses from the gastrointestinal tract (22). compared to group B (p=0.024). A need for early In this regard, Gupta and Lis in their review stu- reintervention was noticed in 17 (22.7%) patients dy of the epidemiological literature found in 26 with hypoproteinemia, and in only two (6.3%) (from 29) studies better survival of patients with patients without hypoproteinemia. Thus, in the gastrointestinal cancer if they had serum albumin patients with preoperative hypoproteinemia (gro- >35 g/L (8). Worryingly, in our study, the group of up A) surgical reinterventions were significantly patients with hypoalbuminemia had significantly more often performed (p=0.038). lower mean serum albumin value (29 g/L) than the However, the average length of hospitalization indicated cut off value for better survival. did not statistically differ between the two ob- The role of preoperative serum albumin as a pro- served groups of patients (p=0.113). The average gnostic tool in survival of colorectal cancer pati- length of hospital stay in patients with hypopro- ents was demonstrated by many studies (23,24), teinemia (group A) was 18 days (ranging from 13 but investigations of association of hypoprotei- to 25 days), while the average length of hospital nemia with the different stages of CRC are very stay in group B with normal values of serum pro- limited. In a Turkish study, albumin levels among tein was 14.5 days, ranging from 12 to 21 days. patients with preoperative metastatic disease Values of total serum proteins, albumins and were lower compared to those who were meta- Table 3. Prognostic value of serum proteins in the assess- stasis-free as a part of a systemic inflammatory ment of colorectal cancer (CRC) stage* response. Among patients with advanced dise- Variable Median (IR 25-75) B Beta p Total proteins 67.8 (62.5-73.4) 0.071 -0.459 0.647 ase, albumin levels were more reflective of - tu Albumins 34.0 (31-36.5) 0.080 0.065 0.948 mour size, respectively the depth of the CR-wall Globulins 34.0 (29.7-37.9) 0.081 0.666 0.507 invasion rather than the specific tumour stage, *The dependent variable: the stage of CRC; IR, interquartile range; B, regression coefficient; beta, ratio probability with larger tumours having lower serum albu- globulins were not statistically significant in the min levels. The authors suggested that the larger prediction of CRC stage (Table 3). The values volume of tumour cells translates into a higher of serum total proteins, albumins and globulins production of proinflammatory cytokines, which were not statistically significant in the prediction in turn suppress albumin hepatic production (25). of postoperative complications (Table 4). Our study has shown that values of total serum proteins, albumin, and globulin did not prove sta- DISCUSSION tistically significant in the prediction of colorectal Numerous studies have been conducted in order carcinoma stage. Such finding suggests that preo- to find factors with favourable effects, i.e.- fac perative hypoproteinemia and hypoalbuminemia tors that have adverse effects on the survival of are most likely related to some other factors such Table 4. Statistical significance of serum proteins in the prediction of postoperative complications occurrence Statistical significance Variable Infection Surgical reintervention Surgical wound dehiscence Anastomosis dehiscence Abscess Ileus (n=20) (n=19) (n=10) (n=9) (n=4) (n=3) Total proteins 0.967 0.996 0.817 0.492 0.937 0.805 Albumins 0.840 0.996 0.201 0.474 0.097 0.223 Globulins 0.628 0.996 0.201 0.468 0.506 0.597

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as continuous systemic inflammatory response to up with referent levels of serum proteins it did not a malignant tumour that causes loss of body pro- occur. Low preoperative level of albumin might tein in these patients (26). be a risk factor for wound dehiscence. Aksamija Recently, there has been a hypothesis that the et al. have found in a study on 3504 patients ope- albumin-globulin (AGR) ratio could have grea- rative wound dehiscence after laparotomy in 1.25 ter clinical significance and may be used as an patients, of whom 50% had hypoalbuminemia index of disease-status, but since it does not in- (32). In a prospective study on 50 patients with dicate specific proteins that have been altered in wound dehiscence in India, 76% patients had an the ratio, it cannot be used as a specific marker. albumin level of 30-36 g/L (33). Generally, normal AGR is 0.8-2.0, and lower va- In our study, however, we found a link between lues could indicate long-term mortality increase the need for surgical reintervention and preope- in cancer patients (27). In our study this ratio in rative hypoalbuminemia. An investigation of the both groups relative to mean values was in the effects of preoperative hypoalbuminemia and normal range (0.9 and 1.1, respectively). the outcome of open abdominal aortic aneurysm In our study, the average length of hospital stay of surgery (OAR) and endovascular surgery of ab- patients with hypoproteinemia was shorter by 3.5 dominal aortic aneurysm (EVAR) found a strong days (19.4%) comparing to the group without hypo- correlation of significant hypoalbuminemia with proteinemia. Lohsiriwat et al. showed that albumin postoperative complications and the need for re- level of <35 g/L statistically significantly prolonged operation within 30 days (34). hospitalization from 6.8±2.6 days to 9.6±4.7 days The main limitation of the study was that it was (28). Our results were similar to the results of this a retrospective study in a single institution based study in relation to the absolute number of additio- on data obtained from patients’ medical histories. nal days of hospital stay (3.5 vs. 2.8 days). Howe- More recent data could not be included in the stu- ver, the total length of hospital stay of our patients dy because this research was completed as a part of was longer, which could be due to significantly the graduate thesis of the first author at the School more pronounced hypoalbuminemia and its possi- of Medicine, University of Sarajevo, in mid-2018. ble impact on the delayed recovery of our patients. However, this is the first study in Bosnia and Her- Investigating the relationship between hypoalbu- zegovina examining the association between preo- minemia and the occurrence of postoperative com- perative albumin, a stage of colorectal cancer and plications in patients with CRC we did not find postoperative complications in patients undergo- differences between the groups in case of wound ining surgery for primary operable colorectal cancer. fection, anastomosis dehiscence, abscess and ileus. In conclusion, the obtained results show that pre- Abdominal wound dehiscence is a serious posto- operatively measured concentrations of total pro- perative complication with a mortality rate of up teins and albumins in the serum of patients with to 45%, and reported prevalence of 0.4%-3.5% colorectal cancer are not predictors for colorectal (29,30). Important risk factors for wound dehis- cancer stage. cence include age, gender, chronic pulmonary FUNDING disease, ascites, jaundice, anaemia, emergency operations, type of surgery, cough and wound No specific funding was received for this study. infection (31). Wound dehiscence as a postopera- TRANSPARENCY DECLARATION tive complication in our study occurred in 13.3% patients with hypoproteinemia, while in the gro- Conflict of interest: None to declare.

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5. Chang DC, Xu X, Ferrante AW, Jr, Krakoff J. Re- 21. Zietarska M, Krawczyk-Lipiec J, Kraj L, Zaucha duced plasma albumin predicts type 2 diabetes and R, Malgorzewicz S. Nutritional status assessment is associated with greater adipose tissue macrophage in colorectal cancer patients qualified to systemic content and activation. Diabetol Metab Syndr 2019; treatment. Contemp Oncol (Pozn) 2017; 21:157-61. 11:14. 22. Maykel JA. Perioperative nutrition support in co- 6. Truong A, Hanna MH, Moghadamyeghaneh Z, Sta- lorectal surgery. In: Steele SR, Maykel JA, Cham- mos MJ. Implications of preoperative hypoalbumi- pagne BJ, Orangio GR, eds. Complexities in Colo- nemia in colorectal surgery. World J Gastrointest rectal Surgery: Decision-making and Management. Surg 2016; 8:353-62. New York: Springer Science and Business Media; 7. Lai CC, You JF, Yeh CY, Chen JS, Tang R, Wang JY, 2014:29-44. Chin CC. Low preoperative serum albumin in colon 23. Borda F, Borda A, Jimenez J, Zozaya JM, Prieto cancer: a risk factor for poor outcome. Int J Colo- C, Gomez M, Urman J, Ibanez B. Predictive value rectal Dis 2011; 26:473-81. of pre-treatment hypoalbuminemia in prognosis of 8. Gupta D, Lis CG. Pretreatment serum albumin as a resected colorectal cancer. Gastroenterol Hepatol predictor of cancer survival: a systematic review of 2014; 37:289-95. the epidemiological literature. Nutr J 2010; 9:69. 24. Sun LC, Chu KS, Cheng SC, Lu CY, Kuo CH, Hsieh 9. Gibbs J, Cull W, Henderson W, Daley J, Hur K, JS, Shih YL, Chang SJ, Wang JY. Preoperative se- Khuri SF. Preoperative serum albumin level as a pre- rum carcinoembryonic antigen, albumin and age are dictor of operative mortality and morbidity: results supplementary to UICC staging systems in predic- from the National VA Surgical Risk Study. Arch ting survival for colorectal cancer patients undergo- Surg 1999; 134:36-42. ing surgical treatment. BMC Cancer 2009; 9:288. 10. Hennessey DB, Burke JP, Ni-Dhonochu T, Shields 25. Cengiz O, Kocer B, Sürmeli S, Santicky MJ, Soran C, Winter DC, Mealy K. Preoperative hypoalbu- A. Are pretreatment serum albumin and cholesterol minemia is an independent risk factor for the deve- levels prognostic tools in patients with colorectal lopment of surgical site infection following gastro- carcinoma? Med Sci Monit 2006; 12:CR240–7. intestinal surgery: a multi-institutional study. Ann 26. Almasaudi AS, Dolan RD, Edwards CA, McMillan Surg 2010; 252:325-9. DC. Hypoalbuminemia reflects nutritional risk, body 11. Ge X, Dai X, Ding C, Tian H, Yang J, Gong J, Zhu composition and systemic inflammation and is inde- W, Li N, Li J. Early postoperative decrease of serum pendently associated with survival in patients with albumin predicts surgical outcome in patients under- colorectal cancer. Cancers (Basel) 2020; 12:1986. going colorectal resection. Dis Colon Rectum 2017; 27. Alkan A, Koksoy EB, Utkan G. 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Eva- flamm Res 2021; 14:115-29. luation of risk factors of surgical wound dehiscence 18. Hu WH, Eisenstein S, Parry L, Ramamoorthy S. in adults after laparotomy. Med Arch 2016; 70:369- Preoperative malnutrition with mild hypoalbumi- 72. nemia associated with postoperative mortality and 33. Ramneesh G, Sheerin S, Surinder S, Bir S. A pros- morbidity of colorectal cancer: a propensity score pective study of predictors for post laparotomy ab- matching study. Nutr J 2019; 18:33. dominal wound dehiscence. J Clin Diagn Res 2014; 19. Loftus TJ, Brown MP, Slish JH, Rosenthal MD. 8:80-3. Serum levels of prealbumin and albumin for pre- 34. Inagaki E, Farber A, Eslami MH, Kalish J, Rybin operative risk stratification. Nutr Clin Pract 2019; DV, Doros G, Peacock MR, Siracuse JJ. Preoperati- 34:340-8. ve hypoalbuminemia is associated with poor clinical 20. Negrichi S, Taleb S. 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455 ORIGINAL ARTICLE

Comparison of early and delayed lumbar disc herniation surgery and the treatment outcome

Ermin Hadžić1, Bruno Splavski2-5, Goran Lakičević6

1Division of Neurosurgery, Cantonal Hospital “Dr. Safet Mujić”, Mostar, Bosnia and Herzegovina, 2Department of Neurosurgery, ''Sestre milosrdnice'' University Hospital Centre, Zagreb, Croatia, 3School of Medicine, 4School of Dental Medicine and Health; University "Josip Juraj Strossmayer" Osijek, Croatia, 5University of Applied Health Sciences, Zagreb, Croatia, 6Department of Neurosurgery, University Clinical Hospital, Mostar, Bosnia and Herzegovina

ABSTRACT

Aim To evaluate the influence of preoperative symptoms duration on surgical outcome of one-level lumbar disc herniation surgery.

Methods In a prospective randomized study, 67 adult patients with one-level lumbar disc herniation were analysed. The patients whose duration of symptoms was <6 months were included in the case group, while those with the duration of symptoms ˃6 months formed the control group. The investigated preoperative variables were: pain intensity in the back and legs (Visual Analogue Scale - VAS), Sciatica Bothersomeness Index (SBI), index of disability (Oswestry Disability Index - ODI). Postoperative variables were: Corresponding author: pain intensity in the back and legs (VAS), SBI, ODI, and outcome Ermin Hadžić according to the Odom’s criteria (excellent, good, satisfactory and Division of Neurosurgery, poor). Significance level was set at p <0.05. Cantonal Hospital “Dr. Safet Mujić”, Mostar, Bosnia and Herzegovina Results A statistically significant difference was recorded between the groups, showing a better decrease of radicular pain intensity Maršala Tita 294, 88000 Mostar, and sciatica bothersome, as well as patients disability in the case Bosnia and Herzegovina group (p<0.001). According to the Odom’s criteria the outcome Phone +387 36 576 915; was better in the case group, since the difference between the gro- +387 61 227733; ups was statistically significant too (p<0.05). Fax +387 36 576 915; Conclusion Early lumbar disc herniation surgery performed wit- E-mail: [email protected] hin the first 6 months from the start of symptoms is beneficial due ORCID ID: https://orcid.org/0000-0002- to decreases of radicular pain intensity, sciatica bothersomeness, 5072-0095 and patient’s disability.

Original submission: Key words: discectomy, intervertebral disc, pain, radiculopathy, syndrome 13 January 2021; Revised submission: 27 January 2021; Accepted: 23 February 2021 doi: 10.17392/1343-21

Med Glas (Zenica) 2021; 18(2):456-462

456 Hadžić et al. Early and delayed lumbar discectomy

INTRODUCTION The aim of this study was to evaluate the influence of preoperative symptoms duration on the outco- The first publications on lumbar intervertebral disc me of one-level lumbar disc herniation surgery in surgery were made by Mixter and Barr in the first the two groups of patients, as well as to make a half of the last century (1). After that, many studies conclusion about the optimal time of surgical tre- reported success of the surgical treatment and We- atment. The hypothesis was that lumbar disc her- ber was the first one who pointed out better results niation surgery in patients with shorter duration of of the surgical treatment after one year of follow- symptoms (radicular pain) (<6 months) - early dis- up of patients when compared to conservative tre- cectomy, significantly leads to better postoperative atment (2). Although a period between 4 to 8 weeks results and has a positive effect on the treatment for an operation following lumbar radiculopathy outcome compared with patients whose symptoms has been proposed, in the lumbar intervertebral lasted longer (>6 months) - delayed discectomy. disc surgery, the optimal timing of the surgical pro- The purpose of this study was to contribute to fin- cedure is still not aligned considering the onset of ding the optimal time of surgery in relation to the radicular pain syndrome and radicular pain durati- duration of preoperative symptoms using an exten- on (3). According to some studies, the optimal time ded range of different outcome scales. An attempt for surgery, which positively affects the outcome of was also made to highlight a possible impact of treatment ranges from two to twelve months from time of surgery as one of the most important and the onset of symptoms. Some of the reports of pro- least investigated predictors of the favourable tre- longed duration of lumbar radiculopathy related to atment outcome. poor treatment outcome are: Hurme and Alaranta (4) if the duration of pain syndrome is longer than PATIENTS AND METHODS two months, Støttrup et al. (5) if the period was longer than three months, Dvorak et al. (6), Carrage et Patients and study design al. (7), and Siccoli et al. (8) if it was longer than six In this prospective randomised study 67 adult months. Nygard et al. (9) showed that the duration patients of both genders aged 18-65 years ope- of radiculopathy longer than 8 months determined rated in the Cantonal Hospital “Dr Safet Mujić” poorer outcomes of lumbar discectomy while si- Mostar and the University Clinical Hospital of milarly, Ng and Sell (10) showed for a time frame Mostar diagnosed with herniation of the lumbar longer than 12 months. A recent study by Pitsika et intervertebral disc during the period 2013-2017 al. involving 107 patients showed that significantly were included. positive effects of surgery in carefully selected patients can be expected after one and two years from The study included only patients whose neuro- the onset of symptomatology (11). Also, there are radiological findings of single-level lumbar disc some researches that reject the hypothesis that the herniation correlated with neurological symptoms shorter duration of the discogenic lumbar radicular and radicular pain maintained despite conserva- syndrome has a positive effect on the outcome of tive treatment applied. These patients underwent surgery (12,13). surgery and were divided into two groups based on preoperative radicular pain duration: the case There are a few recorded reports on the impact group consisted of 34 patients who underwent of operation time onto the outcome of the lum- early lumbar discectomy (duration of symptoms bar disc herniation surgery in Bosnia and Herze- was <6 months) and the control group consisted govina. Bečulić et al. pointed out that the early of 33 patients who underwent delayed lumbar dis- decompression (operation time of up to 48 hours cectomy (duration of symptoms was ˃6 months). from the onset of symptomatology) was associated with a better outcome considering only pati- All patients had proper medical documentation, ents with cauda equina syndrome caused by lum- performed preoperative diagnostic processing and bar disc herniation (14). Moranjkić et al. have also pre-operative and post-operative check-ups. also mentioned the preoperative pain duration in Pre-operative and post-operative check-ups inclu- order to find a set of available variables that may ded completing a structured survey questionnaire. predict the short-term outcome of lumbar disc The exclusion criteria were: incomplete medical herniation surgery (15). records, the presence of postoperative recurrence

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or residual disc on the same level, extensive neuro- The investigated postoperative variables were: logical deficits cauda( equina syndrome), various pain intensity in the back and legs (VAS), SBI, pathologies of the lumbar spine of another etiology ODI, and outcome according to the Odom’s cri- (previous fractures, infections, spinal tumours and teria (excellent, good, satisfactory and poor) (19). metastases), advanced osteodegenerative patho- Output postoperative data were obtained at least logy in which degeneration of the intervertebral six months after surgical treatment and after per- disc is not a dominant etiological factor (spondylo- forming postoperative control diagnostics (MRI sis, spondylolisthesis, spinal canal stenosis), as well of the lumbar spine). Microdiscectomy was a met- congenital or acquired malformations of the spinal hod of surgical treatment. All patients were treated column. The exclusion criteria were also diagnosed equally during hospitalisation. Everyone was ad- comorbidities in which lumbar disc surgery was vised to continue the physical therapy at home af- contraindicated, previous lumbar disc surgery, as ter discharge from hospital and also avoiding sta- well as other neurological and osteomuscular dise- todynamic loads. Influence of the operation time ases, verified malignancy and pregnancy. on treatment success was analysed by comparing Data obtained were used only for the purposes differences between preoperative and postopera- of this research, and the data and identity of each tive parameters obtained by evaluating the VAS, participant remained anonymous. Before par- SBI, and ODI questionnaires in both of groups. ticipating in the research, the participants were The intensity of pain before and after surgery was informed about the purpose, goals and possible measured and changes in the level of pain intensity scientific contribution of the research. An infor- (improvement), according to all the measured sca- med consent was obtained from all patients and/ les (pain before surgery - pain after surgery) were or their legal representatives, according to the calculated in both groups, as well as the values of local legislation. statistical testing of the obtained results. An ethical approval was obtained from competent Statistical analysis ethics committees of the Cantonal Hospital “Dr. Safet Mujić”, and the University Clinical Hospital Gender differences between the observed groups Mostar, Mostar, Bosnia and Herzegovina. of patients as well as the differences according to age, height, body weight, Body Mass Index (BMI) Methods and operated level of lumbar disc herniation were The data were obtained by prospective analysis calculated using the Fisher exact test and χ2 test. of the medical documentation, as well as on the Comparisons of surgical outcomes between case basis of clinical examinations of the patients and and control group according to changes of valu- analysing results of a written structured survey es of measured scales (VAS for the lower back preoperatively and for at least 6 months after the and leg pain, SBI and ODI scores) were perfor- surgery. Lumbar disc herniation was established by med using the t-test for independent samples. The MRI of the lumbar spine. The investigated preope- difference between the observed groups according rative variables were pain intensity in the back and to the Odom's criteria was done using the Fisher legs (Visual Analogue Scale - VAS) (16), Sciatica exact test. A statistic significance was set at p Bothersomeness Index (SBI) (17), and index of <0.05 and p values that could not be expressed up disability (Oswestry Disability Index - ODI) (18). to three decimal places were reported as p <0.001. The VAS (16) is relating to patient pain perception RESULTS by metric analogue scales 0-10 cm (0 - no pain, 10 - the strongest pain). The SBI (17) 0–6-point scale, There were 21 (61.8%) males and 13 (38.2%) fe- following symptoms according to how botherso- males in the case group of operated patients (du- me they were in the past week, which refers to leg ration of radiculopathy <6 months). In the con- pain, numbness or tingling in the leg, foot or groin, trol group (duration of radiculopathy >6 months) weakness in the leg or foot, back or leg pain whi- were 17 (51.5%) male and 16 (48.5%) female le sitting. The ODI (18), 10-point patient-reported patients (p=0.397). outcome questionnaire, scored from 0 to 5, giving The mean age of 48.50±9.34 years, the ave- a maximum score of 50. rage height 176.94±8.53 cm, and the average

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body weight 82.32±12.88 kg in the case gro- The mean change of the ODI score in the case up of patients was found. In the control group group was 54.00±18.80 whereas in the control the mean age of 50.79±9.80 years, the avera- group it was 28.12±13.28 (p<0.001) (Table 4). ge height 175.73±8.63 cm and average body The mean change of the SBI in the case group weight 81.48±17.70 kg were recorded (p=0.332, was 13.12±4.15 and in the control group it was p=0.565, and p=0.825, respectively) (Table 1). 9.61±4.41 (p<0.001) (Table 4). According to the body mass index (BMI) in the Table 4. Comparison of two patient groups according to the case group there were 12 (35.3%) ideal body mean change of the Oswestry Disability Index (ODI score) weight patients, 18 (52.9%) overweight, whi- and the Sciatica Bothersomeness Index (SBI) le four (11.8%) of them were obese. A total of Group* ODI score Mean (±SD) p SBI Mean (±SD) p <6 months 54.00 (±18.800) 13.12 (±4.154) 16 (48.5%) patients with ideal body weight, 10 <0.001 <0.001 (30.3%) overweight, and 7 (21.2%) obese patients >6 months 28.12 (±13.284) 9.61 (±4.415) *preoperative radicular pain duration; were observed in the control group (p=0.160). According to the Odom’s criteria, there were 21 Table 1. Comparison of two patient groups by age, height and (61.7%) excellent, 9 (26.5%) good and 4 (11.8%) weight according to preoperative radicular pain duration satisfactory results of surgical treatment in the No of Variable Group* Mean (±SD) P case group after at least six months of clinical patients < 6 months 34 48.50 (±9.340) follow-up (poor results were not recorded); in Age (years) 0.332 > 6 months 33 50.79 (±9.800) the control group, eight (24.2%) excellent, 11 < 6 months 34 176.94 (±8.535) Height (cm) 0.565 (33.3%) good, 13 (39.4%) satisfactory and one > 6 months 33 175.73 (±8.632) (3.1%) poor results were recorded (p=0.004). Body weight < 6 months 34 82.32±12.883) 0.825 (kg) > 6 months 33 81.48 (±17.708) *preoperative radicular pain duration DISCUSSION In the case group, 11 (32.4%) patients with L5/ Comparison of the patients according the sex, S1 disc herniation level, 22 (64.7%) with L4/L5 age, height and body weight, was not statistically and one (2.9%) with L3/L4 were operated. In significant indicating the good selection, - consi the control group, 17 (51.5%) patients with L5/ stency and homogeneity of the studied sample. S1 disc herniation level, 13 (39.4%) with L4/ There was no statistically significant difference L5, and three (9.1%) with L3/L4 were operated between the observed groups according to BMI (p=0.107) (Table 2). and the level of disc herniation, although the le-

Table 2. Comparison of groups according to the operated vel L4/L5 was dominant in the case group and the level of lumbar disc herniation level L5/S1 was dominant in the control group. Level of the No (%) of patients in the group Total p It was observed that the patients in the case gro- herniated disc <6 months >6 months up had significantly more pronounced expressi- L3/L4 1 (2.9) 3 (9.1) 4 (6.0) L4/L5 22 (64.7) 13 (39.4) 35 (52.2) on symptomatology preoperatively, VAS leg and L5/S1 11 (32.4) 17 (51.5) 28 (41.8) 0.107 ODI. However, the difference in the assessment Total 34 (100.0) 33 (100.0) 67 (100.0) of the intensity of preoperative lower back pain Reduction the intensity of lower back pain ac- (VAS back) was not statistically significant, nor cording to the mean change of the VAS in the was the difference between the groups according case group was 6.12±2.86 and in the control to SBI. The patients operated within a period of group 4.76±3.13 (p=0.068). Radicular pain re- six months from the onset of symptoms had more duction in the case group was 7.50±2.32 and in pronounced intensity of radicular pain preoperati- the control group 4.79±2.52 (p<0.001) (Table 3). vely and greater disability. This is understandable considering the epidemiological data on the gra- Table 3. Comparison of two patient groups according to the dual regression of lumbar radicular pain in most mean change in the level of the pain intensity in the back and legs (Visual Analogue Scale - VAS) patients and favourable natural course of lumbar Variable Group* VAS mean (±SD) p radiculopathy (20-22). According to preoperati- <6 months 6.12 (±2.868) ve ODI, both groups of patients had symptoms, Back 0.068 >6 months 4.76 (±3.133) which were marked as severe disability as it was <6 months 7.50 (±2.326) Legs <0.001 >6 months 4.79 (±2.522) shown in the most other studies (23-25). For all *preoperative radicular pain duration; tested variables (VAS leg, SBI and ODI), higher

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values of improvements were obtained in the to work (32). It is also questionable whether the case group (early surgery). An improvement was evaluation of results is comparable among studies observed when comparing the changes of lower conducted under different socio-economic condi- back pain intensity (VAS back) in both groups, tions, because the low socioeconomic status can but the difference was not statistically significant. cause an increased number of complications in According to the Odom’s criteria most patients in certain types of spinal surgery (33,34). There were the case group had a significantly higher propor- also differences in the types of statistical methods tion of excellent results. applied in individual studies, and some research The results of the presented study are mostly in line showed a certain discrepancy between the statisti- with the results of the other studies that support the cal significance of the obtained variable (pain, qu- hypothesis that shorter duration of lumbar radi- ality of life) from the perspective of the researcher culopathy symptoms (for a maximum 6 months) and the clinical significance from the perspective leads to better postoperative results (6,7,8,23,26). of the patient (35). Hurme and Alaranta and Rothoerl et al. showed the In the presented research, in evaluating the posto- same regarding the shorter duration of preoperative perative results, unlike many other studies, multi- symptoms (less than two months) (4,27). Støttrup ple rating scales of the treatment outcomes were takes the duration of symptoms less than three used. It also showed that the duration of radiculo- months to achieve better results (5). Nygaard et pathy after which good results of a lumbar discec- al. and Blazhevsky et al. take eight and ten months tomy can be expected is six months and shorter. time frames to achieve better postoperative results, The six-month time limit is also important for the which is longer compared to the presented research prevention of the development the chronic pain (9,28). Blazhevsky et al, however, pointed out that and initiation the complex pathophysiological by far the best operative results are achieved in a actions of processing sensory signals in certain period of up to three months of sciatica duration areas of the brain (36,37); also, optimal surgery (28). Even longer duration of symptoms, up to one time of six months and less cannot be generali- and two years, after which better treatment outco- zed to every patient with lumbar disc herniation. me can be expected has shown researches conduc- Following the experience, it is necessary to adapt ted by Ng and Sell and Pitiska (10,11). Two stu- and individualize the decision to each patient. dies, by Jönsson et al. and Fisher et al. concluded Nowadays, indication for lumbar discectomy and similarly, but they did not highlight a time limit for also the time of surgery are based on the lumbar undertaking the surgery (29,30). radicular syndrome duration, the patient's ability Several limitations in the existing literature make to suffer pain, and on the preferences of surgeons it impossible to produce a solid evidence about the and patients themselves, since a large number of exact time of surgery treatment. There is conside- patients prefer to participate in the decision-ma- rable variability among the studies according to king on the type and time of treatment (38). This diversity of applied methods, inclusion and exclu- means that the patient should make a final decision criteria, as well as the overall quality of the re- sion regarding the type and timing of treatment searches itself. Some studies used different scoring initiation, based on his wishes and individualized systems for outcome evaluation, and also, there is requirements following an open discussion with a question of how to measure the treatment outco- the competent neurosurgeon about the benefits as me. In other words, the classification of treatment well as the existing risks of the surgery itself. outcomes was made by different instruments for Like the other examples of limitations from the assessing the severity of pain syndrome and there existing literature, presented study included a re- have also been validated several questionnaires for latively small number of subjects. No more com- measuring outcomes in spine surgery in the past plex statistical analysis was performed conside- decade (31). Moreover, the self-assessment of the ring the heterogeneity of the sample and a large severity of a painful syndrome does not allow amount of data related to radicular pain syndro- conclusions to be drawn about daily functioning me such as the age, body weight and others. The of the patient, his/her quality of life, or his/her re- inclusion of these factors with operation time of integration into the social community and return shorter than six months in a more complex stati-

460 Hadžić et al. Early and delayed lumbar discectomy

stical analysis could be the subject of future re- ACKNOWLEDGEMENTS search and a more accurate selection of patients We thank the staff of Division of Neurosurgery, who will have most benefit from surgery. Cantonal Hospital “Dr. Safet Mujic”, Mostar, and In conclusion, our results are in line with most Neurosurgery Clinic of the University Clinical research reporting that the shorter duration of the Hospital of Mostar, Bosnia and Herzegovina, for lumbar radicular pain has a positive effect on the their help with the local organization of the study results of lumbar discectomy. An operation perfor- and obtaining data used in the study. med within the first six months after the onset of symptoms (early surgery) is useful for reducing the FUNDING intensity of pain as well as the disability of patients. No specific funding was received for this study. The optimal timing of surgery (up to six months) cannot be generalized to every patient with lumbar TRANSPARENCY DECLARATION disc herniation. It is necessary to adapt and indi- Competing interests: None to declare. vidualize the decision considering preferences and opinion of a surgeon and a patient himself.

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22. Hofstee DJ, Gijtenbeek JM, Hoogland PH, van 30. Fisher C, Noonan V, Bishop P, Boyd M, Fairholm D, Houwelingen HC, Kloet A, Lotters F, Tans JT. We- Wing P, Dvorak M. Outcome evaluation of the ope- steinde Sciatica Trial: randomized controlled study of rative management of lumbar disc herniation causing bedrest and physiotherapy for acute sciatica. J Neuro- sciatica. J Neurosurg 2004; 100:317-24. surg 2002; 96(Suppl 1):45-9. 31. Teles A, Khoshhal K, Falavigna A. Why and how 23. Rihn JA, Hilibrand AS, Radcliff K, Kurd M, Lurie J, should we measure outcomes in spine surgery? J Blood E, Albert TJ, Weinstein JN. Duration of symp- Taibah Univ Med Sci 2016; 11:91-7. toms resulting from lumbar disc herniation: Effect 32. Coretti S, Ruggeri M, McNamee P. The minimum on treatment outcomes: Analysis of the Spine Patient clinically important difference for EQ-5D index: a Outcomes Research Trial (SPORT). J Bone Joint Surg critical review. Expert Rev Pharmacoecon Outcomes Am 2011; 93:1906-14. Res 2014; 14:221-33. 24. Gerbershagen K, Gerbershagen HU, Lindena GU, 33. Lieber AM, Boniello AJ, Kerbel YE, Petrucelli P, Lachenmayer L, Lefering R, Schmidt CO, Kohlmann Kavuri V, Jakoi A, Khalsa AS. Low socioeconomic T. Prevalence and impact of pain in neurological in- status is associated with increased complication rates: patients of a German teaching hospital. Clini Neurol are risk adjustment models necessary in cervical spine Neurosurg 2008; 110:710-17. surgery. Global Spine J 2020; 10:748-53. 25. Omidi-Kashani F, Ghayem Hasankhani E, Kachooei 34. Abdallah A, Seyithanoğlu MH. Evaluation of the AR, Rahimi MD, Khanzadeh R. Does duration of demografic and socioeconomic factors affecting recu- preoperative sciatica impact surgical outcomes in rrence of lumbar disc herniation: a prospective study. patients with lumbar disc herniation? Neurol Res Int J Turk Spinal Surg 2019; 30:121-6. 2014; 2014:565189. 35. Mattei T. “Statistically significant” does not -nece 26. Silverplats K, Lind B, Zoëga B, Halldin K, Gellerstedt ssarily mean ‘clinically different’ on pain/quality of M, Brisby H, Rutberg L. Clinical factors of importan- life scales: opportune remarks on clinical outcomes ce for outcome after lumbar disc herniation surgery: measures in cervical spondylotic myelopathy. Neuro- long-term follow-up. Eur Spine J 2010; 19:1459-67. surgery 2012; 71:518-21. 27. Rothoerl RD, Woertgen C, Brawanski A. When sho- 36. Coderre TJ, Katz J, Vaccarino AL, Melzack R. Con- uld conservative treatment for lumbar disc hernia- tribution of central neuroplasticity to pathological tion be ceased and surgery considered? Neurosurg pain: review of clinical and experimental evidence. Rev 2002; 25:162-5. Pain 1993; 52:259-85. 28. Blazhevski B, Filipche V, Cvetanovski V, Simonov- 37. Zhou F, Zhao Y, Zhu L, Jiang J, Huang M, Zhang Y, ska N. Predictive value of the duration of sciatica Zhuang Y, Gong H. Compressing the lumbar nerve for lumbar discectomy. Contribut Sec Biol Med Sci root changes the frequency-associated cerebral am- 2008; 29:325-35. plitude of fluctuations in patients with low back/leg 29. Jönsson B. Patient-related factors predicting the pain. Sci Rep 2019; 9:2246. outcome of decompressive surgery. Acta Orthop 38. Rätsep T, Abel A, Linnamägi Ü. Patient involvement Scand 1993; 251:69-70. in surgical treatment decisions and satisfaction with the treatment results after lumbar intervertebral discectomy. Eur Spine J 2014; 23:873-81.

462 ORIGINAL ARTICLE

Single-centre experience of emergency hernia surgery during COVID-19 pandemic: a comparative study of the operative activity and outcomes before and after the outbreak

Adnan Malik, Mohamed Zohdy, Aftab Ahmad, Charalampos Seretis

Department of General Surgery, George Eliot Hospital NHS Trust, Warwickshire, United Kingdom

ABSTRACT

Aim The outbreak of COVID-19 pandemic in January 2020 affected largely the elective operating for non-urgent surgical pathologies, such as hernias, due to periodical cancellations of the operating lists on a worldwide scale. To the best of our knowledge, the long-term impact of the COVID-19 pandemic in relation to the emergency hernia surgery operative workload and postoperative outcomes remains largely unknown.

Corresponding author: Methods Retrospective research of admission, operation and in- patient records of all patients who underwent emergency surgery Charalampos Seretis over a 2-year period (2019-2020) was done. George Eliot Hospital NHS Trust College Street, Nuneaton, CV10 7DJ, Results An 18% increase in terms of emergency hernia surgery United Kingdom operating volume, with a 23% increase of visceral resections due to unsalvageable herniated content strangulation was found. Ove- Phone: +44 24 76351351; rall morbidity did not increase during the pandemic period and Fax: +44 24 76865175; there was no postoperative mortality or occurrence of COVID-19 E-mail: [email protected] related complications. Adnan Malik ORCID ID: 0000-0002- Conclusion Emergency operative management of acutely sympto- 8892-6574 matic hernias can be safely performed even during the COVID-19 infection peak waves; hernia taxis should be reserved only for patients unfit or unwilling to undergo upfront surgery.

Key words: abdominal hernia, coronavirus, SARS, general sur- Original submission: gery 31 March 2021; Revised submission: 14 June 2021; Accepted: 21 June 2021 doi: 10.17392/1383-21

Med Glas (Zenica) 2021; 18(2):463-467

463 Medicinski Glasnik, Volume 18, Number 2, August 2021

INTRODUCTION PATIENTS AND METHODS The outbreak of COVID-19 pandemic without Patients and study design a doubt affected the provision of emergency and elective surgery on a global scale. In con- The Department of General Surgery, George Eli- trast though to elective procedures, emergency ot Hospital NHS Trust (district general) has been surgery can hardly be postponed due to the providing continuously uninterrupted emergency associated increased morbidity and mortality general surgery services in the region of Warwick- risks; hence the public was globally encouraged shire, UK, during the COVID-19 pandemic to seek medical attention upon the development outbreaks. Of note, our hospital’s wider capture of acute symptoms (1,2). However, in our area (West Midlands), has been in the epicentre anecdotal institutional experience, and being of the pandemic in the UK with continuous strict in accordance to recently published data, there lockdown/isolation measures during the pande- was a reduction in the volume of the emergency mic waves. We performed a retrospective resear- hospital admissions, with a notable increase ch of emergency surgical admissions between 01 of the complexity of the presenting problems January-31 December 2020 and 01 January - 31 due to delayed presentation of the patients to December 2019, comparing during those two pe- the acute hospital services (3). In addition, for riods regarding the overall emergency hernia ope- intermittent time periods during the COVID-19 rative workload (number and type of operations), peak waves, we were obliged as surgical depar- the visceral resection rates due to strangulation of tment to proceed with occasional cancellations herniated contents, as well as the postoperative 30- of elective operations (excluding surgical on- day morbidity and mortality rates, overall length cology procedures), in order to maximise ava- of stay and the occurrence of hospital/community- ilability of critical care beds, theatre staff and acquired COVID-19 infection in the 2020 emer- medical doctors to areas of the hospital where gency hernia admissions. We also identified from the pressure of COVID-19 admissions was be- their electronic clinical history records the elapsed coming overwhelming. As a result, and focu- time interval between the onset of the hernia-rela- sing in particular on hernia surgery, the delays ted acute symptoms and the time the patients pre- in performing elective hernia repairs in a timely sented to our emergency surgical service. manner, in combination with the general trend of the public to avoid a hospital admission, led Methods to an increase of the emergency hernia wor- The relevant information was extracted from the kload and presentation of patients with neglec- electronic records of the daily emergency admi- ted acutely symptomatic hernias (4,5). ssions, which were maintained by each one of the Under this notion, we attempted to assess the as- admitting general surgery firms, as well as from pects regarding emergency hernia surgery in our the electronic patient records and operation notes institution since the outbreak of the COVID-19 / emergency theatre records. The occurred posto- pandemic (January–December 2020) and compa- perative complications were classified according re the relevant data and outcomes from the pre- to the modified Clavien-Dindo classification (6), vious year (January-December 2019). The aim of with the complication capture period extending up this study was to capture differences between the to 30 days post the emergency hernia procedures. observation periods in terms of actual operative With respect to our institutional policy regarding volume changes, features of clinical presentati- screening for COVID-19 infection status, all emer- on, postoperative outcomes and hospital-acqu- gency surgical admissions were required to have a ired COVID-19 infection rates among the pati- swab taken from the oropharynx and nostrils for ents who underwent emergency hernia surgery in polymerase chain reaction (RT-PCR) essay; upon our institution. To the best of our knowledge, no clinical suspicion of concurrent respiratory tract previous study has specifically evaluated to date symptoms, the patients would be assessed with the impact of COVID-19 pandemic on the emer- a computed tomography (CT) scan of the chest gency hernia surgery outcomes. or plain chest radiograph as per discretion of the admitting surgical team. Regarding the preopera-

464 Malik et al. Emergency hernia surgery during COVID-19

tive investigations of our patients who underwent any inflammatory lung changes). Also, none of the emergency hernia surgery during both study pe- performed preoperative chest radiographs or chest riods, all had undergone baseline blood tests CT scans for the entire 2020 cohort detected any including full blood count, baseline biochemical changes in consistency with possible COVID-19 assessment of liver and renal function and clotting infection. Moreover, no patient acquired the virus screening. Pre-operative CT of the abdomen and during their in-hospital stay, resulting in a 0% ove- pelvis was performed at the discretion of the ad- rall COVID-19 infection rate in the entire 2020 mitting surgical firm. Concerning the attempt for patient group. taxis of the incarcerated hernia under analgesia or Despite a small increase in the number of visce- sedation, in our institutional practice we avoid to ral resections in the 2020 cohort, four (12.5% in proceed with this manoeuvre if the hernia is not 2019 vs six (15.4%) in 2020, the 30-day posto- easily reducible due to the inability to clinically perative morbidity rates were similar in the two exclude the presence of evolving strangulation of study periods (36% for 2019 vs 32% for 2020, the herniated viscera. respectively), with 0% postoperative mortality Our study did not require approval from our local during both observation periods (Table 2). Ethics Committee, due to its retrospective and non-invasive nature. Input from medical statisti- Table 2. Patients’ hernia types and repair details cian was not required due to the performance of Operation types and hernia Number during the year essentially descriptive analysis and presentation location 2019 2020 Open suture umbilical/paraumbilical 6 2 of our data and clinical outcomes. Open mesh umbilical/paraumbilical 4 8 Open mesh inguinal 9 13 (one recurrent) RESULTS Open suture inguinal (laparotomy) 3 1 Open mesh incisional 4 4 A total of 71 patients had undergone emergency Open suture femoral 3 5 hernia surgery during the entire 2-year observati- Open mesh (plug) femoral - 1 on period, with 39 patients being operated in 2020 Open mesh Spigelian 1 3 Open mesh other ventral 2 1 and 32 in 2019, indicating an increase of 18% in Laparoscopic mesh incisional - 1 terms of workload after the COVID-19 outbre- 6 (15.4%) 4 (12.5%) (2 right hemico- ak. The patient groups matched in terms of mean Visceral resections (4 small bowel lectomies /4 small resections) age and American Society of Anaesthesiologists bowel resections) (ASA) status full name of the abbreviation (7), but there was a noted delay of the admitted patients in It has to be noted that from the 2020 study group terms of seeking medical attention since the onset only one patient was admitted to intensive care of the acute hernia-related symptomatology (Table unit (ICU) postoperatively, having undergone ab- 1). With respect to the patients’ COVID-19 infecti- dominal wall reconstruction in the form of com- on status in the 2020 cohort, 37 (out of 39) patients ponent separation including visceral resection had a negative PCR test on admission, while two (strangulated hernia, complex incisional hernia patients were not screened (these two had preope- with loss of domain, performance of right he- rative imaging of the chest, one had plain radio- micolectomy due to strangulation). In addition, graph and the other CT thorax, which did not show none of the patients who were operated in 2020 had an unplanned return to ICU for respiratory tract complications or need ward-based non-in- Table 1. Patients demographic features on admission vasive ventilation. Regarding need for re-opera- Variable 2019 2020 tions, two patients from the 2019 period had to Number of emergency hernia operations 32 39 Male / female 17 / 15 26 / 13 undergo an additional procedure under general Mean age (years) 68.4 65.5 anaesthetic (hematoma evacuation post open ASA grade (% from year’s admissions) mesh inguinal hernia repair, orchidectomy due to 1 2 (6.3) 3 (7.7) 2 13 (40.6) 14 (35.9) severe ischemic orchitis post open mesh inguinal 3 14 (43.8) 20 (51.3) hernia repair), while none of the 2020 patients 4 3 (9.4) 2 (5.2) had an unplanned return to the operating theatre Duration of symptoms prior to admission (days) 1.31 2.03 within the 30-day follow-up period (Table 3). ASA, American Society of Anaesthesiologists

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Table 3. Postoperative outcomes, including 30-day morbidity/mortality, length of in-hospital stay and admission to Intensive Care Unit Postoperative outcome (No) Variable 2019 2020 Clavien-Dindo classification II 10 7 RTI-abx (1) urinary retention-catheter (2) wound urinary retention-catheter (3) infection-abx (3) seroma-cons. Mx (2) seroma-cons. Mx (1) wound infection-abx (3) ischemic orchitis -cons. Mx (1) CD colitis-abx (1) IIIa 0 4 seroma - I/R drain (2) pelvic collection - I/R drain (1) wound dehiscence -VAC (1) IIIb 2 0 (re-operations: hematoma evacuation / orchidectomy for ischemic orchitis) IV-V 0 0 Average postoperative in-hospital stay (days) 4.7 6.8 Planned postoperative ICU admission 0 1 (CAWR patient) Unplanned readmission to ICU 0 0 RTI, respiratory tract infection; abx, antibiotics; cons., conservative; CAWR, complex abdominal wall reconstruction; I/R, Interventional Radio- logy, cons. Mx, conservative management; CD colitis, Clostridium difficile colitis; VAC, vacuum-assisted closure DISCUSSION had been postponed on a rolling basis for a cumulative period of three months since January 2020. The outbreak of COVID-19 pandemic in 2020 has had a major impact in the delivery of electi- As suspected, in 2020 we noted an 18% incre- ve hernia surgery, with cancellations of non-urgent ase of total emergency hernia operative volume elective procedures and subsequent build-up of compared to 2019, with a moderate increase in longer waiting lists (8). Combining the latter with the number of visceral resections due to strangu- the tendency of the public to avoid hospital admi- lated hernia contents. The latter could be attri- ssions unless grossly symptomatic due to the fear buted to the noted delayed presentation of the of acquiring COVID-19 infection, previous reports patients with acutely symptomatic hernias to our demonstrated a reduction of emergency hernia sur- emergency surgical service. Having a 0% hospi- gical activity during the first wave of the pandemic tal-acquired COVID-19 rate in our 2020 study in 2020 (9). However, to the best of our knowled- group, with also absence of major postoperative ge, no published study has evaluated in the long respiratory complications, we demonstrated that run the emergency hernia surgery outcomes since emergency hernia surgery could be safely perfor- the declaration of the pandemic by the World He- med during the pandemic waves. alth Organisation in January 2020. Our anecdotal Therefore, we strongly advocate upfront surgery experience, in accordance to previously published of patients with acutely symptomatic hernias, re- multi-centre studies (10), regarding the characteri- serving taxis under analgesia/sedation only for stics of the acute surgical admissions in general has patients who are unfit or unwilling to undergo clearly shown a significant increase in 2020 of the surgery. We also strongly believe that the involved number of patients who come with features of ne- surgical societies and medical regulating authoriti- glected peritonitis secondary to perforated viscus, es should send a clear message to the patients who locally advanced obstructing tumours, as well as have acutely symptomatic hernias or are currently complicated biliary pathologies, such as gallblad- on surgical waiting lists, advising them to seek der empyemas, etc. To an extent the same acco- medical advice upon any relevant concerns wit- unted for the acute presentations of symptomatic hout delay, even during the pandemic peak waves. hernias, with an observed higher overall number of emergency hernia operations carried out during the FUNDING last year. It has to be noted that our emergency sur- No specific funding was received for this study gery services have been continuously fully functioning since the outbreak of COVID-19 pandemic, in TRANSPARENCY DECLARATION contrast to our elective hernia operating lists, which Conflict of Interest: None to declare.

466 Malik et al. Emergency hernia surgery during COVID-19

REFERENCES

1. Hussain PM, Kanwal A, Gopikrishna D. Resuming 6. Dindo D, Demartines N, Clavien PA. Classification of elective operations after COVID-19 pandemic. Br J surgical complications: a new proposal with evaluati- Surg 2020; 107:e549. on in a cohort of 6336 patients and results of a survey. 2. Poeran J, Zhong H, Wilson L, Liu J, Memtsoudis SG. Ann Surg 2004; 240:205–13. Cancellation of elective surgery and intensive care 7. Saklad M. Grading of patients for surgical procedu- unit capacity in New York State: a retrospective co- res. Anesthesiology 1941; 2:281-4. hort analysis. Anesth Analg 2020; 131:1337-41. 8. Köckerling F, Köckerling D, Schug-Pass C. Electi- 3. Cano-Valderrama O, Morales X, Ferrigni CJ, Mar- ve hernia surgery cancellation due to the COVID-19 tín-Antona E, Turrado V, García A, Cuñarro-López pandemic. Hernia 2020; 24:1143-5. Y, Zarain-Obrador L, Duran-Poveda M, Balibrea JM, 9. Lima DL, Pereira X, Dos Santos DC, Camacho D, Torres AJ. Acute care surgery during the COVID-19 Malcher F. Where are the hernias? A paradoxical pandemic in Spain: changes in volume, causes and decrease in emergency hernia surgery during CO- complications. A multicentre retrospective cohort stu- VID-19 pandemic. Hernia 2020; 24:1141-2. dy. Int J Surg 2020; 80:157-61. 10. Reichert M, Sartelli M, Weigand MA, Weigand 4. Patriti A, Baiocchi GL, Catena F, Marini P, Catarci MA, Doppstadt C, Hecker M, Reinisch-Liese A, Ben- M; FACS on behalf of the Associazione Chirurghi Os- der F, Askevold I, Padberg W, Coccolini F, Catena pedalieri Italiani (ACOI). Emergency general surgery F, Hecker A, WSES COVID-19 emergency surgery in Italy during the COVID-19 outbreak: first survey survey collaboration group et al. Impact of the SARS- from the real life. World J Emerg Surg 2020; 15:36. CoV-2 pandemic on emergency surgery services-a 5. Burgard M, Cherbanyk F, Nassiopoulos K, Malekza- multi-national survey among WSES members. World deh S, Pugin F, Egger B. An effect of the COVID-19 J Emerg Surg 2020; 15:64. pandemic: significantly more complicated appendici- tis due to delayed presentation of patients! PLoS One 2021; 16:e0249171.

467 ORIGINAL ARTICLE

Epidemiology of hospitalized patients with peripheral arterial disease in Bosnia and Herzegovina

Akif Mlačo1 , Nejra Mlačo2 , Mevludin Mekić3, Alen Džubur4

1Department of Angiology, Clinic for Heart, Blood Vessel and Rheumatic Diseases, Clinical Centre University of Sarajevo, 2Health Centre, Visoko, 3Department of Rheumatology, Clinic for Heart, Blood Vessel and Rheumatic Diseases, Clinical Centre University of Sarajevo, 4Department of Cardiology, Clinic for Heart, Blood Vessel and Rheumatic Diseases, Clinical Centre University of Sarajevo; Bosnia and Herzegovina

ABSTRACT

Aim To investigate a profile of patients with peripheral artery disease (PAD) in Bosnia and Herzegovina.

Methods This observational study included 1022 patients hospitalized at the Clinical Centre University of Sarajevo in a 5-year period, 2015 to 2019.

Results Disease prevalence rises sharply after the age of 50. Most patients, 797 (78%) had proximal PAD; 658 (64.4%) were Corresponding author: males. The death occurred in 73 (7.1%) patients, more often in Akif Mlačo females (66- 10%), and in patients with chronic kidney disease (10- 23.8%). Amputation occurred in 153 (15%) patients, where Department of Angiology, Clinic for Heart, 102 (66.7%) patients had diabetes. Other surgical procedures were Blood Vessel and Rheumatic Diseases, more common in males and smokers. Necrosis and phlegmon on Clinical Centre University of Sarajevo lower extremities were found in 563 (55.1%) and 43 (4.2%) pati- Bolnička 25, Sarajevo, ents, respectively. History of tobacco use was noted in 620 (60.2%) Bosnia and Herzegovina patients, and 414 (40.8%) patients were current smokers. More Phone: +387 33 297 521; than a half of patients had hypertension and diabetes, 596 (58.3%) and 513 (50.2%), respectively. One in 10 patients had a history of Fax: +387 33 297 805; myocardial infarction or stroke. Most patients had high fibrinogen @ E-mail: mlaco.akif gmail.com and blood glucose and low high-density lipoprotein (HDL). ORCID ID: https://orcid.org/0000-0002- 1907-9017 Conclusion Patients with PAD have multiple comorbidities and risk for various complications. Primary and secondary prevention of risk factors is the mainstay of treatment.

Original submission: Key words: atherosclerosis, diabetes mellitus, risk factors, to- 10 May 2021; bacco use Revised submission: 21 June 2021; Accepted: 29 June 2021 doi: 10.17392/1397-21

Med Glas (Zenica) 2021; 18(2):468-474

468 Mlačo et al. Peripheral artery disease epidemiology

INTRODUCTION Data were collected from medical records of all patients hospitalized for PAD: gender, age, Peripheral arterial disease (PAD) is a complete or PAD location, number of hospitalizations, lethal partial obstruction of blood flow in major syste- outcome, Leriche syndrome, Buerger syndrome, mic arteries other than those of the cerebral and ulcer, phlegmon, necrosis, history of smoking coronary circulation. The most common cause cigarettes (packs of cigarettes smoked per day is atherosclerosis, while other, such as vasculitis, x years; 1 pack= 20 cigarettes), comorbidities, thrombosis, thromboembolism, are infrequent (1). amputation, other surgical procedures and labo- The estimated prevalence of this disease in Euro- ratory values. pe is around 5.3% (40 million) (2). The PAD has a strong correlation with coronary artery disease The study was completed in compliance with the (CAD) and cerebrovascular disease, as atheroscle- Helsinki Declaration (last revised in 2013) and rosis is the basis of all these conditions (1). Accor- approved by the Institute for Scientific Research dingly, the main risk factors for PAD include smo- and Development of the Clinical Centre Univer- king, diabetes, hypertension, hypercholesterolemia sity of Sarajevo. (3). The presence of three or more risk factors Methods increases the risk of PAD ten times and smoking alone increases that risk two to four times (2,4). The diagnosis of PAD was confirmed by ultraso- The prevalence of PAD is increasing, due to the und VI-VID S5 with 12 L linear probe (General ageing of the global population and larger detec- Electric, Boston, Massachusetts, United States) or tion of asymptomatic cases (2). Studies show that Logiq Book XP with 8L curvilinear probe (Gene- patients with symptomatic PAD with no prior ral Electric, Boston, Massachusetts, United Sta- myocardial infarction (MI) or stroke had around tes), or CT angiography (Toshiba Aquilion Prime 2 times higher risk of cardiovascular events than 160 Slice CT), or digital subtraction angiography those with prior MI or stroke but without sympto- (Siemens Axiom Artis or General Electric Innova). matic PAD (5). This condition remains underdi- Laboratory analysis included fibrinogen (1.8-3.8 agnosed and undertreated, and it is a very impor- g/L), blood glucose (3.3-6.1 mmol/L), glycated tant public health problem in all countries (2,4). haemoglobin (HbA1C) (<6.5%) and lipoprotein The aim of this study was to present epidemio- fractions - total cholesterol (3.1-5.2 mmol/L), logical data on PAD in Bosnia and Herzegovina high-density lipoprotein (HDL; 1.06-1.94 (B&H) as a middle-income country, and empha- mmol/L, low-density lipoprotein (LDL; 1.4-3.4 size the importance of the disease and the range mmol/L), and triglycerides (0.11-1.70 mmol/L). of associated conditions. Such data have not been Reference values were according to the Clinical previously published in our institution and sparse Centre University of Sarajevo. data are available in the region. When tobacco use was compared with other conditions, both current and former smokers are con- PATIENTS AND METHODS sidered as smokers, while non-smokers are those who have never smoked, unless otherwise noted. Patients and study design Statistical analysis This observational retrospective study included 1022 patients hospitalized at the Department of The results of the descriptive analysis were dis- Angiology, Clinic for Heart, Blood Vessel and played in frequencies and percentages. Differ- Rheumatic Diseases, Clinical Centre University ences between parameters were assessed using of Sarajevo from 2015 to 2019. Inclusion criteria Mann Whitney U test. The correlation between were patients with confirmed PAD. parameters was assessed by the χ2 test of inde- PAD was defined as atherosclerotic disease in pendence with Yates’ Correction for Continuity: the lower extremities and classified as proximal the phi coefficient is a measure of association if occlusion/stenosis involves arteries above the between two binary variables, ranging from -1 knee and distal if the obstruction only involves to 1 (higher number shows stronger correlation popliteal arteries or those below the knee. between two variables). The p≤0.05 was used for statistical significance.

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RESULTS Most patients had proximal PAD (797; 78%), including patients with diabetes mellitus Out of 1022 patients hospitalized with PAD in (72.1%) and smokers (84%) (p=0.00; phi=-0.14; the 5 year-period, 658 (64.4%) were males and p=0.00; phi=0.18, respectively). The significan- 364 (35.6%) were females. Overall average age ce of PAD location was proven in other groups was 68.5 (±11.3) (range 18-96) years: 66.7±10.5 of patients (Table 2). (range 37-93) years for males, and 71.8±11.8 (range 18-96) years for females. Table 2. Peripheral artery disease (PAD) location and its significance among groups of patients according to comorbidities One in ten patients, 110 (10.8%), were hospi- No (%) of patients in the PAD group Patients group p (phi*) talized more than once in the five-year period; Proximal Distal among those, 66 (60%) were hospitalized twice, All 797 (78) 225 (22) 25 (22.7%) three times, 13 (11.8%) four times, Diabetes mellitus 370 (72.1) 143 (27.9) 0.00 (-0.14) six (5.5%) patients five or more times. Annually, Necrosis 414 (73.5) 149 (26.5) 0.00 (-0.13) Leg ulcer 38 (63.3) 22 (36.7) 0.008 (-0.09) 189 (18.5%) patients were hospitalized more Buerger’s disease 6 (27.3) 16 (72.7) 0.00 (-0.18) than once, and most of them twice, 145 (76.7%). Current smokers 348 (84) 66 (16) 0.00 (0.18) Amputation 109 (71.2) 44 (28.8) 0.05 (-0.07) Lethal outcome was registered in 73 (7.1%) hos- *The Phi Coefficient is a measure of association between two binary pitalized patients (Table 1), significantly more variables, it is in the range from -1 to 1, higher number shows stron- females than males, 10% and 5%, respectively ger correlation between two variables (p=0.001; phi=-0.12). More deaths occurred in Necrosis was more common in patients with non-smokers than smokers, 10.7% and 4.8%, a history of stroke (67.6% vs 56.2% in others), respectively (p=0.001; phi=-0.12). More deaths p=0.03; phi=0.07. Phlegmon was more common occurred in patients who had lower extremity ne- in non-smokers (7.5%) than smokers (2.1%) crosis than in patients without necrosis (76.5% vs. (p=0.00; phi=-0.13) (Table 1). 23.5% (p=0.002; phi=0.10). Lethal outcome was Tobacco use was declared by 620 (60.2%) pa- more frequent in patients with chronic kidney di- tients: 414 (40.8%) were current smokers at sease (CKD) comparing the ones without CKD, the time of hospitalization, while 207 (20.2%) 23.8% and 6.4%, respectively (p=0.00; phi=0.13). were former smokers; four patients were coun- ted in both categories, due to different smoking Table 1. Conditions and outcome associated with peripheral artery disease (PAD) status during rehospitalization. Among 138 patients with available data on the smoking habit, No (%) of No (%) of patients* p patients pack-years were in the range from 10 to 200. Variable p (phi)§ (phi)§ Smo- Non- Over 90% of patients smoked 30 pack-years and Total† Females Males kers‡ smokers 60.1% at least 50 pack-years; males significantly Lethal 73 36 33 0.001 30 43 0.001 surpassed females (mean 76 vs 51 pack-years) outcome (7.1) (10) (5) (-0.12) (4.8) (10.7) (0.12) Leriche 29 14 15 0.21 27 2 p=0.001 (p=0.002); 81 (7.9%) were categorized as passi- syndrome (2.8) (3.8) (2.3) (-0.045) (93.1) (6.9) (0.11) onate smokers, without quantification (data not Buerger’s 22 5 17 0.29 21 1 p=0.002 shown). Most of our former smokers (83.5%) disease (2.2) (1.4) (2.6) (0.04) (95.5) (4.5) (0.11) 563 233 355 0.003 303 260 p=0.00 quit smoking cigarettes within 20 years. Necrosis (55.1) (64.1) (53.9) (-0.09) (53.8) (46.2) (-0.16) More than a half of the patients had hypertension 43 12 31 0.36 13 30 p=0.00 Phlegmon (4.2) (3.3) (4.7) (0.034) (2.1) (7.5) (-0.13) and diabetes, 596 (58.3%) and 513 (50.2%) res- Lower-extre- 60 21 39 1.00 29 31 0.06 pectively, more frequently non-smokers (63.4% mity ulcer (5.9) (5.8) (5.9) (0.003) (48.3) (51.7) (-0.06) and 63.7%, respectively) than smokers (55.0% History of 620 154 466 0.00 N/A N/A N/A and 47.32%, respectively) (p=0.009; phi=-0.08 tobacco use (60.2) (42.3) (70.8) (0.28) 153 49 104 0.36 85 68 0.19 and p=0.00; phi=-0.22, respectively). Amputation (15) (13.5) (15.8) (0.03) (55.6) (44.4) (0.04) History of 117 (11.5%) and 116 (11.4%) patients Other surgical 124 25 99 0.00 99 25 0.00 procedures (12.1) (6.9) (15) (0.12) (16) (6.2) (0.15) revealed MI and stroke, respectively. Hypertensi- *Percentages are shown within gender for females and males, and on has been more often in females, while MI has within primary condition for smokers and non-smokers; †Number of been more often in males (Table 3). patients with available data; ‡Current or former smokers; §The Phi Coefficient is a measure of association between two binary variables, A total number of 153 (15%) patients had some it is in the range from -1 to 1, higher number shows stronger correlation between two variables; N/A, non applicable; form of amputation on the lower extremities (Ta-

470 Mlačo et al. Peripheral artery disease epidemiology

Table 3. Comorbidities of patients with peripheral artery Surgical procedures (Table 1) were more common disease (PAD) in males (99- 15%) versus females (25- 6.9%)

* No (%) of No (%) of patients * (p=0.00; phi=0.12), as well in smokers (99- p patients p Comorbidity § § 16%) versus non-smokers (25- 6.2%) (p=0.00; (phi) Smo- Non- (phi) Total† Females Males kers‡ smokers phi=0.15). Patients with hypertension and a history 596 235 361 0.003 341 255 0.009 of MI had more often surgical procedures (p=0.01; Hypertension (58.3) (64.6) (54.9) (-0.09) (57.2) (42.8) (-0.08) phi=0.08 and p=0.00; phi=0.17, respectively). Diabetes 513 186 327 0.72 257 256 0.00 mellitus (50.2) (51.1) (49.7) (-0.01) (50.1) (49.9) (-0.22) High fibrinogen level was detected in 657 Myocardial 117 31 86 0.04 73 44 0.76 (82.8%) patients. Patients with a history of am- infarction (11.5) (8.5) (13.1) (0.07) (62.4) (37.6) (0.013) putation and patients with distal PAD had higher 116 48 68 0.20 62 54 0.12 Stroke (11.4) (13.2) (10.3) (-0.04) (53.4) (46.6) (-0.05) fibrinogen values than patients without a history Hyperlipide- 43 14 29 0.79 35 8 0.007 of amputation (p=0.00; p=0.014; respectively) mia (4.2) (3.8) (4.4) (0.01) (81.4) (18.6) (0.09) (Table 5). Chronic kid- 42 9 33 0.07 23 19 0.52 ney disease (4.1) (2.5) (5.0) (0.06) (54.8) (45.2) (-0.02) Table 5. Laboratory values of patients hospitalized with 25 4 21 0.06 21 4 0.03 AAA peripheral artery disease (PAD) (2.4) (1.1) (3.2) (0.06) (84) (16) (0.08) No (%) of patients* with Popliteal ar- 18 2 16 0.05 13 5 0.44 Parameter Mean Range tery aneurysm (1.8) (0.5) (2.4) (0.07) (72.2) (27.8) (0.03) (reference values) (±SD) High Normal Low value value value 7 1 6 0.43 6 1 0.33 Malignancy (0.7) (0.3) (0.9) (0.04) (85.7) (14.3) (0.43) Fibrinogen 4.9 657 134 2 0.8-13.5 Pulmonary 5 3 2 0.50 1 4 0.16 (1.8-3.8 g/L) (±1.6) (82.8) (16.9) (0.3) Blood glucose 3.3-6.1 9.8 665 327 3 embolism (0.5) (0.8) (0.3) (-0.04) (20) (80) (-0.06) 2.5-37.5 AIC aneu- 5 5 5 0 (mmol/L) (±5.7) (66.8) (32.9) (0.3) 0 N/A N/A HbA1C 8.4 227 64 rysm (0.5) (0.8) (100) (0) 4.5-14.8 N/A Deep vein 4 1 3 2 2 (<6.5%) (±1.93) (81.2) (18.8) N/A N/A Total cholesterol 4.2 168 570 191 thrombosis (0.4) (0.3) (0.5) (50) (50) 1.7-16 AFC aneu- 2 1 1 2 0 (3.1-5.2 mmol/L) (±1.38) (18.1) (61.4) (20.6) N/A N/A rysm (0.2) (0.3) (0.2) (100) (0) HDL cholesterol 0.98 9 224 444 0.1-2.9 *Percentages are shown within gender for females and males, and (1.06-1.94 mmol/L) (±0.37) (1.3) (33.1) (65.6) † LDL cholesterol 2.43 94 482 93 within comorbidity for smokers and non-smokers; Number of patients 0.6-5.7 with available data; ‡Current or former smokers; §The Phi Coefficient is (1.4-3.4 mmol/L) (±0.89) (14.1) (72) (13.9) a measure of association between two binary variables, it is in the Trigycerides 1.85 374 552 0.5-14.56 0 range from -1 to 1, higher number shows stronger correlation between (0.11-1.70 mmol/L) (±1.2) (40.4) (59.6) two variables; AAA, abdominal aortic aneurysm; AIC, arteria iliaca *Within patients with available data; HbA1C, glycated haemoglobin; communis; AFC, arteria femoris communis; N/A, non applicable HDL, high-density lipoprotein; LDL, low-density lipoprotein; N/A, ble 1), during hospitalization or earlier in history, not applicable and 102 (66.7%) had diabetes (p=0.00; phi=0.14). Blood glucose ˃6.1 mmol/L was detected in 665 The most common site was partial foot amputa- (65.1%) patients; patients with amputation had tion seen in 85 (8.4%) patients, followed by tran- higher blood glucose level (mean 5.80 mmol/L) sfemoral and transitibial amputation in 44 (4.3%) than the ones without amputation (mean 4.83 and 34 (3.3%) patients, respectively. mmol/L) (p=0.00). A correlation between necrosis and amputation HbA1C ≥6.5% was determined in 277 (81.2%) was found (p=0.038; phi=0.19). patients; 76 (21.9%) patients had HbA1C ≥10%. Regarding other surgical procedures, peripheral Most patients had total and LDL cholesterol in artery bypass surgery was noticed in 67 (6.5%) the reference range, 570 (61.4%) and 482 (72%) cases (Table 4). (mean value of 4.2 mmol/L and 2.43 mmol/L, Table 4. Surgical procedures in patients hospitalized with respectively). Lower HDL cholesterol was found peripheral artery disease (PAD) in 444 (65.6%) patients; 552 (59.6%) patients Procedure No (%) of patients* had triglycerides in the reference range (Table 5). Bypass procedure 67 (6.5) PTA +/- stenting 33 (3.3) DISCUSSION CABG 29 (2.8) AAA repair 7 (0.7) PAD is more common among the elderly popu- PAA repair 1 (0.1) lation. After the age of 50, the prevalence of this Hip replacement 1 (0.1) *of the total number of patients; AAA, abdominal aortic aneurysm; disease increases sharply (6), similarly to our re- CABG, coronary artery bypass grafting; PAA, popliteal artery aneu- sults. Most of our patients were ≥50 years old (a rysm; PTA, percutaneous transluminal angioplasty

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mean age of 68 years), similar to the age distribu- the clinical setting of this study including more tion in other studies (2,7,8). severe patients. Most of our diabetic patients had Fewer PAD-related hospitalizations and procedu- proximal PAD, which is inconsistent with earlier res were found in females, as it was previously evidence that diabetes is related to atherosclero- reported (9). Females had higher mortality than sis in arteries distal to the knee (14). males in our study. Several studies have reported Males in our study were more likely to have a hi- higher mortality in females who undergo a vas- story of MI than females. Similarly, prior studies cular procedure (9-11). On the other hand, some demonstrated more males with a history of CAD studies found higher rates of amputation in males (10,11). Prior studies also demonstrated a higher (9,12), however, we did not prove this. prevalence of diabetes in males (10,11), which More than 40% of our patients were current smo- we did not prove. kers, which is much more compared to the Rotter- Approximately one in ten patients with PAD has dam study where around 24% and 17% males and a history of stroke, considering that 11.6% pati- females, respectively, were current smokers, and ents in our study and 11.2% patients in NHANES compared to the National Health and Nutrition had a history of stroke (7). Examination Survey (NHANES) where around Chronic kidney disease was associated with PAD 34% were current smokers (7,8). If we consider in several studies and now is considered one of that these studies were published at the beginning the independent risk factors for the disease (16- of the 21st century, when the prevalence of to- 18). More lethal outcomes are reported in pati- bacco use in the West countries was higher than ents with concomitant CKD (16,19,20), which today and that over 90% of our patients had 30 was proved in our study (almost four times more or more pack-years, the tobacco use frequency in deaths in patients with CKD). Some studies also B&H is an even a more worrying fact. Our stu- reported a higher risk for limb amputation in pa- dy showed a much lower percentage of former tients with CKD (21-23). We did not prove this smokers in relation to the other studies (7,8). The association, neither did a French study which risk for PAD associated with the smoking rema- concluded that CKD was not an independent pre- ins elevated even 20 years after cessation, altho- dictor of limb amputation (20). One study deter- ugh this risk is significantly reduced 10 years af- mined that only patients receiving dialysis have a ter smoking cessation (13). Most smokers in our higher risk for amputation, and not patients with study had proximal PAD, which is consistent with milder kidney disease (21). earlier data (14). Lethal outcome and phlegmon Limb amputation was noted in 15% of our pati- were more common in non-smokers in our study; ents and two-thirds of those patients had diabe- a possible explanation is that non-smokers had tes. This association was significant and repor- diabetes and hypertension more often, which are ted in other studies (7,8,24,25). Diabetes is the the main risk factors for PAD as well. most common cause of non-traumatic amputati- Studies, including ours, reported that more than on (24). One study reveals that diabetic patients half patients with PAD have hypertension (8). with PAD have a five times higher risk to have an Half of our patients had diabetes, which is a amputation (26). much higher percentage than in other studies, Several studies determined a strong positive around 8% and 26% in the Rotterdam Study and association between fibrinogen level and PAD the NHANES, respectively (7,8). The preva- (8,27,28). We confirmed high fibrinogen level in lence of diabetes in the USA is estimated to be more than 80% of our patients. Fibrinogen has a higher than in B&H, which makes this data more possible role as a biomarker for the detection of unclear, while the prevalence of diabetes in the subclinical arterial disease (29). Most of our pa- Netherlands is lower, but not enough to explain tients had normal total cholesterol with a mean this discrepancy (15). A possible explanation is value of 4.2 mmol/L compared to around 6.5 poorer diabetes control in B&H due to lack of mmol/L in the Rotterdam Study (weak association population awareness, lack of educational pro- between PAD and serum cholesterol), which is in- grams for people with diabetes, inability to afford compatible (8). Normal total cholesterol, LDL and medication and others. Another reason could be

472 Mlačo et al. Peripheral artery disease epidemiology

triglycerides in our study could be explained by In conclusion, the impact of PAD is higher an effect of statin therapy. Other studies determi- than the importance attached to it. These are ned that high HDL cholesterol level protects aga- commonly the patients with multiple comorbi- inst PAD (8,29), accordingly, most of our patients dities and risk for various complications. These had lower HDL level. Many studies confirm that findings highlight the importance of primary and any arterial disease is significantly associated with secondary prevention of atherosclerosis. Smo- high HbA1C and plasma glucose (30,31); we fo- king cessation, exercise and other lifestyle mo- und those values to be high in most patients. difications, as well as usage of antihypertensives, The role of elevated triglycerides as an indepen- statins, antiplatelets and other medication, dependent risk factor for PAD has been rejected in some ding on comorbidities, is a mainstay of treatment. studies (27,32), but has been confirmed in others This study is, to the best of our knowledge, the (33,34). This question remains controversial. first study that clearly describes the profile of -pa tients with PAD in Bosnia and Herzegovina. The main limitation of this study is that patients were from the clinical setting from one institution FUNDING and established epidemiological data could overe- stimate the burden of the disease in the general po- No specific funding was received for this study. pulation. Our results represent severe cases of this TRANSPARENCY DECLARATION disease in need of hospitalization, not considering those with subclinical or asymptomatic PAD. Conflicts of interest: None to declare.

REFERENCES 1. Conte SM, Vale PR. Peripheral arterial disease. Heart 10. McCoach CE, Armstrong EJ, Singh S, Javed U, An- Lung Circ 2018; 27:427-32. derson D, Yeo KK, Westin GG, Hedayati N, Amster- 2. Olinic DM, Spinu M, Olinic M, Homorodean C, Tata- dam EA, Laird JR. Gender-related variation in the ru DA, Liew A, Schernthaner GH, Stanek A, Fowkes clinical presentation and outcomes of critical limb G, Catalano M. Epidemiology of peripheral artery di- ischemia. Vasc Med 2013; 18:19-26. sease in Europe: VAS Educational Paper. Int Angiol 11. Vouyouka AG, Egorova NN, Salloum A, Kleinman L, 2018; 37:327-34. Marin M, Faries PL, Moscowitz A. Lessons learned 3. Fowkes FG, Aboyans V, Fowkes FJ, McDermott from the analysis of gender effect on risk factors and MM, Sampson UK, Criqui MH. Peripheral artery di- procedural outcomes of lower extremity arterial dise- sease: epidemiology and global perspectives. Nat Rev ase. J Vasc Surg 2010; 52:1196-202. Cardiol 2017; 14:156-70. 12. Feinglass J, Kaushik S, Handel D, Kosifas A, Martin 4. Firnhaber JM, Powell CS. Lower extremity periphe- GJ, Pearce WH. Peripheral bypass surgery and ampu- ral artery disease: diagnosis and treatment. Am Fam tation: northern Illinois demographics, 1993 to 1997. Physician 2019; 99:362-9. Arch Surg 2000; 135:75-80. 5. Tada H, Usui S, Sakata K, Takamura M, Kawashiri 13. Joosten MM, Pai JK, Bertoia ML, Rimm EB, Spiegel- MA. Low-density lipoprotein cholesterol level cannot man D, Mittleman MA, Mukamal KJ. Associations be too low: considerations from clinical trials, human between conventional cardiovascular risk factors and genetics, and biology. J Atheroscler Thromb 2020; risk of peripheral artery disease in men. JAMA 2012; 27:489-98. 308:1660-7. 6. Allison MA, Ho E, Denenberg JO, Langer RD, 14. Haltmayer M, Mueller T, Horvath W, Luft C, Poelz Newman AB, Fabsitz RR, Criqui MH. Ethnic-specific W, Haidinger D. Impact of atherosclerotic risk factors prevalence of peripheral arterial disease in the United on the anatomical distribution of peripheral arterial States. Am J Prev Med 2007; 32:328-33. disease. Int Angiol 2001; 20:200-7. 7. Selvin E, Erlinger TP. Prevalence of and risk fac- 15. The World Bank Data. Diabetes prevalence (% of po- tors for peripheral arterial disease in the United Sta- pulation ages 20 to 79) 2019. https://data.worldbank. tes: results from the National Health and Nutrition org/indicator/SH.STA.DIAB.ZS (01 March 2021) Examination Survey, 1999-2000. Circulation 2004; 16. Liew YP, Bartholomew JR, Demirjian S, Michaels 110:738-43. J, Schreiber MJ. Combined effect of chronic kidney 8. Meijer WT, Hoes AW, Rutgers D, Bots ML, Hofman disease and peripheral arterial disease on all-cause A, Grobbee DE. Peripheral arterial disease in the mortality in a high-risk population. Clin J Am Soc elderly: The Rotterdam Study. Arterioscler Thromb Nephrol 2008; 3:1084-9. Vasc Biol 1998; 18:185-92. 17. O'Hare AM, Glidden DV, Fox CS, Hsu CY. High pre- 9. Egorova N, Vouyouka AG, Quin J, Guillerme S, Mo- valence of peripheral arterial disease in persons with skowitz A, Marin M, Faries PL. Analysis of gender- renal insufficiency: results from the National Health related differences in lower extremity peripheral arte- and Nutrition Examination Survey 1999-2000. Circu- rial disease. J Vasc Surg 2010; 51:372-8.e1. lation 2004; 109:320-3.

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18. O'Hare AM, Vittinghoff E, Hsia J, Shlipak MG. Renal 27. Murabito JM, Evans JC, Nieto K, Larson MG, Levy insufficiency and the risk of lower extremity periphe- D, Wilson PW. Prevalence and clinical correlates ral arterial disease: results from the Heart and Estro- of peripheral arterial disease in the Framingham gen/Progestin Replacement Study (HERS). J Am Soc Offspring Study. Am Heart J 2002; 143:961-5. Nephrol 2004; 15:1046-51. 28. Allison MA, Criqui MH, McClelland RL, Scott JM, 19. Pasqualini L, Schillaci G, Pirro M, Vaudo G, Sie- McDermott MM, Liu K, Folsom AR, Bertoni AG, pi D, Innocente S, Ciuffetti G, Mannarino E. Renal Sharrett AR, Homma S, Kori S. The effect of novel dysfunction predicts long-term mortality in patients cardiovascular risk factors on the ethnic-specific odds with lower extremity arterial disease. J Intern Med for peripheral arterial disease in the Multi-Ethnic Stu- 2007; 262:668-77. dy of Atherosclerosis (MESA). J Am Coll Cardiol 20. Lacroix P, Aboyans V, Desormais I, Kowalsky T, 2006; 48:1190-7. Cambou JP, Constans J, Rivière AB, COPART inve- 29. Ridker PM, Stampfer MJ, Rifai N. Novel risk factors stigators. Chronic kidney disease and the short-term for systemic atherosclerosis: a comparison of C-reac- risk of mortality and amputation in patients hospita- tive protein, fibrinogen, homocysteine, lipoprotein(a), lized for peripheral artery disease. J Vasc Surg 2013; and standard cholesterol screening as predictors of 58:966-71. peripheral arterial disease. JAMA 2001; 285:2481-5. 21. O'Hare AM, Sidawy AN, Feinglass J, Merine KM, 30. Beks PJ, Mackaay AJ, de Neeling JN, de Vries H, Daley J, Khuri S, Henderson WG, Johansen KL. In- Bouter LM, Heine RJ. Peripheral arterial disease in fluence of renal insufficiency on limb loss and morta- relation to glycaemic level in an elderly Caucasi- lity after initial lower extremity surgical revasculari- an population: the Hoorn study. Diabetologia 1995; zation. J Vasc Surg 2004; 39:709-16. 38:86-96. 22. O'Hare AM, Bertenthal D, Sidawy AN, Shlipak MG, 31. Criqui MH, Browner D, Fronek A, Klauber MR, Co- Sen S, Chren MM. Renal insufficiency and use of re- ughlin SS, Barrett-Connor E, Gabriel S. Peripheral vascularization among a national cohort of men with arterial disease in large vessels is epidemiologically advanced lower extremity peripheral arterial disease. distinct from small vessel disease. An analysis of risk Clin J Am Soc Nephrol 2006; 1:297-304. factors. Am J Epidemiol 1989; 129:1110-9. 23. Landray MJ, Thambyrajah J, McGlynn FJ, Jones HJ, 32. Fowkes FG, Housley E, Riemersma RA, Macintyre Baigent C, Kendall MJ, Townend JN, Wheeler DC. CC, Cawood EH, Prescott RJ, Ruckley CV. Smoking, Epidemiological evaluation of known and suspected lipids, glucose intolerance, and blood pressure as risk cardiovascular risk factors in chronic renal impair- factors for peripheral atherosclerosis compared with ment. Am J Kidney Dis 2001; 38:537-46. ischemic heart disease in the Edinburgh Artery Study. 24. Beckman JA, Paneni F, Cosentino F, Creager MA. Am J Epidemiol 1992; 135:331-40. Diabetes and vascular disease: pathophysiology, cli- 33. Katsilambros NL, Tsapogas PC, Arvanitis MP, Tritos nical consequences, and medical therapy: part II. Eur NA, Alexiou ZP, Rigas KL. Risk factors for lower Heart J 2013; 34:2444-52. extremity arterial disease in non-insulin-dependent 25. Abbott RD, Brand FN, Kannel WB. Epidemiology of diabetic persons. Diabet Med 1996; 13:243-6. some peripheral arterial findings in diabetic men and 34. Bainton D, Sweetnam P, Baker I, Elwood P. Peripheral women: experiences from the Framingham Study. vascular disease: consequence for survival and asso- Am J Med 1990; 88:376-81. ciation with risk factors in the Speedwell prospective 26. Jude EB, Oyibo SO, Chalmers N, Boulton AJ. Pe- heart disease study. Br Heart J 1994; 72:128-32. ripheral arterial disease in diabetic and nondiabetic patients: a comparison of severity and outcome. Dia- betes Care 2001; 24:1433-7.

474 ORIGINAL ARTICLE

Anatomy of septocutaneous blood vessels of the anterior forearm

Darko Jović1, Mirza Bišćević2, Milan Milisavljevic3, Zoran Aleksić4, Milica Jakovljević1, Nevena Tešović1, Mićo Kremenović1

1Surgical Hospital "S.tetik", 2Department of Orthopaedics, General Hospital Sarajevo; Bosnia and Herzegovina, 3 Institute of Anatomy, School of Medicine, Belgrade, Serbia, 4Department of Surgery, School of Medicine Banja Luka, Bosnia and Herzegovina

ABSTRACT

Aim To measure a calibre of radial and ulnar septocutaneous perforators at the anterior forearm, and to count its number in proximal, middle and distal thirds.

Methods The study was conducted on 50 fresh amputated forearms (trauma, tumours) in the period between January 2012 and December 2021. Forearms were collected from several hospitals in Belgrade, and analysed at the Institute of Anatomy, Medical School, University of Belgrade, Serbia. Injection of ink-gelatin and fine dissection of autopsy material was performed on 30 forearms, and corrosion method with injecting methyl methacrylate Corresponding author: for 3D analysis on the other 20 forearms. Darko Jović Surgical Hospital "S.tetik" Results A mean calibre of septocutaneous perforators on the radial artery was 0.53±0.46 mm (0.2-0.85). Averagely, there were Patrijarha A. Čarnojevića 2b 8.1 radial artery septocutaneous perforators - two perforators on 78000 Banja Luka, the proximal third, 3.7 on the middle third, and 2.7 on the distal Bosnia and Herzegovina third. The mean calibre of ulnar artery perforators was 0.65±0.35 Phone: +387 65 657 037; mm (0.18-1.8). The average number of septocutaneous perforators Fax: +387 51 430 889; of the ulnar artery was 5.6; 1.2 on the proximal third, two on the E--mail: [email protected] middle third, and 2.2 on distal third. ORCID ID: https://orcid.org/0000-0002- Conclusion Determination of the origin, calibre and spreading di- 1357-8210 rections of the arterial septocutaneous perforators on the anterior forearm provide quantification of data about arborisation of radial and ulnar septocutaneous perforators at the anterior forearm. Clinical relevance of those anatomical data is in defining of safe Original submission: locations and dimensions of forearm fasciocutaneous flaps in plastic surgery. 07 April 2020; Revised submission: Key words: flap, perforator, radial artery, ulnar artery 16 April 2021; Accepted: 10 May 2021 doi: 10.17392/1386-21

Med Glas (Zenica) 2021; 18(2):475-478

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INTRODUCTION logical analysis were performed at the Institute of Anatomy, School of Medicine, University of The anatomy, number and origin of septocuta- Belgrade, Serbia. The Ethical Committee of the neous vessels of the anterior forearm are of huge School of Medicine, University of Belgrade, importance in the surgery of fasciocutaneous fo- approved the investigation in compliance with rearm flaps. Forearm flaps are numerous, varia- the Helsinki Declaration. ble in origin, dimensions and shape (proximally or distally based, pedicle or free). However, their Methods common characteristic is that they are based on a unique vascular system - the septocutaneous vas- The arterial system was injected by ink-gelatin, cular system (1-3). with latter microsurgical dissection of septocutaneous arteries in 30 forearms (10). For the re- Septocutaneous perforators are blood vessels maining 20 preparations the corrosion method originating from magistral vessels, radial and of injecting methyl methacrylate was applied ulnar artery at the anterior forearm. They pass to produce resin casts of blood vessels (11), through the intermuscular septa (fascia duplica- which allowed an accurate spatial analysis of the tures), perforate the deep fascia, and branching branching of perforating vessels. out, forming so-called fascial plexus (4,5). The most commonly used flap among fasciocuta- Separation of vascular cutaneous territories of neous forearm flaps is definitively a so-called perforators of the ulnar and radial arteries was Chinese flap, described by Yang Guofan et al. preceded by a longitudinal incision 10 cm long in 1981 (2,3). It belongs to “type C fasciocuta- in the cubital fossa and the dissection of the neous flaps”, arborizing from the radial artery brachial artery tree and its terminal branches. to supply the fascial plexus and the covering Two catheters were placed in the ulnar and ra- skin. Another important anterior forearm fas- dial arteries, secured with a hook and loop fa- ciocutaneous flap is the ulnar forearm flap, stener. After the arteries were rinsed by warm located in the medial intermuscular septum, water, 20 mL of 5% melted gelatin was simul- between m. flexor carpi ulnaris and m. flexor taneously injected, coloured by a blue and red digitorum superficialis (2, 6-8). fine ink. The injection lasted until the skin was stained without overlapping the vascular cuta- Good knowledge of the anatomy, topography and neous territories. After gelatin had hardened, the calibre of septocutaneous forearm arteries is cru- dissection was approached. cial for clinical success (aesthetic consideration, flap survival, donor-site morbidity) of forearm In the corrosion method (10), two-component flap-raising techniques (9). To the best of the aut- methyl methacrylate was used to produce resin hors’ knowledge, it is the first study of this kind blood vessel casts. After mixing the monomers in our region. and polymers and adding special dyes, acrylate was injected into the arteries of the forearm thro- The aim of this study was to investigate a calibre ugh placed catheters. In order for the acrylate of radial and ulnar septocutaneous perforators at injected by perfusion to fill the arterial network the anterior forearm, and to count its number in and to avoid deformations of the forearm, the proximal, middle and distal thirds. injection was performed in a tub filled with MATERIAL AND METHODS warm water. The injection itself was preceded by careful ligation of all blood vessels affected Study design and materials by amputation. It took around five hours for acrylate components to bind and for it to soli- The study of septocutaneous perforating dify. Corrosion of forearm tissue was performed branches of the anterior forearm arteries was in 40% NaOH solution, and the corrosion per conducted on 50 fresh amputated forearms. preparation lasted for a month. The blood vessel Upper limbs were mostly amputated due to trau- cast was ready for analysis after a thorough rin- ma and tumours in several hospitals in Belgrade sing with hot water (Figure 1). (Serbia) during the period January 2012 - De- cember 2021. Forearm’s preparation and histo-

476 Jović et al. Anterior forearm septocutaneous vessels

Topographical anatomy of septocutaneous perforators of the anterior forearm arteries was rather typical - shortly passing through the forearm fascia, the perforators form two branches which continue to branch out and extend in the longitudinal direction.

DISCUSSION Quality of soft tissue flaps is important not only in plastic-reconstructive surgery, but also in traumatology - soft tissue coverage is a prerequi- site for bony healing of radius/ulnar fractures and non-unions (12,13). Septocutaneous perfora- Figure 1. Single septocutaneous perforator, greatly enlarged (corrosion preparation) (Jović D, 2018. y, Institute of Anatomy, tors of radial and ulnar artery basically connect School of Medicine, University of Belgrade, Serbia) forearm’s magistral blood vessel with the fascia Statistical analysis and the flap itself, forming the basis of its vascularization. Therefore, its number, calibre and Descriptive statistic method (average, standard morphology are very interesting to plastic surge- deviation, range) and Student’s t-test for com- ons - the greater the number of septocutaneous parison of two groups with level of significance perforators involved in vascularization and the p=0.05 were used. larger their calibre, the more certain is flap survi- RESULTS val (14). The vascular territories of the radial and ulnar arteries complement the view of the blood The calibre of 324 septocutaneous perforators on supply of forearm skin zones. Also, the dominan- the total of 50 radial arteries at the site of origin ce in vascularization of particular blood vessel was 0.53±0.46 mm (0.20-0.85), and 0.65±0.35 contributes to define a limit of forearm raising mm (0.20-1.80) at the 224 septocutaneous perfo- flap. Thus, the radial forearm flap can be raised rators on the total of 50 ulnar arteries. In relation from the elbow to the hand flexion toward radial to the thirds (levels) of the forearm, the mean ca- side (15,16), while the ulnar forearm flap can be libres of radial septocutaneous perforators were raised over the same area, toward ulnar side (17). 0.95 mm in the proximal, 0.47 mm in the middle, and 0.14 mm in the distal third, while mean ca- The mean diameter (calibre) of septocutaneous libres of ulnar perforators were 0.95 mm in the perforators of the radial artery was 0.53 mm (0.20- proximal, 0.73 mm in the middle, and 0.42 mm 0.85) and 0.65 mm (0.18-1.80) of the ulnar artery in in the distal third. our study. If we observe peroforators’ dimensions in relation to the thirds (levels) of the forearm, ulnar The mean number of radial arteries septocuta- radial septocutaneous perforators were about 50% neous perforators was 8.1 – in average two perfo- wider in middle and distal thirds in comparison to rators in the proximal, 3.7 in the middle, and 2.7 the radial septocutaneous perforators. According to in the distal third, while on the ulnar artery there Cormack’s and Raspanti’s research, the calibre of was a total of 5.6 septocutaneous perforators - septocutaneous blood vessels of the radial artery 1.2 in the proximal, two in the middle, and 2.2 in does not exceed 0.5 mm, and there were about six the distal third (Table 1). perforators, the most proximal is the largest one, while distally their size decreases (1,2). However, Table 1. Septocutaneous perforators in proximal, middle and distal forearm thirds (levels) small-calibre blood vessels (<0.8 mm) are not sui- Septocutaneous Mean (minimal-maxi- Average number of sep- table for microsurgical transfer (14). perforators mal) calibre (mm) tocutaneous perforators The mean number of radial arteries septocutaneous Radial perforators in our study was 8.1, and 5.6 septocuta- Proximal 0.95 (0.55-1.77) 2 Middle 0.47 (0.20-1.20) 3.7 neous perforators of the ulnar artery. The most Distal 0.30 (0.18-0.80) 2.4 proximal perforator was located at the beginning Ulnar of radial artery, while others appeared on each 1-2 Proximal 0.95 (0.50-1.80) 1.2 Middle 0.74 (0.25-1.60) 2 cm, along the artery. Therefore, the middle third of Distal 0.43 (0.18-0.90) 2.4 the forearm was the richest in terms of the number

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and concentration of radial artery perforators, im- In conclusion, most septocutaneous perforators plying that flap raising is the safest in this region. of the radial and ulnar arteries are located in the Radial artery has averagely almost twice more middle thirds. The calibre of septocutaneous per- septocutaneous perforators in proximal and forators of the radial and ulnar arteries decreases middle thirds in comparison to the ulnar artery in the proximal-to-distal direction. Due to a large (statistically significant p<0.05), similar as in number of septocutaneous blood vessels and a Bell’s and Huang’s studies (18,19). good network of anastomoses between them, radial and ulnar fasciocutaneous flaps can be safely ra- Our results imply that majority of ulnar perfora- ised from practically all parts of the anterior fore- tors are located in the distal third, while majo- arm. The selection of a flap should be based on the rity of the radial forearm septocutaneous perfo- need for pedicle length, flap bulk, concerns about rators are located in the middle third. Cormack radial or ulnar dominance, and surgeon’s comfort. and associates found that ulnar artery had six to seven perforators (1), while Arnstein and Lewis FUNDING found two to four perforators (20), which partially support our findings. In comparison to the No specific funding was received for this study. radial artery septocutaneous perforators, ulnar TRANSPARENCY DECLARATION artery perforators have larger calibres, but their number is on average significantly smaller than Conflicts of interest: None to declare. the number of radial artery perforators.

REFERENCES 1. Weinzweig N, Chen L, Chen ZW. The distally based ra- 11. Ilic M, Milisavljevic M, Malikovic A, Laketic D,Eric dial forearm fasciosubcutaneous flap with preserva- D, Boljanovic J, Dozic A, Stimec BV, Manojlovic R. tion of the radial artery: an anatomic and clinical ap- The superficial palmar branch of the radial artery: a proach. Plast Reconstr Surg 1994; 94:675-9. corrosion cast study. Folia Morphol (Warsz) 2018; 2. Raspanti A, Delcroix L, Ghezzi S, Innocenti M. Study 77:649-6. of the tendinous vascularization for the compound ra- 12. Rollo G, Luceri F, Bisaccia M, Lanzetti RM, Luceri dial forearm flap plus flexor carpiradialis tendon. Sur A, Agnoletto M, Llaquet-Leiva AA, Mangiavini L, Radiol Anat 2016; 38:409-5. Meccariello L. Allograft versus autograft in forearm 3. Altiparmak M. Fasciocutaneus and/or myocutaneus aseptic non-union treatment. J Biol Regul Homeost flaps versus perforator flaps: systematic review and Agents 2020; 34:207-5. meta-analysis for reconstruction of ishial pressure so- 13. Rollo G, Luceri F, Pasquino A, Pichierri P, Tomarchio res. Eur J Plast Surg 2020; 43:211-7. A, Bisaccia M, Garagnani L, Biserni M, Agnoletto 4. Xie RG. Medial versus lateral approach to harvesting of M, Marmotti A, Mangiavini L, Meccariello L. Bone anterolateral thigh flap. J Int Med Res 2018; 46:4569-8. grafiting combined with Sauvé-Kapandji Procedures 5. Kerr RP, Hanick A, Fritz M.A. Fascia lata free flap re- for the treatment of aseptic distal radius non-union. J construction of limited hard palate defects. Cureus Biol Regul Homeost Agents 2020; 4(Suppl. 3):213-5. 2018 21;10:e2356. 14. Kastenbauer E, Tardy EM. Aestetische und Plastische 6. Hwang K, Han JY, Chung IH. Hypothenar flap based Chirurgie an Nase, Gesicht und Ohrmuschel. 2nd ed. on a cutaneous perforator branch of the ulnar artery: Stuttgart, New York: Georg Thieme Verlag, 2002. an anatomic study. J Reconstruction Microsurg 2005; 15. Daya M, Nair V. Free radial forearm flap lip recon- 21:297-4. struction: a clinical series and case reports of techni- 7. Hyuon HH, Yong SCh, In BK, Sang HK, Young JJ. A cal refinements. Ann Plast Surg 2009; 62:361-7. perforator from the ulnar artery and cutaneous nerve 16. Lane JC, Swan MC, Cassell OC. Closure of the ra- of the hypothenararea: An anatomical study for clini- dial forearm donor site using a local hatchet flap: cal application. Microsurgery 2017; 37:49-6. Analysis of 45 consecutive cases. Ann Plast Surg 8. Mathy JA, Moaveni Z, Tan ST. Perforator anatomy of 2013; 70:308-4. the ulnar forearm fasciocutaneus flap. J Plast Recon- 17. Hakim SG, Trenkle T, Sieg P, Jacobsen HC. Ul- str Aesthet Surg 2012; 65:1076-5. nar artery-based free forearm flap: review of specific 9. Chang EI, Liu J. Prospective comparison of donor-site anatomic features in 322 cases and related literature. morbidity following radial forearm and ulnar ar- Head Neck 2014; 36:1224-9. tery perforator flap harvest. Plast Reconstr Surg 2020; 18. Bell RA, Schneider DS, Wax MK, Superfiatial ulnar 145:1267-7. artery; a contraindication to radial forearm free tissue 10. Camuzard O, Foissac R, Clerico C, Fernandez J, transfer. Laryngoscope 2011; 121:933-6. Balaguer T, Ihrai T, de Peretti F, Baqué P, Boileau P, 19. Huang JJ, Wu CW, Lam WL, Nguyen DH, Kao HK, Georgiou C, Bronsard N. Inferior cubital artery per- Lin CY, Cheng MH. Anatomical basis and clinical forator flap for soft-tissue coverage of the elbow: ana- application of the ulnar forearm free flap for head and tomical study and clinical application. J Bone Joint neck reconstruction. Laryngoscope 2012; 122:2670-6. Surg Am 2016; 98:457-8. 20. Arnstein PM, Lewis JS. Free ulnar artery forearm flap: modification.Br J Plast Surg 2002; 55:356-7.

478 ORIGINAL ARTICLE

Reamputation stumps below knee Viktor I. Shevchuk, Yurii O. Bezsmertnyi, Yankai Jiang, Halyna V. Bezsmertna, Tetyana V. Dovgalyuk

Scientific Department, Research Institute of Rehabilitation of National Pirogov Memorial Medical University, Vinnytsia, Ukraine

ABSTRACT

Aim To investigate rehabilitation outcomes of patients with malformed tibial stumps.

Methods Observations included 421 patients with residual limb diseases and malformations (extensive inactive scars adhered to the bone, excessively long or short stumps, bone filing, osteomyelitis of the stump, muscle attachment to the skin scar, excessive mobility and deviations of the fibula, improper filing). Four hundred and thirty-six (436) reconstructive surgeries were performed. A follow-up period was from 6 months to 15 years. Radiological, ultrasonic, tensometrical, and histological methods were used.

Results Due to the frequent combination of several malformations and diseases in the same patient, non-free skin grafting with displaced dermal-subcutaneous flaps, which cover rather large Corresponding author: defects, was widely used. The surgeries were performed simul- Yurii O. Bezsmertnyi taneously and allowed for reconstruction without shortening the Scientific Department, Research Institute bone lever stump. Complications in the form of marginal necrosis of Rehabilitation of National Pirogov were obtained in three (0.71%) patients. The improved technique Memorial Medical University, of muscle grafting with the fixation of muscles to the bone provi- Vinnytsia, Ukraine ded an elastic stump covering the bone filing. The authors have developed original methods of surgery to create a bone block of the Khmelnytsky highway, 104, Vinnytsia, tibia, which make it possible to obtain painless, highly functional 21029, Ukraine stumps using partial support and ensure long-term use of modern Phone: +38 097 281 5160; prosthesis designs in 100% of cases. E-mail: [email protected] Conclusion Non-free dermal plasty with cutaneous-subcutaneous ORCID ID: https://orcid.org/0000-0002- flap is the method of choice for closing skin defects on the residu- 1388-7910 al limb. Muscle-bone fixation permits to eliminate some residual limb defects and to form an elastic muscular residual limb with closed bone filaments. Synostosis formation in different ways con- Original submission: siderably improves functional quality of the residual limb. 21 March 2021; Key words: bone synostosis, reconstructive surgery, stump Revised submission: 05 April 2021; Accepted: 26 May 2021 doi: 10.17392/1374-21

Med Glas (Zenica) 2021; 18(2):479-486

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INTRODUCTION PATIENTS AND METHODS Currently, there is some decline in the interest in Patients and study design amputations and reconstructive interventions on limb stumps. However, increasing traumatism, A total of 421 patients with lower leg residu- thrombo-vascular diseases, military conflicts in al limb malformations and diseases were under some cases result in amputations. After them, observation in the clinic of Research Institute due to a number of objective and subjective rea- of Rehabilitation of National Pirogov Memorial sons, as well as irrational prosthetics, stump di- Medical University (Vinnytsia, Ukraine) from seases and malformations occur, the elimination 2010 to 2021. The patients' age ranged from 18 of which is sometimes more difficult than the to 70 years. The period since the initial amputa- primary amputation. In recent years, a number tion ranged from 6 months to 15 years. The cau- of studies (1-13) have highlighted particular ses of primary amputation were: trauma in 391, issues in amputations, describing residual limb chilblains in 12, burns in one, tumours in six and diseases and malformations, post-amputation vascular diseases in 11 patients (Table 1). pain syndrome, and nerve treatment techniques. Table 1. General material characteristics The works (14,15) devoted to the creation of a No of Number (%) reamputations Other ope- bone bridge using the Ertl technique present the Type of pathology of patients Bone rations* results of these operations. The essence of the Myoplastic grafting method is the formation of a synostosis between Painful neuromas, 8 (1.90) 1 - 7 the ends of the tibia and fibula in the form of a ligature fistulas coiled periosteum with part of the cortical bone Extensive, low-mobile 96 (22.81) 54 36 6 layer; above the bridge the muscles are sutured scars fused to the bone Excessively long and 38 (9.02) 1 30 7 (15). The method is good, but in practice it is short stumps both difficult and prone to complications, ran- Bone filing stand-off 59 (14.02) 39 20 - ging from displacement of the muscle and the Osteomyelitis of 18 (4.27) 6 - 12 flap to an irregularly shaped or missing bridge the end of the stump (14). In our practice, we perform bone-plastic Bursitis 15 (3.56) 8 7 - formation of the synostosis with possible exten- Excessively tapered 18 (4.28) 7 9 2 sion of the tibial section plane to optimize the stumps use of partial support, because only with axial Attachment of muscles 66 (15.68) 30 36 - load there are hydrodynamic effects that ensure to the skin scar normal trophism. Fibula deviation 39 (9.26) 21 18 -

Excessive mobility Using the ideas of the Polish scientist Weiss M. 16 (3.80) 2 6 8 (16), we have successfully used the technique of the fibula Incorrect filing 48 (11.40) 20 18 10 of muscle-bone fixation for many years and of the tibia have improved it somewhat for reconstructive Total 421 (100.00) 189 180 52 interventions on the residual limb. The functio- *resection of neuroma; excision of fistula, bursa, ulcer, scar, excess of soft tissues; resection of osteophytes nality of a truncated limb depends not only on the muscle and bone, but also on the condition An informed consent was obtained from all pa- of the skin. Therefore, when performing recon- tients. structive interventions, their rational combinati- The Ethics Committee of Pirogov Memorial Me- on should be used. The development of prosthe- dical University, Vinnytsia, Ukraine approved tics makes it possible to significantly improve this investigation. the rehabilitation of persons with amputations and to return them to active work, everyday life, Methods culture and sport. In addition to the general condition, the clinical The aim of the study was to improve the rehabi- examination included assessment of the level of litation results of persons with tibia residual limb amputation, shape of the residual limb, degree of malformations. soft tissue coverage, skin, muscle, muscle strength, bone, postoperative scar, blood circulation,

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joint mobility, and the presence and nature of pain. The duration of the prosthesis use during the day was assessed. Digital radiography was performed as standard in two projections. Ultrasound polypositioning of the junction area with the graft was performed using Sonoline- SL450 (Siemens, Germany). The scanning was performed in longitudinal and transverse sections at the level of contact of the graft with the tibia bones. A convex sensor (3.5-4.5 MHz) was used to overcome soft tissue thickness. Stump support was determined using magneto- strictive transducers that were fixed between the Figure 1. Schematic representation of myoplastic reamputation (Shevchuk V, 2020) skin and the prosthesis receiving cavity. The tibial nerve, superficial and deep peroneal Morphological examination was performed on nerve and the posterior cutaneous nerve were ne- material taken from 22 patients at the time of cessarily shortened. reamputation and from two patients who died 5 and 9 years after osteoplastic reamputation from In the case of a short residual limb with scars concomitant diseases. fused to the bone, the latter were excised and reamputated by shortening the bone if necessary, Methods of reconstructive operations. During with muscle grafting and fixation of the muscles myoplastic stabilization of an excessively mobile to the bone. To lengthen the residual limb, a tibial fibula residual limb, the long fibula muscle was osteotomy was performed, followed by distracti- fixed transosseously, first to the residual fibula on and regeneration. After achieving the required and then to the outer surface of the tibia. length and adequate strength of the regenerate, In the case of a protruding tibial ridge, the bursa prosthetics were performed. was excised. The ridge was exposed and sawed With osteomyelitis of the end of the stump, 1% off at a 45° angle during internal rotation of the brilliant green solution with 3% hydrogen pe- residual limb. A 0.6-1 cm long transverse groove roxide solution was injected into the fistulous was made in the upper part of this saw cut with a opening. An incision was made, followed by drill, without penetrating the medullary canal. A excision of the scar and necrectomy. resorbable thread was passed through the canal. One free end was stitched laterally to the anterior In the presence of a long residual limb, in some edge of the tibia muscle and medially to the edge cases with a lower-third residual limb with of the medial head of the calf muscle. The liga- malformed scarring in the distal region, given the ture was tied, obtaining the closure of the ridge. excessive length of the residual limb, reamputa- After that, one loose end of the suture was passed tion was performed on the border of the middle through the lateral and the other through the me- and lower thirds of the tibia. More often it was dial (Figure 1) heads of the calf muscle in a U- necessary to perform an economical reamputa- shape returning to the anterior edge of the tibialis tion with simultaneous skin grafting. The skin muscle. The sutures were tied. An elastic muscle incision was made atypically, according to the stump was obtained on the operating table. In the location of the scarred tissue. After dissecting case of excess muscle, a high resection of the ca- them and excising the scars, skin plasty was melback muscle was performed. started. The latter was performed with a massive dermal-subcutaneous flap on a feeding base, cut Bone reconstruction involved formation of the off and displaced from the adjacent areas of the tibial synostosis by free plasty using autografts residual limb, the knee area and the thigh. Befo- from the removed part. rehand, intensive preparation was always carried Ligature fistulas were eliminated by excision af- out to create greater mobility and extensibility of ter a dye injection. the donor skin area.

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On the middle third of the tibia stump, if the skin defect was located on the anterior-thoracic or posterior-thoracic surface, the skin with subcutaneous tissue was separated on the anterior or, respectively, on the external surface, and it was adjusted to the size of the defect to be excised. The opposite edge of the wound was mobilized along the entire incision. Sutures were applied without tension. If there were extensive defects on the anterior-thoracic surface of the residual limb in the upper third, a flap was moved from the anterior-external surface of the femur to close them. Thus, it was possible to close defects up to Figure 2. Synostosis 2 70-80 cm in size with full-fledged skin. Simul- formation using a fibula taneously with skin grafting, other reconstructive graft (Shevchuk V, 2019) interventions were performed: removal of osteophytes, resection of nerve trunks above the loa- trapezoid-shaped graft was taken from the part of ding zone of the residual limb in the prosthesis, the tibia to be removed. The sides of this trape- sawing down the ridge, smoothing bone fillets, zium corresponded to the cross-sectional planes reamputation with muscle and skin grafting. of the tibia and fibula and the length of the larger base was equal to the distance between their Depending on the conditions, several techniques lowest points. The graft was placed between the for synostosis of the tibia bones with different grafts fillets of the bones, tightly closing the openings were used. A tubular graft was prepared from the of the medullary canals. A wire with a support amputated segment of the fibula and its length was pad was passed through the ends of both bones equal or slightly shorter than the width of the inter- and the graft on the fibula side using an electric tibial space. The graft was placed perpendicular to drill. Fixation was performed with the Ilizarov the bone between the lateral surfaces of the bone apparatus (Kurgan, Russia). stumps. On the fibula side, a spoke with a support pad was inserted through the end of the fibula, the Another variant of this operation was also used, medullary canal of the graft and the tibia. A second which allowed for a slight, up to 1 cm, shortening spoke was crossed over to the first one through the of the residual limb and obtaining synostosis. The tibia. The spokes were fixed in the ring of the Ili- tibia was sawed obliquely from top to bottom and zarov apparatus, creating compression. A base ring from outside to inside, forming a trapezoidal graft was placed in the upper third of the tibia, where the with the smaller base corresponding to the width spokes were also crossed. The rings were connected of the intertibial space and the larger base corres- with screws. This technique eliminates the possibi- ponding to the distance between the inner edges of lity of graft displacement. Bone synostosis occurs the tibia and fibula. The resulting graft was rotated 6-7 weeks after the amputation (Figure 2). 180°, aligning the lateral sides of the trapezium with the fillet of the tibia and the lateral surface The fibula graft can be fixed with an autograft of the fibula. Fixation was performed with an Ili- taken from the tibia or fibula. For this purpose, a zarov apparatus as in the previous technique. The channel was made from the outer surface of the healing time was 6-7 weeks (Figure 3). fibula, which is inserted into the autograft. The pin was inserted into the channel, achieving a The following method of synostosis formation stable fixation. In some cases, an indentation was using a tibial bone graft did not require additional made on the outer surface of the tibia for better means of fixation. retention of the graft. The medial and lateral por- A fragment whose length was equal to the distan- tions of the calf muscle were fixed to the tubules ce between the outer and inner points of the tibia formed in the graft. bones was taken from the part of the tibia to be If the residual limb was long enough, the bone removed. An aperture was cut into it, the width of was sawn off obliquely from the inside back. A which corresponded to half of the anteroposteri-

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Figure 3. Synostosis formation using a trapezoid Figure 4. Synostosis for- graft (Shevchuk V, 2019) mation by grafting the tibia on the residual limb of the or dimension of the tibia. A groove with a width fibula (Shevchuk V, 2019) equal to the thickness of the cortical layer of the graft and a length of 1 cm was formed in the fron- a Z-shaped lengthening of the bicep’s tendon was tal plane at the end of the tibia. The transplant was performed. An electric drill with a drill corres- inserted into the groove. The posterior surface of ponding to the thickness of the graft was used to the tibia was inserted into the groove; the stump form a transverse blind canal on the outer surface of the fibula was also placed in the groove. This of the tibia and inner surface of the fibula. The method allows the graft to be held firmly in place. graft was inserted into the canals. Muscle plasty Additional fixation is not required for the tibial bone was performed and the wound was sutured. synostosis with a tubular graft placed on the fibula. Synostosis of the tibia bones with short stumps A 3-4 cm long continuous tubular graft was cut can be performed by compression of the fibula to from the part of the tibia to be removed, freed the tibia with the Ilizarov apparatus or by placing from soft tissues and firmly placed on the resi- the fibula stump in a groove on the external-late- dual limb of the fibula. The graft was placed so ral surface of the tibia. that one of its three sides was tightly adjacent to the external-lateral surface of the tibia. The ante- RESULTS rior tibial and calf muscles were fixed to the graft. The excision of ligature fistulas with wound closu- The time to fusion was 6-8 weeks (Figure 4). re and drainage and removal of the bursa in the area The next technique involves synostosis of the ti- of the tibial crest was ended with wound healing in bia bones with a tibial trough graft. A navicular all patients. The elimination of excessive mobility graft was formed from the part of the tibia to be of the fibula was achieved in all patients too. removed using a saw and chisel, which was fitted Percutaneous reconstruction of the middle and under the crest of the tibia and overlapped with the upper third residual limbs was complex. The fibula. A firm block is formed after the operation. stumps and functionally very important adjacent In the case of a short tibial residual limb with a areas were covered with scar changed tissues, remnant of the fibula, which is always deflected which were tightly fused with the underlying for- backwards and outwards by the permanent tracti- mations and easily injured at the slightest load. on of the biceps muscle, creating a club-like shape The patients had long-term non-healing wounds, and making prosthetics impossible, one of the painful or ulcerative scars and trophic ulcers, are- following bone grafting operations was performed. as of altered skin with signs of atrophy and dege- One of them involves forming a synostosis with neration, and tightening scars causing a deformed a thin autograft from the crest of the tibia. The residual limb. Partial necrosis of the flap margins remainder of the fibula was brought into the occurred in three (0.71%) patients, which was physiological position. If this was unsuccessful, corrected by resection and suturing of the defect.

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The formation of the residual limb by fixing the of the tibia bones and the increase in the bearing muscles to the file resulted in 100% resilient surface, are slightly better than conventional muscular residual limbs with well-closed bones. stumps because the diameter of the plane is lar- In some cases (15%), it was difficult to tighten ger. These stumps are considered not to be very the contracted calf muscle against the crest of the good, but since we created an enlarged pain-free tibia. It was lengthened by serrated incisions of distal region, prosthesis in the prosthesis socket two-thirds thickness to avoid tension. with full contact was satisfactory. Indications for osteoplastic reconstructi- The tibial stumps in the middle third are well co- ve surgery include excess soft tissue, muscle vered by the muscles. The increased cross-secti- attachment to the skin scar, incorrect bone filing, onal surface area allows for a permissible load of outward deviation and excessive mobility of the up to 60% of the body weight. The residual limbs residual fibula, overly long residual limb, scar ti- are smooth, elastic and painless. ssue, tightening scars and areas of skin alteration. The residual limb in the upper third of the tibia, More often than not, the same patient had more thanks to synostosis and enlargement of the re- than one malformation. sidual limb area, enables good contact with the In almost all cases of osteoplastic surgery, muscle prosthesis. grafts were used to cover the bones and good In no case had the radiographical graft displace- functioning of the residual limb. No serious comment or non-unionization been observed. plications were noted with their use. Postopera- After 4 weeks, the ultrasound picture was cha- tive hematomas, which occurred in some cases, racterized by a decrease in diastasis and crevice indicated the need for more active drainage. depth. An increase in echo positive inclusions Recovery was achieved in all cases of reconstruc- with their transformation into linear echo positive stump interventions. Individual weighted tive structures oriented longitudinally along the approach, reasonable combination of skin, mus- stump axis was observed in the fissure structure. cular, bone plasty, careful attitude to muscles, In some observations, along with the change of their fixation to fillets prevents the formation of the junction structure, "bone bridges" emana- high standing truncated muscles, protrusion of ting from the bone fragment in the form of a thin bone fillet under the skin, excess of soft tissues hyperechogenic line were determined. under bone fillet. Careful smoothing and cleaning After 8 weeks the ultrasound picture was con- of the irregularities of the bone filing and the for- tinuous throughout, in some cases there was an mation of a bone block of the tibia bones help to uneven hyperechogenic fusion line with the pre- prevent possible diseases and malformations of sence of acoustic shadow, i.e. there was a com- the residual limbs. plete fusion of the graft with the bone. All patients are fitted with lightweight prosthe- Tensometric tests carried out show significantly sis constructions of the half-length prosthesis greater support and uniformity of the end-pie- type. These prostheses are aesthetically plea- ce pressure on the socket of the residual limb sing, lightweight and easy to use. They allow for following bone grafting of the synostosis. All pa- a maximum contact between residual limb and tients used the prosthesis for 12-14 hours per day. prosthetic socket as well as the end-face pressure. The blood circulation in the residual limb DISCUSSION is good. There were no trophic disorders. They The restoration of a functionally sound skin on the exclude the presence of chronic distal residual residual limb is of great importance. Skin graf- limb oedema. Atrophy of the thigh muscles is ting also plays an important role in muscular and insignificant. Rotation in the knee joint is free. bone graft reconstruction. Its use in reamputations Almost all patients use a prosthesis. allows bone length to be maintained, which has When assessing the results of amputations in a positive effect on the functionality of the resi- terms of prosthetics, the varying degree of suppor- dual limb in prosthetic reconstructions. The skin tability at different levels must be emphasized. transferred to the defect area is homogenous with Stumps above the ankles, due to the connection the surrounding skin, it retains natural blood cir-

484 Shevchuk et al. Reamputation stumps below knee

culation and innervation. The most important role periosteal deposits was detected. With good fixa- is played by the subcutaneous fat included in the tion, graft-bone fusion occurs as a primary bone contour flap, because tissues can be easily moved fusion within 6-12 weeks. and deep defects can be compensated. The skin Earlier studies (5) found that in bone grafting with with subcutaneous fat must be incised at the same bone synostosis, the durability and degree of loa- level within the healthy tissue and separated from ding of the residual limb was more than four times the fascia with one flap. If there is tension, skin higher than without it. The latter is particularly im- incisions should be made without damaging the ti- portant because functional loading creates elastic ssue. The closure of tibial residual limb defects by deformations in the bones, causing a hydrodyna- moving a skin-subcutaneous flap from the anterior mic effect that contributes to normal intraosseous surface of the knee joint and thigh is particularly microcirculation and hence bone trophism. effective if preparation is done to train skin mobi- The performed morphological studies testify that lity and extensibility. the processes of physiological reorganization with There is currently no doubt about the usefulne- a full balance of osteogenesis and osteoresorption ss of muscle grafting in tibia reamputation (16). proceeded in the bone tissue of the residual limb Fixation of the muscles to the bone allows for the ends. The tibial bone block even 6-9 years after covering of the fillets, the crest of the bone and amputation is represented by a compact structure the tight closure of the medullary canal by tight of mature bone tissue, which confirms full functi- stitching of the fixed muscles. The rational com- onal compliance of the residual limb. bination of stabilising the residual fibula with the The use of methods of non-free bone grafting, long fibula muscle and restoring the anterior and myoplasty and free bone grafting allows to obta- posterior intermuscular septa and creating a layer in elastic muscle stumps with the possibility of covering the crest of the tibia with fixation of the long-term complete functioning. calf muscle to it enables a resilient muscle resi- In conclusion, non-free skin grafting with a der- dual limb to be created. Fixation of the muscles mal-subcutaneous flap is the method of choice to the bone during bone grafting of the residual for closing skin defects on the residual limb. limb fulfils the same role (4). During subsequent Fixation of muscles to bone allows to eliminate residual limb formation, the end-stem muscles some residual limb defects and to form an ela- are converted into a dense, organ-shaped fibro- stic muscular residual limb with closed bone fi- us tissue that serves as a firm gasket between laments. Synostosis formation in different ways the bone and the residual limb. The muscles on significantly improves the functional quality of the lateral surfaces of the residual limb undergo the residual limb. atrophy, the extent of which depends on the use of the prosthesis. FUNDING When evaluating bone grafting techniques, it sho- The study was conducted within the framework uld be noted that the technique is not complicated. of the research work ''Revealing regularities in The wide variety of options allows the surgeon the formation of post-amputation pain syndro- to choose the most appropriate one for the parti- me", funded by the Ministry of Health of Ukraine cular case. The reparative process generally pro- with the state budget (No 0120U101372). ceeds smoothly. Closure of the medullary cavity creates the conditions for a normal course of the TRANSPARENCY DECLARATION reparative process. No additional bone formati- Conflicts of interest: None to declare. on along the lateral bone surfaces in the form of

REFERENCES 1. Ahmed A, Bhatnagar S, Mishra S, Khurana D, Jos- 2. Ahuja V, Thapa D, Ghai B. Strategies for prevention hi S, Ahmad SM. Prevalence of phantom limb pain, of lower limb post-amputationpain: A clinical narra- stumppain, and phantom limb sensation among the tive review. J Anaesthesiol Clin Pharmacol 2018; amputated cancer patients in India: a prospective, 34:439-49. observational study. Indian J Palliat Care 2017; 23:24-35.

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3. Allami M, Faraji E, Mohammadzadeh F, Soroush 9. Dumanian GA, Potter BK, Mioton LM, Ko JH, MR. Chronic musculoskeletalpain, phantom sensa- Cheesborough JE, Souza JM, Ertl WJ, Tintle SM, tion, phantom andstumppainin veterans with uni- Nanos GP, Valerio IL, Kuiken TA, Apkarian AV, lateral below-kneeamputation. Scand JPain 2019; Porter K, Jordan SW. Targeted muscle reinnervation 19:779-87. treats neuroma and phantompainin major limb am- 4. Bezsmertnyi YO, Shevchuk VI, Bezsmertna HV, putees: a randomized clinical trial. Ann Surg 2019; Shevchuk SV. Krovoobrashhenie v kul'te kosti pri 270:238-46. razlichnyh sposobah amputacionnoj plastiki (Circu- 10. Gilmore C, Ilfeld B, Rosenow J, Sean Li, Desai M, lation in stump of bone at various methods of ampu- Hunter C, Rauck R, Kapural L, Nader A, Mak J, tation plastics) [in Russian] Likarsʹka Sprava 2018; Cohen S, CrosbyND, Boggs JW. Percutaneous pe- 7-8:141-8. ripheral nerve stimulation for the treatment of chro- 5. Bezsmertnyi YO, ShevchukVI, Grushko OV, Tymchyk nic neuropathic postamputation pain: a multicenter, SV, Bezsmertna HV, Dzierżak R, Dassibekov K. In- randomized, placebo-controlled trial. Reg Anesth formation model for the evaluation of the efficiency PainMed 2019; 44:637-45. of osteoplasty performing in case of amputations on 11. Grubor P, Milicevic S, Grubor M, Meccariello L. below knee. Proc. SPIE 10808, Photonics Applicati- Treatment of bone defects in war wounds: retrospec- ons in Astronomy, Communications, Industry, and tive study. Med Arch 2015; 69:260-4 High-Energy Physics Experiments 2018, 108083H 12. Rollo G, Falzarano G, Ronga M, Bisaccia M, Grubor https://www.spiedigitallibrary.org/conference-pro- P, Erasmo R, Rocca G, Tomé-Bermejo F, Gómez- ceedings-of-spie/10808/108083H/Information-mo- Garrido D, Pichierri P, Rinonapoli G, Meccariello del-for-the-evaluation-of-the-efficiency-of-osteopla- L. Challenges in the management of floating knee sty/10.1117/12.2501558.full?SSO=1 (1 October 2018) injuries: results of treatment and outcomes of 224 6. Bosanquet DC, Glasbey JC, Stimpson A, Williams consecutive cases in 10 years. Injury 2019; 50(Suppl IM, Twina CP. Systematic review and meta-analysis 4):S30-S38. of the efficacy of perineural local anaesthetic cathe- 13. Tosun B, Selek O, Gok U, Tosun O. Medial ga- ters after major lower limb amputation. Eur J Vasc strocnemius muscle flap for the reconstruction of Endovasc Surg 2015; 50:241-9. unhealed amputation stumps. J Wound Care 2017; 7. Buch NS, Qerama E, Brix Finnerup N, Nikolajsen 26:504-7. L. Neuromas and postamputation pain. Pain 2020; 14. Plucknette BF, Krueger CA, Rivera JC, Wenke JC. 161:147-55. Combat-related bridge synostosis versus traditional 8. Buchheit T, Hsia HJ, Cooter M, Shortell C, Kent M, transtibial amputation: comparison of military-spe- McDuffie M, Shaw A, Buckenmaier CT, Van de Ven cific outcomes. Strategies Trauma Limb Reconstr T. The impact of surgical amputation and valproic 2016; 11:5-11. acid on pain and functional trajectory: results from 15. Taylor BC, Poka A. Osteomyoplastic Transtibial the veterans integrated pain evaluation research Amputation: The Ertl technique. J Am Acad Orthop (VIPER) randomized, double-blinded placebo-con- Surg 2016; 24:259-65. trolled trial. Pain Med 2019; 20:2004-17. 16. Weiss M. Amputacja fiziologiczna (Physiological amputation) [in Polish] Warszawa: PZWL, 1972.

486 ORIGINAL ARTICLE

Relapses of traumatic peroneal tendons subluxation already treated surgically: a new surgical approach

Alessandro Tomarchio1, Luigi Meccariello2, Dariush Ghargozloo3, Andrea Pasquino4, Enrico Leonardi1

1Orthopaedic and Trauma Unit, Department of Surgery, "S. Croce e Carle" Hospital, Cuneo, 2Department of Orthopaedics and Trauma- tology, “AORN San Pio”, Benevento,3Department of Orthopaedics and Traumatology, “Esine Hospital”, Valcamonica (Bs), 4Department of Orthopaedics and Traumatology, “Vito Fazzi” Hospital, Lecce; Italy

ABSTRACT

Aim To illustrate the surgical treatment of relapses of traumatic peroneal tendons subluxation.

Methods We came across a young woman, who sustained a sprain in her dominant ankle after a trauma; we noticed subluxation of the peroneal tendons during eversion and extension of the foot. She referred to a previous accident some years before with peroneal tendon subluxation treated by superior peroneal retinaculum (SPR) sutures with a synthetic braided absorbable material. We prescribed conventional radiography, magnetic resonance imaging (MRI) and performed surgery: we removed scar tissue, reattached the retinaculum using suture anchors strengthening it with an acel- Corresponding author: lular dermal matrix allograft patch. Alessandro Tomarchio Results Periodic clinical follow-ups until 24 months were perfor- Orthopaedic and Trauma Unit, med evaluating the stability of the ankle, checking the range of Department of Surgery, movement, and the Visual Analogic Scale (VAS) and American "S. Croce e Carle" Hospital Orthopedic Foot and Ankle Society Score (AOFAS) was admi- Via M. Coppino 26, Cuneo 12100, Italy nistered. At the first check the subluxation was resolved and the Phone: +39 3206014935; ankle was stable. The VAS scale had the value of 0 at the 3-month follow-up maintained until the final check. Fax: +39 0171642208; E-mail: [email protected] Conclusion Relapsing traumatic peroneal tendons subluxation is ORCID ID: https://orcid.org/0000-0002- rare, as well as the possibility of a re-intervention years later. This 6894-3924 technique seems to guarantee an excellent result even in the long term, allowing resolution of pain and joint stability. In fact, the use of acellular dermal patch is an already commonly described technique for the augmentation in rotator cuff and hip capsular repair; no reports are available in literature in relation to the use of graft Original submission: for the repair of the superior peroneal retinaculum. 02 February 2020; Revised submission: Key words: ankle sprains, allografts, suture anchors, suture techniques, tendon injuries 29 March 2021; Accepted: 12 April 2021 doi: 10.17392/1354-21

Med Glas (Zenica) 2021; 18(2):487-492

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INTRODUCTION plantar flexion to a position of eversion and dorsiflexion (10). The diagnosis is essentially clinical. Instability of the peroneal tendon complex is a fa- Further radiologic studies that corroborate the di- irly uncommon pathology, mostly involving young agnosis are dynamic ultrasound and MRI. Radio- athletes. The pathology consists of an acute or chro- graphs could show an avulsed fragment from the nic condition of subluxation or luxation of the pe- fibular insertion of the SPR, but are normally not roneal tendons, often imputable to a rupture of the needed in chronic cases (11). Tendoscopy may be superior peroneal retinaculum (SPR), a thin fibrous beneficial, and should be reserved, in absence of structure the binds the tendons and keeps them loca- positive findings in ultrasound and MRI imaging ted on the postero-lateral aspect of the fibula (1). (12). The most well-known and shared classificati- Peroneus longus (PL) and Peroneus brevis run on on of SPR lesion has been purposed by Oden (13). the postero-lateral shape of the fibula and curve A well-timed diagnosis and a prompt treatment in anteriorly towards their insertions on the malleo- the acute/early stage is important as it is useful to lus, passing through a fibrocartilaginous groove. avoid the long-term sequelae as chronic tendino- They share a common sheath proximally, while pathy or tendon tears (the PB is more often invol- distally they own their proper. At the level of the ved). In addition, acute injuries have a better hea- SPR, PB runs anteriorly and slightly medial to ling tendency, and while it is assessed that chronic the PL, and its mio-tendinous junction is usually lesions of the SPR need surgical treatment, in acute more distal than that of the PL. (2) settings it is advisable to attempt a conservative The most studied mechanisms that lead to acute treatment even if it has a recurrence of tendon in- peroneal dislocation are spontaneous powerful stability rate of about 50% (7,14). Since in chronic reflex contractions of peroneal muscles, while the injuries a shortening of the tendon is often obser- foot is set in dorsiflexion or a forced dorsiflexion ved, the first-line treatment includes a deepening of in the everted foot (1). the peroneal groove, allowing for a greater stability Predisposing factors for dislocation are a flattened and a theoretically inferior risk of recurrence. The or convex shape of the peroneal groove, a conge- aim of the intervention is to prevent further perone- nital ligamentous laxity, an overcrowding of the al dislocation, repairing the SPR or correcting the fibular groove (3-5). Ankle sprains are common predisposing factors increasing the volume or con- events, especially in young athletes (1). The injury tinence of peroneal tunnel. Many techniques have mechanism, sometimes common to those descri- been described to fix the SPR, including periostal bed above, can provoke the disruption of the SPR, flap retinaculplasty (15,16), open or tendoscopic beyond other lesions to peroneal tendons complex simple or anchor suture repair (17-19), grafting (6). While lesions of the tibio-tarsal ligament com- (20), transposition (21,22). Although first surgical plex are usually well recognised and diagnosed, an attempt failure is not rare and already described acute peroneal tendon instability is often misdia- eventuality, we found just one study in literature gnosed at the onset leading to chronic pathologic facing up the issue, purposing an antero-medial re- conditions (7). The disruption of the SPR may be routing of the PB tendon (23). associated with a small avulsion fracture at the The aim of this study was to describe a new sur- attachment of the retinaculum to the fibula, or to gical procedure in case of relapses of traumatic the rupture of the fibrocartilage ridge of the gro- peroneal tendons subluxation already treated ove (8). In acute settings patients often describe a surgically with a SPR suture. This occurrence is “pop” or snapping sensation, followed by tender- quite rare and not described in the literature. In ness and swelling on the posterior profile of late- particular we have introduced the use of graft for ral malleolus (1,9). If associated ankle ligament or the repair of the superior peroneal retinaculum. osseous lesions are present, the patient could be unable to weight bearing. Physical examination PATIENTS AND METHODS reveals variable pain and swelling depending on the acuteness of the injury, functional impotence in Patient and study design active evertion. Dislocation or subluxation may be We came across a 39-year old woman, who was observed through manoeuvres of ankle rotation or referred in the outpatient clinic of Orthopaedic forcing the foot from a position of inversion and and Trauma Unit of “S. Croce e Carle" Hospital

488 Tomarchio et al. Peroneal tendons subluxation recurrence

(Cuneo, Italy) in January 2019. She presented with pain in the right dominant ankle after a sprain one week before. She reported "shooting sensation" during different ankle movements as well as severe pain that made walking impossible. No fractures history was referred but a previous accident sixteen years before after a fall with peroneal tendon subluxation. At that time she was surgically treated by superior peroneal retinaculum (SPR) Figure 2. Physical examination: subluxation of the peroneal tendons during eversion (right) and extension (left) of the foot © sutures with a Panacryl and immobilized with and their reduction during the opposite movement (Tomarchio cast for 30 days. After this treatment she referred A, 2019) a good functional outcome, the absence of pain and subluxation symptoms; a complete return to work after about two months and sport activities like skiing and running six months later. Physical examination showed swelling and ecchymosis around the ankle joint especially on lateral side. Also significant tenderness on lateral malleolus was noted on palpation of the ankle joint. Neurovascular function of the foot and ankle was normal. On passive dynamic evaluation we noticed the subluxation of the peroneal tendons during eversion and extension of the foot (Figure 1). Conventional radiography, including standing lateral, dorsoplantar, anteroposterior and oblique views of the ankle, did not show fractures, bone Figure 3. A) Superior peroneal retinaculum (SPR) longitudinal loss or avulsion; MRI showed the injury to the lesion; B) After removing synovial-like scar tissue, during the SPR and the presence of greatly altered tissue placement of the Arthrex mini corkscrew; C) Suture of SPR; D) Acellular dermal matrix allograft patch positioning; E) First (Figure 2), probably as a sign of previous surgery. modelling and start of suture, then finished and modelled (To- marchio A, 2019)

Methods A surgical treatment was performed twelve days after the trauma in spinal anaesthesia under tour- niquet control: the patient was placed supine with a bolster under the ipsilateral buttock. A slightly curvilinear skin incision was made, extending from about 4 cm proximal to 1 cm beyond the tip of the lateral malleolus along the posterolateral edge of the fibula and along the course of the peroneal tendons (almost on the previous surgical wound). After removing synovial-like scar tissue and after doing a meticulous subcutaneous preparation, the SPR and peroneal tendon sheaths were explored. The SPR complete longitudinal injury was visu-

Figure 1. Magnetic resonance (MRI) right dominant ankle: alized and the peroneal tendons were dislocated evidence of previous suture scar tissue in sagittal (A, B) and running passive dynamic evaluation in eversion coronal (C) planes; in axial plane (D) the peroneus longus dis- and extension of the foot. The tendons returned to location can be observed (1), peroneus brevis (2) and previous the site performing the opposite movements. After superior peroneal retinaculum remnant (3) (Tomarchio A, 2019) reducing the tendons the repair was easily perfor-

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med; sutured the SPR using suture anchors (Ar- Table 1. Results of clinical follow-ups: Visual Analogic Scale threx Mini Corkscrew FT 2.7 mm x 7 mm) to its (VAS) and American Orthopedic Foot and Ankle Society Ankle-Hindfoot Score (AOFAS) Scale, ankle stability fibular insertion reinforcing it and then sutured the Peroneal tendons Follow up time VAS score AOFAS score two heads of the SPR (knots tied on lateral edge subluxation of trough and same suture passed through anterior 1 month 2 68 NO portion of SPR). Finally it was strengthened with 3 months 1 83 NO an acellular dermal matrix allograft patch sutured 6 months 0 86 NO 12 months 0 89 NO between SPR and subcutaneous tissue. Immedia- 24 months 0 90 NO tely after surgery a short leg cast with the foot in The patient returned to office work 2 months neutral to slight inversion was applied holding the after surgery. We decided not to perform X-ray, ankle in 90°, maintained for 4 weeks. The patient CT scan or MRI during the follow up. Overall, was kept non-weight-bearing for 4 weeks and then the result of surgery was considered excellent as started to wean out the patient from boots with confirmed by the degree of satisfaction reported indication for the dressing after 7 days and the by the patient. No adherence of the tendons and removal of the stitches after 15 days. At the remo- recurrence of the subluxation were observed until val of the short leg cast, structural physical the- the final follow-up. rapy program was started, focusing on the range of motion followed by proprioceptive training and DISCUSSION strengthening exercises. We performed periodic clinical follow-ups at one month post-operatively, Dislocating or subluxing peroneal tendons is a as well as 3, 6, 12 and 24 months. relatively infrequent injury, even rarer being fa- ced with relapses of dislocation after years from A range of movement, the stability of the ankle was the first episode treated surgically (1). Clinical evaluated using the Visual Analogic Scale (VAS) assessment, X-ray and MRI help to elaborate the that is a psychometric response scale consisting of correct diagnosis (5,11) a 100-mm horizontal line on which the patient’s pain intensity is represented by a point between Surgical treatment attempts to restore structural the extremes of “no pain at all” and “worst pain stabilization of the peroneal tendon and retina- imaginable (24) and the American Orthopedic cular complex (26). The surgical techniques Foot and Ankle Society Ankle-Hindfoot Score vary and depend largely on the surgeon’s cli- (AOFAS) that is among the most commonly used nical experience and preference, even if there instruments for measuring the outcome of tre- is no experience described in literature. In our atment in patients who sustained a complex ankle case before repairing the SPR, we had removed or hindfoot injury. It combines a clinician-reported synovial-like scar tissue, probably due to the use and a patient-reported part (25). of specific type of synthetic braided absorbable material (Panacryl). This is considered as a long- RESULTS term suture; it is a slow degradable suture with high concentration of polylactide acid. In inter- The surgical scar was perfectly healed and the national literature the question has been asked stitches were removed fifteen days after the sur- about the possible synovitis determined by this gery. At the first check the subluxation was resol- material but no scientific evidence has ever been ved and the ankle stable; subluxation of peroneal highlighted (27,28). We have introduced the use tendons was no longer caused by active or passi- of the grafting. There are many classes of bio- ve movement of the ankle and foot. Similar re- logical matrices currently available, including sults were obtained by administering the AOFAS dermal allografts, dermal xenografts, resorbable scale. After one month, immediately after the cast and nonresorbable fabrics, and numerous other removal, VAS was 2 and AOFAS 68. During the collagen and synthetic products (29 ). Our choi- follow-up, according to the progress of physical ce, considering previous clinical patient’s history therapy, we observed a progressive improvement was the use of an acellular dermal matrix allograft of the AOFAS score and decrease in pain. The patch. This is an acellular cryopreserved human improvements observed at the follow-up in the dermal graft prepared by removing the epidermis third month were maintained until the final check and all cellular components: the letter is compo- at 24 months (Vas score: 0; AOFAS 90) (Table 1).

490 Tomarchio et al. Peroneal tendons subluxation recurrence

sed of several types of collagen, chondroitin sul- of any kind of graft for the repair of the superior fate, elastin, proteoglycans composing a matrix peroneal retinaculum. suitable for an early revascularization and with The surgical technique described in this article a high load resistance (30,31). In fact, the use of is largely successfully used with a high satisfac- acellular dermal patch is an already commonly tion rate. described technique for the augmentation in rotator cuff repair (32-34) and hip capsular repa- FUNDING ir (35). Many studies indicate that it improves No specific funding was received for this study. tendon healing and clinical outcomes compared with repair without graft (30). No reports or data TRANSPARENCY DECLARATION are available in literature in relation to the use Competing interests: None to declare.

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26. Espinosa N, Maurer MA. Peroneal tendon dislocati- 32. Cai Y-Z, Zhang C, Jin R-L, Shen T, Gu P-C, Lin X-J, on. Eur J Trauma Emerg Surg 2015; 41:631-7. De Chen J. Arthroscopic rotator cuff repair with graft 27. Clavert P, Warner JJ. Panacryl synovitis: fact or ficti- augmentation of 3- dimensional biological collagen on? Arthroscopy 2005; 21:200-3. for moderate to large tears: a randomized controlled 28. Burger C, Kabir K, Rangger C, Mueller M, Minor T, study. Am J Sports Medicine 2018; 46:1424–31. Tolba RH. Polylactide (LTS) causes less inflammati- 33. Derwin KA, Badylak SF, Steinmann SP, Iannotti JP. on response than polydioxanone (PDS): a meniscus Extracellular matrix scaffold devices for rotator cuff repair model in sheep. Arch Orthop Trauma Surg repair. J Shoulder Elb Surg 2010; 19:467–76. 2006;126: 695-705. 34. Chalmers PN, Tashjian RZ. Patch augmentation in ro- 29. Chen FM, Liu X. Advancing biomaterials of human tator cuff repair. Curr Rev Musculoskelet Med 2020; origin for tissue engineering. Prog Polym Sci 2016; 13:561-71. 53:86-168. 35. Jacobsen S, Guth JJ, Schimoler PJ, Kharlamov A, 30. Jones CR, Snyder SJ. Massive irreparable rotator cuff Giordano BD, Miller MC, Christoforetti JJ. Bio- tears: a solution that bridges the gap. Sports Med Ar- mechanical response to distraction of hip capsular re- throsc Rev 2015; 23:130-8. construction with human acellular dermal patch graft. 31. Coons DA, Alan Barber F. Tendon graft substitutes- Arthroscopy 2020; 36:1337-42. rotator cuff patches. Sports Med Arthrosc Rev 2006; 14:185–190.

492 ORIGINAL ARTICLE

Factors related to anxiety among resident doctors assigned to emergency room during the COVID-19 pandemic: a multivariate study at Sumatera Utara Affiliated Teaching Hospital

Elmeida Effendy, Ariwan Selian, Julius Martin Siagian

1Department of Psychiatry, Faculty of Medicine, Universitas Sumatera Utara, Indonesia

ABSTRACT

Aim To evaluate and assess knowledge and perception, as well as factors related to the occurrence of anxiety among frontliners, especially resident doctors working in emergency room (ER).

Methods This multivariate study was conducted with cross-sectional approach involving 80 eligible subjects (based on inclusion and exclusion criteria) that are consecutively assigned and assessed with GAD-7 questionnaire. The study was held in ER of Uni- versitas Sumatera Utara affiliated teaching hospital from May to Corresponding author: August 2020. Elmeida Effendy Department of Psychiatry, Results Our study found that variables such as nuptial status (p=0.032), seniority level (p=0.037), history of direct exposu- Faculty of Medicine, re to COVID-19 patients (p=0.001) and weekly work duration Universitas Sumatera Utara (p=0.002) were all statistically significant to correlate with the Jalan Dr. Mansyur No.66, occurrence of anxiety among resident doctors assigned to work Medan, 20155, Indonesia in ER. Phone: +62 8211638; Conclusion Acknowledgement of these factors might lead to pro- E-mail: [email protected] per and targeted support system strategies to address the anxiety ORCID ID https://orcid.org/ 0000-0002- issues among doctors, particularly those who work in ER during 9382-3609 COVID-19 pandemic.

Key words: anxiety, mental health, physicians

Original submission: 02 March 2021; Revised submission: 19 April 2021; Accepted: 29 June 2021 doi: 10.17392/1365-21

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INTRODUCTION Therefore, doctors, nurses, paramedics and other health care workers are actually the real Severe Acute Respiratory Syndrome-Corona Vi- fighters and heros to combat this pandemic. rus 2 (SARS-Cov-2) has spread worldwide and Thus, mental and physical health of these pe- escalated to become world’s pandemic as anno- ople should have been a concern to ensure unced by the WHO on 30 January 2020. CO- that they are able to carry their responsibility VID-19 is known to affect respiratory system, appropriately. Unfortunately, there are only and is transmitted from man to man rapidly. The few studies particularly in developing countri- infection can be asymptomatic and may involve es to investigate and assess factors related to multiple organs that intensifies its complexity. psychological problems that may affect health The spread of this virus for the first time was care workers. Acknowledgement of these fac- in Tiongkok, China in December 2019 and has tors may lead to proper and targeted support spread to countries in Europe, America, and Asia system strategies to address the anxiety issues ever since finally on 11 March 2020, the WHO among doctors, particularly those who work in declared COVID-19 as a global pandemic (1-3). emergency room (ER) during the COVID-19 This pandemic leads into intense fear in soci- pandemic. Therefore, we believe that our study ety, which may affect mental health. Front liners, is necessarily important to address this issue. including doctors, nurses, and paramedics are even The aim of this study was to evaluate and asse- more vulnerable of getting infected as they are ss knowledge and perception, as well as factors exposed directly to COVID-19 infection. Insuffi- related to the occurrence of anxiety among front cient health care system, social isolation, uncer- liners, especially resident doctors working in tainty (as a number of developed countries, even emergency room. those with prominent and great health care system failed to survive and ended up in collapsing) are EXAMINEES AND METHODS possible factors related to the occurrence of mental issues among health care workers. To make matters Population and study design worse, illogical and misuse of personal protective equipment (PPE) by society results in even grea- This multivariate study was conducted with ter anxiety among health care workers as they are cross-sectional approach involving 80 eligible worried of not having enough equipment (4). doctors (based on inclusion and exclusion criteria) that are consecutively assigned and asse- A previous study also reported that junior resident ssed with Generalized Anxiety Disorder 7-item doctors, trainees, and interns are the ones that get (GAD-7) questionnaire (after informed consent even heavier pressure because most of immedia- is given). The study was conducted in the ER of te or emergency cases are put on them (5). This Universitas Sumatera Utara (USU) affiliated te- causes not only physical burden because they have aching hospital from May to August 2020. Inclu- to work overtime, but also result in psychological sion criteria were resident doctors who were 25 problems. Issues related to confirmation of - CO – 40 years of age, cooperative, and were able to VID-19, difficult ethical decisions for the - pati understand the Indonesian language. Exclusion ents, and uncertainty in terms of working rotation, criteria were any resident doctors with psychia- contribute to even greater risk of mental problem try disorders or any comorbidity. In this context, among those health care workers (5). Stress reac- comorbidity was defined as any medical history tion such as anxiety, depression, somatization as that may expose the subjects to a greater risk of well as hostility have been reported in 10% of he- experiencing anxiety. Subjects with psychosis alth care workers during this pandemic. A study were already initially excluded from the study. form Taiwan reported that hospital staff experien- In addition, work duration was measured as to- ced acute stress (5%), stigma (20%), worked re- tal hours spent on duty for a week. luctancy (9%) and even considered resigning (2). During the pandemic in China, depression, anxi- Methods ety and stress were found to reach 50.7%, 44.7% The GAD-7 (6) was used to assess anxiety level and 73.4%, respectively (2). as well as other types of anxiety, including panic

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disorder, social anxiety, and post-traumatic stress statistically not significant until the statistically disorder. It exerts reliability and adequate inter- fittest model (fit model) was found (8). nal consistence, as well as good criteria, factorial, and procedural validity (7). RESULTS The determination of the minimum sample Median age of 80 eligible doctors was 31 (26- size was based on a preliminary study, and the 38) years. The variables such as resident's nup- number of subjects was also in accordance with tial status (p<0.001), seniority level of resident the minimum sample size and the appropriate (p=0.037), history of direct contact with CO- analysis test was carried out (analysis validity). VID-19 sufferers (p<0.001) and the duration of This study also managed to get 80 research su- work week (p=0.02) were related to the GAD-7 bjects (external validity 1b), therefore, at least score of residents serving in ER. Therefore, these the results of this study could be generalized to variables deserve to be considered as a factor that the target population. Restriction for confoun- can cause anxiety disorders in residents serving ding variables were also applied so that internal in ER (Table 1). validity could be fulfilled. A total of 42 (52.5%) doctors were male resident; 48 (60%) were already married. Most were Statistical analysis found to live along with family, 42 (52.5%), Linear regression model was used in this study and admitted not to have any comorbidity, 61 after ensuring that each acquirement of using li- (76.3%). Junior residents were represented with near regression model was achieved. As for the 42 (52.5%); 54 (67.5%) had no family member independent variable, it had no multicollinearity infected with COVID-19. More than a half, 41 (as proved with Pearson correlation and tolerance (51.2%), showed no sign and symptoms rela- test). Both independent and dependent variables ted to COVID-19 and 42 (52.5%) admitted not showed linearity (as proved with scatter graph). having any direct contact with COVID-19 pati- Categorical independent variables were analyzed ents (Table 1). by using descriptive statistics and normality test. Table 1. Demographic characteristics of study participants Bivariate analysis using t test independent and Variable No (%) Mann Whitney U test were also conducted and Gender Pearson test was used for numerical independent Male 42 (52.5) variables. Multivariate analysis was also carried Female 38 (47.5) Nuptial status out for both variables. Kolmogorov-Smirnov was Married 48 (60) carried out to test the normality of data distributi- Not married 32 (40) on in both dependent and independent variables, Living with Alone 38 (47.5) and Pearson test was used if the distribution of With Family 42 (52.5) the data was found to be abnormal. Variables that Physical comorbidity were allowed to be analyzed with linear regressi- Present 19 (23.8) Not Present 61 (76.3) on model were those with p<0.25 (8). History of family infected with COVID-19 The total of 11 independent variables in our Present 26 (32.5) study consisted of 8 continuous and 3 catego- Not Present 54 (67.5) Residency Level rical variables, hence we used linear regressi- Senior 38 (47.5) on to conduct the multivariate analysis. Prior to Junior 42 (52.5) multivariate analysis, we conducted bivariate COVID-19 related sign or symptoms Present 39 (48.8) analysis only variables with p<0.25 that wo- Not Present 41 (51.2) uld be eligible for further analysis with linear Median sleep duration (hours) / (min-max) 38 (47.5) regression model. When multivariate regression Median work duration/week (hours) / (min-max) 42 (52.5) linear analysis was applied with a predictive conceptual framework, it was suggested to use Maximum and minimum age of residents was backward method, which means that the SPSS 38 and 26 years old, respectively. Multivariate program would filter the multicollinearity value analysis for age was not significant (p> 0.05) of independent variable and the ones that were (Table 2).

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Table 2. Bivariate analysis of categorical independent variables Table 4. Multivariate analysis summary Number of Correlation Regression Variable Mean±SD Median p Variable p participants coefficients multivariate β Gender Constant 4.51 0.032 Male 42 6.76 ± 3.77 Married compared to not 0.016 – 0.259 -2.31 <0.001 Female 38 9.13 ± 473 married Nuptial Status Junior resident compared to 0.154 1.35 0.037 Married 48 9 (3-19) senior resident <0.001 Not married 32 5 (2-17) History of direct contact with Living With COVID-19 patient compared 0.489 0.29 <0.001 Alone 38 6.16 ± 3.28 to no history of contact <0.001 With family 42 9.45 ± 4.71 Resident’s duration of work – 0.251 -0.22 0.002 Comorbidity per week YES 19 11.05 ± 4.62 2 <0.001 Adjusted R 71.4 % NO 61 6.90 ± 3.85 History of family infected with COVID-19 YES 26 12 (2-19) appeared especially stressful for younger adults <0.001 NO 54 6.5 (2-14) (<35 years) (9). It is known that getting older, the Residency level risk for anxiety is getting smaller (10). Senior 38 5.39 ± 2.90 <0.001 Junior 42 10.14 ± 4.31 In terms of living with family members, the result COVID-19 related signs and symptoms of this study contradicted with the study by Liu et YES 39 12 (2-19) <0.001 NO 41 6 (2-13) al. showing that there was a relationship between History of direct contact with COVID-19 patients anxiety symptoms reported by the subject themse- YES 38 11 (5-19) <0.001 lves and the status of not living alone (11). In addi- NO 42 2 (2-12) tion, resident gender was found different from a In this study, median sleep duration was 4 hours Pakistani study suggesting that female residents (min. of 4 hours, max. of 8 hours) and median were at more risk of experiencing symptoms of work duration was 36 hours (min. of 16 hours depression, anxiety and acute stress (5), which is and max. of 68 hours) (Table 3). also in contrast to the US study in which females experienced more stress (12). Table 3. Bivariate analysis of numerical independent variables Family history of COVID-19 infection in our according to Generalized Anxiety Disorder 7-item (GAD-7) scale study is in accordance with a study conducted in Variable Value r p China using the self-rating anxiety scale, self-ra- Age (years) 31 (26-38) 0.182 0.106 Median sleep duration per day ting depression scale and Pittsburgh sleep quality 4 (4-8) -0.363 0.001 (hours) (min-max) index measuring instruments: for 307 COVID-19 Median working duration/week 36 (6-15) 0.747 < 0.001 patients, in the multivariate analysis there was no (hours) (min-max) relationship between family history of confirmed r, correlation coefficient COVID-19 and anxiety scores (13). By using backward analysis method, GAD-7 The results of COVID-19 related symptoms score of 4.51 –2.31 was found for married vs analysis in our study were in contrast to a study not married, +1.35 for junior vs senior residents conducted by Wathelet et al. which suggested that +0.29 for working duration per week, –0.22 for those who had symptoms similar to COVID-19 history of direct contact. Our study confirmed reported mental health complaints (14). In addi- that all variables had a significant correlation tion, a study by Badahdah et al. showed about a with GAD-7 score (p<0.05) (Table 4). third of healthcare workers caring for COVID-19 patients who were hospitalized experienced mo- DISCUSSION derate to severe symptoms of anxiety caused by poor sleep quality (15). The results of multivariate analysis for age of our doctors were found not to be significantly related In this study, only comorbidities had the result to GAD 7 scores. This result is different from the of multivariate analysis (not significant). It was study conducted by Pieh et al., in which 19% of different from a study by Galindo-Vázquez et al. patients who experienced symptoms of anxiety in Spain, which found that history of comorbiditi- and lock-down during the COVID-19 pandemic es had a relationship with anxiety and depression

496 Effendy et al. Anxiety during pandemic

scores. Patients who had two physical symptoms senior staff reported more symptoms of depre- were more likely to experience symptoms of de- ssion, anxiety and acute stress (4). pression and/or anxiety related to the severity of The results of multivariate analysis for the dura- the disease (16). tion of work per week showed that there was a Resident nuptial status and history of direct con- significant relationship for the duration of work tact with COVID-19 patients showed significant per week with an anxiety score, which is in results, which is consistent with a study conduc- accordance with Giusti et al. study that showed ted by Liu et al., which stated that nuptial status that the working hours, which caused burn out to was associated with symptoms of anxiety related 330 health professionals on duty, resulted in sco- to family burdens including responsibility and re state anxiety that became significantly above finance (11). In addition, Kannampallil et al. in the cut-off (17). Furthermore, a study conducted the United States reported that residents who had by Amin et al. in Pakistan also found that su- direct contact with COVID-19 patient reported bjects who even worked 20 hours /week or less significantly higher level of stress and were more were exposed to an increased risk of anxiety and likely to burn out (12). The fear of being unpro- depression levels in physicians (4). tected contributes to the anxiety score of those Our study suggests that nuptial status, seniority le- who work in the front lines and direct contact vel, history of direct contact, and duration of work with COVID-19 patients, which is even worse per week are contributing factors of anxiety among when PPE is insufficient (5). resident doctors working in emergency room. Du- Seniors were represented with 47.5% and juniors ring COVID-19 pandemic similar studies may be with 52.5% in this study, and resulted in a rela- abundant, but vast majority of studies are focusing tionship between the seniority level variable and only on the patients. Meanwhile, our study put the GAD-7 score (very weak correlation stren- healthcare workers as the focus of the study. This gth and positive direction). In our study, being study is currently the first one in Medan that used junior residents showed a positive correlation GAD 7 as an instrument. By knowing contributing to higher GAD 7 scores, which means that risk factors for anxiety, preventive strategies can junior residents were more at risk of experi- be taken as early as possible. encing symptoms of anxiety. In this study, the high level of anxiety among junior residents may FUNDING be caused by lack of knowledge and experience No specific funding was received for this study. when dealing with patients, as well as new working environment that they have to adjust to. In TRANSPARENCY DECLARATIONS contrast to our results, in the Pakistani study Competing interest: None to declare.

REFERENCES

1. Dhahri AA, Arain SY, Memon AM, Rao A, Mian 5. Imran N, Masood HMU, Ayub M, Gondal KM. MA. The psychological impact of COVID-19 on Psychological Impact Of COVID-19 pandemic on medical education of final year students in Pakistan: postgraduate trainees: a cross-sectional survey. BMJ a cross-sectional study. Ann Med Surg 2020; 60: 2020; Online ahead of print. 445-450. 6. Johnson SU, Ulvenes PG, Oktedalen T, Hoffart 2. Elbay RY, Kurtulmus A, Arpacioglu S, Karadere E. A. Psychometric properties of the general anxiety Depression, anxiety, stress levels of physicians and disorder 7-item (GAD-7) scale in a heterogenous associated factors in COVID-19. Elsevier 2020; psychiatric sample. Front Psychol 2019; 10:1713. 1781:31203-8. 7. Spitzer RL, Kroenke K, Williams JB, Lowe B. A 3. Conti C, Fontanesi L, Lanzara R, Rosa I, Porcelli P. brief measure for assessing generalized anxiety dis- Fragile heroes, the psychological impact of the CO- order: the GAD-7. Arch Intern Med 2006; 166:1092- VID-19 pandemic on health-care workers in Italy. 7. Plos One 2020; 15:e0242538. 8. Sastroasmoro S, Ismael S. Dasar-Dasar Metodologi 4. Amin F, Sharif S, Saeed R, Durrani N, Jilani D. CO- Penelitian Klinis (Fundamentals of Clinical Rese- VID-19 pandemic-knowledge, perception, anxiety arch Methodology) [Indonesian]. Jakarta: Sagung and depression among frontline doctors of Pakistan. Seto, 2016. BMC Psychiatry 2020; 20:459. 9. Pieh C, Budimir S, Probst T. The effect of age, gender, income, work, and physical activity on mental

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health during coronavirus disease (COVID-19) 14. Wathelet M, Duhem S, Vaiva G, Baubet T, Habran E, lockdown in Austria. J Psychosom Res 2020; Veerapa E, Debien C, Molenda S, Horn M, Grandge- 136:110186 1-9. nevre P, Notredame CE, D’Hondt F. Factors associa- 10. Faravelli C, Alessandra Scarpato M., Castellini ted with mental health disorders among university G, Lo Sauro C. Gender differences in depression students in France confined during the COVID-19 and anxiety: the role of age. Psychiatry Res 2013; pandemic. JAMA Network Open 2020; 3:1-13. 210:1301–3. 15. Badahdah AM, Khamis F, Al Mahyijari N. Sleep 11. Liu Y, Chen H, Zhang N, Wang X, Fan Q, Zhang Y. quality among health care workers during the CO- Anxiety and depression symptoms of medical staff VID-19 pandemic. J Clin Sleep Med 2020; 16:1635. under COVID-19 epidemic in China. J Affect Disord 16. Galindo-Vázquez O, Ramirez-Orozco M, Costas- 2020; 278:144-8. Muniz R, Mendoza-Contreras LA, Calderillo-Ruiz 12. Kannampallil TG, Goss CW, Evanoff BA, Strickland G, Meneses-Garcia A. Symptoms of anxiety, depre- JR, McAlister RP, Duncan J. Exposure to COVID-19 ssion and self-care behaviors during the COVID-19 patients increases physician trainee stress and bur- pandemic in the general population. Gac Mad Mex nout. PLoS One 2020; 15:1-12. 2020; 156:298-305. 13. Dai LL, Jiang TC, Li PF, Wang Y, Wu SJ, Jia LQ, 17. Giusti EM, Pedroli E, D’Aniello GE, Badiale CS, Pi- Liu M, An L, Cheng Z. Anxiety and depressive etrabissa G, Manna C, Badiale MS, Riva G, Castel- symptoms among COVID-19 patients in Jianghan nuovo G, Molinari E. The psychological impact of Fangcang Shelter Hospital in Wuhan, China. PLoS the COVID-19 outbreak on health professionals: a One 2020; 15:1-11. cross-sectional study. Front Psychol 2020; 11:1684.

498 ORIGINAL ARTICLE

Knowledge, attitudes and practices during the second wave of COVID-19 outbreak: a cross-sectional study from various perspectives

Armin Šljivo1, Sutanay Bhattacharyya2, Ahmed Mulać1, Arian Abdulkhaliq3, Rexhep Sahatçiu4

1School of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 2Medicine Safdarjung Hospital, New Delhi, India, 3School of Medicine, Iuliu Hatieganu University of Medicine and Pharmacy Cluj-Napoca, Cluj-Napoca, Romania, 4School of Medicine, University of Pristina, Pristina, Kosovo

ABSTRACT

Aim To investigate knowledge, attitudes and practice towards CO- VID-19 among selected population.

Methods An anonymous online questionnaire based on a Chine- se study was distributed via online social media platforms among general population of Bosnia and Herzegovina, Germany, India, Kosovo and Romania.

Results In total 1032 subjects, predominately females, 615 (59.6%) with a mean age of 31.23±12.94 years, single, 705 (68.3%), with high school degree or lower, 469 (45.4%), students, 528 (51.1%) Corresponding author: and living in an urban environment, 824 (79.8%), have completed the survey. The median knowledge score was 10.0 (range 0-12). Armin Šljivo Being male (β: -0.437; p=0.003) and older (β: -0.028; p<0.001) School of Medicine, were associated with lower knowledge scores, while being sin- University of Sarajevo gle (β: 1.026; p<0.001) and mental labour employee (β: 0.402; Čekaluša 90, Sarajevo, p=0.032) were associated with higher knowledge scores. The vast Bosnia and Herzegovina majority of subjects had not visited crowded places, 630 (61.0%) Phone: +387 33 226 478; and wearing masks when they were going out, 928 (89.9%). Be- ing female (OR=0.731; p=0.022), having higher knowledge sco- +387 33 203 670; res (OR=0.929; p=0.017) and being a mental labour employee E-mail: [email protected] (OR=0.713; p=0.031) decreased the exposure to crowded places. ORCID ID: https://orcid.org/0000-0003- High school or lower education level (OR=0.616; p=0.024) decre- 2865-0446 ased the action of wearing a mask in public places, while higher knowledge scores (OR=1.112; p=0.013) increased it.

Conclusion Our study suggests that residents of the selected regions have had good knowledge, pessimistic attitudes and relatively Original submission: appropriate practices towards COVID-19 during the second wave 17 May 2021; of the outbreak. Revised submission: Key words: attitude, COVID-19, epidemiology, knowledge, pu- 28 May 2021; blic health Accepted: 06 June 2021 doi: 10.17392/1378-21

Med Glas (Zenica) 2021; 18(2):499-504

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INTRODUCTION evaluate the public awareness about spreading, symptomatology, treatment and outcome of in- Coronavirus disease 2019 (COVID-19) is an infection with coronavirus. By evaluating the pu- fectious disease caused by the highly contagious blic knowledge, attitude and practices towards novel severe acute respiratory syndrome corona- COVID-19, patterns of responsive behaviour virus 2 (Novel SARS-CoV-2) (1). Even though and applying healthy practices may be studied. this infection may be asymptomatic, the disease Moreover, lack of information and maximizing usually presents with mild symptoms such as on-going prevention measures are possible (8,9). fever, dry cough, fatigue, myalgia, shortness of breath and loss of sense of smell and taste, but Individual studies have been done in various it can also progress to lethal forms with severe populations, and in different geographical areas pneumonia, acute respiratory distress syndrome including Bosnia and Herzegovina (B&H) (9- and even fatality (2). This emerging respiratory 11). This multicentre research was conducted in infection that was first discovered in December order to gather not only local (B&H) but global 2019 in Wuhan city has infected more than 25 COVID-19 KAP data from various population 602 665 patients and resulted in more than 852 and to compare them. 758 deaths as of 2 September 2020 (3). During The aim of this study was to investigate knowled- COVID-19 pandemic, government responses ge, attitudes and practice towards COVID-19 varied from doing little to nothing, laissez-faire among selected population from Bosnia and Her- strategy, to more aggressive measures which li- zegovina, Germany, India, Kosovo and Romania. mited even population’s liberty (4). PARTICIPANTS AND METHODS In the examined region of Bosnia and Herzego- vina, Germany, India, Kosovo and Romania from Participants and study design the beginning of the pandemic to 2nd September 2020 there have been 4 194 095 reported cases This observational cross-sectional study conduc- with death ratio ranging from 2.0% to 4.1%: Bo- ted from 15th July to 2nd September 2020 was done snia and Herzegovina 20 234 (3.0%), Germany in the form of an online questionnaire-based sur- 246 808 (4.0%), India 3 823 449 (2.0%), Kosovo vey in order to respect the norms of social distan- 13 713 (3.9%) and Romania 89 891 (4.1%) (5). cing and lockdown in various areas in the study Pandemic spread of COVID-19 is an undefined setting. Subjects across Bosnia and Herzegovina, medical challenge and unprecedented measures Germany, India, Kosovo and Romania were pro- have been made worldwide. Being a novel infec- vided with a KAP questionnaire adapted from a

tious agent, healthcare professionals are constantly similar Chinese study (9) via e-mail, WhatsApp, challenged in order to apply the most efficient pre- Facebook and other social networking media. vention measures, treatment schemes and avoid the The questionnaire informed the subjects about long-term complication of the disease. Knowled- the objectives of the study, their voluntary and ge, attitude and practices studies (KAP) find their anonymous participation, including online infor- unique importance in the selected topic (6). med consent and details of how to fill up the questionnaire. Exclusion criteria were being younger KAP surveys usually apply in the first step of a than 18 years, not being a resident of one of the co- clinical trial or research in order to collect data untries and not completing the questionnaire. The about a chosen topic from the general population. study was approved by the Ethics Committee of The investigation may be developed at any point the University of Sarajevo and all procedures were during control activities, but it proves its maxi- followed in accordance with the Helsinki Declara- mum utility and efficiency in the early phases of tion and subsequent amendments. a novel project. Therefore, the data obtained from a KAP survey are useful to orientate the resource Methods allocation, to develop the project design itself or The questionnaire consisted of two parts. The to obtain baseline information which will be used first part assessed demographic characteristics for comparison with post-interventional data (7). of subjects such as gender, age, marital status, KAP research is a powerful tool in order to education level, current occupational status and

500 Šljivo et al. KAP during the second wave of COVID-19

living environment. The second part, KAP asse- expect in Kosovo, (106; 59.5%) where the ma- ssment, consisted of 16 questions, divided in 3 jority held a bachelor’s degree, 528 (51.1%) sections: knowledge test, attitudes towards CO- were students, and 824 (79.8%) lived in an ur- VID-19 and practices towards COVID-19. ban environment (Table 1). Knowledge test had 12 questions with each question having as response ‘True’, ‘False’ and ‘I Table 1. Demographic characteristics of the subjects presented in five countries don’t know’. The questions primarily were re- No (%) of participants in the country garding the main symptoms of COVID-19, the Variable B&H India Romania Kosovo Germany mode of transmission, treatment and prevention (n=132) (n=150) (n=265) (n=178) (n=307) 103 57 161 113 181 principles of the disease. Correct answers were Female (78.0) (38.0) (60.7) (63.5) (58.9) given 1 point, while incorrect and unknown res- Gender 29 93 104 65 126 Male ponses were assigned 0 points. A total of 12 score (22.0) (62.0) (39.3) (36.5) (41.1) was attributed to the knowledge test of KAP with 128 24 191 138 175 18-29 higher scores denoting better understanding of (96.9) (16.0) (72.1) (77.5) (57.0) 3 49 58 35 106 Age 30-49 the disease by the subject. (2.3) (32.7) (21.9) (19.6) (34.5) 1 77 16 5 26 Attitude aspect towards COVID-19 was asse- 50+ (0.8) (51.3) (6.0) (2.9%) (8.5) ssed by 2 components, whether COVID-19 can 120 24 221 133 207 Single be ultimately controlled or not; and whether they Marital (90.9) (16.0) (83.4) (74.7) (67.4) status 12 126 44 45 100 have the belief that their respective countries can Married achieve this goal. (9.1) (84.0) (16.6) (25.3) (32.6) High 55 87 94 34 199 school or The subject’s practice in preventing disease tran- (41.7) (58.0) (35.5) (19.1) (64.8) smission was assessed by whether they had gone lower Bachelor’s 44 16 92 106 67 Educati- to a crowded place recently and whether they had degree (33.4) (10.7) (34.7) (59.5) (21.8) on level worn masks while going out. Master’s 25 47 62 36 38 degree (18.9) (31.3) (23.4) (20.2) (12.3) PhD 8 17 2 3 Statistical analysis 0 degree (6.0) (6.4) (1.2) (1.1) Categorical variables were presented in frequ- 103 27 157 106 135 Student encies and percentages, while numerical va- (78.0) (18.0) (59.2) (59.5) (43.9) Mental 20 54 73 25 94 riables by arithmetic mean±standard deviation Occupa- labour (15.1) (36.0) (27.5) (14.0) (30.6) (SD) for normally distributed data, or by median tion Physical 2 53 17 14 48 (25th; 75th quartile) for not normally distributed labour (1.5) (35.3) (6.4) (5.2) (15.6) Unem- 7 16 18 33 30 data. Binary logistic regression was performed ployed (5.4) (10.7) (6.9) (21.3) (9.9) to assess predictors of knowledge test scores, 118 123 232 142 209 Living Urban (89.4) (72.0) (87.5) (79.8) (66.1) attitudes and practices towards COVID-19. Ho- envi- 14 27 33 36 98 ronment Rural smer-Lameshow goodness of fit test of binary (10.6) (18.0) (12.5) (20.2) (33.9) logistic regression models were not significant B&H, Bosnia and Herzegovina; (p>0.05) indicating good fit of the models, while the Nagelkerke R2 variation showed effect The correct answer rates on questions 1 to 12 size regarding our models. of the COVID-19 knowledge test were between 60.6% and 91.8%: 89.8%, 61.1%, 75.9%, 76.4%, RESULTS 60.6%, 77.1%, 87.1%, 64.9%, 82.9%, 86.8%, 88.1% and 91.8%, respectively, with a median A total of 1069 subjects have completed the score of 10.0 (8.0, 11.0). Overall knowledge test survey. After excluding 37 subjects because of scores varied across different countries ranging the exclusion criteria, the final sample consi- from lowest knowledge test scores in India of 7.0 sted of 1032 subjects. The dominant characte- (6.0, 8.0), to the highest test scores in Romania ristics of the sample were: 615 (59.6%) were of 11.0 (10.0, 11.0) (Table 2). females except India where the majority were males (93; 62.0%), a mean age of 31.23±12.94 A multiple linear regression model showed that years, 705 (68.3%) single, 469 (45.4%) had being male (β: -0.437; p=0.003) and older age high school degree or lower education level group (˃30) (β: -0.028; p<0.001) were associ-

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Table 2. Knowledge test, attitudes and practices question- knowledge scores (OR=1.112; p=0.013) increased naire results in five countries it. The model showed no statistical significance B&H India Romania Kosovo Germany Parameter (p=0.290); it explained 3% (Nagelkerke R2) and N=132 N=150 N=265 N=178 N=307 correctly classified 89.7% of cases (Table 3). Knowledge test results 10.0 7.0 11.0 10.0 10.0 Table 3. Multiple linear regression model in association (median, 25th, (8.5, 11.0) (6.0, 8.0) (10.0, 11.0) (9.0, 11.0) (9.0, 11.0) 75th percentile) with knowledge test scores and independent predictors determined by logistic regression model in association with Answer No (%) of participants per country various practices towards COVID-19 A1: Agreeing that COVID-19 will finally be contained 47 60 158 88 122 K: Multiple linear regression model in association with knowled- Yes (35.6) (40.0) (59.6) (49.4) (39.7) ge test scores 39 74 36 35 93 Variable β Coefficient t p No (29.5) (49.3) (13.6) (19.7) (30.3) Gender (male vs female) -0.437 -3.002 0.003 46 16 71 55 92 Age (>30 vs younger) -0.028 -3.602 <0.001 I don’t know (34.9) (10.7) (26.8) (30.9) (30.0) Education (Master’s degree and 0.331 0.091 A2: Agreeing that the country will win the fight above vs other) 1.690 against COVID-19 Marital status (single vs married) 1.026 5.188 <0.001 70 94 179 108 265 Employment (mental labour vs Yes 0.402 2.149 0.032 (53.0) (62.7) (67.5) (60.7) (86.3) other) 62 56 86 70 42 Variable OR 95% CI p No (47.0) (37.3) (32.5) (39.3) (13.7) P1: Independent predictors in association with visiting crowded P1: Visiting crowded places places 42 70 107 60 123 Gender (female vs male) 0.731 0.559-0.955 0.022 Yes (31.8) (46.7) (40.4) (33.7) (40.1) Knowledge score (high vs low) 0.929 0.875-0.987 0.017 90 80 158 118 184 Employment (mental labour vs No 0.713 0.525-0.969 0.031 (68.2) (53.3) (59.6) (66.3) (59.9) other) P2: Wearing face masks outside home P2: Independent predictors in association with wearing face 121 136 250 171 250 masks in public places Yes (91.7) (90.7) (94.3) (96.1) (81.4) Education (high school and lower 0.616 0.405-0.937 0.024 11 14 15 57 vs other) No 7 (3.9) (8.3) (9.3) (5.7) (18.6) Knowledge scores (high vs low) 1.112 1.023-1.210 0.013 B&H, Bosnia and Herzegovina; t, t-test; OR, Odds ratio; CI, Confidence interval P1: The model was not statistically significant (p=0.301); it explained ated with lower knowledge scores, while being 2.4% (Nagelkerke R2) and correctly classified 62.2% of cases P2: The model was not statistically significant (p=0.290); it explained single (β: 1.026; p<0.001) and being employed 3% (Nagelkerke R2) and correctly classified 89.7% cases; in mental labour sector (β: 0.402; p=0.032) were associated with higher knowledge test scores. Regarding attitudes, 716 (69.3%) of the subjects DISCUSSION agreed that their country would win the fight ver- Our results showed that most of the respondents sus COVID-19 and only 475 (46.0%) agreed that were aware of the existence of COVID-19 with the virus would be finally contained (Table 3). No knowledge questions correct rates varying betwe- independent predictors were determined for any en 60.6% and 90.8%, similarly with studies from of the attitudes included in the questionnaire. other regions of the world (9,12) and with studi- The vast majority of the participants had not vi- es relating to other epidemics (13,14). Variations sited any crowded place, 630 (61.0%), and wore between correct rates of knowledge questions masks when they were going out, 928 (89.9%) in regarding the novel coronavirus are explained by recent days. Binary logistic model showed that be- the variability of the population included in the ing female (OR=0.731; p=0.022), having higher study. Our study sample was mostly represented knowledge scores (OR=0.929; p=0.017) and being with females (except in India where the majo- a mental worker (OR=0.713; p=0.031) were asso- rity were males) and had a high school degree or ciated with decreased exposure to crowded pla- lower (except in Kosovo where the majority held ces. The model showed no statistical significance a bachelor’s degree). Furthermore, not all subjects (p=0.301); it explained 2.4% (Nagelkerke R2) and had the same access to new platforms, Internet, te- correctly classified 62.2% of cases (Table 3). levision and other mass media, which could lead High school or lower level of education to lack of knowledge regarding COVID-19. (OR=0.616; p=0.024) decreased the action of Knowledge test results varied through the popu- wearing a mask in public places, while higher lation studied by geographical area, which could

502 Šljivo et al. KAP during the second wave of COVID-19

be a consequence, the same one as for knowled- Our study had several limitations. Firstly, we per- ge correct rates. We identified age and marital formed a cross sectional questionnaire-based stu- status as strong predictors for higher knowled- dy and were unable to assess whether the practi- ge test scores, as well as the gender, which was ces and attitudes of the study population changed also significantly associated with the grade of over time as the disease spread subsequently. Our information about the COVID-19. These results study was also limited by the fact that we were are supported by other research that demonstra- unable to reach out those patients who did not ted that females and older age are more aware have internet access and thus could not participate and informed about infectious diseases (13,15). in the study. Apart from the restricted sample re- Moreover, data from the literature described an presentativeness the lack of a standardized KAP association between knowledge, high-income questionnaire for COVID awareness was another and education (16). Therefore, it is essential to limitation of the study. A population based stan- target risk groups represented by young people, dardized KAP questionnaire for COVID-19 needs low-educated, low-income, and males. to be made and implemented on a larger scale with Concerning infectious diseases and their spre- easy access and understanding. Also, rural and ad, other studies showed that higher knowledge often neglected sections of the society need to be scores are associated with positive attitudes and attended. Further, such studies are recommended lower risk of dangerous practices (17,18). to look into the KAP of COVID-19 especially, in low socioeconomic and low-income vulnerable Regarding attitudes, our subjects mostly agreed population groups. The KAP studies find their gre- that their country would win the fight versus CO- at utility in the development of policy strategies VID-19 and less than half agreed that the virus and healthcare programmes (21). would be finally contained. Our results showed a more pessimistic attitude regarding the potenti- The results of our study showed that males were al control of the virus when compared with Sau- associated with lower knowledge scores, while di Arabia (94%) and China (90.8%) (9,12). One older aged, being single and being employed in possible explanation of the phenomenon may be mental labour sector are associated with a higher that the previously published studies analysed data knowledge test score. The results are similar to after a short evolution of the spread of the disease; the data obtained in other research regarding in- moreover, these results showed a higher level of fection with SARS or MERS, showing that ma- confidence for the specified country control over les used significantly lower preventive measures the disease, 97.0 % for Saudi Arabia and 97.1% than females (22,23). Regarding COVID-19 for China (9,12). The two countries took unprece- pandemic, older age males are considered at risk ded measures for protecting the population, with population and vulnerable in front of the disease. extended lockdowns, multiple restrictions for in- Other authors suggest that an efficient way of tar- dividuals and conducted intensive awareness cam- geting this segment of the population is through paigns through news channels, which may explain females from their community or families (22). the differences between our results and their res- The particularity of presented work consists of ponses (9). Another important factor that may in- heterogeneity of the population by choosing fluence subjects’ attitudes is different psychologi- countries with different profiles, and its unicity cal types of the subjects: it has been demonstrated is given by the chosen time of evaluation, after that during a pandemic or a natural disaster, peo- approximately one year from the appearance of ple tend to control their negative emotion less and the pandemic, leading to valuable information express anxiety that may affect their attitude (19). about the evolution of perception from the gene- The level of knowledge and the attitude dictate ral population toward the chosen topic. the practice of a population concerning a selected In conclusion, our results imply that the health theme (20). A proportion of 61% of the subjects education may be more efficient if addressed included in our study did not visit any crowded to certain subgroups of population, taking into place and 89.9% of the people wore masks while consideration the evaluated parameters. In par- going out indicating a relatively appropriate level ticular, these targeted programs may be develo- of adherence to preventive practices. ped for people with lower level of education, for

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males or for older age groups in order to obtain FUNDING maximum results and better knowledge, positive No specific funding was received for this study. attitudes and improved practices towards CO- VID-19 pandemic. TRANSPARENCY DECLARATION Conflicts of interest: None to declare. REFERENCES 1. Amawi H, Deiab GIA, Aljabali AAA, Dua K, Tam- 11. Ferdous MZ, Islam MS, Sikder MT, Mosaddek ASM, buwala MM. COVID-19 pandemic: an overview of Valdivia JAZ, Gozal D. Knowledge, attitude, and epidemiology, pathogenesis, diagnostics and potential practice regarding COVID-19 outbreak in Banglade- vaccines and therapeutics. Ther Deliv 2020; 11:245– sh: An online-based cross-sectional study. PLoS One 68. 2020; 15:e0239254. 2. Uddin M, Mustafa F, Rizvi TA, Loney T, Suwaidi HA, 12. Al-Hanawi MK, Angawi K, Alshareef N, Qattan Al-Marzouqi AHH, Eldin AK, Alsabeeha N, Adrian AMN, Helmy HZ, Abudawood Y, Alqurashi M, TE, Stefanini C, Nowotny N, Alsheikh-Ali A, Senok Kattan WM, Kadasah NA, Chirwa GC, Alsharqi O. AC. SARS-CoV-2/COVID-19: Viral genomics, epi- Knowledge, attitude and practice toward COVID-19 demiology, vaccines, and therapeutic interventions. among the public in the Kingdom of Saudi Arabia: Viruses 2020; 12:526. a cross-sectional study. Front Public Health 2020; 3. World Health Organization. WHO Coronavirus Dise- 8:217. ase (COVID-19) Dashboard. https://covid19.who.int/ 13. Al-Mohrej OA, Al-Shirian SD, Al-Otaibi SK, Tamim (13 October 2020) HM, Masuadi EM, Fakhoury HM. Is the Saudi public 4. Desvars-Larrive A, Dervic E, Haug N, Niederkroten- aware of Middle East respiratory syndrome? J Infect thaler T, Chen J, Di Natale A, Lasser J, Gliga D, Roux Public Health 2016; 9:259–66. A, Sorger J, Chakraborty A, Ten A, Dervic A, Pacheco 14. Aldowyan N, Abdallah AS, El-Gharabawy R. A, Jurczak A, Cserjan D, Lederhilger D, Bulska D, Knowledge, Attitude and Practice (KAP) Study Berishaj D, Tames E, Álvarez F, Takriti H, Korbel J, about Middle East respiratory syndrome coronavirus Reddish J, Grzymała-Moszczyńska J, Stangl J, Had- (MERS-CoV) among population in Saudi Arabia. Int ziavdic L, Stoeger L, Gooriah L, Geyrhofer L, Ferreira Arch Med 2017; 10. M, Bartoszek M, Vierlinger R, Holder S, Haberfellner 15. Bawazir A, Al-Mazroo E, Jradi H, Ahmed A, Badri S, Ahne V, Reisch V, Servedio V, Chen X, Pocasan- M. MERS-CoV infection: Mind the public knowled- gre-Orellana X, Garncarek Z, Garcia D, Thurner S. A ge gap. J Infect Public Health 2018; 11:89–93. structured open dataset of government interventions in 16. Beier ME, Ackerman PL. Determinants of health response to COVID-19. Sci Dana 2020; 7. knowledge: an investigation of age, gender, abilities, 5. Worldometer. Coronavirus Cases. https://www.worl- personality, and interests. J Pers Soc Psychol 2003; dometers.info/coronavirus/ (13 October 2020) 84:439–48. 6. Zhou P, Yang X, Wang X, Hu B, Zhang L, Zhang W, 17. Xiu Z. Analysis on mental health status of community Si H, Zhu Y, Li B, Huang C, Chen H, Chen J, Luo Y, residents in Hefei during SARS spread. Chin J Dis Guo H, Jiang R, Liu M, Chen Y, Shen X, Wang X, Control Prev 2003; 7:280–2. Zheng X, Zhao K, Chen Q, Deng F, Liu L, Yan B, 18. Jiao J, Tang X, Li H, Chen J, Xiao Y, Li A. Survey Zhan F, Wang Y, Xiao G. Shi Z. A pneumonia outbre- of knowledge of villagers in prevention and control ak associated with a new coronavirus of probable bat of SARS in Hainan Province. Chin Trop Med 2005; origin. Nature 2020; 579:270–3. 5:703–5. 7. Taber KS. The use of Cronbach’s alpha when deve- 19. Blendon RJ, Benson JM, Desroches CM, Raleigh loping and reporting research instruments in science E, Taylor‐Clark K. The public’s response to severe education. Res Sci Educ 2017; 48:1273–96. acute respiratory syndrome in Toronto and the United 8. Nooh HZ, Alshammary RH, Alenezy JM, Alrowaili States. Clin Infect Dis 2004; 38:925–31. NH, Alsharari AJ, Alenzi NM, Sabaa HE. Public awa- 20.. Centers for Disease Control and Prevention. Corona- reness of coronavirus in Al-Jouf region, Saudi Arabia. virus Disease 2019 (COVID-19). https://www.cdc. Z Gesundh Wiss 2020;1-8. gov/coronavirus/2019-ncov/index.html (13 October 9. Zhong B-L, Luo W, Li H-M, Zhang Q-Q, Liu X-G, 2020) Li W-T, Li Y. Knowledge, attitudes, and practices 21. Johnson EJ, Hariharan S. Public health awareness towards COVID-19 among Chinese residents during knowledge, attitude and behavior of the general pu- the rapid rise period of the COVID-19 outbreak: a qu- blic on health risks during the H1N1 influenza pande- ick online cross-sectional survey. Int J Biol Sci 2020; mic. J Public Health 2017; 25:333-37. 16:1745–52. 22. Leung GM, Lam T, Ho L, Ho S, Chan B, Wong I, 10. Šljivo A, Kačamaković M, Siručić I, Mujičić E, Hedley AJ. The impact of community psychological Džubur Kulenović A. Knowledge, attitudes, and prac- responses on outbreak control for severe acute respi- tices towards COVID-19 among residents of Bosnia ratory syndrome in Hong Kong. J Epidemiol Commu- and Herzegovina during the first stage of COVID-19 nity Health 2003; 57:857–63. outbreak. Ann Ig 2021; 33:371-380. 23. Moran KR, Valle SYD. A meta-analysis of the Asso- ciation between Gender and Protective Behaviors in Response to Respiratory Epidemics and Pandemics. Plos One 2016; 11.

504 ORIGINAL ARTICLE

A measurement of irradiance of light-curing units in dental offices in three Croatian cities

Ante Dundić1, Valentina Rajić Brzović2, Gloria Vlajnić3, Danijela Kalibović Govorko4,5, Ivana Medvedec Mikić5,6

1Medical Care Facility A2 Dental, Trogir, 2Department of Endodontics and Restorative Stomatology, School of Dental Medicine, Univer- sity of Zagreb, 3Private Dental Clinic, Zapresic, 4Department of Orthodontics, Study Programme of Dental Medicine, School of Medicine, University of Split, 5University Hospital of Split, 6Department of Endodontics and Restorative Dental Medicine, Study Programme of Dental Medicine, School of Medicine, University of Split; Split, Croatia

ABSTRACT

Aim To determine irradiance of light-curing units (LCUs) in dental offices in three Croatian cities and to compare irradiance values with the age and model of LCUs.

Methods Private and public dental offices in three most prominent cities in Croatia (Rijeka, Split and Zagreb) were included in this study. In total, 195 LCUs were tested, using radiometer Ivoclar Bluephase Meter 2 for irradiance (mW/cm2). The minimum acceptable value was set at 400 mW/cm2. The age, model and difference between declared and measured irradiance of the LCUs were also determined. Of the total of 195 LCUs, 190 (98%) were LED Corresponding author: (light-emitting diode) and 5 (2%) were QTH (quartz-tungsten – Ivana Medvedec Mikić halogen). School of Medicine, Study Programme of Dental Medicine, Results The mean age of tested LCUs was 4.43±3.4 years; the oldest was in Rijeka, 5.2±3.8 years. The overall mean irradiance University of Split for all three cities was 806.4 mW/cm2 (p=0.0004). Of all LCUs, Šoltanska 2, 21000 Split 11.3% were considered clinically unacceptable with irradiance of Phone: +385 21 557 846; less than 400 mW/cm2. Of all tested LCUs 42% (p=0.0005) had a E-mail: [email protected] 30% lower value of irradiance than the manufacturer of the LCU Ante Dundić ORCID ID: https://orcid. declared. In 73% tested LCUs, there was a matching between me- org/0000-0002-1439-0711 asured and declared irradiance. The age and model of LCUs had the most significant impact on irradiance.

Conclusion The most commonly used LCU included in dental offices was LED. Mean irradiance was good enough to secure adequate polymerization of resin-based materials. Irradiance de- Original submission: creases with usage time of LCU. 01 December 2020; Key words: composite dental resin, dental curing lights, polyme- Revised submission: risation 12 February 2021; Accepted: 10 March 2021 doi: 10.17392/1323-21

Med Glas (Zenica) 2021; 18(2):505-509

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INTRODUCTION gon ion lasers are quite useful in polymerizing RBCs, but small light size (spot size) prolongs Light-activated resin-based composites (RBC) the curing cycle of RBCs. In order to enhance the are the most commonly used restorative mate- clinical success of composite resin restorations, rials because of their easy handling, excellent dental manufacturers have focused on the deve- aesthetics and good physical and mechanical lopment of new LCUs (11). properties (1). They are used in everyday clinical practice not only as restorative materials but An appropriate intensity of light with the maxi- also as liners or as luting agents for cementation mum absorption wavelength of photoinitiators is of inlays, onlays, crowns, veneers and orthodon- the main factor in the polymerization of RBCs tic brackets (2). An adequate polymerization of (12). According to Lopes et al. (13), two decades RBCs is essential for the ultimate success of re- ago, irradiance of 300 mW/cm 2 is low and sho- storations (3). The quality of the energy delivered uld be compensated by curing time of 60 s for a to RBC restorations can be affected by many fac- 2-mm increment. With new curing units, usually 2 tors such as the exposure duration of light from 1000 mW/cm , we would not suggest this curing light-curing units (LCUs), intensity of the light time because of the possibility of burning soft ti- output, wavelength range, the distance between ssue and pulp. the surface of the restoration and the curing tip, The reduction of light intensity of the LCUs can operators technique, beam profile, internal/exter- reduce the success rate of the restorative treatment nal diameter, time of exposure (3,4). by reducing the degree of conversion of composi- Incomplete polymerization produces adverse tes (14) with abovementioned consequences. biological effects, increasing water absorpti- As the LCUs usage time, the irradiance diminis- on, composite solubility and reduced hardness, hes due to deterioration of the components (7) which clinically results in discolouration, low re- leading to the reduction of LCUs effectiveness. sistance to wear, marginal breakdown, cytotoxi- There are many models of LCUs in the dental city and increased microleakage (5), irreversible market, and each manufacturer in datasheets pro- pulpal damage and allergic reaction (6). vided by the manufacturer states maximum irra- There are four types of LCUs used by dentists diance for their device. Every model of LCUs has to polymerize RBCs: quartz-tungsten – halo- different characteristics and limitations, and that gen (QTH), light-emitting diode (LED), Plasma is why we wanted to determine the relationship Arc (PAC) and Argon-Lasers units (7). QTH between the model of LCU and irradiance. As to and LED are the most commonly used types of our knowledge a similar study was conducted ten LCUs in dental offices. QTH produces blue- li years ago but only in Zagreb (15). ght in the 400-500 nm region, and the intensity The aim of this study was to assess irradiance of ranges between 400 and 1600 mW/ cm2. Some LCUs used in the dental offices in Split, Rijeka limitations of QTH LCUs are producing a broad- and Zagreb, as well as the differences between spectrum light energy, including the infrared ra- the manufacturer declared and measured irradi- diation range, which is responsible for excessive ance and the relationship between the LCSs’ age heat generation and short service life (bulb, re- and irradiance were assessed. flector or filter degradation) (8). LED - polyme rization units (third-generation) produce blue MATERIALS AND METHODS light between 450 and 470 nm and delivering no Materials and study design light below 420 nm (monowave) and polywave that produce two or more distinct emission ban- A list of public and private dental offices and cli- ds from 440 to 470 nm and another below 420 nics in selected three cities was obtained from the nm. Newer high power LED LCUs light intensity Croatian Dental Chamber registration. A total of approach or go above 3000 mW/ cm2 (9). PAC 65 dental offices in the cities of Rijeka, Split and light sources provide light intensity more than Zagreb (Croatia) were randomly selected. A total 2000 mW/ cm2 and very rapid polymerization, of 195 LCUs in 65 dental offices were tested for but studies showed inadequate polymerization their irradiance in the period between April and resulting with microleaking of RBCs (10). Ar- August 2019. After explaining the methodology

506 Dundić et al. Irradiance of light-curing units

and the principle of the study, an informed con- in Rijeka (713.3 mW/cm2) then in Split (818.8 mW/ sent was obtained from doctors. cm2 ) and Zagreb (880.8 mW/cm2) (p=0.0406). Table 1. Distribution of light curing units (LCU) in dental of- Methods fices in three cities in Croatia No (%) Radiometer Ivoclar Bluephase Meter 2 (Ivoclar City Vivadent, Schaan, Principality of Liechtenstein) LED QTH Rijeka 59 (30.25) 3 (1.53) was used for evaluation of irradiance of LCUs me- Split 66 (33.84) 2 (1.02) asuring the LCUs light intensity of the wavelength Zagreb 65 (33.33) 0 between 385 nm and 515 nm and it was used solely Total 190 (97.45) 5 (2.55) LED, light - emitting diode; QTH, quartz - tungsten – halogen; for the round-peak devices of the light conductor. The same person conducted the reading procedure in Rijeka, Split and Zagreb (for each city there was one person, in total three persons). All investigators were trained in taking sample readings by the same experts. The tip of each LCU was cleaned with an alcohol swab and visually inspected to ensure that no debris was present. This was followed by placing the tip of the LCU in direct contact with Figure 1. Usage time of the measured light- curing units the sensor of the radiometer. (LCUs) in three different cities and in total expressed in years Three measurements were performed on each de- The overall mean irradiance for all three cities was 2 vice (at the beginning, in the middle and at the 806.4 mW/cm . Of 195 LCUs, 21 (11.3%) were end of the curing process), and the average was considered clinically unacceptable with irradian- 2 calculated. The standard program and 20 s curing ce of less than 400 mW/cm . A high number of time was used. Minimally required irradiance LCUs had the 30% lower value of irradiance than was 400 mW/cm2 (16). the manufacturer of the LCU declared (Table 2). The type of LCU, their usage time and declared Table 2. Mean intensity and intensity 30% lower than declared of the tested light curing units (LCUs) values from the manufacturer of the LCU were City also recorded. Declared values were found in unit Intensity Total p Split Rijeka Zagreb instructions online. Mean (mW/cm2) 818.3 718.3 880.8 806.4 0.0402* 30% lower than declared (%) 39.7 50.8 36.9 42.21 0.0005* Statistical analysis *statistically significant The frequency tables were used for presentati- Declared irradiance of LCUs ranged from 500 – on of each categorical variable. The presence/ 3200 mW/cm2 with a mean value of 1200 mW/ absence of a statistically significant difference cm2 in all three cities. Measured irradiance ran- between the three cities was determined using ged from 0 – 2050 mW/cm2 with mean value of the Kruskal-Wallis ANOVA test. For continuous 875.6 mW/cm2. The ratio between declared and variables, the basic statistical parameters were measured irradiance indicated a statistically si- calculated, and the statistical significance of the gnificant difference (p=0.0002) in values betwe- difference was tested using the variance analysis en three cities. The lowest overlap of measured and the Newman-Keuls test. The p<0.05 was and declared irradiance was in Rijeka, than in considered statistically significant. Split, and highest in Zagreb (Figure 2). As for the model of LCUs, the highest irradiance between RESULTS In total, 195 LCUs were tested. Five of them were QTH and 190 were LED LCUs (Table 1). The overall mean usage time of the tested LCUs was 4.43±3.4 years (Figure 1). The oldest LCUs were found in Rijeka (5.2±3.8 years) then in Split ( 4.1±3.4) and Zagreb (4.1±2.9) (p=0.1668). The Figure 2. Difference between declared and measured irradi- mean irradiance of tested LCUs' values was lowest ance of tested light – curing units LCUs

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Table 3. Dependence of mean measured intensity on selected predictor variables Total Rijeka Split Zagreb Variable β p β p β p β p Model of LCU 0.16 0.0131* 0 0.9682 0.03 0.7888 0.16 0.2269 LED/ QTH 0.11 0.1206 0.31 0.0444* 0.2 0.1652 - - Usage time of LCU 0.38 0.0000* 0.46 0.0001* 0.61 0.0000* 0.14 0.2805 *statistical significance; LCU, light - curing unit; LED, light - emitting diode; QTH, quartz-tungsten – halogen; β, beta coefficient of the individual contribution of a single variable to the overall; all measured LCUs of 2050 mW/cm2 had Mini cm2 and in half of them 30% lower than the ma- LED Ortho 2 (Acteon, Mont Laurel, NY), than nufacturer of LCU declared. These results are 1000 mW/cm2 on Elipar Freelight 2 (3M ESPE, in accordance with another study (20), where Germany). However, the largest match between 27.4% of LCUs without minimally required irra- the measured and declared irradiance values of diance were reported. Matosevic et al. (15) repor- 99% was shown by the LCU Bluephase style ted that 44% of tested LCUs had irradiance lower and 95% by the Woodpecker LED.B. than 400 mW/cm2. Results from Split and Zagreb Usage time of the LCUs seems to have the most were similar and in accordance with the results significant impact on irradiance (p=0.0000), es- of an Iranian study (12). In a Brasilian study, six pecially in Rijeka (p=0.0001). Also, the type of out of 22 LCUs delivered inadequate irradiance tested LCU had a statistically significant impact in the posterior region (7). on irradiance in total (p=0.0131) and again in Ri- Every manufacturer of the LCU declares maxi- jeka (p=0.0001) and Split (0.0000) (Table 3). mum irradiance in the manufacturer provided datasheets. In this study, we wanted to assess if DISCUSSION that declared intensity was in accordance with Light-activated RBCs are the available and esthe- the measured one. Declared irradiance of tested tic solution for dental restorative treatments. Ina- LCUs models varied from 800 to 3000 mW/cm2, dequate handling or use of improper equipment which is quite a range of values. A large number such are LCUs can result in unesthetic restorati- of models of LCUs with different characteristics ons, secondary caries and pulpal irritation (17). and maximum irradiance are available in the Our results showed that the most commonly used market which, as the results of this study show, LCUs were LEDs (97.43%), which present a high had an impact on measured irradiance. Similar percentage if we compare it with the results from results were reported by Omidi et al. (12). A com- recent literature. Similar results with 88.5% LED prehensive study conducted by the University of LCUs were reported by Alquira et al. (18). LED Mainz in dental practices in the Rhine-Main area LCUs were least represented in Rijeka (30.25%), in 2005 also showed that many curing lights do Split and Zagreb had similar ones (33.84% and not achieve the specified light irradiance stated 33.33% respectively). This difference could have by the supplier. In extreme cases, they did not resulted from the small sample number since in even achieve half of the stimulating power (21). tested dental offices in Rijeka, a very high num- Our results showed matching between measured ber of LED LCUs was in use. and declared irradiance in 73% of LCUs in total. Mean irradiance in this study was 806.4 mW/ The results in Rijeka showed matching of 55.3%, cm2, which is much higher than in the study of and in Zagreb, it was 75.1%. Al Shaafi (4), and similar to the results reported The effect of the LCUs usage time on irradiance by Alquira (18). Only five LCUs had irradiance was also tested. The mean usage time was four higher than 1000 mW/cm2. Similar results were years, and LCUs in Rijeka were a bit older than reported by Al Shaafi et al. (19) in their study. average (5.2 years). These results follow results of The lowest values were recorded in Rijeka, whe- a few studies (14,22). Our results suggest that the re mean irradiance was acceptable but the lowest age of the LCUs had the most significant influence of all three tested cities. Perhaps the frequency on irradiance considering all other tested parame- of using LCUs in Rijeka is higher than in the ters (the type of the LCU and model). Other studi- other two cities. Also, in one-quarter of the te- es reported similar conclusions (14,22). However, sted LCUs, the irradiance was less than 400 mW/ Javaheri and Ashreghi (20) found no significant

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correlation between clinical age and light inten- composite. The age and model of LCUs have si- sity. Two possible reasons for the difference in gnificant impact on irradiation and thus have im- the results of different studies could be the use of pact on the quality and durability of resin-based different models of LCUs in different studies and composite fillings. different levels of device awareness by participating dentists. Testing and regular servicing are ACKNOWLEDGEMENTS critical for light-curing units to ensure adequate The authors would like to thank professor Ivana irradiance. LCUs should be tested, and their com- Brekalo-Pršo for help in organizing the collecti- ponents should be replaced regularly (4). on of data on light-curing units in Rijeka. In conclusion, the results of this study showed mostly LED LCUs in dental offices. The light in- FUNDING tensity of the tested curing units was lower than No specific funding was received for this study. expected, but still, most of them have satisfactory irradiance that enables adequate and quality TRANSPARENCY DECLARATION polymerization of light-activated resin-based Competing interest: None to declare.

REFERENCES 1. Pratap B, Gupta RK, Bhardwaj B, Nag M. Resin based 12. Omidi BR, Gosili A, Jaber-Ansari M, Mahdkhah restorative dental materials: characteristics and future A. Intensity output and effectiveness of light cu- perspectives. Jpn Dent Sci Rev 2019; 55:126-38. ring units in dental offices. J Clin Exp Dent 2018; 2. Knezevic A, Zeljezic D, Kopjar N, Duarte S Jr, Tarle 10:e555-60. Z. In vitro biocompatibility of preheated giomer and 13. Lopes GC, Vieira LCC, Araujo E. Direct composite microfilled- hybrid composite. Acta Stomatol Croat resin restorations: a review of some clinical procedu- 2018; 52:286-97. res to achive predictable results in posterior teeth. J 3. Mazhari F, Ajami B, Moazzami SM, Baghaee B, Esteth Restor Dent 2004; 16:19-31. Hafez B. Microhardness of composite resin cured 14. Price RBT. Light curing in dentistry. Dent Clin through different primary tooth thicknesses with North Am 2017; 61:751-78. different light intensities and curing times: in vitro 15. Matosevic D, Panduric V, Jankovic B, Knezevic A, study. Eur J Dent 2016; 10:203-9. Klaric E, Tarle Z. Light intensity of curing units in 4. AlShaafi MM. Factors affecting polymerization of dental offices in Zagreb, Croatia. Acta Stomatol Cro- resin-based composites: a literature review. Saudi at 2011; 45:31-40. Dent J 2017; 29:48-58. 16. Madhusudhana K, Swathi TW, Suneelkumar C, La- 5. Oztur B, Cobanoglu N, Cetin AR, Gunduz B. Con- vanya A. A clinical survey of the output intensity of version degrees of resin composite using different light curing units in dental offices across Nellore ur- light sources. Eur J Dent 2013; 7:102-9. ban area. J Res Dent Sci 2016; 7:64-8. 6. Alkhudhairy F, AlKheraif A, Naseem M, Khan R, 17. Ajaj RA, Nassar HM, Hasanain FA. Infection control Vohra F. Degree of conversion and depth of cure of barrier and curing time as factors affecting the irradi- Ivocerin containing photo-polymerized resin luting ance of light-cure units. J Int Soc Prev Community cement in comparison to conventional luting agents. Dent 2018; 8:523-8. Pak J Med Sci 2018; 34:253-9. 18. Alquira T, Al Gady M, MclinDent KA, Ali S. Types 7. Soares CJ, Rodrigues MP, Oliveira LRS, Braga SSL, of polymerization units and their intensity output in Barcelos LM, Silva GRD, Giannini M, Price RB. An private dental clinics of twin cities in eastern pro- evaluation of the light output from 22 contemporary vince, KSA- a pilot study. J Taibah Univ Med Sci light curing units. Braz Dent J 2017; 28:362-71. 2018; 14:47-51. 8. Mahant RH, Chokshi S, Vaidya R, Patel P, Vora A, 19. AlShaafi MM, Harlow JE, Price HL, Rueggeberg Mahant P. Comparison of the amount of temperature FA, Labrie D, AlQahtani MQ, Price RB. Emission rise in the pulp chamber of teeth treated with QTH, characteristics and effect of battery drain in "budget second and third generation LED light curing units: "curing lights. Oper Dent 2016; 41:397-408. an in vitro study. J Lasers Med Sci 2016; 7:184-91. 20. Javaheri M, Ashreghi M. Evaluation of curing light 9. Price RB, Ferracane JL, Shortall AC. Light-curing intensity in private dental offices. J Qazvin Univ units: a review of what we need to know. J Dent Res Med Sci 2009; 12:50–5. 2015; 94:1179-86. 21. Ernst CP, Busemann I, Kern T, Willershausen B. 10. Singh TK, Ataide I, Fernandes M, Lambor RT. Light Feldtest zur lichtemissionsleistung von polymeri- curing devices-a clinical review. J Orofac Res 2011; sationsgeräten in zahnärztlichen praxen. Deutsche 1:5-19. Zahnärztliche Zeitschrift 2006; 61:466-71. 11. Sartori N, Knezevic A, Peruchi LD, Phark JH, Du- 22. Al Shaafi MM, Maawadh AM, Al Qahtani MQ. Eva- arte S Jr. Effects of light attenuation through dental luation of light intensity output of QTH and LED tissues on cure depth of composite resins. Acta Sto- curing devices in various governmental health insti- matol Croat 2019; 53:95-105. tutions. Oper Dent 2011; 36:356-61.

509 ORIGINAL ARTICLE

Assessment of mothers’ satisfaction with health care during childbirth in a tertiary-level maternity ward

Adriana Haller1, Albert Haller1, Dejan Tirić1,2, Vajdana Tomić1-3

1School of Medicine, University of Mostar, 2Department of Obstetrics and Gynaecology, University Clinical Hospital Mostar, 3Faculty of Health Studies, University of Mostar

ABSTRACT

Aim To evaluate satisfaction of mothers who gave birth at term with received hospital care and to find areas for improvement at a tertiary hospital.

Methods A cross-sectional study at the Department of Obstetrics & Gynaecology at the University Clinical Hospital Mostar was conducted by an anonymous survey using a questionnaire designed exclusively for this study. A total of 100 mothers were included in the study.

Results Satisfaction with midwives’ communication and their approach to the women during their stay in the delivery room was rated significantly higher (4.7±0.6) when compared to ob- Corresponding author: stetricians-gynaecologists (4.5±0.8) (p=0.02). Midwives were ra- Vajdana Tomić ted better in providing breastfeeding information (4.5±0.8) than University Clinical Hospital Mostar for the speed of arrival after a call bell (average grade 4.2±1.0). Bijeli brijeg b.b., 88000 Mostar, Respondents were least satisfied with the hygiene (toilet, shower Bosnia and Herzegovina and rooms) and the quality of food (average grades 3.8±1.1 and 3.9±1.0, respectively). Mothers with previous experience in child- Phone: +387 36 336-231; birth at the same hospital rated current stay with a similar level of Fax: +387 36 336 211; satisfaction. E-mail: [email protected] Adriana Haller ORCID ID: https://orcid. Conclusion Good communication skills of medical and non-me- org/000-0003-1481-872X dical staff are a recommended step to maintain mothers′ childbirth satisfaction, while improvement in quality of nutrition and hygiene should be mandatory.

Key words: delivery room, health care, hygiene, midwifery, obstetrics Original submission: 17 March 2021; Revised submission: 23 April 2021; Accepted: 07 June 2021 doi: 10.17392/1373-21

Med Glas (Zenica) 2021; 18(2):510-515

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INTRODUCTION PATIENTS AND METHODS Patient satisfaction with the provided hospital Patients and study design care is recognized as one of the key factors in the assessment of the health care system, and it is A cross-sectional study of mothers’ satisfaction defined as the difference between an expectation with the received medical care at the Perinato- and experience by health care users (1). logy Ward of the Department of Obstetrics & Assessing patient satisfaction with hospital ser- Gynaecology at the University Clinical Hospital vices is an important determinant for taking me- Mostar was conducted in the period from 9 No- asures to improve health care (2). The quality of vember 2019 to 10 February 2020. care provided by any hospital can be assessed A total of 100 mothers with live and term babi- and monitored in several ways (3). One of the es regardless of the mode of delivery (vaginal methods, which are not ordinarily used, involves delivery or caesarean section) were included in expressing patients' subjective perceptions about the study, while women with preterm delivery or the services provided to them. Assessments and stillbirth were excluded. perceptions by health care providers and admi- All mothers were informed about the study in a nistrators of the quality and standards of service written form and signed a consent form for inclu- provided can often be completely different from sion in the study. patients’ perceptions on the same services (4). An approval to conduct the survey was previou- The quality of communication with health care sly obtained from the Ethical Committee of the professionals and health care providers could University Clinical Hospital Mostar. affect patients of all ages and genders, but women of childbearing age and especially pregnant Methods women and women in labour are more prone Anonymous questionnaires specifically designed to develop depression due to their social and for this study were applied. The participation of psychological sensitivity (5). This requires a mothers was voluntary and at any time they could more empathetic and collaborative approach that have stopped completing the questionnaire. can be achieved with good communication skills. Mothers who agreed to the survey received the Obstetricians and midwives need to put more questionnaires and completed them independently. effort into quality communication (6). In some centres, guidelines are defined to achieve good The questionnaire consisted of questions on ge- communication such as providing a comfortable neral and sociodemographic data, followed by environment, quick response, appropriate attitu- five separate groups of questions. The answers de, and good knowledge of clinical skills (7) were rated as follows: 1 - very bad, 2 - bad, 3 - good, 4 - very good, or 5 - excellent. The first According to the recommendations of the National group of questions focused on the birth experien- Institute for Health and Care Excellence (NICE), ce (Table 1), and the second and third group on measuring the satisfaction of mothers with intrapar- the experience and satisfaction with health care at tum care helps to identify problems whose solution the delivery room (Table 2) and maternity ward, will improve the quality of obstetric services (8). respectively, referring to the satisfaction of inte- Maternal satisfactions as one of the important raction with all health professionals who came quality indicators of health care could be used into contact with the mothers (obstetricians- for comparison between hospitals, public (score gynaecologists, paediatricians, midwives and 4.46) and private (score 4.60) one (9). There is a nurses). The fourth group of questions referred lack of research on the maternal satisfaction with to the satisfaction with the hygiene of the space hospital care in Bosnia and Herzegovina. in which the mothers stayed, the quality of the The aim of the study was to evaluate mothers’ food they received and the attitude of the support satisfaction with received hospital care during staff (Table 4). The fifth group of questions con- childbirth and early puerperium at the Depar- sisted of the overall grades for the Department’s tment of Obstetrics and Gynaecology, University medical staff as well as the overall quality of the Clinical Hospital Mostar and to determine areas stay in the delivery room and maternity hospital, in which it could be improved. the total quality of the previous stay (if the res-

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pondent had a previous childbirth at the same de- midwives was rated significantly higher than the partment) and the question on personal readiness average grade (p=0.0214). Also, the midwifes’ to take care of the child after discharge from the approach towards the mothers’ view of the way maternity hospital (Table 5). of giving birth was rated significantly higher when compared to the obstetricians - gynaecolo- Statistical analysis gists’ approach (p=0.0145) (Table 2). Statistical analysis included a calculation of des- Table 2. Mothers’ satisfaction during the labour and delivery criptive data, and the arithmetic mean and stan- Mothers’ satisfacti- Number of Question on rating (arithme- dard deviation (SD) were calculated. A χ2 test mothers tic mean±SD)* was used for frequency analysis. A non - parame- Communication with an obstetrici- 100 4.5±0.8 tric Wilcoxon test was used to analyse the inter- an– gynaecologist val scale of independent samples, while a t - test Information received during the labour and delivery from an obstetri- 99 4.4±0.9 was used to analyse the mean values. The level of cian - gynaecologist statistical significance was p < 0.05. Obstetrician - gynaecologist's 98 4.5±0.8 approach during labour and delivery RESULTS Communication with the midwife 97 4.7±0.6 Personal qualities of midwives The majority of mothers belonged to the age (decency, respect, sensitivity, tender- 100 4.7±0.6 group between 20 and 39 years old (mean age ness, patience) 30.6±4.5), finished secondary school, lived in the Approach of midwives towards the city, had average socioeconomic status, no pre- mother view of the way of giving 96 4.7±0.6 birth (alternative way of giving birth) vious delivery experience, and a spontaneous on- Attention of midwife paid for more 100 4.7±0.6 set of labour with vaginal delivery (Table 1). The pleasant experience mean stay in hospital was 4.2±2.3 days. *The answers were rated as follows: 1 - very bad, 2 - bad, 3 - good, 4 - very good, or 5 - excellent; SD, standard deviation Communication with obstetricians - gynaecologists and midwives in delivery room was rated The mothers rated communication with the he- with high grades. However, communication with alth care staff, the attention they received and the care they received with relatively high grades. Table 1. General and obstetric characteristics of mothers Variable (No of mothers) No (%) of mothers Midwives received better average grade for pro- Age (years) (n=93) viding information on breastfeeding than paedia- 20 – 29 42 (45.2) tricians (p=0.0026) (Table 3). 30 – 39 49 (52.7) 40 – 49 2 (2.1) Table 3. Mothers’ satisfaction during stays at the Maternity Ward Education degree (n=100) Mothers’ satisfaction Primary school 0 Number of Question rating (arithmetic High school 51 (51.5) mothers mean±SD)* Undergraduate studies 16 (16.2) Communication with a paedia- Graduated (University) 33 (32.3) 100 4.2±1.0 Marital status (n=100) trician Information obtained on the child Married 98 (98.0) 100 4.2±1.0 Unmarried 2 (2.0) health from a paediatrician Information on breastfeeding Residence (n=100) 99 4.2±1.2 Village 45 (45.0) obtained from a paediatrician Town 55 (55.0) Information obtained on a mor- Socioeconomic status (n=99) ning round from an obstetrician– 99 4.2±1.1 Below average 0 gynaecologist Average 93 (93.0) Information on breastfeeding 100 4.5±0.8 Above average 6 (6.1) obtained from midwives Parity (n=99) The attention that midwives paid 99 4.3±0.9 First delivery 40 (40.4) for a more pleasant experience Second delivery 39 (39.4) Comfort and support received 99 4.3±1.0 Third and more deliveries 20 (20.2) from midwives Onset of labour (n=100) Personal qualities of midwives Spontaneous, contraction 47 (48.0) (decency, respect, sensitivity, 99 4.3±0.9 Spontaneous membrane rupture 14 (14.3) tenderness, patience) Induction of labour (oxytocin – misoprostol) 29 (29.6) Specific breastfeeding assistance 97 4.3±1.0 Elective caesarean section 8 (8.1) from midwives Type of delivery (n=100) Call bell response time 92 4.2±1.0 Vaginal 73 (72.0) *The answers were rated as follows: 1 - very bad, 2 - bad, 3 - good, Caesarean section 27 (27.0) 4 - very good, or 5 – excellent; SD, standard deviation

512 Haller et al. Mothers’ satisfaction with health care

The attitude of support staff towards the mothers at the Perinatology Ward of University Clinical was rated better than tidiness and cleanliness of Hospital Mostar. We should emphasize that there rooms (p <0.0001), quality of food (p = 0, 0001) are high grades for satisfaction with communica- and tidiness and cleanliness of showers and toi- tion with midwives during the stay in the delivery lets (p <0.0001), which have the lowest average room and slightly lower grades for satisfaction grade in this study (Table 4). with the stay at the Maternity Ward after childbirth, as well as for the grades on satisfaction Table 4. Mothers’ satisfaction with hygiene, food and attitude of support staff during their stay at the Perinatology Ward with communication with obstetricians - gynae- Number of Mothers’ satisfaction ra- cologists and paediatricians. Question * mothers ting (arithmetic mean±SD) The quality characteristics of health care inclu- Tidiness and cleanliness 100 3.9±1.0 de the quality of interpersonal skills of health of rooms Tidiness and cleanliness of care providers, competencies of health care 100 3.8±1.1 showers and toilets staff, physical environment and arrangement of Number of meals 99 4.2±0.9 the institution, accessibility of medical services, Quality of food 99 3.9±1.0 continuity of care, hospital characteristics and Relationship of support staff 100 4.3±0.9 therapeutic outcomes - all strongly related to pa- *The answers were rated as follows: 1 - very bad, 2 - bad, 3 - good, 4 - very good, or 5 – excellent; SD, standard deviation tient satisfaction (10). This supports a number of theories and models on health service quality, The satisfaction of the mothers with midwives in suggesting that health service quality indicators, the delivery room was statistically significantly including indicators of health service processes higher when compared to obstetricians-gynae- and outcomes, play a key role in patient satisfac- cologists (p=0.0007). There was no significant tion (10,11). Among the determinants related to difference in the satisfaction of the respondents the service, the strongest positive correlation was with obstetricians-gynaecologists and paediatri- found between interpersonal skills of healthcare cians (p=0.345) (Table 4). workers and patient satisfaction (12). For this Table 5. Mothers’ satisfaction during their stay at the Perina- reason, it is necessary to try and define patient tology Ward satisfaction on the basis of several health service Mothers’ satisfaction Number of quality indicators and the way patients develop Question rating (arithmetic mothers mean±SD)* their satisfaction with health services (12). Mo- Obstetricians - gynaecologists in reover, if patient satisfaction is a central issue of 100 4.5± 0.7 the delivery room health services, the first step would be to -esta Midwives in the delivery room 99 4.7 ± 0.5 blish or strengthen the education of medical and Paediatricians in the maternity ward 99 4.4.± 0.8 health students on interpersonal skills to increase Nurses for newborns in the mater- 99 4.5± 0.8 nity ward communication and empathy skills, as well as en- Overall quality of the stay in the sure continuity of on-the-job training for health 99 4.4.± 0.8 delivery room workers (13). Overall quality of the stay in the 99 4.2± 0.9 maternity ward Satisfaction is also greatly affected by the cle- Overall quality of the previous stay 67 4.3± 0.8 anliness and tidiness of the space in which the *The answers were rated as follows: 1 - very bad, 2 - bad, 3 - good, patients stay during hospitalization (14). In this 4 - very good, or 5 – excellent; SD, standard deviation study, the average grades are relatively lower in A stay at the Perinatology Ward at the time of deli- relation to tidiness, cleanliness and quality of very and at the time of early puerperium were rated patient rooms, showers and toilets. This issue is with a high average grade (Table 5). However, the also observed by other studies, especially those stay in the delivery room was rated significantly from third world countries (15). In a study from better (p=0.0023). Mothers with previous experi- Pakistan, satisfaction with cleanliness was recor- ence in childbirth in the same hospital rated current ded in only 13% of cases (15). This problem, es- hospitalisation with a similar score of satisfaction pecially in state institutions, requires appropriate with health care as in the previous stay (p=1.0). measures to address, as well as to maintain the DISCUSSION standard once they are achieved, which ultimately depends not only on finances but also on or- The results of this study confirm high satisfac- ganizational measures and control over its imple- tion of mothers with the received hospital care mentation (16).

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Additional factors that potentially limit this stu- conducted during the winter months where only dy should be taken into account. Published data a short period, from Christmas (25 December) to have shown evidence that sociodemographic the Three Kings Day (6 January), was covered by factors affected patient satisfaction with health a reduction in the number of medical staff. services and therefore these variables should be In conclusion, the mothers showed high satisfac- considered when comparing patient satisfaction tion with received health care at the Perinatology between certain groups or countries (17). The Ward of University Clinical Hospital Mostar analysed group of mothers in our study repre- which represents an obligation for the continu- sented a homogeneous structure with appropriate ation of the development of good clinical prac- education and optimal age for birth with average tice with an emphasis on the implementation of socio-economic status. The effect of the length of communication skills of medical and non-me- stay can also cause changes in satisfaction outco- dical staff. Areas that require improvement are mes (18). In a Japanese study, the factors influen- hygiene and nutrition and they are largely depen- cing the patient satisfaction experience differed dent on the overall organization at the level of the significantly in those hospitalized for less than entire hospital. This study, the first of its kind at one week, between one and four weeks, and the University Clinical Hospital Mostar, Bosnia those hospitalized for more than a month (18). and Herzegovina, should be the starting point Additionally, patient illness severity could also for future studies on satisfaction of mothers with contribute to a possible impact on psychological hospital care during childbirth and early postpar- wellbeing (19). In our study, the length of hospi- tum period. In the future studies, it is necessary tal stay, in most cases, was within 6 days, which to include specific procedures during hospital makes the group compact and reduces possible care in perinatology and compare them with the variations in the expressed satisfaction. expressed satisfaction of health care users. The seasonal fluctuation in hospitalization of patients and the completion of satisfaction que- FUNDING stionnaires can further affect outcomes of patient No specific funding was received for this study. satisfaction (20). The unwillingness of patients to come to the hospital during the summer months TRANSPARENCY DECLARATION must be taken into account (20). Our survey was Conflict of interest: None to declare. REFERENCES

1. Xesfingi S, Vozikis A. Patient satisfaction with the he- 6. Corscadden L, Callander EJ, Topp SM, Watson DE. althcare system: assessing the impact of socio- eco- Experiences of maternity care in New South Wales nomic and healthcare provision factors. BMC Health among women with mental health conditions. BMC Serv Res 2016; 16:94. Pregnancy Childbirth 2020; 20:286. 2. Mutaganzwa C, Wibecan L, lyer HS, Nahimana E, 7. Goldstein E, Farquhar M, Crofton C, Darby C, Gar- Manzi A, Biziyaremye F, Nyishime M, Nkikabahizi finkel S. Measuring hospital care from the patients’ F, Hirschhorn LR, Magge H. Advancing the health of perspective: an over view of the CAHPS hospital women and newborns: predictors of patient satisfac- survey development process. Health Serv Res 2005; tion among women attending antenatal and maternity 40:1977-95. care in rural Rwanda. Int J Qual Health Care 2018; 8. National Institute for Health and Clinical Excellence 30:793-801. (NICE). Intrapartum care for healthy women and babi- 3. Handely SC, Bell S, Nembhard IM. A systematic re- es. Clinical guideline [CG190]https://www.nice.org. view of surveys for measuring patients-centered care uk/guidance/cg190/resources/intrapartum-care-for- in the hospital setting. Med Care 2021; 59:228-37. healthy-women-and-babies-pdf-35109866447557 4. Munavi-Babigumira S, Glenton C, Lewin S, Frethe- (05 March 2021) im A, Nabudere H. Factors that influence the provisi- 9. Okumu C, Oyugi B. Clients’ satisfaction with quality on of intrapartum and postnatal care by skilled birth of childbirth services: A comparative study between attendants in low‐ and middle‐income countries: public and private facilities in Limuru Sub-County, a qualitative evidence synthesis. Cochrane Database Kiambu, Kenya. PLoS One 13:e0193593 Syst Rev 2017; 11:CD011558. 10. Batbaatar E, Dorjdagva J, Luvsannyam A, Amenta P. 5. Passarelli VC, Lopes F, Merighe LS, Araujo RS, No- Conceptualisation of patient satisfaction: a systema- mura RMY. Satisfaction of adolescent mothers with tic narrative literature review. Perspect Public Health childbirth care at a public maternity hospital. J Obstet 2015; 135:243–50. Gynaecol Res 2019; 45:443-9.

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11. Lazzerini M, Mariani I, Semenzato C, Valente EP. 15. Hussain SS, Pervez KF, Izzat F. To assess patient sa- Association between maternal satisfaction and other tisfaction in gynaecology and obstetrics in Tertiary indicators of quality of care at childbirth: a cross-sec- care hospital and to highlight the areas of improve- tional study based on the WHO standards. BMJ Open ment. J Postgrad Med Inst 2015; 29:93-6. 2020;10: e037063. 16. Nisa M, Sadaf R, Zahid M. Patient satisfaction survey 12. Sanchez-Piedra CA, Prado-Galbarro FJ, Gar- in an obstetrics and gynaecology ward of a Tertiary cia-Perez S, Santamera AS. Factors associated care hospital. J Med Sci 2012; 20:142-5. with patient satisfaction with primary care in Euro- 17. Venn S, Fone DL. Assessing the influence of socio- pe: results from the EU prime care project. Qual Prim demographic factors and health status on expression Care 2014; 22:147–55. of satisfaction with GP services. Clinical Governan- 13. Burgener AM. Enhancing communication to improve ce: An International Journal 2005; 10:118–25. patient safety and to increase patient satisfaction. He- 18. Tokunaga J, Imanaka Y. Influence of length of stay on alth Care Manage (Frederick) 2017; 36:238-43. patient satisfaction with hospital care in Japan. Int J 14. Xie A, Rock C, Hsu YJ, Osei P, Andonian J, Scheeler Qual Health Care 2002; 14:493–502. V, Keller SC, Cosgrove SE, Gurses AP, Centers for 19. Alfaro Blazquez R, Ferrer Ferrandiz E, Gea Caballe- Disease Control and Prevention (CDC) Prevention ro V, Corchon S, Juarez-Vela R.Women's satisfaction Epicenter Program. Improving daily patient room cle- with maternity care during preterm birth. Birth 2019; aning: an observational study using a human factors 46:670-7. and systems engineering approach. IISE Trans Occup 20. Salin S, Kaunonen M, Aalto P. Explaining patient sa- Ergon Hum Factors 2018; 6:178–91. tisfaction without patient care using data-based nurse staffing indicators. J Nurs Adm 2012; 42:592–7.

515 ORIGINAL ARTICLE

Mortality associated with seasonal changes in ambient temperature and humidity in Zenica-Doboj Canton

Suad Sivic

Institute for Health and Food Safety Zenica, Bosnia and Herzegovina

ABSTRACT

Aim To determine the relationship between seasonal changes in ambient temperature, humidity and general and specific mortality rates in the area of Zenica-Doboj Canton.

Methods Changes in the average monthly mortality in the period from 2008 to 2019 were analysed (linear regression) in relation to the average temperatures and humidity in those months in the same time period in Zenica-Doboj Canton.

Results Overall mortality increased from 7.9 ‰ in 2008 to 10.2 ‰ in 2019. Overall and specific mortality rates for cardiovascular, malignant, respiratory and metabolic diseases followed seasonal Corresponding author: change of ambient temperature and humidity. The monitoring Suad Sivić trend showed strong determination degree for overall mortality Institute for Health and Food Safety Zenica and mortality for cardiovascular, malignant and respiratory diseases, while for metabolic diseases it was somewhat lower. The Fra Ivana Jukića 2, 72000 Zenica, highest mortality rates were found in January (cold month), and Bosnia and Herzegovina in August (warm month); the lowest one was in May, September Phone: +387 32 448 030; and October. There was a strong significant negative correlation Fax: +387 32 448 000; between temperature and mortality rates, while the correlation E-mail: [email protected] between humidity and mortality rates was not significant. ORCID ID: http://www.0000-0001-5696- Conclusion As we have proven that mortality rates followed se- 8676 asonal changes in ambient temperature and determined months with the least mortality rate, the community must take measures to ensure microclimatic conditions for the survival of patients with cardiovascular, malignant, respiratory and metabolic diseases.

Key words: air temperature, humidity, mortality rate Original submission: 25 December 2020; Revised submission: 02 March 2021; Accepted: 24 March 2021 doi: 10.17392/1337-21

Med Glas (Zenica) 2021; 18(2):516-521

516 Sivic S. Mortality in seasonal climate changes

INTRODUCTION transitions suitable for adaptation (10,11). Man's adaptive capacities depend on technologies that The human body is under a constant influence of can improve the living and working environment, environmental factors. These factors are nume- as well as internal adaptive capabilities that lar- rous and affect the quality of life, age of healthy gely depend on a person's age. life and health conditions. Among environmental factors are also climatic ones, and their impact on There is no information of the impact on the mor- human health can occur in different ways. The- tality in the temperate continental and mountain se can be indirect effects such as the spread of climate regions, which is characteristic of Zeni- vector-borne diseases, or direct effects of extre- ca-Doboj Canton. me weather events such as flood, drought or heat. The aim of this research was to determine how Numerous studies indicate the impact of climate seasonal climate changes in the area of Zenica- change on health of the population (1,2). Global Doboj Canton affect general and specific morta- impacts of climate change on population health lity of the population. are studied in large multicentre studies, and possible outcomes of change are predicted (3,4). MATERIALS AND METHODS

Among the factors that significantly affect human Materials and study design health and whose changes are constantly present are seasonal climate environmental factors such Zenica-Doboj Canton is located in the central as temperature and humidity. The physiologi- part of Bosnia and Herzegovina and has a tem- cal consequences of human exposure to tempe- perate continental and mountain climate. The rature changes and its ability to adapt are well temperate continental climate is characterized by known (5). Research also provides data on the harsh winters, short springs and warm and humid short-term and long-term cumulative impact of summers. The mountain climate is characteri- exposure to changes in ambient temperature. It zed by low fluctuation of air temperature with a has been proven that short-term as well as longer fluctuation average of about 20 °C (12). exposure leads to an increased risk of death from Data on mortality, population and meteorological cardiovascular and respiratory diseases (6). It has conditions in Zenica-Doboj Canton were collec- also been shown that low temperatures during the ted from the Federal Statistical Office (13), the winter months lead to increased mortality from Federal Hydrometeorological Institute (14) of the cardiovascular disease (7,8). Federation of Bosnia and Herzegovina, as well as In addition to the change in temperature during at the Institute for Health and Food Safety Zenica. the change of seasons, relative humidity also The impact of annual climate changes on morta- changes. Increased humidity in the warm months lity (for general, cardiovascular, malignant, me- contributes to heat stress due to the difficult tabolic and respiratory diseases) in Zenica-Doboj exchange of heat in the human body, which is Canton for the period 2008-2019 was analysed. also a factor that increases mortality. Sweating is Methods the main process by which the body dissipates metabolic heat into the environment and beco- After data collection and extraction, average mes significantly less efficient if relative humi- monthly values for both temperature and humidity of the environment is high, resulting in the dity and overall and specific mortality were cal- accumulation of heat in the body (9). culated, and obtained values were presented by How much the impact of changes in heat and hu- months of the year. Values for air temperature midity will affect human health depends on the in- and humidity were used from meteorological au- tensity of the change and adaptive capabilities of tomatic measuring station in Zenica city. a person. According to data from meteorological Statistical analysis stations around the world and according to projections of experimental models, the frequency and A settlement of average deaths is required due intensity of extreme hot waves have increased. to the unequal number of days for each month. Sharper changes are also noticeable without slight In the observed period, each month had an ave-

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Figure 1. Overall average monthly values of deaths

rage of 30.44 days, and this value was calcula- The overall mortality rate in the observed period ted using an equation with one unknown for the showed an increasing trend from 7.9‰ in 2008 to monthly average of deaths (Figure 1). 10.2‰ in 2019. Statistical calculations determined the differen- The average of overall total mortality by months ces between general and specific mortality - ra ranged from 233 in September to 336 in January. tes in each month, on the basis of which it was These values were within the parabolic trend of concluded how climate change affected mortality the second degree (Figure 1). The highest mor- rates. To determine the interdependence of gene- tality was noticed in January (when the average ral and specific mortality rates and humidity and air temperature was the lowest), then mortality air temperature, statistical methods for determi- decreased in parallel with an increase of the tem- ning the regression (R2) and correlation coeffici- perature to an average of 16 °C in May; when ents (r) were used. Regression trends are shown the average temperature exceeded 20 °C (from by a parabolic trend of the second degree. The le- June to August) mortality was again increased. vel of statistical significance of p<0.05 was used. In September (average temperatures falling to For an estimation of the significance of the stren- around 16 °C), the lowest mortality rate was gth of regression coefficient (R), the Chaddock noticed. Further drop in average temperatures scale (15) was used, where the strengths were resulted in a rise of mortality rate in December. arranged as follows: 0

518 Sivic S. Mortality in seasonal climate changes

Figure 2. Average monthly values of air temperature nation showed high values, i.e. a strong correla- cyclical changes in the intensity of factor action tion between the numerical values of temperatu- associated with seasonal climate changes only re (R2=9.927) and humidity (R2=0.849) and the for temperature and humidity was found in our month of the year (Figure 2). study. Therefore, we hypothesized that seasonal Similar trends were found for specific mortality cyclical changes in temperature and humidity of cardiovascular, malignant, metabolic and res- were probably the cause of cyclical changes in piratory diseases; Pearson correlation coefficient the number of deaths. was negative and significant. The coefficient of There is ample evidence that air temperature determination for regression analysis was also si- may affect morbidity and mortality rates (17). An gnificant and high. The regression curve was spe- increase in ambient temperature by 1 °C incre- cific and characteristic with two peaks of increa- ases cardiovascular mortality by 3.44%, respi- sed morbidity in January and August. ratory mortality by 3.60% and cerebrovascular Diabetes mellitus was a cause of death in 94.4% mortality by 1.40% in the population older than of metabolic diseases cases. The correlation co- 65 years of age, according to a meta-analysis of efficient showed a strong significant inverse a systematic review of epidemiological evidence association of deaths caused by metabolic dise- in 2016 (17). Our research has shown a strong ases and air temperature. Following the trend of dependence of mortality on the year's month and mortality by months of the year, the coefficient the strong correlation with the air temperature. of determination of medium strength was noticed We also found a strong association between ambient (Table 1 and Figure 3). temperature and humidity with specific mortality from cardiovascular disease, respiratory and ma- DISCUSSION lignant diseases. Seasonal variations in cardiovas- The results of this study showed the cyclic rise cular diseases were identified in study by Stewart et and fall in the rate of deaths associated with the al. in Australia; the variations had a similar pattern seasons. These cycles in the twelve-year period of occurrence as in our study with peaks in the col- were repeated in the same way each year, so that dest and warmest months (18). A study done in the highest total mortality rate was determined in Taiwan found an inverse correlation between tem- January, and the lowest one in September. perature rise and mortality from cardiovascular and respiratory diseases (19). Unlike our study, a study A large number of environmental factors can conducted in Kuwait did not establish an associa- affect mortality rates (16), but the characteristic

Table 1. Association of general and specific mortality with monthly temperature and humidity Percentage of overall Determination coefficient of the quadratic Mortality category Air temperature* p Air humidity* p mortality (%) polynomial regression (R2) Overall 100.0 -0.60 0.04 0.23 0.47 0.90 Cardiovascular disease 53.0 -0.66 0.01 0.31 0.32 0.89 Malignant neoplasms 20.4 -0.70 0.01 0.38 0.23 0.84 Metabolic diseases 4.7 -0.60 0.04 0.20 0.54 0.65 Respiratory diseases 4.1 -0.73 0.01 0.29 0.37 0.81 *Pearson's correlation coefficient, r;

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Figure 3. Trendline of specific mortality data by months tion between deaths from malignant diseases and September. This research can be of great practi- seasonal changes in temperature (20). cal importance in advising patients with chronic Following the trend of mortality by months of diseases to avoid exposure to extremely high or the year for metabolic diseases, the coefficient of low temperatures. The community can also be determination of medium strength was found in encouraged to provide favourable microclimatic our study. Similar results were obtained in a 2004 conditions for the chronically ill. Future research study conducted in Japan (21). should answer the question of how to provide a favourable environment to reduce mortality asso- Humidity as a single factor did not show a signi- ciated with seasonal temperature changes. ficant effect in all examined causes of death in our study. FUNDING In conclusion, the most favourable average am- No specific funding was received for this study. bient temperatures in Zenica-Doboj Canton for the survival of patients with cardiovascular, ma- TRANSPARENCY DECLARATION lignant, respiratory and even metabolic disea- Conflict of interest: None to declare. ses are around 16 ℃, which occur in May and REFERENCES 1. World Health Organization (WHO). Quantitative Heaviside C,Vardoulakis S, Hajat S, Haines A, Arm- Risk Assessment of the Effects of Climate Chan- strong B. Projections of temperature-related excess ge on Selected Causes of Death, 2030s and 2050s. mortality under climate change scenarios. Lancet Geneva, Switzerland: World Health Organization Planet Health 2017; 1:e360–e367 (WHO), 2014. https://apps.who.int/iris/bitstream/ 4. Rossati A. Global warming and its health impact. Int handle/10665/134014/9789241507691_eng.pdf (01 J Occup Environ Med 2017; 8:720. December 2020) 5. Hanna EG, Tait PW. Limitations to thermoregulati- 2. Kjellstrom T, McMichael AJ. Climate change threats on and acclimatization challenge human adaptation to population health and well-being: the imperative to global warming. Int J Environ Res Public Health of protective solutions that will last. Glob Health Ac- 2015; 12:8034-74. tion 2013; 6:20816. 6. Basu R, Malig B. High ambient temperature and 3. Gasparrini A, Guo Y, Sera F, Vicedo-Cabrera AM, mortality in California: exploring the roles of age, Huber V, Tong S, Coelho MSZS, Saldiva PHN, La- disease, and mortality displacement. Environ Res vigne E, Correa PM, Ortega NV, Kan H, Osorio S, 2011; 111:1286-92. Kyselý J, Urban A, Jaakkola JJK, Ryti NRI, Pascal 7. Mercer JB. Cold--an underrated risk factor for he- M, Goodman PG, Zeka A, Michelozzi P, Scortichini alth. Environ Res 2003; 92:8-13. M, Hashizume M, Honda Y, Hurtado-Diaz M, Cruz 8. Fowler T, Southgate RJ, Waite T, Harrell R, Kovats JC, Seposo X, Kim H, Tobias A, Iñiguez C, Forsberg S, Bone A, Doyle Y, Murray V. Excess winter deaths B, Åström DO,Ragettli MS, Guo YL, Wu C, Zano- in Europe: a multi-country descriptive analysis. Eur betti A, Schwartz J, Bell ML, Dang TN, Van DD, J Public Health 2015; 25:339-45.

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9. Chen X, Li N, Liu J, Zhang Z, Liu Y. Global heat 15. Zolotarev KV, Belyaeva NF, Mikhailov AN, Mikha- wave hazard considering humidity effects during the ilova MV. Dependence between LD50 for rodents 21st century. Int J Environ Res Public Health 2019; and LC50 for adult fish and fish embryos. Bull Exp 16:1513. Biol Med 2017; 162:445-50. 10. Wehner M, Castillo F, Stone D. The impact of mo- 16. Curtiss JR, Grahn D. Population characteristics and isture and temperature on human health in heat environmental factors that influence level and cau- waves. Natural Hazard Science 2017; 26. https:// se of mortality. A review. J Environ Pathol Toxicol oxfordre.com/naturalhazardscience/view/10.1093/ 1980; 4:471-511. acrefore/9780199389407.001.0001/acrefore- 17. Bunker A, Wildenhain J, Vandenbergh A, Henschke 9780199389407-e-58 (02 October 2020) N, Rocklöv J, Hajat S, Sauerborn R. Effects of air 11. Vogel MM, Zscheischler J, Wartenburger R, Dee D, temperature on climate-sensitive mortality and mor- Seneviratne SI. Concurrent 2018 hot extremes acro- bidity outcomes in the elderly; a systematic review ss northern hemisphere due to human-induced cli- and meta-analysis of epidemiological evidence. Ebi- mate change. Earths Future 2019; 7:692-703. oMedicine 2016; 6:258-68. 12. Federalni hidrometeorološki zavod. Klima Bosne i 18. Stewart S, Keates AK, Redfern A, McMurray JJV. Hercegovine (Federal Meterological Institute of Bo- Seasonal variations in cardiovascular disease. Nat snia and Herzegovina. Climate monitoring) https:// Rev Cardiol 2017; 14:654-64. www.fhmzbih.gov.ba/latinica/KLIMA/klimaBIH. 19. Yang LT, Chang YM, Hsieh TH, Hou WH, Li CY. php (02 September 2020) Associations of ambient temperature with morta- 13. Federalni zavod za statistiku. Stanovništvo i regi- lity rates of cardiovascular and respiratory diseases star. Hercegovine (Federal Institute of Statistics of in Taiwan: a subtropical country. Acta Cardiol Sin Bosnia and Herzegovina. Population and register) 2018; 34:166-74. http://fzs.ba/index.php/publikacije/godisnji-bilteni/ 20. Douglas AS, al-Sayer H, Rawles JM, Allan TM. stanovnistvo-i-registar/ (06 October 2020) Seasonality of disease in Kuwait. Lancet 1991; 14. Federalni hidrometeorološki zavod. Meteorološ- 337:1393-7. ki godišnjaci (Federal Meterological Institute of 21. Nakaji S, Parodi S, Fontana V, Umeda T, Suzuki K, Bosnia and Herzegovina. Yearbook) http://www. Sakamoto J, Fukuda S, Wada S, Sugawara K. Seaso- fhmzbih.gov.ba/latinica/KLIMA/godisnjaci.php (06 nal changes in mortality rates from main causes of October 2020) death in Japan (1970--1999). Eur J Epidemiol 2004; 19:905-13.

521 POSEBNA PONUDA INTESA SANPAOLO BANKE

INTESA SANPAOLO BANKA JE NOVI POSLOVNI PARTNER LJEKARSKE KOMORE ZENIČKO DOBOJSKOG KANTONA Intesa Sanpaolo Banka je kreirala posebnu ponudu za sve članove Ljekarske komore Zeničko-dobojskog kantona. Naša posebna ponuda nije posebna samo zbog najpovoljnijih uslova finansiranja koje možete naći na tržištu nego je posebna i po tome što želimo da budemo pouzdan poslovni partner svim članovima komore i omogućimo Vama, kao članovima komore, poseban status u našoj Banci. Poseban status omogućava brojne pogodnosti u poslovanju s Bankom a prednosti su brojne: Posebna ponuda svih usluga i proizvoda Banke, Individualan pristup svakome od Vas, Ušteda vremena jer imate mogućnost dogovora sastanaka putem telefona i mailom u terminu koji Vama odgovara, Izrada personaliziranih ponuda po posebnim cijenama kreiranim za članove komore, Savjetovanje od strane naših uposlenika u cilju pronalaženja adekvatnog rješenja svih Vaših finansijskih pitanja.

ISP ELBA OMOGUĆAVA: TEKUĆI RAČUN Uvid u sve proizvode koje koristite u našoj Banci OMOGUĆAVA: (računi, štednja, krediti i kartice) Usluge plaćanja u BiH i inostranstvu Usluge transfera novca i konverzije Korištenje Visa Inspire kartice Upravljanje uzorcima i paketima Korištenje dva limita na jednoj kartici - redovni i dodatni limit Online zahtjeve za proizvode Banke Korištenje redovnog limita po tekućem računu, maksimalno do 10.000 KM Privremenu blokadu i deblokadu debitnih kartica Korištenje dodatnog limita za plaćanje na 12 rata bez kamata i naknada Niz dodatnih opcija i pogodnosti Podizanje gotovine uz otplatu do 12 rata na POS uređajima u poslovnicama m-INTESA Korištenje elektronskog bankarstva 3 mjeseca bez naknada m-Intesa je usluga Banke koja korisniku omogućava obavljanje finansijskih Isplata gotovine bez naknade na bankomatima transakcija, pregled stanja na računima putem mobilnog uređaja (mobitela) Intesa Sanpaolo Banke i Intesa Sanpaolo Grupe širom svijeta kao i blokadu i deblokadu debitnih kartica. Aplikaciju možete preuzeti i Beskontaktno plaćanje instalirati sa sljedećih marketa: Dodatnu sigurnost za online plaćanje uz 3D Secure code

Niže naknade za sve obavljene transakcije putem elektronskog i/ili mobilnog bankarstva! Korištenje elektronskog i mobilnog bankarstva 3 mjeseca bez naknada

Kako biste iskoristili sve pogodnosti koje Vam se nude i olakšali pristup svim potrebnim informacijama pozivamo Vas da kontaktirate Voditelja Vama najbliže poslovnice.

Niže naknade za sve obavljene transakcije putem elektronskog i/ili mobilnog bankarstva! Korištenje elektronskog i mobilnog bankarstva 3 mjeseca bez naknada

Kako biste iskoristili sve pogodnosti koje Vam se nude i olakšali pristup svim potrebnim informacijama pozivamo Vas da kontaktirate Voditelja Vama najbliže poslovnice.

POSLOVNICA ZENICA POSLOVNICA PARK POSLOVNICA NOVA ZENICA POSLOVNICA ŽEPČE Silvana Peričević Indira Dajić Arnela Hadžiomerović Jozo Tomas Londža 81 M.Tita bb Dr.Abdulaziza Aske Borića 27 Ulica prva bb 061/763-283 061/138-848 062/890-516 061/894-361 silvana.pericevic indira.dajic arnela.hadziomerovic jozo.tomas @intesasanpaolobanka.ba @intesasanpaolobanka.ba @intesasanpaolobanka.ba @intesasanpaolobanka.ba POSLOVNICA JELAH POSLOVNICA BREZA POSLOVNICA KAKANJ POSLOVNICA VISOKO Sanja Halilović Nermana Čabaravdić Erna Kozlo Hašim Handžić Trg Branilaca BiH 22 Bosanska bb Alije Izetbegovića 77 Alije Izetbegovića 1 061/894-168 061/199-941 062/210-634 061/723-947 sanja.halilovic nermana.cabaravdic erna.kozlo hasim.handzic @intesasanpaolobanka.ba @intesasanpaolobanka.ba @intesasanpaolobanka.ba @intesasanpaolobanka.ba STAMBENI KREDITI Pregled rata stambenog kredita: Nominalna kamatna stopa već od 1,88% (EKS 2,08%)* IZNOS KM 5 GODINA 10 GODINA 15 GODINA 20 GODINA Krediti do 300.000 KM 30.000 524,26 274,43 205,88 170,92 Rok otplate do 30 godina 50.000 873,77 457,39 343,13 284,87 Mogućnost odabira fiksne ili promjenjive 100.000 1.747,53 914,77 686,26 569,74 kamatne stope do 10 godina 150.000 2.621,30 1.372,16 1.029,39 854,60 Bez naknade za obradu kredita 200.000 3.495,06 1.829,54 1.372,52 1.139,47

*Efektivna kamatna stopa (EKS) za stambeni kredit izračunata je na iznos 200.000 KM i rok otplate kredita 120 mjeseci, za klijente Banke. U izračun EKS ulaze svi troškovi koji mogu pasti na teret korisnika kredita (naknada za vođenje tekućeg računa, naknada za obradu i održavanje kredita, naknada za CRK izvještaj, troškovi mjenica, ovjere potrebne dokumentacije, troškovi police osiguranja, ZK izvatka, procjene vrijednosti nekretnine i troškovi notara). Za izračun EKS uzeti su sljedeći iznosi troškova instrumenta osiguranja, koje ne definiše Banka, a ovise o aktima ovlaštenih institucija: trošak mjenica 10 KM, trošak ovjere neophodne dokumentacije 10 KM, trošak notarskih usluga 450 KM, trošak zasnivanja hipoteke 550 KM, polisa osiguranja nekretnine od osnovnih opasnosti 70 KM na godišnjem nivou, procjena vrijednosti nekretnine 200 KM, trošak ostale dokumentacije 70 KM. Ukupan iznos troškova koji pada na teret korisnika kredita, a koji ulaze u izračun EKS, po promjenjivoj kamatnoj stopi za iznos 200.000 KM i rok otplate 120 mjeseci iznosi 22.000,00 KM. Stambeni krediti se odobravaju u iznosu do 300.000 KM na rok do 360 mjeseci u KM valuti. Kredit se odobrava sa fiksnom i promjenjivom kamatnom stopom u KM valuti do 10 godina. Kredit se odobrava sa promjenjivom kamatnom stopom i valutnom klauzulom u EUR prema važećem kursu Centralne Banke, preko 10 godina.

NENAMJENSKI KREDITI Pregled rata nenamjenskog kredita:

Nominalna kamatna stopa već od 1,00% (EKS 2,26%)* IZNOS KM 1 GODINE 3 GODINA 7 GODINA 10 GODINA Krediti do 50.000 KM 10.000 837,85 303,72 146,04 110,97 Rok otplate do 10 godina 20.000 1.675,71 598,43 282,58 212,03 Mogućnost odabira fiksne ili promjenjive 30.000 2.513,56 889,19 415,61 309,47 kamatne stope do 10 godina 40.000 3.351,42 1.185,59 554,15 412,63 Bez naknade za obradu kredita 50.000 4.189,27 1.481,99 692,68 515,79

*Efektivna kamatna stopa (EKS) za nenamjenski kredit izračunata je na maksimalan iznos 50.000 KM i rok otplate 12 mjeseci za klijente Banke. U izračun EKS ulaze svi troškovi koji mogu pasti na teret korisnika kredita (naknada za vođenje tekućeg računa, naknada za obradu i održavanje kredita, naknada za CRK, troškovi polise osiguranja, troškovi mjenice i ovjere potrebne dokumentacije). Za izračun EKS uzeti su sljedeći iznosi troškova instrumenta osiguranja, koje ne definiše Banka, a ovise o aktima ovlaštenih institucija: trošak mjenica 10 KM, trošak ovjere neophodne dokumentacije 10 KM, premija za osiguranje otplate kredita 250 KM. Ukupan iznos troškova koji pada na teret korisnika kredita, a koji ulaze u izračun EKS, po fiksnoj kamatnoj stopi za iznos 50.000 KM na rok 12 mjeseci iznosi 612,25 KM. Kredit se odobrava sa fiksnom i promjenjivom kamatnom stopom u KM valuti u iznosu do 50.000 na rok do 120 mjeseci.

AMERICAN MASTERCARD E X P R E S S KARTICA KARTICA

Mogućnost jednokratnog plaćanja s beskamatnom odgodom do 50 dana Dodatna zaštita uz obavezan unos PIN-a ili plaćanje do 12 rata, bez kamata i naknada za svaku transakciju na POS i ATM uređajima Asistenciju na putovanjima u inostranstvo – 24 sata medicinska, Beskontaktno plaćanje, bez obaveze unosa PIN-a do 60,00 KM pravna i putna pomoć Dodatna sigurnost online plaćanja uz 3D Secure code Osiguranje za vrijeme putovanja u inostranstvo – u slučaju nesretnog Limiti do 10.000 KM slučaja/trajne invalidnosti Minimalna uplata 3% utrošenih sredstava My account usluga – online pregled i ispis računa te nefakturisanih troškova Svim novim korisnicima Mastercard kartice poklanjamo vaučer Beskontaktno plaćanje u vrijednosti od 25 KM koji se može iskoristiti prema izboru na Sigurna online kupovina uz 3D Secure code Hifa pumpama ili Bingo hipermarketima širom BiH

ŽIVOTNO OSIGURANJE PREKO NAŠEG POSLOVNOG PARTNERA VIENNA OSIGURANJE

PREDNOSTI ŠTEDNJE U INTESA SANPAOLO BANCI

Članstvo u Agenciji za osiguranje depozita na iznose do 50.000 KM Sigurnost Vašeg novca garantujemo dugogodišnjim poslovnim uspjehom i kontinuitetom bankarskog poslovanja Intesa Sanpaolo Banka BiH je članica Intesa Sanpaolo Grupe koja je prisutna u 30 zemalja širom svijeta

Pratite nas: www.intesasanpaolobanka.ba Ovaj letak je informativnog karaktera i ne predstavlja obvezujuću ponudu za Intesa Sanpaolo Banku d.d. BiH. Bank of