Romanian Journal of Military Medicine

Founded 1897 • New Series Romanian Journal of Vol. CXXII • No. 3/2019 • December Military Medicine

REVISTA DE MEDICINĂ MILITARĂ

• About malpraxis, with love • Current therapeutic strategies for erectile function recovery after radical prostatectomy – literature review and meta-analysis • Toxoplasma Gondii infection and the signs of fetal affection during pregnancy as seen on ultrasound • High values of procalcitonin in non-septic patients with thermal and airway burns • Management of war-related vascular injuries. A civilian surgeon experience in the treatment of war casualties at a secondary care hospital • 3D echo in everyday life: Could it reset our threshold for interventions? • Aptamer as a proper alternative instead of monoclonal antibody in diagnosis and neutralization of menacing biological agents • Alteration of levels of thyroid hormones in acute ischemic stroke and its correlation with severity and functional outcome • Characteristics and complications of supernumerary permanent teeth in a sample of patients examined in a university pedodontics clinic • Development of quantitative real-time RT-PCR assay for detection and viral load determination of Crimean-Congo Hemorrhagic Fever (CCHF) virus • Prioritisation In delivering health services – a military health system example • Why cancer/terminal ill diagnosis unsuccessful in India: a qualitative analysis • Simulation and dynamic analysis of military marching using lower limbs anthropometric data • Drug allergies interpretation based on patient’s history alone may have therapeutic consequences in hospital setting • New approaches regarding the protection forces’ health against the effects of some toxic substances • Pregnancy outcomes in a patient with Fontan circulation for single ventricle congenital heart disease and aberrant praevia placental lobe • A simple approach for risk stratification in the plan of exercise in older adults • Can we still manage the relationship with patients? • The safety of genetic reserves in bioterrorism from the passive defense perspective • Secure wireless system based on reconfigurable devices for human biomedical parameters monitoring

Journal included in Web of Science, Emerging Sources Citation Index, Index Copernicus International, National Library of Medicine Catalog, Ulrich’s Periodicals Directory database, Directory of Open Access Journals, Directory of Research Journals Index, Eurasian Scientific Journal Index, Science Library Index and Open Academic Journals Index.

www.revistamedicinamilitara.ro

Editorial Board of Romanian Journal of Military Medicine Under the patronage Romanian Association of Military Physicians and Pharmacists Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Honorary Editor Acad. Victor Voicu MD, PhD Editors-in-Chief Florentina Ioniță Radu MD, PhD, MBA Dan Mischianu MD, PhD Executive Editors Daniel O. Costache MD, PhD, MBA Victor L. Purcărea PhD, MBA Associate Editor Mariana Jinga MD, PhD, MBA Redactors Raluca S. Costache MD, PhD, MBA – Bucharest Mihail S. Tudosie MD, PhD – Bucharest Editorial Assistants Ioana Bratu MD Cristina Solea Technical Secretary Oana Ciobanu Ionuț M. Olteanu Publisher Carol Davila University of Medicine and Pharmacy Publishing House

International Editorial Board Natan Børnstein (Israel) Mihai Moldovan (Denmark) Ioanel Sinescu (Romania) Silviu Brill (Israel) Ioan Opriș (USA) C. Ionescu Târgovişte (Romania) Cris S. Constantinescu (UK) Gerard Roul (France) Radu Ţuţuian (Switzerland) Daniel Dănilă (USA) Erwin Santo (Israel) Shyam Varadarajulu (USA) Stergios Ganatsios (Greece) Adrian Săftoiu (Denmark) Peter Vilmann (Denmark)

Scientific Publishing Committee Adrian Barbilian (Bucharest) Raluca S. Costache (Bucharest) Tudor Nicolaie (Bucharest) Anda Băicuş (Bucharest) Dragoș Cuzino (Bucharest) Ana Maria Oproiu (Bucharest) Cristian Băicuş (Bucharest) Camelia Diaconu (Bucharest) Carmen Orban (Bucharest) Andra R. Bălănescu (Bucharest) Mircea Diculescu (Bucharest) Bogdan A. Popescu (Bucharest) Mircea Beuran (Bucharest) Lidia Dobrescu (Bucharest) Aurelian E. Ranetti (Bucharest) Ovidiu Bratu (Bucharest) Cosmin Dobrin (Bucharest) Corneliu Romanițan (Bucharest) Daciana Brănișteanu (Iași) Dumitru Constantin Dulcan (Bucharest) Mugurel Rusu (Bucharest) Dragoș Bumbăcea (Bucharest) Silviu Dumitrescu (Bucharest) Andrada Seicean (Cluj Napoca) Marian Burcea (Bucharest) Carmen G. Fierbințeanu (Bucharest) Carmen A. Sîrbu (Bucharest) Mihai Ciocârlan (Bucharest) Cristian Gheorghe (Bucharest) Silviu Stanciu (Bucharest) Sofia Colesca (Bucharest) Liana S. Gheorghe (Bucharest) Ion Țintoiu (Bucharest) Gabriel Constantinescu (Bucharest) Viorel Jinga (Bucharest) Sorin G. Țiplica (Bucharest) Silviu Constantinoiu (Bucharest) Carmen Moldovan (Bucharest) Daniel Vasile (Bucharest) Dan Corneci (Bucharest) Ovidiu Nicodin (Bucharest) Dragoş Vinereanu (Bucharest)

REDACTION

B-dul Eroii sanitari, Nr.8, Sector 5, București, Tel/fax 021/318.07.59, tel. 021/318.08.62/Int. 199; Email [email protected] Romanian Journal of Military Medicine (RJMM) is included in Romanian College of Physicians Medical Publications Index.

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Romanian Journal of Military Medicine, New Series, vol. CXXII, No 3/2019, December ISSN-L 1222-5126; eISSN 2501-2312; pISSN 1222-5126

Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine

RJMM Founded 1897 • New Series Romanian Journal of Military Medicine Vol. CXXII • No. 3/2019 • December Edited by the Romanian Association of Military Physicians and Pharmacists.

Contents

EDITORIAL Dan Mischianu ● About malpraxis, with love 5

SYSTEMATIC REVIEW Cătălin Belinski, Alexandru Aungurenci, Dragoș Marcu, Dan Spînu, Ovidiu Bratu, Dan Mischianu ● Current therapeutic strategies for erectile function recovery after radical prostatectomy – literature review and meta-analysis 9

REVIEW ARTICLE C.A. Ispas, M. Bujor-Moraru, A. Bibire, F. Isopescu, M.J. Gardescu, C. Teodorescu, A. Edu ● Toxoplasma Gondii infection and the signs of fetal affection during pregnancy as seen on ultrasound 18

ORIGINAL ARTICLES Bogdan I. Pavel, Luisa M.V. Vătășoiu, Adrian T. Stanculea, Adina M. Milos, Alina A. Păstrăv, Adrian E.I. Roșca, Ana-Maria S. Zăgrean, Adrian M.E. Otoiu, Mihaela A. Covrig, Ileana C.P.V. Boiangiu ● High values of procalcitonin in non-septic patients with thermal and airway burns 22 Bariş Akça ● Management of war-related vascular injuries. A civilian surgeon experience in the treatment of war casualties at a secondary care hospital 29 Maria M. Gurzun, Silviu Stanciu, Adrian Gabără, Adrian Ionescu ● 3D echo in everyday life: Could it reset our threshold for interventions? 36 Hadi E.G. Ghaleh, Mojtaba Sharti, Mohammad S. Hashemzadeh ● Aptamer as a proper alternative instead of monoclonal antibody in diagnosis and neutralization of menacing biological agents 42 Brinder M.S. Lamba, Shalini Sharma, Pulin Gupta ● Alteration of levels of thyroid hormones in acute ischemic stroke and its correlation with severity and functional outcome 52 Mihaela Tănase, Aneta Munteanu, Ioana A. Stanciu, Elena C. Coculescu, Carmen Nicolae, Gabriela D. Bălan ● Characteristics and complications of supernumerary permanent teeth in a sample of patients examined in a university pedodontics clinic 64 Mojtaba Sharti, Mohammad S. Hashemzadeh, Ruhollah Dorostkar ● Development of quantitative real-time RT-PCR assay for detection and viral load determination of Crimean-Congo Hemorrhagic Fever (CCHF) virus 69

Ünal Demirtaș ● Prioritisation In delivering health services – a military health system example 76 Suantak D. Vaiphei, Devendra S. Singh ● Why cancer/terminal ill diagnosis unsuccessful in India: a qualitative analysis 84 Abolfazl Shakibaee, Alireza Asgari, Kamal Mostafavi, Gholamhossein Pourtaghi, Zeynab Ebrahimpour ● Simulation and dynamic analysis of military marching using lower limbs anthropometric data 92 Polliana M. Leru, Andreea Leontescu, Ion Ștefan, Irena Nedelea, Iuliana Ceaușu, Edu Antoine ● Drug allergies interpretation based on patient’s history alone may have therapeutic consequences in hospital setting 101 Pavel Otrisal, Camelia Diaconu, Ovidiu Bratu, Florentina Ioniță Radu, Zdenek Melicharik, Simona G. Bungău ● New approaches regarding the protection forces’ health against the effects of some toxic substances 106

CLINICAL PRACTICE Diana I. Voicu, Octavian Munteanu, Maria Sajin, Adrian Dumitru, Roxana O. Darabonț, Cătălin Cîrstoiu, Monica M. Cîrstoiu ● Pregnancy outcomes in a patient with Fontan circulation for single ventricle congenital heart disease and aberrant praevia placental lobe 110

VARIA Mehmet I. Naharci, Oznur Buyukturan, Ilker Tasci ● A simple approach for risk stratification in the plan of exercise in older adults 115 Liana Manolache ● Can we still manage the relationship with patients 117 Hadi E.G. Ghaleh, Mohsen A. Farajzadeh, Mosa Jafari, Nader N. Shadbad ● The safety of genetic reserves in bioterrorism from the passive defense perspective 120 Ionuț Rădoi, Lidia Dobrescu, Ștefan C. Arseni, Florin Răstoceanu, Florent M. Roman, Dragoș Dobrescu, Stela Halichidis ● Secure wireless system based on reconfigurable devices for human biomedical parameters monitoring 128

Guidelines for authors 133

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EDITORIAL

About malpraxis, with love… for your information

Dan Mischianu

The title of this article for the Romanian Journal of Military I do not pretend to be an expert Medicine, is inspired from the title of an American movie in the field of "medical from 1967, ''To Sir with love'', starring Sidney Poitier in the malpractice" but being lead role. Maybe some of you still remember it!... It is said involved and exposed to it in that once, when school did not oscillate between "variable my daily activities, I welcome concepts" and "palpable realities", between protocols and some considerations on this procedures, a hydrogeology professor, in a practical work topic, which I have found out Gral (R) Prof DAN MISCHIANU with his students, asked them if they knew the significance from books, from the media, Chief of Urology Clinic, Carol Davila of the walnuts planted near the houses of the Romanian and also from journalistic University Central Emergency peasants. All the students were amazed by the question and practice. Military Hospital kept silence. Nobody made any gesture. The professor knew Faculty of General Medicine, This article also wishes to draw Carol Davila University of Medicine that this would be the reaction of the students - because this attention of the medical and Pharmacy, Bucharest, Romania was the answer to the following question: "Why does the community and to make it world need teachers?" The professor answered the more aware of the responsibilities that we have when we questions and taught the students, as much as a professor dare to practice this noble profession. All of us – physicians, can teach, that ''a walnut needs plenty of water to grow and nurses, stretcher-bearers, technical staff – have civil liability the roots of the walnut are very deep, and that is why houses (and here is the context of a possible allegation of medical that have walnut trees next to them will not have problems malpractice), disciplinary liability (in the relationships we with dampness...'' have as full members of our professional organizations) and This parable, as understood by our mind, aims on one hand criminal liability (most often as defendants, sometimes as to draw the reader’s attention to the fact that "malpractice investigated parties or as witnesses). is defined as an improper or negligent treatment applied by I will present you several cases that have been the subject of a doctor to a patient, which causes the latter any kind of disciplinary, civil and criminal investigations, all of them in harm, in relation to the degree of damage of his physical and the “urological filed”. mental capacity", according to DEX, and on the other hand to state that the "WALNUT" – that is, the Teacher, in the way Case 1 he teaches his students will keep the "house" (i.e. the A 55-year-old patient is admitted in the Surgery Department workplace) safe against possible future malpractice charges. for urgent mumps and blurred urine. Diagnosis at admission: And after all, malpractice can even be compared to ''Urinary infection. Prostate adenoma. Hematuria.” dampness... for any professional organization. A superb Diagnosis after three days since admission: ''Medium allegory! I consider it a proper allegory and that is why I periurethral adenoma with urethral fistula microabs. wanted to introduce it to you. Piohematuria. Urinary Infection." Under antibiotic

treatment, the symptomatology improves and an open The Territorial Discipline Committee ordered the action to surgery is performed – with the patient's consent – be discontinued, but the patient filed an appeal with the practicing transvesical adenomectomy with favorable post- Supreme Discipline Commission. The latter did not find any surgery progress. irregularities neither in terms of the technique used nor of the complication that has arisen, not even regarding the 14 days after surgery, the urinary incontinence at effort is postsurgery treatment administered by the urologist, that is, detected, which subsequently worsens with the occurrence Driptane. The lack of the informed consent from the of episodes of urinary tract infection recorded on a posterior patient's file was the only and actually the capital irregularity urethral stricture. Urethrotomy and TUR-P for remaining found, for which the physician was sanctioned. adenomatous tissue are performed within urological university department. The histopathological result revealed The medical deontology code in Romania is very clear: "for prosthetic adenocarcinoma microfocus and chronic any diagnostic or therapeutic medical intervention, the urethritis. Unfortunately, the functional diagnosis is informed consent of the patient is necessary". Moreover, probative: High overall deficiency, Invalidity degree II. the consent is given only after informing the patient about his/her diagnosis, prognosis, therapeutic alternatives, along The patient's complaint was lodged against the surgery after with their risks and benefits. [2] which the patient was diagnosed with urinary incontinence that can only be cured by means of an artificial sphincter Case 4 costing about 5,000 euros and retirement due to illness. In the same spirit, there is also the case of a 39-year-old The Superior Disciplinary Commission of the Romanian patient admitted in emergency with the diagnosis: right College of Physicians considered that the moment when kidney colic. Two days after the admittance in hospital, surgery was performed and the surgical approach were surgery is performed: right pielolithotomy with internal inappropriate, therefore it decided to sanction the physician. drainage for calculus included in the right skin-ureteral [2] junction and secondary uropionefrosis. The immediate postoperative progression was favorable up to the occurence of Case 2 a cardio-respiratory stop during the first post-surgery night The son of a patient lodges a complaint against his family which was found to be irreversible. The cause of death was physician for the unauthorized delay of the diagnosis of his a massive pulmonary thromboembolism with probable father's renal carcinoma. The physician treated him for a causes either the lithotomy position, or more likely the year and six months for macroscopic hematuria and a so- presence of urinary sepsis. called renal colic. After one year and 6 months, the patient The Superior Discipline Commission decided to sanction the is diagnosed with renal neoplasm, he goes through surgery physician for the lack of informed consent, lack of and dies one year later. anticoagulation therapy and poor post-surgery follow-up [2, The Superior Discipline Commission analyzes the facts and 3] notes the following: Hematuria is a diagnostic urgency and Case 5 therefore its etiology needs to be confirmed as soon as possible by an urologist, the diagnosis of renal carcinoma is The following is the case of a 54-year-old patient with a not the responsibility of the family doctor, therefore the transient ischemic stroke, recently treated in a hospital from applied sanction has remained in force. the country, where a Foley autostatic bladder ureter was mounted. From the patient’s personal history, we mention: Case 3 type II diabetes, hypertension and ischemic heart disease. This is a case distinct from the others due to a capital error. Approximately 8 (eight) days after urethro-bladder probe It involves a 74-year-old patient admitted in a Urology was mounted (according to the patient's report), physicians department and diagnosed with: prostate adenoma, found the presence of an irreducible paraphimosis. Upon complete urine retention, prostate litiation. Twenty-four admission to the urology clinic, glandular necrosis, lack of hours after admission, the urologist intervenes and performs local sensitivity, denuded penile body, penile scar tissue and transvesic adenomyctomy. necrosis, present at the base of the penis, free cutaneous mucous suture, skin remnants, cavernous body having an Immediate post-surgery progression is favorable with the indurated appearance at palpation are found. The subsequent occurrence of a third degree urinary consultation of plastic and reparative surgery is entirely incontinence. Two years after the surgery, the patient lodges consistent with the urological clinical examination a complaint against the doctor.

6 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine performed. Figure 2: Postoperative final aspect; ureteral bladder and definitive perineal urethrostoma Figure 1: Preoperative aspect; the devitalization of the gland and the partially necrotic areas of the tegument can be seen

A surgical intervention is performed, after the patient gave his written consent, consisting of the following: noncrectomy, lavage, double drainage both by the uretro - bladder probe and by suprapubic cistostomy.

After limiting the infectious process, a new surgical intervention is performed consisting of definitive perineal urethrostomy and closure of the penile arch. Post-operative evolution is surgically favorable [4]

The patient contacted the media, the criminal investigation organs, the territorial discipline commission, and is in charge of the Superior Discipline Commission.

CONCLUSIONS

We wanted to point out, against all inconsistencies, errors, to think about. mistakes and malpractice, which perhaps appear at first The old Romanian saying teaches us: "The good deed speaks sight incriminating up to the contrary proof, that the for itself", but here, in Romania, there is a new saying which presence during the professional training of a ''Teacher'' of a has been quoted for about thirty years, in my opinion “Walnut'' absorbing all ''professional dampness'' is defining without any substantial background, which says: "No good for the next years of professional maturity. deed remains unpunished!" In relation to all the exposed cases, some of them not so However, I have the absolute conviction that although you relevant anymore, others still ''incendiary'' on which the have performed 10,000 perfect surgeries, if you make a public opinion and the indigenous media keep a close eye, I mistake on your last day of surgical practice, that mistake believe this journalistic warning should give us all something will not be forgiven!

References:

1. Beatrice-Gabriela Ioan, Andrei Codruț Nanu, Irinel Rotariu: 3. Med Right Experts, Andrei Nanu- Malpraxisul medical- 7 Răspunderea profesională în practica medicală, Editura Junimea, instrucțiuni de sigurantă, București, 2013 Iași, 2017 4. Dan Mischianu, Ovidiu Bratu, Alex Aungurenci, Dragoș Marcu, 2. Comisia Superioară de Disciplină a Colegiului Medicilor din Agnes Ciucă, Robert Popescu, Dan Spînu: Talking About Malpractice Romania: Răspunderea Disciplinară a Medicilor din Romania, 2011 A Possible Future Case, Medicina Modernă, 2015, vol. 22, No. 2: 183- (www.cmr.ro) 186

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Article received on March 4, 2019 and accepted for publishing on July 19, 2019. SYSTEMATIC REVIEW

Current therapeutic strategies for erectile function recovery after radical prostatectomy – literature review and meta-analysis

Cătălin Belinski¹, Alexandru Aungurenci¹, Dragoș Marcu2, Dan Spînu2, Ovidiu Bratu2,3, Dan Mischianu²,3

INTRODUCTION injections or intraurethral applications of prostaglandin E1 (PGE1), vacuum or vibratory treatments. Though there are Radical prostatectomy is the most commonly performed many options for ED treatment, to this date there is no treatment option for localised prostate cancer. In the last definitive protocol for penile rehabilitation (PR) after decades the surgical technique has been improved and prostatectomy. [5, 7] In this regard we have made a modified in order to satisfy both oncological safety and literature review and meta-analysis of the most important postoperative functional results. Urinary incontinence and randomised trials published in the last 5 years in order to find erectile dysfunction (ED) are the main postoperative the best strategies of penile rehabilitation after radical functional impairments in patients that undergo radical prostatectomy. prostatectomy (RP). [1]

According to the EAU Guidelines erectile dysfunction is MATERIALS AND METHODS defined as “the persistent inability to attain and maintain an We have systematically reviewed and collected data from erection sufficient to permit satisfactory sexual randomised trials published in the last 5 years involving performance”. To this moment it is estimated that 25-75% patients diagnosed with prostate cancer who experienced of the patients who underwent RP develop ED with major ED after radical prostatectomy. In order to find the necessary implications in postoperative quality of life. [2] Various risk trials we have searched PubMed and ScienceDirect factors can be related to post-prostatectomy ED, among databases and used several key words like: erectile which the most important are the patient’s age, dysfunction, penile rehabilitation, phosphodiesterase 5 comorbidities, preoperative erectile function, but also inhibitors, erectile functional recovery, prostate cancer, surgical approach (robot-assisted, laparoscopic, open) and prostatectomy and nerve sparing – in various combinations. nerve-sparing technique (inter/intrafascial). [2, 3] Among The first aspect for our meta-analysis inclusion criteria was physiopathological modifications that concur to ED, the to identify randomized (prospective or retrospective) nerve damage (neuropraxia) appears to be the main domino placebo-controlled trials involving patients with localised piece that triggers other alterations like structural changes prostate cancer that benefited of nerve sparing radical of the smooth muscle, arterial damage and veno-oclusive prostatectomy and developed postoperative ED. The key dysfunction which lead to cavernosal oxygenation aspect of our research was the penile rehabilitation strategy impairments. [4–6] The clinical results of physiopathological which included the therapeutic means used for erectile alterations are shortening of penile size, impairment of orgasm and ejaculation achievement which have tremendous impact on psychological and mental health. [5] 1 Dimitrie Gerota Emergency Hospital, Bucharest, Romania 2 Dr. Carol Davila University Central Emergency Military Hospital, Various treatment options include oral medication with Bucharest, Romania 3 phosphodiesterase 5 inhibitors (PDE5i), intracavernosal Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

function recovery and the treatment duration. Other aspects matching our criteria for meta-analysis that were thoroughly followed by us regarded the means of ED assessment, the full text reviewed. Seven out of the 15 were prospective surgical means and invasiveness and the rate of treatment randomised trials, 2 of them being double-blind, placebo- success reported to ED severity. controlled. Another 2 were nonrandomised prospective trials and the rest of 6 articles were retrospective review. In this regard we have conducted our research for each year Although some studies included by us followed-up both the between January 2014 and December 2018 and reviewed a ED and postoperative incontinence problem, we have total of 631 articles published on this theme collected from concerned strictly on erectile dysfunction issue and collected both search engines. For a time-efficient assessment we the necessary data. have read the title and abstract of each article and included only those articles that matched our criteria. We have For statistical analysis we have used ReviewManager 5.3 and excluded the reviews and meta-analyses and also the trials Microsoft Excel software with data of treatment strategies of ED that was not related to prostate cancer surgery. That and drug dosages collected from the studies. Although there left us 194 publications from which to select. We have also was less heterogeneity between the study designs of the excluded the trials withdrawn from database for various included articles we found important data about reasons and articles that didn’t followed the therapeutic postoperative EF recovery. These data allowed us to include means of ED recovery. Finally we have selected 15 articles the studies in subgroups in order to analyse them.

Table 1. Distribution of included studies

Patient Mean Cardiovascular Trial author Design Diabetes no. age comorbidities

Vickers Randomised prospective 2162 64.2 NoS NoS et al. [8] Montorsi Randomised double- 423 58 NoS NoS et al. [9] blind placebo-control Seo 40 Retrospective review 92 67.9 14 (15.2%) et al. [10] (43.5%) Nakano Retrospective review 103 63.4 NoS NoS et al. [11] Fode Randomised prospective 68 62 NoS NoS et al. [12] control Stolzenburg Randomised double- 422 59 NoS NoS et al. [13] blind Canat 42 Randomised prospective 112 63 18 (16.07%) et al. [14] (37.5%) Yiou 30 6 Retrospective review 75 59.4 et al. [15] (40%) (8%) Haglind Prospective controlled 2625 63 NoS NoS et al. [16] nonrandomised Kim D. Randomised prospective 97 54 NoS NoS et al. [17] placebo-control Capogrosso Retrospective review 2364 61 NoS NoS et al. [18] Sooriakumaran Prospective non- 2545 63.3 556 (35.2%) 99 (6.2%) et al. [19] randomised

RESULTS most encountered comorbidity was the cardiovascular disease (37.5% - 100%) followed by diabetes mellitus (6.2% A total of 11,831 patients were enlisted in the 15 studies we – 16.07%). For most ED patients the presence of have included for analysis. Mean age of all patients was cardiovascular comorbidities creates the premises of disease 61.91 years with a range between 54 and 67.9 years. (Table trigger, followed by diabetes and obesity. [] All patients were 1) Although not all the studies specifically mentioned the

10 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine diagnosed (by means of PSA, prostate biopsy and pelvic MRI) prostatectomy (n = 4,929). (Table 2) Most patients benefited with localised prostate cancer mainly T1c - T2c, with Gleason of bilateral nerve sparing (BNS) dissection of the nerve score ranging between 6 and 7 and PSA values below 10 bundles (n = 6,852) by al known technical procedures ng/ml. (Table 2) These data were correlated with the (interfascial/intrafascial), which represented 63.68% of the postoperative pathology findings which in some cases operated patients. When case did not impose BNS changed the actual diagnostic stage according to d’Amico dissection, unilateral nerve sparing technique was criteria. As it can be seen in Table 2 in some cases based on attempted (n = 3,209) which was performed in 29.82% of the the degree of invasion the cancer stage was changed from operated patients. The rest of 6.5% of the patients either had localised to locally-advanced (T3b-T4) and based on Gleason not benefited of NS dissection either the information about score from low or intermediate risk to high risk (Gleason 8). the NS status was not mentioned. [16, 18, 19] The NS status at the end of the surgery was crucial because it ensured the All patients were proposed for radical prostatectomy using premises for postoperative ED recovery although most of various approaches: open surgery (n = 4,460), conventional the patients experienced postoperative neuropraxia. laparoscopy (n = 1,371) or robot assisted laparoscopic

Table 2. PCa status and surgical approach for each study

PCa clinical Gleason Surgical approach Nerve sparing Trial author stage Score OP CLP RALP BNS UNS

Vickers 859 546 757 657 1498 T2a – T3 6 - 8 et al. [8] (40%) (25%) (35%) (30%) (70%) Montorsi 68 29 31 423 T1c – T2c 6 - 7 - et al. [9] (48.9%) (20.9%) (22.3%) (100%) Seo 92 T2a – T2c 4 - 8 - - 57 (62%) 35 (38%) et al. [10] (100%) Nakano 24 79 T2 – T3 6 - 7 NoS NoS NoS et al. [11] (23.3%) (76.7%) Fode 7 61 37 31 T2 – T3 NoS - et al. [12] (10.3%) (89.7%) (54.41%) (45.58%) Stolzenburg 189 88 115 422 T2-T3 ≤ 7 - et al. [13] (44.78%) (20.85%) (27.25%) (100%) Canat T2a-T2c 5 - 8 NoS NoS NoS 112 - et al. [14] Yiou 75 75 T2 - T3 6 - 8 - - - et al. [15] (100%) (100%) Haglind T1 - T3 6 - 8 778 - 1847 1318 750 et al. [16] Kim D. 58 36 96 1 T1c – T3 6 - 8 - et al. [17] (61.7%) (38.3%) (98.96%) (1.04%) Capogrosso 614 633 1117 2253 78 T2 - T4 NoS et al. [18] (26%) (27%) (47%) (95%) (3%) Sooriakumaran 1792 753 1283 737 T2 – T4 NoS - et al. [19] (70.41%) (29.58%) (50.41%) (28.95%) Kim S. 95 95 T2a – T2c 4 - 8 - - - et al. [20] (100%) (100%) Kwon NoS NoS NoS NoS NoS NoS NoS et al. [21] Jo 120 T2a – T2c 5 - 7 - - NoS NoS et al. [22] (100%) NoS – no specification; OS – open surgery; CLP – conventional laparoscopic prostatectomy; RALP – robot assisted laparoscopic prostatectomy; BNS – bilateral nerve-sparing; UNS – unilateral nerve-sparing.

Table 3. Preoperative EF assessment and postoperative EF recovery means and results Type of EF Preop. mean Means of Trial Trial author Subgroups Recovery rates questionnaire EF score penile recovery duration

Vickers IIEF-6 22 - 937 PDE5i (NoS) - 2 years 36% (NoS) et al. [8] (59%) PGE1 ICI

Montorsi IIEF-EF 22 - 423 PDE5i Tadalafil 5 mg – OaD – 139 9 Tadalafil 5 mg – 25.2% et al. [9] (100%) (32.86%) months Tadalafil 20 mg – 19.7% Tadalafil 20 mg – PRN – Placebo – 14.2% 143 (33.8%) Placebo – 141 (33.3%) Seo IIEF-5 22 - 92 PDE5i Tadalafil 5 mg – OaD 1 year Tadalafil 5 mg – 13.2% et al. [10] (100%) Non-Tadalafil Non-Tadalafil – 7.7% Nakano IIEF-5 17.7 PDE5i Vardenafil 10 mg – OD – 1 year Vardenafil 10 mg + et al. [11] 30 (29.12%) Vardenafil 20 mg – 21 (60%) Vardenafil 20 mg – PRN – No treat – 26 (38.2%) 5 (4.85%) No treat – 68 (66.01%) Fode IIEF-5 25 PDE5i PDE5i + PVS - 30 1 year PDE5i + PVS – 16 (53%) et al. [12] PVS PDE5i - 38 PDE5i – 12 (32%)

Stolzenburg IIEF-5 22 – 422 PDE5i Tadalafil 5 mg – OaD - 102 9 Tadalafil 5 mg – OaD – 29 et al. [13] (100%) Tadalafil 20 mg – PRN - months (28.43%) 112 Tadalafil 20 mg – PRN – 24 Placebo - 106 (21.42%) Placebo – 27 (25.4%)

Canat IIEF-6  22 PDE5i Tadalafil 5 mg – OpW - 38 1 year Tadalafil 5 mg – OpW – et al. [14] Tadalafil 20 mg – PRN - 40 19.89 (mean IIEF) Non-Tadalafil - 34 Tadalafil 20 mg – PRN – 15.8 (mean IIEF) Non-Tadalafil – 13.47 (mean IIEF)

Yiou IIEF-EF  24 PDE5i PDE5i + PGE1 ICI – NoS 2 years M12 – 19.6 (mean IIEF – et al. [15] EHS  2 PGE1 ICI No treatment - NoS treat.) M12 – 18.07 (mean IIEF – no treat.) M24 – 4.63 (mean IIEF – treat.) M24 – 4.92 (mean IIEF – no treat.)

Haglind IIEF-5  21 - RRP 1 year RRP – 124 (15.93%) IIEF-5  et al. [16] RALP 21 RALP – 339 (18.35%)

Kim D. IIEF-EF  21 (100%) PDE5i Sildenafil 50 mg - OaD + 13 Sildenafil 50 mg - OaD + et al. [17] (Mean 28.1) Sildenafil 100 mg - PRN months Sildenafil 100 mg – PRN – 15 Placebo + (32.4%) Sildenafil 100 mg - PRN Placebo + Sildenafil 100 mg – PRN – 13 (29%)

Capogrosso IIEF-6  24 PDE5i 12 months - 1779 2 years 12 mo – 483 (27%) et al. [18] (Mean 27) PGE1 ICI 24 months - 1095 24 mo – 377 (34%)

Sooriakumaran Penile stiffness 1702 (66.9%) PGE1 ICI RARP 2 years RARP – 377 (21%) et al. [19] Morning erection PPP. PGE1 IUr RRP RRP – 106 (14%) Kim S. IIEF-5 22.4 PDE5i Tadalafil 5 mg – OaD – 2 2 years Tadalafil 5 mg OaD – 2 yr. – et al. [20] years 16.1 (mean IIEF) Tadalafil 5 mg – OaD – 1 Tadalafil 5 mg OaD – 1 yr. – year 13.5 (mean IIEF) No Tadalafil No Tadalafil – 9.4 (mean IIEF)

12 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine

Kwon Penile lenght NoS PDE5i Complete recovery 1 year CR – 318 (60.2%) et al. [21] SHIM (Sildenafil, Incomplete recovery IR – 210 (39.8%) Vardenafil, Tadalafil)

Jo IIEF - 5  17 PDE5i Early treatment - 58 1 year ET – 24 (41.4%) et al. [22] (Sildenafil 100 (3 wk. postop.) DT – 11 (17.7%) mg) Delayed treatment - 62 (3 mo. postop.)

PPP – preoperative potent patients; IIEF – International Index of Erectile Function; OaD – oance a day; PRN – pro re nata (on demand); ICI – intracavernosal injections; IUr – intrauretral;

For pre and postoperative EF assessment the IIEF recovery was the administration of PDE5i which was tested questionnaire was preferred in most of the studies in with various ways of administration and dosages and different forms: the classical 15 questions IIEF-EF or the compared with placebo or no treatment control groups. The shorter IIEF-5/IIEF-6 questionnaires. most commonly used drug in the included trials was Tadalafil [9, 10, 13, 14, 20], followed by Sildenafil [17, 22] and Most authors preferred the IIEF-5 questionnaire due to its Vardenafil. [11] Some trials studied the single use treatment reliability and rapid way of determining the EF status. (Table of PDE5i compared with combinations of PDE5 with PGE1 ICI 3) In most of the studies that used the IIEF questionnaire a [8, 15, 18] or penile vibratory stimulation (PVS) devices. [12] cut-of value of 22 points was established for preoperative There were also particular studies that did not compare the potent men [8–10, 13, 14] while others established close cut- method of PDE5i administration but rather the rate of EF of values. [16, 20] Only two studies used other means of EF recovery [8, 21], the moment of treatment initiation [22] or assessment. Sooriakumaran et al. used two questions about the length of treatment administration. [18] Haglid et al. penile stiffness and morning erections, both having single compared the EF recovery based only on the surgical choice answer from 5 possible variants. [19] In a particular approach type (open vs. RALP) and not interfering with any case Kim et al. compared the penile length both pre and medication in this regard while Sooriakumaran et al. postoperative and additionally used the SHIM questionnaire compared the same surgical approaches using PGE1 for ED appraisal. [20] treatment setting. [16] The most frequently used treatment method for penile

Figure 1: Multivariate comparison of PDE5i OaD and PRN treatment compared to control group

Although the articles included in our meta-analysis had recorded at the end of the trial for each group (OaD, PRN and dichotomous criteria of study design we could find control). As it can be observed in fig. 1 the higher rates of similarities between some of them, which allowed us to recovery are in favour of PDE5i OaD group compared to organise subgroups of trials. On multivariate analysis we control group (CI 95%, P < 0.0001). On a lower scale, the have included trials in which PDE5i was administered as OaD same result could be observed when PDE5i OaD treatment and PRN treatment compared to a pacebo control group. was compared to PDE5i PRN although in this analysis only 2 The variables included in analysis were the mean IIEF scores studies had the necessary data to be included (CI 95%, P =

0.48). (CI 95% P = 0.40). Also when we have compared PDE5i OaD with PRN group the better results were slightly higher in In a univariate analysis we also compared the trials were only favour of OaD group thus resulting that the benefit of PDE5i the percentage of recovered patients after PDE5i treatment OaD compared to PRN treatment was minor (CI 95%, P = was given. (Fig 2) As in previous assessment the PDE5i OaD 0.95). group had higher recovery rates compared to control group

Figure 2: Recovery rates in trials using PDE5i OaD and PRN treatment compared to control group

DISCUSSION Two trials included in our meta-analysis studied the rate of ED occurrence and penile recovery degree after RALP Erectile dysfunction is a major postoperative complication compared to RRP in high volume centres from Sweden. In a for men who undergo radical prostatectomy, alongside 2015 prospective study, Haglid et al. found a slightly higher urinary incontinence. At this moment prostate cancer is the rate of penile recovery for RALP, but the differences were second most diagnosed neoplasia in men due to the not significant for these two surgical approaches. [16] In a systematic determination of PSA. [23] Radical prostatectomy recently conducted prospective trial, Sooriakumaran et al. remains the most important therapeutic procedure for (2018) found earlier recovery rates of EF for RALP (21%) localised PCa having a postoperative life-expectancy of 10 compared to RRP (14%) due to a more precise identification years. [2] Although the technical enhancements of and preservation of nerve bundles during RALP. Also the prostatectomy reduced the rates of postoperative rates of post-surgical positive margins were similar to open complication, ED still remains one of the most complex surgery which is an important aspect for oncological problem for PCa patient’s quality of life. radicality. [19] Both studies were prospective non- Even if the nerve sparing dissection technique of the randomised. prostate bundles partially improved the ED rates, there are One key aspect of ED diagnosis is the use of pre and still problems regarding the postoperative penile postoperative EF assessment questionnaires. As we have rehabilitation. Neuropraxia is the main pathological shown above the most frequently used questionnaire was alteration that leads to major structural changes of the IIEF-5 which is the short version of the IIEF-EF questionnaire. penile tissue. This can also lead to veno-oclusive Other questionnaires like Sexual Health Inventory for Men dysfunctions of blood drainage. Due to the constant state of (SHIM) or Expanded Prostate Cancer Index Composite hypo-oxygenation the penile smooth muscles suffers a (EHGS) have similar predictive results to IIEF and should be process of apoptosis and fibrosis which in time are producing routinely used for postoperative ED diagnosis. [25, 26] irreversible penile malformations such as penile shortening of 2-3 cm and Peyronie disease. [5, 24] In this regard it is It is a known fact that older age, cardiovascular advisable that urologists should discuss with their patients comorbidities and diabetes mellitus are the most important about the possibility of postoperative ED in various rates – predictive factors for ED onset. [27, 28] On the other hand from temporary to permanent. [25] the younger age and the lower body mass index are

14 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine demonstrated protective factors for ED recovery. [2] Even introduced and studied by Prof. Montorsi et al. [31] The most though not all the studies included in our meta-analysis important advantage of PGE1 ICI is that is very effective in reported the rate of cardiovascular disease and diabetes any type of surgical approach even if nerve sparing was not mellitus we could emphasize that in the post prostatectomy indicated, though the cost, the need to refrigerate the setting these two comorbidities could have significant product, the invasiveness of the treatment and the side impact on ED onset and the subsequent penile recovery. [10, effects are a majos counterbalance for treatment initiation. 14, 15, 19, 20, 21] [24] In our meta-analysis, Vickers et al., Yiou et al. and Capogrosso et al. have used PGE1 ICI in combination with Penile rehabilitation is the cumulus of therapeutically means PDE5i treatment for penile recovery. The results achieved by which includes medication, medical devices or actions, Yiou at 12 respectively 24 months of combined treatment simple or in various combination recommended for erectile were very similar in both drug-administered and control function recovery. [5] PDE5i are the most frequently used group. [15] On the other hand Capogrosso et al. found that drugs in clinical practice for penile rehabilitation and to this at 24 months of combined PGE1 ICI and PDE5i treatment the date is the most studied therapeutic method administered rates of recovery were higher than at 12 months (34% vs. to ED patients. The main advantage of PDE5i therapy is that 27%). [18] there are quick and easy to administer but it requires the integrity of at least 1 cavernosal nerve bundle. Also the Vacuum and vibratory devices are non-invasive and very treatment cost and side effects (headache, flushing and effective in EF recovery especially when combined with palpitations) are not negletable. [24] PDE5i treatment. Fode et al. demonstrated in their randomised prospective trial that EF recovery could be very In their common trial Montorsi et al. (2014) and Stolzenburg effective when penile vibratory stimulation devices are used et al. (2015) studied the effect of Tadalafil administered once in conjunction with PDE5i (53% recovery rate) compared to a day (OaD) and on demand (PRN) compared to a placebo- PDE5i only group (32%). [12] controlled group and found that the OaD administration of Tadalafil 5 mg has the best results for penile recovery after Regarding treatment initiation Jo et al. demonstrated that prostatectomy. [9, 13] Similar results for Tadalafil OaD early penile rehabilitation commencement (at 3 weeks after administration were also found by Seo et al. (2014), Canat et surgery – immediately after urinary catheter removal) has al. (2015) and Kim S. et al. (2018) based on the mean IIEF better recovery results than delayed treatment initiation (at score at the end of the trial. [14, 20] Nakano et al. (2014) and 3 months postoperative). [22] Kim D. et al. (2016) found similar results at the end of their trials although they have studied the effect of Vardenafil CONCLUSIONS respectively Sildenafil. [11, 17] In our multivariate analysis The postoperative EF recovery of PCa patients can be the PDE5i OaD treatment has evident advantages over achieved using a multitude of penile recovery strategies. For placebo and on demand PDE5i administration for this purpose a preoperative assessment is very important, by postoperative penile recovery rate. using erectile function questionnaires. Patients should be Regarding the choice of PDE5i treatment there are no informed about the postoperative ED risk and should be specific trials that compare the efficiency of currently counselled in this area. available substances in postoperative ED. In a 2005 open- Nerve-sparing robot assisted laparoscopic prostatectomy is label randomised trial Eardley et al. emphasized that the most protective surgical approach for EF preservation. Tadalafil is the preferred PDE5i used for ED treatment PDE5i is the main therapeutic approach for postoperative because of long-lasting effect in time compared to Sildenafil penile recovery. that allowed patients to have less concerns about spontaneity of erection. [29] Further more Hyndman et al. Tadalafil 5 mg once a day was the most effective long term demonstrated in their randomised trial that Sildenafil could therapy for ED treatment both single use or in combination have major impact over urinary continence, thus impairing with PGE1 and other devices (vacuum or vibratory the overall recovery of PCa operated patients. [30] stimulation). Penile prosthesis implants should be regarded as last resort treatment option for cases when no penile Prostaglandin E1 intracavernosal injection (PGE1 ICI) was the recovery strategy has been successful. first type of treatment for postoperative EF recovery,

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16 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine multicentre, randomized, crossover study comparing sildenafil randomized trial of nightly vs on-demand dosing regimens. J. Can. citrate and tadalafil for treating erectile dysfunction in men naïve to Urol. Assoc. 2015;9(11-12December):414-419. doi:10.5489/ phosphodiesterase 5 inhibitor therapy. BJU Int. 2005;96(9):1323- cuaj.3169. 1332. doi:10.1111/j.1464-410X.2005.05892.x. 31. Salonia A, Adaikan G, Buvat J, et al. Sexual Rehabilitation After 30. Hyndman ME, Bivalacqua TJ, Mettee LZ, Su LM, Trock BJ, Treatment for Prostate Cancer—Part 2: Recommendations From the Pavlovich CP. Nightly sildenafil use after radical prostatectomy has Fourth International Consultation for Sexual Medicine (ICSM 2015). adverse effects on urinary convalescence: Results from a J. Sex. Med. 2017;14(3):297-315. doi:10.1016/j.jsxm.2016.11.325.

Article received on March 25, 2019 and accepted for publishing on September 21, 2019. REVIEW ARTICLE

Toxoplasma Gondii infection and the signs of fetal affection during pregnancy as seen on ultrasound

C.A. Ispas2, M. Bujor-Moraru2, A. Bibire1, F. Isopescu2, M.J. Gardescu1, C. Teodorescu1, A. Edu2

Abstract: Toxoplasma Gondii is a protozoan parasite which causes multisystem affections and unusual clinical outcomes. Although primo- infection takes place frequently during childhood, there is a possibility for it to be found during pregnancy, when it could lead to congenital toxoplasmosis, with serious ocular and cerebral lesions (that can be identified during ultrasound). The positive diagnostic for the mother is made after a serologic test and for the fetus the parasite can be found in the amniotic fluid during amniocentesis. A prompt treatment can avoid the presence of cerebral lesions (hydrocephalus, ventriculomegaly, intracerebral calcifications). Keywords: protozoan, pregnancy, serology, amniocentesis, hydrocephalus, ventriculomegaly, intracerebral calcifications

INTRODUCTION The incidence of maternal infection during pregnancy varies between 1-8 ‰ [1, 5]. Toxoplasma Gondii is a protozoan parasite that affects the human body in many ways; Infection generally occurs in In the acute phase the infection is usually asymptomatic. If infancy or adolescence, with declining prevalence in symptoms occur, these are nonspecific, such as fatigue, developed countries in the temperate climate [1, 2]. During fever, headache, malaise, and myalgia [6]. an initial infection, an immunocompetent body will limit the Differential diagnosis is made by clinical appearance of acute spread of the parasite, but it remains cantonized in the Epstein-Barr infection, CMV, HIV, Treponema, sarcoidosis, nervous and muscular tissues, and cannot be completely Hodgkin's disease or lymphoma [1]. eliminated [3]. Positive diagnosis of maternal infection in pregnancy When the infection occurs during pregnancy, there is a risk involves at least two blood test results with seroconversion of transmission to the fetus, resulting in congenital from negative to positive for Toxoplasma-specific IgM or IgG. toxoplasmosis that can be manifested by ocular and Note that the presence of only IgM in the blood does not neurological damage. Proliferation of the parasite may automatically involve acute infection, with data showing continue, and cerebral tissue destruction may occur even that only 20% of patients with IgM positive had acute after an important maternal immune response, including IgG infection with Toxoplasma [7]. production [1, 4]. Screening for Toxoplasma is performed in some European countries on a monthly basis or at 3 months during 1 Nicolae Malaxa Clinic Hospital, Bucharest, Romania Corresponding author: F. Isopescu MD 2 “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania [email protected]

18 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine pregnancy, with the major advantage of rapidly detecting recent infection requires additional tests [ 13]. A very useful such infection and increasing the effectiveness of therapy option is testing IgG avidity, which is very high in old [8]. The US, Canada and UK do not routinely test pregnant infections, but may remain at a low level for some patients women because the prevalence of primo-infection during for some years [14]. IgG titration may be a variant, but is pregnancy appear to be sufficiently low and the costs seem insufficiently evaluated and requires a reference laboratory to outweigh the benefits [5,9]. On the other hand, repeated for good quality of the results [1]. tests increase the number of false positive results, resulting in an unnecessary elevation in the number of prenatal MATERIAL AND METHOD invasive investigations and the number of treated patients. Fetal infection occurs by transplacental transmission after These drawbacks should be weighed against the potential maternal primo-infection. The risk of transmission to the benefit of early-onset therapy, which appears to be present fetus increases drastically with increasing gestational age only in the case of serious but rare cases of neurological [15]. In a meta-analysis of 1438 patients in 26 cohorts by sequelae of congenital toxoplasmosis [1,11]. individual patient data, it is estimated that the risk of IgM antibodies are positive in the first week of infection. maternal-fetal transmission is 15% when maternal Diagnosis of the primo-infection is much more precise when seroconversion occurs at 13 weeks, 44% when the initial testing shows IgM positive and negative IgG, and seroconversion takes place at 26 weeks and 71% when at the 14-day retest both of them become positive. This seroconversion prevails at 36 weeks [15]. The study showed combination of results represents the safest diagnosis of that early therapy seems to reduce the risk of developing Toxoplasma primo-infection two weeks ago and eliminates a congenital toxoplasmosis, but the strength of this assertion possible false-positive result for IgM. If IgG retests remained is low [15]. negative, most likely positive IgM was only a false positive, and recent or older infection is excluded. An important RESULTS problem is a positive IgG and IgM result in the first trimester As mentioned earlier, the same meta-analysis has shown because once IgG is present, IgG remains Positive for life [1]. that with increasing gestational age to seroconversion, the Depending on the laboratory, the real risk of infection after risk of maternal-fetal transmission of T Gondii infection conception is only 1-3% [12]. Since IgM is only positive after increases drastically, but the risk of cerebral fetal sequelae only two weeks but can remain positive for years and IgG decreases and eye damage is not influenced by this factor reaches a maximum titer at 6-8 weeks and then decreases [15]. slowly over the next two years, the positive diagnosis of

Figure 1: A: Cerebral calcifications, B: Hydrocephaly, C: Vetriculomegaly

Ultrasound signs in Toxoplasma Gondii infection are another, most of the schemes being based on old, poor generally nonspecific. The most common signs are quality studies [15]. Another possible reason for carrying out intracerebral calcifications, intracerebral hyperecogenic amniocentesis is the exclusion of a possible fetal infection, seals and ventriculomegaly, usually medium or severe which implies an easier to follow treatment by the patient bilateral symmetry. The existence of brain pathological with lower drug doses and reduced side effects. Last but not images usually means a gloomy prognosis with numerous least, positive diagnosis can help the patient to make an possible impairments: corioretinitis, psychomotor informed decision about a possible discontinuation of retardation, blindness, epilepsy, postnatal hydrocephalus pregnancy [15], knowing that a positive diagnosis [16]. In fact, in a study of 8 confirmed children with accompanied by signs of fetal cerebral affection is associated congenital toxoplasmosis, only the absence of any with an unfavorable prognosis. On the other hand, a pathological brain imaging or the existence of only a mild negative diagnosis of amniocentesis accompanied by a lack ventriculomegaly were associated with favorable postnatal of any signs of fetal harm will limit the need for aggressive prognosis [16]. Another study found that only 43% of fetuses postnatal antibiotic treatment and will also have a beneficial with signs of impaired brain had a poor prognosis [17]. effect on the mother's psyche.

This study involving 32 fetuses with confirmed congenital CONCLUSION toxoplasmosis showed that evolution intrauterine lesions is usually very fast, on the order of days [17]. However, the T Gondii infection during pregnancy occurs in approximately cranial pathological images were identified only after 21 1-8 ‰ of the population and is usually asymptomatic. weeks of pregnancy in all the studies quoted [16, 17]. Diagnosis of this infection is done either by testing for Prognosis also appears to be more unfavorable in cases nonspecific symptoms such as fatigue, fever, headache, where seroconversion occurred earlier in pregnancy, or the malaise and myalgia, or through dedicated screening tests patient did not receive treatment for this infection [16, 17]. performed in pregnancy. There is a great variation in assessing the need for screening for this infection in the Besides cerebral signs, fetal toxoplasmosis can be world, so only some countries offer such programs. manifested along with other signs, but lacking specificity for this pathology. Thus intrahepatic calcifications, ascites, Diagnosis is performed by detecting specific IgM and IgG placental hyperecogenic images with thickened placenta, or immunoglobulins in the serum, the proportion of which rarely pleural and pericardial effusions [17, 18] can be varies depending on the age of the infection. Generally, IgM discovered. is an acute phase control, and IgG indicates an old infection that will not cross the transplacental barrier. Testing IgG Restriction of intrauterine growth and microcephaly can be avidity against the parasite seems to help increase the associated with this fetal infection, but they are not sensitivity and specificity of acute infection detection. characteristic [16, 17]. Maternal infection with Toxoplasma Gondii during Spontaneous fetal death of the fetus with the termination of pregnancy may lead to fetal infection with this protozoan by pregnancy in evolution does not seem to be more common transplacental transmission. Fetal infection does not in affected fetus [17]. A slight tendency towards early necessarily imply an unfavorable prognosis, but the childbirth appears to be influenced by obstetrical attitudes occurrence of suggestive echographic signs for this and not necessarily by fetal harm [17]. pathology is a serious factor. There are no signs of this kind Prenatal diagnosis of this fetal infection can only be achieved of fetal infection, but the affected faces usually present by performing an amniocentesis with PCR analysis of the some cerebral pathological echographic imaging, including resulting amniotic fluid. Amniocentesis carries a risk of cerebral calcifications, medium or severe symmetrical abortion or premature birth, a small but still present risk bilateral ventriculomegaly or hyperrecognized intracerebral [15]. Patients should be informed of this risk and of the seizures. There are other non-specific signs of fetal damage. potential benefits of this diagnostic approach [15]. The positive diagnosis of fetal infection is done by testing The main benefits of fetal infection confirmation with amniotic fluid. This diagnosis will help in choosing the Toxoplasma Gondii refer to the subsequent therapeutic required treatment regimen, although there are no solid course in choosing the right drug combination. One problem, randomized studies indicating the use of a particular however, is that there are no randomized studies showing regimen in a given situation. the benefit of a treatment regimen at the expense of

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1. Gilbert R, Petersen E, “Toxoplasmosis and pregnancy”, UptoDate, 11. Cortina-Borja M, Tan HK, Wallon M, Paul M, Prusa A, Buffolano accessed 23.02.2017 W, Malm G, Salt A, Freeman K, Petersen E, Gilbert RE,” Prenatal treatment for serious neurological sequelae of congenital 2. Welton, NJ, Ades,” A model of toxoplasmosis incidence in the UK: toxoplasmosis: an observational prospective cohort study.”, evidence synthesis and consistency of evidence”, Ades, JRSS (C) European Multicentre Study on Congenital Toxoplasmosis Applied Statistics. 2005; 54:385 (EMSCOT), PLoS Med. 2010;7(10) 3. Remington JS, McLeod R, Thulliez P, Desmonts G., 6th ed, 12. Gras L, Gilbert RE, Wallon M, Peyron F, Cortina-Borja M,” Remington JS, Klein J, Wilson CB, Baker CJ (Eds), Elsevier Saunders, Duration of the IgM response in women acquiring Toxoplasma Philadelphia 2006. p.947,” Toxoplasmosis. In: Infectious Disease of gondii during pregnancy: implications for clinical practice and cross- the Fetus and Newborn Infant” sectional incidence studies”, Epidemiol Infect. 2004;132(3):541 4. Ferguson DJ, Bowker C, Jeffery KJ, Chamberlain P, Squier W,” 13. Montoya J, Boothroyd J, Kovacs J., Mandell, Douglas,” Congenital toxoplasmosis: continued parasite proliferation in the Toxoplasma gondii.”, Bennett's Principles and Practice of Infectious fetal brain despite maternal immunological control in other Diseases, Seventh Ed, 2010. Vol 2, p.3495. tissues.”, Clin Infect Dis. 2013 Jan;56(2):204-8. Epub 2012 Nov 9 14. Villard O, Breit L, Cimon B, Franck J, Fricker-Hidalgo H, Godineau 5. Gilbert RE, Peckham CS,” Congenital toxoplasmosis in the United N, Houze S, Paris L, Pelloux H, Villena I, Candolfi E,” Comparison of Kingdom: to screen or not to screen?”, J Med Screen. 2002;9(3):135 four commercially available avidity tests for Toxoplasma gondii- 6. Gilbert R, Gras L,” Effect of timing and type of treatment on the specific IgG antibodies.”, French National Reference Center for risk of mother to child transmission of Toxoplasma gondii.”, Toxoplasmosis Network, Clin Vaccine Immunol. 2013 Feb;20(2):197- European Multicentre Study on Congenital Toxoplasmosis, BJOG. 204. Epub 2012 Dec 12 2003;110(2):112 15. Thiébaut R, Leproust S, Chêne G, Gilbert R, Lancet,” 7. Dhakal R, Gajurel K, Pomares C, Talucod J, Press CJ, Montoya JG, Effectiveness of prenatal treatment for congenital toxoplasmosis: a J Clin Microbiol,” Significance of a Positive Toxoplasma meta-analysis of individual patients' data”, SYROCOT (Systematic Immunoglobulin M Test Result in the United States”, 2015 Review on Congenital Toxoplasmosis) study group, Nov;53(11):3601-5. Epub 2015 Sep 9 2007;369(9556):115 8. Wallon M, Peyron F, Cornu C, Vinault S, Abrahamowicz M, Kopp 16. Malinger G, Werner H, Rodriguez Leonel JC, Rebolledo M, CB, Binquet C,” Congenital toxoplasma infection: monthly prenatal Duque M, Mizyrycki S, Lerman-Sagie T, Herrera M,” Prenatal brain screening decreases transmission rate and improves clinical imaging in congenital toxoplasmosis.”, Prenat Diagn. 2011;31(9):881 outcome at age 3 years”, Clin Infect Dis. 2013 May;56(9):1223-31. 17. Hohlfeld P, MacAleese J, Capella-Pavlovski M, Giovangrandi Y, Epub 2013 Feb 28 Thulliez P, Forestier F, Daffos F,” Fetal toxoplasmosis: 9. Paquet C, Yudin MH,” Toxoplasmosis in pregnancy: prevention, ultrasonographic signs.”, Ultrasound Obstet Gynecol. 1991;1(4):241 screening, and treatment”, Society of Obstetricians and 18. Liana Ples, Romina-Marina Sima, Cristina Moisei, Marius Moga, Gynaecologists of Canada, J Obstet Gynaecol Can. 2013;35(1):78 Laura Dracea,” Abnormal ultrasound appearance of the amniotic 10. American College of Obstetricians and Gynecologists,” Practice membranes – diagnostic and significance: a pictorial essay”, Med bulletin no. 151: Cytomegalovirus, parvovirus B19, varicella zoster, Ultrason 2017:0, 1-5 ISSN 2066-864 and toxoplasmosis in pregnancy.”, Obstet Gynecol. 2015;125(6):1510.

Article received on May 20, 2019 and accepted for publishing on August 23, 2019. ORIGINAL ARTICLE

High values of procalcitonin in non-septic patients with thermal and airway burns – an early severity predictor?

Bogdan I. Pavel1,2, Luisa M.V. Vătășoiu1, Adrian T. Stanculea1, Adina M. Milos1, Alina A. Pastrav1, Adrian E.I. Roșca2, Ana- Maria S. Zăgrean2, Adrian M.E. Otoiu3, Mihaela A. Covrig3, Ileana C.P.V. Boiangiu1

Abstract: Purpose – Combined airway burns with cutaneous burns is a further negative prognostic factor in burn patients. In this study we tried to identify the most relevant negative prognostic factors at admission in a relatively homogeneous population. Methods – In this study, ten young patients who suffered from skin and airway burns following a fire in a club, admitted to our ICU department, were included. At the intake, ABSI and APACHE II scores were calculated, and a series of biomarkers were also collected. The correlation and associations between gravity scores and biomarkers was then calculated using the Spearman correlation and Chi square tests. Results – Based on data analysis, we found very high values of ABSI 8.9 ± 1.4 and APACHE II 25.4 ± 2.79 scores, procalcitonin (46.16 ± 68.18 ng/ml), white blood cells (41.26 ± 15.66 × 103/mm3), hematocrit (51.6 ± 7.31 %), and low corrected albumin values (22.08 ± 4.66 g/l). The statistical analysis revealed a good correlation between gravity scores and values of PCT and low albumin levels (p< 0.05). The mortality was also associated with the need for vasopressor support from the beginning (p < 0.05). Conclusions – Adverse prognostic factors that we have identified in this group of young patients with skin burns and airway include: increased values of the severity scores and PCT, low values of corrected albumin and vasopressor support necessary at admission.

INTRODUCTION surprisingly high in the developing countries, including Europe’s burn centers, where it ranges between 1.4% and Burns represent one of the most aggressive injuries of the 18% [5]. The high mortality is associated with a number of human body known for high mortality, thus raising major factors, including long-term severity of burn driven public health issues such as increased length of inflammatory response of the host, secondary human body hospitalization and recuperation, along with psychological frailty and abnormal response to further injuries like sepsis and physiological traumas, all these leading to increased [6, 7]. The association of airways’ and cutaneous burns costs [1-4]. induces a supplemental increase of the inflammatory Burns-induced mortality rate is globally increased, and response, difficult to quantify because of the inaccurate evaluation of the burns from the surface of the lungs and burn degree same time [8]. Consequently, in the case of 1 Clinical Emergency Hospital of Plastic Reconstructive Surgery and Burns, Bucharest, Romania mixed cutaneous-and-airway burns the mortality increases, 2 Carol Davila University of Medicine and Pharmacy, Division of Physiology and Neurosciences, Bucharest, Romania Corresponding author: Bogdan I. Pavel, MD, PhD 3 The Bucharest University of Economic Studies, Department of Statistics and Econometrics, Bucharest, Romania [email protected]

22 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine despite the usage of advanced techniques to support lung following the fire in a club in Bucharest, Romania and we function (Extracorporeal membrane oxygenation – ECMO) studied their correlation with mortality. [9-12]. On the other hand, in the beginning, it is unclear whether a patient has both cutaneous and airway burns, as MATERIAL AND METHOD for the latter it might take up to 48 hours to observe any This study includes the victims of a fire at a club in Bucharest, clinical manifestations. As such, the airways' burns in the night of 29-30 October 2015, all of them hospitalized assumption is made based on anamnesis and correlation that night in the Intensive Care Unit of the Emergency between the high incidence of airways burns and the Hospital for Plastic Reconstructive Surgery and Burns, in increased surface of cutaneous burns [11]. If one can Bucharest. All admitted patients underwent surgery (non- clinically suppose an airways burn (in this case a excisional debridement) and subsequently, they were bronchoscopy is usually performed for a certain diagnosis), connected to artificial breathing devices, central venous and there is no toll to quantify its’ impact on the inflammatory urinary catheters being placed on them too. response of human body. Currently, there is just an indirect estimation of their aggression on the human body using Hydroelectrolytic rebalancing started from the operating either dedicated markers or severity scores. Despite the room with a crystalloid solution (Ringer lactate), following wide availability of severity scores as Acute Physiology And the Parkland protocol (4 ml/kgc/Total Burn Surface Area- Chronic Health Evaluation (APACHE II and APACHE III), TBSA), while in patients who could not maintain a Mean Abbreviated Burn Severity Index (ABSI), and biomarkers Arterial Pressure (MAP) > 65 mmHg, epinephrine was (such as microalbuminuria, serum albumin, C-reactive administered in continuous perfusion from the beginning. protein, procalcitonin (PCT)), both used to estimate the Patients were admitted to ICU between 00:00 and 03:00 on severity of cutaneous burn injuries, they are not sufficiently 30 October, and the first set of blood tests was harvested at standardized for the estimation of mortality risk in case of 6:00 on 30 October. To determine the quantitative combined thermal and airway burns [13-16]. procalcitonin (PCT) values, the testers arrived after about 48 hours from admission. Considering the increased mortality and likeliness of combined burns complications, it is paramount to know the We estimated the severity of the patients’ burns using ABSI severity degree and the prognosis of the burn evolution from and APACHE II scores. the beginning. In, order to estimate the appropriate level of serum albumin In the current study, we evaluated the absolute values of under hypovolemic conditions, we corrected it in serological markers and severity scores upon admission to accordance to the hematocrit (Ht) level, using the following the Intensive Care Unit (ICU) in a group of young adult formula: patients who presented both cutaneous and airway burns 푛표푟푚푎푙 퐻푡 푐표푟푟푒푐푡푒푑 푎푙푏푢푚𝑖푛 푙푒푣푒푙 = 푚푒푎푠푢푟푒푑 푎푙푏푢푚𝑖푛 푙푒푣푒푙 × 푚푒푎푠푢푟푒푑 퐻푡 where normal hematocrit (Ht) is 45% for men and 40% for relative frequencies, respectively for the qualitative women. variables. Batch variance analysis was performed with the Levene test. For the analysis of the difference between the For the determination of the blood cells count, the Ac-T 5 diff environments the t-student test was used together with the CP (Beckman Coulter, Germany) was used. The biochemical calculation of the confidence intervals. For the correlation markers were measured using the Konelab-20i/20XTI analysis Spearman correlation coefficient and chi-square (Thermo Electron Corporation, Finland). Coagulation nonparametric test were employed. In addition, for the parameters were determined using the ACL-7000 correlation analysis we performed Percentile Bootstrap instrument (Instruments Laboratory, Italy). PCT was Correlation in order to calculate the Pearson parametric assessed using the VIDAS, bio Merieux VITEK, France. correlation coefficient. Confidence intervals for the value of The conduct of this study was approved by the Ethics the t test and the Pearson correlation coefficient were Committee of the Emergency Hospital for Plastic obtained through the bootstrap method with 10,000 and Reconstructive Surgery and Burns (number 5/10 July 2017). 1,000 replications, respectively. The results were considered statistically significant for a p value <0.05. For the statistical Statistical analysis analysis IBM SPSS 18.0 and several R packages, such as The data is presented as the average ± standard deviation wBoot, were used. for quantitative variables, and absolute frequencies and

RESULTS one month and one year are also provided in Table 1. Blood tests revealed: high levels of white blood cells (WBC) of During the night of 29-30 October 2015, ten patients (seven 41.26 ± 15.66/mm3, elevated hematocrit (Ht) level51.6 ± male and three women) were admitted in our ICU. The deep 7.31%, albumin values of 25.95 ± 4.52 g/dl (uncorrected) and cutaneous burn lesions (3rd degree) ranged between 5% and 22.08 ± 4.66 g/dl (corrected), creatinine level of 1.39 ± 0.53 50% of the body surface area and the age range of admitted mg/dl, glutamic oxaloacetic transaminase (TGO) values of patients was 18 to 40 years old. Their ABSI scores were 70.8 ± 38.31 mg/dl and glutamate-pyruvate transaminase between 7 and 11, associating a mortality between 10% and (TGP) levels of 54 ± 43.28 mg/dl. 60 - 80% and the APACHE II score ranged between 23 and 30, which is associated with a 46% to 70.3% mortality at Forty-eight hour procalcitonin (PCT) values were extremely admission. In Table 1 we report demographical data, several elevated: 49.85 ± 67.34 ng/ml (PCT was measured in eight laboratory measures describing the admission conditions patients). The coagulation parameters activated partial and, for some of them, the measures sampled 30 hours after thromboplastin time (APTT), international normalized ratio admission. (INR), and platelets (PLT) we report in Table 1 were measured at 30 hours from admission (the second set of Table 1. Characteristics of the patients included in the study analysis) because at the first set there were no changes (first set: APTT = 29.3 ± 3.01, INR = 1.15 ± 0.18, PLT = 306.2 ± Values 108.47 ). So we found: APTT = 40.27 ± 10.76 seconds, INR = WBC (1000/ml) 41.26 ± 15.66 1.87 ± 0.45, PLT = 142.5 ± 50*103/ml. The pH values of 7.04 PLT (1000/ml) 142.5 ± 50 ± 0.11 presented in the table were obtained from central APTT (sec) 40.27 ± 10.76 venous blood samples after the central venous catheters were placed. The temperature at admission had a mean INR 1.87 ± 0.45 value of 35.95 ± 0.61 Celsius degrees. Of the ten patients in pH 7.04 ± 0.11 ICU eight needed vasopressor support at admission and also Temperature (0C) 35.95 ± 0.61 during the next days.

Albumin (g/l) 25.95 ± 4.52 Table 2 presents values of serological markers, Total Burn Corrected Albumin (g/l) 22.08 ± 4.66 Surface Area 3rd degree in terms of mean ± standard PCT (ng/dl) 46.16 ± 68.18 deviation, and the number of patients who required vasopressor support with epinephrine (norepinephrine from Creatinine (mg/dl) 1.39 ± 0.53 the second day) from the admission, considering the total TGO (mg/dl) 70.8 ± 38.31 study group and the subgroup of those who did not survived. TGP (mg/dl) 54 ± 43.28 Table 2. Values of serological markers, total burn surface area One month mortality (%) 80% (TBSA) and norepinephrine status in the study group and One year mortality (%) 90% subgroup WBC (1000/ml) 41.26 ± 15.66 Total Non Survivors p value

PLT (1000/ml) 142.5 ± 50 Albumin (g/l) 25.95 ± 4.52 24.63 ± 3.97 0.26 APTT (sec) 40.27 ± 10.76 Corrected 22.08 ± 4.66 21.25 ± 4.62 0.35 INR 1.87 ± 0.45 Albumin (g/l) pH 7.04 ± 0.11 PCT (ng/ml) 46.16 ± 68.18 65.88 ± 71.52 0.35 Temperature (0C) 35.95 ± 0.61 Hematocrit (%) 51.6 ± 7.31 51.19 ± 5.07 0.43 Albumin (g/l) 25.95 ± 4.52 WBC (1000/ml) 41.26 ± 15.66 44.00 ± 16.22 0.36 Norepinephrine 8 (10) 8 0.03 Note: TBSA III = Total Burn Surface Area 3rd degree, WBC = white blood cells, PLT = platelets, APTT = activated partial thromboplastin time, INR = TBSA III (%) 25.7 ± 16.73 29.62 ± 16.27 0.30 international normalized ratio, Corrected Albumin represents corrected values of albumin according to hematocrit, PCT = procalcitonin, TGO = glutamic Note: WBC = white blood cells, PCT = procalcitonin, Corrected Albumin oxaloacetic transaminase, TGP = glutamate-pyruvate transaminase. represents corrected values of albumin according to hematocrit. TBSA III = Total Burn Surface Area 3rd degree. Data are presented as mean ± standard deviation. For these variables, the values presented are mean ± standard deviation. Gender distribution and mortality after In the case of norepinephrine requirements association chi-

24 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine square test were performed. values for those that died. Due to the existence of abnormal values for TBSA and PCT 48 hrs, we have eliminated TBSA Statistical significance is considered for a p value < 0.05. We values exceeding 8, and PCT 48H values under 200. Table 3 performed parametric t-tests for the above variables in presents two versions of the t-tests: regular tests assuming order to assess whether there are sizeable differences unequal variances, and bootstrap tests with 10000 between the values recorded for all patients versus the replications.

Table 3. T-tests and bootstrap confidence intervals Bootstrap confidence interval limits Parameter T-test value P-value Lower 95% Upper 95% Lower 90% Upper 90%

Corrected albumin 0.37421 0.7134 -2.90235 4.7619 -2.52655 4.1034 TBSA -0.50211 0.6227 -17.32 9.86 -2.3798 4.0939 PCT -0.38325 0.7093 -105.1441 8.8855 -100.0322 5.32 WCB (divided by 100) -0.36157 0.7228 -16.404 8.845175 -13.9097 7.57075 TBSA>8 -0.28615 0.7793 -15.71875 11.5 -13.75 9.25 PCT48h <200 -0.4898 0.6367 -37.916 19.39814 -32.02486 14.458 Albumin 0.65438 0.5223 -2.195 4.57 -1.575 4.21 Hematocrit 0.16848 0.8683 -4.50495 5.85595 -3.6257 5.1709

All results show that differences between the two samples Table 4. Spearman correlations are non-significant at 90% confidence level. Further, we Correlation investigated the correlations between severity scores (ABSI Correlation p value Coefficient and APACHE II), procalcitonin (PCT) and corrected albumin using the Spearman non-parametric (rank) correlation ABSI - APACHE II 0.54 0.053 coefficient. Table 4 shows that these correlations are APACHE II - Corrected Albumin -0.59 0.036 significant at 10% significance level. We pursued a more in- ABSI - PCT 0.73 0.019 depth analysis of the correlations between the above APACHE II - PCT 0.73 0.019 variables calculating the Pearson parametric correlation coefficient using the Percentile Bootstrap Correlation Table 5 presents the values of the correlation coefficient, the Method available in the package wBoot of R. intervals standard error as well as the 90% or 95% confidence obtained after 1000 replications.

Table 5. Pearson correlations: results from the Percentile Bootstrap Correlation Method, 1000 replications

Mean Standard Confidence interval Variables coefficient error Lower Limit Upper Limit Level

ABSI , Apache II 0.4904211 0.2582198 0.008991 0.8477 90% ABSI , PCT 0.5594826 0.1655465 0.2769 0.8614 95% Apache II, PCT 0.5487429 0.208531 0.1969 0.9677 95% Apache II, Corrected Albumin -0.5645653 0.2334818 -0.8677 -0.151 90%

Bronchoscopic aspects at 48 hours did not reveal severe distress syndrome (ARDS), nine patients presented mild changes, most of them related to mild or moderate ARDS and only one moderate ARDS upon admission to ICU. impairment of the lungs (Grade 0-2), only one exception with We have obtained a good correlation between the APACHE severe lesions (Grade 3) being identified. Following the II score at admission and the procalcitonin values (at 48 Berlin criteria for the classification of acute respiratory hours), ABSI score and procalcitonin and also between the

APACHE II score and albumin (Table 3). so at a burned surface of 60%, serum albumin (at admission) is around 1.95 ± 0.02 g/l [33, 34, 35]. In a recent study, it was One month after admission, two of the ten patients survived found that in patients with burn driven injuries an albumin and after one year, one patient survived. value < 2 g/l is correlated with a mortality > 80% [36]. For our patients, the value of serum albumin on admission was 25.95 DISCUSSION ± 4.52 g/dl and corrected albumin was 22.08 ± 4.66 g/dl. We Upon arrival, patients showed signs of increased burn consider it more useful to report corrected albumin values severity including high values of PCT (at 48 hours from considering that patients have high hematocrit variations as admission), WBC, APACHE II & ABSI scores, and Ht, as well as most of them are hemoconcentrated at admission. In all of low levels of albumin. Eight of the ten admitted patients the studies we considered, the serum albumin values were required vasopressor support from the beginning. presented without being adjusted to the hematocrit value.

It is known that PCT, a molecule secreted by the thyroid, The APACHE II score is known as a severity score that is used intestine, liver, kidney, muscle and adipocytes under the to estimate mortality in intensive care units [37]. Its action of TNFα and IL-1β has elevated values especially in correlation with mortality is applicable to burn patients [38, bacterial sepsis [17] and not in viral infections [18] and also 39] and also to acute respiratory distress syndrome (ARDS) has a rapid kinetic growth from the first few hours of injury patients [40, 41]. In our group, the high APACHE II scores to a maximum of 12 hours [19]. PCT is also increased in septic have been interpreted as being induced by an extreme burned patients. Several studies were performed for inflammatory response probably induced by thermal and identifying a cut-off value for sepsis in burned patients, the airway burns. The ABSI score is a good indicator of the yielded values ranging from 0.5 ng/ml to 1.5 ng/ml [20-23]. severity of a thermal injury and seems to estimate quite well The highest PCT values reported for non septic patients were the burning severity in patients with airway burns [42, 43]. 2.3 ± 3.7 ng/ml [20]. ABSI score has a similar prognostic value compared to APACHE II in estimating the severity of patients with both Nevertheless, PCT was also found to be increased in non- cutaneous and airway burns. ABSI and APACHE II predict a septic states by systemic inflammatory response syndrome- similar mortality and the correlation of their scores is at the SIRS, which is logical considering that its secretion is p = 0.05 limit. In our patients, the WBC count at admission stimulated by TNFα and IL-1β. Additionally, it has been was extremely high 41.26 ± 15.66 *103/ml. These elevated noticed that in complex visceral liver/spleen plus thoracic values can be only partially explained by hemoconcentration trauma the PCT values go up to 9.37 ± 2.71 ng/ml [24], because the mean value of the hematocrit was just slightly consequently elevated values were also found in other SIRS elevated. WBC count is also increased in other situations states [25, 26]. In burns it was found that PCT grows even in with a systemic inflammatory response but not to those the absence of infection, so patients who associate PCT values reported in our group [44, 45]. values > 2 ng/ml within the first 48 hours of admission have a mortality of 65% at 50 days [27]. According to the American The mean serum creatinine value, at 6 hours after admission, Burns Association criteria (ABA) [28], we had just one patient was 1.39 mg/dl, which may be due to hypovolemia induced who presented sepsis criteria in the first 48 hours, so the by renal hypoperfusion. On the other hand, elevated high PCT values observed in our patients (46.16 ± 68.18 transaminases with predominantly TGO/TGP increased ratio ng/ml) were explained by an extremely aggressive may be explained by their release from the burn cutaneous inflammatory response to burn. Our opinion is that this area rather than by hepatic hypoperfusion. The triad: reaction was induced not only by skin burns but also by acidosis, hypothermia and coagulopathy has a poor pulmonary injury so that high PTC values on admission may prognosis in polytraumatized patients and in burn patients also indicate an associated airway burn in patients who have [46]. This triad is particularly presents in patients with such a suspicion. cutaneous and airway burns. All the patients with skin and airway burns showed severe metabolic acidosis at Serum albumin is a marker of severity in critical patients and admission. Coagulopathy did not immediately appear but is associated with an increased mortality [29, 30]. Low levels emerged in the next 30 hours. Hypothermia (temperature of serum albumin and urinary albumin occur by increasing under 35.5°C degrees) was present in just two patients at vascular permeability induced by pro-inflammatory admission, and the mean value was about 35.95 Celsius mediators in SIRS or sepsis [31, 32]. Patients with burn degrees. The absence of hypothermia can be explained by injuries lose their albumin by increasing the capillary the very short time until the patients were brought to the permeability induced by pro-inflammatory mediators and hospital, the fire being located in a club in Bucharest, the serum albumin value correlates with the burned surface,

26 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine relatively close to the hospital. Thus, out of the three obtained with regard to vasopressor support, so all eight parameters of the triad, in our patients, only 2 of them were patients requiring vasopressor support at the admission died present. in the first month.

These changes of PCT, WBC and high APACHE II and ABSI The patients who survived one month from the incident, had scores can be explained by an extreme inflammatory PCT values at 48 hours of 3.14 ng/ml and 0.41 ng/ml, reaction or septic shock. This inflammatory reaction could be respectively, corrected albumin of 22.3 g/l and 28.45 g/l, caused by thermal cutaneous lesions and/or airway burns. respectively, while the APACHE II values were 24 and 23 Although we found a good correlation between the APACHE respectively, and ABSI scores were 8 and 7 respectively. II score & PCT & corrected albumin, and also between ABSI These were also the only patients who did not require score & PCT, we did not find a statistically significant vasopressor support at admission. correlation between the burn lesions surface or depth and the biological parameters. So we can state that the CONCLUSIONS inflammatory reaction was determined by a mixed cause The association of airway burns to the cutaneous thermal represented by the thermal cutaneous lesions and airway lesions is linked to an increased mortality even in young burns. For patients with airway burns, the etiology may be patients. There is no dedicated marker or score for the thermal or chemical. In most of our cases, bronchoscopy did assessment of the combined cutaneous and airways burns not reveal significant structural changes in the upper aggression on the human body but an association of markers airways, which led us to the idea that pulmonary injury was and scores could improve the evaluation of the burn mixed, thermal and chemical. Although we did not obtain severity. The negative prognostic factors we identified in our statistically significant changes, we found an increase in group were high PCT values at 48 hours, low corrected WBC, PCT and TBSA III values in the case of non-survivors albumin levels, vasopressor requirements and elevated compared to the mean values obtained in the entire group APACHE II and ABSI scores. The WBC count and TBSA could of patients. Yet, a statistically significant difference was be also considered in establishing the severity of the cases.

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451-60. of microalbuminuria with acute physiology scores after 24 hours. 17. Simon L, Gauvin F, Amre DK, Saint-Louis P, Lacroix J. Serum Crit Care Med. 2003 Jan; 31 (1): 98-103. procalcitonin and C-reactive protein levels as markers of bacterial 32. Waxman K, Rebello T, Pinderski L, O'Neal K, Khan N, Tourangeau infection: a systematic review and meta-analysis. Clin Infect Dis. S, Himes E, Cordill K. Protein loss across burn wounds. J Trauma. 2004 Jul 15; 39 (2): 206-17. 1987 Feb; 27 (2): 136-40. 18. Brunkhorst FM, Heinz U, Forycki ZF. Kinetics of procalcitonin in 33. Pérez-Guisado J, de Haro-Padilla JM, Rioja LF, Derosier LC, from iatrogenic sepsis. Intensive Care Med. 1998 Aug; 24 (8): 888-9. Torre JI. Serum albumin levels in burn people are associated with 19. von Heimburg D, Stieghorst W, Khorram-Sefat R, Pallua N. the total body surface burned and the length of hospital stay but not Procalcitonin—a sepsis parameter in severe burn injuries. Burns. the initiation of oral / enteral nutrition. Int J Burns Trauma. 2013 July 1998 Dec;24(8):745-50. 8; 3 (3): 159-63. 20. Lavrentieva A, Kontakiotis T, Lazaridis L, Tsotsolis N, Koumis J, 34. Cucereanu-Badica I, Luca-Vasiliu I, Grintescu I, Lascar I. The Kyriazis G, Bitzani M. Inflammatory markers in patients with severe correlation between burn size and serum albumin level in the first burn injury. What is the best indicator of sepsis? Burns. 2007 48 hours after burn injury. Romanian Journal of Anesthesia Intensive Mar;33(2):189-94. Epub 2007 Jan 9. Therapy 2013 Vol.20 Nr.1, 5-9. 21. Barati M, Alinejad F, Bahar MA, Tabrisi MS, Shamshiri AR, 35. Aguayo-Becerra OA, Torres-Garibay C, Macias-Amezcua MD, Bodouhi NO, Karimi H. Comparison of WBC, ESR, CRP and PCT serum Fuentes-Orozco C, Chávez-Tostado Mde G, Andalon-Dueñas E, levels in septic and non-septic burn cases. Burns. 2008 Espinosa Partida A, Alvarez-Villaseñor Adel S, Cortés-Flores AO, Sep;34(6):770-4. González-Ojeda A Serum albumin level as a risk factor for mortality in burn patients. Clinics (Sao Paulo). 2013 Jul; 68 (7): 940-5. 22. Mokline A, Garsallah L, Rahmani I, Jerbi K, Oueslati H, Tlaili S, Hammouda R, Gasri B, Messadi AA. Procalcitonin: a diagnostic and 36. Godinjak A, Iglika A, Rama A, Tancica I, Jusufović S, Ajanović A, prognostic biomarker of sepsis in burned patients. Ann Burns Fire Kukuljac A. Predictive value of SAPS II and APACHE II scoring systems Disasters. 2015 Jun 30;28(2):116-20. for patient outcome in a medical intensive care unit. Acta Med Acad. 2016 Nov; 45 (2): 97-103. 23. Maier M, Wutzler S, Lehnert M, Szermutzky M, Wyen H, Bingold T, Henrich D, Walcher F, Marzi I. Serum procalcitonin levels in 37. Douglas HE, Ratcliffe A, Sandhu R, Anwar U. Comparison of patients with multiple injuries including visceral trauma. J Trauma. mortality predictive models in ICU patients in Pinderfields Hospital 2009 Jan; 66 (1): 243-9. over 3 years. Burns. 2015 Feb; 41 (1): 49-52. 24. Meisner M, Adina H, Schmidt J. Correlation of procalcitonin and 38. Martynoga R, Fried M. APACHE II score may predict mortality in C-reactive protein to inflammation, complications, and outcome patients with Critical Care 2009, 13 (Suppl 1): P504. during the intensive care unit of multiple trauma patients. Crit Care. 39. Wu WH, Niu YY, Zhang CR, Xiao LB, Yeh HS, Pan DM, Zeng M. 2006 Feb; 10 (1): R1. Combined APACH II score and arterial blood lactate clearance rate 25. Wojtaszek M, Staśkiewicz G, Torres K, Jakubowski K, Rácz O, to predict the prognosis of ARDS patients. Asian Pac J Trop Med. Cipora E. Changes of procalcitonin level in multiple trauma patients. 2012 Aug; 5 (8): 656-60. Anesthesiol Intensive Ther. 2014 Apr-Jun; 46 (2): 78-82. 40. Luecke T, Muench E, Roth H, Friess U, Paul T, Kleinhuber K, 26. Kim HS, Yang HT, Hur J, Chun W, Ju YS, Shin SH, Kang HJ, Lee KM. Quintel M. Predictors of mortality in ARDS patients referred to a Procalcitonin levels within 48 hours after burn injuryas a prognostic tertiary care center: a pilot study. Eur J Anaesthesiol. 2006 May; 23 factor. Ann Clin Lab Sci. 2012 Winter; 42 (1): 57-64. (5): 403-10. 27. Greenhalgh DG, Saffle JR, Holmes JH, Gamelli RL, Palmieri TL, 41. Forster NA, Zingg M, Haile SR, Künzi W, Giovanoli P, Horton JW, et al. American Burn Association Consensus Conference Guggenheim M. 30 years later - does the ABSI need revision? Burns. on Burn Sepsis and Infection Group. American Burn Association 2011 Sep; 37 (6): 958-63. consensus conference to define sepsis and infection in burns. J Burn 42. Cachafeiro L, Sanchez M, Herrero E, Fernández L, Irazabal M, Care Res. 2007 Nov-Dec;28(6):776-90. Hernandez M, Agrifoglio A, Garcia de Lorenzo A, and Lendinez M. 28. Lyons O, Whelan B, Bennett K, O'Riordan D, Silke B. Serum Inhalation injury in critical burn patients. Critical Care 2013, 17 albumin as a result predictor in hospital emergency medical (Suppl 2): P11834. admissions. Eur J Intern Med. 2010 Feb; 21 (1): 17-20. 43. Santucci CA, Purcell TB, Mejia C. Leukocytosis as a predictor of 29. Herrmann FR, Safran C, Levkoff SE, Minaker KL. Serum albumin severe injury in blunt trauma. West J Emerg Med. 2008 May;9(2):81- level on admission as a predictor of death, length of stay, and 5. readmission. Arch Intern Med. 1992 Jan; 152 (1): 125-30. 44. Belba M, Aleksi A, Nezha I, Filaj V. Impact of Severe Burns in 30. McClain CJ, Hennig B, Ott LG, Goldblum S, Young AB. Hematological Parameters, AJMHS 2015; Vol. 46 (3): 59-69 Mechanisms and implications of hypoalbuminemia in head-injured 45. Sherren PB, Hussey J, Martin R, Kundishora T, Parker M, patients. J Neurosurg. 1988 Sep; 69 (3): 386-92. Emerson B. Lethal triad in severe burns. Burns. 2014 Dec; 31. Gosling P, Brudney S, McGrath L, Riseboro S, and Manji M. 40(8):1492-6. Mortality predictions at admission to intensive care: a comparison

28 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine

Article received on July 29, 2019 and accepted for publishing on September 28, 2019. ORIGINAL ARTICLES

Management of war-related vascular injuries: A civilian surgeon experience in the treatment of war casualties at a secondary care hospital

Bariș Akça1

Abstract: Background: This study presents the management of patients with war-related injuries and early results of vascular injuries treated at a secondary care hospital neighboring to civil war. Methods: Between December 2013-December 2014 ninety-six war-related injury patients evaluated by a single vascular surgeon and participating disciplines according to components of injury in polytrauma patients. Thirty-six patients who underwent vascular surgery were retrospectively registered to the study. Demographics, treatment and early results of patients reviewed. Results: Mean age was 26.69±11.80 (7-66) years and 91.66% (33/36) were males. Injuries were caused by shrapnel wound due to bombing 61.11% (22/36), stab wound 8.33% (3/36), gunshot wound 30.55 % (11/36). The number of patients with polytrauma was 29, seven patients had isolated vascular injury. Among 42 arterial and 31 venous injuries primary suture repair, autologous vein graft interposition and prosthetic graft interposition performed to 20.54% (15), 28.76% (21), 19.17% (14) respectively. Amputation and all-cause mortality were %8.3 (3/36), 13.8% (5/36) respectively. Conclusions: Vascular injury with concomitant trauma is the main issue of war-related injuries. Surgeons should be cautious about patients who were initially deemed not to need surgery for vascular injury and then were found to need it. Repetitive physical examination is very important in case of any suspicion, especially in limited conditions for diagnostic tools like CT angiography. A rapid, careful multidisciplinary evaluation of experienced surgeons for the diagnosis and the treatment priorities in war-related injury patients may lead to increase life and limb salvage rates with a better quality of life. Keywords: vascular system injuries, war-related injuries, mortality, amputation, multiple trauma

INTRODUCTION Early intervention is considered crucial for a successful outcome, although the management of vascular injuries is Vascular injuries reported as varying rates of 2-3% of civilian clinically challenging even for experienced surgeons when trauma [1, 2], while about 7% of war-related trauma [3]. resources are limited, particularly in polytrauma patients, as War-related vascular injuries frequently result in life- imaging methods like computed tomography angiography threatening hemorrhage or limb loss, and while the (CTA) or angiography is necessary for prompt diagnosis, as amputation rate was 60 % in the past, with ligation being the well as for prompt and appropriate treatment. Ideally, war- preferred technique for arterial injuries, today this rate has related injuries should be treated by surgeons with military decreased to as low as 1 % through developments in early surgery experience, although civilian surgeons may find transport, damage control methods providing hemodynamic themselves in practicing military surgery without prior stability, diagnosis and complex treatment techniques [4, 5].

Corresponding author: Baris Akca 1 Department of Cardiovascular Surgery, Inonu University Faculty [email protected] of Medicine, Malatya, Turkey

training or experience in this field [6]. referred to a center where further investigations could be made. If there was no such opportunity, these patients were As a result of ongoing civil war in Syria, an increasing number evaluated by DUS, if possible, or mostly through repetitive of patients with war-related polytrauma have been managed physical examinations, for clinical findings of ischemia, in a frontier secondary care hospital which neighboring the cyanosis, the absence of arterial pulse and hemorrhage. CTA civil war, in Kilis, Turkey. This study presents the experiences or angiography was essential for diagnosis and the of a single civilian surgeon with war-related injury treatment options in 19 (19.79 %) polytrauma patients with management strategies and the early results of vascular uncertain physical examination findings, hence they referred injuries treated at a secondary care center with limited to the relevant centers. resources. Among 96 patients evaluated for vascular injury, a total of 36 METHODS (37.5%) patients operated by vascular surgeon enrolled in the study. All patients received a tetanus vaccine and dual Between December 2013 and December 2014, 96 patients antibiotics prior to surgery. The initial surgical exploration with war-related injuries were evaluated for suspected was usually performed by the vascular surgeon, both for vascular injury through a multidisciplinary approach better bleeding control and arterial and venous repair, involving a single vascular surgeon and other disciplines, especially in injuries of the extremities, based on who were involved depending on the components (cranial, hemodynamic status and duration of ischemia. The chest, abdomen, bone fracture and soft tissue loss) of injury exploration of injured vessels was performed via standard in polytrauma patients. Data of patients were recorded for incisions if in the same course as the injury area; otherwise, retrospective analysis in real time. This retrospective study proximal and distal control of the vessels was obtained was conducted after obtaining the Ethical Committee directly from the injury area. In patients with extremity approvals of Inonu University and where the work was injuries (bone, nerve, and soft tissue damage, whether carried out. individually or together) vascular repair was performed All patients were transferred to our hospital from the place immediately after proximal and distal control of the vessels where the injury occurred. In accordance with Advanced to achieve better bleeding control and stable hemodynamic Trauma Life Support protocols and damage control status in cases where extremity-threatening critical ischemia principles [7, 8], tourniquets were routinely applied to all existed. Otherwise, vascular repairs were made after bone patients to achieve rapid hemorrhage control, and stabilization with external fixators. permissive hypotension (massive red blood cell, plasma, and Systemic Standard heparin was used intra-operatively, minimal crystalloid infusion) was used to initiate a based on the level of bone and soft tissue damage/loss in hemostatic resuscitation plan aimed at correcting metabolic non-coagulopathic patients. Thrombectomy was performed imbalances. Subsequently, patients were evaluated for routinely with an embolectomy catheter after the routine vascular and other injuries. In the absence of angiography assessment of proximal inflow and distal backflow. Also, the and CTA, diagnosis of vascular injury was made almost distal segment of the artery was cleansed with heparinized entirely through physical examination findings, with a saline in patients for whom systemic standard heparin could Doppler ultrasound (DUS) used when possible. If a patient not be used. presented with only hard signs of vascular injury, such as active pulsatile hemorrhage, absent distal arterial pulse, Vascular injuries were managed via simple repairs, end-to- ischemia, expanding or pulsatile hematoma with no end anastomoses, interpositions of the autologous concomitant injury, immediately transported to the saphenous vein graft or polytetrafluoroethylene (PTFE) graft operating theater for vascular repair. After the and simple ligation methods. The vascular repair method multidisciplinary agreement about the benefit of vascular was determined based on an intraoperative assessment of repair and concomitant surgical interventions, and an initial the injured vessels. The decision of ligation was based on life- surgery decision according to clinical status at admission, threatening hemorrhage and distal arterial circulation with patients with polytrauma were sent immediately to the more than one artery (Ulnar/radial and one of the tibia operating theater. However, patients presenting with soft arteries). Venous injuries of large veins, especially to the signs of vascular injury (non-pulsatile and non-expanding femoral, popliteal and axillary veins, were repaired via hematoma, palpable but diminished or unequal distal simple repair, vein graft or PTFE graft methods, when arterial pulses, bone fracture, peripheral nerve deficit and possible. Other minor venous injuries were ligated. history of bleeding that subsided spontaneously) and with During the initial evaluation in the emergency room, soft no immediate threat to life or limb, were preferred to be

30 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine tissue defects and necrotic tissues in patients with limb reviewed, and the studied variables included age, gender, injuries were scrubbed with 2000–3000 cc of Ringer solution clinical signs and symptoms on admission, anatomical region and cleansed from foreign bodies prior to the operation. of vascular injury, mechanism of injury, concomitant injuries Subsequently, the necrotic tissue debridement, foreign body in polytrauma patients, Mangled Extremity Severity Score removal and soft tissue reconstruction performed after (MESS) [9], type of vascular repair and clinical outcome. forceful irrigation with Ringer solution in the operating The data were analyzed using PASW Statistics software (SPSS theater. Inc, USA), and was presented as mean (SD), range or Low molecular weight heparin was given to all patients numbers and percentage (%), as appropriate. postoperatively for anticoagulation, and a dual or triple antibiotic regimen was applied postoperatively, based on RESULTS the contamination and severity of the injury. The mean age was 26.69±11.80 (7-66) years and 33 (91.66%) Fasciotomy was performed by orthopedists intra operatively were male. The mean MESS score was 8.47±2.64 (3-14), and or during intensive care unit follow up, according to the extensive tissue defect/loss was observed in 12 (33.33%) follow up of patient and operative management of injury. patients. The cause of injuries were shrapnel wound due to Nerve injuries were repaired either in the same session or the bombing, stab wounds and gunshot (mostly rifle) delayed for two to three weeks, by orthopedists or wounds in 22 (61.11%), 3 (8.33%) and 11 (30.55%) patients neurosurgeons. respectively.

The demographics, treatment and early results (in hospital The anatomical localization of injuries presented in Table 1. period; up to postoperative seven days) of patients were

Table 1. Anatomical localization and repair methods of injured 73 vessels in 36 patients Repair method n Primary repair Prosthetic graft Vein graft Ligation Vessel (73) (15) (14) (21) (23) Carotid artery 1 1 Subclavian artery 1 1 Brachial artery 4 1 3 Radial artery 3 2 1 Ulnar artery 3 1 2 Common Femoral artery 5 2 3 Superficial femoral artery 8 2 3 3 Profunda femoral artery 3 1 1 1 Popliteal artery 5 3 2 Anterior Tibial artery 5 1 3 1 Posterior Tibial artery 4 3 1 Internal jugular vein 1 1 Subclavian vein 1 1 Common femoral vein 7 4 1 2 Profunda femoral vein 3 1 2 Superficial femoral vein 7 3 3 1 Popliteal vein 4 2 2 Anterior tibial vein 4 4 Posterior tibial vein 4 4 Total (%) 20.54 19.17 28.76 31.50

Of the patients, high-energy injuries were observed in 24 injury distribution of patients presented in Table 2. (66.6%), medium-energy injuries in 2 (5.55%), low energy In the study, 7 (19.44%) patients had isolated vascular injury; injuries in 3 (8.33%) patients and 7 (19.44%) patients had 12 (33.33%) patients underwent vascular surgery with crush injuries (rescued from beneath the wreckage of concomitant active bleeding; 17 (47.22%) patients collapsed buildings after the bombing). The number of presented with hypovolemic shock upon arrival at the patients with polytrauma was 29 (80.55%) and concomitant

emergency room; ischemia signs were observed in 24 graft thrombosis. The amputation rate was 8.3% (3/36). Two (66.66%) patients, and the mean ischemia time was 5.45 ± (5.55%) patients had secondary amputation after vascular 5.20 (1-24) h. repair and one patient with mangled extremity underwent primary amputation via an intraoperative multidisciplinary Table 2. Concomitant injury distribution of 36 patients enrolled in decision in order to save life. All three patients who the study underwent amputation had injuries of the popliteal artery n % including trifurcation. Cranial injury 3 8.3 The mortality rate was 13.8% (5/36), four of them had high- Neck injury 1 2.7 energy injuries and one had medium-energy injury. Chest İnjury 5 13.8 Furthermore, two patients with concomitant abdomen Abdominal injury 7 19.4 injuries died from sepsis, one patient with concomitant Pelvic injury 4 11.1 cranial injury died due to intracranial hemorrhage. Two Lower extremity injury 28 77.7 patients in hypovolemic shock with concomitant large soft Upper extremity injury 11 30.5 tissue defect underwent surgery with preoperative early Large tissue defect 12 33.3 intubation and died from acute respiratory distress syndrome and multiple organ dysfunction syndrome. A Of the patients, 25 (69.44%) had single extremity injuries, 9 fasciotomy was applied to three patients, of which two were (25%) had double extremity injuries and one patient had closed and one was open. three extremity injuries. In 20 (55.55%) patients, extremity injuries were accompanied by bone fractures, of which 17 DISCUSSION (47.22%) were open fractures. As reported variously, the incidence of war-related vascular Initial physical examinations revealed motor loss in 20 injuries has been increased gradually from World war II (0.2- (55.55%) patients. Intraoperative nerve injury was mostly 4%) to the wars in Afghanistan and Iraq (6.8 %), also a recent determined as a full-thickness incision in 12 (33.33%) study from the Lebanon war reported that the rate of patients. Three (8.33%) patients with polytrauma had vascular injuries increased to 7.6% of all casualties, and to postponed surgery decision after a follow-up period for 10.8% in the soldiers [4, 10-12]. This increase is possibly repetitive physical examinations due to uncertain initial correlated with shortened evacuation time, improved physical examination findings. immediate resuscitation, damage control by the progressive A total of 42 arterial and 31 venous injuries were observed and effective use of tourniquets and temporary shunts. in the operating theater. The most common repair method Furthermore, these developments over years have brought was graft interposition with autologous saphenous vein and improved life and limb rescue rates in war-related injuries PTFE. Ligation was the second preferred method, especially [13-15]. In this civilian study, however, the distance to the for venous injuries, and 31.50% of total injuries were ligated. war zone (15 min. away by road to the hospital) was the The details of repair methods presented in Table 1. most important factor affecting the increasing admittance of war-related trauma patients. Postoperative complications are listed in Table 3. Recent experiences have triggered the management of such Table 3. Postoperative complications of patients injuries, assessing especially the effect of explosives on n % injury patterns and treatment strategies [3]. Explosive Graft Thrombosis 4 11.1 weapons are designed to increase the number and energy of Amputation 3 8.3 casing fragments, leading to multiple penetrating wounds Mortality 5 13.8 [16]. In the present study, most common injuries were Reperfusion injury 1 2.7 shrapnel wounds from bombing and rifle wounds, hence ARDS 1 2.7 these kinds of high energy injuries includes both penetrating MODS 1 2.7 and blast effects on tissues as designed. Therefore, vascular ARDS: Acute respiratory distress syndrome, MODS: Multiple organ dysfunction injuries associated with multiple trauma often lead to high syndrome mortality rates, unless prompt and appropriate surgical management is made [17], hence war-related injury Thrombectomy and/or embolectomy performed for all treatment should be provided by surgeons with military postoperative graft thrombosis patients. Three patients had surgery experience [6]. autologous vein graft thrombosis and one had a prosthetic

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Vascular injuries of extremities are the most common type It should not be forgotten that the possibility of arterial of injury in war-related trauma patients [3, 12-14], as is the trauma still exists, particularly in the upper extremities, with case in this study and it may lead to limb loss or mortality. palpable distal arterial pulses [18-22], also impaired Bleeding may lead to hypovolemic shock, as was the case perfusions with absent distal arterial pulses may not be with 47.2% of the patients in the present study at the noticed due to of collateral circulation. admittance. Consequently, the primary aim is to avert In this study, ligation of vessels was considered as the final exsanguination and death, and then to restore blood flow to option especially in hemodynamically stable patients, prevent amputation. although ligation is a good strategy in selected vessels that The initial physical examination of patients who present with do not cause distal extremity ischemia, and in cases of life- hypovolemic shock may be misleading about vascular injury threatening bleeding. due to diminished or absent distal arterial pulse and Similar to other studies [3, 23, 24], superficial femoral, inadequate perfusion of extremities. In patients suffering common femoral, popliteal and brachial arteries were the from hypovolemic shock, recurrent physical examinations most commonly injured arteries in this study. Vein injuries are important, as in patients with soft signs, and also should are mostly accompanying with the adjacent artery injuries. be carried out after appropriate resuscitation of The most common repair method of this study was hypovolemic shock. Three polytrauma patients of this study saphenous vein graft interposition, as recommended in most underwent surgery through recurrent physical examination studies [3, 25]. Applying a prosthetic graft is still a findings. controversial issue, increased risk of thrombosis and If the initial evaluation of a patient revealed hard signs of infection, thus poor outcome with a high secondary vascular injury with a single entry and/or exit wound, and amputation rate [17, 23, 26]. In this study, contrast with the with no concomitant injury, there is consensus on the previous studies the absence of early infection despite the decision to make an immediate vascular exploration after high ratio use of prosthetic grafts may be attributed to the resuscitation in the emergency room. However, there is a factors like; a large amount of wound irrigation as an initial lack of consensus regarding the management of patients response in the emergency department and also in the with only soft signs in polytrauma patients complicated with operating room, careful and aggressive debridement of concomitant injury, multiple extremity injuries or single necrotic and contaminated tissues, additionally use of dual extremity multiple segment injuries with soft or no signs of or triple antibiotic regimen for the follow up. In contrast with vascular injury [18]. For this grey zone of patients with soft expectations, the number of autologous vein graft signs, additional diagnostic methods such as angiography or thrombosis was higher than prosthetic grafts. CTA are required to be applied prior to surgery in Revascularization of these polytrauma patients was carried hemodynamically stable patients, both for diagnosis and out initially, followed by bone and soft tissue operations. choice of treatment [19-21]. Of these, angiography is the Therefore, thrombosis developed due to inadequate length optimum diagnostic method, although CTA has gained (short/long) of the graft. No temporary vascular shunting increasing value in diagnosis and treatment planning, was used in this study. therefore some studies reported that CTA should be The amputation ratio relies on many variables, including the considered as the first line modality [19]. severity of limb injury, mechanism of the injury, ischemia In this study, patients underwent immediate vascular time, existence of accompanying injuries, and disaster surgery on the basis of physical examination findings and conditions when dealing with mass injuries [13]. In this hard signs of arterial injury with the goal of shortening the study, three patients with popliteal artery injuries ischemic period. As neither angiography nor CTA was underwent amputation, one pediatric patient who had an present, hemodynamically stable patients with soft signs of ischemia time of 2h with MESS score of 9 underwent primary arterial injury were referred to a center where these amputation by an intraoperative multidisciplinary decision in examinations could be made. In a very small number of order to save life. The ischemia times of these three patients patients DUS could be used. In the absence of contrast were found to be 2h, 4h, and 8h. The MESS scores of patients imaging, physical examination findings with hard signs of who underwent amputation were 9, 10 and 11 while the vascular injury seem to be an effective means of diagnosis, mean MESS score of this study was 8.47±2.64. During the although the need for contrast imaging methods for patients treatment of mixed skeletal, vascular and soft tissue injuries, with uncertain physical examination findings of vascular limb ischemia specifies the treatment primacy, and also high injury is undisputed. limb loss rates related with popliteal artery involvement [19]. The amputation rate of this study is close to previous

studies reported in varying rates (3-20%) [27, 28]. Mortality patients should be operated by two different disciplines at rate (13.8%) of this study was higher than the varying rates the same time, in the same session; and also which type of (1.5% - 6%) have been reported in previous studies [28, 29]. injury can be delayed. These are challenging situations for even most experienced surgeons in patients with complex Retrospective design of the study, short follow up period of polytrauma accompanying with systemic effects. Therefore, patients and relatively small number of patients are the basic a multidisciplinary approach should be used when limitations of this study. establishing treatment priorities.

CONCLUSIONS Civilian surgeons unexpectedly finding themselves acting war surgery with no background, hence may tend to make When dealing with war-related injuries, especially in the same traditional mistakes that military surgeons have polytrauma patients, occult vascular injuries have been already learned from previous wars, nevertheless a rapid, possibly missed due to uncertain initial physical examination careful multidisciplinary evaluation of experienced surgeons findings of vascular injury and frequently presents later. for the diagnosis and treatment priorities in war-related Therefore, the definite diagnosis of vascular injury should be injury patients may lead to increase life and limb salvage revealed by angiography or CTA. Following the diagnosis, rates. some critical decisions arising such as: which patient should be operated first, among who applied at the same time and requiring emergency surgical intervention; which one of the Acknowledgement injury components should be operated initially in patients Between 2013 and 2015 the author was working at the secondary care with polytrauma; which type of injuries of polytrauma hospital where the study was carried out.

References:

1. Compton C, Rhee R. Peripheral vascular trauma. Perspect Vasc Registry. J Am Coll Surg 2007; 204:625-32. Surg Endovasc Ther 2005; 17:297-307. 12. Nitecki SS, Karram T, Ofer A, Engel A, Hoffman A. Vascular 2. Sugrue M, Caldwell EM, D’Amours SK, Crozier JA, Deane SA. injuries in an urban combat setting: experience from the 2006 Vascular injury in Australia. Surg Clin North Am 2002; 82:211-9. Lebanon war. Vascular 2010; 18:1-8. 3. Fox CJ, Gillespie DL, O’ Donnell SD, et al. Contemporary 13. Starnes BW, Beekley AC, Sebesta JA, Andersen CA, Rush RM Jr. management of wartime vascular trauma. J Vas Surg 2005; 41:638- Extremity vascular injuries on the battlefield: tips for surgeons 44. deploying to war. J Trauma 2006; 60;432-42. 4. DeBakey ME, Simeone FA. Battle injuries in World War II: an 14. Fox CJ, Starnes BW. Vascular surgery on the modern battlefield. analysis of 2,471 cases. Ann Surg 1946; 123:534-79. Surg Clin North Am 2007; 87:1193-211. 5. Onur Hanedan M, Ketenciler S, Murat Ercişli A, et al. Civil War 15. Fox CJ, Gillespie DL, Cox ED, et al. The effectiveness of a damage Surgical Experience: Gun Shot or Shell Fragment Injuries. Damar Cer control resuscitation strategy for vascular injury in a combat support Derg 2014; 23:1-4. hospital: results of a case control study. J Trauma 2008; 64:S99- 6. Behbehani A, Abu Zidan F, Hasaniya N, Merei J. War injuries S107. during the Gulf War: experience of a teaching hospital in Kuwait. 16. Champion HR, Holcomb JB, Young LA. Injuries from explosions: Ann R Coll Surg Engl 1994; 76:407-11. Physics, biophysics, pathology, and required research focus. J 7. Tsang B, McKee J, Engels PT, Paton-Gay D, Widder SL. Trauma 2009; 66:1468-77. Compliance to advanced trauma life support protocols in adult 17. Jawas A, Abbas AK, Nazzal M, Albader M, Abu-Zidan FM. trauma patients in the acute setting. World J Emerg Surg. 2013; 8:39. Management of war-related vascular injuries: experience from the 8. Fox LCJ, Kreishman MP. High-energy trauma and damage second gulf war. World J Emerg Surg 2013; 8:22. control in the lower limb. Semin Plast Surg. 2010; 24:5-10. 18. Aydın H, Okçu O, Dural K, Sakıncı U. Management of 9. Johansen K, Daines M, Howey T, Helfet D, Hansen ST Jr. community-based shotgun injuries of the extremities: impact of Objective criteria accurately predict amputation following lower emergent vascular repair without angiography. Ulus Travma Acil extremity trauma. J Trauma 1990; 30:568-72. Cerrahi Derg 2011;17:152-8 10. Rai KM, Mohanty SK, Kale R, Chakrabarty A, Prasad D. 19. Nitecki SS, Karram T, Ofer A, Engel A, Hoffman A. Management Management of vascular injuries in a forward hospital. Med J Armed of Combat Vascular Injuries Using Modern Imaging: Are We Getting Forces India, 2006; 62:246–51. Better? Emerg Med Int 2013; 2013:689473. 11. Clouse WD, Rasmussen TE, Peck MA, et al. In-theater 20. Mataracı İ, Polat A, Songur M, et al. Amputation-free treatment management of vascular injury: 2 years of the Balad Vascular of vascular trauma patients. Turk Gogus Kalp Dama 2010; 18:17-22.

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21. Van Waes OJ, Van Lieshout EM, Hogendoorn W, Halm JA, North Am 2001; 81:1199-215. Vermeulen J. Treatment of penetrating trauma of the extremities: 26. Wolf YG, Rivkind A. Vascular trauma in high-velocity gunshot ten years’ experience at a Dutch level 1 trauma center. Scand J wounds and shrapnel-blast injuries in Israel. Surg Clin North Am Trauma Resusc Emerg Med 2013; 21:2. 2002; 82:237-44. 22. Orcutt MB, Levine BA, Gaskill HV, Sirinek KR. Civilian vascular 27. Dua A, Patel B, Desai SS, et al. Comparison of military and civilian trauma of the upper extremity. J Trauma 1986; 26:63-7. popliteal artery trauma outcomes. J vasc Surg. 2014; 59:1628-32. 23. Hafez HM, Woolgar J, Robbs JV. Lower extremity arterial injury: 28. Peck MA, Clouse WD, Cox MW, et al. The complete Results of 550 cases and review of risk factors associated with limb management of extremity vascular injury in a local population: A loss. J Vasc Surg 2001; 33:1212-9. wartime report from the 332nd Expeditionary Medical Group/Air 24. Woodward EB, Clouse WD, Eliason JL, et al. Penetrating Force Theater Hospital, Balad Air Base, Iraq. J Vasc Surg 2007; femoropopliteal injury during modern warfare: Experience of the 45:1197-204. Balad Vascular Registry. J Vasc Surg 2008; 47:1259-64. 29. Sohn VY, Arthurs ZM, Herbert GS, Beekley AC, Sebesta JA. 25. Rich NM, Rhee P. An historical tour of vascular injury Demographics, treatment and early outcomes in penetrating management: from its inception to the new millennium. Surg Clin vascular combat trauma. Arch Surg 2008; 143:783-7

Article received on May 16, 2019 and accepted for publishing on September 30, 2019. ORIGINAL ARTICLES

3D echo in everyday life: Could it reset our threshold for interventions?

Maria M. Gurzun1, Silviu Stanciu1, Adrian Gabără1, Adrian Ionescu2

Abstract: Background Left ventricular ejection fraction (LVEF), the single most important metric in cardiology, is the cornerstone on which prognosis is estimated and costly decisions such as whether to implant an ICD are based. LVEF is most often assessed by 2D echocardiography (2DE), although 3D echocardiography (3DE) has been shown repeatedly to be more accurate. Aim of the study. We set out to assess whether using 3DE would reclassify the severity of LV impairment in patients with LVEF < 35% by 2DE. Setting. Tertiary cardiac centre serving a population of one million, and performing approximately 100 ICD implants/year. Methods. Successive patients in sinus rhythm, with good endocardial border definition and LVEF<35% by 2D Simpson’s method, had scans according to the BSE protocol. 3D loops were acquired from the apical 4-chamber view and were analysed off-line using for regional wall motion abnormalities (RWMAs) and LVEF. The patients were classified in subgroups according to EF value: less than 20%, 20-25%, 25-30%, 35% and more than 35%. Moderate LV systolic was defined as LVEF between 35% and 45% and severe as LVEF ≤35%. Results. We studied 100 patients (78 M, mean age (SD) 69.94 (13.54) years). 2DE had been requested for decision-making regarding ICD implantation in 86 patients (86%) and for LVEF measurement after acute coronary syndromes in 14 patients (14%). Regional wall motion abnormalities (RWMAs) were present in half of patients (55%, 51 pts – 56% in LAD territory, 31% in RCA/CX territory and 12% multiple territories). 3D LVEF regrouped 67% of patients: 10% to a lower EF and 57% to a higher EF subgroup. Twenty nine patients (29%) were reclassified from severe LV systolic dysfunction by to 2DE to moderate LV systolic dysfunction by 3DE. Patients with RWMAs were more often reclassified than patients without RWMAs (p=0.006). The LV dimensions were lower for the reclassified patients. The image quality had no effect on reclassification. Conclusion Measuring LVEF by 3DE reclassifies the severity of LV systolic impairment in a substantial proportion of patients with 2D LVEF<35% and RWMAs, which may have important clinical and financial implications by resetting thresholds for costly interventions such as ICD implants. Keywords: biological attack, biological warfare, biological agents, international legislation, medical protection

BACKGROUND The clinical importance of echocardiography in ejection fraction measurement is undoubtedly, left ventricle systolic Left ventricular ejection fraction (LVEF), the single most function evaluation being the most common reason for important metric in cardiology, is the cornerstone on which referring a patient for an echocardiogram [2]. In our prognosis is estimated and costly decisions (defibrillator department, from 117 patients scanned during one week, implantation, resynchronisation therapy) [1] are based. 60% were referred for LV systolic function evaluation. The main advantages of echocardiography comparing to more 1 “Carol Davila” University Central Emergency Military Hospital, Corresponding author: Maria Magdalena Gurzun Bucharest, Romania 2 Morriston Regional Cardiac Centre, Swansea, Wales, UK [email protected]

36 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine accurate methods (like MRI or CT (3)) are availability, department for left ventricle systolic function evaluation. inexpensive character, rapidity of performance, portability The study was developed in a tertiary cardiac centre serving and radiation freedom. a population of one million, and performing approximately 100 ICD implants/year. Two-dimensional echocardiography (2DE) remains the most commonly used modality for EF estimation in clinical All patients had transthoracic 2D echocardiography obtained practice. The current guidelines recommend Simpson’s discs with harmonic imaging (Philips iE33, Philips Medical System, method for LVEF calculation. However this method is subject Holland). Left ventricle end-diastolic volume (LVEDV), left to errors due to foreshortening and assumptions about LV ventricle end-systolic volume (LVESV) and LVEF was shape (stacked disks with varying diameters). measured on-line using Simpson’s method of disks (using four and two chamber apical views). Patients with LVEF Three-dimensional echocardiography (3DE) overcomes ≤35% were included in further analyse. The value of 35% was some of this limitation by capturing entire volumes, arbitrary selected because it is the cut-off limit for important excluding foreshortening and geometrical assumption (very and expensive treatments: ICD implant or cardiac important in deformed ventricle). In addition, the resynchronisation therapy (1). LV systolic dysfunction was endocardial position is measured at hundreds of points over defined as severe for LVEF <35% and as moderate for LVEF the LV surface comparing to only two planes in conventional 35-45%. 2DE. Therefore, the calculation of LV volume is more accurate and reproducible. 3D echocardiography was performed in the same time, using a matrix array ultrasonic transducer (X3-1 transducer, Philips OBJECTIVE iE33, Philips Medical System, Holland). An apical full volume The primary aim of this study is to evaluate if LV EF image focused on LV, over four cardiac cycles was acquired. assessment by 3DE in routine clinical practice can change the Measurement of 3DE LV volumes and ejection fraction was clinical decision making for patients with LV systolic performed offline, using the ‘mesh method’ (Q- Lab 9, Philips dysfunction and if routine 3DE is useful and worthy versus Medical Systems) blinded to the 2D measurements. The largely used 2DE. semi-automated LV border detection was used for contour tracing, after first identifying the apex and the four mitral METHODS annular points (septal, lateral, anterior and inferior) (Figure 1A). A 3D endocardial shell was than produced and used for We prospectively analyzed successive patients in sinus LV volumes and LVEF calculation (Figure 1B). rhythm with good endocardial border referred to our echo

Figure 1. Full volume 3DE, 4 beats image of the left ventricle in a four chamber equivalent (apical probe position) and the corresponding end-diastolic four chamber (), two chamber () and short axis () view (A). The resulting end-diastolic volume (mesh), end-systolic volume (shell) and calculated 3DE after sequences analysis (B).

Statistical analysis: Agreement between 2DE and 3DE were performed using χ2 test for categorical data and measurements was assessed by performing Bland-Altman Independent/Paired-Samples T test for continuous data with analisys (MedCalc Software, version 12. 7.0, Acacialaan 22, a two-tailed p value < 0.05 for statistical significance (IBM B-8400 Ostend, Belgium). Means and standard deviations SPSS Statistics v.21, Armonk, NY). (SD) were calculated for continuous variables. Comparisons

RESULTS Figure 3. Graphic representation of LVEDV and LVEDS distribution by 2DE vs 3DE. During the enrollment period 106 patients were eligible. 6 patients were excluded due to inadequate quality of 3D acquisition (4 patients presented stitch artifacts and 2 patients had severely dilated LV, impossible to be included in 3D volume). The analyzed group was composed by 100 patients, 78 male, mean age (SD) 66.94 (13.54) years.

Transthoracic echocardiography had been requested for decision-making regarding ICD implantation in 86 patients (86%) and for LVEF measurement after acute coronary syndromes in 14 patients (14%). Regional wall motion abnormalities (RWMAs) were present in half of the cases: 51 patients (51%). The affected territory was corresponding to anterior descending artery in 29 patients (56%), to right Reclassification by 3DE analysis coronary artery/circumflex artery in 16 patients (31%) ant to multiple vessels in 6 patients (12%). The QRS complex was According to EF value, the patients were classified in four narrow in 52% of patients and large in 48 patients (75% subgroups: <20%, ≥20% and <25%, ≥25% and <30%, ≥30% presented bundle branch block and 25% paced rhythm). and ≤35%. Only for EF calculated by 3DE there was a new Thirty two patients (32%) had left ventricle dilated (LVED subgroup formed by patients with EF >35%. The 2DE and 3DE volume >155ml). LVEF value for all patients is presented in Figure 4. The distribution of patients among subgroups comparing 2D to Comparing 2DE and 3DE data 3DE is illustrated in Figure 5. Considering the patients The mean value (SD) for LVEF calculated by 2DE (Simpson’s classification in these subgroups, 67% were reclassified by method) was 27.36 (5.8%) and for LVEF calculated by 3DE 3DE comparing to 2DE: 57% in a group with higher EF and was 30.53 (6.9%), p<0.001. The correlation between the two 10% in a group with lower EF. methods was good (Spearman’s coefficient 0.79). On Bland- Twenty-nine patients (29%) were reclassified by 3DE from Altman analysis the bias was -3.2% and the agreement severe LV systolic dysfunction (EF ≤35%) to moderate LV interval -4.1, -2.4 (Figure 2). systolic dysfunction (EF>35%). Eight patients reclassified from severe to moderate LV systolic dysfunction had 2DE EF Figure 2. Bland-Altman plot representing the agreement between 25-30% and 21 had EF 30-35% (Figure 4). two methods for EF estimation: 2DE and 3DE.

Figure 4. Graphic representation of 2DE EF (blue dots) vs 3DE EF (green dots) for all analyzed patients, grouped by 2DE in four subgroups (as previously mentioned).

The mean value (SD) for LVEDV was 144.37 (69.2) ml by 2DE and 143.6 (67.2) ml by 3DE, p=0.8, Spearman’s coefficient

0.86. For LVESV the mean value (SD) was 106.26 (59.53) ml by 2DE, 103.24 (55.02) ml by 3DE, p=0.051, Spearman’s The difference between LVEF evaluated by 2DE and 3DE was coefficient 0.85 (Figure 3). more than 5% in 40 patients (40%). The mean (SD) difference EF was 3.2 (4.2) %. The difference between EF by 3DE and by 2DE was not significantly different among the 2DE defined

38 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine four subgroups (mean value was 5.73% for EF <20%, 4.04% Figure 5. Graphic representation of number of patients from each for EF 20-25%, 4.3% for EF 25-30% and 4.53% for EF 30-35%, subgroup by 2DE and 3DE. p =0.4).

Characteristics of reclassified patients a) Patients regrouped among subgroups by 3DE

The ejection fraction was not different between patients who were regrouped among the previously described subgroups and patients who remained in the same subgroup after 3DE. The end-diastolic and end-systolic LV volume was significantly lower for patients who were regrouped. The RWMAs and the QRS width did not influenced significantly the regrouping among subgroups (Table 1).

Table 1. Regrouped patients Non-regrouped patients p value (subgroups) (subgroups)

EF (SD)% 28.03 (5.09) 25.69 (7.0) 0.006 LVEDV (SD) ml 133.56 (57.58) 166.30 (85.22) 0.026 LVESV (SD) ml 97.25 (47.17) 127.60 (75.77) 0.016 Yes 72.5% 27.5% RWMA 0.02 No 61.2% 38.8% narrow 65.4% 34.6% QRS 0.7 large 68.8% 31.3%

Table 2. Non-reclassified Reclassified patients patients p value (LVEF by 3DE>35%) (LVEF by 3DE≤35%)

LVEDV (SD) ml 106.27 (37.39) 159.93 (73.34) 0.000 Dilated LV 6.3% 93.8% 0.001 (LVEDV>155ml) Normal LV(LVEDV 39.7% 60.3% <155ml) LVESV (SD) ml 71.14 (26.43) 122.02 (63.00) 0.000

Image average 30% 70% 0.82 quality good 28% 72% Yes 41.2% 58.8% RWMA 0.006 No 16.3% 83.7% narrow 40.4% 59.6% QRS 0.009 large 16.7% 83.3%

b) Patients reclassified from severe to moderate LV systolic dysfunction who were reclassified as moderate systolic dysfunction by 3DE dysfunction after 3DE. The reclassified patients had more often regional wall motion abnormalities comparing to non- The end-diastolic and end-systolic LV volume was reclassified patients (Table 2). The number of reclassified significantly lower in patients with severe LV systolic patients was 16.3% form subjects with no regional wall

motion abnormality, 48.3% for subjects with regional wall was not reported previously [12]. In our study, from 106 abnormality in LAD territory, 37.5% for RCA/Cx territory and patients with adequate echo images quality for Simpson’s 16.7% for multiple territory (p=0.018). evaluation, 6 patients (0.056%) could not be analyzed by 3DE. DISCUSSIONS Do we really need 3DE in clinical practice? Can we trust 3DE? What do we know from cardiac magnetic In nowadays clinical cardiology, when expensive decision resonance comparing studies? makings are relied on ejection fraction value, its accurate A large number of studies during the last years have shown determination becomes mandatory. The recent studies that 3DE is superior to 2DE for left ventricle quantification showed repeatedly that 3DE is superior to 2DE for LV (LV volumes and EF) comparing to MRI. The superiority of quantification. However the EF estimation 2DE vs 3DE were the method is related to a better accuracy and a better not significantly different comparing to MRI in some studies reproducibility. The accuracy and the repeatability of 3DE [9]. Therefore the advantage of 3DE on 2DE in clinical are comparable with cardiac MRI for LV volumes and EF for echocardiography is uncertain. unselected patients [4] or for patients with regional wall Our data showed that routine 3DE use for LVEF estimation in motion abnormalities [5, 6]. In a follow up study of patients every day practice can change the decision making in a with previous myocardial infarction the 3DE LV parameters significant number of patients. Twenty nine patients (29%) changes were better correlated with MRI results comparing were reclassified from EF≤35% by 2DE to EF>35% by 3DE. to 2DE [7]. Moreover, the 3DE estimation of LV volumes and Therefore, the clinical management of these patients EF was superior to 2DE measurement in predicting major (regarding ICD implant or CRT) should be reanalyzed in the cardiovascular events [8]. A recent meta-analysis showed view of the new data obtained by 3DE. The patients with 2DE that in studies comparing 2DE, 3DE and MRI LV severe LV systolic dysfunction and nondilated LV and measurements the 2DE/MRI pooled Bland Altman biases +/- RWMAs were often reclassified to 3DE moderate LV systolic SD were -48.2+/-55.9ml, -27.7+/-45.7ml and 0.1+/-13.9% for dysfunction. LVEDV, LVESV and EF. The 3DE/MRI pooled Bland Altman biases +/-SD were -15.7+/-31ml, -9.6+/-25.8ml and 0.0+/- LIMITATIONS 9.2% for LVEDV, LVESV and EF. The differences in biases was not statistically significant for EF (p=0.42) [9]. However, most The main limitation of the study is the lack of ‘gold standard’ of the previous studies included only patients with good measure. However, the superiority of 3DE over 2DE for LV quality of echo pictures. In our study, we included all assessment was previously demonstrated and the purpose patients for which the EF calculation by Simpson’s method of our study was to evaluate the possible clinical impact of was feasible, including patients with average quality of echo 3DE in clinical practice. images. CONCLUSIONS Is 3DE evaluation a feasible method in clinical practice? Measuring LVEF by 3D echo reclassifies the severity of LV The feasibility of 3DE for LV parameters analysis was 89% in systolic impairment in a substantial proportion of patients a large study, including unselected normal subjects [10]. In with 2D LVEF<35% and RWMAs, which may have important routine clinical practice 3DE was technically feasible in 83% clinical and financial implications by resetting thresholds for of unselected patients [11]. The percentage of patients costly interventions such as ICD implants. properly examined by 2DE that could not be analyzed by 3DE

References:

1. ESC Guidelines for the diagnosis and treatment of acute and Comparison of Left Ventricular Function Assessment with 64-Row chronic heart failure 2012. McMurray J, Adamopoulos S, Anker S. Computed Tomography,Biplane Left Cineventriculography, and Eur Heart J. 2012; 33:1787-1847. Both2- and 3-Dimensional Transthoracic Echocardiography. J Am 2. Lang RM, Bierig M, Devereux RB, et al. Recommendations for Coll Cardiol. 2012;59:1897-1907 chamber quantification: a report from the American Society of 4. Jenkins C, Leano R, Chan J et al. Reconstructed versus real-time 3- Echocardiography's Guidelines and Standards Committee and the dimensional echocardiography: comparison with magnetic Chamber Quantification Writing Group, developed in conjunction resonance imaging. J Am Soc Echocardiogr. 2007; 20:862-868. with the European Association of Echocardiograph. J Am Soc 5. Marsan NA, Westenger JJ, Roes SD. Three dimensional Echocardiogr. 2005: 1440-63. echocardiography for the preoperative assessment of patients with 3. Greupner J., Zimmermann E, Grohmann A. et al. Head-to-Head left ventricular aneurysm. Ann Thorac Surg, 2011; 91:113-21.

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6. Pouleur AC, le Polain de Waroux JB, Pasquet A et al. Assessment 9. Dorosz J., Lezotte D., Weitzenkamp D. Performance of 3- of left ventricular mass and volumes by three-dimensional Dimensional Echocardiographi in Measuring Left ventricular echocardiography in patients with or without wall motion volumes and Ejection Fraction. J Am Coll Cardiol, 2012: 1799-1808. abnormalities: comparison against cine magnetic resonance. Heart, 10. Chahal N, Tiong L, Jain R et al. Population-Based References 2008; 94:1050-1057. Values for 3D Echocardiography LV volumes and ejection fraction. J 7. Jenkins C, Bricknesll K, Chan J et al. Comparison of two- and three Am Coll Cardiol Img, 2012; 5:1191-7. dimensionla echocardiography with sequentail magnetic resonance 11. Hare JL, Jenkins C, Nakatani S et al. Feasibility and clinical imaging for evaluating left ventricular volume and ejection fraction decision-making with 3D echocardiography in routine practice. over time in patients with healed myocardial infarction. Am J Heart, 2008; 94:440-445. Cardiol, 2007; 99:300-6. 12. Ruddox V, Mathisen M, Bækkevar M. Is 3D echocardiography 8. Caselli S, Canali E, Foschi ML et al. Long-term prognostic superior to 2D echocardiography in general practice?: A systematic significance of three-dimensional echocardiographic parameters of review of studies published between 2007 and 2012. 2013, Int J the left ventricle and left atrium. Eur J Echocardiogr, 2011; 11:250- Cardiol, p. in press. 6.

Article received on May 15, 2019 and accepted for publishing on September 3, 2019. ORIGINAL ARTICLES

Aptamer as a proper alternative instead of monoclonal antibody in diagnosis and neutralization of menacing biological agents

Hadi E.G. Ghaleh1, Mojtaba Sharti1, Mohammad S. Hashemzadeh1

Abstract: Of the major threats to contemporary mankind, is the use of very dangerous and lethal biological agents as the biological weapons. The first step in confronting with this serious threat after prevention, is the accurate and rapid detection of this agents and neutralization of them. In this article, the role of molecules known as aptamer, has been studied in biological defense against these menacing biological agents. Traditional methods for detection of these agents are based primarily on immuno-affinity assays and the use of antibody molecules. While the modern methods, based on aptamer-affinity assays, are being replaced with traditional methods, due to the abundant advantages of them. The selection and preparation method of specific aptamer with high binding affinity to these biological agents is known as SELEX and the use of magnetic nanoparticles to perform this procedure (Mag-SELEX) is very common. The isolated aptamers with high specificity can also be used in neutralization and inhibition of menacing agents function, in addition to, quick and accurate diagnosis of these agents, utilizing them in nano-biosensors, based on aptamers (as the nano- aptasensors). Keywords: Aptamer, SELEX, nano aptasensor, diagnosis and neutralization, menacing biological agents

INTRODUCTION this article we will first introduce aptamer and chemical modifications possible to make on this molecule to approach Nowadays virulent and dangerous biological agents which certain purposes, and compare them to antibody molecules can be used as biological weapons are becoming a as a conventional tool in diagnoses and neutralization of the considerable threat to us [1]. Some of very important agents biological and viral agents or their exclusive products (such used in bioterrorism are namely botulinum neurotoxins as toxins and etc.) and we will interpret concisely the works which are some of the strongest toxins known to men and done around the world. produced by the anaerobic bacteria called clostridium botulinum [2]. Botulinum neurotoxins, especially three types APTAMER, AS A NEW MOLECULAR DEVICE of A, B and E, have been known as dangerous biologic weapons. Among these three serotypes, serotype A is more Aptamer is generated from the Latin word of “Aptus” which potentially virulent [3]. The first action against these very means “to fit”. In fact, aptamers are oligopeptides or single dangerous biological agents, after prevention is to identify stranded oligonucleotides (DNA or RNA) with high affinity for and detect the agent accurately and rapidly and then to binding to target molecule (Figure 1). They create a bond neutralize it [4]. Modern methods of identification and with high specificity through their third structure to a target neutralization is based on molecules called aptamer [5]. In molecule [6]. In this article we discuss about oligonucleotide aptamers, especially DNA aptamers. By development of

1 Applied Virology Research Center, Baqiyatallah University of Corresponding author: Mohammad S. Hashemzadeh PhD Medical Sciences, Tehran, Iran [email protected]

42 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine molecular sciences and setting up the SELEX technique, the be situated in that position with the same probability [11]. specific DNA/RNA aptamers have been separated for binding These fragments are prepared by frequent synthetizations, to many molecules. These molecules are consisted of nucleic with mixture of four types of nucleotides, coincidentally and acids, amino acids, sugars and their polymers, and also by synthetization machine (will extendedly be discussed in mineral compounds, enzymes, growth factors, antibodies, results and discussion section) [12]. the molecules binding to cells, chitins and even viral Aptamer library screening by SELEX cycles components [7]. In this stage, fragments with the highest affinity and Figure 1. A schematic representation of the binding of aptamer specificity for target protein is selected from the aptamer molecule to the target protein library, through several SELEX cycles (usually 13-15 cycles). Repetition of these cycles is required for attaining the suitable affinity [13].

Using magnetic nano-particles for screening by SELEX cycles (Mag-SELEX)

Some of Mag-SELEX benefits include: 1) No requirement for special facilities or equipment, 2) Easiness of gathering and separation of aptamers by SELEX process only using a magnet or a simple centrifugation, 3) Possibility of using nano particles for two purposes: stabilization of target protein and also making aptamers

single stranded in each cycle, Natural aptamers in living systems 4) Possibility of performing the methods such as flow cytometry for evaluation of affinity, Naturally, aptamer is found in bacteria, archaea bacteria, 5) Needing to little amount of protein in this technique fungi and plants, and acts as a part of riboswitch structures (around 100 micrograms), and often plays a role in regulation of expression of the 6) Possibility of coating nano particles with streptavidin for genes involved in production of vitamins (co-enzymes) and very strong interaction with biotin and thus prevention of in some cases is efficient in regulation of amino acids’ wastage of stabilized protein during the washing process in production [8]. which the conditions becomes tougher by rising cycle Evolution of aptamers in non-living systems number of SELEX, 7) Expansion of protein contact surface with existent In 1990, Gold and Tuerk attained RNA fragments which were aptamers in the solution and rise in SELEX efficiency and able to bind to T4 bacteriophage DNA polymerase. These abate of errors, oligonucleotide fragments were selected by a new 8) Easiness of stabilizing protein on these Nano particles. combinatory technique called SELEX (systematic evolution of ligands by exponential enrichment). The meaning of this It is worth mentioning that the main procedure of SELEX in expression is that among a collection of aptamers with different methods such as cell SELEX, CE SELEX, microfluidic various sequences, it is possible to attain the specific SELEX (M-SELEX) and etc. are the same [14-16]. sequence with high binding affinity to target molecule Corrections and chemical rearrangements of aptamer through some screening steps [9]. molecules

Aptamer library (AptLib) For increasing the stability and resistance of these molecules The initial library for performing the SELEX process includes in clinical use, labeling or increasing the performance of single stranded DNA (or RNA) oligonucleotides with a central these molecules, some modifications are made on these core (approximately 40 bases) containing random and aptamer oligonucleotides. variable sequences (more than 1,015 different fragments), in The most important modifications include: which every oligonucleotide is consisted of certain flanking sites in both ends that are fixed positions for primer 1) Corrections on carbon 2’ of sugar including: a) Adding attachment [10]. In the middle of these constant regions (i.e. fluorine and amino group to pyrimidine bases. b) Adding the central core), sequences of 15 to 75 (e.g. 40) nucleotides hydroxyl group to purine bases. c) Adding o-methyl group to are randomly situated and every four base (A, G, C, T) may purine and pyrimidine bases. In clinical applications, 2’-O-

methyl oligomers are in priority, for they are less toxic than little non-immunogenic molecules; and possibility of other corrections on carbon 2’ (these structures exist aptamer production regarding to these molecules. 9) Factors naturally in ribosomal RNAs) [17]. like pH variation, salt concentration, heat, and chelating agents may help the process of aptamer reactivation and 2) Capping of 3’-terminus of oligonucleotide including: a) creation of functional structures in them. But for antibodies Attachment of inverted thymidine (3’-idT) to 3’-end of these agents would cause denaturation of them. 10) oligonucleotide and creation of 3’-3’ connection. b) Stabilization of aptamers and labeling them are easier than Attachment of biotin to the 3’-end [18]. 3) Capping of 5’- antibodies. terminus of oligonucleotide including: a) Adding alkyl-amine group to 5’-end phosphate. b) Attachment of 5’-end of These prominences have caused them to be widely used as oligonucleotide to polyethylene glycol or on surface of molecular detectors in biosensors based on aptamer liposome. c) Adding cholesterol, fatty acids and protein [19]. molecules (apta-sensors) and nanobiosensors based on 4) Correction and chemical rearrangement of phosphate aptamer molecules (nano-apta-sensors) [24]. group which means replacement of sulfur group instead of the oxygen that doesn’t attend in phosphodiester bond AN OVERVIEW OF THE RESEARCH WORKS DONE UNTIL (thio-aptamer). 5) Adding signal peptides to aptamers such NOW, IN RELATION TO APTAMERS AND THEIR as penetrating peptides including Tat and antennapedia and APPLICATIONS fusogenic peptides [20]. 6) Labeling aptamers with Selection of aptamers for binding to antibodies, proteins fluorescent molecules (for example derivatives of and growth factors fluorescein such as FITC, FAM and etc.) for flow cytometry or with biotin molecules for stabilizing aptamer on solid surface - In 1996, Wiegand et al. succeeded in isolating aptamers) coated with streptavidin (for performing ELISA or in single stranded DNA and also RNA with 2’-amino chemical biosensors design) [21]. arrangement) against human IgE to inhibit the interaction of this molecule with “FcεRI” receptor on surface of basophilic It's rarely seen that the corrections and chemical leukemia cells of rat (as a new drug for the treatment of rearrangements made for resistance improvement are allergic diseases) [25]. effective on the main features, such as binding affinity and third structure. That's while in performance most of these - In 1995, Doudna et al. succeeded in selecting a RNA modifications are possible in primary library before SELEX aptamer to detect the main epitope of insulin receptor [22]. antigen on surface of human cells [26].

Comparison of aptamers with antibody molecules - In 1995, Geling et al. selected RNA aptamers (with 2’- amino chemical arrangement) to inhibit the activity of 1) Antibodies’ half-life is limited, while aptamers are very fibroblast growth factor [27]. stable despite of the chemical rearrangements of their nucleotides. 2) Antibodies unlike aptamers are sensitive to - In 2002, Lupold et al. isolated the RNA aptamer detecting environmental conditions such as temperature, pH and etc. prostate specific membrane antigen (PSMA) on surface of 3) Antibodies have irreversible denaturation, that's while prostate cancer cells and labeled that aptamer by aptamers have denaturation and renaturation capacity fluorescent molecules to observe its ligand-binding function because of their nucleic acid structure. 4) Antibodies unlike in condition of “in vivo” [28]. aptamers are immunogenic and this causes their rapid Disruption of translation pathway signs of lymphocytes by elimination in the body in clinical applications. 5) Size of Aptamar antibodies unlike aptamers is massive (around 150 KDa) which causes long-term pause of them in blood and slow - In 1997, Kubic et al. separated the binding and inhibiting penetration in tissues and as a result, causes bone marrow RNA aptamer against the interferon gamma (INF-γ) which toxicity in radioactive form [23]. 6) The procedure of caused the inhibition of MHCI and ICAMI expression antibody production is very difficult and for aptamers it is induction by INF-γ [29]. only difficult in the first time, during the panning steps of Selection of anti-viral aptamers SELEX and then by sequencing of the aptamer selected from library, it is possible to synthesize the sequence attained Some of these aptamers resulted in the elimination of virus easily. 7) Possibility of using aptamers in the chips that are cell cycle by interaction with the proteins necessary for virus based on DNA, so that in protein staining process aptamers proliferation and some of them were able to block surface do not get color. 8) Impossibility of antibody production for

44 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine binding proteins and consequently resulted in the single wall carbon nanotubes (SWNT) [41]. prevention of attachment and entry of viruses into cells [30]. - In 2012, Jeffery Degrasse et al. isolated a selective DNA - In 1995, Pan et al. separated the detecting and aptamer for detection of Staphylococcus aureus enterotoxin neutralizing RNA aptamer against Rose Sarcoma virus to B (SEB) for designing nano-aptasensor on the basis of surface inhibit the binding of the virus [31]. plasmon resonance (SPR) (Figure 2 – as seen in the figure, biotinylated apatamers have been stabilized on streptavidin- Selection of specific aptamers for binding and inhibiting the coated gold nanoparticles, and the above nanoparticles are function of enzymes also fixed onto gold discs and by use of corresponding - In 1998, Brindonau et al. succeeded in isolating 2’-amino device, signals from surface plasmon resonance (SPR) RNA aptamer by SELEX (as a new drug) for inhibition of changes are measured after detecting the target ligand by phospholipase A2 enzymatic activity in patients [32]. aptamer.) [42]. - In 2011, Tzu-Wang Chang et al. succeeded in isolating the Figure 2. A schematic representation of aptamer molecule RNA aptamer specified for inhibition of enzymatic activity of application in the design of nano-aptasensor. botulinum neurotoxin type A [33].

Selection of aptamers for detection of small molecules

- In 1998 and some years later, one DNA aptamer for Sulforhodamine B, two RNA aptamers for sulforhodamine B and fluorescein and one RNA aptamer for malachite green were separated by Wilson and Szostak [34].

Aptamers for cell diagnosis

- In 1999, Bruno et al. detected Bacillus anthracis spore by APTAMER AS A PROPER TOOL IN MODERN METHODS OF a single stranded DNA (ssDNA) aptamer using magnetic DIAGNOSIS beads [35]. Nowadays traditional methods based on immune-affinity - In 2005, Qin Pan et al. isolated the RNA aptamer detecting assays, in which antibody molecules are used, while the strains of Solmonella enterica serovar Typhi containing transposing with new methods based on aptamers-affinity type IVb pili [36]. assays [43].

- In 2007, Fan Chen et al. isolated the ssDNA aptamer Functions and features of modern determination methods detecting a virulent strain of Mycobacterium tuberclosis based on immune-affinity assays: called H37Rv [37]. 1. Aptamers are produced in vitro conditions and they do not - In 2006, Vivekananda et al. isolated 25 sequences of the require any living organisms, to which they allow us to use ssDNA aptamer identifying surface bacterial antigens of any molecules regardless of it being toxic for the organism Francisella tularensis japonica [38]. and separate exclusive aptamers for determination.

- In 2007, Ikanovic et al. designed an optical biosensor 2. After separation and selection of exclusive aptamers by based on aptamer for detection of Bacillus thuringiensis SELEX, we could easily reproduce and mass-produce it in bacteria [39]. high purity of specified aptamers.

Aptamers as tools for drug delivery 3. Mass production expenses are very low and they have proved to be cost effective. - In 2006, Farrokhzad et al. prepared nano-capsules of poly lactic acid (PLA) containing drug and linked to them the 4. Nucleic acids (including DNA-aptamers) by having some special aptamer related to prostate cancer cells through the functional groups can more easily be adapted to their target poly-ethylene glycol linkage (PEG) [40]. exclusive ligand. Without any malfunctions or lessening in tendency for binding with exclusive ligand in the specified Using aptamers for designing biosensors (apta-sensors) and domain. nano-biosensors (nano-aptasensors) 5. Aptamers are originally stable in a broader domain of - In 2011, Lakshmi et al. designed a chemical-resistant conditions. nano-aptasensor, for detection of anthrax PA toxin based on

6. Aptamers have very high tendency to synthesize and Separating the aptamer against the catalytic domain of dedicate compared to antibodies. botulinum neurotoxin for recognizing and counteracting this toxin 7. Aptamers with high affinity can be used for determination and detection of different sorts of ligands such as toxins, Choosing the catalytic site from other operational sites of allergens, sugars (that are weak immunogens), haptens, botulinum neurotoxin is important according to reasons nucleic acids, proteins and different kinds of little biological mentioned below: and non-biological molecules like morphine and etc. can be 1. Results gained from conducted studies around the world separated and used. It is while in previous methods there show that this operational site is very suitable for getting the were many limitations regarding to this matter [44]. recognizing and counteracting goals.

USING APTAMERS MOLECULES IN DETECTION AND - In 2011, Tzuu-Wang Chang et al. separated RNA-aptamer NEUTRALIZATION (INHIBITION) OF BOTULINUM exclusively for inhibiting botulinum neurotoxin type A NEUROTOXINS enzymatic activity [50].

As latterly mentioned, until today there have been many - In 2009, Fang Wei et al. used separated aptamer for researches around the globe on these oligonucleotides with recognizing botulinum neurotoxin type A in designing detection and neutralization purposes specifically on electrochemical sensors [51]. botulinum neurotoxins (such as type A). - In 2009, Xinhul Lou et al. separated the special conjugative Reports have indicated that aptamers molecules separated aptamers to catalytic domain of recombinant botulinum from library are suitable and proper for this matter [45]. neurotoxin type A for recognizing this neurotoxin [52].

Diagnostic purposes 2. The main and operational part of this neurotoxin is the catalytic part and the quality of this neurotoxin is basically In diagnosis of botulism agent as the one of biologic threat related to this part, in a way that said toxin will have no factors, we can separate some aptamers with high binding effect or special toxic function without this part [53]. So in tendency with SELEX method. Aptamers separated to conclusion the diagnosis and neutralizing value of this interact with botulinum toxin and also with different location will be much higher than other ones. And neurotoxin regions including catalytic domain can be very recognizing this location will lead to the certain recognition effective in diagnosis purposes. But catalytic domains are of infection to this type of neurotoxin, whereas recognizing more efficient and useful in diagnosis purposes for other locations won’t necessarily mean that and it’s possible neurotoxin types specially type E [46]. that other locations be used as recombinant with other Inhibitory goals factors [54].

In inhibiting and neutralizing this extremely lethal factor we 3. The unique structure of botulinum neurotoxin type E and can get our hands on molecules with inhibiting and the importance of epitopes situated on this location and neutralizing effects on operational sites, by investigating symmetric bond to the connector site, in a more general inhibiting effects of separated aptamers for exclusive sense epitope makes the selection of this site for the toxin connections with high tendency to catalytic site [47]. for this serotype exclusively more suitable (Figure 3 – the red represents the catalytic region, the green color indicates the These aptamers might hinder the activation of this translocating region and the yellow color indicates the operational site by connecting to de-activated catalytic site binding region of the neurotoxin. As can be seen, both the of the toxin’s body or by binding themselves to the freed catalytic and binding regions are located on one side of the catalytic site and cause the enzyme activity to stop [48]. In translocating region, not on two sides as seen in the other fact, inhibiting this position’s enzymatic function leads to types.) [55]. restraining the toxin’s function. But antidotal aptamers can do the neutralizing process by connecting to other sites like RESULTS AND DISCUSSION connector sites and actually prevent the toxin from connecting to the cell and entering it [49]. Studies have shown that aptamers with central core of 75- 100 base length are not suitable aptamers and on the other hand aptamers with the central core of lesser than 15-20 base also cannot be suitable aptamers [56]. If the length of aptamers fragments is longer than the above:

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Figure 3. A schematic representation of second structures of be seen as smear. neurotoxin botulinum type E and a symmetric relationship between catalytic and binding domains of toxin. 3. Also the larger of these fragments be their particular operationally will also be decreased. Because after the formation of the 3rd structure there will be more binding sites to bind to target protein that each of which will have different affinity and can bind to our protein from different sites and positions and one of these position might have a high infinity to our target protein but no other position. So in conclusion the average affinity will drop and this means the decrement of specificity (affinity and specificity are two completely separate parameters) [57-59].

If the length of aptamers fragments is shorter than the above:

1. We’ll witness a significant decrease in genetically variety of aptamer fragments and this will significantly limit our screening stages.

2. On the other hand the 3rd structures and conformation of aptamer which is the basis of aptamers binding to biomolecules will not form properly or won’t form at all and this means the inefficiency of aptamer. 3. Another problem is the drawback in performing PCR and 1. The possibility of creating similar sequences with primer evaluating the target binding which distinguishing it from in the central core is increased and with binding primer to dimer primer is not easily achievable. this central parts and amplification, no particular fragments with shorter length will be the outcome. 4. On the other side we’ll face up to a huge problem in the purification of a production with such dimensions. Studies 2. On the other hand the possibility of connecting flanking have shown that lengths about 40 base will be adequate and sites of 2 sides (primer binding sites) with central sequences as we advance to larger or smaller numbers we will of various fragments, is increased and will cause an encounter the problems mentioned above more frequently unhindered length increase and generation of fragments although we are free to choose between 15-75 (20-100 with larger sizes and PCR product of these two processes will according to some sources) (Figure 4) [60-62].

Figure 4. A schematic form of the single-stranded oligonucleotide structure of aptamer with a central core of 40 nucleotides. Scientifically and statistically, there is the probability of having a variety of 440 aptamer fragments (which is 1024 fragments) with a central core of 40 nucleotides, but optimistically virtually this variation is about 1015 aptamer fragments.

In Figure 5 the schematic form of SELEX cycle on RNA removing the non-original strand the next cycle is again aptamer library is shown. The primary library made of DNA continued by transacting on the product mentioned above is firstly turned into a RNA library during a transcription and creation of a RNA aptamer library [63-65]. It is necessary process and after being added to nano particles covered with to perform a stage of negative selection in the presence of target proteins and performing the stages of washing and similar proteins. reunifying by performing the process of “asymmetric RT- Figure 6 shows the increment of aptamers’ affinity as with PCR”, is transformed to c-DNA and after single threading and the progress of SELEX stages, which is evaluable using the

ELASA method (aptamer-based ELISA). Then we can perform Figure 7. A schematic picture of two types of aptamers. 7a: An a software based test on aptameric sequences using M-fold aptamer library with a central core containing a fixed region that software. is obtained by software evaluating by the M-Fold software. 7b: A simple aptamer library with a completely variable core

Figure 5. A schematic representation of SELEX cycle on RNA aptamer library

In Figure 8 the pattern of aptamer nucleotides labeled with digoxigenin (DIG) recognizing the target molecule is shown, so in part A the flow cytometry is used for identifying the interaction between aptamer and target molecule and in part B the chemiluminescence is used. Part C shows the chemical formula of digoxigenin [69].

This investigation will give us the chance to understand how Figure 8. A) Digoxigenin labeled aptamer identifying the target nucleotide changes in the final aptameric sequences causes molecule and measuring the binding by flow cytometry B) Digoxigenin labeled aptamer identifying the target molecule and a change in operational structure [66]. On the other hand, measuring the binding with the ELISA method C) The chemical the software itself will predict different kinds of possible formula of digoxigenin. operational structures for that aptamer molecule and it will also suggest the best possible form. Sometimes the result of such investigations will show us the existence of a necessary operational site in the central core, which will have a key role in binding to the target protein [67].

Figure 6. Binding affinity of aptamers increases with the progress of the SELEX steps

In a more accurate investigation for getting aptamers with higher affinities and higher specificity for binding to target In Figure 9 an image of the function of flow cytometer protein we can put this specified operational site in the machine is shown. In a way that the sample containing the central core of a new aptamer library and perform the SELEX aptamers marked with fluorescent (marked aptamers bound stages on that library again (Figure 7a and 7b) [68]. to nanoparticles) is passed through a fluorescent sensor and after radiating laser light on that the number of marked

nanoparticles are counted [70].

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Figure 9. Flow cytometer device operation attributes in this biomolecule for the means of getting to the considered goal and the beneficial role of this molecule as a powerful tool for recognizing and neutralizing (inhibiting) the threatening factors shows great promise. These days the traditional methods for recognizing the biological factors based on immuno-affinity assays in which antibody molecules are used are being replaced with the new methods based on aptamer-affinity assays. The selected aptamers during the different stages of SELEX, with the ability of creating exclusive bonds and high tendency to interact with operational sites of biologic factors, in addition to having precise and fast recognition of this factors e.g. utilizing them in biological nano-sensors based on aptamer CONCLUSION (nano-aptasensor), they could also be used in neutralizing The most recent discoveries related to aptamer molecule and inhibiting the function of these threatening biological shows the existence of particular properties and unique agents.

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Article received on May 15, 2019 and accepted for publishing on August 13, 2019. ORIGINAL ARTICLES

Alteration of levels of thyroid hormones in acute ischemic stroke and its correlation with severity and functional outcome

Brinder M.S. Lamba1, Shalini Sharma1, Pulin Gupta1

Abstract: Introduction – With increasing life expectancy and changing trends in diseases, lifestyle diseases come forward and become a major concern. Ischemic stroke is an emerging public health issue leading to significant morbidity and mortality. To improvise positive outcome in acute ischemic stroke (AIS) and optimizing healthcare resources, accurate and timed prognostication of functional outcome is important. Various studies implied derangement of Hypothalamo- pituitary-thyroid (HPT) axis in AIS, thereby advocates neuroprotective properties to these hormones. This is, still, an open field for active research. Thyroid hormones can, therefore, be used as a surrogate tool for outcome predilection in AIS. This prospective observational cohort study aimed to investigate the alteration of levels of thyroid hormones in AIS and simultaneously correlate their levels in assessment of clinical severity and predicting the functional outcome. Materials and methods – We had collected data from 60 patients of, radiologically confirmed new cases of AIS who sought medical help within 72 hours of initiation of stroke symptom and had no previous history of thyroid disease. Blood samples for thyroid function test [TSH, FreeT3 (FT3), and FreeT4 (FT4)] were collected within 24 hours after admission and subsequently at 7th and 30th day of discharge .On admission neurological clinical severity was assessed by using National Institute of Health Stroke Scale (NIHSS). Functional outcome was assessed by Modified Rankin Scale (mRS) at 7th and 30th day of discharge. Result – Out of 60 AIS patients, 40 were having Non thyroidal illness Syndrome (NTIS). Low T3 was most common NTIS observed. Negative correlation was observed between FT3 levels and on admission NIHSS score and mRS score on follow up. Conclusion – This study thereby concludes that low T3 levels were associated with clinical severity and poor functional outcome in AIS. Keywords: stroke, thyroid profile, correlation, NIHSS, mRS, NTIS

INTRODUCTION the fourth most common cause of death behind the disease of heart, cancer and chronic lower respiratory disease [2]. Stroke, described by the WHO, as the clinical syndrome of Globally, stroke is the second leading cause of death above rapid onset of focal (or global, in SAH) neurological deficit, the age of 60 years [3]. persisting more than 24 hrs or leading to death with no apparent cause other than a vascular one. [1] After dementia, CVA is the second chief cause of disability. Disability comprise of deprivation of speech and /or vision, It is an emerging health problem all over the world. confusion and paralysis [3]. Worldwide every year, 15 million people suffer a stroke. It is Burden of stroke is increasing, which can be, due to

1 Post Graduate Institute of Medical Education and Research, Dr. Corresponding author: Brinder Mohan Singh Lamba MD R.M.L. Hospital, New Delhi, India [email protected]

52 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine increasing longevity, smoking, changing habits &lifestyles especially within the normal range, on hospital admission and type A personality. Beside these, other known risk provide worthy prognosticative information. A better factors include hypertension, obesity, diabetes mellitus and understanding of this issue can be used for risk stratification metabolic syndrome. in AIS and hence better healthcare resource utilization.

In comparison to developed countries, where the incidence The basic aim of our study was to find out whether there of stroke has been decreased or reached a plateau, burden occurs any alteration in thyroid hormone levels in AIS and of stroke has been increasing in developing countries [3, 4, also to correlate their levels in assessment of stroke severity 5]. India, like other developing countries, is in the midst of and functional outcome as evaluated by using NIHSS and epidemic of stroke. modified Rankin scale (mRS) respectively.

In rural areas, the estimated adjusted stroke prevalence rate MATERIALS AND METHODS range 84-262/ 100,000 and 334-424/100,000 in urban areas. The incidence rate is 119-145/100,000 based on recent This study was performed at the Department of Medicine at population based studies. [6] In India, nearly one fifth of PGIMER (Post Graduate Institute of Medical Education and patients with first ever stroke admitted to hospital is Research), Dr RML Hospital, New Delhi from November 2016 estimated to be of age 40 years or less [7]. Globally, a third to March 2018. It was a prospective observational study in of all strokes occur in 20-64 year age group [8]. which a cohort of 60 patients of AIS was admitted and followed up at 7th and 30th day after discharge. Stroke can be classified into ischemic and hemorrhagic type. The Indian Collaborative Acute Stroke Study (ICASS) Inclusion criteria reported that up to 80% stroke were of the ischemic type [9]. Any patient of age 18 years or older & with no previous The ICMR has advocated tobacco use, diabetes mellitus, history of thyroid hormone abnormality, with first ever hypertension and low concentration of hemoglobin as main radiologically confirmed ischemic CVA presented within 72 risk factors for ischemic CVA [10]. hours of onset of stroke symptoms. Review of the literature reveals a significant derangement in Exclusion criteria thyroid hormones in patients with critical illness, specifically during the acute phase [11]. Euthyroid Sick Syndrome or Non 1. Patients with known thyroid disease or biochemically Thyroidal Illness Syndrome (NTIS) is a condition in which defined overt thyroid disease, derangement of thyroid hormones occurs in association with 2. Patients with transient ischemic attack, or past history of severity of disease and stress like surgeries [12] The most stroke, common pattern found under NTIS is low T3 level with normal level of T4 and TSH [13,14]. 3. Those using medications that can affect thyroid functions,

In acute ischemic stroke (AIS), HPT axis are significantly 4. Those with cancer, severe renal or liver failure, deranged. Various studies advocate low T3 levels as an inflammatory or infectious diseases and hematological indicator of poor prognosis in AIS [15, 16]. A reduction of diseases. serum T3 without elevation of TSH is associated with severity The enrolment of patients was done after informed consent. and worse clinical outcome in stroke [15, 17, 18]. Whether All the issues, including ethical issues of the study had been the prognosis is direr in AIS patients than those with the reviewed by the Institutional Review Board and approved. same disease but without the low T3 syndrome remain unknown. In AIS, implication of thyroid hormones is hence, The patient was first evaluated clinically which include a controversial. detailed history and examination.

Low T4 syndrome, a condition found in severe illness, in Baseline characteristics, include demographic data such as which T3 and T4 levels are reduced while TSH level does not age, gender, and concurrent diseases, thyroid disorder show expected pituitary thyroid axis reactivity [13, 14] related medication use, risk factors of stroke, such as a history of diabetes mellitus, coronary artery disease After acute stroke, better survivals were reported even hypertension, smoking and TIA. when the cut-off point used was the median T3 levels [15], showing that lower T3, even it is in the normal range, may Acute ischemic CVA was confirmed by using radiological be associated with poorer prognosis. However, the modality NCCT head /CECT head/MRI brain was used. association was not confirmed by other studies [19-21]. On admission, stroke severity was assessed by using NIHS Therefore, it is not clear if, in ischemic CVA, lower T3 levels,

scale and was divided into 3 categories:- severity and functional outcome was evaluated by Pearson • Mild NIHSS score <8 correlation. The chi square and independent sample t test • Moderate NIHSS score 8-14 were used to compare respective categorical and continuous • Severe NIHSS score ≥ 14 variables between different outcome groups. Variables with significant difference between different outcome groups Blood samples for assessment of the thyroid profile was were included in multiple logistic regression analysis. The drawn within the first 24hrs of presentation and then criterion of statistical significance was set at p value < 0.05. subsequently during follow up at 7th day and 30th day after discharge. RESULTS FT3, FT4 and TSH was measured by using chemiluminescent The mean age of the study population was 63.75 ±11.755 SD micro particle immunoassay. years with maximum age reported was 87 years and the Normal range in our laboratory is minimum age was 38 years (Figure 1). Out of 60 study • TSH 0.5-5.0 U/ml subjects 14 were female and 46 were male (Figure 2), • Free T3 (FT3) 2.0-4.4 pg/dl suggesting ischemic stroke was more frequent in male • Free T4 (FT4) 0.7-2.0 ng/dl (76.7%) as compared to female (23.3%) and male to female ratio was 3.28:1. On the basis of thyroid profile, the patients were divided into five categories: Figure 1. 1. NTIS was diagnosed as a low FT3, low or normal FT4, and low or normal TSH. Low T3 syndrome was defined as low FT3 levels with normal TSH and FT4 levels. [19, 22, 23, 28]

2. Euthyroid was diagnosed as normal FT3, FT4, and TSH. [19, 22, 23]

3. Subclinical hypothyroid was diagnosed as normal FT3, normal FT4, and high TSH. [24, 25, 26, 27, 29] Figure 2. 4. Hyperthyroid was diagnosed as low TSH with or without high FT4 and FT3 [22, 30, 31]

5. Hypothyroid was diagnosed as high TSH with low FT4 and normal FT3. [23, 30, 31]

Functional outcome was evaluated by using the Modified Rankin Scale (mRS) at the first follow up clinic visit at 7th day and subsequently 30th day after discharge. The mRS [32, 33, 34] is a scale to determine the disability after stroke. It has a score of 0-6, with 0 referring to no limitations or symptoms, The incidence of stroke increases with presence of risk 5 being severe disability requiring constant care and 6 being factors. Common vascular risk factors, as shown in Table 1, dead included in the study were hypertension (n=40, 66.7%), diabetes (n=23, 38.3%), smoking (n=38, 63.3%) and On the basis of this scale, patients were divided into 2 dyslipidaemia (n=17, 28.3%). outcome groups: • Poor functional outcome (mRS > 2) Figure 3. • Good functional outcome (mRS ≤ 2)

Data was analysed by using SPSS version 21.0 Qualitative & categorical or nominal variables were expressed as numbers (percent) or proportion. Quantitative & continuous or ordinal variables were expressed as medians or mean ± standard deviation (SD) Association and comparison of two categorical variables was done by using Chi square. The At admission, 19 patients (31.7%) had mild, 23 (38.3%) had association between thyroid hormone levels and stroke moderate, whereas 18 (30%) had a severe stroke on NIHS

54 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine scale (Figure 3). There was no statistical significant difference found between NIHSS score and age, gender, vascular risk factors for stroke studied (p value >0.05).

Table 1: Distribution of risk factors according to age group Age (year) <49 50-59 60-69 70-79 >80 Total p-Value

Hypertension HTN 3 11 10 8 8 40 >0.05 (HTN) non HTN 1 8 3 6 3 20 Diabetes 1 9 5 4 4 23 Diabetes (DM) >0.05 non DM 3 10 8 10 6 37 Smoker 1 12 8 11 6 38 Smoker >0.05 non 3 7 5 3 4 22 Dyslipidemia 1 2 5 5 4 17 >0.05 Dyslipidemia non 3 17 8 9 6 43

Figure 4: Comparison between thyroid patterns observed

Figure 5: NIHSS score and thyroid dysfunction on admission

Table 2: Characteristics of AIS with low T3 & normal T3 values low T3 group normal T3 group p value (n=40) (n=20) Clinical variables Age (years) 65.75 65.50 0.819 Sex (% male) 31 (77.5%) 15 (75%) 0.829 Hypertension 27 (67.5%) 13 (65%) 0.846 Diabetes 16 (40%) 7 (35%) 0.707 Smoking 25 (62.5%) 13 (65%) 0.850 Dyslipidemia 8 (20%) 9 (45%) 0.064 NIHSS (median) 11 6 0.000 Baseline NIHSS score 12.35±4.5 7.15±2.3 0.000 Distribution of NIHSS scores Mild 5 (12.5%) 14 (70%) 0.000 Moderate 17 (42.5%) 6 (30%) 0.000 Severe 18 (45%) 0 0.000 Thyroid function test (on admission) TSH 2.40±1.12 3.81±2.87 0.05 FT3 1.28±0.50 2.37±1.0 0.05 FT4 1.60±0.64 1.57±0.77 0.05 mRS_7 (median) 3 2 0.000 Pt with mRS_7 <2 8 (20%) 16 (80%) 0.000 mRS_30 (median) 3 2 0.000 Pt with mRS_ 30 <2 13 18 0.000

Thyroid pattern observed in this study (Figure 4) was low T3 Table 3. Correlation between FT3 & NIHSS scores on admission syndrome (n=40, 66.7%), euthyroid (n=8, 13.3%), subclinical NIHSS FT3_P hypothyroid (SCH, n=4, 6.7%), hyperthyroid (n=3, 5%) & hypothyroid (n=5, 8.3%) during admission. Pearson Correlation 1 -0.712** NIHSS Sig. (2-tailed) 0.000 A statistically significant (r=0.436, p value <0.05, Figure 5) direct relationship found between NIHSS score and the N 60 60 number of patients with the low T3 syndrome. Pearson Correlation -0.712** 1 FT3_P Sig. (2-tailed) 0.000 Figure 6. Scatter plot graph between FT3 values and NIHSS scores on admission. Each circle represents a patient N 60 60 ** Correlation is significant at the 0.01 level (2-tailed). FT3_P: free T3 levels on presentation /admission

In order to study the impact of T3 levels of severity & outcome of stroke in our study, we divided cohort of 60 patients into two groups – low T3 and normal T3 group and also analyze the distribution of risk factors among them (Table 2). No significant difference observed between age, gender & vascular risk factors between these two groups.

There were significant negative correlations between free T3

(FT3) levels and NIHSS scores on admission (n=60, r= -0.712,

56 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine r2=0.507, p=0.000, 99%CI, Figure 6, Table 3) among all admission as well as 7th and 30th day of discharge (Table 4 – patients. This implies, after acute stroke, that the lower FT3 correlation is significant at the 0.01 level 2-tailed). NIHSS values are, the worse neurological impairment will be. Scores were shown a negative correlation with FT4 values only on follow up. Although the strength of the correlation NIHSS Score was negatively correlated with TSH on was not as stronger as it was with FT3 levels.

Table 4: Associations between thyroid hormones and NIHSS score on admission and on 7th and 30th day of discharge Pearson Correlation Sig NIHSS r2 (r ) (2 –tailed) TSH_P -0.227 -0.227 0.051 0.041 FT3_P -0.712 -0.712 0.507 0.000 FT4_P -0.043 -0.043 0.002 0.747 TFT_P -0.402 -0.402 0.162 0.001 TSH_7 -0.259 -0.259 0.101 0.044 FT3_7 -0.753 -0.753 0.567 0.000 FT4_7 -0.264 -0.264 0.070 0.042 TFT_7 -0.455 -0.455 0.207 0.000 TSH_30 -0.343 -0.343 0.117 0.007 FT3_30 -0.787 -0.787 0.619 0.000 FT4_30 -0.365 -0.365 0.133 0.004 TFT-30 -0.479 -0.479 0.229 0.000

ASSESSMENT OF FUNCTIONAL OUTCOME outcome had mRS Score 6 (dead) constituting 13.7 % of patients with mRS score>2. Retrospective analysis of these Final outcomes were evaluated at 7th and 30th day of patients revealed that they had a severe stroke on NIHS discharge and the mRS score so obtained was compared with scale. Analysis of thyroid hormone pattern on admission and on admission NIHSS Score as well as thyroid hormone levels at 7th day of discharge were suggesting that they belong to measured concurrently. the low T3 syndrome. They had very low levels of free T3. Out of 60 patients, 36 patients (60%) had a poor functional TSH and free T4 levels were also low. These mortalities outcome (mRS>2) at 7th day of discharge in comparison to thereby emphasize severe derangement of hypothalamo- 29 patients (48.3%) who still had a poor functional outcome pituitary-thyroid axis. at 30th day of discharge. Univariate analysis showed that mRS scores at first and No mortality either in hospital or hospice was observed on second follow up visit, were not associated with age, gender first following up clinic visit. However, on second follow up and vascular risk factors like hypertension, smoking, clinic visit, 4 patients, among those with poor functional diabetes & dyslipidaemia (p>0.05, Table 5).

Table 5: Demographic and vascular risk factors according to outcome at 7th & 30th day of discharge Modified Rankin Age Sex Smoking HTN Diabetes Dyslipidemia Scale (mRS) (years) (male %) (n, %) (n, %) (n, %) (n, %) TSH_P mRS≤2 (n=24) 64±11.4 19 (79.2%) 20 (83.3%) 13 (54.1%) 07 (29.2%) 09 (37.5%) FT3_P FT4_P mRS>2 (n=36) 63.6±12.1 27 (75.0%) 18 (50.0%) 27 (75.0%) 16 (44.4%) 08 (22.2%) p value > 0.05 (NS) TFT_P mRS≤2 (n=31) 61.4±12.6 23 (74.2%) 22 (70.9%) 19 (61.2%) 09 (29.0%) 10 (32.3%) TSH_7 TFT-30 mRS>2 (n=29) 66.2±10.3 23 (79.3%) 16 (55.2%) 21 (72.4%) 14 (48.3%) 07 (24.1%) p value > 0.05 (NS)

Table 6: Admission clinical and lab findings according to outcome at 7th & 30th day of discharge Modified Rankin scale Modified Rankin scale P value at 7th day at 30th day mRS_7≤2 mRS_7>2 mRS_30≤2 mRS_30>2

(n=24) (n=36) (n=31) (n=29) Baseline NIHSS 6 13 6 15 <0.05 (median) Baseline NIHSS 6.83±2.26 13.14±3.92 7.23±2.14 14.24±3.56 <0.05 (mean±SD) Distribution of NIHSS score Mild (n) 17 2 19 0 <0.05 Moderate (n) 6 17 12 11 <0.05 Severe (n) 1 17 0 18 <0.05 Thyroid hormone levels at admission TSH 3.15±2.23 2.69±1.81 3.27±2.29 2.45±1.28 <0.05 FT3 2.16±0.83 1.30±0.72 2.08±0.87 1.18±0.59 <0.05 FT4 1.60±0.61 1.50±0.73 1.57±0.73 1.62±0.64 <0.05 Thyroid hormone levels at 7th day of discharge TSH 3.09±2.21 2.62±1.59 3.19±2.29 2.40±1.16 <0.05 FT3 2.20±0.84 1.33±.73 2.13±0.86 1.20±0.61 <0.05 FT4 1.56±0.65 1.47±0.64 1.60±0.72 1.42±0.45 <0.05 Thyroid hormone levels at 30th day of discharge TSH 3.17±2.05 2.66±1.47 2.42±1.36 2.40±1.23 <0.05 FT3 2.30±0.81 1.42±0.82 3.31±2.01 1.24±0.71 <0.05 FT4 1.58±0.61 1.35±0.54 2.30±0.81 1.24±0.43 <0.05

Patients with poor functional outcome were having high patients with poor functional outcome there might be baseline & median NIHSS Score. The majority of patients decreased peripheral conversion of FT4 to FT3 which lead to with poor outcome had moderate to severe score on NIHS low levels of FT3. Scale (p value <0.05, Table 6). Free T3 levels were significantly lower in them during first as well second follow Table 7. Correlation between FT3_7 values and mRS_7 scores at up visit (p value <0.05, Table 6). 7th day of discharge mRS_7 FT3_7 Figure 7. Scatter plot between FT3 values and mRS_7 scores at Pearson Correlation 1 -0.692** 7th day of discharge mRS_7 Sig. (2-tailed) 0.000 N 60 60 Pearson Correlation -0.692** 1 FT3_7 Sig. (2-tailed) 0.000 N 60 60 **Correlation is significant at the 0.01 level (2-tailed). mRS_7: modified Rankin scale at 7th day of discharge FT3_7: free T3 levels at 7th day of discharge

On the 7th day of discharge, there was a significant negative correlation between mRS scores and free T3 levels These findings, consistent with the findings reported by Xu [correlation between mRS & FT3 on admission (n=60, r= - and his colleagues [21], which is due to the fact that in 0.599, r2=0.301, p<0.05, 99%CI, Table 8) and at 7th day of

58 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine discharge (n=60, r= -0.692, r2=0.479, p<0.05, 99% CI, Figure On 2nd follow up clinic visit, similar trends of negative 7, Table 7)] correlation was observed between mRS score and free T3 levels (Figure 8, Table 9). Table 8: Associations between thyroid hormones on admission & at 7th day of discharge and mRS scores at 7th day This implies that AIS patients with the low T3 syndrome tend to have poor outcome at follow up. From Table 8, it was mRS _7 Pearson Correlation Sig r2 scores (r ) (2 –tailed) inferred that at first follow up visit, no statistically significant correlation found between mRS score and FT4 levels (neither TSH_P -0.215* -0.215* 0.046 0.009 an admission nor at 7th day of discharge). There were a FT3_P -0.599** -0.599** 0.301 0.000 statistically significant negative correlation between TSH FT4_P -0.021* -0.021* 0.0004 0.874 levels (on admission, at 7th and 30th day of discharge) and TFT_P -0.384** -0.384** 0.147 0.002 mRS score at 30th day of discharge (p value <0.05, Table 9). No relation was seen between FT4 levels (on admission, & at TSH_7 -0.242* -0.242* 0.058 0.006 7th day of discharge) and mRS score at 2nd follow up visit (p FT3_7 -0.692** -0.692** 0.479 0.000 value >0.05, Table 9). But a negative relationship seen FT4_7 -0.150* -0.150* 0.023 0.254 between the two at 30th day of discharge (r = -0.387, CI 95%, TFT_7 -0.445** -0.445** 0.198 0.000 p value <0.05, Table 9).

Figure 8. Scatter plot graph between FT3_30 values and mRS_30 Table 9: Associations between mRS_30 scores and thyroid scores at 30th day of discharge. hormones on admission and on 7th and 30th day of discharge mRS_30 Pearson Correlation Sig r2 scores (r ) (2 –tailed) TSH_P -0.250* -0.250* 0.063 0.054 FT3_P -0.600** -0.600** 0.360 0.000 FT4_P -0.095* -0.095* 0.009 0.570 TFT_P -0.376** -0.376** 0.141 0.003 TSH_7 -0.281* -0.281* 0.466 0.029

FT3_7 -0.683** -0.683** 0.028 0.000 FT4_7 -0.166* -0.166* 0.042 0.204 This findings can be explained by the fact that even with the TFT_7 -0.432** -0.432** 0.186 0.001 severity of stroke, levels of T3 decrease immediately, but it TSH_30 -0.376* -0.376* 0.141 0.003 requires some time for T4 levels to show changes in their FT3_30 -0.724** -0.724** 0.524 0.000 levels. FT4_30 -0.387* -0.387* 0.149 0.002 On multiple logistic regression analysis (Table 10), lower FT3 TFT-30 -0.477** -0.477** 0.228 0.000 concentration (odds ratio OR=1.166, 95%CI, p value <0.05) **Significance of Correlation at the 0.01 level (2-tailed). remained independently related to poor outcomes. * Significance of Correlation at the 0.05 level (2-tailed). mRS_30: mRS score at 30th day of discharge

Table 10: Multiple logistic regression analysis NIHSS* TSH* FT3* FT4* OR (95% CI), OR (95% CI), OR (95% CI), OR (95% CI), p p p p 7th day outcome 0.641 0.936 1.166 0.803 mRS ≤2 vs mRS >2 (0.457-0.845) 0.661-1.326 (0.425-3.200) (0.289-2.229) or death P<0.05 P<0.05 P<0.05 P>0.05 30th day outcome 0.418 1.125 1.296 0.255 mRS ≤2 vs mRS >2 (0.241-0.725) (0.744-1.701) (0.276-6.087) (0.052-1.258) or death p<0.05 p<0.05 p<0.05 p>0.05 * values at the time of admission

Table 11: ΔmRS score vs Δthyroid pattern during follow up Δthyroid pattern Total no change euthyroid ΔmRS improvement 10 4 14 Pearson chi square= 3.08 same statuts 27 11 38 p value 0.215 detoriation 8 0 8 Total 45 15 60 ΔmRS = change in functional outcome (mRS_30 score - mRS_7 score) Δthyroid pattern = change in thyroid pattern

Figure 9. Thyroid hormones pattern observed at various time interval in this study

In addition to the FT3, the only other variables from either trauma or hemorrhagic CVA, low T3 has been independently associated with poor functional outcome reported [38, 39]. The present study showed thyroid were NIHSS score (OR=0.641, 95% CI, p value <0.05) and TSH hormones derangement in AIS and most common type of it levels (OR=0.936, 95%CI, p value <0.05). was NTIS/low T3 syndrome.

Of 40 patients with the low T3 syndrome, 13 patients An inverse relationship between T3 levels and NIHSS score became euthyroid on follow up (Figures 4 & 9, Table 11). This was observed in our study. To follow up, patients with poor can be explained by the fact that during disease recovery, functional outcomes were found to have low free T3 levels. thyroid hormones and TSH concentrations return to normal Thus, an inverse relationship observed between FT3 value [35]. This emphasize the need to follow up of such patients and NIHSS score as well as mRS score, thereby strengthening to confirm suspected cases of sick euthyroid. Similarly, on its predictive role in estimating the severity and functional follow up, a very small percentage had shown improvement outcome in AIS. in their functional outcome. However, this finding was not The degree of decrease in T3 levels is related to worse statistically significant (p>0.05). neurological impairment. Thus, monitoring levels of FT3 could potentially be an easy, quick and surrogate marker in DISCUSSIONS the future if other studies confirmed it further. The Conduction of this study was done with an objective to observations and findings delineated in the present study decipher the relationship between thyroid hormone levels were similar to studies done by Alvezaki et al [17] which and functional outcome in AIS. Observations recorded were stated that low T3 is a possible independent predictor of similar to studies conducted by Alvezaki et al [17] and Zhang stroke outcome. et al [19]. In hospitalized patients, especially in ICUs, the In severe illness, T3 levels would decline to spare energy, most common type of NTIS seen is low T3 [36, 37]. In ICH considering it as an adaptive response to stressful

60 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine conditions. [40-44]. prognosis can have higher or normal T4 levels.

Similar to other systemic severe diseases, a decline in serum On univariate analysis, during follow up, no relationship was T3 with no increment in TSH levels seem to be related to observed between mRS score and baseline parameters, i.e. stroke severity and poor clinical outcome [17, 20, 45-47]. age, gender and vascular risk factors. It was observed that AIS patients with high moors scores had low levels of free T3 Possible mechanisms include the following: and also had a high NIHSS score, both baseline and median. (1) Change in the peripheral metabolism of thyroid These findings, consistent with the findings reported by Xu hormones due to alterations in enzyme activity involved in and his colleagues [21], which is due to the fact that in peripheral T4 to T3 conversion, [42, 48] patients with poor functional outcome there might be decreased peripheral conversion of FT4 to FT3 which lead to (2) Involvement of pro-inflammatory cytokine action, [49] low levels of FT3. (3) A disturbed shift in thyroid hormone distribution or At first follow up clinic visit, only other thyroid parameters change in binding proteins, [41, 49, 50] with which mRS score can be correlated was TSH. Similarly, (4) In severe illness, excessive glucocorticoids release, which on 2nd follow up visit, mRS score was negatively correlated lead to inhibition of hypothalamic-pituitary- thyroid axis with TSH levels. A negative correlation was observed activities and T4 to T3 conversion. [51] between free T4 and 2nd follow up visit mRS score only at 30th day of discharge. To sum up, apart from free T3, TSH Similar to various studies conducted in ICUs, AIS patients levels would be next important parameter to determine with low T3 or combination with low T3 and T4 found to have functional outcome in AIS. However, free T4 would not show a worse prognosis. [15, 43, 48, 52-54] any significant change in its level immediately, but ultimately Interestingly, studies showed that derangement in T3 levels its levels also decrease in those with severe stroke and poor is not related with regional distribution of stroke (anterior vs outcome which would be observed only on follow up. posterior circulation). This change in thyroid hormone levels In the present study, no significant difference observed can, therefore, be due to disturbance in their metabolism between age, gender and vascular risk factors among AIS instead of any structural defect induced due to abnormality patients with low T3 and those with normal T3 levels. Zhang in blood supply to hypothalamic-pituitary-thyroid axis. [19, et al [19] study, however, showed that low T3 group patients 23] were slightly but not significantly older. Alvezaki et al [17] A significant negative correlation observed between NIHSS study showed that patients under low T3 group were score and TSH (p value < 0.05) on admission and on follow significantly older. In elderly, baseline thyroid function is up. On admission, no correlation was observed between usually at the lower side and when stressful events like NIHSS score and free T4, however, negative correlations stroke occurs, they tended not to compensate fully. This was observed between them only to follow up. Unlike, NIHSS the explanation offered by his study. score and FT3 levels, strength of association between NIHSS Except low T3 subgroup, none of the other subgroups had score and TSH & T4 levels were not strong. shown any mortality. In this subgroup on follow up, 4 After CVA onset, within hours, the decline in serum T3 levels patients (10%) were expired. Retrospective analysis of these and degree of alteration in its levels is associated with the patients showed that they had a severe stroke on NIHS Scale severity of stroke.[48 ,55-57] In severely ill patients, levels of and had very low levels of FT3.TSH and free T4 levels were T4 also decrease [55, 57]. A drop in FT4 levels represents a also at the lower range. These mortalities emphasize a continuum of the NTIS spectrum in which as illness severe derangement in HPT axis. These findings were progresses in its severity, levels of FT4 would decrease. consistent with studies which advocate that long term Therefore, low FT4 levels also correlated with bad prognosis. survival is poor in patients with the low T3 syndrome and However, no relationship of NIHSS score with FT4 levels on emphasized that ischemic stroke patients with the low T3 admission can be explained by the fact that with the severity syndrome were at higher risk of poor outcome on follow-up of stroke there occurs decrease in the peripheral conversion that was arranged from 2 to 4 weeks after ischemic CVA and of T4 to T3 as a result of which T3 levels would decrease but 1-year survival was remarkably worse in CVA patients with T4 levels would either normal or increases. Similar findings the low T3 syndrome. The outcomes were supported by were reported by Xu and his colleagues in their study [21] other studies. [17, 19] that T4 levels were found to be increased in patients with We, in the end, conclude that there is alteration in levels of higher scores on NIHS scale and hence those with poorer thyroid hormones in AIS and low T3 levels are predictive of

severity and functional outcome in these patients. However, LIMITATIONS OF THE STUDY the understanding of pituitary-thyroid axis, factors Although the research was prepared carefully, there were controlling action of thyroid hormone at the cellular level some inevitable limitations such as small size of the sample, and explanation of different arrays of thyroid function tests shortfall of long term follow up, selection biases, etc. Due to may be considered cardinal in the management of ischemic time constraint, this study was conducted on a small sample CVA. size, but it is advised to conduct it on large population so that better results and better comprehension of subjects can be CONCLUSIONS made. In acute ischemic stroke, there is the major impact of thyroid dysfunction. In such patients non thyroidal illness (NTIS) is not uncommon and reflect the increase in physical stress The work was carried out in Post Graduate Institute of Medical Education and associated with the insult. The present study showed that Research (PGIMER), Dr. R.M.L. Hospital, New Delhi – 110001. No financial severity of initial (functional) impairment measured by an assistance or funding was taken from any source. NIH Stroke scale on admission, and free T3 & TSH levels had The Authors thank the Director and Dean of Post Graduate Institute of Medical an independent predictive role in determining the functional Education and Research (PGIMER), Dr. R.M.L. Hospital New Delhi for granting permission to conduct the work. outcome.

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Article received on December 19, 2018 and accepted for publishing on June 23, 2019. ORIGINAL ARTICLES

Characteristics and complications of supernumerary permanent teeth in a sample of patients examined in a university pedodontics clinic

Mihaela Tănase1, Aneta Munteanu1, Ioana A. Stanciu1, Elena C. Coculescu2, Carmen Nicolae2, Gabriela D. Bălan3

Abstract: Aim. The assessment of distribution of supernumerary permanent teeth (SPT) and the disturbances caused by them. Material and method. Cross-sectional study upon a sample of 85 children (58 boys) aged between 3 and 18 years (mean age=9.08±3.09 years) with at least one SPT. Were analyzed: patients’ distribution according to gender, age and number of dental units; SPT distribution according to arch and topography and also the disturbances noticed. Results. a) boys:girls ratio=2.14:1; b) the 85 patients had 107 SPT, mean number=1.25; c) 76.47% of patients had one SPT; d) 89.72% of SPT were on maxillary arch, maxilla:mandible ratio = 8.7:1; d) 92.52% of SPT were localized in anterior region, anterior:posterior region ratio = 12.3:1; e) mesiodens –71.96%, supernumerary lateral incisor – 19.62%; f) disturbances caused by SPT: ectopic eruption of permanent teeth – 36.47%; delay or failure of eruption of permanent teeth – 21.17%; crowding – 20%. Conclusions. 1. Supernumerary teeth were most frequent noticed in boys, in maxillary anterior region. 2. Most often they caused eruption disturbances of permanent teeth. Keywords: supernumerary permanent teeth, distribution, complications

INTRODUCTION out on the SPT [4]. These focused more on the distribution of SPT, but less on local disturbances caused by their SPT are defined as any tooth or odontogenic structure that presence. This study is important because it helps to identify is formed from a tooth germ in excess of the usual number early signs of the presence of SPT and thus prevent loss of of teeth for any region of the dental arch [1, 2]. In permanent permanent teeth or further complications. dentition, this dental anomaly is 5 times more frequent than in primary dentition, the prevalence values reported ranging The aim was to analyze the distribution of SPT and to assess from 0.1 to 6% [2, 3]. the disturbances produced by them, in a group of children examined and treated in a University Pedodontics Clinic. In Romania, there are few recent studies have been carried

MATERIAL AND METHODS 1 Pedodontics Department, Faculty of Dental Medicine, University of Medicine and Pharmacy ”Carol Davila”, Bucharest, A retrospective cross-sectional study was conducted on a Romania sample of 85 children (58 boys, 27 girls) aged 3 to 18 years 2 Oral Medicine/Oral Pathology Discipline, Faculty of Dental (mean age = 9.08 ± 3.09 years), with at least one SPT. Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest Children were examined in the Pedodontics Department, 3 Department of Physiology, Faculty of Dental Medicine, “Carol Corresponding author: Aneta Munteanu DDS, PhD Davila” University of Medicine and Pharmacy Bucharest, Romania [email protected]

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Faculty of Dental Medicine, Carol Davila University, permanent tooth in excess – 65 (76.47%), 18 (21.17%) had 2 Bucharest. Criteria for choosing the study sample were: SPT and only 2 patients (2.35%) had 3 SPT. patients aged up to 18 years old, without genetic diseases, chronic diseases or lip-palate clift. Pre-existing conditions, Figure 1. Age distribution of the sample according to the age at such as cardiac pathology, were evaluated [5-8]. which the anomaly was diagnosed

Data on dental status were obtained from dental records of patients examined and treated by a single dentist. The diagnosis of the number anomaly was made on clinical and radiological examinations. For each patient diagnosed with this anomaly were noted: age, gender, permanent teeth erupted, presence, position and shape of the SPT and disturbances produced by them.

The data obtained were collected and processed using SPSS- version 20.0 for Windows (SPSS Inc, Chicago, IL, USA).

The aim of the research was to analyze: the distribution of patients with SPT according to sex, age and number of SPT distribution according to their topography affected teeth; the distribution of SPT according to the affected arch and the topography of the tooth, and the Most of the SPT were in the upper arch, in the front area disturbances produced. (Table 1). Maxilla: mandible ratio was 8.7:1, and the anterior: posterior area ratio was 12.3:1. Note that multiple SPT have RESULTS AND DISCUSSION only met in the upper front area.

Sex distribution of the sample Table 1. Distribution of SPTs according to their topography The percentage of boys diagnosed with this anomaly was Maxilla Mandible Total

68.23% and for girls - 31.77%. The boys:girls ratio was 2.14:1. n % n % n %

It was noted that in 3 male patients it was observed that the Anterior 92 85.98 7 6.54 99 92.52 SPTs were associated with the congenital absence of other Posterior 4 3.74 4 3.74 8 7.48 permanent teeth (Hyper-Hipodontia syndrome). Total 96 89.72 11 10.28 107 100 Age distribution of the sample

Most often, the number anomaly was noticed at the age of SPT distribution according to the type and shape of the 9 years (Figure 1). teeth

Patients’ distribution according to the number of affected The conical-shaped mesiodens was the most common teeth oversized tooth, followed by lateral incisor with a similar shape to the normal-series tooth (Table 2). The 85 children had a total of 107 teeth, with an average number of 1.25 teeth. Most commonly patients had a single

Table 2. Distribution of SPT according to type and morphology

Conical Tuberculate Total Shape Supplementary Odontoma shape shape n %

Mesiodens 9 47 17 4 77 71.96 Lateral incisor 19 1 1 0 21 19.62 Parapremolar 4 0 0 0 4 3.73 Paramolar 1 2 0 0 3 2.80 Distomolar 1 0 0 0 1 0.93 Canine 0 0 1 0 1 0.93 Total 33 50 19 4 107 100

Of the 77 mesiodens, 12 (15.58%) were inverted, being discovered Distribution of the disorders caused by SPT by chance on the X-rays. Most of mesiodens determined the deflection of the permanent tooth eruption from the normal series, while the lateral incisors caused or worsened the crowding (Table 3).

Table 3. Disorders caused by SPT

Lateral Parapremolar Total Disturbances Mesiodens Para-molar Canine incisor distomolar n %

Ectopic eruption 29 1 0 1 0 31 36.47 Failed eruption 16 2 0 0 0 18 21.17 Crowding 2 15 0 0 1 17 20 No consequences 8 2 0 0 0 17 20 It can't be appreciated 2 0 5 2 0 2 2.35 Total 77 21 5 3 1 85 100

DISCUSSIONS the interval for early diagnosis and prevention of malocclusions caused by hyperdontia is around the age of 9 In the literature there are numerous studies of this anomaly [13]. More than ¾ between children had one extra tooth, that analyze their frequency, shape variations and resulting similar to values reported by other researchers [9- disturbances that can be associated with different 14]. population groups. Most studies were conducted on samples of patients from orthodontic clinics. But early Regarding the topography, the SPT were identified in the diagnosis of SPT helps to achieve an appropriate treatment present study of approximately 90% in the upper jaw, a plan and to prevent further complications. percentage similar to that reported by Zhu et al. [19] or Hattab et al. [20]. Instead, Fernández-Montenegro et al. In permanent dentition, the prevalence of the disease varies (2006) [21] found that 74.5% of the SPT were at the maxilla, between 0.1 and 2.1% in Caucasian, between 2.4 to 6% in while Leco-Berrocal et al. (2007) – a percentage of 79.2% [9]. Chinese, Japanese and black Americans [2, 5]. In Romania, the prevalence value was 3.04% [4]. In the present study, 92.52% of the SPT were located in the front area. Other studies report in adult populations a higher Analyzing the distribution by sex, the boys:girls ratio of the frequency of SPT in the posterior jaw area. It is agree that in SPT was 2.14:1. This result is similar to that reported by children the most frequent location is in the front area, and numerous studies conducted in international groups of in adults in the posterior area [9]. children or adults [9-14]. Instead, Bäckman and Wahlin (2001) found a predilection of SPT in females [15], which was In the group studied in the Pedodontics Clinic from highlighted by Mitchell (1989), who found a female:male Bucharest, mesiodens appeared in 71.96% of cases, followed ratio of 2:1 [16]. Unlike them, Dominguez and Mendoza by lateral incisor (19.62%). Also, Fernández-Montenegro et (1995) did not discover any difference between the sexes al. (2006) found that mesiodens was the most commonly [17]. diagnosed, followed by parapremolar and distomolar [21], Salcido-Garcia et al. (2004) found the order: mesiodens, Rani et al. (2017) noted that SPT are more frequently parapremolar and lateral incisor [18] and Bereket et al.: diagnosed in people aged the first 3 decades of life [2]. In a mesiodens, distomolar, parapremolar [10]. On the other study conducted by analyzing 2241 X-rays, Salcido-García et hand, in the study of Khandewal et al. (2018), parapremolars al. (2004) found that from the total number of patients with were most commonly met [14], and at Leco-Berrocal et al. SPT 47.6% were in the third decade of life, followed by those (2007) [9] and Rani et al. (2017) – distomolars [2]. in the first decade of life in a percentage of 28.5% [18]. This study, conducted on a group of children, showed that the Most of the authors found that the conical shape is the most average age to which this anomaly was diagnosed was 9 common, this fact also emerged in the present research [10- years. Also, Alberti et al. (2006) conducted a study on a lot of 12, 14, 21, 22]. In second place, supplementary teeth were patients aged between 6 and 10 years and concluded that observed, as in the study conducted by Fernandez-

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Montenegro et al. (2010) [21], while Shah et al., and (2015) found the lack of any consequences in a large Schmukli et al. (2010) found the tuberculate shape [11, 22]. percentage – 57.9%, respectively 61.64% [10, 21].

As far as the consequences are concerned, in this study in Most of the results from the present study were similar to most cases the deflection of the teeth eruption of the normal the values reported in the literature. Differences may be series was encountered, followed by the failed of permanent caused by racial type and the mean age of the sample. teeth eruption and crowding. Leco-Berrocal et al. reported that the most common complications were mechanical CONCLUSION accidents, namely the deflection of adjacent teeth -39%, 1. Hiperdontia was seen more frequently in boys compared root resorption -12.5% and failed of eruption-4.1%, but also to girls (2.14:1). 19% follicular cysts [9]. Rajab and Hamdan were observed 2. The SPT have been located most frequently in the upper that 51.47% of meziodentes failed the eruption of front area. permanent incisors [12]. 3. The most common type of SPT was mesiodens (71.61%) No consequences were observed in 20% of cases. Instead, followed by the upper lateral incisor (19.62%). Fernández-Montenegro et al. (2006) and Bereket et al. 4. In most cases, the SPT resulted in the deflection of a tooth from the normal series.

References:

1. Garvey JB, Hugh MB. Supernumerary Teeth -An Overview of 738. Classification, Diagnosis and Management. J Can Dent Assoc. 11. Schmuckli R, Lipowsky C, Peltomäki T. Prevalence and 1999;65:612-616. morphology of supernumerary teeth in the population of a Swiss 2. Rani A, Arvind KP, Rakesh KV, Rakesh KD, Jai PG, Rani A. community. Short communication. Schweizer Monatsschrift fur Prevalence of Supernumerary teeth in North Indian Population: A Zahnmedizin. 2010;120(11): 987-990. Radiological Study. Int J Anat Res. 2017; 5(2.2):3861-3875. 12. Rajab LD, Hamdan MAM. Supernumerary teeth: Review of the 3. Mallineni SK, Kumar S. Supernumerary Teeth: Review of the literature and a survey of 152 cases. Int J Paediatr Dent. 2002;12(4): Literature with Recent Updates. Conf Pap Sci. 2014: 1-6. 244-254. 4. Ionescu E. Anomaliile Dentare. Editura Cartea Universitară, 13. Alberti G, Mondani PM, Parodi V. Eruption of supernumerary București 2005: 40-68. permanent teeth in a sample of urban primary school population in 5. Coculescu BI, Dincă GV, Bălăeţ C, Manole G, Bălăeţ M, Stocheci Genoa, Italy. Eur J Paediatr Dent. 2006;7(2): 89-92. CM. Myeloperoxidase, a possible biomarker for the early diagnosis 14. Khandelwal P, Rai AB, Bulgannawar B et al. Prevalence, of cardiac diastolic dysfunction with preserved ejection fraction, J Characteristics, and Morphology of Supernumerary Teeth among Enzyme Inhib Med Chem., 2018, 33(1):1292-1298. Patients Visiting a Dental Institution in Rajastan. Contemp Clin Dent 6. Coculescu BI, Dincă GV, Manole G, Purcărea VL, Stocheci CM. 2018; 9(3): 349-356. Serum concentration of hsCRP - possible marker for therapy 15. Bäckman B, Wahlin YB. Variations in number and morphology evaluation in left ventricular dysfunction with preserved ejection of permanent teeth in 7-year-old Swedish children. Int J Paediatr fraction, Rev. de Chimie (Bucharest), 2018, 69(10): 2885-2890. Dent. 2001; 11(1): 11-17. 7. Bălăeț C, Coculescu BI, Bălăeț M, Manole G, Dincă VG. 16. Mitchell L. Supernumerary teeth. Dent Updat. 1989; 16(2): 65- Haemolytic anaemia and hepatocitolysis associated with 69. hypermagnesaemia by repeated exposures to copper calcium 17. Domínguez A, Mendoza A FH. Estudio retrospectivo de dientes fungicides, J Enzyme Inhib Med Chem., 2018, 33(1): 184-189. supernumerarios en 2045 pacientes. Av Odontoestomatol. 1995; 11: 8. Bălăeț C, Coculescu BI, Manole G, Bălăeț M, Dincă VG, Gamma- 575-582. glutamyltransferase, possible novel biomarker in colon 18. Salcido García JF, Ledesma Montes C, Hernández Flores F, Pérez diverticulosis: a case-control study, J Enzyme Inhib Med Chem., D, Garcés Ortíz M. Frecuencia de dientes supernumerarios en una 2018, 33(1): 428-432. población mexicana. Med oral 2004;9(5): 403-409. 9. Leco Berrocal MI, Martín Morales JF, Martínez González JM. An 19. Zhu J, Marcushamer M, King D HR. Supernumerary and observational study of the frequency of supernumerary teeth in a congenitally absent teeth: A literature review. J Clin Pediatr Dent. population of 2000 patients. Med Oral Patol Oral Cir Bucal. 1996;20: 87-95. 2007;12(2): 96-100. 20. Hattab F, Yassin O RM. Supernumerary teeth: Report of three 10. Bereket C, Çakir-Özkan N, Şener I, Bulut E, Baştan A. Analyses of cases and review of the literature. J Dent Child. 1994;61:382-393. 1100 supernumerary teeth in a nonsyndromic Turkish population: A 21. Fernández Montenegro P, Valmaseda Castellón E, Berini Aytés retrospective multicenter study. Niger J Clin Pract. 2015; 18(6): 731-

L, Gay Escoda C. Retrospective study of 145 supernumerary teeth. 22. Shah A, Gill DS, Tredwin C, Naini FB. Diagnosis and management Med Oral Patol Oral Cir Bucal. 2006;11(4):240-245. of supernumerary teeth. Dent Update. 2008;35(8): 510–512, 514– 516, 519-520.

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Article received on June 15, 2019 and accepted for publishing on October 23, 2019. ORIGINAL ARTICLES

Development of quantitative real-time RT-PCR assay for detection and viral load determination of Crimean-Congo Hemorrhagic Fever (CCHF) virus

Mojtaba Sharti1, Mohammad S. Hashemzadeh1, Ruhollah Dorostkar1

Abstract: Background and Aim: The CCHF (Crimean-Congo hemorrhagic fever) virus causes a severe disease in human with a case fatality rate of up to 50%. Since, there is no specific treatment or approved vaccine against CCHF viral infections, an accurate and early detection as well as a reliable surveillance and quantitative determination of viral load is necessary for patient improvement and case management. In this research, our aim was to develop a probe based one-step real- time reverse-transcription polymerase chain reaction (rRT-PCR) assay for in-house quantitative detection of CCHF virus. Methods: At first, the highly conserved S-fragment sequence of CCHF virus genome was adapted from GenBank and the specific probe and primers targeting this region were designed. Then, viral RNAs were extracted from 37 blood samples of different patients from east of Iran (Zahedan). The specificity and sensitivity of the probe and primers were also evaluated in positive blood samples, confirmed to have CCHF virus. A standard (PTG19-T vector containing S-fragment) for quantization was also constructed and the viral load was determined in some of positive samples. Results: From a total of 37 suspicious blood samples, 15 samples were confirmed to be positive for CCHF virus by this probe based one-step rRT-PCR assay and no false-positive result was detected according to sequencing data. The predicted fragment of 176 bp was also confirmed in all positive samples by gel-based electrophoresis analysis. The assay was linear between 10 to 103 copy numbers per each microliter of extracted plasmid for this technique and the viral load determined in one of patient blood samples was 55,000 viral particles per each milliliter, for example. Bioinformatics and experimental evaluations approved the specificity of this assay. The LOD of the assay was 10 (or fewer) copy numbers of viral genome per each microliter of the extracted genome. Conclusions: This research showed that the developed probe based one-step rRT-PCR assay is a specific, rapid, sensitive and the simple tool for detection and viral load determination of the CCHF virus. Keywords: CCHF Virus, Real-time RT-PCR, Quantitative, viral load, S-region

INTRODUCTION or approved vaccine against CCHF viral infections, an accurate and early detection as well as a reliable surveillance The CCHF (Crimean-Congo hemorrhagic fever) is a and quantitative determination of viral load is necessary for widespread disease caused by Nairovirus (a tick-borne virus) case management, patient improvement, and protection of of the Bunyaviridae family. The CCHF virus causes a severe medical staffs. disease (a zoonotic viral hemorrhagic fever) in human and is endemic in Africa, the Balkans, Asia, and Europe [1-6]. In Several different laboratory assays can be used for CCHFV Iran, this virus has also caused serious challenges in the diagnosis, including enzyme-linked immune sorbent assay Sistan-va-Baluchestan province [7-15]. The fatality rate is (ELISA), serum neutralization, antigen detection, virus about 10-50% [16, 17]. Since, there is no specific treatment

Corresponding author: Ruhollah Dorostkar PhD 1 Applied Virology Research Center, Baqiyatallah University of [email protected] Medical Sciences, Tehran, Iran

isolation by cell culture and reverse-transcriptase Cascade, CO, USA) and GeneRunner (Hastings Software, polymerase chain reaction (RT-PCR) assay [4, 18-21]. CCHFV Hastings, NY, USA). RNA from different clinical samples is a proper diagnosis The probe sequence designed for S-region internal sequence target during the acute phase of the infection or even before was FAM-5'-CAA AAC AGG ATC TAC ATG CAC CCT GCC-3'- the beginning of disease when detection of antibody is BHQ as well as the forward and reverse oligonucleotide untrustable or impossible [22-24]. primers for S-region internal sequence were 5′-TGG GGK Unlike the traditional two-step RT-PCR technique, the one- AAG AAR CTY TAT GAG CT-3′ and 5′-ATG GAC TTG GTR TGY step real-time RT-PCR (rRT-PCR) assay does not need to pre- CCA GAT CC-3′, respectively. K, R and Y are the wobble bases. PCR (post-RT) sample handling, avoiding the PCR product Eventually, the probe and primers were synthesized by the dependent probable contamination transmission and Bioneer Company (Korea). resulting in more sensitive, rapid and also higher efficient One-step real-time RT-PCR assay assays. On the other hand, the probe based rRT-PCR assays have a very high specificity in addition to the mentioned The probe based one-step rRT-PCR assays were developed features. Therefore, it has become an accepted recognition by use of QuantiTect® Probe RT-PCR Kit (Qiagen, Hilden, method for detecting many microbes [25]. However, in this Germany) using both the Applied Biosystem (ABI) 7500 (USA) research we developed a quantitative probe based one-step and Corbet (Rotor-Gene) 6000 (Germany) real-time PCR rRT-PCR assay for detection and viral load determination of instruments. The amplification process was performed in 60 the CCHF virus in suspected blood samples of the Sistan-va- minutes within 20 µl reaction mixtures including 5 µl of the Baluchestan prov¬ince of Iran. The aim of this experimental viral RNA template, 1 µl (10 pmol) of both primers (0.5 µM study was to develop a probe based one-step rRT-PCR assay in the reaction) and 0.5 µl of the probe (0.25 µM in the for in-house quantitative detection of the CCHF virus. reaction). The conditions of real-time cycler were as follows: two single steps of 30 minutes at 50°C (required for reverse METHODS transcriptase activity) and 15 minutes at 95°C (required for HotStarTaq DNA polymerase activation); followed by 50 Collection of sample cycles of 15 seconds at 94°C; and 60 seconds at 60°C To perform this study, we provided 37 blood samples of (collecting the fluorescence data was accomplished at the different CCHF suspicious patients from the Zahedan city in end of each 60°C step). So, the real-time PCR products were the southeast of Iran. A few samples had already been detected trough an increase in the fluorescence intensity confirmed to be positive for CCHF viral RNA existence by rRT- from cycle to cycle. Cycling curves were evaluated with PCR analysis (Pasteur Institute, Tehran, Iran) that were also respect to negative and positive controls (as the quality used as the positive control and they had been stored at - controls of the process) in the real-time analysis. The rRT- 70°C. PCR products were then approved by gel-based electrophoresis. Extraction of viral genome Evaluation of sensitivity and specificity of the assay Viral genomes were extracted from 0.2 ml of blood samples using the High Pure Viral RNA Kit (Roche-Germany) in 30 Analytical sensitivity minutes, according to manufacturer’s instructions. Then, To determine the LoD (limit of detection) of the assay, serial each one of the extracted genomes were dissolved in 50 µl dilutions containing 1, 10, 100 and 1000 copies of the of RNase free water and stored at -70°C until rRT-PCR positive control sample (plasmid) per each microliter of the analysis (of which only 5 µl was required in each assay). It is extracted sample were analyzed. The highest dilution of noteworthy that all of manipulations were performed in a sample at which 100% of rRT-PCR replicates were positive biological hood with class II biosafety. was considered as the LoD of the assay. In this assay, the LOD Designing and synthesizing the specific probe and primers was 10 or fewer copy numbers of viral genome per each microliter of the extracted genome (2500 or fewer copy The nucleotide sequence of S-region of the available CCHFV numbers of viral genome per each milliliter of patient blood strains was adapted from the NCBI database (GenBank) and sample). the highly conserved sequences in this region were determinated and specified from the alignment data analysis Analytical specificity via in silico prediction and rRT-PCR assy obtained from MEGA 7 software. Then the specific probe As previously mentioned, none of the probe and primer and primers targeting this region were designed using the sequences demonstrated genomic cross-reactivity with Oligo software, version 7.0 (Molecular Biology Insights,

70 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine other viruses, human genome and other likely interfering using both the Applied Biosystem (ABI) 7500 (USA) and genomes in a BLAST search analysis and only detected a 176 Corbet (Rotor-Gene) 6000 (Germany) real-time PCR bp fragment of the CCHFV S-region in bioinformatics analysis instruments. (data not shown). On the other hand, in the experimental Quantitative determination of viral load in positive samples rRT-PCR assay, some of the accessible RNA viruses such as norovirus, flu A, HCV, WNV and CCHF (as the positive Construction of a standard for quantization: The fragment of control) were also evaluated in terms of specificity of the 176 bp within the S-region sequence of CCHF virus genome assay. was ampliﬁed by rRT-PCR and the amplicon was cloned into the PTG19-T vector using a T/A cloning Kit (Vivantis, Linearity range and repeatability of the assay Malaysia). The plasmid was then purified using a plasmid Linearity of the assay was evaluated by analysis of serial purification kit (Qiagen, Hilden, Germany). After the dilutions containing 10 to 105 copy numbers of positive performance confirmation of cloning process by PCR, the control sample per each microliter of extracted plasmid and accuracy of this process and the sequence of cloned the assay was linear between 10 to 103 copy numbers per fragment was confirmed by sequencing. Determination of each microliter of extracted plasmid for this technique recombinant plasmid concentration was then performed by (Figure 1 – Ct was plotted against the quantity of plasmid a NanoDrop. DNA (common logarithmic scale). Measurement of the copy For preparation of a standard curve, the plasmid was diluted number of CCHF virus was estimated from Ct by quantitative serially from 103 to 10 copy numbers per microliter as a rRT-PCR< ■ [in Ct = 30.69] represents unknown patient master of standard positive control. As soon as optimizing sample and ■ [in Cts = 35.36, 31.87 and 28.42] represents the the assay conditions, we obtained a standard curve with a standard dilution). These assays were evaluated and linear range across at least three logs of DNA (recombinant repeated by at least three different users and three times by plasmid) concentration, from which the Ct values can be each one of them on different days and the repeatability of referred to the virus copy number (Figure 1). the assays was approved. Also the assays were developed

Figure 1. The standard curve with a linear range across three logs of different concentrations of the recombinant plasmid DNA containing the S-region sequence of CCHFV genome.

Determination of the Ct values and construction of the RESULTS standard curve were performed using the Rotor-Gene 6000 Diagnostic assay of CCHF viral RNA in suspicious blood software. In all real-time PCR assays, the correlation samples coefficient of the standard curve was greater than 0.980. Ct was plotted against the quantity of plasmid DNA (common From a total of 37 suspicious blood samples, 15 cases were logarithmic scale). approved to be positive and 22 were negative for CCHF viral RNA by use of this technique and no false positive was Measurement of the copy number of CCHF virus in unknown detected in the results of sequencing. The predicted length blood samples from different patients was estimated from of 176 bp resulted by rRT-PCR amplification was approved in Ct by quantitative rRT-PCR. Since, the viral genome was all the positive samples by electrophoresis analysis (Figure extracted from 200 µl of a patient blood sample, on the 2A). The amplification curve related to some of the other hand, the extracted genome was dissolved in 50 µl of suspicious blood samples evaluated is shown in Figure 2B. RNase-free water, as well as, 5 µl was only used in each quantitative assay, the final viral load in 1 ml of blood sample Evaluation of analytical sensitivity of the assay would be 50 folds. The serial dilutions prepared containing 1, 10, 100 and 1000

copies of the positive control sample (plasmid) per each LOD, which in this assay was 10 (or fewer) copy numbers of microliter of the extracted genome were analyzed in rRT-PCR viral genome per each microliter of the extracted genome or for the analytical sensitivity evaluation of the assay. As 2500 (or fewer) viral particles per each milliliter of patient shown in table 1, the highest dilution at which 100% of rRT- blood sample. PCR assays were positive (underlined) is considered as the

Figure 2. A) The predicted fragment with the length of 176 bp, resulted by rRT-PCR amplification. Lad is a 50 bp DNA Ladder (SinaClon, Iran). B) The rRT-PCR amplification curve related to some of the suspicious blood samples considered (■ represents NTC [Not template control] or negative control sample, ■ [in Ct = 23.29] represents positive control sample, ■ and ■ represent negative patient sample, ■ [in Ct = 28.12] and ■ [in Ct = 26.85] represent positive patient sample) A B Lad

Table 1. CCHFV rRT-PCR assay limit of detection with positive control sample Evaluation of analytical specificity via in silico prediction and rRT-PCR assay Serial dilution 퐏퐨퐬퐢퐭퐢퐯퐞 퐭퐞퐬퐭퐬 (%) As shown in Figures 3A and 3B, the highly conserved (copies/microliter) 퐬 − 퐫퐞퐠퐢퐨퐧 sequences (yellow) in the S-region were determinated from 1 variable the alignment data analysis obtained from the MEGA 7 software which were quite specific for CCHF virus, as 10 100 previously mentioned and the specific primers targeting this 100 100 region were designed using the mentioned softwares 1,000 100 (bioinformatics data for the probe is not shown).

Figure 3. The alignment data resulted from the MEGA 7 software related to the S-region of CCHFV. A) The Yellow zone is the highly conserved sequence used for designing the specific forward primer. B) The Yellow zone is the highly conserved sequence used for designing the specific reverse primer.

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The results of experimental rRT-PCR assay on the accessible 21.44] represents the positive control [CCHFV] sample, ■ RNA viruses such as Norovirus, Flu A, HCV, WNV and CCHFV represents the negative result from Norovirus sample assay, (as the positive control) showed that this assay was quite ■ represents the negative result from Flu A sample assay, ■ specific for CCHFV and other viruses were not detected by represents the negative result from HCV sample assay and ■ the designed probe and primers (Figure 4 - ■ represents NTC represents the negative result from WNV sample assay). [Not template control] or negative control sample, ■ [in Ct =

Figure 4. The rRT-PCR amplification curve related to some of the accessible RNA virus samples

Figure 5. Representative amplification plot of the developed CCHFV quantitative rRT-PCR assay (quantitative curve) showing three ten- fold dilutions of the standards (Cts = 35.36, 31.87 and 28.42) and one unknown sample (Ct = 30.69).

Quantitative determination of viral load in positive samples each milliliter of patient blood sample.

The copy number of CCHF virus in different patient blood It is notable that viral load decreased slowly in most of cases samples was estimated from Ct by the rRT-PCR assay (Table in subsequent samplings (severe form of disease) and in 2), based on the quantitative curve (Figure 5). For example, others (moderate form of disease) decreased very faster. the viral load determined in patient blood sample-1 was 220 The results of subsequent evaluations is not reported in this copy numbers of viral genome per each microliter of the article. extracted genome or 55,000 (250 folds) viral particles per

Table 2. The calculated viral load of CCHF virus in several patient blood samples based on the quantitative curve and Ct, NTC is "not template control" or "negative control" sample Given Conc. Calc. Conc. Calc. Conc. No. Color Name Type Ct (copy number/µl) (copy number/µl) (viral particle/ml)

1 Sample 1 Unknown 30/69 ------220 55,000

2 Sample 2 Standard 28/42 1,000 994 248,500

3 Sample 3 Standard 31/87 100 101 25,250

4 Sample 4 Standard 35/36 10 10 2,500

5 Sample 5 NTC ------

DISCUSSION Green based rRT-PCR assay that was also developed in our previous study [22]. Therefore, it has become an accepted The CCHF (Crimean-Congo hemorrhagic fever) is an acute diagnosis method for detecting many microbes [25] and also widespread zoonotic illness, caused by Nairovirus (a tick- has greater repeatability when the need for post- borne virus), which is characterized by a severe and often amplification processing (analysis by agarose gel hemorrhagic course of disease, with a fatality rate of about electrophoresis) is eliminated, thus avoiding the transfer 10-50% [16, 17]. contamination, difficulty and time-consuming [26, 27]. The CCHF viral RNA from various clinical samples is a proper The assay was linear between 10 to 103 viral genomes per tracing target during the acute phase of the infection, or each microliter of the extracted genome for this technique even before the beginning of the disease when detection of and the bioinformatics and experimental evaluations antibody is impossible or unreliable [22-24]. As there is no approved high specificity of this assay. On the other hand, specific treatment or approved vaccine against CCHF virus, the LOD of the assay was 10 (or fewer) copy numbers of viral an accurate and early detection as well as a reliable genome per each microliter of the extracted genome. surveillance and quantitative determination of viral load is Measurement of the CCHF virus copy number and necessary for case management, patient improvement, and quantitative determination of viral load in positive samples protection of medical staffs. was estimated from Ct via the rRT-PCR on the basis of the Several different laboratory assays can be used for CCHFV quantitative curve. It is notable that construction of a diagnosis, including enzyme-linked immune sorbent assay standard for quantization of this assay is necessary and very (ELISA), serum neutralization, antigen detection, virus difficult, which was devoted a lot of time in this study. isolation by cell culture and reverse-transcriptase polymerase chain reaction (RT-PCR) assay [4, 18-21]. RT-PCR CONCLUSIONS is the best molecular diagnostic techniques in clinical This research showed that the developed probe based one- laboratories owing to its simplicity, high specificity and step rRT-PCR assay is a specific, reliable, rapid, sensitive, sensitivity [24]. Unlike the traditional two-step RT-PCR repeatable and simple tool for detection as well as technique, the one-step real-time RT-PCR (rRT-PCR) assay quantitative determination of viral load of the CCHF virus in does not need to pre-PCR (post-RT) sample handling, various suspicious patient samples. avoiding the PCR product dependent probable contamination transmission and resulting in more sensitive, Acknowledgements rapid and also higher efficient assays. On the other hand, the We thank the Baqiyatallah Hospital (Tehran, Iran) for its contribution. probe based rRT-PCR assay has a very high specificity in This study was supported by grants from the Baqiyatallah University of addition to the mentioned features, compared with the SYBR Medical Sciences (Tehran, Iran).

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Drosten C, Gottig S, Schilling S, Asper M, Panning M, Schmitz H, PCR assays for detection of 17 species of food- or waterborne et al. Rapid detection and quantification of RNA of Ebola and pathogens in stools. J Clin Microbiol. 2003; 41(11): 5134–5146. Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever 18. Altamura LA, Bertolotti-Ciarlet A, Teigler J, Paragas J, virus, Rift Valley fever virus, dengue virus, and yellow fever virus by Schmaljohn CS, Doms RW. Identification of a novel C-terminal real-time reverse transcription-PCR. J Clin Microbiol. 2002; 40(7): cleavage of Crimean-Congo hemorrhagic fever virus PreGN that 2323–2330. leads to generation of an NSM protein. J Virol. 2007; 81(12): 6632– 5. Estrada-Pena A, Vatansever Z, Gargili A, Buzgan T. An early 6642. warning system for Crimean-Congo haemorrhagic fever seasonality 19. Mehravaran A, Moradi M, Telmadarraiy Z, Mostafavi E, Moradi in Turkey based on remote sensing technology. Geospat Health. AR, Khakifirouz S, et al. Molecular detection of Crimean-Congo 2007; 2(1): 127–135. haemorrhagic fever (CCHF) virus in ticks from southeastern Iran. 6. Vorou RM. Crimean-Congo hemorrhagic fever in southeastern Ticks Tick Borne Dis. 2013; 4(1-2): 35–38. Europe. Int J Infect Dis. 2009; 13(6): 659–662. 20. Chinikar S, Goya MM, Shirzadi MR, Ghiasi SM, Mirahmadi R, 7. Mostafavi E, Chinikar S, Bokaei S, Haghdoost A. Temporal Haeri A, et al. Surveillance and laboratory detection system of modeling of Crimean-Congo hemorrhagic fever in eastern Iran. Int J Crimean-Congo haemorrhagic fever in Iran. Transbound Emerg Dis. Infect Dis. 2013; 17(7): e524–528. 2008; 55(5-6): 200–204. 8. Chinikar S, Mojtaba Ghiasi S, Moradi M, Goya MM, Reza Shirzadi 21. Fakoorziba MR, Golmohammadi P, Moradzadeh R, M, Zeinali M, et al. Phylogenetic analysis in a recent controlled Moemenbellah-Fard MD, Azizi K, Davari B, et al. 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Article received on April 3, 2019 and accepted for publishing on June 23, 2019. ORIGINAL ARTICLES

Prioritisation in delivering health services: A military health system example

Ünal Demirtaș1

Abstract: Objectives: The aim of this study is to use of available resources effectively and efficiently, to determine the situation of recource allocation, priority setting and to offer an insight into future planning in delivery of health services in Turkish Armed Forces (TAF). Study Design: This study was conducted using qualitative focus group method and it is a descriptive and qualitative study. Questions to be asked in the focus group discussions, the location and the time of discussions and the participants was determined by review of the literature. Methods: Focus group interviews lasted between approximately 90-120 minutes, respectively, to each focus group, 9 chief phsycians, 13 doctors /dentists, 6 nurses, 8 health noncommissioned officers, 7 officers, 6 noncommissioned officers and 6 health administrators were attended. Results: The result of focus group discussions; priority settings in military health services must be done and to preventive health care would be prioritised. The military hospitals in Internal Security Operation Area should be strengthened in terms of medical personnel and medical supplies and be full-fledged hospital. There was found that secondary care should be met from civilian hospitals in the other regions. Conclusion: It is evaluated that the study will be contributed to set of priorities in TAF military health services take the investments in the public or private health sectors and operational requirements into consideration, the studies carried out by TAF Medical Command in order to restructure of the military hospitals, to be employed the recources allocated for health services and health personnel graduated from Gulhane Military Medical Academy such as physicians, health noncommissioned officer and nurses in real requirement positions. Keywords: Military hospital, prioritisation, primary health services, focus group, health personnel

INTRODUCTION services which are the secondary purpose of health services; and the third purpose is to give rehabilitation services to Health services have great importance in protecting human individuals who have not fully recovered [2]. health and according to the World Health Organization, they include all services necessary for diagnosis and treatment of Facilitating the access to health services, improving the all diseases, maintenance and improvement of health [1]. scope and quality depend on resources such as money, personnel, material and medicine. Primary purpose of health services is to protect the society from diseases and to ensure all individuals are healthy during During the last 50 years, GPD expenditure on health in OECD their lifetimes within the scope of preventive health services; countries has increased from 3.7% to 9.4%. Growth in health when it is not possible, to provide diagnosis and treatment expenditures has nearly exceeded the economic growth

1 Department of Health and Veterinary Medicine, Gendarmerie Corresponding author: Ünal Demirtaș General Command, Ankara, Turkey [email protected]

76 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine nearly in all OECD countries in the last 15 years. Since current and features into consideration, shedding light on studies resources are insufficient compared to this growth and it is conducted with regards to restructure of military hospitals, difficult to meet all health needs in an optimum level with revealing data to take a good turn in terms of resources current means, it is concluded that health services need to allocated for armed services. Also, it is to contribute to the be limited and prioritization concepts in health services have employment of military health personnel, especially doctors, begun to be studied [3]. medical non-commissioned officers and nurses, in the areas where there is actual need. Prioritisation in health services is a complicated decision process which includes many dynamics and decision-makers MATERIALS AND METHODS such as plans/plannings regarding which services, regions, programs, hospitals, patients or diseases the resources Study of prioritisation in delivering health services is realized allocated to health service should be divided into primarily. through focus group meeting which is a qualitative method. Focus group meetings were realized from September 2014 Prioritisation in health services is realized by receiving to April 2015. The universe of this study consists of sides of opinions of all sides and analysing data regarding service health services which are service providers (doctors, nurses demands. These are findings regarding politicians and health and medical non-commissioned officers), service recipients managers, service providers (doctor and other health (commissioned officers, non-commissioned officers, personnel), service recipients (society) and cost and specialized sergeants/privates and their families) and health efficiency of health needs and proper responses/treatments managers (hospital chief surgeons, health administrators [4]. and headquarters planners). Participants are selected from Methods such as public opinion survey, opinion researches, chief surgeons and hospital administrators who work in public meetings, focus group studies are used to determine different military hospitals; doctors, nurses and medical non- the priorities in health services. commissioned soldiers who have worked in different garrisons and have experience in terms of troop and Prioritisation and resource allocation in health service can be headquarters, willing personnel of commissioned officers used in many criteria such as respect for humans, necessity, and non-commissioned officers who received health service individual responsibility, efficiency, the number of persons from primary care and secondary care health organisations affected as well as fundamental criteria such as lifesaving, – who accept to participate to focus group meeting. cost- effectiveness, cost- benefit and equality [5]. Focus group meetings were realized in four stages in this Determination of priority studies have been realized in many study. In the first stage, the subject of prioritisation in health countries for many years. National Health Accounts (NHA) services is presented through a wide scale literature review. and National Burden of Disease (NBD) studies were realized In the second stage whom to be interviewed was decided. for prioritisation in health services in Turkey [6], but no study Groups who participated to the focus group meeting and regarding the prioritisation in national and international their numbers are presented in Table 1. military health services has been found. After reviewing the literature, the questions to be asked in Military health services include controls for compatibility focus group meetings were prepared with contributions and suitability to the task and periodical or mandatory from the Department of Military Health Services, the controls, health certificate control, prevention and removal Department of Biostatistics and Epidemiology faculty of medical threats, discharge, treatment, health logistics, members who have worked in different positions including blood services and other needs with regards to military administration in different levels of health system. Which health [7]. titles were to be used and which questions to be asked in The aim of military health system is to provide sustainability which order during the meeting were determined for soldiers to be healthy, prevent the weakening of combat beforehand, the questions were prepared by abstaining power due to epidemics, discharging the wounded in time from directing the interviewee. and successfully and saving the limbs and life of the injured. In the third stage, the place and time of the meeting was In short, it is to increase the desire and confidence for determined. Meetings were minimum 60 minutes and warfare of each soldier by facilitating well-coordinated and maximum 120 minutes. 9 chief surgeons, 6 doctors, 6 nurses, not-failing health system, keeping militant power fit [8]. 8 medical non-commissioned officers, 7 commissioned Purpose of this study is to determine priorities regarding officers, 6 non-commissioned officers and 7 doctors/dentists health services by taking military operational requirements participated to the meeting, respectively.

Table 1. Focus groups and number of participants in group consultation. Female Male Total Status N % N % N %

Chief Surgeon 0 0 9 100 9 100

Administrative Chief 2 33.0 6 67.0 8 100 Medical/Dentist Officer 2 15.3 11 84.7 13 100 Nurse 7 100 0 0 7 100 Health Non-commissioned Officer 0 0 8 100 8 100

Healthcare Providers Healthcare Total 11 34 45 100 Officer 0 0 7 100 7 100

Non-commissioned Officer 0 0 6 100 6 100

Takers

Healthcare Healthcare Total 0 0 13 100 13 100

In the final stage, the meeting was summarised, content views are tried to be distinguished by benefitting from the analysis was performed on the acquired data and the perspective of each group meeting. meeting results were presented. Data acquired in this study is reviewed and interpreted by way of content analysis FINDINGS method, researchers compared data acquired from each Distribution of participants who participated to focus group focus group with other focus groups. Opposing and similar meetings by force are presented in Table 2 and Table 3.

Table 2. Distribution of participants who provide health service by force (there is no force separation regarding nurses). Land Forces Naval Forces Air Forces Gendarmerie Total Status N % N % N % N % N %

Chief Surgeon 7 78.0 1 11.0 1 11.0 0 0 9 100 Administrative Chief 5 62.5 1 12.5 1 12.5 1 12.5 8 100 Medical/Dentist Officer 7 53.8 2 15.3 2 15.3 2 15.3 13 100 Health Non-commissioned Officer 3 37.5 1 12.5 3 37.5 1 12.5 8 100 Nurse 7 100

Table 3. Distribution of participants who received health service. Land Forces Naval Forces Air Forces Gendarmerie Total Status N % N % N % N % N %

Officer 5 71.4 1 14.3 1 14.3 0 0 7 100 Non-commissioned Officer 3 50.0 0 0 1 16.6 2 33.4 6 100 Total 8 61.8 1 7.6 2 15.3 2 15.3 13 100

When the answers of participants are reviewed in focus operational requirements, civil health care means, cost- group meetings, most participants stated that there is “a effectiveness status; nurses stated regional needs; medical requirement for prioritization” to be able to present non-commissioned officers stated regional needs, disease available resources and means in an effective and efficient burden, frequent diseases whereas service recipients put way in terms of delivering health service, 1 of the nurses and emphasis on efficiency and cost-effectiveness. 2 participants who receive health service stated that “all When asked who should play a role in prioritization, people services must be provided without any prioritization”. Chief who provide healthcare service stated that especially surgeons stated health care needs of armed forces as doctors should play a role, opinions of those who receive prioritization criteria; administrators and doctors stated service should be obtained, also opinions of patients who

78 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine receive healthcare service and their families as service When asked which areas of expertise should be available in recipients must be received and they also stated that military hospitals, chief surgeons stated that “psychiatrists opinions of health personnel are important. Hospital are essential” due to the increase in the number of administrators stated that the prioritization is needed to be personnel and specialized sergeants/privates who have realized according to operational requirements and opinions psychological problems in the recent period, they also stated of generals who decide the operation are also needed to be that there is a need for branches such as orthopaedics, received. A participant in the service recipient group stated general surgery, anaesthesia for operations; administrators that prioritization should be done according to data in the presented their opinions that there should be evaluations database by establishing a council for health science. specific to the regions, there is a need especially for psychiatry, emergency medicine, surgical branches and When asked if preventive health service, diagnosis- anaesthetists; doctors/dentists and nurses stated that there treatment services or rehabilitation services should be is a need for internal medicine, psychiatry, emergency prioritized in terms of health care delivery of armed forces, medicine, anaesthesia and surgical branches; medical non- health care providers stated their opinions that “preventive commissioned officers stated that there is no need for health service” and “primary health service delivered within gynaecology and obstetrics and paediatrics, instead there unit” should be at the forefront by taking operational should be surgical branches; some service recipients stated requirements into consideration since armed forces that all branches should be available and the others stated personnel mostly consist of able-bodied, young men and that there should be branches that can treat the wounded health service is given to a group healthier compared to the personnel. society. The opinion that duty of armed forces is to deliver primary health service and secondary and tertiary health are RESULTS possible to receive from civil health organisations is stated especially by doctors and medical non-commissioned When studies on this subject realized in countries such as officers. Nurses who mostly work in hospitals and healthcare USA, England, Germany, Holland and Sweden, it is recipients stated that diagnosis-treatment services should determined that a basic benefit package is created to be prioritized. provide a balance between limited health resources and healthcare needs, to prevent parameters such as treatment When the current structure of military hospitals is costs, medicine and surgery expenses within the scope of considered, chief surgeons stated that the most suitable general health insurance from imposing an excessive burden thing is “to reduce the number of military hospitals and turn on national economy. Committees are formed to determine them into a general structure in terms personnel and health services which are to be included in basic benefit medical devices” and to enable the existence of military packages. These committees consist of the representatives hospitals only in the regions where there are operations of healthcare providers (such as doctors, nurses) and against terrorism and “to enable other regions to receive healthcare recipients (society). Even though these secondary health service from civil hospitals”. Hospital committees differ in each country, they prepare guides in administrators stated that the hospitals are currently which priorities in delivering health services are determined inadequate and they are in no state to compete with civil by meeting with people from all levels of society and taking health organisations in terms of service quality and delivered health data of countries into consideration. Whether healthcare. Doctors, nurses and medical non-commissioned disease-treatment (medicine, surgery etc.) expenses are to officers stated that actions should be in accordance with be covered by health insurances is decided according to operational requirement, thus military hospitals outside of these guides. Within this framework, studies such as the region where there are operations against terrorism International Health Accounts Household Health Expenses should be closed since secondary health service can be and Disease Burden Projects are realized in our country in received from civil hospitals and military hospitals in the the recent time. regions where operations against terrorism are realized should be fully-organised and in a level enough to compete Military health services are primary health services provided with civil hospitals. Service recipients pointed out that especially for operation. It is determined by all participants military hospitals are not currently cost efficient and there is in the focus group meetings that primary health service no high-quality health service in these hospitals, they also should be prioritized. It is pointed out in the focus group stated that there are only need to be military hospitals in the meetings that there must be doctors and assistant health regions where operations against terrorism are realised so personnel in primary health organisations; it is not enough as to provide high-quality health service. to have only health personnel and laboratory facilities and

medical devices and materials should be re-enforced. emphasised that military hospitals in operation areas where Hospital administrators and doctor’s point to the increase in fights against terrorism happen should be fully-organised the number of personnel and specialized sergeants/privates with sufficient personnel, medical device and equipment who have psychological problems in the recent years in and can provide service for 24 hours in a day with ambulance troops, psychological counselling and guidance services helicopters and health teams. The fact that many military which are mostly given by reserve officers could be more hospitals are currently in an idle state and they can only efficient if they are provided by regular personnel or civil provide service as polyclinics, the number of in-patients and psychologists. surgeries are very low is exemplified. There are similar problems in the United States of America. Existence of In the studies realized by Wiseman and Rosén, transparency, military hospitals and how efficient and effective they are in equality, justice, efficiency, responsiveness of health system their current state have become highly questionable. and health personnel’s approach towards patients are stated Pentagon military health administrations state that primary as priority determination criteria in health services. In the mission of hospitals is military needs, doctors and nurses study realized by Mamas, priority determination criteria in must develop their abilities for combat requirements. Two health services is stated as maintaining trust and thirds of USA Military Hospitals treated 30 or less patients accountability between patients and doctors and daily in 2013 and less than one-third of them treated as establishing patient-centered approach [9, 10, 11]. In some many patients as civil hospitals treated. Nine hospitals studies, criteria are determined as benefit of patient, cost treated 10 or less patients [14]. The military worked in more effectiveness and severity of disease [12, 13]. In the PHD than half of domestic hospital network until today. Pentagon thesis by M. Top, the principles of equality, health need, analysts suggested that 15 of 25 hospitals are to be reduced, cost- effectiveness are placed near the top [5]. In this study, but the list has been reduced to eight due to complaints by prioritization criteria are listed as operational requirements Land, Naval and Air Forces [15]. of the region, status of civil health facilities in the garrison, benefit of patient and cost-effectiveness. When criteria such as operational requirements of military hospitals, status of civil health organisations in the region are Parties who are required to take part in delivering health considered, it is stated that areas of expertise that are service are stated as doctors, patients and their families, required to have in military hospitals first of all must be to respectively, by the doctors and as patients and their meet the operational needs; afterwards, psychiatry, internal families, doctors and health authorities, respectively, by the medicine and anaesthesia, surgical branches such as patients who receive service in a study realized in Greece. orthopaedics, general surgery are listed by the participants. Both groups stated that politicians should play no part [11]. Common opinion of all groups who participated to the focus In the study realized by Wiseman and others, it is stated that group meeting is that there is no need for branches such as doctors, health service administrators and patients and their paediatrics, gynaecology and obstetrics in military hospitals families should especially take part in determination of in the regions where there are operations against terrorism priorities [9]. In the studies realized by Bowling and and these services can be delivered by civil health Kneeshaw, the result was that doctors, local health organisations. When the literature is examined, it is seen authorities and public should be a great part of the that 66% of injuries happening in theater of operations are prioritization studies [11]. In harmony with the literature, orthopaedics, 21% is general surgery, 6% are of head and the opinion regarding health personnel and especially neck injuries, 5% are of burn injuries [16]. In another study doctors is stated by service providers whereas service based on the experiences of English military health recipients had the opinion with regards to patients and their personnel got in Afghanistan, it is determined that 93 of 124 families and doctors in this study. people who had been wounded in battle lost their lives When military hospitals are evaluated according to their because of injuries in upper thigh, inguen and pelvic. Most current state, it is seen that there are no specialists in many of the casualties are because of pelvic injuries (17). Almost branches of many hospitals and there is only one doctor 11% of 7,856 trauma patients who applied to English Role-3 when there is a specialist in a branch. All participants who Sahara Hospital in Afghanistan from 2003 to 2011 applied participated to the focus group meeting stated that because of chest trauma and almost 14% of them lost their secondary health service delivered in many military hospitals lives. Most of the wounded people whose systolic blood fall behind of civil health services and it would be suitable if pressures were detected very low had head and abdominal military hospitals outside of the region where there are injuries and cardiac arrest and they caused mortality [18]. In operations against terrorism should be closed and secondary another study, 83% of 271 people who lost their lives health service is received from civil hospitals. It is because of war injuries died because of brain injuries. 80%

80 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine of 287 people who were potentially survivable, It is emphasised that the priority of armed forces should be haemorrhage caused by major trauma caused mortality. primary health services and preventive health services and When bleeding site was examined in these people, it is primary health service is a must for armed forces. determined that 48% of bleeding happened in torso, 31% Considering regional features and operational requirements, extremity and 21% neck, axilla and inguen [19]. In a study it is common opinion of all parties that secondary health realized by Owens and others, extremity injuries in 1281 service can be received from civil health organisations. soldiers are examined and it is seen that 53% of them have Health boards in military hospitals stated that specific health penetrating soft tissue injury, 26% have fractures and these services such as health service for operational requirement fractures are localized similarly in upper and lower must be focused on and it is more appropriate for extremities [20]. Of injuries except thoracic injuries of 258 rehabilitation services to continue as they are currently. patients treated for thoracic injuries happened with It is determined that military hospitals cannot be utilized in weapons with high kinetic energy between 1996 and 2005, an efficient and effective way due to the fact that personnel 13% are intra-abdominal injuries, 5% are fractures, and 3.5% and their families prefer civil health organisations, there is are major vascular injuries [21]. The most frequent injuries not sufficient specialists, medical devices and materials are of patients who are brought to an emergency ward in a lacking, physical conditions of hospitals are worse compared hospital in Turkey after getting injured in the civil war in Syria to civil hospitals, there are problems in logistics and the are extremity injuries, internal diseases; chest, abdominal system responds to the needs very slowly. and head injuries with similar rates and eye injuries, respectively [22]. The importance of providing efficient, correct and quick primary health service is emphasised. Within this scope, it is In the light of this information, as stated by the participants, pointed out that there must be a doctor regarding primary it is important to have specialists on surgical branches such service, but it is not enough only to have doctors, there as orthopaedics, general surgery, brain surgery, cardio- should also be laboratory facilities which consist of complete vascular surgery, thoracic surgery and branches such as blood, urine, roentgen devices and primary health services anaesthesia, internal medicine, radiology to be able to treat must be supported. the wounded people effectively and increase the motivation of the personnel in the military hospitals in the regions Recently, there is an increase in the number of personnel where there are operations against terrorism. and specialized sergeants/privates who apply to Psychological Counselling and Guidance Centers and it is DISCUSSION determined that to deliver more productive service to these patients, regular personnel or civil officers should work The following results are obtained through this study instead of reserve officers so that the service becomes more realized with focus group meetings to determine priorities in effective and efficient. delivering health service to armed forces. When military hospitals are evaluated according to their Almost all parties and participants who participated to the current state, it is seen that there are no specialists in many study pointed out the necessity for determining priority in branches of many hospitals and there is only one doctor military health system to use resources allocated to armed when there is a specialist in a branch, especially in the forces effectively and efficiently since resources are limit, surrounding hospitals. Participants are in agreement requirements are many. While making prioritization, it is regarding the fact that the military hospitals in the regions stated that criteria such as operational requirements, where there are operations against terrorism must be restructure of civil health organisations in the region and enforced and turned into hospitals which have a fully- military health organisations being utilized by personnel and organised structured and several doctors in each branch to their families is needed to be considered because of unique continue delivering service, hospitals except this must be structure of armed forces. closed or turned into health centers suitable to the unit As a result of focus group meetings, it is determined that the needs. opinions of doctors especially are needed to be at the Parties that participated to the group meetings emphasised forefront during prioritisation as well as opinions of health the importance of quick and efficient patient/wounded personnel, hospital managers such as chief surgeon, discharge regarding operations is emphasised. Afterwards, it assistant chief surgeon, hospital administrator, privates who is concluded that having an ambulance helicopter which receive service and civil patients. have regular health personnel who provide service in accordance with 24-hour principle within military hospitals

and having first aid and emergency medical technician provided especially for trainings units to deliver more working in units which take part in operations improve the efficient and effective primary health services and to make efficiency of health services. these services more suitable to unit needs.

The following suggestions are developed in the light of these For health board needs, regional hospitals, military hospitals results: outside of the region where there are operations against terrorism should be transformed into proper health centers, Health system of armed forces is required to be restructured other military hospitals should be structured in accordance in a way that efficiency and economy criteria are taken into with the criteria of the Ministry of Health through personnel consideration, medical board processes specific to armed and medical device savings with this transformation. forces can be realized, diagnosis-treatment abilities and capacities focused on unit and operation needs, in As specialist quota is determined, planning should be made accordance with national health legislation are increased by considering operational requirements of armed forces, without losing specific features and by re-evaluating vision, current status of staff in health board branches, especially mission, current situation and the point the national health branches such as general surgery, orthopaedics, system has reached. anaesthesia, internal medicine, psychiatry, and radiology should be focused on. Health Services Policy Determination and Assessment Board should be organised to give scientific support to When the fact that there is an increase in the number of restructuring studies of Health System, one member from personnel and specialized sergeants/privates who have the Departments of Public Health, Military Health Services, psychological problems in the recent periods, regulations General Surgery, Internal Medicine, Psychiatry, Infection regarding regular personnel recruitment should be made to Diseases and Family Medicine and Health Services solve problems arising from conducting psychological Management and the Department of Epidemiology, 5 Chief counselling and guidance services in the troops especially Surgeons from Military Hospital and 3 general practitioners with reserve officers. should be assigned.

Inpatient treatment facility and laboratory facilities which Author Statements: Ethical Approval was given by Ethics Commitee consist of complete blood, urine, roentgen devices should be in Gulhane Military Medical Academy. There is not any sources of funding and competing interests.

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Article received on May 2, 2019 and accepted for publishing on July 28, 2019. ORIGINAL ARTICLES

Why cancer/terminal ill diagnosis unsuccessful in India: a qualitative analysis

Suantak D. Vaiphei1, Devendra S. Singh1

Abstract: The increasing of the modern technologies requires the setting up of multiple factories and industries that produce smokes and chemicals, which polluted the air, water, and the environment resulting in putting lives in dreaded conditions. In fact, the coming of the modern era and the modern lifestyle gives birth to numerous deadly diseases like cancer and its related diseases, which increasingly takes the lives of many in the country. The cold-blooded killer ‘cancer’ becomes the major public health concern in India as a whole. Among all the problems existed across the world, the problems or the issues associated with cancer or terminal illness becomes the worldwide phenomenon that produces several unwanted worst experiences. Human polluting the water, soil, and the air continued, and the global burden of cancer affected populations rapidly increasing year after year. Unfortunately, India turns out to be the world most densely cancer populated regions and a cancer hub. The reason behind the rapidly increasing of cancer illness and its mortality rates in India is mainly due to people lack awareness on cancerous factors, no proper preventive measures, and the late detection of the symptoms at the advanced stages when cure is not possible.Thus, concerning the helplessness conditions of the cancer effected people of in general, the current analytical study has been forms with an aim to spread awareness on the cold-blooded killer cancer across the country. Keywords: cancer, terminal illness, awareness, mortality rates, diagnosis

Objectives: The underlying aim of the study is to investigate Cancer Centers, the underlying reasons behinds the rapid growths of cancer • National Family Health Survey of India (NFHS-3), NICPR- populations and the cancer mortality rates in the present National Institute of Cancer Prevention and Research, ICMR- India. Indian Council of Medical Research, Indo-Asia News Service The study emphasis mainly on the cancerous factors, the (IANS), and Zee Media Bureau. The study also utilized the underlying barriers behind the unfruitfulness of the terminal available journals database, along with WHO database. The diagnosis, and the propose solutions or preventive measures researcher also accesses to the government’s data and in traditional ways. hospitals documents on cancer statistics and their reports.

Methodology: The current study is an analytical study on the Results: The modern turns out to be a cancer hub and the collected data and reports of the following reliable sources: world largest contributor to cancer mortality rates. The numbers of cancer effected people increases every year, • The Population-Based Cancer Registries Data’ of the while the government had minimal inputs towards the Central and State Governments. preventive measures against cancer/terminal illness. • The Data from National Cancer Registry and Regional

Corresponding author: Suantak Demkhosei Vaiphei 1 Bhupal Nobles’ University, Udaipur, India [email protected]

84 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine

INTRODUCTION The cervical and stomach cancer symptoms populations in the Indian state of Mizoram alone is equal with the total Looking at the current cancer statistics, it is visible that numbers of cancer affected populations of Japan and is the Indian as a whole fought against the cold-blooded killer highest cancer populated region in the world.[3] ‘terminal illness’ in the most unsuccessful way. The failures of the health care systems of the country in its terminal The Zee Media Bureau Report, among the 8.8 million global diagnosis resulted in making the larger groups of the cancer deaths, in which India is consider as the world largest patients facing the worst ill experiences one had ever gone contributor to cancer mortality rates.[4] In another finding, through in life. India is the world largest contributor to around 505,428 to 500,000 cancer patients in India died per cancer mortality with around 500,000 deaths per year, year. The main reason for the high cancer mortality rates in mainly due to people unawareness of the cancer and its India is mainly due to people unawareness of the treatment symptoms diagnosis policy. However, no proper preventives procedures of cancer and its symptoms. This being the measures or actions had not been taken up by the reason the cancer affected people consulting their government, though the country is densely populated with Oncologists mostly when they are in the terminal stages, terminal illness. Moreover, 80% of the cancer populations in which is in the fourth stage’ of cancer symptoms that is India lives in rural undeveloped areas with low economic impossible to cure.[5] Thus, around 80% of the deaths from status, who were not able to afford for their cancer diagnosis cancer’s cannot be prevents in India today. and on the other hand, higher diagnosis fees in India. Thus, However, consulting their clinicians in the second stages of undergoes painful terminal ill experiences and live a life of cancer diagnosis patients could possibly have at least 60%, suffering hell without accesses to any hospitals or clinical for cure, while the interventions of cancer diagnosis in its diagnosis. The Indian Council of Medical Research, New third stages the cure possible rates reduces to 30%, but with Delhi, predicted that the numbers of terminal deaths in the the possibility of stage four of cancer within a short time. country would increases significantly in the coming years. Stating by 2020 around 1,700,000 Indian would be However, there are no chances for survivor when it comes diagnosing this cool-blooded killer and its related deadly to the stage four of cancer that gives a maximum life spends disease. Moreover, the percentages of cancer patients of 5-7 years. Basing on the latest findings of NICPR report, would likely to be increases by 20% in India, if the Breast Cancer and Cervix Uteri is the two most common government did not take up any immediate quality Cancer killers among Indian women, while Lip/Oral Cavity preventive measures against this cool-blooded killer. and Lung Cancer becomes the two most common killers among Indian men, killing around 253,521 Indians every INDIA IN THE BATTLE OF TERMINAL ILL ILLNESS: THE year.[6] The last 20 years were the periods where cancer PRESENT SCENARIO’S mortality rates has seen rapidly increasing in the country, while the numbers of cancer incidence has been decreasing In the definitions of World Health Organization (WHO), in many western countries. Out of all the cancer mortalities, cancer or terminal illness is the uncontrolled growth of the cervical cancer remains the leading cause of deaths deadly cells with unstoppable spreads, which destroys every among Indian men and women follow by Breast cancer portion of human body organ one after another. Once the mortality.[7] The following tableshows the five most deadly cells increase and affected the patient’s start losing common deadly cancer symptoms, its causes, and the the functioning of his/her body sites that usually leads to a numbers of terminal deaths in a year in India as a whole paralyses condition. These deadly cancer cells expanded (Table 1). through invading the surrounding tissues and spreads to other parts of the body by metastasis leading to 8.2 million Looking at the current cancer statistics, it is visible that India global deaths a year at present. In which India becomes the as a whole is fighting with the cold-blooded killer “Terminal world top contributor to cancer mortality rates with around Illness” in the most unsuccessful way. India at present is in 556,400 terminal deaths per year.[1] The modern India turn critical conditions with rapid increasing rates of one-lakh out to be a cancer hub with 2.5 million cancer-affected cancer populations per year and minimal cancer care center. people, which would be expected to increase by 50% in There are around only 300 regional cancer care centers, 2020, if no immediate action plan had been done by the which is not enough even to treat the one thirds of the governmental and non-governmental agencies. At present a cancer populations in the country. Moreover, India today premature death through non-communicable diseases is the has only 1000 oncologists, which is in the ratio of 1:2000 leading causes of deaths in India like cardiovascular (one oncologist per two thousand cancer patients). This ailments, chronic respiratory problems, and diabetes. [2] constituted the underlying reason why modern India turned

into a cancer hub. The fighting with cancer will continues as The following statistic is formed to explain in detail about the the numbers of affected people raises up every year.[8, 9] current cancer status in India (Table 2).

Table 1: The common types of cancers and the numbers of death in a yearly basis. [4-6] Numbers No. Cancer symptoms Main causes of death per year

1 Gallbladder cancer 230,000 Unhealthy lifestyle, genetic, poor prevention 2 Breast cancer 5,522,000 Unhealthy lifestyle, genetic, alcohol 3 Head & neck cancer 575,000 Alcohol, tobacco, cigarettes 4 Throat cancer 115,200 Poor nutrition, smoking, alcohol, genetic syndromes 5 Lung cancer 22,900 Smoking, tobacco 6 Oral cancer 245,800 Smoking, tobacco, alcohol, hpv, sunlight, weak immune system 7 Cervical cancer 67,477 HPV, birth control pills, smoking, unhealthy sex

Table 2: Current cancer statistic in India. Adapted from NICPR statistical report on August 23, 2018. No. of Cancer Yearly Cancer No. of Deaths Common Age’s of Women Men Percentage Affected Registration In a Year Death 39-60 Yrs 2.5 Million 7 Lakh 556,400 395,400 195,300 200,100 71%

In India, at present, one woman dies of Cervical Cancer every the common types of cancer in the country. The uses of 8 minutes and for every newly diagnosis with breast cancer tobacco cause 100% poor oral health with 90% of mouth one out of two women dies in India. Moreover, around 2,500 cancer, 80% of lung cancer, 50% of all human cancers, 70% died per day due to the cancerous factors like tobacco and of lung diseases, and 60% of heart attacks.[11] The above bitternut. While smoking, which is the most common mention cancerous factors were the most common practices practices in India cause 1 in 5 death amongst men and 1 in in the Indian Sub-Continent at present, leading to the rapid 20 death amongst women that constituted around 930,000 growth of cancer populations in the country. deaths in 2010. In which the most productive age period Another reason for India being the top cancer populated in could be highlights in the ages between 30-60 years, which the country is mainly due to people unawareness of need a special consideration and these ages grouped need cancerous components, its preventive measures, and the to be targeted the most.[6] treatment policy. Due to the lack of awareness, majority of Why cancer/terminal diagnosis unsuccessful in India the cancer effected patients detected their cancer today? symptoms only when the physical pain becomes unbearable, which is in the terminal stages (third or fourth stages) that is The main cause of cancer is through the internal factors impossible to cure. Late detection of cancer symptoms and (inherited mutations, hormones, and immunes conditions) late cancer diagnosis becomes the underlying reasons for and external/environment factors (tobacco, insufficient diet, the 80% of the cancer population failing to cure in India unhealthy food, organism, and chemical with radiations). today. However, early detections of the symptoms in its first Among all these components for the causes of cancer and its stage there is 80% chances to cure. On the other hand, deadly symptoms, there is a closed link between unhealthy undergoing cancer diagnosis in the second stages could food and insufficient diets with cancer disease as observed possibly have 60%, chances for cure, and in the third stage by many experts.[2] Unhealthy lifestyles with alcohol and the cure possibility rates reduce to 30%, but with the smoking are the second most common cancerous factors possibility of stage four of cancer within a short period of leading to chronic disease, cardiovascular, lung, kidney, time. However, in the fourth stage, which is the terminal throat, esophagus, and breast cancer. The third common stage there is no chances to cure, rather to live with the factor for the causes of cancer is visible in excessive painful symptoms for maximum life spends of 5-7 years.[9] consumptions of red meats and salted fish leading to heart and breast cancer.[10] At present as per the National The general causes of stomach and cervical cancer Tobacco Control Programmed, the tobacco related cancer symptoms is due to the excessive drinking of alcohol, illness like Heart Attack, Lung Diseases, and Stroke etc. were cigarette smoking, and chewing bitternuts. While, lung and

86 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine oral cancer that hugely affect the Indian men and women nasopharyngeal cancer is mainly causing by excessive eating populations, is mainly due to the excessive uses of tobacco, of meat, fish, salted fish, uses of firewood in the house and smoking, hookah, and the consumption of local made other environmental related factors like the eating of alcohol. Another prevailing cancer symptom in India today is bitternuts (Kuwa or Komkuwa) with or without tobacco the nasopharyngeal cancer, which is a malignant cells products. Mostly the women populations were mainly disease form in the tissues of the nasopharynx. The affected by nasopharyngeal cancer comparing to men. nasopharyngeal cancer (NPC) is the rarest type of cancer Moreover, within 15-20 years the nasopharyngeal affected around the world; accept in the South East Asia, North Africa, women populations will be increasing, if the uses of tobacco, and Arctic. The NPC is claims to be a Chinese origin, which is bitternuts, and the uses of firewood in cooking are not being largely affecting the North East Indian states of Nagaland, under-controlled.[3, 10] The table below will clearly show Manipur, Mizoram, and to some extend to Meghalaya. The the various factors that are responsible for the causes of state of Nagaland has the highest age adjusted with cancer as a whole (Table 3). 19.5/100,000, followed by the state of Manipur. The

Table 3: The cancerous factors in India as a whole. Liquids Particles Cultivations Environment Components Components

polycyclic aromatic metals or diesel exhaust toxin from smoking & hydrocarbures pahs coated with metals particles fungi hookahs’ vinyl chloride and excessive unhealthy drinking chewing of junk benzedrine meats water bitternuts food

Apart from food habits, the population explosion, rapid would be increases by five-fold in India. The rapid increasing industrialization, and genetics, which include mutations, rates of cancer in the country are mainly due to lifestyle risk hormonal and lack of immunity are also responsible for the factors like uses of tobacco, alcohol, low fiber in diet, rapid growth of terminal illness in India as a whole. If proper increasing body weight, minimal physical activities, and the awareness had not given mainly to the rural undeveloped reproductive risk factors regarding age at first pregnancy, areas of the country, there is a possibility of increasing and higher numbers of children breastfeeding’s. Out of many cancer populations to 19% in the next five years. In which cancer mortalities, the two leading deaths are from cervical women have more chances of affected with cancers than cancer (HPV), hepatitis C (liver), and gastric H. pylori men in India, as per the findings of many. Moreover, the (stomach).[15] mortality rate of cancers in India is visible higher among the illiterate people groupin the rural areas of the country than THE PROPOSE SOLUTIONS AND PREVENTIVE MEASURES the educated, and the maximum deaths in India are mainly Terminal death is the top causes of Death in India, in the year due to poor prevention strategies and no proper diagnosis 2000 India was in seventh position among the world cancer as a whole [13] populated country. However, due to the rapid increasing of According to the latest NICRP reports, 122,844 women are cancer populations from 2006-2018, India successfully stood diagnoses with cervical cancer every year out of which as the highest contributor to world terminal fertility rates, in 67,477 women died from cervical cancer per year. In a which the numbers of effected populations is currently population of 432.2 million women in India at present, those visible increasing with 100,000 per year. In recent research women who are at the age of 15 and above and or between finding, only 5-10% the cancer is from genetics, in which the 15- 40 years are at risk of developing cervical cancer.[14] The other 90-95% of the cancer and its related deadly diseases health scientists on the other hand, were not able to identify are from hormonal and environment factors.[16] Which the processes of how the risk factors like genetic, hormonal, means, the 90% of the cancer and its related diseases can be and environment factors works together to cause normal prevented effectively through proper medical interventions cells to become cancerous tumor for the cancer symptoms. in its early stages and hygienic lifestyle. Moreover, another [10] Thus, cancer becomes the leading causes of deaths in 10% genetic cancerous symptoms can also be prevented India, with 2.5 million cancer populations, with 1 million through early detection and immediate diagnosis. In short, cancer patients added every single year. It could be cancer of any types is curable if detected in its early stages predicting that the numbers of cancer patients by 2025 and through proper diagnosis. The followings are the

qualitative preventive measures in order to control the rapid fruits are less in calories, sodium, and fats. Fruits are the growths of cancer in the country: [2, 10, 16] sources of essential nutrients like potassium, dietary fibers, vitamin C, and folate (folic acid), which prevents deficiency, 1. Regular medical checked-up, though being in a healthy birth defect, and helps a person to growth with healthy condition, negligees of regular medical checked-up is visible blood pressure. as the underlying reason for the rapid growths of cancer in the country for the past ten years. 13. In order to have fair skins some avoid exposing to sunlight, wearing protective clothing during 10 am-4 pm, in 2. Educating people with proper awareness strategies, offer which the excessive uses of sunscreen cream need to be effective public health concerned in the schools and avoid. Avoiding of exposing to sun or UV resulted in many organizing a social meeting for spreading cancer awareness. women diagnoses with skin cancer. On the other hand, Especially in the rural areas of the country. excessive exposing to sunlight or UV ray is also dangerous, 3. Early detection of the symptom and early medical especially for those having genetic cancerous symptoms. intervention, because when it comes to third and fourth 14. Immediate medical intervention on virus and bacterial stages there is no possibility for cure. infections, otherwise the bacteria diseases virus cells usually 4. Public awareness on active physical activities, exercise at hide inside the cells and turning out to be a terminal virus least for 30 minutes per day, the healthier a person is, the Moreover, the rapid increasing of industrializations and cells in the body can effectively fight against the invader urbanizations are visible as the two factors leading to new virus and bacteria. Regular exercise prevents oneself from lifestyle of many Indians, which resulting in increasing the colon and breast cancer too. cancer affected populations in the country. Concerning the 5. Minimizing the uses of alcohol products, smoking, current polluted environment, the burden of cancer tobacco, and chewing of bitternut products, which will incidences will gradually keep on increasing, as majorities of minimize the risk of having lung, kidney, throat, esophagus, the Indians were not aware of cancerous preventive and breast cancer. It is better to develop a moderate way of measures. The only ways to prevent the Indian men and consuming with 2-3 glass of alcohol products per day, which women from the rapid growth of cancer and its deadly will keep oneself away from heart attacked. symptoms is to detect the symptoms at the early stages with immediate medical interventions. It is also important to 6. Immunization against hepatitis B virus to the infants of prevent the water and the environment from polluted by one to sixth months old without failed. Neglecting medical industries and chemicals. However, it can only be possible treatment in hepatitis B & C can causes chronic illness and only if the men and women in the rural areas were being liver cancer. educated on cancer awareness like preventive measures, 7. Developing healthy and safe sexual practices to avoid and overall treatment policies, which needs multiple efforts cancer genesis, unhealthy sexual practices can give birth to from the government agencies and other non-governmental cancerous cells. agencies. Through such awareness programs, men and women should realize the risk factor and to identify the 8. Avoiding obesities, as being grossly fat or overweight have symptom through screening by physical examination or by negative effects on health and the makes cells less effective. self-cancerous symptom examination, in which if the rick 9. Developing healthy diets, a healthy diet has scientifically substances where found than certain carcinogenic proven with numerous health benefits and reducing the risk substances need to be reduces or eliminated. Moreover, from chronic diseases. there are over 85,000 synthetic chemicals that were easily 10. Reducing occupational and environmental exposures, as available in the market of the country today like cosmetic excessive exposures to chemical and its related heavy metals items to flame-retardants, plasticizers in water bottles to produces ill health. It also effected for the future offspring pesticides in fruits and vegetables’ etc.[13, 16] In the findings and produces toxicity. of the researcher, 80% of the cancer patients in the country were associated with environment factors like exposes to 11. Avoiding excessive consumption of red meats, salt and contaminants, unhealthy lifestyle, food, and exposing to long preserved food. High consumptions of red meat, salt ionizing radiations. In the urban areas, the cleaning of and preserved food leads to diabetes, breast cancer, and contaminated drainages without any proper preventive obesity. measures, polluting the river to the maximum with many 12. Developing the habits of eating fruits regularly, most forms of chemical and eating those fishes from the river produces several cancerous symptoms. The using the

88 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine polluted water from the factories or industries for impossibility rates as the cancer metastasis accelerates in agricultural farming also produces several cancerous higher speeds from one body part to another. However, if components leading in rapid increasing of cancer the stage three patient being diagnosis by special medical populations through consuming theses agricultural team in a well to do clinical setting, it has around 60% products. Thus, maintaining healthy environment, healthy possibility chances to cure. But does not guarantee a lifestyle, healthy food, proper diets, daily exercise, staying complete free from cancerous cells, as there is a possibility away from tobacco, and smoking, with decreasing alcohol of being in terminal stage after three to five months. The consumptions can decreases the rates of cancer-affected reason being the cancer cells have the possibility of hiding populations. Moreover, in the rural villages the practice of under another cells that is hard to detect, resulting in leading using of those water which were uses in deeping hot metal one’s life a disability-adjusted life years (DALY). or iron in the blacksmithfor washing hand and leg need to be Nevertheless, the detection of the cancer symptoms in the avoided, as it contains the component that is cancerous. fourth stage have no possible way to cure by any means, which is term as the ‘terminal stage’ that is 100% impossible CANCER TREATMENT POLICY AND TERMINAL DIAGNOSIS to cure in any clinical practices. Thus, resulting in leaving the patient with disability-adjusted life for five to seven years The WHO (2017) stated that, the cancer patients need until the inevitable death strikes him/her. immediate access to the modern equipments like: 3D Conformal Radiation Therapy, Intensely Modulated and These 5-7 years period of terminal experience is the most Therapy (IMRT), Image Guided Radiation (IGRT), VMAT and crucial moment for every terminal patients, which is also Rapid Arc-Volumetric Modulated Arc Therapy (VMAT), Low considers as the worst moment in a terminal experience with Dose Rate Brachyntherapy (LDR), High Dose Rate heartful of emotional sufferings and mindful of mental Brachyntherapy (HRD), Deep Inspiration Breath Hold (using disharmony that needs special considerations to the most. the goggles or snorkel technique), and Stereotactic Radiation The acknowledgment of the psycho-emotional symptoms Therapy.[17] However, it is sad to say that, due to the alongside the treatments of physical pain is very essential in unavailabilities of the above mention modern cancer any terminal diagnosis in the clinical practices. However, at treatment equipments, cancer patients in India could not present India turns out to be the worst place to die or a place undergo such treatments. Failing the interventions of not to die. The reasons being the undergoing cancer/ modern equipments in terminal diagnosis, resulted in terminal diagnosis core emphasis is on the physical pain uneffective diagnosis in many cases. The quality treatment treatment alone, leaving the psycho-emotional pain and plan and policy are the core components for successful suffering untreated. The psychological suffering and mental cancer metastasis diagnosis, which is also visible ineffective disharmony are the bi-products of the terminal illness to the minimal. The main emphasis of a cancer diagnosis is undergoes by every terminal patients were considered the to cure the symptom of the patient or to prolong the lives’ symptoms that need to be treated in the clinical practices in of the patient through ensuring quality of life.[1] However, India. Thus, failing to acknowledge and leaving the psycho- the greatest challenges in the Indian cancer diagnosis is the emotional symptoms untreated resulted in worsening the ensuring of the patient quality of life, which is not visible in patient’s conditions. The psycho-emotional and mental well- the clinical practices of the country as a whole. Unnecessary being is for the successful treatment of the patient physical prolonging of the patient lives with an aim to increases the pain symptoms. (Suantak & Sisodia, 2018). Well being of the numbers of days spend in the hospital ward for more bills. whole body requires a psycho-emotional and mental well- The patient’s value and dignity were unconcern the most by being to the most; a psychological well-being can give the clinicians’ and the patient undergoing treatment against positive responses to physical pain treatment to the their will/choices need to be rectifying immediately in the maximum. health care systems of the country as a whole. In India as a whole, the clinicians tend to forgot that even The early detection and immediate treatment intervention when cure is not possible in terminal diagnosis, there is a are the two most effective way of cancer diagnosis to control possibility of delivering healing as an alternative to cure. the cancer metastasis and to deliver total cure. As per the However, only through a person centered meaning making above findings, the medical intervention in stages one and psychotherapy in the clinical practices. The psychological two has higher possibilities rates to cure, which has around self-reflective and life review therapeutic approach in 80% cure possibilities as per the finding. While detecting the terminal diagnosis can make oneself aware that he/she is cancer symptom and consulting the oncologists in stage still in the condition of limitless achievements. Which will in three is visible in curtail conditions, having higher turn helps the patient to recreate his/her life goals, and set

new life goal that could be achievable, also able to make preventive measure interventions. Majority of the cancer beautiful memories with his/her loved ones that delivers the patient in rural regions failed to maintain their diets, while quality of life in terminal experience. Above all, it will make proper dietary is the core to successful diagnosis. Moreover, dying as normal as birth, which will deliver peaceful and eliminating the usages of tobacco, cigarettes and chewing of meaningful death (Suantak & Sisodia, 2018). Thus, in bitternuts can successfully reduces the rates of oral lip, terminal diagnosis, healing can be deliver as an alternative breast, mouth, cervical, head and neck, and the to cure, even when total cure is impossible in the clinical nasopharyngeal (NPC) cancer into at least 40-50% as per the practices. However, sadly, in the clinical practices of India as researcher findings. Another urgent need is to develop social a whole, the clinicians alone are the core medical team in the awareness on the causes of cancer, and treatment policies cancer/terminal diagnosis, in the absences of professional by educating the people mainly those leaving in the rural clinical psychologists and clinical social workers. The areas of the country. Gearing up for health awareness and terminal diagnosis requires a multidisciplinary team to strengthening the health care team at the community level deliver quality of life and quality end-of-life care. The by addressing the cancer preventive measures will clinicians were responsible for treated the physical pain effectively reduces the growing cancer populations. symptoms and its related, while the clinical psychologists Hygienic living with healthy food awareness also the urgent and clinical social workers were responsible for the patient requirement to fight against the deadly diseases, with the psycho-emotional suffering and mental disharmony appointed staff, conducting seminar in each village at least treatments, which the trained medical doctors or nurses once in a year. Training more oncologists in the country cannot handles in the clinical practices. would also be an effective ways to successfully fight against the ongoing cancer mortality in the country. However, as of CHALLENGES AND CONCLUSION now the ratio of oncologists and cancer patients in the Recently, Prime Minister Narendra Modi inaugurate country is 1:2,000, and that is next to impossible for an “Ayushman Bharat Health Scheme” on September 23, 2018, oncologist treating 2,000 patients in a day. The Centre for which would be effective by 25 December 2018 in Ranchi Cancer Epidemiology in the rural areas of the country is at with a primary aims to provide free health care to those present minimal and majority of the cancer care centres are underprivileged living in the rural areas, who are access not not functioning properly, whichis the major challenges for to any health care thus far. It means there are Indian who the government to take immidiate initial action. It is a high does not received any medical facilities thus far, which time for India to take up the necessary actions against cancer remain as the most challenging and root causes to maximum mortality as the rates of mortality increased year after years cancer/terminal illness in the country. As per the findings, with 70-90% per 100,000 populations, which constituted 500,000 Indian died of cancer every single day and the around 2,500,000 (2.5 millions) with 800,000 new cases government with the least concerned on the awareness every year and 5,50,000 deaths in a year. Moreover, in the programe and in establishing regional cancer care centers. battle of terminal illness fighting back and defending people This being the underlying reasons India successfully becomes is the only option that India had. For the love of humanity, the largest contributor to cancer mortality rate. Another let the terminal diagnosis acknowledge the psycho- reason for the rapid increasing mainly rates of cancer and its emotional suffering and mental disharmony of the patient mortality rates is mainly due to people unawareness on the alongside the physical pain symptoms to deliver the whole causes of cancerous factors, treatment policy, unhealthy person treatment in the clinical practices. lifestyles, and unhealthy food consumptions with no proper

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1. World Health Organization. (2018). Global Action Against http://www.epao.net/epsubpageExtractor.asp?src+education.Heal Cancer. Available at: http://www.who.int/cancer/media/ th_Issue.Scenario_in_Northeast_India. Accessed October 25, 2018. GlobalActionCancerEnglfull.pdf? Accessed October 25, 2018. 4. Zee Media Bureau. (Feb. 7, 2017). India World Largest 2. Sangita, P Ingole. Aruna, U Kakde. &Priti, B Bonde. A Review on Contributor To Cancer Deaths; Doctors Say 70% Patients Avoid Statistics of Cancer in India. IOSR Journal of Environmental Science, Concultation Till Terminal Stage. Available at: Toxicology and Food Technology 2016; 10: 107-116. DOI: http://zeenewsindia.com/health/india-worlds-largest-contributor- 10.9790/2402-100701107116. to-cancer-deaths-doctors-say-70%-patients-avoid-concultation-till- 3. Singh Tomcha Th. Cancer Scenario in Northeast India. Available terminal-stage-1973803. Accessed October 25, 2018. at: 5. Bhowmick Sourya. 500,000 Indians Died of Cancer Last Year.

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And More Shock Figures. Available at: http://www.catchnews.com/ Kalpana Sharma, and Narinder Kumar Mehra. Nasopharyngeal india-news/500,000-indians-died-of-cancer-last-year-and more- carcinoma in the Northeastern states of India. Chin J Cancer 2011; shock-figures. Accessed October 23, 2018. 30: 106–113. 6. National Institute of Cancer Prevention and Research. India 13. Bhattacharjee, Abhinandan A. Chakraborty, P. Purkaystha. Against Cancer: Statistics. Available at: Prevalence of Head and Neck Cancers in the North East -An http://cancerinindiadia.org.in/statistics/. Accessed November 12, Institutional Study. Indian Journal of Otolaryngology and Head and 2018. Neck Surgery 2006; 58: 15-19. 7. Dikshit R, Gupta PC, Ramasundarahettige C, Gajalakshmi V, 14. National Institute of Cancer Prevention and Research. Cancer Aleksandrowicz L, Badwe R, Kumar R, Roy S, Suraweera W, Bray F, Hits More Women in India than Men, But More Men Die of It. Mallath M, Singh PK, Sinha DN, Shet AS, Gelband H, Jha P. Cancer Available at: http://www.nicpr.res.in /images/imp_ban.png. mortality in India: A nationally representative survey. The Lancet. Accessed September 12, 2018. Available at: https://www.researchgate.net/publication/ 15. Dhillon, Preeet. Cancer – A Looming Threat In The North East. 223989113. Accessed October 27, 2018. The Shillong Times. Available at: http://www.theshillongtimes. 8. Sreedevi, Aswathy. Javed, Reshma. & Dinesh, Avani. com/2016/07/26/cancer-a-looming-threat-in-the-north-east/. Epidemiology of Cervical Cancer with Special Focus on India.Int J Accessed October 30, 2018. Womens Health 2015; 7: 405–414. DOI: 10.2147/IJWH.S50001. 16. Anand P, Kunnumakkara AB, Sundaram C, Harikumar KB, 9. Deccan Chronicle. Cancer Kills 10 Million In India. Available at: Tharakan ST, Lai OS, Sung B, Aggarwal BB. Cancer is a Preventable https://www.deccanchronicle.com/lifestyle/health-and-wellbeing/ Disease that Requires Major Lifestyle Changes. Journal of 120918/cancer-to-kill-10-mn-in-2018-despite-better- Pharmaceutical Research 2008; 25: 2079-2116. DOI: prevention.html. Accessed September 30, 2018. 10.1007/s11095-008-9661-9. 10. Varghese Cherian. Cancer Prevention and Control in India‖ 50 17. World Health Organization. WHO List of Priority Medical Years of Cancer Control in India. Available at: Devices for Cancer Management: WHO Medical Device Technical https://mohfw.gov.in/sites/default/files/Cancer%20Prevention % Series 2017. Available at: http://apps.who.int/iris/ 20And%20Control%20In%20India.pdf. Accessed October 27, 2018. bitstream/handle/10665/255262/9789241565462eng.pdf;jsession 11. National Tobacco Control Programme. Manipur Has The Highest d=4A6BE3B164041D129A49E2F006282717?sequence=1. Accessed Tobacco Consumption In India. Available at: December 8, 2018. https://www.ifp.co.in/page/items/8126/8126-manipur-has-the- 18. Vaiphei Demkhosei Suantak. & Singh Sisodia Davendra. highest-tobacco-consumption-in-india/. Accessed November 1, Psychotherapeutic Intervention in Terminal Diagnosis: An Over 2018. View. Journal of Indian Health Psychology 2018; 13: 20-34. 12. Amal Chandra Kataki, Malcolm J. Simons, Ashok Kumar Das,

Article received on August 1, 2019 and accepted for publishing on October 13, 2019. ORIGINAL ARTICLES

Simulation and dynamic analysis of military marching using lower limbs anthropometric data

Abolfazl Shakibaee1, Alireza Asgari2, Kamal Mostafavi3, Gholamhossein Pourtaghi4, Zeynab Ebrahimpour5

Abstract: Background: Gait analysis is receiving increasing attention due to various applications in athletic performance, man-machine interfaces and especially in military services. This analysis involves the analysis of human locomotion augmented by body movements and biomechanics of joints. The kinematic motion of the body during a gait cycle capturing by cameras is then used as the desired target for modelling the motion of body segments. By taking advantage of gait analysis concept, this study aims to model the military marching, using anthropometric data with the focus on lower limbs while introducing top candidates with better healthy conditions in lower limb joints during a cycle of marching. Methods: Using 100 anthropometric data from military soldiers, equations of motion for the model are derived by applying Lagrangian methods in an inverse dynamic approach. In this model, the joints are simulated using springs and dampers while the actuators, simulated the muscles, acted like motors and applied enough torque on joints so that the model motion replicates normal military marching. Finally, all the springs and dampers coefficients are driven from optimization process. Results: Hip, knee and ankle torques were calculated after the optimization process for all 100 soldiers and then 5 candidates among them were established with less suffering forces and torques in their joints. Conclusions: In this study using biomechanics basics and anthropometry data at the same time, a standard could be evaluated to select the soldiers based on healthy condition of lower organs. Keywords: marching, anthropometric data, gait analysis, biomechanics, torque, equations of motion, optimization

INTRODUCTION human gait cycle is required in which we should benefit from priori biomechanical knowledge on human motion and Marching is a specific sample of daily human gait [1]. That anthropometric data at the same time [2]. The gait cycle being said, in order to simulate marching, simulation of consists of complex functional tasks requiring interaction between lower limb joints of the body [3]. Motion of body segments during this interaction is studied as a part of 1 Exercise Physiology Research Center, Life style institute, Baqiyatallah University of Medical Sciences, Tehran, Iran kinematics without considering any forces. Typically, camera 2 Faculty of Aerospace and Submarine Medicine AJA Medical systems and electromagnetic devices are being used to Sciences University, Tehran, Iran record the motion of the body during a cycle [4]. 3 Research Assistant, Mechanic and Material Engineering department, Western University, London, Ontario, Canada While the body segments are considered as a mechanism, 4 Health Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran joints in hip, knee and ankle provide degrees-of-freedom 5 Department of Physical Education & Sport Sciences, Faculty of Human Sciences, Qaemshahr Branch, Islamic Azad University, Corresponding author: Kamal Mostafavi, PhD Qaemshahr, Iran [email protected]

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(DOF) of the system. The joint reaction forces and muscle net The appropriate method in studying the simulation of torques convert this problem into a dynamic analysis in marching is by applying dynamic models. Using this method, which inverse dynamic method is being used commonly. the model was considered as a rigid body with several links. Then, equations of motion relate the kinematics of the body Considering exact and complete number of variables for motion to the forces and torques that are causing those dynamic simulation complicates the model and motion motions [5]. Friction force calculation completes this set of equations, therefore in this study some assumptions were equations as an external force. This force during a walking employed to simplify the model. These assumptions include depends highly on the foot-ground contact simulation and 1) the upper section of the body (head, trunk and hands) was modeling. Some of the studies used additional kinematic considered as one link only, 2) friction was neglected in joint while other used temporal fixing to the support foot. joints, 3) the weights of the elements were equally But in some studies, realistic models for this section have distributed, 4) modelling and solving equations of motion been modeled which can simulate the reaction close to the conducted in sagital plane (2D), 5) due to 2D modeling, joints real foot-ground contact [2, 6]. Some studies conduct the were from hinge type and not spherical for hip joint. whole gait simulation by taking advantage of three The model for rigid body motion of a soldier contains seven dimensional models [7, 9] while others consider two links that include trunk, two thighs, two legs and two feet. dimensional models [3,10,11] for simulation [4,8]. Stability The model was defined with nine generalized coordinates as of such models is significantly important. Active feedback consisting of seven links with vertical angles and hip control systems [12] and simple PDI controller [10] are coordinates in Cartesian coordinate system (Figure 1). In this common methods used as a controller for the balance of gait model, the angles are trunk, right thigh, right leg, left thigh, models during a cycle. left leg, right foot and left foot angles with vertical direction Application of gait analysis in military services mainly limits while the joints are hinge joints. to assessing the influence of boot [11, 13] and carrying a backpack of soldiers during a gait cycle. Rare studies like [1] Figure 1. The model used in simulation with seven angles and hip demonstrated one of the first researches on marching coordinates locomotion. As other applications of gait analysis, there have been some studies about modeling the gait analysis in order to analyze the dynamic of prosthesis during a gait cycle [3, 10, 14, 18, 19, 20].

Towards simulation of marching, this paper conducts this analysis by computing the internal actuator forces and dynamic analysis of 100 soldiers. The major part of this simulation is to replicate normal kinematics of marching when subjected to normal muscle forces and joint torques during a cycle. In an inverse dynamic approach, using One of the most important issues in this dynamic simulation Lagrangian method the net joint torques is calculated from a is modeling the reaction force of the ground to the body. The sample military marching benchmark and external forces in modeling of this section was developed based on using a a condition that muscles are the actuators of the spring and a damper in contact points of foot to the ground musculoskeletal system. The optimization process then is so that the equivalent of damper and spring forces reflects needed to compute the forces and the torques in a way to the ground reaction force to the body [16]. This estimation generate the desired kinematic motion. helps to calculate this reaction force during dynamic simulation and to simulate the viscoelastic contact of the METHOD foot. As it can be seen in Figure 2, a series of vertical springs From the engineering point of view, marching is in fact body and dampers were attached to the contact points to segment movements in a mechanism way with couple of generate this vertical reaction force [10]. In the case of no degrees of freedom (DOF). This mechanism consists of hip contact between foot and ground, the spring and damper joint, knee joint and ankle joint. There is friction force contacts are disconnected and no force is reacted and between foot and ground which provides movement of the whenever the contact between foot and ground occurs whole body. In this simulation, bones are considered as rigid again, the spring and damper contacts are connected and so elements while supporting the weight of the body and the force is reacted. muscles act as elastic stimulators and displace the bones.

Figure 2. Foot-ground contact force simulated by spring and Table 2. Standards for mass calculation for body segments damper Center of mass/segment length Density Segment Proximal Distal (Kg/l)

foot 0.5 0.5 1.1 leg 0.433 0.567 1.09 thigh 0.433 0.567 1.05 trunk 0.5 0.5 1.3

While the anthropometric data varies from one soldier to In this model, the muscles are neglected and instead, another, the kinematic data is the same for all soldiers. The actuators are employed in hip, knee and ankle joints. The kinematic data must be considered the same for all since the torque being applied on the joints is a function of current ending result of joint torques for the model varies for various angle in joint, desired angle and their derivatives. kinematic data but this study targets to analyze the joint torques only based on anthropometric data, so that as a Indeed, springs and dampers are used in joints so that the result a standard of anthropometric data could be actuators act as motors which are linked with these springs concluded. and dampers. Therefore, appropriate modelling is needed for calculating the optimized variables for springs and Therefore kinematic data of a marching benchmark (Figure dampers. The general equation for torque in the joints is 3) was considered for all soldiers. 푚 푑 ̇푚 ̇푑 휏푖 = 퐾i(휃푖 − 휃푖 ) + 퐶i(휃푖 − 휃푖 ) Figure 3. Marching benchmark used in kinematic section of this study m ̇ m In which Ki and Ci are constant coefficients, θi and θi are 푑 angle and angular speed of links in each time frame, 휃푖 and ̇푑 휃푖 are desired angle and angular speed of the links. Spring and damper are key factors in stability of the model during marching. If these factors are neglected, any small disturbance in the model at the contact time leads to instability of the whole system. On the other hand, calculating the optimized coefficients requires desired kinematic data and stability of whole model.

Table 1. Statistical parameters of all 100 anthropometric data of soldiers Waist height Knee height Weight Height Data (mm) (mm) (kg) (mm)

Minimum 820 410 52 1,615

Maximum 1,152 602 78 1,875 Average 985.91 516.91 67.55 1,738.55 SD 58.12 35.94 7.07 60.17

In this study, anthropometric data of 100 military soldiers were analyzed through the dynamic simulation method. Table 1 shows the values of min, max, average and standard deviation parameters for all 100 soldiers. Using waist height and knee height, thigh length was calculated and based on the lengths of segments, the weights of segments were approached [15] (Table 2).

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As this figure depicts, right thigh and leg are raised to specific Kinematic data includes, 1) angular speed and angle of hip height in an extension movement and then in a felxion joint 2) angular speed and angle of knee joint 3) angular movement, right foot makes contact with the ground and speed and angle of ankle joint 4) angular speed and angle of then left segments follow the same path alternatively. Knee trunk. Figure 4 shows the angle of hip and ankle during one angle was considered the same as hip angle which means cycle of marching. Fourier function was used in order to that the knee by itself does not have any flexion-extension generate a curve passing through acquired kinematic data movement. points.

Figure 4. Hip and ankle angles during a gait cycle of the benchmark

In order to complete the modeling of marching, adding a total torque in this torsion spring and damper is transferred parameter to dynamic simulation is needed for keeping the to each hip joint to keep balance of the whole model. balance of the model. To accomplish this step, a torsion As it was mentioned in previous section, the equations of spring and a linear damper are applied to the trunk as a motion for this 2D model follow Lagrangian method. General function of simulated and desired kinematic of trunk. formulation of the Lagrangian method is Whenever the model is contacting the ground half of the

d ∂L ∂L  By having the angles of the model and desired angles ( ) + = 푄푘 dt ∂q̇ k ∂qk (from the literature), the objective function is found,  The genetic algorithm comes to play and starts to In which k is number of general coordinates which is 9 in this minimize the objective function by finding the optimized model, L is the difference between kinetic and potential values for spring and damper coefficients of the whole energy of the whole system, Qk reflects non-conservative model forces. In order to use these equations, initially kinetic and  Once the optimized coefficients are found from the step potential energy of the whole system were calculated and 6, the values are replaced with coefficients of step 2 until then after 9 equations of the order of 2 were produced. For final optimized converged coefficients are obtained. the dynamic simulation using this method, initial conditions  Return to step 3. and angular speed of the links were provided.

Constant coefficients in all joint torques (discussed in RESULTS previous sections) could be generated in an optimization The numerical programming for dynamic simulation of a process. The goal of optimization is to calculate these soldier was accomplished in MATLAB software. After coefficients in the condition that the discrepancy between providing all necessary parameters such as initial conditions, flexion-extension angles of joints in simulated and desired kinematic data, equations of motion and optimization, the circumstances is minimal. The desired (normal) values for whole program focused to generate the constant this simulation were obtained from [17]. In order to conduct coefficients using the optimization method for a sample this optimization, genetic algorithm method was applied to anthropometric data. Then, whole 100 anthropometric data decrease the difference between angles in desired and was given to the program to calculate the joint torques in simulated modes. While in this optimization variables are hip, knee and ankle. In fact this torque value is equivalent to the constant coefficients, the objective function is the maximum total torques acting on that specific joint. Table 3 summation squared of the difference between these angles. shows the statistic about resultant torque values. Below are the steps that the genetic algorithm takes to find the optimization coefficients: Table 3. Resultant torques in hip, knee and ankle joints  Initial parameters are provided as the initial condition of Hip torque Knee torque Ankle torque the problem, Segment (N.m) (N.m) (N.m)  Initial guess of the spring and damper coefficients of the model is provided, Min 20.96 0.06 1.73  Based on the values from steps 1 and 2, the equation of Max 22.94 1.37 3.50 the motion is formed, Average 21.65 0.27 2.61  From this equation of the motion, the angles function of STD 0.33 0.19 0.37 the model is derived,

Table 4. Top 10 candidates with lowest toque amount in hip joint Hip torque Knee torque Ankle torque Waist height Knee height Weight Height (N.m) (N.m) (N.m) (mm) (mm) (Kg) (mm)

20.98 0.15 2.42 990 480 70 1,775 20.99 0.12 2.36 975 500 62 1,705 21.00 0.12 2.36 990 480 70 1,735 21.03 0.31 1.88 880 490 60 1,632 21.04 0.35 2.98 970 550 75 1,797 21.05 0.11 2.55 995 500 66 1,730 21.08 0.33 2.25 1,110 575 70 1,780 21.09 0.21 2.13 1,015 570 75 1,788 21.10 0.55 2.61 1,040 500 52 1,700 21.11 0.11 3.11 1,100 565 85 1,895

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In the initial analysis of this huge data, we focused on hip Moreover, Figure 5 shows how the waist height and knee data. Since in this study having healthy joints are more height vary by the torque in the hip. It is interesting that both desirable, we sorted the data from small to large values and heights increase by gradually increasing of the hip torque. then just consider the first top 10 as good candidates for This fact is confirming that higher waist and knee heights can healthy joints (Table 4). This narrowing down of the results lead to larger torques in the hip. helps us to analyze the data more consistently.

Figure 5. Variation of waist and knee heights by hip torque increasing in top 10 candidates with lowest torque amount in hip joint

In the next step, knee torque was assessed by the numerical selection for the hip. Indeed, this study approached the joint programming code in MATLAB and the results were again torques behaviour separately and then concludes all the sorted from small to large and first 10 candidates were resultant data together. The corresponding knee data can be selected. This selection was done independently of previous found in Table 5.

Table 5. Top 10 candidates with lowest torque amount in knee joint Hip torque Knee torque Ankle torque Waist height Knee height Weight Height (N.m) (N.m) (N.m) (mm) (mm) (Kg) (mm)

22.15 0.02 3.74 1,152 602 79 1,915 22.04 0.03 4.27 1,030 530 73 1,805 21.79 0.04 3.25 900 490 72 1,705 21.80 0.05 3.69 980 500 72 1,743 22.05 0.05 4.07 1,080 520 68 1,805 21.60 0.06 3.35 975 480 63 1,642 21.83 0.06 3.48 1,000 480 78 1,745 21.25 0.06 2.69 1,000 540 65 1,780 21.25 0.07 2.69 998 510 57 1,695 21.93 0.07 3.91 1,000 525 75 1,710

Comparing the torque values in knee joint to other two Therefore, for the ending result of this study, knee did not joints, it can be concluded that for this specific kinematic play a major role and that is because flexion – extension data for marching, the torque in knee is negligible. movement was not considered for the knee.

Ankle torques is the next criteria considering as a result. cycle of marching for the top 10 candidates who their ankle Table 6 represents the torques in ankle joint during a full torques were minimal independent from other joint torques.

Table 6. Top 10 candidates with lowest torque amount in ankle joint Hip torque Knee torque Ankle torque Waist height Knee height Weight Height (N.m) (N.m) (N.m) (mm) (mm) (Kg) (mm)

21.22 0.08 1.75 1,070 520 78 1,835 21.03 0.31 1.88 880 490 60 1,632 21.36 0.47 1.95 1,148 600 75 1,900 21.63 0.11 1.98 994 500 71 1,766 21.63 0.50 2.08 945 475 72 1,680 21.09 0.21 2.13 1,015 570 75 1,788 22.07 0.13 2.15 1,040 530 56 1,708 21.58 0.35 2.18 930 480 69 1,720 22.01 0.22 2.23 1,010 535 80 1,758 21.08 0.33 2.25 1,110 575 70 1,780

Comparing the variation of the ankle torque versus waist and variation of ankle torque (Figure 6). In fact the amount of knee heights depicts the fact that ankle torque is highly ground force reaction plays the major role in the torque of dependent on the force being applied by the ground, since the ankle. the waist and knee heights are almost constant with the

Figure 6. Variation of waist and knee heights by ankle torque increasing in top 10 candidates with lowest torque amount in ankle joint

DISCUSSION anthropometric data have been introduced, therefore it is needed to define another factor in order to select from those In order to assess the healthy joints under marching candidates, those with better healthy conditions in terms of condition, 2 factors were considered in this study: 1) hip minimum torque value in their joints. Due to less torque rate torque and 2) ankle torque. Then based on the torque data in knee comparing to hip and ankle, a new factor called of each joint, the whole data was sorted from smallest to “Minimal Summation Torque” is defined for this study which largest value three times. Then, first 10 candidates in each follows below equation, considering the fact that the section were highlighted and analyzed. In the first look, it maximum torque in hip is 22.39 N.m and in ankle is 4.35 N.m. seems like 20 different candidates with identical

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ℎ𝑖푝 푡표푟푞푢푒 푎푛푘푙푒 푡표푟푞푢푒 푚𝑖푛𝑖푚푎푙 푠푢푚푎푡𝑖표푛 푡표푟푞푢푒 = + 푚푎푥𝑖푚푢푚 ℎ𝑖푝 푡표푟푞푢푒 푚푎푥𝑖푚푢푚 푎푛푘푙푒 푡표푟푞푢푒

By using the maximum values of torques we normalized our other factors for all 100 data and sort again based on torque data. Then, we needed to calculate this factor beside minimum value of torques. Table 7 shows this result.

Table 7. Top 10 candidates with lowest Minimal Summation Torque Minimal Hip torque Knee torque Waist height Knee height Weight Height Summation (N.m) (N.m) (mm) (mm) (Kg) (mm) Torque

21.22 1.75 1.35 1070 347 78 1835 21.03 1.88 1.37 880 325 60 1632 21.36 1.95 1.40 1148 325 75 1900 21.63 1.98 1.42 994 347 71 1766 21.09 2.13 1.43 1015 337 75 1788 21.63 2.08 1.44 945 336 72 1680 21.08 2.25 1.45 1110 348 70 1780 21.58 2.18 1.46 930 334 69 1720 21.34 2.26 1.47 980 296 72 1710 21.23 0.33 2.28 1005 330 66 1722

Analyzing Tables 4, 6, 7 reveals that there is a common point marching. We highlighted these candidates in all tables and between all of them. There are some soldiers that exist in all allocated one colour to each of them. Table 8 represents all those tables, showing that these candidates are common of them in one separate table containing corresponding among all other candidates. This approves that this set of anthropometric data. candidates contain a healthier body during a cycle of

Table 8. Top 5 candidates with healthier joint condition during a cycle of marching Hip Knee Ankle Waist height Knee height Weight Height circumference circumference circumference (mm) (mm) (Kg) (mm) (mm) (mm) (mm)

880 490 990 416 230 60 1632 1148 600 990 460 230 75 1900 1015 570 940 380 250 75 1788 945 475 940 400 250 72 1680 1110 575 980 410 270 70 1780

In fact, in this study using biomechanics basics and Authors' contributions anthropometry data at the same time, a standard could be Dr. Shakibaee provided assistance for all experiments and collecting anthropometric data. Dr. Asgari supervised this study and provided assistance concluded to select the soldiers based on healthy condition in manuscript preparation. Dr. Kamal Mostafavi carried out the modelling, of lower organs. coordinated the study, completed the analysis, extracted the results and prepared the manuscript. Dr. Pourtaghti and Dr. Ebrahimpour assisted and This study is one of the first studies being conducted in the coordinated the processing of data collection. All authors have read and area of specifying a standard for soldiers based on their approved the content of the manuscript. lower organ healthy condition during a cycle of marching. Acknowledgement This paper has been supported by Exercise Physiology Research Center of Conflict of interest Baqiyatallah University of Medical Sciences. The authors have no conflicts of interest.

References:

1. Flesher MM: Repetitive order and the human walking Selvamurthy W:Temporal spatial parameters of gait with barefoot, apparatus: Prussian military science versus the Webers' locomotion bathroom slippers and military boots. Indian journal of physiology research. Annals of Science 1997, 54:5, 463-487. and pharmacology 2006, 50(1): 33 2. Wojtyra M: Dynamical analysis of human walking. 15th 12. Bauby CE, Kuo AD: Active control of lateral balance in human European ADAMS users’ Conference 2000, Warsaw. walking. Journal of biomechanics 2000, 33(11): 1433-1440. 3. Pejhan S, Farahmand F, Parnianpour M: Design optimization of 13. Cikajlo I, Matjačić Z: The influence of boot stiffness on gait an above-knee prosthesis based on the kinematics of gait. Kinematics and kinetics during stance phase, Ergonomics 2007, 50 Engineering in Medicine and Biology Conference 2008, Vancouver, (12): 2171- 2182. 4274-4277. 14. Faure F, Debunne G, Cani MP, Multon F: Dynamic analysis of 4. Aggarwal JK, Cai Q: Human Motion Analysis: A Review. human walking. In 8th Eurographics Workshop on Computer Computer Vision and Image Understanding 1999, 73 (3): 428–440. Animation and Simulation 1997,53-65. 5. Winter DA: Biomechanics and motor control of human 15. http://www.ele.uri.edu/faculty/vetter/BME207/anthropometr movement. Wiley, 2009 ic-data.pdf 6. Peasgood ME: Stabilization of a dynamic walking gait 16. J. L. Sutherland, D. H. Sutherland, et al., “Case study forum: gait simulation. J Comput Nonlinear Dynam 2007, 2(1): 65. comparison of two prosthetic knee units”. J Prosth Ortho, 9(4), 168 7. Anderson FC, Pandy MG: Dynamic optimization of human (1997). walking. Journal of biomechanical engineering 2001, 123:381-390. 17. D.A. Winter, “Biomechanics of human movement”. John Wiley 8. Winter DA: Biomechanics of human movement. John Wiley & & Sons Inc. London (1979). Sons Inc.1979. 18. Zhao, Y., Zhang, W., Ge, W., & Li, S. (2013). Finite Element 9. Eng JJ, Winter DA: Kinetic analysis of the lower limbs during Simulation of Soldier Lower Extremity Exoskeleton. Journal of walking: what information can be gained from a three-dimensional Multimedia, 8(6), 705-711. model? Journal of Biomechanics 1995, 28(6): 753-758 19. Tang, Z. Y., Tan, Z. Z., & Pei, Z. C. (2013). Design and Dynamic 10. Akbari SM, Farahmand F, Zohour H: Dynamic simulation of the Analysis of Lower Extremity Exoskeleton. Journal of System Biped normal and amputee human gait, 12th International Simulation, 6, 033. Conference on Climbing and Walking Robots and the Support 20. Clark, Hannah D. Military load carriage during prolonged Technologies for Mobile Machines (CLAWAR )2009, 1113-1120, 9-11 marches on lower extremity mechanics: Influence of gender. State September 2009 - Istanbul, Turkey. University of New York at Buffalo, 2013. 11. Majumdar DH, Banerjee PK, Majumdar D, Pal M, Kumar RA,

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Article received on May 13, 2019 and accepted for publishing on September 30, 2019. ORIGINAL ARTICLES

Drug allergies interpretation based on patient’s history alone may have therapeutic consequences in hospital setting

Polliana M. Leru1,2, Andreea Leontescu3, Ion Stefan1,4, Irena Nedelea5, Iuliana Ceausu6, Edu Antoine1,3

Abstract: Background and aim. Adverse drug reactions (ADRs) are common events in medical practice, representing a matter of concern for both outpatient and in hospital care. Understanding and management of ADRs depend on their mechanism and clinical picture, which are complex and highly heterogeneous. Drug hypersensitivity reactions (DHRs) are immunologically-mediated ADRs, which are considered allergies after demonstrating evidence of either drug-specific antibodies or T-cells. The correct diagnosis of a drug allergy in hospital setting is essential for the outcome of treated disease and for the patient future in terms of therapeutic needs. Patients may be labeled as being drug allergic, based on their history and vague symptoms that do not correspond to a true allergic reaction. The aim of our study was to evaluate consequences of drug allergy labelling of female patients hospitalized in the department of obstetrics and gynecology from a university hospital during one year, in terms of medical attitude and therapeutic approach. Method and Results. We found that 159 out of 2395 patients hospitalized in the obstetrics-gynecology department declared a history of drug allergy on admission, but only 3 patients had medical documents and adequate recommendations from the allergist. Hospital medical attitude was significantly influenced in all declared allergic patients, mainly in terms of antimicrobial and anti-inflammatory therapy. Conclusion. We concluded that overestimation of drug allergies in hospital care has important consequences in terms of current and future recommended therapy that should be based on standardized allergist evaluation. Keywords: drug allergies, hospital setting, patient’s history, therapeutic errors

INTRODUCTION No reliable data about incidence of ADRs in Romania could be found. Understanding and management of ADRs depend Adverse drug reactions (ADRs) are common events in on their mechanism and clinical picture, which are complex medical practice, representing a matter of concern for both and highly heterogeneous [3]. outpatient and in hospital care. The ADRs definition accepted by the World Health Organization is any noxious The pharmacological classification of ADRs includes two and unintended response to a medication that occurs at major subtypes: type A which are dose-dependent and normal doses used for prophylaxis, diagnosis and/or predictable reactions, with non-immunological mechanism, treatment [1]. ADRs severity range from minor common side effects to potentially life-threatening medical conditions. 1 Carol Davila University of Medicine and Pharmacy, Bucharest The reported incidence of ADRs in general population varies 2 Colentina Clinical Hospital, Bucharest between countries and may account for 2-3 % of all hospital 3 Nicolae Malaxa Clinical Hospital, Bucharest admissions in some regions, as reported from Australia [2]. 4 Carol Davila Emergency Central Military Hospital, Bucharest 5 Iuliu Hatieganu University of Medicine and Pharmacy, Cluj Corresponding author: Polliana Mihaela Leru Napoca [email protected] 6 Ioan Cantacuzino Clinical Hospital, Bucharest

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commonly termed intolerance and type B, which are one-year period and we recorded diagnosis of drug allergies unpredictable, not dose-dependent and have immunologic based on patient’s history, as well as medical events mechanism [4]. Drug hypersensitivity reactions (DHRs) are considered as drug allergy and medical therapeutic attitude immunologically-mediated ADRs, considered allergies after during hospitalization. demonstrating evidence of either drug-specific antibodies or A total number of 2,395 female patients have been T-cells [5]. Therefore it is recommended to use the term hospitalized during twelve months between January- allergy only to describe reactions for which an December 2016 in the Obstetrics-Gynecology department of immunological mechanism has been demonstrated. The Nicolae Malaxa Clinical Hospital from Bucharest, with 1,817 majority of ADRs (>85%) are type A, while drug allergies patients hospitalized for childbirth and 578 patients comprise up to 15 % of ADRs [4]. It is considered that drug hospitalized for gynecological diseases. allergies affect more than 7 % of the general population [6]. The correct classification of an adverse drug reaction, by Patients were aged between 18-67 years, with a mean age medical professionals, as allergy (due to immunological of 33 years and about 80% lived in urban area. We recorded mechanism) or intolerance (non-immunological mechanism) medical history documented in the patient file, including any has important medical implications [3]. Both underdiagnosis, type of reported drug allergies, written information due to underreporting and overdiagnosis, due to an overuse regarding diagnosis confirmation and tolerated medication, of the term „allergy” are common. There are still many concomitant diseases, other personal or familial allergies, controversies in drug allergy and recognized need for current illness and medication, outcome and status at standardized and improved approaches of patients who hospital discharge, particular recommendations regarding report prior drug allergy [7]. Mislabeling of a drug allergy in drug allergies. In cases who had medical events considered patients records may result in unnecessary avoidance of an drug allergies during hospitalization, we noted type of effective drug and medical recommendation of a second-line reaction, severity, the possible culprit drug, relevant therapy, possibly less effective or more expensive. This laboratory tests, therapeutic attitude and outcome. situation is more frequent in case of antimicrobial drugs and may be also responsible for increased bacterial resistance to RESULTS frequently used antimicrobials. Despite lack of reliable We found that 159 out of 2395 patients, meaning 6.63% of evidence, if an allergy label is noted into a patient medical female patients hospitalized in the Obstetrics-Gynecology record, it will remain for the rest of his life in most of the department declared a history of drug allergy on admission. cases [8]. Maintaining the diagnosis of a drug allergy in From this group, 116 were pregnant patients hospitalized for hospital setting is essential for the outcome of treated childbirth and 43 patients were hospitalized for gynecologic disease and for the patient future in terms of therapeutic diseases. needs. We noticed that 26 patients out of 159 (16.35%) were The aim of our study was to evaluate medical recording and current smokers. The possible culprit drugs based on consequences of drug allergy labeling of female patients patient’s declaration were: antibiotics in 76 patients hospitalized in the department of Obstetrics and Gynecology (47.79%), non-steroidal anti-inflammatory drugs (NSAIDs) from a university hospital during one year, mainly in terms and analgesics in 41 patients (25.78%) and other drug of current medical attitude, clinical outcome and therapeutic allergies in 42 patients (26.41%) (Figure 1). approach. Only three patients out of 159 (1.88%) could show medical MATERIAL AND METHOD documents with diagnosis confirmation of drug allergy and clear recommendations to follow in case of future events. We performed a retrospective analysis of hospital data over

Figure 1. Culprit drugs based on patient’s declaration

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Clinical episodes considered drug allergies were recorded ergometrine, drotaverine. during hospitalization in another 9 patients from the total The possible offending drug was avoided in all cases who 2395 hospitalized group (0.37%), who have not declare any reported drug allergies. No medical events attributable to drug allergy on hospital admission. Eight patients had acute anesthetics have been recorded. urticaria and one patient had respiratory symptoms. All these events have been successfully treated with In case of patients who declared allergy to NSAIDs, no drug antihistamines and systemic corticosteroids, based on from this class was administered, but also analgesics and allergist recommendation and resolved within short time. In antipyretics were avoided. No data referring to influence of all cases who declared history of drug allergy, the possible drug allergy on duration of hospital stay could be found. culprit drug was not administered and medication from another pharmacological class was considered. No DISCUSSION allergological work-up could be performed during Our study showed that drug allergy is overestimated in hospitalization, but this was recommended after hospital hospital setting, based on patient’s history alone and most discharge. From the 76 cases who declared allergies to of the patients have no evaluation or diagnosis confirmation antibiotics, the incriminated classes were: beta-lactam from allergist. Hospital data referring to possible allergy antibiotics in 48 cases, macrolides in 9 cases, tetracyclines in were limited and some laboratory tests that might support 5, sulphonamides in 14 cases (Figure 2). The recommended atopy or certain symptoms of allergy, such as total serum IgE antimicrobial therapy during hospitalization was quinolone and blood eosinophilia were missing [9]. The main in all cases (ciprofloxacin and levofloxacine). consequence for hospitalized patients who declare having drug allergy is change of the antimicrobial medication with a Figure 2. Antimicrobials reported as culprit drugs different class and refrain from any anti-inflammatory and Sulphona painkiller medication. The situation might be more mides Beta-Lactam 18% antibiotics complicated and has to be more seriously considered for Tetracycli 63% patients who declare multiple drug allergies to various and nes unrelated molecules. 7% The term „allergy” is frequently used and one study showed Macrolide that more than 50% of reported allergies to antimicrobials s 12% have no immunologic pattern [10]. Generally, patients tend to overestimate adverse drug reactions and to interpret erroneously new symptoms or signs that occur during From the 41 cases who reported allergies to non-steroidal treating their disease. anti-inflamatory drugs (NSAIDs) and analgesics, the Data from the literature showed that up to 25% of patient’s incriminated substances were: metamizole in 16 cases, self-report having drug allergies, most commonly induced by aspirin in 8, acetaminophen in 3, diclofenac in 2, ibuprofen antimicrobials, mainly penicillins, followed by sulfonamides, in 5, indomethacin in 3, nimesulide in 4 cases. (Figure 3) macrolides and cephalosporines [11]. Beta-lactam allergy,

Figure 3. NSAIDs and analgesics reported as culprit drugs recorded in up to 15 % hospitalized population and leading to increased use of broad-spectrum antibiotic use, is Acetamino Diclofenac confirmed in only 5 % of patients [12]. phen 5% Ibuprofen 7% 12% Having a label of drug allergy or intolerance in the medical Aspirin Indomethacin record may lead to withholding of first line therapy or even 20% 7% avoidance. It was proved that about 30% of patients having the label of drug allergy to at least one antimicrobial drug Nimesulide Metamizole 10% received another drug that did not reflect the standard of 39% care [13]. Alternative antibiotic therapy may be less effective or may have less favorable safety profile, leading to treatment failure and increased risk of antibiotic resistance. Other possible culprit drugs reported by patients who Trends in antibiotic prescription rates show an increase use considered themselves to have drug allergies were: of broad-spectrum antibiotics, such as azithromycin and tolperisone, metoclopramide, clexane, vitamines, quinolones [14]. It was also proved that total hospital costs

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with medication were increased for patients caring the label performed in Romania, our study has some limitations. The of a drug allergy, with 63% higher antibiotic costs for patients main limitation is the retrospective analysis of medical with beta-lactam allergy [15]. Despite the potential records, with relative few and variable information about deleterious consequences of selecting an alternate drug allergies, depending on the level of knowledge of medication and the lack of validity of most allergy labels, doctors in training, students or nurses who filled in the many clinicians do not take time to complete a patient’s files. We had no way to verify the proportion of comprehensive drug allergy history before prescribing an patients from our study with true versus false drug allergy antimicrobial. It was found that only 4% of patients who label, based on standardized diagnosis criteria. No follow-up declared being allergic to antimicrobial medication on of the patients who experienced allergic reactions during hospital admission had documentation detailing the specific hospitalization was performed. reaction [16]. This finding may be due to the erroneous perception that therapeutic decisions based on an allergy CONCLUSIONS label will not influence the patients’ clinical outcome. The We concluded that the impact of drug allergy label is ability of health professionals to discriminate between underestimated in hospitalized patients and diagnosis is allergy and intolerance, to evaluate the severity of an ADR generally based on patient’s history. and the degree of contraindication depends on their level of knowledge in the field of allergic diseases and was evaluated The allergy label is overused, most of the patients who self- in recent studies and publications [17]. A published survey of report drug allergies have no confirmation from specialist physicians practice and knowledge in the field of drug and no medical documentation. The main consequences are allergies at a university medical center showed that those switch of antimicrobial medication to another class, with any internal medicine training performed significantly generally quinolones and avoidance of anti-inflammatory or better [18]. analgesic medication.

A recent study evaluating drug allergies in primary care We consider that information referring to drug allergy practice in Romania concluded that the medical attitude and should be taken by medical staff with minimal training in this competence of family doctors in the field of drug allergies, field and recorded more detailed in patient’s files on hospital as well as their collaboration with allergists are not admission. Improved collaboration between specialists, standardized and updated according to guidelines and including allergists and primary care physicians is needed. experts’ recommendations [19]. Since primary care physicians play a key role in evaluating patients with possible drug allergies, there is a clear need for pre-and postgraduate Conflict of interest allergy educational programs and training [20]. The authors declare no conflict of interest in relation with this study and manuscript.

LIMITATIONS OF THE STUDY Disclosure statement The authors declare no funding or financial grants. Despite being the only study on drug allergy labeling

References:

1. World Health Organization International drug monitoring: the Organization, October 2003. J Allergy Clin Immunol 2004;113:832- role of the hospital. Geneva: The Organization, 1996. 836. 2. Roughead EE, Semple SJ, Rosenfeld E. Australian Commission on 6. Gomes ER, Demoly P. Epidemiology of hypersensitivity drug Safety and Quality in Health Care. Literature review: medication reactions. Curr Opin Allergy Clin Immunol 2005;5:309-316. safety in Australia. Sydney: ACSQHC, 2013 7. Chiriac AM, Banerji A, Gruchalla RS, Thong BYH, Wickner P. 3. Smith W. Adverse drug reactions. Allergy? Side effects? Mertes PM et al. Controversies in Drug allergy: Drug allergy Intollerance? Aust Fam Phys 2013;42:12-6. Pathways. J Allergy Clin Immunol Pract 2019;7:46-60. 4. Demoly P, Adkinson NF, Brockow K, Castells M, Chiriac AM, 8. Borch JE, Anderson KE, Bindslev-Jensen C. The prevalence of Greenberger PA, et al. International consensus on drug allergy. suspected and challenge-verified penicillin allergy in a university Allergy 2014;69:420-437. hospital population. Basic Clin Pharmacol Toxicol 2006;98:357-62. 5. Johansson SG, Bieber T, Dahl R, Friedmann PS, Lanier BQ, Lockey 9. Leru PM. Eosinophilia and Hypereosinophilic disorders – update RF et al. Revised nomenclature for allergy for global use: Report of on etiopathogeny, classification and clinical approach. Rom J Intern the Nomenclature Review Committee of the World Allergy Med 2015;53(4):289-295. DOI: 9.1515/rijm-2015-0049.

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10. Kerr JR. Penicillin allergy: a study of incidence as reported by 16. Lee CE, Zembower TR, Fotis MA, Postelnick MJ, Greenberger patients. Br J Clin Pract 1994;48:5-7. PA, Peterson LR et al. The incidence of antimicrobial allergies in 11. Charneski L, Deshpande G, Smith SW. Impact of an antimicrobial hospitalized patients. Arch Intern Med 2000; 160:2819- 22. allergy label in the medical record on clinical outcomes in 17. Shakib S, Caughey GE, Fok JS, Smith W. Adverse drug reaction hospitalized patients. Pharmacotherapy 2011;31:742-7. classification by health professionals: appropiate discrimination 12. Sacco KA, Bates A, Brigham TJ, Imam JS, Burton MC. Clinical between allergy and intolerance? Clin Transl Allergy.2019;9-18 outcomes following inpatient penicillin allergy testing: a systematic 18. Sturm JM, Temprano A. A survey of physician practice and review and metanalysis. Allergy 2017;72:1288-96. knowledge of drug allergy at a university medical center. J Allergy 13. Lutomski DM, LaFollette JA, Biaglow MA, Haglund LA Antibiotic Clin Immunol Pract.2014;2:461-4. allergies in the medical record: effect on drug selection and 19. Leru PM. Drug allergies in primary care practice in Romania: A assessment of validity. Pharmacotherapy 2008; 28:1348-53. questionnaire –based survey. Allergy Asthma Clin Immunol (AACI). 14. Grijalva CG. Antibiotic prescription rates for acute respiratory 2014;10:16 tract infections in US ambulatory settings. JAMA 2009;302:758-66. 20. Dona I, Caubet JC, Brockow K, Doyle M, Moreno E, Terreehorst 15. MacLaughlin EJ, Saseen JJ, Malone DC. Costs of beta-lactam I, et al. An EAACI task force report: recognizing the potential of the allergies: selection and costs of antibiotics for patients with a primary care physician in the diagnosis and management of drug reported bet-lactam allergy. Arch Fam Med 2009;9: 722-6. hypersensitivity. Clin Transl Allergy. 2018;8:16 https//doi.org/ 10.1186/s13601-018-0202-2.

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Article received on August 12, 2019 and accepted for publishing on October 28, 2019. ORIGINAL ARTICLES

New approaches regarding the protection forces’ health against the effects of some toxic substances

Pavel Otrisal1, Camelia Diaconu2, Ovidiu Bratu3, Florentina Ionita Radu3, Zdenek Melicharik4, Simona G. Bungau5

Abstract: In the past, finding out the chemical resistance of the barrier materials designated for the production of individual protective equipment ofthe protection forces (policemen, firefighters, soldiers) was based on the need to respond to protection against weapons of mass destruction. Simple chemical methods based on indicating the colorimetric reactions were used. It was necessary to switch to more universal methods due to a change in the security of the environment and the need to focus on toxic industrial materials. The universality is due to using sensitive QCM sensors equipped with a special detection layer and some special carbon electrodes. The paper deals with new methods that are used to determine the chemical resistance of the barrier materials against the permeation of toxic substances, primarily in field conditions. However, they can also be used in stationary chemical laboratory conditions. Keywords: Permeation, barrier material, PIEZOTEST, KONDUKTOTEST, SORPTEST

INTRODUCTION cope with specific interventions. Exposure to various harmful factors (fire, extreme heat, chemicals, pollutants of Public health is and must be a major interest for the policies all kinds, etc.) must take place in the optimum parameters, and legislation of a country. Maintaining and improving it ensuring the best performing materials, with maximum level must be done by implementing appropriate measures and of protection [1]. Direct contact with these substances programs, by educating the population and by raising the (drugs, chemicals, flammable substances, poisons, narcotics, standard of living. A particular sector within this field of various medical and pharmaceutical waste) [2-4] which can health is represented by the health insurance of the usually be found in nature and especially in cases of protection forces (police, firefighters, soldiers). In addition to emergency (fire, war, explosions) can often be lethal, psychological training, physical training, proper nutrition, through harmful compounds that are released or which, in etc., they must be provided with the optimal equipment to direct contact with the skin can cause severe burns, major infections, dizziness, fainting, headache, choking, serious 1 Nuclear, Biolological and Chemical Defence Institute, University pathologies as cancer [5, 6] etc., and finally – death. of Defence in Brno, Brno, Czech Republic 2 University of Medicine and Pharmacy ”Carol Davila”, Bucharest, The interest to produce protective materials impervious to Romania possible contamination agents has increased in recent years. 3 University Emergency Central Military Hospital, Bucharest, The political decision makers, as well as the economic agents Romania and the manufacturing companies (that are producing 4 Faculty of Physical Culture, Palacký University Olomouc, harmful substances, pollutants, as well as those who's Olomouc, Czech Republic 5 Faculty of Medicine and Pharmacy, University of Oradea, Corresponding author: Simona Bungau Oradea, Romania [email protected]

106 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine activity field involve obtaining the performant barrier of 1 ng of a toxic substance. This sensitivity is quite enough materials) concentrate their activity more and more for operating the measuring device in both field and intensely on this aspect of the development of the protective laboratory conditions. Application and software equipment, equipment with high quality, reduced cost, with high which is an integral part of the measuring equipment, allows demand on the market and which generate waste as little as on-line monitoring of a wide range of standardized possible following the production process [7-14]. parameters. The most important data is the breakthrough time (or also the resistance time) when the permeation In the last decade, there has been a shift from relatively mass's (cumulative amount) value is reached for a particular simple chemical colorimetric methods for testing the (subtracted) value based on the permeation mass resistance of the barrier materials against the permeation of (cumulative mass) versus time graph. However, it can also be toxic substances to sophisticated methods based on a specified as the normalized breakthrough time, but only if it principle of sensor technologies [15-21]. This change was relates to the normalized permeation weight (cumulated called by a gradual re-evaluation of the security threats amount) specified by the relevant standard. For fast data experienced by military professionals in military operations evaluation without the need for application and software [22, 23]. Representatives of the North Atlantic Treaty equipment, it is possible to use the value of protection time Organization declared that high use of weapons of mass (chemical resistance time) expressed as lag-time, to which destruction is currently unlikely [24]. Industrial chemicals are the value of diffusion coefficient is automatically now considered to be a significantly more important security recalculated. The time to reach the maximum permeated threat because their leakage can cause many losses and can concentration, which is sometimes referred to as the time of cause contamination of large areas of interest [25, 26]. New half diffusion flow, is also significant for the purposes of methods that enable the testing of the chemical resistance assessing the capture efficiency of the test chemicals of the barrier materials against the permeation of toxic through barrier materials. The permeation calculator substances find their practical application also in testing of automatically recalculates the value of the respective already established final products and also of the barrier diffusion coefficient to this value, which creates a space for materials that may find their use in the future [27, 28]. New comparing the penetration rate of the test chemical through methods also allow very fast evaluation of the permeation the tested barrier material. The time to reach the maximum data found and meet all requirements set by military and permeation rate (when the lag-time is reached) and the European standards [29]. The concept of research, maximum reached permeation rate (when the steady state development, and production of permeation devices using permeation rate is reached) is very important for the sensor technologies has been designed to meet the evaluation of the breathable barrier materials is very requirements for their use in field conditions and to enable significant. Results of the calibration measurements showed obtaining permeation data with minimal attendance and that it is possible to achieve high-quality dependencies of the service [30-32]. QCM sensor working frequency increase, thus the All these aspects have led to a gradual decrease in the concentration of test chemical over time, even though this efficiency of work with colorimetric methods, applicable method was not primarily intended as analytical. only for determining the chemical resistance of toxic substances (used in wars) under simulated laboratory KONDUKTOTEST method conditions. These methods did not allow a detailed analysis The KONDUKTOTEST method is based on the basic analytical at the level of the North Atlantic Treaty Organization nor at method working on electrochemistry, thus dissociation of the European Union level. ions. It introduces practical knowledge of conductometry into the implementation testing practice and adapts them to PIEZOTEST method the conditions for determining the chemical resistance of The PIEZOTEST method uses a QCM (Quartz Crystal barrier materials against the permeation of toxic substances. Microbalance) based piezoelectric sensor to detect A significant benefit of the used measuring device is the permeated toxic substances. These sensors, which find their replacement of very expensive platinum electrodes with application in many technical and industrial applications, glass electrodes. These electrodes are characterized by a have been modified to detect the permeation of the toxic very good sensitivity and minimal maintenance and substances through the barrier materials by applying the operation requirements. Their further advantage is that they appropriate detection layer to guarantee the functionality of do not experience problems with memory effect, which is an this sensor as a gravimetric detection device. The 1 Hz important and limiting factor for the electroanalytical sensor's response is reported to correspond to the detection methods. The elimination of this effect is based on the actual

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needs of measurement when working with very dilute calibration, actual measurement and desorption of QCM solutions. In practice this means that a very low sensor. Before starting and during the measurement, it is concentration of dissociated ions is sufficient to form a possible to adjust the temperature, which ensures the permeation curve, which serves as a basis for the reading of transfer of the test chemical into the gas phase and the standard values. This concentration is so low that it does not temperature of the Peltier´s cooler. Permeation measuring produce the memory effect of the used carbon electrodes cells are tempered separately. A biological incubator has even after their repeated use. The KONDUKTOTEST method proved to be a suitable device. The SORPTEST measurement uses the dependence of electrical conductivity increaseover device allows the generation of the same standardized time to generate the resulting permeation data and evaluate permeation data. This approach respects the principle of the effectiveness of barrier materials to withstand to a direct comparability of the observed permeation data across the contact with toxic substances. The other standard values are older used measurement methods. Application and software the same as in the previous case. The results of the equipment of this device enables creation of an output calibration measurements confirmed the theoretical report from measurements. It includes results of calibration considerations associated with classical conductometry, measurements and individual graphs characterizing the which in practice means that high quality results have been course of monitored standard quantities. The report also achieved. includes data concerning the chemical test that was used, its basic tabulated characteristics and toxicological data. SORPTEST method CONCLUSIONS The SOPRTEST method suitably complements the abilities of the above two methods. Its primary use can be seen to Obtaining permeation data characterizing barrier materials determine the chemical resistance of barrier materials under in accordance with European standards enables their dynamic conditions. Due to the combination of the unambiguous description not only at the national level but conductometric measurement and the measurement based also at the European level.This approach will allow on the use of QCM sensors, a unique device has been describing the barrier properties not only during their use, constructed. It enables the combination of methods and but also during their development and quality verification. used barrier materials. The permeation cells can be adapted The combination of methods for the implementation the to the particular type of measurement and the test material. knowledge of physics, sensor technologies, classical In addition to breathable and insulating barrier materials, analytical chemistry and toxicology has resulted in the the sorption properties of the bulk sorbent layers can also be construction of very modern and sophisticated measuring tested. By partial modification of the measuring system it is systems that meet the highest demands on the quality of the also possible to characterize filters of protective masks and obtained data while maintaining economic rationality. thus to study their dynamic and static sorption capacity. The source of the gas phase of the test chemical is a test tube fitted with a Peltier´s cooler (condenser). The device enables Compliance with Ethics Requirements The authors declare no conflict of interest regarding this article continuous regulation of air flow during its use within

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Article received on August 22, 2019 and accepted for publishing on October 12, 2019. CLINICAL PRACTICE

Pregnancy outcomes in a patient with Fontan circulation for single ventricle congenital heart disease and aberrant praevia placental lobe

Diana I. Voicu1, Octavian Munteanu1,2, Maria Sajin1, Adrian Dumitru1, Roxana O. Darabont2, Catalin Cirstoiu1,2, Monica M. Cirstoiu1,2

Abstract: Single ventricle heart (SVH) is a very rare congenital heart disease. However, since the introduction of Fontan's intervention, the survival rate of patients with SVH has been significantly improved, increasing the number of women who may become pregnant in this condition. It has been already reported, in isolated case presentations or in some series of patients, that the pregnacy in the context of SVH and Fontan circulation carries a very high risk for first trimester miscarriage, maternal cardiovascular or obstetric complications and for an increased rate of pre-term birth. Therefore, it is an open field of debate weather or not to allow pregnancy in this category of patients. We are presenting the case of a 22 years old patient who underwent two surgeries for SVH: a Glenn procedure when she was 2 years old and the implantation of a Fontan circuit at the age of 16 years. Since the last surgery she was under permanent internal cardiac stimulation. During the pregnancy the patient was hemodynamically stable, with a normal resting oxygen saturation and a good ejection fraction of the single ventricle. However, the evolution of this patient has become even more challenging due to an aberrant praevia placental lobe. This condition brought her to hospital for several episodes of metrorrhagia and uterine contractions. She delivered at 33 weeks of gestation by emergency Caesarian section with a good evolution until discharge, for mother and the newborn. This case is supporting the opinion that pregnancy can be tolerated without important cardiovascular events in patients with SVH and Fontan circulation, unless heart failure or cardiac arrythmia are associated. Nevertheless, the obstetrical complications remain an important problem in pregnancy overcome of patients with Fontan circulation for SVH that can be superimposed with other unfavorable conditions, like aberrant praevia placental lobe in this case. During the management of such particular clinical settings difficult decisions must be taken, including those addressing the antithrombotic therapy that will be covered in the presentation of this case. Keywords: Pregnancy outcome; Cardiovascular pregnancy complications; Fontan procedure; Delivery, obstetrics

INTRODUCTION surgical management of tricuspid atresia in 1971, the Fontan procedure has become the definitive palliative surgical A single ventricle is a heart defect in which one lower treatment of SVH. Its principal consists in the deviation of chamber does not develop resulting in a one pumping the systemic venous return directly to the pulmonary artery ventricle only and cyanosis. Single ventricle heart (SVH) without passing through a subpulmonary ventricle. defects are rare. They occur in about five out of every 100 Contemporary cohorts of surviving patients with SVH in 000 live births [1]. There have been distinguished more adulthood have undergone modified procedures compared features of SVH, but the most frequently encountered is with the original version, of which the most common is a double-inlet left ventricle [2]. Since its description for the Dacron by-pass connecting the inferior caval vein to the inferior aspect of the right pulmonary artery [3]. Since the 1 University Emergency Hospital Bucharest, Romania 2 “Carol Davila” University of Medicine and Pharmacy, Bucharest, Corresponding author: Octavian Munteanu Romania [email protected]

110 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine introduction of Fontan's intervention, the survival rate of anticoagulation should be considered in pregnant women patients with SVH has significantly improved, increasing the with Folan circulation, but balanced with the risk of bleeding number of women who may become pregnant in this [13]. condition. Overtime, patients with SVH and Fontan circulation are developing complications characterised by CASE PRESENTATION atrial arrhythmias, thrombo-embolic events and myocardial We are presenting the case of a 22 years old patient who and even hepatic dysfunction [4-7]. In particular, the lack of underwent two surgeries for SVH: a Glenn procedure in subpulmonary ventricle predisposes to a low cardiac output order to connect the superior vena cava to the right state with pulmonary blood flow dependent on adequate pulmonary artery when she was 2 years old, and the preload [8]. This fragile balance can be overcome during implantation of a Fontan circuit at the age of 16 years. Since pregnancy, when basal oxygen consumption increases by 50 the last surgery she was under permanent internal cardiac mL/min at 40 weeks, requering a 30-80% increase in cardiac stimulation. output [9]. Patients with Fontan circulation have limited resources to take over a volume overload or to increase Due to the fact that patients with Fontan circulation are adequately their cardiac output [10]. Moreover, it has been considered as having high and very high risk in pregnancy shown that pregnacy in the context of SVH and Fontan (WHO risk class III or IV) (13) the patient was clearly informed circulation carries a very high risk for first trimester of the possible complications that might occur. However, miscarriage, maternal cardiovascular or obstetric she gave her written consent for keeping the pregnancy, complications and for an increased rate of pre-term birth despite the repeated warnings of the cardiogists and [11]. Therefore, it is an open field of debate wether or not obstetritions. to allow pregnancy in this patients with SVH and Fontan The patient had a double-inlet left ventricle with an ejection circulation. fraction of 57% and a small regurgitation of the right atrio- Patients with Fontan circulation have a high rate of ventricular valve (Figure 1). Throughout of pregnancy the thromboemebolic events, recorded in about 20% of cases, patient remained hemodynamically stable, with a normal which seems to be similar in all variants of this operation, resting oxygen saturation and a good function of the left with most of the thrombs located in within the venous ventricle, including during delivery. No arhythmia have been circulation [12]. However, current guidelines of the recorded. She received antiplatelet therapy with European Society of Cardiology adressed to cardiovascular acetylsalicylic 100 mg per day, up to 31 weeks of pregnancy, diseases in pregnancy consider that therapeutic in order to prevent thromboembolic events.

Figure 1. Transthoracic echocardiography indicating the one pumping chamber with double inlet left ventricle and a part of the Glenn- Fontan circuit

Based on echographic parameters in conjunction with challenging due to an aberrant praevia placental lobe. This maternal serum biochemistry, the estimated risk for trisomy condition brought her to hospital for several episodes of 21, 18 and 13 was low. The thrombophilic work-up revealed metrorrhagia and uterine contractions. MTHFR C677T homozygote mutation associated with PAI-1 At 33 weeks of gestation the patient was admitted for 4G/5G. An infection with Ureaplasma Urealyticum was uterine contractions and vaginal bleeding. She delivered by detected, that required antibiotic treatment at 17 weeks of emergency Caesarean section for placenta praevia and pregnancy. pelvic presentation. The intervention was particularly However, the pregnancy overcome has become more difficult considering the emergency state of the procedure

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and the massive bleeding, but during delivery and heparines. After 12 days from the Cesarian section the afterwords, in the Intensive Care Unit, sufficient volume patient was suspicioned for an intrauterine residual mass replacement was added in order to maintain adequate confirmed at echography (Figure 2) for which a guided preload of Fontan circulation. The postoperative outcome instrumental revision was performed. The preterm neonate, was satisfactory, without further bleedings under weighting 1700 g, was registered with 7 on Apgar scale, but prophylactic anticoagulation with low molecular weight with good evolution until discharge.

Figure 2. Ultrasonographic aspect of intrauterine residual mass (transverse section), measuring 27 mm

All the fragments extracted underwent an extensive Figure 5. Postpartum endometritis leading to decidual abscess, histopathological analysis which concluded their placental massive inflamation and necrotic debris. HE 10x origin with postpartum endometritis and decidual abcesses (Figures 3-5).

The patient received antibiotic therapy with Carbapenems for 10 days. She was discharged on post-operative day 25 in a good condition and without cardiovascular manifestations.

Figure 3. Acute villitis and intervillositis diffuse neutrophilic infiltration of intervillous space. HE 10x

DISCUSSIONS

Since the introduction of Fontan's intervention, the survival rate of patients with SVH has significantly improved, increasing the number of women who may become pregnant in this condition. Based on a report from UK it is expected in the next decade a rise of the adults’ number with this condition by 60% [14]. It has been already reported, in isolated case presentations or in some series of patients, Figure 4. Massive inflamation and infection of the decidua. HE 20x that the pregnacy in the context of SVH and Fontan circulation carries a very high risk for first trimester miscarriage, maternal cardiovascular or obstetric complications, including perinatal haemorrhage and for an increased rate of pre-term birth or small for gestational age [15-20]. Due to these reports many authors advised against pregnancy in women with Fontan procedure for SVH. However, in some series of patients with this condition have been identified some categories of patients which might safely evolve during pregnancy, in particular those without heart failure or arrythmias and with a normal resting oxygen

112 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine saturation [11, 21-23]. The presented case is supporting the superimposed with an aberrant praevia placental lobe and opinion that pregnancy can be tolerated without important repeated episodes of metrorrhagia. In this context, the cardiovascular events in patients with SVH and Fontan obstetritions and the cardiologists involved in the circulation in selected situations. Age might have an management of this patient have chosen to give her important role in pregnancy overcome, as long as the risk of acetylsalicylic 100 mg per day, because the risk of cardiovascular complications increases with the duration of hemorrhages was too high under heparin therapy. This time from the palliative intervention. decision was taken according to the single multicenter, randomized, controlled study which has revealed no Antepartum haemorrhage complicates 2–5% of pregnancies, differencies in the thrombosis rate of Fontan patients of which approximately one‐third are caused by abnormal between the use of aspirin and heparin/warfarin [27]. placental implantations [24]. Placenta previa is characterised Moreover the postpartum period was complicated by the by the partial or total covering of the internal os [25]. In expulsion of an intrauterine mass. The extracted fragments certain situations, like in the presented case, only an proved to be placental and infected. Following the prompt aberrant lobe may have this aberrant location. Placenta guided instrumental revision and the antibiotic cure with praevia is an obstetric condition that can induce massive Carbapenems, septic complications that could particularly antepartum vaginal bleeding. The risk is even higher when affect Fontan's circulation have been avoided. accreta and percreta invasion are occuring [26]. It has been reported that the incidence of placenta praevia and CONCLUSIONS abnormal placental invasion has increased in the last years, probably as a result of the large number of C-sections This case is supporting the opinion that pregnancy can be worldwide [25]. However, in our case the patient was tolerated without important cardiovascular events in primiparous and did not undergo any other previous surgical patients with SVH and Fontan circulation, unless heart interventions, but the risk of antepartum bleeding was failure or cardiac arrythmia are associated. Nevertheless, the augmanted by the cardiovascular condition of this patient. obstetrical complications remain an important problem in Data accumulated over the two past decades are indicating pregnancy overcome of patients with Fontan circulation for that the maternal and fetal obtetrical complications are high SVH that can be superimposed with other unfavorable in patients with Fontan circulation, even in those with an conditions, like aberrant praevia placental lobe in this case. estimated low score of cardiovascular complications during During the management of such particular clinical settings pregnancy: a rate of about 50% for obsterical complications difficult decisions must be taken, including those addressing and between 70-80% of preterm delivery [11]. the antithrombotic therapy, that must be carried on by a multidisciplinary team in order to reduce as much as possible Patients with Fontan procedure have a high-risk of the maternal and fetal morbidity and mortality. thromboembolic events. In pregnancy this risk can be augmented. For this reason our patient had an indication of therapeutic anticoagulation with low molecular weight Acknowledgment heparine [13], but her pregnancy overcome was All authors have participated equally in developing this study.

References:

1. Bernstein D. Cyanotic congenital heart disease. In: Behrman RE, 5. Diller GP, Giardini A, Dimopoulos K, et al: Predictors of Kliegman RM, Jenson HB, Stanton FB, editors. Nelson Textbook of morbidity and mortality in contemporary Fontan patients: Results Pediatrics. 18th ed. Philadelphia: WB Saunders; 2008. 1918-30. from a multicenter study including cardiopulmonary exercise testing 2. Weigel TJ, Driscoll DJ, Michels VV. Occurrence of congenital in 321 patients. Eur Heart J. 2010; 31:3073. heart defects in siblings of patients with univentricular heart and 6. Stephenson EA, Lu M, Berul CI, et al: Arrhythmias in a tricuspid atresia. Am J Cardiology, 1989; 64: 768-71. contemporary Fontan cohort: Prevalence and clinical associations in 3. Webb GD, Smallhorn JF, Therrien J, Redington AN. Congenital a multicenter cross-sectional study. J Am Coll Cardiol. 2010; 56:890. Heart Disease. In: Mann DL, Zipes DP, Libby P, Bonow RO, editors. 7. Wu FM, Ukomadu C, Odze RD, et al: Liver disease in the patient Braunwald’s Heart Disease. A textbook of cardiovascular medicine. with Fontan circulation. 10th ed. Philadelphia: Elsevier Saunders; 2015. 1392-1445. 8. Congenit Heart Dis. 2011; 6:190. 4. Fontan F, Kirklin JW, Fernandez G, Costa F, Naftel DC, Tritto F, 9. Gewillig M. The Fontan circulation. Heart. 2005; 1: 839-46. et al. Outcome after „perfect” Fontan operation. Circulation. 1990; 10. Hunter S, Robson SC. Adaptation of maternal heart in 81: 1520-36. pregnancy. Br Heart J. 1992; 68: 540-3.

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11. Le Gloan L, Mercier LA, Dore A, Marcotte F, Mongeon FP, circulation: a multicentric observational study. Int J Cardiol, 2015; Ibrahim R, et al. Pregnancy in women with Fontan physiology. Expert 187: 84-89. Rev Cardiovasc Ther. 2011; 9: 1547-56. 21. Bonner SJ, Asghar O, Roberts A, Vause S, Clarke B, Keavney B. 12. Arif S, Chaudhary A, Clift PF, Morris K, Selman TJ, Bowater SE et Cardiovascular, obstetric and neonatal outcomes in women with al. Pregnancy outcomes in patients with Fontan circulationand previous Fontan repair. Eur J Obstet Gynecol Reprod Biol. 2017; 219: protocol for a risk-scoring system: single centre experience. J Cong 53-56. Cardiol. 2017; 1: 10. 22. Pundi KN, Pundi K, Johnson JN, Dearani JA, Bonnichsen CR, 13. Rosenthal DN, Friedman AH, Kleinman CS, Kopf GS, Rosenfeld Phillips SD, et al. Contraception practices and pregnancy outcome in LE, Hellenbrand WE. Thromboembolic complications after Fontan patients after Fontan operation. Congenit Heart Dis. 2016; 11:63– operations. Circulation. 1995; 92: 287-293. 70. 14. Regitz-Zagrosek V, Roos-Hesselink JW, Bauersachs J, 23. Zentner D, Kotevski A, King I, Grigg L, dU Y. Fertility and Blomström-Lundqvist C, Cıfkova R, De Bonis M. 2018 ESC Guidelines pregnancy in the Fontan population. Int J Cardiol. 2016; 208:97–101. for the management of cardiovascular diseases during pregnancy. 24. Cauldwell M, Steer PJ, Bonner S, Asghar O, Swan L, Hodson K, et Eu Heart J. 2018; 39: 3165-31241 al. Retrospective UK multicentre study of the pregnancy outcomes 15. Coats L, O'Connor S, Wren C, O'Sullivan J. The single-ventricle of women with a Fontan repair. 2018; 104: 401-406. patient population: a current and future concern a population-based 25. Oppenheimer LW, Farine D. A new classification of placenta study in the North of England. Heart. 2014;100: 1348–53. previa: measuring progress in obstetrics. American Journal of 16. Canobbio MM, Mair DD, van der Velde M, Koos BJ. Pregnancy Obstetrics & Gynecology. 2009 Sep 1;201(3):227-9. outcomes after the Fontan repair. J Am Coll Cardiol. 1996; 28:763– 26. Yang JI, Lim YK, Kim HS, Chang KH, Lee JP, Ryu HS. Sonographic 7. findings of placental lacunae and the prediction of adherent 17. Hoare JV, Radford D. Pregnancy after fontan repair of complex placenta in women with placenta previa totalis and prior Cesarean congenital heart disease. Aust N Z J Obstet Gynaecol. 2001; 41:464– section. Ultrasound in Obstetrics and Gynecology. 2006 Aug 8. 1;28(2):178-82 18. Drenthen W, Pieper PG, Roos-Hesselink JW, van Lottum WA, 27. Klar M, Michels KB. Cesarean section and placental disorders in Voors AA, Mulder BJ, et al. Pregnancy and delivery in women after subsequent pregnancies–a meta-analysis. Journal of perinatal Fontan palliation. Heart. 2006; 92:1290–4. medicine. 2014 Sep 1;42(5):571-83. 19. Chugh R. Management of pregnancy in women with repaired 28. Monagle P, Cochrane A, Roberts R, Manlhiot C, Weintraub R, congenital heart disease or after Fontan procedure. Curr Treat Szechtman B, et al. A multicenter, randomized trial comparing Options Cardiovasc Med. 2013; 15: 646-62. heparin/warfarin and acetylsalicylic acid as primary 20. Gouton M, Nizard J, Patel M, Sassolas F, Jimenez M, Radojevic J, thromboprophylaxis for 2 years after the Fontan procedure in et al. Maternal and fetal outcomes of pregnancy with Fontan children. J Am Coll Cardiol 2011;58:645-51.

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Article received on April 2, 2019 and accepted for publishing on August 28, 2019. VARIA

A simple approach for risk stratification in the plan of exercise in older adults

Mehmet I. Naharci1, Oznur Buyukturan2, Ilker Tasci1,3

Keywords: aged; exercise; risk

Letter to the Editor range 0-6) [4, 5].

The benefits of physical activity (PA) in decreasing morbidity 1. Low-risk individuals: Those with no or a single chronic and increasing life span have been proven at all ages, even disease, no cognitive impairment (MMSE score ≥ 28), no in later life [1]. In addition, PA not only improves the quality functional impairment (KATZ score ≥ 5), and no history of of life but also contributes to maintenance of social and falls in past 12 months. psychological well-being [2]. 2. Medium-risk individuals: Those with two or more chronic There are a lot of specific PA programs for adults aged over diseases, mild cognitive impairments (MMSE score ≥ 24 and 65 years, with various recommendations and ≤ 27), mild functional disabilities (e.g., using a cane or implementations. However, adherence and persistence in walker) (KATZ index score 3 or 4) or positive history of falls PA are generally not at desired levels in these individuals [3]. without injury in past 12 months. While numerous causes can decrease the compliance in PA, 3. High-risk individuals: Those with two or more chronic a set recommendations specific to person’s condition and diseases, significant cognitive impairment (MMSE score ≤ needs can successfully be used to individualize the exercise 23), advanced functional disability (KATZ index score ≤ 2), or plan in older adults. Comorbid conditions and functional positive history of falls with injury in past 12 months. impairment can reduce engagement in PA. Therefore, these should be reviewed in the first step of a systematic approach Table 1 suggests some exercise recommendations for the by the physicians, especially in the primary care setting. risk groups [6].

In order to improve the efficacy of PA plan offered to an Encouraging older adults to participate in PA is an important older adult, we intended to test a novel approach in small issue at the primary care level. There are still problems to sample of community dwelling elderly. The proposed tool classifies the subjects in three categories based on a geriatric 1 University of Health Sciences, Gulhane Faculty of Medicine & evaluation that includes review of chronic diseases and 12- Gulhane Training and Research Hospital, Division of Geriatrics, month fall history, assessment of cognitive status [assessed Ankara, Turkey by Mini-Mental State Examination (MMSE), range 0-30], and 2 Ahi Evran University, School of Physical Therapy and screening for the functional status (assessed by KATZ index, Rehabilitation, Kirsehir, Turkey 3 University of Health Sciences, Gulhane Faculty of Medicine & Corresponding author: Mehmet Ilkin Naharci Gulhane Training and Research Hospital, Department of Internal [email protected] Medicine, Ankara, Turkey

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constitute an activity plan in this population. Therefore, need to be assessed to recommend a suitable exercise plan comorbidities and physical activity status of older adults and achieve reasonable outcomes.

Table 1. Examples of exercises according to the risk groups Risk groups Exercise types Frequency Intensity

• Aerobic • ≥ 30 min/day and ≥ 5 days/week • Moderate to high Low • Strength • ≥ 2 days/week • Moderate to high • Flexibility/Balance • ≥ 10 min and ≥ 2 days/week • Moderate to high • Aerobic • ≥ 30 min/day and ≥ 5 days/week • Low to moderate Medium • Strength (In selected subjects) • 1-2 days/week • Low to moderate • Flexibility/Balance (In selected subjects) • 1-2 days/week • Low to moderate • Active, active assistive, and passive (an • 20-30 min/day and ≥ 3 days/week • Low High individualized PA plan should be created) PA, physical activity. Circle or check the boxes of the conditions that apply. “Low Risk” column refers to only individuals in Low Risk; “Medium Risk” column refers to only individuals in Medium Risk; “High Risk” column refers to only individuals in the High Risk (Please see the text).

Disclosure statement: No potential conflict of interest was reported by the authors.

References:

1. Tasci I. Persistent physical activities in leisure time over decades practical method for grading the cognitive state of patients for the improve late life CVD markers. Atherosclerosis. 2018; 269:256-257. clinician. J Psychiatr Res. 1975; 12:189-198. 2. Penedo FJ, Dahn JR. Exercise and well-being: a review of mental 5. Katz S, Ford AB, Moskowitz RW, et al. Studies of illness in the and physical health benefits associated with physical activity. Curr aged: the index of ADL: a standardized measure of biological and Opin Psychiatry. 2005; 18:189-193. psychosocial function. JAMA. 1963; 185:914-919. 3. Nelson ME, Rejeski WJ, Blair SN, et al. Physical activity and 6. Morey MC. Physical activity and exercise in older adults. In: public health in older adults. Recommendation from the American Givens J (Ed), UpToDate, 2018. Retrived March 5, 2019, from College of Sports Medicine and the American Heart Association. https://www.uptodate.com/contents/physical-activity-and- Circulation. 2007; 116(9):1094-1105. exercise-in-older-adults 4. Folstein MF, Folstein SE, McHugh PR. “Mini-mental state”: a

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Article received on May 6, 2019 and accepted for publishing on August 28, 2019. VARIA

Can we still manage the relationship with patients?

Liana Manolache1

Eighteen years ago, I started my out-patients’ experience as Time is also a burden for the doctor, because the real time specialist dermatologist. I worked from the very first day for consultation has dramatically reduced in favor of with National Health Insurance System. It was a chance to bureaucratic issues. Official papers seem to be more build a huge experience (more than 120,000 visits since important than people. It is a constant effort to remain then). Over time, relationships with patients have changed, emphatic and to really listen to patient’s history. The stories influenced by many factors. To preserve good relationships that we are ignoring for lacking time could give us important we have to reflect and to use different approaches. clues for diagnosis, approach, could enforce the relationship and give trust, could relief the worries. In 2001, my stamp and my signature were enough for a consultation. The cell phone era was at the beginning. Over “You are the 5th dermatologist I am seeing”- a young woman the years, the process has become more and more told me the other day. I looked at her: she was around 20, complicated. Today, if the computer, internet, different some pimples on her jaw (not too many) and some tiny scars types of applications, software, other devices (printer, card on her forehead covered by a lot of makeup. Not quite a reader, etc.) are not working, my knowledge becomes good start for a relationship. Her expectations were not absolutely necessary, but not sufficient for a consultation. fulfilled by visiting the other doctors. Maybe the So, the doctor is now, totally dependent on IT issues. Besides expectations were not realistic, maybe she was fed up trying instant information, cell phones induce a lack of intimacy. different things. “How can I propose something new or Everyone could reach you everywhere, every moment. You miraculous when she tried probably “everything”? - I asked are almost “forced” to give advice on whatsapp, e-mail, myself. I spent at least half an hour discussing about acne, messenger and people expect you to answer on-site, therapeutic options and adjusting the expectations to our otherwise you are not “reachable”. limits. It was more a counseling session than a dermatologic consultation. Time is the first pressure that we are feeling. Patients want and expect a consultation as soon as possible even you have Today, patients are coming in our office very informed. They appointments for the next few weeks. I try my best not to “know” the diagnosis and sometimes even the treatment push later than 10 days, if it is a new ordinary consultation. they want or need. After “Dr. Google” you can be a second Sometimes, it is possible for me to see the patient on-site (a opinion. Dermatology is underestimated even by other child, an emergency, pregnant women, etc.). In the physicians that feel competent enough to prescribe literature, time for appointment could vary from 7 working medication inducing iatrogenesis [6]. So, what to expect days in Brazil [1], to median 41 days in Canada (Ontario) [2] from patients? It is a good thing to have an informed patient. or median 45 days in US (Pennsylvania) [3] to a certain It will save you a lot of time and energy in expanded dramatic situation waiting list of 57 weeks in a unit in UK [4]. explanations. But, there is also a lot of incorrect or The number of consultations tends to increase. It is not only a perception, but a reality, revealed also by a French study 1 Dali Medical, Bucharest, Romania (21% increase from 2000 to 2010) [5].

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misunderstood information, prejudices from other’s Debate Series regarding medical students’ selection [9]. experience shared on forums, incorrect auto-diagnosis or Another pressure point is the constant fear of errors. With even incorrect auto-medication. It is our duty to listen and all the efforts of protocols for reducing the risk of mistakes to correct, as much as we can. Patients have to be taught or misconducts, unfortunately there are lots of gaps and how to choose important information from the constant debates. The fear of error and malpraxis leads to excess of “soup of news feed”. medication and investigations, sometimes too expensive The flux of information is huge, also for doctors as for and useless. patients. Thousands of opinions, articles, and new Doctors should not be scared by the abundance of products, approaches invade our space daily. A good selection criteria instruments, techniques, aggressively promoted. They have for useful, relevant data is absolutely necessary. to be more flexible, more intuitive and more eager to try, A “happy” patient seems to be the one with a certain making personal experience and not taking results for diagnosis, with clear treatment plan, including explaining granted. Many of these products will not pass the test of side effects and with a contact number in case of recurrence time, even they are presented as “miraculous”. Sometimes, [7]. The access of any information being so instant, patients patients’ needs are the trigger for experimenting new expect a rapid response of the treatment. The result has to methods and push us to progress faster. be now and definitive. The pressure of quick response is not Not only patients are in a rush, doctors too. The race for EMC very subtle, the doctor feeling it as a burden. It takes time to points is making the doctor more informed, but we have to explain and to understand the progression of chronic be careful not to become too superficial. Even a doctor is illnesses. getting a diploma after a “3 days course”, it doesn’t mean Sometimes, the expectations are to get well with any that he/she will be an expert in that field, not even treatment, eventually without any changes of their life style, competent. It will take a lot of time and energy to really get even when doctor explains that some habits could aggravate the expertise, that course being only the very first step on or maintain the lesions. Taking responsibility for some the road. The “diplomas wall”, real or virtual is a false goal. adjustments is an important part to discuss with patient, as In the end, the real skills are more important than a sublime part of therapeutic approach. Paternal, omniscient doctor’s image and it will take time to get them. image is no longer in actuality and patient is an important Sometimes, vanity makes the teamwork harder. This will be part of the relationship. Instead of an “infantile” patient, unproductive for both doctors and patients. It is not a shame coming for any transitory rash or any mosquito bite, it is to refer the patient to an expert on a field when you feel that better to “grow” him/her as a self-confident “partner”. The you have reached a limit. new and healthy bond has to transform patient from the passive, sometimes passive-aggressive role, into an People are hardly trying to change our state from patient to assumed, informed, pro-active one. Cooperation is the key client, that new status being debatable. Being a client means of healthy relation. Patient has the right to ask for to take some responsibilities as in a contract. That is the explanations, to discuss therapeutic options, to refuse good part. But, fortunately, remaining a doctor means more treatments. Patients have opinions that have to be than providing services. Practicing medicine is a state of respected and sometimes corrected if they are distorted. On knowledge, art, experience, intuition, with magic touches the way to get therapeutic alliance and long-term sometimes. cooperation, the doctor-patient relationship has to be Fortunately, with all the changes during the last years, personalized, giving value to it. This kind of relationship will dermatologists seem to remain satisfied with their specialty. make the difference in the end and even the direction is to A recent Mexican study shows that 93% of dermatologists involve more high tech and robots. Face-to-face relationship (with an average of 16 years of practice) were happy with will not be replaced by anything and it will be highly their professional life, more than 98% choosing it once again appreciated after the “speed” condition passes. More social [10]. skills are often required, doctors not being known as the best communicators. Sometimes, doctors are not aware of Maintaining certain levels of professional and personal patients’ perception regarding communication [8]. I have got happiness, keeping informed and open-minded, avoiding my social skills working day by day, no special courses were burn-out, trying to fulfill patients’ expectations, doctors are made during faculty, unfortunately. “We need fewer not in a battle, but in strong alliance with patients. memorizers and more thinkers and communicators in modern medicine” is the most recent conclusion of Canadian

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References:

1. Miot HA, Miot LD. Time needed to schedule dermatological 6. Fuentes-Suárez A, Domínguez-Soto L. [Underestimation of consultations in Brazil. An Bras Dermatol. 2013 Jul-Aug;88(4):563-9. dermatology and the efforts of dermatologists to benefit patients]. 2. Neimanis I, Gaebel K, Dickson R, Levy R, Goebel C, Zizzo A, Rev Med Inst Mex Seguro Soc. 2015 Sep-Oct;53(5):536-7. Woods A, Corsini J. Referral processes and wait times in primary 7. Harun NA, Finlay AY, Piguet V, Salek S.Understanding clinician care. Can Fam Physician. 2017 Aug;63(8):619-624. influences and patient perspectives on outpatient discharge 3. Jayakumar KL, Samimi SS, Vittorio CC, Micheletti RG, Steele KT, decisions: a qualitative study. BMJ Open. 2017 Mar 6;7(3):e010807. Ogunleye TA, Richardson V, Chiesa ZC, Rosenbach M. Expediting 8. Burt J, Abel G, Elliott MN, Elmore N, Newbould J, Davey A, patient appointments with dermatology rapid access clinics. Llanwarne N, Maramba I, Paddison C, Campbell J, Roland M The Dermatol Online J. 2018 Jun 15;24(6). Evaluation of Physicians' Communication Skills From Multiple 4. Appleby A, Lawrence C. From blacklist to beacon, a case study Perspectives. Ann Fam Med. 2018 Jul;16(4):330-337 in reducing dermatology out-patient waiting times. Clin Exp 9. Steeves JM, Petrie DA, Atkinson PR. CJEM Debate Series: Dermatol. 2001 Sep;26(6):548-55. #BetterSelection - Medical school acceptance tests select the wrong 5. Mirkamali A, Ingen-Housz-Oro S, Valeyrie-Allanore L, Bouvresse doctors: We need fewer memorizers and more thinkers and S, Duong TA, Chosidow O, Wolkenstein P. Dermatological communicators in modern medicine. CJEM. 2018 Jul;20(4):495-500. emergencies: a comparative study of activity in 2000 and 2010. J Eur 10. Fierro-Arias L, Simón-Díaz P, Ponce-Olivera RM, Arenas-Guzmán Acad Dermatol Venereol. 2013 Jul;27(7):916-8. R. Dermatologists happiness and satisfaction. Gac Med Mex. 2018;154(1):26-35.

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Article received on September 4, 2019 and accepted for publishing on October 28, 2019. VARIA

The safety of genetic reserves in bioterrorism from the passive defense perspective

Hadi E.G. Ghaleh1, Mohsen A. Farajzadeh2, Mosa Jafari3, Nader N. Shadbad1

Abstract: Several studies have shown that some countries are among the main users of structural, molecular and physiologic information of human, animal, plant, and microorganisms cells (genetic and biological reserves) against living of organisms, especially humans. This method used as a mechanism for removing their rivals and opponents to achieve their goals. This descriptive-analytical study was designed to define the concept of bioterrorism, passive defense, and genetic resources and its historical development process, with emphasis on efforts to control these types of threats over time. In the present review, a wide range of literature through the search on Google Scholar, PubMed, Medline, and Scopus databases, extracted without regard to time constraints and focusing on the keywords listed above with three valid criteria and new sources of resources. The explanation of the material was based on the data obtained from the review of the sources and based on the inferences of the researchers from them. Given the importance of genetic information and the lack of clear prospective research in this area, the confidentiality of genetic data has been particularly respected among researchers. Eliminating data security can lead to unplanned advertising and costs. Therefore, the existence of laws and protective measures against coping with the misuse of enemies from genetic information is more than other country's prior demand. Keywords: bioterrorism, passive defense, genetic resource, threat

INTRODUCTION humans, animals and plants with a prior intent to terrorize, threaten, or coerce a state or group of people. Acting or Today, the hostile use of science and technology has led fulfilling political or social demands [3]. But with the astray in the field of medical and human health that has led advances in basic science and medicine, especially in the to the emergence of biological weapons for the purposes of field of molecular biology, genetic engineering, biological assassinations [1]. The definition of bioterrorism is biotechnology, synthetic biology, bioterrorism has recently the misuse of microbial, viral, parasitic agents or their taken on a new meaning [4]. In the meantime, we should not products for the purpose of intimidating or killing humans overlook the widespread developments of the biological and destroying livestock or plants [2]. According to the sciences in the past few decades and the widespread International Police Bioterrorism definition, it is the release scientific ability to manipulate and recombine genes and of biological or toxic agents with the intent to kill or harm identify molecular pathogens to increase the virulence of deadly toxins [5]. The possibility of increasing drug 1 Applied Virology Research Center, Baqiyatallah University of resistance and antibiotic resistance and increasing the Medical Sciences, Tehran, Iran epidemic of disease has led hostile military researchers to 2 Marine Medicine Research Center, Baqiyatallah University of research, procure, and produce hazardous agents in Medical Sciences, Tehran, Iran 3 Faculty of Management, Tehran Central Branch, Islamic Azad Corresponding author: Nader Nezamdost Shadbad, Msc University, Tehran, Iran [email protected]

120 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine contravention of international laws and conventions diseases such as cancer, cardiovascular disease, genetic prohibiting the development of biological weapons. Of diseases, diabetes, neurological disorders, etc. are a rich course, they do so with justifications such as trying to make source for future research [13]. A collection of samples and a vaccine or knowing the causes and pathogens or gathering genetic information related to clinical trials provides very scientific information [6]. On the other hand, genetic good opportunities for analyzing genetic factors and helping resources or hereditary flora and fauna are one of the most to respond to treatment. Genetic information includes valuable national wealth and basic resources of any country. information on genes and genetic characteristics [4]. Considering that some countries are a rich country due to What are the goals of Genetic Banks? fossil fuel oil and gas reserves, the abundance of unique plant and animal genetic resources is also a major and Genetic banks generally pursue two basic goals: strategic asset of countries and unfortunately has been  Storage and storage of biological substances such as neglected [7]. The economic function of genetic reserves is ؛ blood, bone marrow, etc. for future therapeutic purposes greater than that of other reserves in the countries, and these valuable reserves should be exploited. The genetic  Storing and keeping samples and genetic information of resources available in the countries are very rich and we can different individuals to identify disease-causing genes [14]. replace them with other resources and if we do not use them Uses of biological and genetic samples: we will lose them in many ways. Even today in the world it is 1. Diagnosis of genetic diseases and clinical care. believed that genetic reserves in compared of gold and oil 2. Genetic epidemiological studies. reserves are also more valuable and strategic [8]. Genetic 3. Genetic mapping. reserve information is therefore the most important 4. Applied Studies. strategic part of a country that may be exploited if recently 5. Development of diagnostic and therapeutic goals. based on research conducted in the Netherlands and Japan, 6. Drug design and pharmacogenomics. a new and modified strain of influenza virus has been studied 7. Gene Therapy. for its respiratory transmission studies in many reflective laboratory animals [9]. And raised concerns among the Genetic banks depend on the following organizations for scientific and security community about the potential for various applications, including: exploiting it in the production of dangerous biological 1. Forensic Medicine Organization; agents. Finally, after long discussions and agreeing to publish 2. Army; it, the relevant researchers agreed that part of the technical 3. Universities and study centers; knowledge and techniques of gene manipulation and 4. Business Organizations [5]. production factors remove from this article [10, 11]. Biodiversity

METHODS One of the major threats in the discussion of threats is BioDiversity. The discipline is currently one of the safest This descriptive-analytical study explores the concept of biology disciplines in the world, and it is witnessed that there bioterrorism, non-performing defense and its genetic is no student admission to the field from third world reserves and its historical developmental process, with countries. However, most of the funding in biology is emphasis on efforts to control these threats over time. In devoted to this sector. In fact, the diversity within and this review study, a wide range of sources were extracted by between organisms in a region is considered to be the searching Google Scholar, PubMed, Medline, and Scopus biodiversity of that region; the high biodiversity means the databases without considering time constraints and focusing high diversity of genes that increases production, enhances on the keywords listed with three valid criteria and the product quality, and increases pest resistance [15]. This newness of the sources. Explanation was based on the data biodiversity has evolved over very long periods of time, and obtained from the literature review and based on the any human manipulation of it will alter and destroy the researchers' inferences. biological cycles of the creatures in an ecosystem that will have irreversible effects. For example, paddy fields in the THEORETICAL FOUNDATIONS north of the country have been destroyed by the Human Genetics Association, an organization called the introduction of fossils of Azolla by a Japanese researcher Genetic Bank, announced that it would collect human from Japan (16). Azola ferns in Japan make the rice genetic samples and data and store them for future scientific unnecessary for nitrate fertilizer and multiply its production research [12]. Genetic specimens obtained from people with efficiency, but there is an insect that controls the population

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by eating ferns. The inability to prevent the growth of this agriculture to the conditions ahead is of particular fern in the northern fields and in fact the biomass of that importance and the need for a new variety that can with region is different from the northern region and the lack of stand these challenges has been strongly considered. It is attention to the biodiversity of each region has had a major essential not only to reduce hunger but also to strengthen impact on domestic production [17]. Biodiversity is world food security in the medium and long term [24]. important in several respects, which is limited two resean: Biodiversity Conservation First, high biodiversity leads to higher production. Today, the Biodiversity loss is one of the most important environmental issue of food security and production is one of the most concerns today. According to some reports, as the current important issues in the world. The annual income of one trend continues, half of the plant species will be at risk of Chinese medicinal plant, JinSingh, is about half of some extinction. Among the endangered species are wild countries annual oil sales. This high biodiversity can lead to ancestors of crops, which carry valuable traits that can be more diverse crops as well as increased production [18]. transferred to crops [25]. It is estimated that six percent of Secondly, biodiversity is considered a source of human wild cereal ancestors (wheat, maize, rice, sorghum, etc), 18% knowledge today. Biotechnology and future work on genes of legume species (wild ancestors of beans, chickpeas and with insufficient genome resources, such as having proper lentils) and 13 percent of vegetable and safflower species, knowledge of oil, but lacking oil storage [19]. India is the including potatoes, tomatoes, eggplants and peppers, are at largest country to make huge profits from the blood trade. risk. Clever use of genetic diversity in breeding activities can However, due to the lack of proper laws in some country, be significantly effective in protecting the environment many plant, animal and microbial species go abroad for animal varieties that are susceptible to disease can reduce sequencing [20]. the need to use harmful drugs and increase production efficiency[26]. More varieties of plants with more branching EFFECTIVENESS OF GENETIC RESOURCE CENTERS ability can compete better with weeds and reduce the ACTIVITIES IN THE ECONOMIC AREA amount of herbicide needed. Drought-tolerant plants help conserve less water, varieties with deeper root systems can Providing food security contribute to greater soil stability and stability and varieties Food security is a very complex issue, not only involving the with lower soil nutrient intake reduce fertilizer requirement production and processing of food products but also its [27]. accessibility for people to maintain a healthy and active life. Improving nutrition adequacy Diversity is the basis of food security and today's production supplier and the prerequisite for ensuring tomorrow's Genetic diversity not only guarantees stability and stability production in the face of the rapidly changing world [21]. in providing adequate amounts of feed (protein and energy), Achieving food security and ending hunger is one of the but also plays a key role in ensuring its quality. Diversity, great challenges facing the world in the coming centuries. which is a direct product of diversity in crops, nutritionists Population growth, resource depletion, and habitat say it is optimal and the presence of vital nutrients (vitamins degradation add to the deterioration [22]. The greater and minerals) can be enhanced by choosing the right genetic diversity of genetic resources in gene banks and their diversity [28]. New varieties can, through breeding, have availability through an effective protection system helps higher nutritional quality in terms of vitamins, iron and other ensure food security at lower costs. This will help provide the essential nutrients and have higher protein quality and less genetic feedstock for breeding activities to produce diverse antifouling or toxic substances [29]. and nutritious foods and increase access for poorer Poverty Reduction populations to healthier, more nutritious foods to combat malnutrition [23]. Genetic diversity is one of the most powerful weapons to combat hunger and poverty. Agriculture is an economic Climate Change Adaptation infrastructure in many countries, and is a potential source of Climate change will put unprecedented pressure on our economic growth for developing countries agricultural ability to produce food, and these effects will be especially growth is more beneficial for economic development and severe in developing countries. Research has shown that the poorer and by providing affordable food, This benefits more trend of rising temperatures will continue in the next few than 70 percent of the world's poor, living in rural areas, And centuries and the conditions for agriculture will be very those people whose agriculture is their main livelihood [30]. different from the current situation, therefore, adaptation of Agricultural insurance can play a key role that requires a

122 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine range of reforms, including the cultivation of higher value NEW SCIENCE AND TECHNOLOGY AND NEW BIOLOGICAL crops, the promotion of value-added activities, For example, FACTORS improved product processing, expanding market access and Non-lethal biological agents lowering prices through increased production, higher processing and marketing efficiency[31]. The basis of all The some Department of Defense is already increasingly these possible solutions is genetic diversity, a variety that turning to non-lethal weapons (NLWs), these weapons enables the farmer and breeder to produce higher yield designed specifically, the amount of deadly and serious varieties and higher quality. They can produce varieties that damage to the low end and damage to the city, property and are tailored to specific processing methods and more the environment and they don’t harm the environment portable and maintainable [32]. They can also produce some countries, of course, have been using crippling varieties that are resistant to pests and diseases and weapons for a long time. But these non-lethal weapons of environmental stresses, which protect the crop from biological origin can have different effects [39]. damage and reduce the risk. The role of agriculture in Non-microbial biomaterials in cells and organs of various combating poverty is very complex, but it cannot reach its animals (plants and animals) and humans that do not fall into potential without the proper exploitation of genetic diversity the category of microorganisms and biological toxins, but [33]. despite their properties transmitted through an aerosol or Sustainable Agriculture Guarantee through Respiratory and mucosal tissues absorbed If used in wars or bioterrorist operations, they can cause clinical signs Harnessing the genetic diversity in plant and animal and symptoms in humans, or cause economic damage to a resources is an important strategy through which farmers country's strategic plants and animals [40]. can develop their farming systems with the least environmental impacts. The global off-site conservation These include a variety of brain regulators (Bio regulators) system is a key component of the effort to conserve these and some gastroenteritis-producing enterotoxins, as well as resources and make them available to farmers in all a variety of substances that affect the human senses, such as countries [34]. In the 21st century, agriculture faces many non-lethal bioweapons, calmative and malodorants Agents, challenges, as it needs to provide more food to meet the etc. to be [41]. Other non-lethal biological agents can affect needs of a growing population living mainly in urban areas, various materials and military and civilian equipment and while dependent on the shrinking rural labor force [35]. cause them to corrupt, destroy or decompose in addition to causing economic damage to the country but also disrupting In addition, climate change is a threat that could increase the the political affairs and activities of the military. And number of hungry people in the future and pose more becomes civilian the existence of oncogenes (carcinogenic complex agricultural challenges. While the effects of climate genes) is no exception [42]. change are slowly becoming apparent, the future impacts will be significant if not taken properly [36]. Increased Recombinant and Synthetic Agents exploitation of plant genetic diversity is essential to address By identifying the genome of different microorganisms or these threats and future challenges. Therefore, despite such identifying a part of one's genome structure and using strategic effects on genetic resources, the protection and genetic engineering techniques of microbes' genes to security of such centers require special attention to prevent increase their pathogenicity such as drug resistance, enemy misuse [9]. pathogenic genes, carcinogenic genes, and altered escape structure. The new recombinant germs are made from the RECENT ADVANCES IN HUMAN GENETIC STUDIES diagnosis, vaccine-resistant that has created a new, Human genetic studies have provided cognitive insights and recombinant weapon with special features [43]. mental disorders are the genetic basis of several inherited Ethnic race pathogen agents disorders, such as Down syndrome and Tay-Sachs disease, and other associations such as the role of APOE in The susceptibility or resistance of different human races to Alzheimer's disease are still extensively studied [37]. different diseases, the abundance of hereditary diseases in However, despite advances in understanding the human specific human races, as well as research to discover and genome, there are concerns about the privacy of genetic identify infectious substances and agents that can cause a data and possible discrimination resulting from its particular race are bioterrorist efforts. Recently disclosed disclosure, and there is incomplete monitoring of genetic documents indicate a widespread program in the former of testing [38]. some countries racist regime to provide specific infectious

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black agents that could be used against those of the New achievements and development of various programs predominantly population [8,9]. for the prevention and control of bioterrorism factors have been made, including: Interfering RNA iRNA and SiRNA or Oligo Antisense • The existence of new vaccines preventing biological agents Technology • Antioxidants effective against toxicological biological Interfering RNAs silence genes, and are used in medicine by agents inactivating genes that cause diseases including cancer, viral • The presence of potent antibiotics as drug prophylaxis in infections and other diseases. Gene silencing methods are preparation, highly needed because of their ability to treat human • Developing a national, provincial, municipal and health diseases [44]. What puts the repressor RNA above all other care crisis response plan competitors is its proprietary function in eliminating only • Provide vaccine equipment, medicines and other items and inappropriate genetic information and that other healthy prepare for delivery genes are immune to its effects[45]. Identification and • Training as the most important element of preparation synthesis of molecules that inhibit gene activity. This small • Using virtual training systems to train all forces fragment of RNA binds to or suppresses gene activity by • Utilizing the ability of information networks to coordinate binding to its complement locus in the process of gene and execute maneuvers at the confrontation stage, new activity. The technology is widely used in medicine as a technologies play an important role in the control and crisis vaccine. Also, using nanotechnology can produce bio- management of modern war events such as: particles that are designed to stop vital activities of humans • New systems of rapid alert the threat of a new war quickly or other organisms, which can range from minor damage alerts crisis management and non-operational defense include a serious injury [46]. centers. • The use of new technologies and new materials for Genetic Engineering disinfection that eliminates a large number of casualties, Genetic engineering of food, one of the branches of equipment and facilities without any adverse effects. biotechnology, involves targeted alterations to the genome • Modern information systems are rapidly bringing forces of plants or animals. Most foods that are consumed today and people into crisis. are either genetically modified to be termed GMOs or • Crisis management operates much faster due to new contain components derived from genetic engineering information technologies. technology [47]. Genetic engineering and plant manufac- • Use modern systems of field hospitals that can be installed turing and production of plants resistant to pests, diseases in the shortest possible time and have the necessary facilities and the need for the use of dangerous pesticides have made to help and treat modern war casualties. a change in agriculture that is comparable only to the Green • New medicines for treatment of modern warfare agents Revolution. Genetic engineering may also be engineered and new therapies based on bioterrorism goals, which in turn require • The use of mobile laboratories and mobile vehicles to monitoring [48]. quickly detect and engage in modern warfare at the scene • New management and information systems at the time of THE ROLE OF NEW TECHNOLOGIES IN PASSIVE DEFENSE confrontation AND CRISIS MANAGEMENT OF NEW THREATS • New systems for transporting the injured to health centers In the anticipation and prevention phase, it is attempted to • New clothes and equipment to protect the health care staff provide the necessary reinforcements by training, for action without contamination developing laws and regulations, strengthening and using • Reconstruction At this stage, given the facilities and safety and security equipment prior to the threat. New equipment of the previous three stages, it is possible to information technologies and new equipment provide great return to pre-crisis mode in less time. help to passive defense, future research as well as disaster • Returning to the pre-crisis phase and control if the crisis preparedness [49]. Production of detectors of biological, has been well managed in the previous three phases [3]. chemical and nuclear agents as well as installing biosensors Currently, some governments, following the approach and in different parts of the country, the system will send the uses of genetic information, have enacted laws protecting system information on the biological risk factors online to this information, requiring that they establish a special the relevant organization the equipment is built using protective location for genetic information to define this modern technologies used in modern warfare and equipped information. And to differentiate it from other forms of with online information networks [50]. information, in particular, personal and health and medical

124 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine information [10]. need for mandatory consent is necessary [11]. Attitudes and practices vary widely across societies. In an interview with DISCUSSION Canadian university administrators, 47% of sites require individual consent to continue their studies, but 45% do not Well-known biological and bioterrorist agents, such as require consent or do not recommend the notification smallpox, plague, anthrax viruses, etc. are naturally process and fix them [12]. One of the major challenges in the pathogenic, but modern biotechnology and genetic field of genetic banking related to national laws and engineering have enabled genetic modifications to control international research policies. Therefore, in matters such as all pathogenic features of an organism that the organism is bioethics, harmonization of medical information privacy genetically manipulated is fully proportionate to its laws and the outcome of individuals, identical terms and purpose[51, 52]. This technology of genetic manipulation interpretations, and how to obtain participant consent, in can be exploited by the enemy if the human, animal, plant countries with access to genetic knowledge and genetic and even genome of a bacterium are identified. In general, information, legislators should be considered to be [60, 61]. recent biological capabilities in the field of weapons production are the result of advances such as the Therefore, passive defense measures should be put in place construction of engineered vectors, precise recognition, to enforce by laws and regulations on genetic information as completion of the human genome project, and the drawing well as how genetic banks operate and prevent any potential of human immunity, etc. [53]. Of course, new biological misuse by the enemy. weapons production technologies provide the middle On the other hand, information networks as a platform for ground from very lethal to non-lethal weapons. And today, data collection and creation of databases on biological, not only is the target of bio-weapons, but also the building chemical and radioactive and nuclear events, epidemic of bio-weapons against the country's economic collection and information systems and biological events infrastructure to hit the country [54, 55]. such as Medysis and Health map and a variety of human and So patients and researchers have long been concerned about animal and plant disease information systems have made it the privacy of health information. In a study report possible for the country and its experts to monitor and participant’s collected medical information during a study, prepare the data online [62]. But there is a gap in protected participants preferred (92%) to request permission before data-collection systems and genetic reserves in the country. using their health information for any purpose other than In addition, conservation of genetic stocks should also medical treatment and 83% want research details before include measures to prevent breeding and genetic diversity being allowed to use their health records. of the country. This study shows that some issues such as family medical CONCLUSION history, genetic disorders, mental illnesses, drug or alcohol related events, lists of previous cases, and current The results of the present study showed that the medications are highly sensitive. There are also ethical confidentiality of genetic data has been considered by complaints about the ability of people with cognitive researchers in different countries, and with the advances of impairment to make informed consent or addicts to molecular and genetic sciences, the possibility of using this participate in studies involving substance use disorders. science as a new bioterrorism is more likely. As a result, Tests to identify addicts or predict the risk of addiction raise defensive measures in the area of non-biodegradable concerns about privacy attacks, third-party misuse of data, defense in order to preserve the genetic reserves and and on the other hand, using data correctly will increase the undiagnosed races by enemies are increasingly common. power of the courts to identify the perpetrators by forcing them to perform such tests [56, 57]. Acknowledgment The confidentiality of genetic data has also been addressed Authors wish to thank all staffs of Applied Virology Research Center; by researchers. Undermining data security can lead to Baqiyatallah University of Medical Science; Tehran; Iran, for their cooperation in implementing experimental procedures and analysis of data. unwanted advertising and costs [58, 59]. Some researcher write that to ensure that privacy laws do not indirectly Conflicts of interest replace observational studies using medical records, The authors declare that they have no conflict of interest. thoughtful decision-making by research ethics boards on the

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Article received on March 20, 2019 and accepted for publishing on August 23, 2019. VARIA

Secure wireless system based on reconfigurable devices for human biomedical parameters monitoring

Ionuț Rădoi1,2, Lidia Dobrescu2, Ștefan C. Arseni3, Florin Răstoceanu1, Florent M. Roman1, Dragoș Dobrescu2, Stela Halichidis4

Abstract: Over the last decade the continuous improvements in sensor technologies, connected with recent hardware reconfigurable devices evolution, enable engineers to merge sensors and reconfigurable devices to develop new applications or to improve the existing ones. The miniaturization and integration of multiple sensors in one chip and the increase of precision, stability and power efficiency allow sensors to play an even more important role in medical technology with the main objective of building more accurate and smaller devices that help medical personal to monitor human biomedical parameters. This paper describes a secured wireless system design and implementation. The proposed system consists in one or more wearable sensor nodes that measure human biomedical parameters and then sends the collected data to a base station in order to be analyzed by qualified personal. This system can be used to monitor patient state-of-health or to supervise military personal in training or even in battles, because the new system uses secured transmission. The typical monitored parameters are body temperature, blood oxygen level, heart rate, respiratory rate, movement and position of the subject, but it can be extended and more other different sensors such as cameras or microphones can be added. Reconfigurable devices are used to process data in both sensor node and base station in an innovative environment. Keywords: biomedical parameters, reconfigurable devices, sensor nodes, wireless

INTRODUCTION innovative health solutions and technologies in medical activity are more than welcome, if the common goal is to Nowadays, electronic industry and healthcare system are save lives. two modern domains, technologically advanced, working together to find solutions for many of the existing medical Heart failure is a major medical problem at global level, due problems. Rapid diagnostic tests, helping patients after to increased prevalence of cardiovascular diseases [3]. Rapid medical interventions and improving the lives of people with diagnosis of heart failure is almost impossible for human disabilities using sensors are authentic examples [1]. Many body subjected to high levels of stress (e.g. gym training reviews in military health care system consider that workout, soldier training and in missions, free running and “medicine is a filed in which evolution ignores borders, parkour, etc.). bureaucratic rules, customs and paradigms” [2] and In such cases diagnosis can be easily accomplished using wearable sensors, incorporated in many wearable devices like: chest belts, cloves, watches, wrist bands, finger clip and 1 Military Equipment and Technology Research Agency, Bucharest, Romania mobile phones. Many electronic integrated circuit 2 Politehnica University of Bucharest, Bucharest, Romania manufacturers offer a variety of sensors capable to measure 3 Military Technical Academy, Bucharest, Romania many biomedical parameters: body temperature, heart rate, 4 Clinical Infectious Diseases Hospital, Constanta, Romania

128 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine oxygen saturation, electrocardiogram, etc. Furthermore, in station/gateway or other sensor nodes; the specific case of heart failure, medical personnel, • encrypts the acquired data and prepares it for supervisors or relatives can be alerted by communications transmission; devices integrated in wearable systems. These systems can • decrypts and process the received data; also provide the exact location of the people in distress in • wakes up the system and puts it back to sleep for power order to be found and rescued, using GPS receivers, also conservation. integrated in a special designed wearable monitoring Due to latest improvements in reconfigurable technology system. regarding power consumption, instead of classic The proposed system architecture is presented in the microcontrollers, reconfigurable devices for both wearable second section of this paper together with a short module and base station are the best alternatives [4]. description of the system’s main module and its components. In the next sections the proposed system Figure 1. System architecture operating mode in a real world scenario is presented together with encryption algorithms that can be implemented on the main controller in order to ensure different levels of security. In the last two sections implementation and experimental results are depicted. Conclusions and future work directions are also indicated, these will enhance the system overall performance and will enlarge application field.

This paper describes an improved solution to the existing health monitoring systems because its main functions are executed using reconfigurable devices such as CPLD or FPGA. Furthermore the new proposed system is secured using the strong advantage of reconfigurable devices capabilities, making it suitable for military applications. Biomedical sensor

SYSTEM ARCHITECTURE In this system different biomedical sensor are used to measure and monitor human body relevant parameters in The overall system architecture is presented in Fig. 1. The order determine its state of health or to prevent human proposed system consists in three main modules: main health degradation (e.g. loss of consciousness, hypo or controller, various biomedical sensors and a base hyperthermia, physical fatigue, etc.) [5]. station/gateway. Biomedical sensors and main controller can be integrated in a chest belt, wristband or a glove making The new system presented in this paper is designed to it a wearable device, thus all the components of the monitor the fallowing physiological parameters: body wearable module must be low-power and small package temperature (T), electrocardiogram (ECG), photo- devices, ideal for this kind of systems. The wristband and the plethysmogram (PPG), heart rate (HR), peripheral capillary glove are added to increase the systems performance in case oxygen saturation (SpO2). After studying numerous types of heart rate or oxygen saturation measurement, because in of biomedical sensor taking into consideration power those areas the veins are located close to the surface of the consumption, monitored parameters, chip dimension and skin and fingers are vascularized organs. The wearable sensor accuracy. As seen in Table 1 chosen sensors are module can be considered as a wireless sensor node and the built specially for wearable devices providing low power base station as a gateway becoming a wireless sensor consumption and most common connection interface to network. communicate with the main controller. In the next paragraph a short description of this sensor is given: Main controller • AFE4900 is an analog front-end (AFE) integrated circuit As the main part of the wearable module, it ensures the built by Texas Instruments designed mainly for (ECG) and following functionalities: (PPG) signal acquisition at data rates up to 1 KHz, but it can • controls and configures the node; also be used for optical bio-sensing applications, such as • collect data from the sensors; heart-rate monitoring (HRM) and saturation of peripheral • establish and maintain communication with the base

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capillary oxygen (SpO2). The ADC codes from the PPG and Transceiver ECG phases can be stored in a 128-sample first in, first out The wearable module must be able to communicate with the (FIFO) block and read out using either an I2C or a serial base station and vice versa. To accomplish this, the license programming interface (SPI) interface;[6] free ISM European 868 MHz band is chosen. Sub GHz wireless transceivers offers many advantages such as Table 1. excellent propagation characteristic at low frequency, is easy Average Power Monitored to use [10] and consumes significant less power than the Part Number consumption Interface Parameter other communications modules (e.g. 2.4 GHz and 5 GHz WiFi Standby Active module). In applications where so called sub GHz ECG, PPG, AFE4900 0µA 30µA I2C, SPI transceivers are used the battery life time is extended by HR, SpO2 48% in comparison with applications where WiFi modules MAX30205 T 1.65µA 600µA I2C are used [11].

MAX86141 HR, SpO2 0.6 µA 10 µA SPI For the system presented in this paper the SPSGRF-868 low power programmable RF transceiver is preferred due to the • MAX30205 uses a sigma-delta analog-to-digital converter low power characteristic and because it is delivered tiny (ADC) with high resolution to convert temperature form factor but providing a complete RF platform with measurements to a digital form. It has a 16 Bit temperature integrated antenna. The module has the capability to resolution (0.0040°C) with 0.1 °C accuracy that meets clinical alternate READY and SLEEP states in order to preserve thermometry specification of the ASTM E1112 when power. Configuration and data transmit and receive is made soldered on the final PCB. The communication with the main through SPI interface. Both wearable module and base controller is made through an I2C-compatible, 2-wire serial station is equipped with this transceiver. interface; [4, 7] Base station • MAX86141 is an ultra low-power optical data acquisition device designed for heart rate (HR) and oxygen saturation In this case power consumption is not a key factor. For this (SpO2) and optimized for wrist, finger or ear location. reason a multitude of reconfigurable devices can be used, but after analyzing a set of this devices and taking into This devices is also an analog front-end (AFE) integrated consideration system’s requirements for data processing circuit with 19 bit analog to digital converter (ADC), ambient speed, reconfigurable resources, communication interface light cancelation and picket fence detect and replace (e.g. SPI, I2C, CAN, etc), the best solution appears to be Xilinx algorithm. The device has an SPI interface to communicate Zynq XC7Z007S SoC. with the main controller [8]. This device has integrated a Single-core ARM Cortex-A9 In Figure 2 a PPG and ECG measurement example is capable of running at maximum frequency of 667 MHz and presented. The displayed data was captured using TIDA- reconfigurable resources are equivalent to Artix-7 FPGA’s. 01580 development board and its associated LabView The base station has the following functions in the system: application [9]. • receive and process data from the sensor nodes; • alerts the medical personnel when measured biomedical Figure 2. PPG and ECG measurement example parameters are outside the defined threshold; • encrypts and decrypts the transmitted or received data; • configures the sensor node (threshold, id, etc); • manage the encryption keys in the wireless sensor network.

Auxiliary components

In addition to the components and modules presented before, each sensor node has other components with less significant roles but without these components the system cannot function or satisfy the system requirements regarding power consumption. These components are:

• TCR2LFXX - low quiescent current (Iq) low dropout

130 Vol. CXXII • No. 3/2019 • December • Romanian Journal of Military Medicine regulator (LDO); preferred to implement the firmware running on CPLD and low-power Spartan 6 FPGA. The design was synthesized and • S25FL127S - 128 Mbit SPI flash memory used do store simulated Xilinx ISE Design Suite 14.7 for CPLD and Spartan 6 firmware in case of nodes with FPGA and other parameter and with Vivado Design Suite 2017.4 in the case of Zynq like network id, node address, pre shared cryptographic XC7Z007S SoC. keys, etc; CPLD consumes only 16 μA at a running clock of 32.768 KHz • 23LCV1024 - 1 Mbit Serial SRAM with Battery Backup use and because in case of temperature measurement only five to store system flags, threshold and other parameters that measurement are needed in 10 minutes of operation. The system needs do store and then quickly access them when main power consummator in this design is the transceiver needed; with 22 mA in TX mode at 11.6 dBm but in shutdown mode • SiT1533 - Ultra-Low Power 32.768 kHz Quartz XTAL to it consumes only 0.1 μA. Thus alternating ACTIVE and clock the system. SHUTDOWN sates significantly reduces the overall power consumption. Figure 3. Operation Modes In normal conditions Spartan 6 is a power consummator, making it inappropriate for our design but Xilinx introduced a low power version of Spartan 6 that consumes with 30-40% less power. In addition to this it too has the possibility to be put in to SLEEP mode furthermore reducing power consumption. After design implementation using AES-256, the following Spartan 6 part number XC6SLX9-L1TQG144C was chosen because it has the necessary logic resources needed for our design. The L later in the part number specifies the low power version of Spartan 6.

The wearable module can be powered from a 3.6V AA battery. Different voltages are adjusted from this power source in order to power up all the system’s devices. The system needs the following voltages to function correctly: 1.8V for CPLD, 1V for Spartan 6 and 3.3V for all other devices.

These voltages are obtained using LDO regulators with 2 μA SYSTEM SECURITY quiescent current.

Due to the use of reconfigurable devices, security for this Table 2 estimates overall power consumption in different system can easily be achieved because this type of electronic operating states. Battery life time is also indicated if a 2400 circuits are notorious when comes to the implementation of mA AA battery is used. cryptographic algorithms. Table 2. Depending on the desired level of security CPLD or Low- Ready Shutdown Battery lifetime Device Power Spartan 6 FPGA can be used as main controller. While State State 5 s/min duty (days) on CPLD a few low level cryptographic algorithms can be CPLD 11 mA 30 μA ≈142 implemented (RC4, DES, Camellia, etc), on Spartan 6 all the advanced cryptographic algorithms can be implemented Spartan 6 40 mA 30 μA ≈37 (AES, SHA, HMAC, RC6, ECC, etc.). CONCLUSION AND FUTURE WORK IMPLEMENTATION AND EXPERIMENTAL RESULTS The design and implementation results presented in this The use of reconfigurable devices was designated as the best paper provides a clear understanding of how engineering choice for this design due to its flexibility in implementation (e.g. electronics and telecommunications) and medical of control functions and most common communication science can work together to design and built advanced protocols (e.g. I2C, SPI, UART, CAN, etc.), [4] and due to biomedical monitoring systems using latest innovations in latest advances in reconfigurable technology regarding sensor technology advantages. It also proves the possibility power consumption. Synthesizable Verilog code has been to use reconfigurable devices in low power designs by

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alternating inactive and active operating states. Using this Adding new devices to the proposed system (e.g. GPS strategy, it becomes possible to integrate such systems in localization, CMOS cameras, microphones or other wearable devices, extending the battery life time for days or biomedical sensors) is also considered for future system even weeks. developments.

References:

1. D. Tudor, L. Dobrescu, D. Dobrescu, “Ultrasonic Electronic System 7. Maxim MAX30205 DataSheet, “Human body temperature for Blind People Navigation” The 5th International Conference on E- sensor”, https://datasheets.maximintegrated.com/en/ds/ Health and Bioengineering, 2015. MAX30205.pdf. 2. F. I. Radu, “Benchmarks of the evolution and revolution of 8. Maxim MAX86140 / MAX86141, “Best-in-Class Optical Pulse military medicine in the XXI century – Tradition, Trust, Oximeter and Heart-Rate Sensor for Wearable Health”, Professionalism”, Romanian Journal of Military Medicine, Vol. CXIX, https://datasheets.maximintegrated.com/en/ds/MAX86140- No. 1, 2016. MAX86141.pdf. 3. R. N. Horodinschi, A. P. Stoian, D. Marcu, R. S. Costache, C. 9. Texas Instruments TIDA-01580, “Wearable, Wireless, Multi- Diaconu, “Heart failure with preserved ejection fraction: A review Parameter Patient Monitor Reference Design”, Romanian Journal of Military Medicine, 2018. http://www.ti.com/lit/ug/tidudo6/ tidudo6.pdf. 4. I. Rădoi, L. Dobrescu, S. V. Pașca, “Low-Power Wireless 10. S. Aust, Ignas Niemegeers, R. V. Prasad, “Performance Evaluation Temperature Sensor for Health Monitoring”, The 10th International of Sub 1 GHz Wireless Sensor Networks for the Smart Grid”, 37th Symposium on Advanced Topics In Electrical Engineering, 2017. Annual Conference on Local Computer Networks, 2012. 5. E. Sardini, M. Serpelloni, “Instrumented Wearable Belt for 11. S. Fudickar, M. Valentin, “Comparing Suitability of Sub 1 GHz and Wireless Health Monitoring”, Procedia Engineering 5, 2010. Wi-Fi Transceivers for RSS-based Indoor Localisation”, International 6. Texas Instruments AFE4900 DataSheet, “AFE4900 Ultra-low Conference on Indoor Positioning and Indoor Navigation, 2014. power, integrated AFE for wearable optical, electrical bio-sensing 12. A. Piedra, A. Braeken, A. Touhafi, “Sensor Systems Based on with FIFO datasheet-(Rev.A)”, 2019, http://www.ti.com/lit/ds/ FPGAs and Their Applications: A Survey”, MDPI Sensor Journal, ISSN symlink/afe4900.pdf. 1424-8220, 2012.

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