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VOLUME 78 - Issue 4October - December 2019

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Copyright © 2019 Cantacuzino National Medico-Military Institute for Research and Development

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Chief Editor: Assoc. Prof. Dr. Anda Băicuș (Deputy Director Cantacuzino N.M.M.I.R.D., Bucharest, Romania)

Deputy Editor: Sorin Dinu (Researcher PhD, Cantacuzino N.M.M.I.R.D., Bucharest, Romania)

Editorial BoardViorel Alexandrescu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania)Gabriela Anton (Şt. S. Nicolau Virology Institute, Bucharest, Romania) Fernando Baquero (Ramón y Cajal Health Research Institute, Ramón y Cajal University Hospital, Madrid, Spain) Daniela Bădescu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Coralia Bleotu (University of Bucharest, Faculty of Biology, Bucharest, Romania) Carmen Chifiriuc (University of Bucharest, ICUB,Bucharest, Romania) Oana Ciofu (University of Copenhagen, Institute of Immunology and Microbiology, Faculty of Health and Medical Sciences, Copenhagen, Denmark) Irina Codiţă (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Daniel I. Cohen (Tel Aviv University, School of Public Health, Sackler Faculty of Medicine, Tel Aviv, Israel)Stefan N. Constantinescu (Université Catholique de Louvain, Belgium)Victor Cristea (Iuliu Haţieganu U.M.Ph., Cluj-Napoca, Romania) Jessica Cusato (University of Torino, School of Medicine, Torino, Italy) Luminiţa Smaranda Iancu (Gr. T. Popa U.M.Ph., Iaşi, Romania) David Y. Graham (Baylor College of Medicine, Houston, Texas, USA) Kianoush Khosravi-Darani (National Nutrition and Food Technology Research Institute, Tehran, Iran)Veronica Lazăr (University of Bucharest, Faculty of Biology, Bucharest, Romania) Andreea-Roxana Lupu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Emilia Lupulescu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Gina Manda (Victor Babeş N.I.R.D., Bucharest, Romania) Grigore Mihăescu (University of Bucharest, Faculty of Biology, Bucharest, Romania) Roxana Moldovan (Victor Babeş U.M.Ph, Timişoara, Romania) Thierry Naas (Université Paris-Sud, APHP, Institut Pasteur, Paris, France) Monica Neagu (Victor Babeş N.I.R.D., Bucharest, Romania) Marian Neguț (Academy of Medical Sciences, Bucharest, Romania)Adrian Onu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Marina Pană (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Giovanni di Perri (University of Torino, School of Medicine, Torino, Italy) Graţiela Grădișteanu Pîrcălăbioru (University of Bucharest, ICUB, Bucharest, Romania) Aurora Sălăgeanu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania)Edit Székely (U.M.Ph Târgu-Mureş, Romania)Codruţa-Romaniţa Usein (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Cem Uzun (Trakya University, Faculty of Medicine, Edirne, Turkey)Weiwen Zhang (Tianjin University, Tianjin, P.R. China)Imola Zigoneanu (University of North Carolina at Chapel Hill, USA)

ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY

209 - CANTACUZINO NATIONAL MEDICO-MILITARY INSTITUTE fOR RESEARCH AND DEVELOPMENT SYMPOSIUM – AbSTRACTS

249 - CURRENT PROgRESS AND PERSPECTIVES Of MALDI-TOf MASS SPECTROMETRY APPLICATIONS fOR THE MEDICAL MICRObIOLOgY fIELD: A MINI-REVIEw Andreia Amzuța, Alina Lenghel, Elena Diana Giol, Mihaela Palela

256 - EXPERIMENTAL bIOfILM MODELS: A REVIEw Ştefania Mădălina Negrea, Raluca Elena Iurea, Costin Ştefan Caracoti, Brînduşa Elena Lixandru, Elena Carmina Drăgulescu, Cristin Coman, Irina Codiță

267 - ULTRA-DEEP SEQUENCINg ASSESSMENT Of gENETIC VARIAbILITY IN HEPATITIS C VIRUS INfECTINg ROMANIAN PATIENTS Sonia Spandole-Dinu, Eugen Radu, Sorin Dinu, Georgeta Cardoş, Grațiela Țârdei, Petre Iacob Calistru, Emanoil Ceauşu, Laurențiu Micu, Simona Ruță, Camelia Sultana, Gabriela Oprişan

286 - SUbJECT INDEX

288 - AUTHOR INDEX

290 - gUIDELINES fOR AUTHORS

CONTENTS

VOLUME 78 Issue 4 OCTOBER - DECEMBER 2019

209

CANTACUZINO NATIONAL MEDICO-MILITARY INSTITUTE FOR RESEARCH AND DEVELOpMENT SYMpOSIUM

SIMpOZION INSTITUTUL NAÞIONAL DE CERCETARE-DEZVOLTARE MEDICO-MILITARÃ CANTACUZINO

25.11.2019

Section I – Impact of reference laboratories on public health / Secţiunea I – Impactul laboratoarelor de referinţă asupra sănătăţii publice

1. Public health genomics: molecular typing of foodborne pathogens / Genomica pentru Sănătate Publică: tipizarea moleculară a patogenilor transmişi prin alimente. Codruţa-Romaniţa Usein, Mihaela Oprea, Mădălina Militaru, Sorin Dinu, Maria Condei, Ramona Iordache, Gabriela Oprişan

2. Diversity of the population of Shigella strains circulating on Romanian territory isolated between 2016-2018 / Diversitatea populaţiei de tulpini de Shigella circulante pe teritoriul României între anii 2016-2018Daniela Cristea, Adriana Simona Ciontea, Mircea Ioan Popa, Mihaela Oprea, Sorin Dinu, Andrei Popa, Melania Mihaela Andrei, Mădălina Zamfir, Maria Condei, Lavinia Zota, Maria Nica, Codruţa Romaniţa Usein

3. C. difficile in paediatric infections. Retrospective study / C. difficile în infecţii pediatrice. Studiu retrospectivIoana S. Macovei

4. Study of Streptococcus pneumoniae strains, resistant to antibiotics, analysed at Cantacuzino National Medico-Military Institute for Research and Development, between 2008-2018 / Studiul tulpinilor de Streptococcus pneumoniae, rezistente la antibiotice, analizate la Institutul Național de Cercetare-Dezvoltare Medico-Militară „Cantacuzino” în perioada 2008-2018 Marina Pană, Anamaria Ilie, Cristina Georgeta Oprea, Irina Nistor, Maria Dorina Crăciun, Raluca Papagheorghe, Nicoleta Popescu, Maria Nica, Olga Dorobăţ

5. The role of the National Reference Laboratory in the diagnosis and surveillance of Viral Respiratory Infections / Rolul Laboratorului Național de Referință în diagnosticul și supravegherea Infecțiilor Respiratorii ViraleCarmen Maria Cherciu, Maria Elena Mihai, Iulia Bistriceanu, Cătălina Mariana Pascu, Luiza Ustea, Mirela Ene, Emilia Dobre, Nicoleta Paraschiv, Oana Vitencu, Mihaela Lazăr

6. Innovation integrated in the process of global eradication of Poliomyelitis / Inovarea integrată în procesul de eradicare globală a Poliomielitei Anda Băicuş

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7. Optimization and validation of diagnostic methods of yellow fever in Reference Laboratory for Vector-Borne Infections from Cantacuzino National Medico-Military Institute for Research and Development / Optimizarea și validarea metodelor de diagnostic al febrei galbene în Laboratorul de Referință Infecții Transmise prin Vectori din Institutul Național de Cercetare-Dezvoltare Medico-Militară „Cantacuzino”Ani Ioana Cotar, Simin Aysel Florescu, Corneliu Petru Popescu, Daniela Bădescu, Cornelia Svetlana Ceianu

8. Eosinophilia in trichinellosis / Eozinofilia în trichinelozăAlexandru Cristian Vasile-Bugarin

9. Biosafety and biosecurity. Basic principles / Biosiguranță și biosecuritate. Principii de bază Cristina Ţărmurean

Section II – Translational immunology studies with an impact on health / Secţiunea II – Studii translaţionale de imunologie cu impact pentru sănătate

10. Characterization of colon tumor cells isolated from tumor specimens / Caracterizarea celulelor tumorale izolate din tumori colorectaleIulia I. Niță, Aurora Sălăgeanu, Iuliana Caraș

11. Cytotoxicity evaluation of some stabilized iron oxide powders and suspensions for biomedical applications / Evaluarea citotoxicității unor pulberi și suspensii stabilizate pe bază de oxizi de fier pentru aplicații biomedicale Dora Domnica Baciu, Iulia Ioana Lungu, Andrei-Mihai Dumitrașcu, Gabriel Prodan, Aurora Sălăgeanu, Florin Dumitrache

12. An in vitro model for testing the influence of the etiologic agents of bacterial meningitis on blood brain barrier / Influenţa agenţilor etiologici ai meningitei bacteriene asupra barierei hematoencefalice: model in vitro Andrei Mihai Dumitraşcu, Ramona Caragheorgheopol, Cătălin Țucureanu, Marina Pană, Andrei Alexandru Muntean, Mădălina Maria Muntean, Veronica Lazăr, Aurora Sălăgeanu

13. Preliminary results in the process of osseointegration of a dental implant in the rabbit tibia / Rezultate preliminare în procesul de osteointegrare a unui implant dentar în tibia de iepure Diana Larisa Ancuța, Jasmina Manolescu, Cristin Coman

14. Clinical and paraclinical changes in acute MnCl2 induced toxicity in BALB/c mice / Modificări clinice și paraclinice induse de intoxicația acută cu MnCl2 pe model murinClaudiu Gal, Mariana Văduva, Mihaela Diaconu, Crina Stăvaru

15. In vitro study of the effect induced by MnCl2 on fibroblast cell line / Studiu in vitro al efectelor induse de MnCl2 pe linii fibroblastice Ştefania Lascăr, Irina Elena Ionescu, Raluca Elena Lăzărescu, Vlad Tofan, Mihaela Diaconu, Cătălin Țucureanu, Ana Șerbănescu, Crina Stăvaru, Adrian Onu

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16. Differentiation and maturation of the murine dendritic cells extracted from the bone marrow. A comparative study between freshly collected and cryopreserved cells / Diferențierea și maturarea celulelor dendritice murine extrase din măduva osoasă. Studiu comparativ între celule proaspăt recoltate și celule crioprezervateIrina Elena Ionescu, Iuliana Caraș, Cătălin Țucureanu, Raluca Elena Lăzărescu, Ana Șerbănescu, Crina Stăvaru, Adrian Onu

17. Mass spectometry characterization of vaccine antigens – application to influenza fragmentation / Caracterizarea prin spectrometrie de masă a antigenelor vaccinale – aplicare la fragmentarea virusului gripalCătălin Țucureanu, Vlad Vasilca, Vlad Tofan, Alina Lenghel, Mihaela Lazăr, Adriana Costache, Crina Stăvaru, Adrian Onu

18. Optimization of bacterial expression of antigens for vaccine development – application in influenza vaccine / Optimizarea expresiei bacteriene a antigenelor din candidații de vaccin – aplicații la vaccinul gripal Laura Andreea Ermeneanu, Vlad Tofan, Cătălin Țucureanu, Vlad Vasilca, Alina Lenghel, Adriana Costache, Crina Stăvaru, Adrian Onu

19. Target Product Profile (TPP) as a central planning tool for biopharmaceutical research: application for the development of a new rabies vaccine for human use / Profilul produsului țintă (TPP – Target Product Profile) ca instrument central de planificare pentru cercetarea biofarmaceutică: aplicare pentru dezvoltarea unui nou vaccin antirabic de uz umanRadu Iulian Tănasă, Paulina Podgoreanu, Ilinca-Mihaela Marandiuc

Section III – Opportunities for methods development and technological transfer /Secţiunea III – Oportunități pentru dezvoltare de metode și transfer tehnologic

20. The main objectives in the production of vaccines/adjuvants and their placing/maintaining on the market. Regulations / Principale obiective în producerea de vaccinuri/adjuvanți și introducerea/menținerea lor pe piață. Reglementări Yuksel Rasit, Cristina Dumitrache, Mariana Văduva

21. Biobanking - trend or necessity? / Biobanca – modă sau necesitate? Andreea-Roxana Lupu, Alexandra Maria Născuțiu

22. Diachronic modulation of antibody-antigen affinity through temperature modulation / Modularea diacronică cu temperatura a afinității anticorpilor față de antigeni Răzvan Stan

Simpozion Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã Cantacuzino

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1. pUBLIC HEALTH GENOMICS: MOLECULAR TYpING OF FOODBORNE pATHOGENS

Codruþa-Romaniþa Usein, Mihaela Oprea, Mãdãlina Militaru, Sorin Dinu, Maria Condei, Ramona Iordache, Gabriela Oprişan

Cantacuzino National Medico-Military Institute for Research and Development, Molecular Epidemiology Laboratory, Bucharest, Romania

ABSTRACTObjectives: Public health microbiology is undergoing a change brought about by the so-called

next-generation sequencing (NGS) technologies that have made whole-genome sequencing (WGS) a realistic alternative to classical subtyping methods for microbial pathogens. Since 2015, the ECDC has been monitoring the level of implementation of WGS-based typing for national public health surveillance and investigation of prioritized diseases at European Union level. In this context, the study addresses the foodborne infections and their approach in the national reference laboratory in concordance with ECDC strategy.

Materials and methods: Identification and typing of microbial pathogens are essential for the diagnosis and surveillance of communicable diseases and the associated epidemiological events. To date, the typing methods currently used for foodborne outbreak investigations are PFGE, MLVA, and MLST. However, the experimental data show that WGS is a more powerful tool to separate sporadic cases from outbreak cases, identify the source of outbreak, and mode of transmission.

Results: To establish a laboratory-based national surveillance in accordance with the ECDC recommendations is a necessity that can no longer be ignored by the public health system, food safety, and veterinary medicine. Since 2017, two thirds of the European countries have been using WGS as first or second line method for public health surveillance of prioritized human pathogens.

Conclusions: Implementation, harmonization, and consolidation of WGS-based typing in the reference laboratories in Romania will enable the pan-European genomic surveillance and enhance our capacity to cope with the continuous multi-country epidemiological threats.

GENOMICA pENTRU SÃNÃTATE pUBLICÃ: TIpIZAREA MOLECULARÃ A pATOGENILOR TRANSMIŞI pRIN ALIMENTE

Codruþa-Romaniþa Usein, Mihaela Oprea, Mãdãlina Militaru, Sorin Dinu, Maria Condei, Ramona Iordache, Gabriela Oprişan

Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino”, Laboratorul de Epidemiologie Molecularã, Bucureşti, România

REZUMATObiective: Microbiologia pentru sănătatea publică trece printr-o schimbare datorată așa-numi-

telor tehnologii de secvențiere de nouă generație (next-generation sequencing, NGS), care au făcut ca secvențierea întregului genom (whole-genome sequencing, WGS) să devină o alternativă reală pentru metodele clasice de tipizare moleculară a patogenilor microbieni. Începând cu anul 2015, Centrul European pentru Prevenția și Controlul Bolilor (European Centre for Disease Prevention and Control, ECDC) a început să monitorizeze stadiul implementării WGS în supravegherea na-țională a sănătații publice și investigarea bolilor prioritizate la nivelul Uniunii Europene. În acest context, lucrarea se adresează infecțiilor cu transmitere alimentară și abordării lor în laboratorul național de referință aliniat la strategia ECDC.

Materiale și metode: Identificarea și tipizarea patogenilor stau la baza diagnosticului și supra-vegherii bolilor transmisibile și evenimentelor epidemiologice asociate acestora. În prezent, metode-

ABSTRACTS / REZUMATE

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le de tipizare moleculară utilizate în mod curent în investigarea epidemiilor declanșate de infecțiile bacteriene cu transmitere alimentară sunt PFGE, MLVA și MLST. Argumentele experimentale dove-desc însă că WGS este un instrument mult mai puternic pentru a discrimina cazurile sporadice de cele epidemice, sursele de contaminare și căile de transmitere.

Rezultate: Realizarea unei supravegheri cu laboratorul, coordonată la nivel național și în acord cu recomandările ECDC, devine o necesitate care nu poate fi ignorată la nivel de sănătate publică, siguranța alimentelor și medicina veterinară. Încă din 2017, în Europa, două treimi dintre țări folo-sesc WGS ca metodă de prima sau a doua linie de supraveghere a patogenilor umani considerați prioritari pentru sănătatea publică.

Concluzii: Introducerea, armonizarea si consolidarea metodelor de tipizare bazate pe WGS în laboratoarele de referință din România, ne vor permite să beneficiem de o supraveghere genomică pan-europeană și să facem față permanentelor amenințări epidemiologice multinaționale.

2. DIVERSITY OF THE pOpULATION OF SHIGELLA STRAINS CIRCULATING ON ROMANIAN TERRITORY ISOLATED BETWEEN 2016-2018

Daniela Cristea1, Adriana Simona Ciontea1, Mircea Ioan Popa1,2, Mihaela Oprea1, Sorin Dinu1, Andrei Popa1, Melania Mihaela Andrei1, Mãdãlina Zamfir1, Maria Condei1, Lavinia Zota3,

Maria Nica4, Codruþa Romaniþa Usein1,2

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2 Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

3National Institute of Public Health, National Centre for Prevention and Control of Communicable Diseases, Bucharest, Romania

4 Dr. Victor Babeş Clinical Hospital of Infectious and Tropical Diseases, Bucharest, Romania

ABSTRACT This aim of this study was the phenotypic and genotypic characterization of the virulence and

antimicrobial resistance profile, as well as the genetic relatedness of the Shigella spp., strains collected during 2016-2018, with the purpose of enriching information on virulence potential, antimicrobial susceptibility and the genetic kinship.

Materials and methods: A total of 86 strains of Shigella spp. were tested phenotypically to establish antibiotic resistance profiles. The analysis of the molecular basis of antibiotic resistance to extended spectrum cephalosporins was performed using a protocol based on the amplification of the ctxM gene, followed by the amplicon sequencing. The ipaH, ipaBCD, ial, sen, set1A, set1B, sat and pic virulence genes were detected by PCR technique. The genetic kinship was established using pulsed field gel electrophoresis (PFGE).

Results: Overall, the resistance most commonly acquired by the autochthonous Shigella strains investigated was to the combination of trimethoprim with sulfamethoxazole (44/86 strains), and ampicillin (41/86 strains). Strains with resistance to extended spectrum cephalosporins (12/86 strains) were detected, as well as strains that displayed resistance to nalidixic acid (3/86 strains) but not to ciprofloxacin. The genes blaCTXM15 (2 strains), blaCTXM 3 (2 strains), blaCTXM 1 (8 strains) were identified. The virulence markers targeted ranged in prevalence from 6% (set1A, set1B, pic) to 100% (ipaH). The most complex virulence genotype was found in S. flexneri serotype 2a strains (ipaH, ipaBCD, ial, sen, sat, set1A, set1B, and pic genes). Molecular typing of Shigella spp. strains allowed the detection of several clusters with a similarity of at least 85%. Eight such clusters were identified among the S. sonnei strains and four clusters among the S. flexneri strains.

Conclusions: Continued laboratory-based surveillance is essential to generate data that help to draw the picture of the public health problem represented by shigellosis in Romania.

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DIVERSITATEA pOpULAÞIEI DE TULpINI DE SHIGELLA CIRCULANTE pE TERITORIUL ROMÂNIEI ÎNTRE ANII 2016-2018

Daniela Cristea1, Adriana Simona Ciontea1, Mircea Ioan Popa1,2, Mihaela Oprea1, Sorin Dinu1, Andrei Popa1, Melania Mihaela Andrei1, Mãdãlina Zamfir1, Maria Condei1, Lavinia Zota3,

Maria Nica4, Codruþa Romaniþa Usein1,2

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino”, Bucureşti, România2Universitatea de Medicinã şi Farmacie “Carol Davila”, Bucureşti, România

3Institutul Naþional de Sãnãtate Publicã, Centrul Naþional pentru Controlul şi Prevenþia Bolilor Transmisibile, Bucureşti, România

4Spitalul de Boli Infecþioase şi Tropicale “Dr. Victor Babeş”, Bucureşti, România

REZUMATScopul acestui studiu a urmărit caracterizarea fenotipică şi genotipică a profilului de virulență

şi de rezistență la antibiotice, precum şi gradul de înrudire genetică a tulpinilor de Shigella spp., colectate în perioada 2016-2018, cu scopul îmbogățirii informațiilor privind potențialul de virulență, antibiorezistența și gradul de înrudirea a acestora.

Materiale si metode: Un total de 86 de tulpini de Shigella spp. au fost testate fenotipic pentru stabilirea sensibilității la antibiotice. Analiza bazei moleculare a rezistenței la cefalosporine cu spectru extins a fost realizată folosind un protocol bazat pe amplificarea genei ctxM, urmată de secvențierea ampliconilor. Genele de virulență ipaH, ipaBCD, ial, sen, set1A, set1B, sat și pic au fost detectate prin tehnica PCR. Gradul de înrudire genetică s-a stabilit prin electroforeză în câmp pulsator (PFGE).

Rezultate: Cel mai comun profil de rezistenţă la antibiotice la Shigella spp. a fost față de trimethoprim/sulfamethoxazole (44/86 tulpini) şi ampicilină (41/86 tulpini). Au fost detectate tulpini cu rezistenţă la beta-lactamine cu spectru extins (12/86 tulpini), precum şi tulpini rezistente la acid nalidixic (3/86 tulpini), dar nu şi la ciprofloxacin. S-au identificat genele blaCTXM15 (2 tulpini) , blaCTXM 3 (2 tulpini), blaCTXM 1 (8 tulpini).

Prevalenţa markerilor de virulenţă a variat între 6% (set1A, set1B, pic) şi 100% (ipaH). Cel mai complex genotip de virulenţă a fost detectat la S. flexneri tip 2 (genele ipaH, ipaBCD, ial, sen, sat, set1A, set1B şi pic).

Tipizarea moleculară a tulpinilor de Shigella spp., a permis detectarea mai multor clustere care prezentau o similaritate de cel puţin 85%. Opt astfel de clustere s-au detectat printre tulpinile de S. sonnei şi patru printre cele de S. flexneri.

Concluzii: Supravegherea continuă bazată pe rezultatele obținute în laborator este esenţială pentru a genera datele care să ajute la înțelegerea problemei de sănătate publică reprezentată de shigelozele din România.

3. C. DIFFICILE IN pAEDIATRIC INFECTIONS. RETROSpECTIVE STUDY

Ioana S. Macovei1

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

ABSTRACTClostridioides difficile infection (CDI), associated with broad spectrum antibiotic therapy (MLSb,

fluoroquinolones, carbapenems, cephalosporins) and often healthcare acquired (5 - 25%), has been reported in Romania since 2010 - 2011, with many severe forms (especially in elderly patients), gen-erated by highly virulent strains, such as PCR ribotype R027 (Popescu et al., 2018). Recently, an


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incidence increase trend has been noted in paediatric CDIs (Nicholson et al., 2016), with recurrence rates similar to those recorded in adults (25%) (Kim et al., 2012). In children, the main risk factor remains the administration of antibiotics, associated with prolonged hospitalization; immunosup-pressive treatment, malignancies (especially hematological), transplantation, inflammatory bowel syndrome or other gastrointestinal disorders may also involve a risk for CDI. Local data regard-ing the frequency of paediatric cases are insufficient. By analyzing local molecular typing data of Romanian isolates collected between 2011 and 2018 (Macovei et al., 2018), a homogeneous diversity of toxigenic PCR ribotypes was observed in paediatric patients (n = 13): R001, R002, R011, R012, R014, R017, RU.02, R027, R046, R053, R087, R106, R115, only one being binary toxin-producing (R027). The prevalent paediatric isolates were TcdA + / TcdB + / CDT− strains, belonging to PCR ribotype R014, frequently reported in Europe (Freeman et al., 2015). R001, R002, R012 and hypervirulent R027 were sporadically identified in our population of strains isolated from children. R014, R002, R012, R017, R046 and R027 were previously defined as multidrug resistant, with similar resistance phenotypes between R014 and R002 (Cefotaxime-Erythromycin-Imipenem, Cefotaxime-Clindamycin), and R017 being unsusceptible to a large number of antibiotic classes (Macovei et al., 2018). As our findings are limited, for an in-depth characterization of the epidemiology of CDI in Romanian children, it would be very useful to study the molecular epidemiology of large populations of geographically repre-sentative C. difficile isolates.

REFERENCES1. Freeman J, Vernon J, Morris K, Nicholson S, Todhunter S, Longshaw C, et al. Pan-European longitudinal

surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes. Clinical Microbiology and Infection. 2015;21(3):248.e9-248.e16.

2. Kim J, Shaklee JF, Smathers S, et al. Risk factors and outcomes associated with severe Clostridium difficile infection in children. Pediatr Infect Dis J 2012;31(2):134-8.

3. Macovei IS, Lemeni D, Șerban R, Niculcea A, Popescu GA, Nica M, et al. Antibiotic susceptibility and resistance profiles of Romanian Clostridioides difficile isolates. Romanian Journal of Laboratory Medicine. 2018;26(2):189-200.

4. Nicholson MR, Thomsen IP, Edwards KM. Controversies surrounding Clostridium difficile infection in infants and young children. Children. 2014;1:40-47.

5. Popescu GA, Șerban R, Pistol A, Niculcea A, Preda A, Lemeni D, et al. The recent emergence of Clostridium difficile infection in Romanian hospitals is associated with a high prevalence of polymerase chain reaction ribotype 027. Balkan Medical Journal. 2018;35(2):191-195.

C. DIFFICILE ÎN INFECÞII pEDIATRICE. STUDIU RETROSpECTIV

Ioana S. Macovei1

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino”

REZUMATInfecţia cu Clostridioides difficile (ICD), consecinţă a administrării îndelungate a antibioticelor cu

spectru larg (MLSb, fluoroquinolone, carbapeneme, cefalosporine), frecvent asociată actului medical (5-25%), a fost semnalată în România încă din 2010-2011, în special la pacienţi vârstnici, cu numeroase cazuri severe, generate de tulpini hipervirulente, precum cele de ribotip PCR R027 (Popescu și colab., 2018). Recent, s-a remarcat tendința de creștere a frecvenţei ICD pediatrice (Nicholson şi colab., 2016), cu rate de recurență similare celei înregistrate la adulți (25%) (Kim și colab., 2012), principalii factori de risc fiind administrarea de antibiotice și spitalizarea prelungită. Tratamentul imunosupresiv, malignitățile (în special hematologice), transplantul, sindromul intestinului inflamator sau alte afecțiuni gastrointestinale pot implica și ele un risc. Datele autohtone privind frecvența cazurilor pediatrice sunt insuficiente. Analizându-se retrospectiv date de tipizare moleculară a izolatelor


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C. difficile românești, colectate în perioada 2011-2018 (Macovei și colab., 2018), s-a remarcat o diversitate omogenă de ribotipuri PCR toxigenice la pacienții pediatrici (n = 13): R001, R002, R011, R012, R014, R017, RU.02, R027, R046, R053, R087, R106, R115, unul singur fiind producător de toxină binară (R027). Tulpinile pediatrice autohtone prevalente au fost de tip toxigenic TcdA+ / TcdB+ / CDT−, aparținând ribotipului R014, frecvent raportat în Europa (Freeman și colab., 2015). R001, R002, R012 și tipul hipervirulent R027 au fost identificate sporadic în populația noastră de izolate pediatrice. R014, R002, R012, R017, R046 și R027 au fost definite anterior ca multirezistente la antibiotice, R014 și R002 având fenotipuri de rezistență similare (Cefotaxim-Eritromicină-Imipenem, Cefotaxim-Clindamicină), iar R017 remarcându-se prin rezistență la un număr mare de clase de antibiotice (Macovei și colab., 2018). Ținând cont de faptul că datele noastre sunt limitate, pentru o caracterizare aprofundată a epidemiologiei ICD la copii în România, poate fi foarte utilă studierea epidemiologiei moleculare a unei populații mari de izolate C. difficile reprezentative geografic.

BIBLIOGRAFIE1. Freeman J, Vernon J, Morris K, Nicholson S, Todhunter S, Longshaw C, et al. Pan-European longitudinal

surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes. Clinical Microbiology and Infection. 2015;21(3):248.e9-248.e16.

2. Kim J, Shaklee JF, Smathers S, et al. Risk factors and outcomes associated with severe Clostridium difficile infection in children. Pediatr Infect Dis J 2012;31(2):134-8.

3. Macovei IS, Lemeni D, Șerban R, Niculcea A, Popescu GA, Nica M, et al. Antibiotic susceptibility and resistance profiles of Romanian Clostridioides difficile isolates. Romanian Journal of Laboratory Medicine. 2018;26(2):189-200.

4. Nicholson MR, Thomsen IP, Edwards KM. Controversies surrounding Clostridium difficile infection in infants and young children. Children. 2014;1:40-47.

5. Popescu GA, Șerban R, Pistol A, Niculcea A, Preda A, Lemeni D, et al. The recent emergence of Clostridium difficile infection in Romanian hospitals is associated with a high prevalence of polymerase chain reaction ribotype 027. Balkan Medical Journal. 2018;35(2):191-195.

4. STUDY OF STREPTOCOCCUS PNEUMONIAE STRAINS, RESISTANT TO ANTIBIOTICS, ANALYSED AT CANTACUZINO NATIONAL

MEDICO-MILITARY INSTITUTE FOR RESEARCH AND DEVELOpMENT, BETWEEN 2008-2018

Marina Panã1, Anamaria Ilie1, Cristina Georgeta Oprea1, Irina Nistor2, Maria Dorina Crãciun2, Raluca Papagheorghe3, Nicoleta Popescu3,

Maria Nica4, Olga Dorobãþ5

1Bacterial Respiratory Infections Laboratory, Cantacuzino National Medico-Military Institute for Research and Development,

Bucharest, Romania2Grigore Alexandrescu Children’s Emergency Clinical Hospital, Bucharest, Romania

3Colþea Clinical Hospital, Bucharest, Romania

4Dr. Victor Babeş Clinical Infectious and Tropical Diseases Hospital, Bucharest, Romania

5Prof. Dr. Matei Balş National Infectious Diseases Hospital, Bucharest, Romania

ABSTRACTIntroduction: Resistance to antibiotics has become a global problem. Pneumococcal infections

remain a major cause of morbidity worldwide. The acquisition of resistance to a wide spectrum of chemically unrelated antibacterial agents has occured in Streptococcus pneumoniae in the recent years in Romania.

Objective: The aim of this study was to analyse the resistance to antibiotics of S.pneumoniae strains, isolated in the recent years.


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Methods: Seven hundred and thirty eight pneumococcal strains were analysed , coming from sputum, trhacheal aspirate, pleural fluid, CSF, blood, sinus and middle ear fluid.

The identification of pneumococci was done in the Bacterial Respiratory Infections Laboratory, by phenotypical tests (optochin, bile solubility, agglutination method and Quellung reaction).

Bacterial strains were tested to 11 antibiotics: penicillin (Pc), erythromycin (Em), cefaclor (Cl), cefuroxime (Cxm), cefotaxime (Ctx), trimethoprim/sulfamethoxazol (Sxt), levofloxacin (Lev), amoxicillin (Amx), tetracycline (Te), linezolid (Lzd), vancomycin (Va), according to EUCAST 2018. Serotyping was done with sera coming from Statens Serum Institute.

Results: Pneumococcal strains isolated from blood, CSF, tracheal aspirate and pleural fluid showed lower resistance levels to antibiotics (8.3 % Cxm and Cl, 6.1% Ctx, 2.7 % Amx, 17 % Em, 15 % Sxt, Te 32 %), against those isolated from sinus and middle ear fluid which revealed higher levels (12 % Cxm and Cl, 10 % Ctx, 3.4 % Amx, 47 % Em, 79 % Sxt, Te 45 % ).

Table 1 reveals resistance percent to penicillin, depending on samples and values of MIC 50 and MIC 90.

Table 1. In vitro activity of penicillin in S. pneumoniae strains, depending on sample

MIC 50 (μg/ml) MIC 90 (μg/ml) Resistance (%)Sputum, T.A. (n = 253) 0.25 2 75Blood, P.F. (n = 212) 0.12 1 39CSf (n = 141) 0.06 2 17Others (n = 132) 1 4 78

Serotyping test revealed the following aspects: ST 19 F (30 %), ST 6 B (24 %), ST 19 A (12 %) and ST 3 (12 %), ST 9 V (8 %), ST 23 F (7 %), ST 14 (3 %), ST 1 (2%) and ST 8 (2%).

Conclusions: The most efficient antibiotics in pneumococci resistant to penicillin were Ctx, Lex, Cxm, Cl and Amx.

No strain resistant to vancomycin and linezolid was detected.These results are strengthening the importance of surveillance programs in order to guide

empirical therapy and interventional control on antimicrobial resistance.

STUDIUL TULpINILOR DE STREPTOCOCCUS PNEUMONIAE, REZISTENTE LA ANTIBIOTICE, ANALIZATE LA INSTITUTUL NAÞIONAL DE CERCETARE-DEZVOLTARE MEDICO-MILITARÃ „CANTACUZINO”,

ÎN pERIOADA 2008-2018

Marina Panã1, Anamaria Ilie1, Cristina Georgeta Oprea1, Irina Nistor2, Maria Dorina Crãciun2, Raluca Papagheorghe3, Nicoleta Popescu3,

Maria Nica4, Olga Dorobãþ5

1Laboratorul Infecþii Respiratorii Bacteriene, Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã „Cantacuzino”,

Bucharest, România2Spitalul Clinic de Urgenþã pentru Copii Grigore Alexandrescu, Bucureşti

, România

3Spitalul Clinic Colþea, Bucureşti, România

4Spitalul Clinic de Boli Infecþioase şi Tropicale Dr. Victor Babeş, Bucureşti, România

5Institutul Naþional de Boli Infecþioase Prof. Dr. Matei Balş, Bucureşti, România

REZUMATIntroducere: Rezistența la antibiotice a devenit o problemă globală. Infecțiile pneumococice au

rămas o cauză majoră de morbiditate în întreaga lume. Achiziția rezistenței la antibiotice cu spectru larg a apărut la tulpinile de Streptococcus pneumoniae, în ultimii ani, în România.


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Obiectiv: Scopul acestui studiu a constat în analiza rezistenței la antibiotice a tulpinilor de S. pneumoniae, izolate in ultimii ani.

Metode: Au fost analizate 738 de tulpini pneumococice izolate din spută, aspirat traheal, lichid pleural, LCR, sânge, exsudat sinusal și exsudat otic.

Identificarea pneumococilor s-a efectuat la Laboratorul Infecții Respiratorii Bacteriene prin teste fenotipice (testul la optochin, solubilitatea la bilă, metoda latex-aglutinare și reacția de umflare a capsulei).

Tulpinile bacteriene au fost testate la 11 antibiotice: penicilină (Pc), eritromicină (Em), cefaclor (Cl), cefuroxim (Cxm), cefotaxim (Ctx), trimethoprim/sulfamethoxazol (Sxt), levofloxacin (Lev), amoxicilină (Amx), tetraciclină (Te), linezolid (Lzd), vancomicină (Va), conform normelor EUCAST 2018. Serotiparea a fost făcută cu serurile provenite de la Statens Serum Institute.

Rezultate: Tulpinile de pneumococi izolate din sânge, LCR, aspirat traheal și lichid pleural au arătat nivele de rezistență la antibiotice mai scăzute ( 8,3 % Cxm și Cl, 6,1% Ctx, 2,7 % Amx, 17 % Em, 15 % Sxt, Te 32 %) față de cele izolate din exsudat sinusal și otic care au demonstrat nivele crescute (12 % Cxm și Cl, 10 % Ctx, 3.4 % Amx, 47 % Em, 79 % Sxt, Te 45 % ).

Tabelul 1 relevă procentele de rezistenţă la penicilină, în funcţie de prelevate şi valorile CMI 50 şi CMI 90.

Tabelul 1. Activitatea in vitro a penicilinei la tulpinile de S. pneumoniae, în funcție de prelevat

CMI 50 (μg/ml) CMI 90 (μg/ml) Rezistența (%)Spute, A.T. (n = 253) 0,25 2 75Sg, L.P. (n = 212) 0,12 1 39LCR (n = 141) 0,06 2 17Alte (n = 132) 1 4 78

În urma reacției de serotipare au fost evidențiate următoarele aspecte: ST 19 F (30 %), ST 6 B (24 %), ST 19 A (12 %) și ST 3 (12 %), ST 9 V (8 %), ST 23 F (7 %), ST 14 (3 %), ST 1 (2%) și ST 8 (2%).

Concluzii: Cele mai eficiente antibiotice asupra pneumococilor rezistenți la penicilină, au fost Ctx, Lex, Cxm, Cl și Amx.

Nu a fost decelată nicio tulpină rezistentă la Vancomicină și Linezolid.Aceste rezultate întăresc importanța programelor de supraveghere pentru ghidarea terapiei

empirice și întărirea controlului intervențional asupra rezistenței antimicrobiene.

5. THE ROLE OF THE NATIONAL REFERENCE LABORATORY IN THE DIAGNOSIS AND SURVEILLANCE OF VIRAL RESpIRATORY INFECTIONS

Carmen Maria Cherciu, Maria Elena Mihai, Iulia Bistriceanu, Cãtãlina Mariana Pascu, Luiza Ustea, Mirela Ene, Emilia Dobre, Nicoleta Paraschiv, Oana Vitencu, Mihaela Lazãr

Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

ABSTRACTIntroduction: The primary role of the national respiratory viruses laboratory is to improve the

surveillance by providing reliable testing to support the goals of the national, regional and global measles, rubella and influenza immunization programmes and to monitor seasonal trends of other respiratory viruses as: syncytial respiratory virus, metapneumovirus, parainfluenza (1, 2, 3, 4), coronaviruses (including MERS-CoV), bocaviruses, adenoviruses, rhinoviruses and enteroviruses.


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The main objectives are strengthening national laboratory capacity and improving preparedness for dealing with diagnostic demand during outbreaks of respiratory virus infections, including pandemic influenza virus.

Core activities: 1. laboratory testing, timely and accurate, to notify early the cases and outbreaks to control the

respiratory viruses transmission;2. virologic and genetic surveillance - critical elements for immunization programmes and the

verification of elimination;3. participation in EQA quality assessment and accreditation programmes;4. communication and documentation of laboratory results and data.Results: Out of 6,280 samples tested for the presence of specific IgM antibodies (commercial

kits), in the ongoing measles outbreak (started in 2016), 5,241 samples were confirmed for measles, including 64 deaths. One hundred ninety virus strains were isolated (VeroSLAM cell line) and genotypes B3, D8 were identified with different chains of transmission.

A total of 3,014 cases from sentinel and non-sentinel counties were tested for influenza during 2018/19 season, 1,597 were positive for influenza A virus (n=1,103 A/H1N1 and 494 AH3N2), 35 other respiratory viruses were detected (parainflunenza, rhinovirus, bocavirus and respiratory syncytial virus). The 95 isolated strains were antigenically similar with A/Michigan/45/2015 that was component of 2018/19 influenza vaccine formula. The genetic analysis of hemagglutinin gene revealed new emerging subgroups of A/H1N1 (6B.1A) and A/H3N2 (3C.2a1b).

Conclusions: The use of timely virus detection, isolation, genotyping, epidemiological field investigation (linked with laboratory analyses) to determine the source of infection could lead to a better understanding of the gaps in national surveillance, the root cause(s) of outbreaks, how future outbreaks can be prevented.

Acknowledgements: We are very grateful to our colleagues from the Romanian local public health departments for sending specimens of possible measles cases.

The financial support was provided from the projects: PN 19 14 02 06, I-MOVE GP, I-MOVE Hospital, PN-III-P1-1.1-PD-2016-1726 (IARNASeq).

REFERENCES1. Manual for the Laboratory-based Surveillance of Measles, Rubella, and Congenital Rubella Syndrome,

Third edition, June 2018.2. Manual for the laboratory diagnosis and virological surveillance of influenza, WHO Global Influenza

Surveillance Network, 2011.

ROLUL LABORATORULUI NAÞIONAL DE REFERINÞÃ ÎN DIAGNOSTICUL ŞI SUpRAVEGHEREA INFECÞIILOR RESpIRATORII VIRALE

Carmen Maria Cherciu, Maria Elena Mihai, Iulia Bistriceanu, Cãtãlina Mariana Pascu, Luiza Ustea, Mirela Ene, Emilia Dobre, Nicoleta Paraschiv, Oana Vitencu, Mihaela LazãrInstitutul Naþional de Cercetare-Dezvoltare Medico-Militarã „Cantacuzino”, Bucharest, România

REZUMATIntroducere: Laboratorul Național de Referință furnizează teste fiabile cu scopul de a sprijini

obiectivele programelor naționale, regionale și globale de supraveghere a infecțiilor respiratorii virale (rujeola, rubeola, gripa, virusuri nongripale: respirator sincițial, metapneumovirus, parainfluenza (1, 2, 3, 4), coronavirus (inclusiv MERS-CoV), bocavirus, adenovirus, rhinovirus și enterovirus).


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Principalele obiective sunt consolidarea și creșterea capacității de răspuns cu laboratorul în ve-derea confirmării cazurilor sporadice, limitarea epidemiilor, identificarea de noi tulpini cu potențial pandemic, precum și a mutațiilor care determină rezistența la antivirale sau creșterea virulenței.

Activități de bază:1. teste de laborator, în timp util și exacte, pentru controlul transmiterii virusurilor respiratorii;2. supravegherea virologică și genetică;3. participarea la programele de evaluare și acreditare a calității EQA;4. comunicarea și documentarea rezultatelor și datelor de laborator.Rezultate: Din 6.280 de probe testate pentru prezența anticorpilor IgM specifici rujeolei (truse

comerciale) în cea mai recentă epidemie de rujeolă cu debut în 2016, 5.241 de probe au fost confirmate, inclusiv 64 de decese. O sută nouăsprezece tulpini de virus au fost izolate (linia celulară VeroSLAM). Genotipurile B3, D8 au fost identificate cu diferite căi de transmitere.

Un număr de 3.014 cazuri din județe santinelă și non-santinelă au fost testate pentru gripă în sezonul 2018/19, 1597 au fost pozitive pentru virusul gripal A (1.103 A/H1N1 și 494 AH3N2), alte 35 de virusuri respiratorii au fost identificate (parainflunenza, rinovirus, bocavirus și virusul sincițial respirator). Nouăzeci și cinci de tulpini au fost izolate, iar caracterizarea antigenică a arătat că sunt asemănătoare cu A/Michigan/45/2015, componentă a vaccinului gripal 2018/19. Analiza genetică a hemaglutininei a relevat noi subgrupuri emergente de A/H1N1 (6B.1A) și A/H3N2 (3C.2a1b).

Concluzii: Identificarea virusurilor respiratorii, caracterizarea antigenică și genetică, corelate cu investigarea epidemiologică, ar putea duce la o mai bună înțelegere a cauzelor principale de apariție a focarelor, prevenția viitoare și îmbunătățirea supravegherii la nivel național și în context european.

Mulțumiri: Suntem foarte recunoscători colegilor din direcțiile de sănătate publică din România pentru trimiterea probelor biologice.

Sprijinul financiar: Ministerul Sănătății, Ministerul Apărării, Ministerul Cercetării: Instalația de Interes Național, proiectul Nucleu PN 19 14 02 06, I-MOVE medici de familie, I-MOVE spitale, PN-III-P1-1.1-PD-2016-1726 (IARNASeq).

BIBLIOGRAFIE1. Manual for the Laboratory-based Surveillance of Measles, Rubella, and Congenital Rubella Syndrome,

Third edition, June 20182. Manual for the laboratory diagnosis and virological surveillance of influenza, WHO Global Influenza

Surveillance Network, 2011

6. INNOVATION INTEGRATED IN THE pROCESS OF GLOBAL ERADICATION OF pOLIOMYELITIS

Anda Bãicuş1,2

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

ABSTRACTIntroduction: The Poliovirus, the prototype strain of the Enterovirus genus, Picornaviridae

family, is the etiological agent of poliomyelitis, an acute paralytic disease. After the decision of the World Health Organization from 1988 to globally eradicate poliomyelitis, there was a reduction in the number of countries where wild poliovirus (PV) was still endemic from 125 to two, Afghanistan


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and Pakistan, in 2019. The Polio Endgame Strategy 2019-2023 offers a review of what activities should continue, what improvements will be implemented and what innovations will be introduced, concerning the new genetically stable vaccine strains for the eradication endgame. In our country, poliomyelitis was controlled mostly by using trivalent oral polio vaccine (TOPV) until 2008 and vaccination with inactivated polio vaccine (IPV) started in 2009. In 2015, the risk for poliovirus importation from south-western Ukraine into Romania was real and the environment enterovirus surveillance was intensified.

Material and methods: Between 2016-2019, in the framework of the research projects the mo-lecular methods (RT- PCR) were evaluated in the Enteric Viral Infections Laboratory in Cantacuzino National Medico-Military Institute for Research and Development, by use of the GeneXpert, the Biofire systems and the virus isolation on cell culture lines, to establish the circulation of enterovi-ruses in sewage water and healthy contacts from at risk areas. The co-existence of enteroviruses with other enteropathogens was assessed in healthy children from at risk areas, with a low level of polio vaccine coverage.

Results: No polioviruses have been detected since 2008 in Romania. The co-existence of enteroviruses with different Gram negative bacteria could prolong viral multiplication in the intestine, with the emergence of the genetically modified strains.

REFERENCES1. WHO. Polio Endgame Strategy 2019–2023: Eradication, integration, certification and containment. Geneva:

World. Health Organization; 2019 (WHO/Polio/19.04).2. ECDC. Outbreak of vaccine-derived poliovirus type 1 (cVDPV1) in Ukraine, August 2015 – 2 September,

Stockholm (2015).3. Băicuș A. Monitoring Enterovirus and Norovirus circulation in sewage water using isolation on cell culture

lines and GeneXpert system. Rom Biotechnol Lett. 2019; 24(5): 820-825. DOI: 10.25083/rbl/24.5/820.8254. Băicuș A. The integration of the molecular methods in the diagnosis algorithm for the poliovirus detection

in the sewage water: comparing concentration and detection methods. A Pilot Study Romanian Journal of Internal Medicine, ISSN (Online) 2501-062X, 2017. DOI: https://doi.org/10.1515/rjim-2017-0028

5. Băicuș A. Could the GeneXpert system be a new tool for poliovirus detection in the sewage water. Roum Arch Microbiol Immunol. 2016;1:52-54.

6. băicuș A, Truică C, Pop R, Zorescu C, Potcovaru. Co-existence of enteroviruses with enteropathogens in healthy children in Romania - a Pilot study. Conference of the polio laboratory network, national poliovirus containment coordinators, national authorities for containment. Book of abstracts 24-26 September 2019.

The investigations were funded by the National Authority of Scientific Research and Innovation, Romania, Core projects PN 16 39 01 03/2016-2017, PN 19 14 02 07/2019-2022.

INOVAREA INTEGRATÃ ÎN pROCESUL DE ERADICARE GLOBALÃ A pOLIOMIELITEI

Anda Bãicuş1, 2

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino” Bucureşti, România2Universitatea de Medicinã şi Farmacie „Carol Davila”, Bucureşti, România

REZUMATIntroducere: Poliovirusul, tulpina prototip din genul Enterovirus, familia Picornaviridae, este

agentul etiologic al poliomielitei, o boală paralitică acută. După decizia Organizației Mondiale a Sănătății din anul 1988 de a eradica poliomielita la nivel mondial, a existat o reducere a numărului de țări în care circulația poliovirusului sălbatic (PV) era endemică, de la 125 de țări, la două, în 2019, Afganistan și Pakistan. Strategia Polio pentru perioada 2019-2023 oferă o trecere în revistă a


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activităților care trebuie să continue, a îmbunătățirilor ce vor fi implementate și a inovațiilor care vor fi introduse în ceea ce privește noile tulpini de vaccin stabile genetic ce vor fi utilizate în etapa post eradicare În România, poliomielita a fost controlată prin utilizarea vaccinurilor polio-oral trivalent (TOPV) până în 2008 și polio inactivat (IPV) din 2009. În 2015, riscul pentru importul tulpinilor de poliovirus din sud-vestul Ucrainei în România a fost real, iar supravegherea circulatiei enterovirusurilor în probe de mediu a fost intensificată.

Material și metode: În perioada 2016-2019, în cadrul proiectelor de cercetare realizate în Laboratorul Infecții Enterice Virale din Institutul Național de Cercetare- Dezvoltare Medico-Militară “Cantacuzino” au fost evaluate metode moleculare de detecție virală (RT-PCR), prin utilizarea sistemelor GeneXpert, Biofire și metoda de izolare a virusului pe liniile de culturi celulare, pentru stabilirea circulației enterovirusurilor în ape uzate și la copii sănătoși, din zone la risc. A fost evaluată coexistența enterovirusurilor cu alți enteropatogeni, la copii sănătoși, cu un nivel scăzut de protecție vaccinală polio.

Rezultate: În România nu au mai fost izolate tulpini de poliovirus din anul 2008. Coexistența enterovirusurilor cu diferite bacterii Gram negative ar putea prelungi multiplicarea virală în intestin, cu apariția tulpinilor modificate genetic.

BIBLIOGRAFIE1. WHO. Polio Endgame Strategy 2019–2023: Eradication, integration, certification and containment. Geneva:

World. Health Organization; 2019 (WHO/Polio/19.04).2. ECDC. Outbreak of vaccine-derived poliovirus type 1 (cVDPV1) in Ukraine, August 2015 – 2 September,

Stockholm (2015).3. băicuș A. Monitoring Enterovirus and Norovirus circulation in sewage water using isolation on cell culture

lines and GeneXpert system. Rom Biotechnol Lett. 2019; 24(5): 820-825. DOI: 10.25083/rbl/24.5/820.8254. Băicuș A. The integration of the molecular methods in the diagnosis algorithm for the poliovirus detection

in the sewage water: comparing concentration and detection methods. A Pilot Study Romanian Journal of Internal Medicine, ISSN (Online) 2501-062X, 2017. DOI: https://doi.org/10.1515/rjim-2017-0028

5. Băicuș A. Could the GeneXpert system be a new tool for poliovirus detection in the sewage water. Roum Arch Microbiol Immunol. 2016;1:52-54.

6. băicuș A, Truică C, Pop R, Zorescu C, Potcovaru. Co-existence of enteroviruses with enteropathogens in healthy children in Romania - a Pilot study. Conference of the polio laboratory network, national poliovirus containment coordinators, national authorities for containment. Book of abstracts 24-26 September 2019.

Investigațiile au fost finanțate de Autoritatea Națională de Cercetare Științifică și Inovare, România, Proiect Nucleu (PN); PN 16 39 01 03 / 2016-2017, PN 19 14 02 07 / 2019-2022.

7. OpTIMIZATION AND VALIDATION OF DIAGNOSTIC METHODS OF YELLOW FEVER IN REFERENCE LABORATORY FOR VECTOR-BORNE INFECTIONS FROM CANTACUZINO NATIONAL MEDICO-MILITARY

INSTITUTE FOR RESEARCH AND DEVELOpMENT

Ani Ioana Cotar1, Simin Aysel Florescu2, Corneliu Petru Popescu2, Daniela Bãdescu1, Cornelia Svetlana Ceianu1

1Laboratory for Vector-Borne Infections, Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

2 Dr. Victor Babeş Clinical Hospital of Infectious and Tropical Diseases, Bucharest, Romania

ABSTRACTIntroduction: Yellow fever virus is a mosquito-borne flavivirus causing a severe disease with a

high fatality. Yellow fever virus is endemic in Africa and South-America and the infection may be


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prevented with a live-attenuated vaccine. Between January 2017-March 2018 a yellow fever outbreak with 1712 cases occurred in Brasil.

Objectives: The main goal of our study was to optimise serological and molecular diagnostic methods and to validate them by confirming case imported from Brasil.

Methods: Indirect immunofluorescence tests were used to determine the presence of antibodies to yellow fever virus in different phases of infection, and four different molecular methods to detect the viral genome.

Results: Seroconversion for IgM and IgG antibodies occurred seven days from disease onset. Three out of four molecular methods tested were adequate for the detection of the viral strain involved in Brasil epidemic.

Conclusion: The use of the serological method is limited by the cross-reactivity of antibodies to other flaviviruses and late seroconversion. The early diagnostic of yellow fever may be performed with molecular methods that have to be continuosly modified, adapted and optimized according to the genotypes of circulating viral strains.

OpTIMIZAREA ŞI VALIDAREA METODELOR DE DIAGNOSTIC AL FEBREI GALBENE ÎN LABORATORUL DE REFERINÞÃ INFECÞII TRANSMISE pRIN VECTORI DIN INSTITITUTUL NAÞIONAL DE CERCETARE-DEZVOLTARE

MEDICO-MILITARÃ “CANTACUZINO”

Ani Ioana Cotar1, Simin Aysel Florescu2, Corneliu Petru Popescu2, Daniela Bãdescu1, Cornelia Svetlana Ceianu1

1Laboratorul Infecþii Transmise prin Vectori, Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã „Cantacuzino”, Bucureşti, România

2 Spitalul de Boli Infecþioase Tropicale „Dr. Victor Babeş”, Bucureşti, România

REZUMATIntroducere: Virusul febrei galbene este un flavivirus transmis de țânțari și produce o boală

severă cu o rată crescută de fatalitate. Virusul este endemic în regiuni din Africa și America de Sud. Infecția este prevenibilă prin vaccinare cu vaccin viu atenuat. Între ianuarie 2017 – martie 2018 în Brazilia a evoluat o epidemie cu 1712 cazuri de febră galbenă.

Obiective: Obiectivul studiului a fost optimizarea metodelor serologice și moleculare de diagnostic al febrei galbene validate prin confirmarea unui caz importat din Brazilia.

Metode: Am testat metoda imunofluorescenței indirecte pentru detecția anticorpilor specifici față de virusul febrei galbene în diferite etape ale infecției, precum și patru metode moleculare pentru detecția genomului viral.

Rezultate: Seroconversia pentru anticorpii IgM și IgG s-a constatat la 7 zile de la debutul clinic. Dintre cele 4 teste moleculare testate, trei au fost adaptate detecției tulpinii virale implicate în epidemia din Brazilia.

Concluzie: Diagnosticul serologic este util, dar prezintă limite legate de reactivitatea încrucișată a anticorpilor față de alte flavivirusuri și de seroconversia târzie, ceea ce împiedică un diagnostic precoce al acestei infecții grave. Diagnosticul timpuriu al febrei galbene poate fi realizat prin metode moleculare, care trebuie permanent modificate, adaptate și optimizate în raport cu genotipurile tulpinilor virale circulante.


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8. EOSINOpHILIA IN TRICHINELLOSIS

Alexandru Cristian Vasile-Bugarin Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

ABSTRACTHuman trichinellosis is a disease produced in our country by the smallest human nematode,

Trichinella spiralis, by consuming infected pork. It seems that after the epidemics of 1990-2000 it is reemerging.

The semiology is not too specific, the immune response is slow and so does eosinophilia, so great care must be taken in differential diagnostic. An elevated eosinophilia between 1500 and 4000, ruled out vascular and autoimmune diseases; the rest of tissular helminthiasis would be rejected by epidemiological findings and, finally, by immunodiagnostic.

Eosinophilia has two distinctive features: a specific production of cytokines, especially IL-5 which facilitates the encystation of larvae; the absence of it, in positive cases, is a bad omen.

EOZINOFILIA ÎN TRICHINELOZÃ

Alexandru Cristian Vasile-Bugarin Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã „Cantacuzino”, Bucureşti, România

REZUMATTrichineloza, boală produsă la om, în România, aproape exclusiv de Trichinella spiralis prin

consumul de carne de porc infestată și insuficient prelucrată termic este reemergentă în ultimii doi ani, după vârfurile epidemice din anii nouăzeci – începutul noului mileniu.

Clinica bolii nu este foarte caracteristică, diagnosticul diferențial făcându-se cu numeroase boli vasculare și autoimune, ca și cu alte boli parazitare. În cazul prezenței eosinofiliei (sau mai corect spus a hipereosinofiliei), diferența față de alte boli produse de helminți va fi presupusă de prezența elementelor epidemiologice. Diagnosticul imunologic va aduce certitudinea.

Eosinofilia în trichineloză, relativ tardivă și la valori mediane (1500-4000/mmc), mai rar peste 70%, are două particularități ce o deosebesc de celelalte helmintice: o producție de citokine, mai ales IL-5 ce protejează miocitele de apoptoză și permite închistarea larvelor; iar absența eosinofiliei anunță un prognostic sumbru.

9. BIOSAFETY AND BIOSECURITY. BASIC pRINCIpLES

Cristina ÞãrmureanCantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

ABSTRACTThe purpose of the presentation was to understand the general principles and issues related to

biosafety and biosecurity, to identify solutions for reducing the biorisk, to establish communication paths between the parties involved and to present responsibilities of the biosafety officer.

According to CWA 15793, the objectives of the biorisk management are as follows: informing all employers about the individual obligations regarding the prevention of the biorisk in the laboratory, risks assessment for different projects and work areas, identifying the aspects that could be improved, promoting the activities of prevention, the use of documented procedures and instructions, the


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continuous improvement of the control measures of biorisk, communication between parties, providing the necessary resources for the education and the training of the laboratory personnel, identifying opportunities and establishing measures and targets for improvement.

The main procedures related to biorisk are: PERSONNEL SELECTION, MANAGEMENT OF THE BIOLOGICAL MATERIAL, IMPLEMENTATION OF SAFE WORK PRACTICES, PROTECTIVE EQUIPMENT, DECONTAMINATION AND DISINFECTION, PROCEDURES OF TRANSPORT OF BIOLOGICAL MATERIAL, INCIDENTS AND ACCIDENTS, RISK ASSESMENT.

Principles of risk mitigation and measures of controlling the biorisk situations are biorisk assessment, identifying ways of reducing the biorisk (use of non-pathogenic strains, the use of safe working practices, the use of individual protective equipment, facility with technical protection equipment, selection of appropriately trained personnel, action ways in different emergency situations), development of various scenarios and evaluating the staff reactions, the equipment and the utilities of the laboratory.

In case of incidents - accidents (spills of contaminated material, cuts with sharp objects, failure of the protective equipment, failure of the laboratory facilities: decontamination system, autoclaving, ventilation systems) the steps to be followed are: Alert, Securization and Organization, Decontamination, Cleaning and waste disposal, Informing the Principal Investigator of the Laboratory and the Biosafety Officer.

REFERENCES1. CEN WORKSHOP AGREEMENT 15793 /20082. Laboratory biorisk management-Guidelines for implementation of CWA 15793:20083. National Biosafety guide for Medical Laboratories /20054. Biosafety Manual IFBA 2018

BIOSIGURANȚĂ ȘI BIOSECURITATE. PRINCIPII DE BAZĂ

Cristina ÞãrmureanInstitutul Naþional de Cercetare-Dezvoltare Medico-Militarã „Cantacuzino”, Bucureşti, România

REZUMATScopul prezentării este înțelegerea principiilor generale și a problemelor legate de biosiguranță

și biosecuritate, identificarea de soluții de micșorare a bioriscului, stabilirea de căi de comunicare între părțile implicate, prezentarea responsabilităților BSO (ofițer de biosiguranță).

Conform CWA 15793, obiectivele managementului bioriscului sunt: informarea tuturor angajaților privind obligațiile individuale cu privire la prevenirea bioriscului în laborator, evaluarea riscurilor pentru proiectele și zonele de lucru, identificarea aspectelor care ar putea fi îmbunătățite, promovarea activităților de prevenire, utilizarea de proceduri și instrucțiuni documentate, îmbunătățirea continuă a performanțelor de control ale bioriscului, comunicarea continuă între părţi, asigurarea resurselor necesare pentru educație și pentru formarea personalului din laborator, inclusiv punerea la dispoziţie de metode și instrumente de îmbunătățire continuă, identificarea oportunităţilor de îmbunătățire și stabilirea de măsuri și ținte de îmbunătățire.

Principalele proceduri legate de biorisc sunt: SELECTAREA PERSONALULUI, GESTIONAREA MATERIALULUI BIOLOGIC, IMPLEMENTAREA DE PRACTICI DE LUCRU SIGURE, ECHIPAMENTUL DE PROTECȚIE, PROCEDURI DE DECONTAMINARE ȘI DEZINFECȚIE, PROCEDURI DE TRANSPORT AL MATERIALULUI BIOLOGIC, PLANURI DE ACȚIUNE ÎN CAZ DE ACCIDENTE-INCIDENTE, EFECTUAREA DE ANALIZE DE RISC.

Principiile reducerii bioriscului și modalitățile de control al situațiilor de risc încep cu evaluarea bioriscului, identificarea de căi de micșorare a bioriscului (folosirea de tulpini nepatogene, folosirea de practici de lucru sigure, utilizarea echipamentului individual de protecție și asigurarea


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laboratorului cu echipament tehnic de protecție, selectarea de personal instruit corespunzător, stabilirea de modalități de acțiune în diferite situații de urgență), stabilirea de căi de urmat în diverse scenarii și evaluarea reacțiilor personalului, a funcționării echipamentului și a funcționării utilităților laboratorului.

În cazul unor incidente-accidente (revărsări de material contaminat, tăieri cu obiecte ascuțite, defecțiuni ale echipamentului de protecție, defecțiuni ale facilităților laboratorului: stație de epurare, autoclav, sisteme de ventilație) etapele de urmat sunt: Alertarea personalului, Securizare și Organizare, Decontaminare, Curățare și eliminare deșeuri, Anunțare Șef laborator și Ofițer de biosiguranță.

BIBLIOGRAFIE1. CEN WORKSHOP AGREEMENT 15793/20082. Laboratory biorisk management-Guidelines for implementation of CWA 15793:20083. Ghid naţional de siguranţă pentru laboratoarele medicale/20054. Biosafety Manual IFBA 2018

10. CHARACTERIZATION OF COLON TUMOR CELLS ISOLATED FROM TUMOR SpECIMENS

Iulia I. Niþã, Aurora Sãlãgeanu, Iuliana CaraşCantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

ABSTRACTIntroduction: The long term primary cultures of human colon cancer cells derived from tumor

specimens serve as in vitro models for the study of the circulating tumor cells (CTC) and circulating stem cells (CSC). These subpopulations of tumor cells play a significant role in monitoring of the therapeutic response and the development of novel drugs.

In culture, individual primary colon tumor cells are organized into spherical 3D structures and paired adherent tumor cells. Primary colon tumor cells derived from tumorspheres are extensively characterized and express epithelial markers (e.g. EpCAM, CK20, panCK) and stem markers (e.g. CD44).

Objectives: This study aims to characterize long term adherent primary tumor cells by examining the expression marker profile.

Methods: The tumor tissue samples collected after colon resection were minced in smaller pieces and cultured at 37°C in a 5% CO2 humidified cell culture incubator. Immunofluorescent staining was performed on human colon tumor cells using antibodies against EpCAM-FITC, panCK-PE, CK20-PE, CD44-APC, CDX2-FITC and nuclear staining with DAPI.

Results: Immunofluorescent assessment of adherent tumor cells displayed the co-expression of epithelial markers EpCAM, panCK and CK20 in a high proportion of the cells. Conversely, the CD44 expression was partially distributed in an extremely low number of cells. Moreover, the expression pattern of CD44 in adherent cells was totally different from CD44 positive colon tumor cells derived from tumorsphere. In addition, CDX2 immunofluorescent staining was absent on both CK20 and CD44 positive adherent tumor cells and expressed alone on a very low number of cells.

Conclusion: The results of our study show that adherent primary tumor cells comprise an abundant proportion of epithelial colon tumor cells and a very low number of differentiated and potential stem like tumor cells.


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CARACTERIZAREA CELULELOR TUMORALE IZOLATE DIN TUMORI COLORECTALE

Iulia I. Niþã, Aurora Sãlãgeanu, Iuliana CaraşInstitutul National de Cercetare-Dezvoltare Medico-Militara “Cantacuzino”, Bucuresti, România

REZUMATIntroducere: Culturile primare ale celulelor umane tumorale de colon provenite din probe de

țesut tumoral servesc ca modele in vitro pentru studiul celulelor tumorale circulante (CTC) și celulelor stem circulante (CSC). Aceste subpopulații de celule tumorale joacă un rol important în monitorizarea răspunsului terapeutic și dezvoltarea de noi medicamente. În cultură, celulele primare tumorale de colon sunt organizate simultan în structuri sferice 3D (sferoizi) și celule tumorale aderente.

Celulele tumorale primare de colon provenite din sferoizi sunt intens caracterizate și exprimă markeri epiteliali (EpCAM, CK20, panCK) și stem (CD44).

Obiectiv: Acest studiu propune caracterizarea celulelor primare tumorale aderente, menținute în cultură timp de mai multe luni, prin examinarea profilului markerilor exprimați.

Metode: Probe de țesut tumoral au fost mărunțite în bucăți foarte mici și cultivate la 37°C într-un incubator 5% CO2. Marcarea imunofluorescentă a fost efectuată pe celule umane tumorale de colon folosind anticorpi împotriva EpCAM-FITC, panCK-PE, CK20-PE, CD44-APC, CDX2-FITC, iar marcarea nucleară s-a făcut cu DAPI.

Rezultate: Evaluarea imunofluorescentă a celulelor tumorale aderente a arătat o coexpresie a markerilor epiteliali EpCAM, panCK si CK20 într-o proporție ridicată de celule. În schimb, expre-sia markerului CD44 a fost parțial distribuită într-un număr de celule extrem de mic. Mai mult, pattern-ul expresiei marker-ului CD44 în celulele aderente a fost complet diferită de celulele CD44 pozitive provenite din sferoizi. În plus, marcarea imunofluorescentă pentru marker-ul CDX2 a fost absentă în ambele tipuri de celule tumorale aderente CK20 și CD44 pozitive, dar exprimat singur într-un număr infim de celule.

Concluzie: Rezultatele studiului nostru arată că celulele primare tumorale aderente cuprind din abundență celule epiteliale tumorale de colon și o populație minoră de celule diferențiate și potențial celule stem.

11. CYTOTOXICITY EVALUATION OF SOME STABILIZED IRON OXIDE pOWDERS AND SUSpENSIONS FOR BIOMEDICAL AppLICATIONS

Dora Domnica Baciu1, Iulia Ioana Lungu2, Andrei-Mihai Dumitraşcu1, Gabriel Prodan3, Aurora Sãlãgeanu1, Florin Dumitrache2

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2 National Institute for Laser, Plasma and Radiation Physics, Bucharest, Romania

3Ovidius University, Constanþa, Romania

Introduction: The nanoparticles involved in this research are designed to be used for medical purposes because they have the ability to be targeted in a magnetic field to the tumor area, where they could be thermally activated to destroy cancer cells.

For this, iron oxide nanoparticles (IONPs) must be biocompatible with the human body.Objectives: The present study concerns two complementary directions: evaluation of cytotoxicity of iron nanoparticles


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evaluation of the inflammatory response triggered by macrophages as a result of treatment with these agents.

Methods: The investigated nanoparticle iron powders were prepared by laser pyrolysis technique.

1. Determination of cell viability using MTT assay2. Evaluation of cytotoxicity by determining nitric oxide concentration3. Evaluation of immunogenicity by quantification of pro-inflammatory cytokinesResults: For both the RAW 264.7 macrophage cell line and the BMDC, cell viability and

proliferation were not affected or were to a very small extent affected by exposure to MNPs, even at high concentrations.

Viability after 24 h for RAW 264.7 treated with various NP +/- LPS 1 μg/ml

NPs are not cytotoxic by inducing NO at concentrations up to 50 µg/mLFor both investigated lines, NPs do not exhibit proinflammatory potential, not inducing the

synthesis of specific cytokines TNFα and IL1ß.Conclusions: Our results demonstrated that two IONPs synthesized by laser pyrolysis technique

are promising candidates for future biomedical applications because they are biocompatible and do not induce the production of inflammatory mediators in macrophages.

EVALUAREA CITOTOXICITÃÞII UNOR pULBERI ŞI SUSpENSII STABILIZATE pE BAZÃ DE OXIZI DE FIER pENTRU ApLICAÞII BIOMEDICALE

Dora Domnica Baciu1, Iulia Ioana Lungu2, Andrei-Mihai Dumitraşcu1, Gabriel Prodan3, Aurora Sãlãgeanu1, Florin Dumitrache2

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino” (INCDMM), Bucureşti, România2Institutul Naþional de Cercetare-Dezvoltare pentru Fizica Laserilor, Plasmei şi Radiaþiei (INFLPR), Bucureşti, România

3Universitatea Ovidius din Constanþa, România

REZUMATIntroducere: Nanoparticulele implicate în această cercetare sunt concepute pentru a fi utilizate

în scopuri medicale, deoarece prezintă capacitatea de a fi direcționate țintit într-un câmp magnetic spre zona tumorală, unde ar putea fi activate termic pentru a distruge celulele canceroase.

Pentru aceasta, nanoparticulele de oxid de fier (IONPs) trebuie să fie biocompatibile cu organismul uman.


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Obiective: Studiul de față vizează două direcții complementare: evaluarea citotoxicității nanoparticulelor de fier evaluarea răspunsului inflamator declanșat de macrofage ca urmare a tratamentului cu acești

agenți.Metode: Pulberile de nanoparticule de fier investigate au fost preparate prin tehnica pirolizei

laser.1. Determinarea viabilității celulare folosind testul MTT2. Evaluarea citotoxicității prin determinarea concentrației de oxid nitric 3. Evaluarea imunogenicității prin cuantificarea unor citokine proinflamatorii

Rezultate: Atât pentru linia de celule macrofage RAW 264.7, cât și pentru BMDC, viabilitatea și proliferarea celulară nu au fost afectate sau au fost afectate într-o foarte mică măsură de expunerea la MNPs, chiar și în concentrații ridicate.

Viabilitatea după 24 h pentru RAW 264.7 tratate cu diverse NP +/- LPS 1 μg/ml

NPs nu sunt citotoxice prin inducerea de NO la concentrații de până la 50 µg/mLPentru ambele linii investigate, NPs nu prezintă potențial proinflamator, neinducând sinteza de

citokine specifice TNFα și IL1ß.

Concluzii: Rezultatele noastre au demonstrat că două IONP sintetizate prin tehnica pirolizei laser sunt candidați promițători pentru aplicațiile biomedicale viitoare, deoarece sunt biocompatibile și nu induc producția de mediatori inflamatori în macrofage.


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12. AN IN VITRO MODEL FOR TESTING THE INFLUENCE OF THE ETIOLOGIC AGENTS OF BACTERIAL MENINGITIS

ON BLOOD BRAIN BARRIER

Andrei Mihai Dumitraşcu1, Ramona Caragheorgheopol1,2, Cãtãlin Þucureanu1, Marina Panã1, Andrei Alexandru Muntean1,3, Mãdãlina Maria Muntean3, Veronica Lazãr2, Aurora Sãlãgeanu1

1Cantacuzino National Medico-Military Institute for Research and Development, Immunology Laboratory, Bucharest, Romania2University of Bucharest, Faculty of Biology, Bucharest, Romania

3Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

ABSTRACTIntroduction: Meningitis is an acute condition characterized by inflammation of the meninges,

which contributes largely to the mortality and morbidity associated with the disease.Objectives: The aim of this study was to investigate the interaction of immune cells with

meningitis-causing pathogens.Methods: An experimental model of co-cultured EA.hy926 endothelial cells and THP-1 monocyte

cells infected with Neisseria meningitidis (Nm) and Streptococcus pneumoniae (Sp) was developed.EaHy cells were grown to confluence on inserts in 24 well plates and labeled with fluorescent

lipophilic cationic indocarbocyanine dye DiI. THP-1 cells labeled with DiO fluorescent dye were added both in the superior and inferior chambers.

Cell cultures were incubated for 24 hours at 37O C and 5% CO2 in the presence or absence of alive or thermally killed Nm and Sp. TLR2 ligand PAM3CSK4 was used as a positive control.

Following incubation, the supernatant was collected, both cell types were fixed with paraformaldehyde and their nuclei stained with DAPI.

TNF-alpha levels from the supernatant of both chambers were measured using ELISA. Cells were microscopically observed in phase contrast and fluorescence to determine the morphological changes induced by stimulation as well as macrophages adherence to endothelial cells.

Results: Following activation by Nm and Sp, THP-1 cells in the inferior chambers differentiated in macrophages and induced adherence of THP-1 cells in the superior chamber onto EaHy layer. The adherence was more pronounced after stimulation with heat inactivated Nm and Sp. At the same time point, of the monolayer was destroyed upon stimulation with live bacteria probably due to the higher cytokine secretion.

Conclusion: The proposed model was suitable to study the interaction of heat killed bacteria while different experimental conditions should be tested for live pathogens.

INFLUENÞA AGENÞILOR ETIOLOGICI AI MENINGITEI BACTERIENE ASUpRA BARIEREI HEMATOENCEFALICE: MODEL IN VITRO

Andrei Mihai Dumitraşcu1, Ramona Caragheorgheopol1,2, Cãtãlin Þucureanu1, Marina Panã1, Andrei Alexandru Muntean1,3, Mãdãlina Maria Muntean3, Veronica Lazãr2, Aurora Sãlãgeanu1

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino”, Laboratorul Imunologie, Bucureşti, Romania2Universitatea Bucureşti, Facultatea de Biologie, Bucureşti, Romania

3Universitatea de Medicinã şi Farmacie “Carol Davila”, Bucureşti, România

REZUMATIntroducere: Meningita este o afecțiune acută, caracterizată prin inflamația meningelor, care

contribuie în mare măsură la mortalitatea și morbiditatea asociate cu boala.


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Obiective: Scopul acestui studiu a fost investigarea interacțiunii celulelor imune cu agenții patogeni care provoacă meningită.

Metode: A fost dezvoltat un model experimental de celule endoteliale EA.hy926 co-cultivate cu celule monocite THP-1, infectate cu Neisseria meningitidis (Nm) și Streptococcus pneumoniae (Sp).

Celulele EaHy au fost cultivate până la confluență pe inserturi în plăci cu 24 de godeuri și marcate cu fluorocromul lipofilic și cationic indocarbocianin DiI. Celule THP-1 marcate cu colorant fluorescent DiO au fost adăugate atât în camerele superioare, cât și în cele inferioare.

Culturile celulare au fost incubate timp de 24 de ore la 37° C și 5% CO2 în prezența și/sau absența Nm și Sp, vii sau inactivate termic. Ligandul TLR2 PAM3CSK4 a fost utilizat ca și control pozitiv.

După incubare, a fost colectat supernatantul, ambele tipuri celulare au fost fixate cu paraformaldehidă, iar nucleii lor au fost colorați cu DAPI.

Nivelul de TNF-alfa din supernatantul ambelor camere a fost măsurat prin ELISA. Celulele au fost observate microscopic în contrast de fază și fluorescență pentru a determina modificările morfologice induse de stimulare, precum și aderarea macrofagelor la celulele endoteliale.

Rezultate: După activarea cu Nm și Sp, celulele THP-1 din camera inferioară au diferențiat în macrofage și au indus aderarea celulelor THP-1 din camera superioară pe stratul de EaHy. Aderența a fost mai accentuată în cazul stimulării cu Nm și Sp inactivate termic. În același timp, o parte a monostratului a fost distrusă ca urmare a stimulării cu bacteriile vii, probabil datorită secreției mai mari de citokine.

Concluzie: Modelul propus a fost potrivit pentru a studia interacțiunea bacteriilor inactivate termic, în timp ce ar trebui testate diferite condiții experimentale pentru agenți patogeni vii.

13. pRELIMINARY RESULTS IN THE pROCESS OF OSSEOINTEGRATION OF A DENTAL IMpLANT IN THE RABBIT TIBIA

Diana Larisa Ancuþa1, 2, Jasmina Manolescu1, Cristin Coman1, 3

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, Bucharest, Romania

3Spiru Haret University, Faculty of Veterinary Medicine, Bucharest, Romania

ABSTRACTOsseointegration of dental implants is one of the important stages of oral surgery, especially

considering the overlapping bacterial infections.The aim of the study is to improve the process of implant osseointegration by preventing the

formation of the bacterial biofilm, which is the main cause of rejection of these devices. The rabbit model has not yet been adapted for the study of osseointegration in correlation with a bacterial source capable of forming biofilms.

In order to achieve the objective, two experiments were performed. The first stage followed the process of osseointegration of Zimmer TMMB10 dental implants that were mounted in the rabbit tibia. A lot of animals was created that underwent surgery as follows: deep anesthesia, toilet of the left hind limb by trimming and antisepsis with Iodine 2%, followed by incision of the skin and subcutaneous tissues up to the level of the cortex. Under continuous jet of pure water a bicortical cavity was executed, with the final diameter of 4 mm into which the dental implant was inserted. The operation was completed by suturing the layers, and the animals received treatment with antibiotics and analgesics. It followed a period of 30 days to monitor the general and local status. The second stage of the study consisted in the reproduction of bone infection using a Staphylococcus epidermidis strain that was isolated from a prosthesis implanted in the human knee. For this


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purpose, two batches of animals were taken in which the bacterial suspension was inoculated in two concentrations: 5x106CFU/mL and 5x108CFU/mL. Cotton meshes were used as a factor for the reproduction of the infection. Each group of rabbits benefited from the same preoperative technique. The area of election was the tibia of the right member, the congener being taken as a witness. At the level of the exposed cortex, two bone defects were created which were augmented with the stems immersed in the staphylococcus culture in the concentrations established per lot, over which 0.1 ml of bacteria was inoculated. The wound was sutured and the animals were treated with analgesics. Monitoring f or 30 days evaluated the installation of the disease through clinical, hematological, microbiological and histopathological examination.

This research work was carried out with the support of the Ministry of Education and Research of Romania under the Projects EuroNanoMed III-ANNAFIB.

REZULTATE pRELIMINARE ÎN pROCESUL DE OSTEOINTEGRARE A UNUI IMpLANT DENTAR ÎN TIBIA DE IEpURE

Diana Larisa Ancuþa1, 2, Jasmina Manolescu1, Cristin Coman1, 3

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã „Cantacuzino”, Bucureşti, România2Universitatea de Ştiinte Agronomice şi Medicinã Veterinarã, Facultatea de Medicinã Veterinarã, Bucureşti, România

3 Universitatea „Spiru Haret”, Facultatea de Medicinã Veterinarã, Bucureşti, România

REZUMATOsteointegrarea implanturilor dentare reprezintă una dintre etapele importante ale chirurgiei

orale, mai ales ținând cont de infecțiile bacteriene suprapuse. Scopul studiului este acela de a îmbunătăți procesul de osteointegrare al implantului prin

prevenirea formării biofilmului bacterian care reprezintă principala cauză de respingere a acestor dispozitive. Modelul de iepure nu a fost adaptat încă pentru studiul osteointegrării în corelație cu o sursă bacteriană capabilă să formeze biofilm.

Pentru realizarea obiectivului, au fost efectuate două experimente. Prima etapă a urmărit procesul osteointegrării implanturilor dentare Zimmer TMMB10, care au fost montate în tibia de iepure. S-a creat un lot de animale care a fost supus intervenției chirurgicale astfel: anestezie profundă, toaleta membrului posterior stâng prin tundere și antisepsie cu Iod 2%, urmată de incizia pielii si a țesuturilor subcutanate până la nivelul corticalei. Sub jet continuu de apă pură, a fost executată o cavitate bicortical, cu diametrul final de 4 mm, în care a fost introdus implantul dentar. Operația a fost finalizată prin sutura straturilor, iar animalele au primit tratament cu antibiotice și analgezice. A urmat o perioadă de 30 de zile de monitorizare a stării generale și locale. Cea de-a doua etapă a studiului a constat în reproducerea infecției osoase prin utilizarea unei tulpini de Staphylococcus epidermidis, ce a fost izolată de la nivelul unei proteze implantate la genunchi uman. Pentru aceasta, au fost luate în lucru două loturi de animale, la care s-a inoculat suspensia bacteriană în două concentrații: 5x106 UFC/mL și 5x108 UFC/mL. Ca factor favorizant pentru reproducerea infecției, s-au utilizat meșele de bumbac. Fiecare lot de iepuri a beneficiat de aceeași tehnică preoperatorie. Zona de elecție a fost tibia membrului drept, congenerul fiind luat drept martor. La nivelul corticalei expuse au fost create câte două defecte osoase care au fost augmentate cu meșele imersate în cultura de stafilococ în concentrațiile stabilite per lot, peste care a mai fost inoculat 0,1 ml bacterie. Plaga operată a fost suturată și animalele au fost tratate cu analgezice. Monitorizarea timp de 30 de zile a evaluat instalarea bolii prin examen clinic, hematologic, microbiologic și histopatologic.

Acest studiu a fost finanțat de către Ministerul Educației și Cercetării din România în cadrul proiectului EuroNanoMed III-ANNAFIB.


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14. CLINICAL AND pARACLINICAL CHANGES IN ACUTE MnCL2 INDUCED TOXICITY IN BALB/C MICE

Claudiu Gal1, Mariana Vãduva1, Mihaela Diaconu1, 2, Crina Stãvaru1

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2University of Bucharest, Faculty of Biology, Bucharest, Romania

ABSTRACTIntroduction: Excess intake of manganese produces dysfunctions of the metabolic and immune

system. In addition, high levels of this compound affect several organs, including brain, liver, pancreas, and kidney.

Objective: The present study aimed to evaluate clinical symptoms, hematological and serum biochemical changes, and histological lesions induced by the MnCl2 dehydrate acute toxicity by intraperitoneal, subcutaneous and oral administration. Groups of five BALB/c female mice received three times per week a dose of 150 mg/kg (body weight) of MnCl2 in sterile solution, injected intraperitoneally for 14 days, subcutaneously for 20 days, and a dose of 500 mg/kg by oral gavage for 28 days, each dose administered three times per week. One control group received subcutaneous injections whereas another, oral gavage of 0.9% sterile saline solution.

Methods: Animal weight and water consumption were monitored three times per week. Blood analysis was performed at the beginning and the end of the experiment, with an intermediary complete blood count for the oral gavage group. Freshly collected tissue samples of liver, lung, heart, pancreas, spleen, kidney, adrenal gland, ovary, uterus, bone, skeletal muscle, stomach, intestine, encephal and cerebellum were formalin fixed, paraffin embedded, 5 µm thick sectioned and hematoxylin-eosin stained.

Results: The parenteral administration groups showed a diminished weight gain and water consumption, a severe local inflammatory effect, reflected also in the serum proteins increased values and the higher number of white blood cells, and an impaired hepatic and pancreatic function by the severe hepatitis and mild pancreatitis. Oral MnCl2 intoxication determined hepatic steatosis, mild to moderate acute gastritis.

Conclusions: High MnCl2 concentration have an irritant effect on the gastric mucosa and a moderate to severe hepatotoxic effect.

This work is supported by the Ministry of Research and Innovation, project number PN-III-P1-1.2-PCCDI-2017-0737.

MODIFICÃRI CLINICE ŞI pARACLINICE INDUSE DE INTOXICAÞIA ACUTÃ CU MnCL2 pE MODEL MURIN

Claudiu Gal1, Mariana Vãduva1 , Mihaela Diaconu1, 2, Crina Stãvaru1

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino”, Bucureşti, România2Universitatea Bucureşti, Facultatea de Biologie, Bucureşti, România

REZUMATIntroducere: Aportul în exces de mangan produce disfuncții ale sistemului metabolic și imunitar.

De asemenea, nivelul ridicat de mangan afectează numeroase organe, incluzând creierul, ficatul, pancreasul sau rinichii.

Obiectiv: Scopul acestui studiu este de a evalua simptomele clinice, modificările hematologice, ale biochimiei serice și histopatologice induse de intoxicația acută cu MnCl2 dihidrat, prin


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administrarea intraperitoneală, subcutanată sau orală. Grupuri a câte 5 femele de șoareci BALB/c au primit de 3 ori pe săptămână o doză de 150 mg/kg (greutate corporală) MnCl2 în soluție sterilă, injectată intraperitoneal timp de 14 zile, subcutanat pentru 20 de zile și o doză de 500 mg/kg prin gavaj, timp de 28 zile. Un grup control a primit injecții subcutanate, iar un altul gavaj cu soluție salină sterilă 0.9%.

Metode: Greutatea animalelor și consumul de apă au fost monitorizate de 3 ori pe săptămână. Analizele de sânge s-au efectuat la începutul și la finalul studiului, cu o recoltare intermediară pen-tru lotul cu gavaj. Probe de țesut proaspăt recoltate din ficat, pulmon, cord, pancreas, splină, rinichi, glande suprarenale, ovar, uter, os, mușchi scheletic, stomac, intestin, encefal și cerebel au fost fixate în formol, incluse în parafină, secționate la o grosime de 5 µm și colorate cu hematoxilină-eozină.

Rezultate: Grupurile cu administrare parenterală au prezentat o creștere în greutate și un con-sum de apă reduse, un răspuns local inflamator sever, observat și în valorile proteinelor serice sau în creșterea numărului de celule albe, precum și o disfuncție hepatică și pancreatică, manifestate prin hepatită severă și pancreatită ușoară. Intoxicația pe cale oral cu MnCl2 a determinat apariția steatozei hepatice și o gastrită acută, ușoară spre moderată.

Concluzii: Concentrațiile ridicate de MnCl2 pot avea un efect iritant asupra mucoase gastrice și un efect hepatotoxic moderat spre sever.

Acest studiu a fost susținut de Ministerul Cercetării și Inovării, proiect PN-III-P1-1.2-PCCDI-2017-0737.

15. IN VITRO STUDY OF THE EFFECT INDUCED BY MnCL2

ON FIBROBLAST CELL LINE

Ştefania Lascãr1, Irina Elena Ionescu1, Raluca Elena Lãzãrescu1, Vlad Tofan1, Mihaela Diaconu1,2, Cãtãlin Þucureanu1, Ana Şerbãnescu1, Crina Stãvaru1, Adrian Onu1,3

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2University of Bucharest, Faculty of Biology, Bucharest, Romania

3Titu Maiorescu University, Faculty of Pharmacy, Bucharest, Romania

ABSTRACTIntroduction: Manganese is an essential microelement for the living organisms, being a cofactor

for manganese-dependent enzymes (arginase, peptidase, manganese superoxide dismutase), thus participating in numerous metabolic processes. At higher concentration, manganese accumulates in various body parts and can interfere with these processes.

Objectives: The main purpose of this study was to investigate the effect of different manganese concentrations on the murine fibroblast cell line NIH/3T3.

Methods: The murine fibroblast cell line NIH/3T3 was cultured in DMEM medium supplemented with 10% FBS, antibiotics, and 2 L-glutamine. Acute exposure of NIH/3T3 cell to manganese was tested using 61 mM - 0.038 mM MnCl2 solutions. After 2h of treatment, the viability test was performed by fluorescent microscopy using acridine orange (AO) and propidium iodide (IP) dyes. The efect of chronic treatment was evaluated by exposing NIH/3T3 cells for 1-5 days to MnCl2 at increasing concentrations (0.0001mM –1mM). The effect induced by MnCl2 was studied by phase-contrast microscopy, Trypan Blue assay, LDH Assay and ELISA test for IFNү and IL6 detection.

Results: The large number of dead cells obtained after short term exposure of cells to manganese indicated that the cytotoxicity range was from 15.25 mM - 3.05 mM MnCl2. After chronic exposure of cells to manganese, at high concentrations (at 0.1mM and 1mM), a decrease of cell viability (at 1 mM) and proliferation (at 0.1mM and 1mM) is observed. At low concentrations (0.0001 mM-0.01 mM), an


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increase in cell proliferation is observed compared with control cells at 72-96 hours post-exposure, a progressive increase of IFNү secretion at 48-96 hours post-exposure and an increase in secretion IL6 at 48 hours after exposure.

Conclusion: The results obtained indicate that the cytotoxic effect of manganese chloride is observed at concentrations greater than 0.1 mM, and below this concentration an immunomodulatory effect of manganese chloride is observed on the murine fibroblast cell line NIH / 3T3 tested.

This work is supported by the Ministry of Research and Innovation, project number PN-III-P1-1.2-PCCDI-2017-0737 (ARTEMIS).

STUDIU IN VITRO AL EFECTELOR INDUSE DE MnCL2

pE LINII FIBROBLASTICE

Ştefania Lascãr1, Irina Elena Ionescu1, Raluca Elena Lãzãrescu1, Vlad Tofan1, Mihaela Diaconu1,2, Cãtãlin Þucureanu1, Ana Şerbãnescu1, Crina Stãvaru1, Adrian Onu1,3

1Institutul Naþional de Cercetare Dezvoltare Medico-Militarã “Cantacuzino”, Bucureşti, România2Universitatea din Bucureşti, Facultatea de Biologie, Bucureşti, România

3Universitatea “Titu Maiorescu”, Facultatea de Farmacie, Buureşti, România

REZUMATIntroducere: Manganul este un microelement esențial pentru toate organismele vii, fiind cofac-

tor pentru enzimele mangan dependente (arginaze, peptidaze, superoxid dismutaza mangan de-pendentă), participând astfel la numeroase procese metabolice. Odată cu creșterea concentrației de mangan, acesta se acumulează la nivelul organismului, interferând cu desfășurarea acestor procese.

Obiective: Principalul scop, al acestui studiu a fost reprezentat de evaluarea efectelor induse de diferite concentrații de mangan asupra liniei fibroblastice murine NIH/3T3.

Metode: În vederea realizării acestui scop s-a utilizat linia celulară fibroblastică murină NIH/3T3 cultivată în mediul DMEM suplimentat cu 10% SFB, antibiotice și 2 mM L-glutamină, aceasta fiind expusă atât acut, cât și cronic la diferite concentrații de clorură de mangan. Expunerea acută, timp de 2h, a celulelor fibroblastice la concentrații de MnCl2 cuprinse între 61 mM - 0,038 mM a fost eva-luată prin microscopia de fluorescență, utilizând doi coloranți vitali: acrinine orange (AO) și iodura de propidiu (IP). Expunerea cronică s-a realizat timp de 5 zile, utilizând concentrații de MnCl2 cu-prinse între 0.0001 mM - 1 mM. Celulele au fost monitorizate la fiecare 24h, utilizând microscopia în contrast de fază, numărătoarea celulară folosind colorantul albastru de tripan, testul lactat dehidro-genazei și tehnica ELISA pentru sinteza de IFNү și IL6.

Rezultate: În urma expunerii acute a celulelor NIH/3T3 la MnCl2 s-a observat apariția unui interval de citotoxicitate cuprins între 15,25 mM - 3,05 mM MnCl2, caracterizat prin prezența unui număr mare de celule moarte. În cazul expunerii cronice la concentrațiile mari, fibroblastele pre-zintă o scădere a viabilitații celulare ( la 1mM), și a capacității proliferative (la 0,1 mM și 1 mM). La concentrațiile mici (0,0001 mM - 0,01 mM), se observă o creștere a proliferării celulare comparativ cu celulele martor la 72-96 ore post-expunere, o creștere progresivă a secreției IFNү la 48-96 ore post-expunere, precum și o creștere a secreției IL6 la 48 ore de la expunere.

Concluzii: Rezultatele obținute indică faptul că efectul citotoxic al MnCl2 se observă la concentrații mai mari de 0.1 mM, iar sub această concentrație se remarcă apariția unui efect imunomodulator al MnCl2 asupra liniei celulare fibroblastice murine NIH/3T3 testate.

Lucrare susținută de Ministerul Cercetării și Inovării, proiect numărul PN-III-P1-2-PCCDI-2017-D737 (ARTEMIS).


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16. DIFFERENTIATION AND MATURATION OF THE MURINE DENDRITIC CELLS EXTRACTED FROM THE BONE MARROW. A COMpARATIVE STUDY

BETWEEN FRESHLY COLLECTED AND CRYOpRESERVED CELLS

Irina Elena Ionescu1, Iuliana Caraş1, Cãtãlin Þucureanu1, Raluca Elena Lãzãrescu1, Ana Şerbãnescu1, Crina Stãvaru1, Adrian Onu1,2

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2Faculty of Pharmacy, Titu Maiorescu University Bucharest, Romania

ABSTRACTIntroduction: Dendritic cells (DCs) represent the most important class of antigen presenting cells

that initiate the immune response during an infection or after vaccination. Due to the activation of naive T lymphocytes and by integrating and generating stimuli and molecules associated with pathogens, DCs are used as experimental model for in vitro evaluation studies of the immunomodulatory/adjuvant properties of vaccine components.

Objectives: To reduce variation between experiments that use DCs and to reduce the number of animals in experiments, an efficient DC differentiation protocol had to be established. Thus, freshly harvested and cryopreserved bone marrow cells were cultured under the same condition. We documented the stages of differentiation and compared the phenotypic expression of DCs from the two groups.

Methods: Differentiation of DCs from isolated bone marrow of Balb/c mice 8 weeks of age was achieved by stimulation with rmGM-CSF (recombinant granulocyte-macrophage colony stimulating factor) for 7 and 9 days. Immature and mature DCs (maturation was obtained by stimulating them with LPS for 48 hours) phenotype was analysed through flow cytometry by staining the cells with specific dendritic cells surface markers (CD11c, MHCII and CD86 - fluorescent labeled antibodies).

Results: Differentiation of precursors into immature DCs showed that cryopreserved cells needed more than 7 days of stimulation with rmGMCSF to fully develop, which was demonstrated by maintaining the cells in culture for 9 days when the phenotype (CD11c +) obtained was comparable to that of DCs obtained from freshly harvested cells. Due to the freezing-thawing process, the number of immature DCs obtained from cryopreserved cells was approximately 8 times lower than the number of DCs obtained from culturing freshly harvested cells.

Conclusion: Cryopreservation of murine bone marrow precursors does not alter the phenotype of DCs at 9 days post differentiation, thus the cells can be used for further experiments, as long as a smaller number of cells is required in the experimental protocols.

The work is supported by the Ministry of Research and Innovation, PCCDI - UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0529 / 62PCCDI ⁄ 2018, from PNCDI III (CONVAC).


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DIFERENÞIEREA ŞI MATURAREA CELULELOR DENDRITICE MURINE EXTRASE DIN MÃDUVA OSOASÃ. STUDIU COMpARATIV ÎNTRE CELULE

pROASpÃT RECOLTATE ŞI CELULE CRIOpREZERVATE

Irina Elena Ionescu1, Iuliana Caraş1, Cãtãlin Þucureanu1, Raluca Elena Lãzãrescu1, Ana Şerbãnescu1, Crina Stãvaru1, Adrian Onu1,2

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino”, Bucureşti, România2Facultatea de Farmacie - Universitatea „Titu Maiorescu” Bucureşti, România

REZUMATIntroducere: Celulele dendritice (CD) reprezintă cea mai importantă clasă de celule prezentatoare

de antigen care duc la inițiarea răspunsului imun în timpul unei infecții sau după vaccinare. Prin activarea limfocitelor T naive și prin integrarea și generarea unor stimuli și molecule asociate cu agenții patogeni, CD sunt folosite ca model experimental în studii privind evaluarea in vitro a proprietăților imunomodulatoare/adjuvante ale componentelor vaccinale.

Scop: Pentru a stabili un protocol de diferențiere a CD din precursori medulari murini cât mai eficient și reproductibil și pentru a reduce numărul de animale utilizate în experimente, celule imediat după recoltare și celule după crioprezervare au fost cultivate în aceleași condiții și comparate din punct de vedere fenotipic.

Metode: Diferențierea CD din celule izolate din măduva osoasă de la șoareci Balb/c în vârsta de 8 săptămâni s-a realizat prin stimularea cu rmGM-CSF (recombinant mouse granulocyte-macrophage colony stimulating factor) timp de 7 și 9 zile. CD au fost comparate din punct de vedere fenotipic, al capacității de maturare prin stimularea acestora cu LPS timp de 48 de ore și din punct de vedere al numărului de celule dendritice (CD) rezultat în urma diferențierii. Pentru controlul diferențierii și maturării s-a analizat expresia unor markeri specifici de suprafață ai celulelor dendritice (CD11c, MHCII și CD86) prin citometrie în flux, folosind anticorpi marcați fluorescent.

Rezultate: Diferenţierea progenitorilor în CD imature a arătat că celulele crioprezervate au nevoie de o perioadă mai mare de 7 zile de diferențiere fapt demonstrat prin menţinerea în cultură, timp de 9 zile, când fenotipul (CD11c+) obţinut a fost comparabil cu cel al CD obţinute din celule proaspăt recoltate. Numărul de CD imature obținute din celule crioprezervate a fost de aproximativ 8 ori mai mic decât numărul de celule dendritice obținute din cultivarea celulelor proaspăt recoltate, acest fapt datorându-se procesului de congelare-decongelare.

Concluzie: Celulele dendritice obținute din măduvă osoasă crioprezervată pot fi utilizate pentru experimente ulterioare, atât timp cât este necesar un număr mai mic de celule în protocoalele experimentale.

Lucrare susținută de Ministerul Cercetării și Inovării, PCCDI - UEFISCDI, nr. proiect PN-III-P1-1.2-PCCDI-2017-0529 / 62PCCDI ⁄ 2018, PNCDI III (CONVAC).


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17. MASS SpECTROMETRY CHARACTERIZATION OF VACCINE ANTIGENS – AppLICATION TO INFLUENZA FRAGMENTATION

Cãtãlin Þucureanu1, Vlad Vasilca1, Vlad Tofan1, Alina Lenghel1, Mihaela Lazãr1,Adriana Costache1, Crina Stãvaru1, Adrian Onu1, 2

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2Faculty of Pharmacy, “Titu Maiorescu“ University, Bucharest, Romania

ABSTRACTIntroduction: While not as efficient as live attenuated viral preparations, split inactivated

vaccines dominate the market due to a better safety profile in sensitive populations (children, elderly, etc.) and fewer/lower magnitude vaccine associated side-effects. Fragmentation conditions are highly dependent on the viral strain involved, minor variations in the aminoacid sequence or glycosylation patterns having a strong impact on splitting efficiency, aggregation and stability of the vaccine preparation.

Objectives: In this study we adapted protein painting, a technique initially developed for the analysis of protein/protein interactions, to probe solvent exposure of viral proteins in whole or split viral preparations in order to allow semiquantitative comparison of fragmentation conditions.

Methods: Whole inactivated or split inactivated NIBRG-14 influenza virus reassortant was incubated with or without remazol brilliant blue - RBB, denatured in SDS, reduced and subjected to tryptic proteolysis. Peptides were separated by Nano-LC, analyzed on a MALDI-TOF/TOF mass spectrometer.

Results: A large number of peptides derived from abundant virion proteins (HA, M1, NP) were identified while low copy number proteins (M2, NA) were less represented, but detectable irrespective of sample treatment conditions. Taken separately, peptides with modified abundance in RBB treated samples were relatively constant in split/unsplit virus preparations for proteins normally exposed on the viral surface, while being significantly less abundant for internal virion proteins in split preparations.

Conclusion: Solvent accessibility of internal viral proteins can be used to assess the degree of fragmentation in split-inactivated viral vaccines.

Work supported by the Ministry of Research and Innovation, CCCDI-UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0529/62PCCDI ⁄ 2018, from PNCDI III.

CARACTERIZAREA pRIN SpECTROMETRIE DE MASÃ A ANTIGENELOR VACCINALE – ApLICARE LA FRAGMENTAREA VIRUSULUI GRIpAL

Cãtãlin Þucureanu1, Vlad Vasilca1, Vlad Tofan1, Alina Lenghel1, Mihaela Lazãr1,Adriana Costache1, Crina Stãvaru1, Adrian Onu1, 2

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã Cantacuzino2 Facultatea de Farmacie, Universitatea Titu Maiorescu, Bucureşti, România

REZUMATIntroducere: Deși nu la fel de eficiente ca preparatele vii atenuate, vaccinurile inactivate/

fragmentate domină piața datorită unui profil de siguranță mai bun la populațiile sensibile (copii, vârstnici etc.) și a efectelor secundare reduse. Condițiile de fragmentare sunt foarte dependente de tulpina virală utilizată, variații minore în secvența de aminoacizi sau de glicozilare având un impact puternic asupra eficienței fragmentării, a tendinței de agregare și a stabilității preparatului vaccinal.


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Obiective: În acest studiu a fost adaptată “protein painting”, o tehnică dezvoltată inițial pen-tru analiza interacțiunilor proteină/proteină, pentru sondarea expunerii la solvent a proteinelor, în preparate virale întregi sau fragmentate și pentru compararea semicantitativă a condițiilor de fragmentare.

Metode: Reasortantul virusului gripal NIBRG-14 inactivat, întreg sau fragmentat, a fost incubat cu sau fără albastru remazol - RBB, denaturat în SDS, redus și supus proteolizei triptice. Peptidele au fost separate prin Nano-LC si analizate pe un spectrometru de masă MALDI-TOF/TOF.

Rezultate: Un număr mare de peptide derivate din proteine abundente in virion (HA, M1, NP) au fost identificate în timp ce proteinele cu număr de copii redus (M2, NA) au fost mai puțin reprezentate, dar detectabile, indiferent de condițiile de tratament ale probei. Luate separat, peptidele cu abundență modificată în urma tratamentului cu RBB, derivate din proteine normal expuse pe suprafața virală, au rămas relativ constante între preparatele cu virus intreg/fragmentat. În schimb, pentru peptidele provenite din proteine interne ale virionului, s-a observat o scădere semnificativă a abundenței în cazul probelor de virus fragmentat tratate cu RBB.

Concluzie: Expunerea la solvent a proteinelor virale interne poate fi utilizată pentru a evalua gradul de fragmentare în vaccinurile virale fragmentate.

Lucrare susținută de Ministerul Cercetării și Inovării, CCCDI-UEFISCDI, număr de proiect PN-III-P1-1.2-PCCDI-2017-0529 / 62PCCDI ⁄ 2018, din PNCDI III.

18. OpTIMIZATION OF BACTERIAL EXpRESSION OF ANTIGENS FOR VACCINE DEVELOpMENT – AppLICATION IN INFLUENZA VACCINE

Laura Andreea Ermeneanu1, Vlad Tofan1, Cãtãlin Þucureanu1, Vlad Vasilca1, Alina Lenghel1, Adriana Costache1, Crina Stãvaru1, Adrian Onu1, 2

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2Titu Maiorescu University, Faculty of Pharmacy, Bucharest, Romania

Introduction: Although no longer a new approach in vaccine manufacturing, bacterial expression of antigenic proteins still possesses difficulties. Due to lack of posttranslational modifications, different codon usage, overwhelming synthesis rate, additional optimization of bacterial expression of antigenic proteins is often required in order to generate large amounts of pure high-quality protein which can be successfully used in immunizations and vaccine preparations.

Objectives: The development of antigenic proteins with immunogenic potential for the Influenza A virus and the formulation of the methodology for cloning, bacterial expression and purification of proteins of potential use for vaccines.

Methods: Starting from Influenza A virus (strain A/Puerto Rico/8/1934 H1N1) hemagglutinin (HA) as a model protein, we aimed to improve antigen solubility to maximize the chance of bacterial expression in its native form. As HA could only be obtained in insoluble form, two truncated forms were engineered that preserve the main epitopes, retain trimerization ability and ensure proper folding: the HA1 segment and the slightly larger ectodomain segment.

The DNA sequences were amplified by Heterostagger PCR reactions and the amplicons were purified from the gel, mixed and realigned by the Ligation-Independent Cloning technique. We tested protein expression in E. coli strains and antigen purification was performed using affinity chromatography.

Results: The PCR amplicons migrated to the corresponding size (approximately 6800 bp). The protein was expressed in the total fraction and in the insoluble fraction in denatured state as inclusion


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bodies. The result of HA-His protein purification shows the presence of high-purity hemagglutinin.Conclusion: This study demonstrated the benefits of optimizing protein sequence to achieve

bacterial expressed antigens for use in vaccine development. Future work will continue the steps of ectodomain segment cloning, protein expression and purification for the shorter hemagglutinin segments.

Work supported by the Ministry of Research and Innovation, CCCDI – UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0529/62PCCDI/2018, from PNCDI III.

OpTIMIZAREA EXpRESIEI BACTERIENE A ANTIGENELOR DIN CANDIDAÞII DE VACCIN – ApLICAÞII LA VACCINUL GRIpAL

Laura Andreea Ermeneanu1, Vlad Tofan1, Cãtãlin Þucureanu1, Vlad Vasilca1, Alina Lenghel1, Adriana Costache1, Crina Stãvaru1, Adrian Onu1,2

1Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino”, Bucureşti, România2Universitatea „Titu Maiorescu”, Facultatea de Farmacie, Bucureşti, România

Introducere: Deși nu mai reprezintă o abordare nouă în fabricarea vaccinurilor, expresia bacteriană a proteinelor antigenice prezintă încă dificultăți. Din cauza incapacităților de modificare post-translațională a gazdei bacteriene, a întrebuințării diferiților codoni, a ratei de sinteză semnificative, este deseori necesară o optimizare suplimentară a expresiei bacteriene a proteinelor antigenice pentru a genera cantități mari de proteine de înaltă calitate, care pot fi utilizate cu succes în imunizări și preparate pentru vaccin.

Obiective: Dezvoltarea proteinelor antigenice cu potențial imunogen pentru virusul gripal A și elaborarea metodologiei de clonare, expresie bacteriană și purificare a proteinelor de interes pentru vaccinuri.

Metode: Pe baza hemaglutininei (HA) virusului gripal A (tulpina A/Puerto Rico/8/1934 H1N1) (PR8) ca proteină model, s-a urmărit îmbunătățirea solubilității antigenului pentru a maximiza șansa de expresie bacteriană în forma ei nativă. Deoarece HA nu a putut fi obținută decât sub formă insolubilă, au fost concepute două forme trunchiate de proteine care păstrează epitopii principali, capacitatea de trimerizare și asigură plierea adecvată: segmentul HA1 și segmentul ectodomeniu ușor mai mare. Secvențele ADN au fost amplificate prin reacții PCR Heterostagger și ampliconii au fost purificați din gel, amestecați și realiniați prin tehnica Ligation-Independent Cloning. Am testat expresia proteinei în tulpini de E. coli și purificarea antigenului a fost realizată folosind cromatografia de afinitate.

Rezultate: Ampliconii PCR au migrat la dimensiunea corespunzătoare (aproximativ 6800 bp). Proteina a fost exprimată în fracția totală și în fracția insolubilă, regăsindu-se în corpii de incluziune în stare denaturată. Rezultatul purificării proteinei arată prezența hemaglutininei de înaltă puritate.

Concluzie: Acest studiu a demonstrat beneficiile optimizării secvenței de proteine pentru a obține antigene exprimate de bacterii pentru utilizare în dezvoltarea vaccinului. Experimentele viitoare vor continua etapele clonării segmentului ectodomeniu, expresiei proteinelor și purificării pentru segmentele de hemaglutinină mai scurte.

Lucrare susținută de Ministerul Cercetării și Inovării, CCCDI - UEFISCDI, număr de proiect PN-III-P1-1.2-PCCDI-2017-0529/62PCCDI/2018, de la PNCDI III.


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19. TARGET pRODUCT pROFILE (Tpp) AS A CENTRAL pLANNING TOOL FOR BIOpHARMACEUTICAL RESEARCH: AppLICATION FOR THE

DEVELOpMENT OF A NEW RABIES VACCINE FOR HUMAN USE

Radu Iulian Tãnasã*, Paulina Podgoreanu, Ilinca-Mihaela MarandiucCantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

Introduction: Essentially, the target product profile (TPP) represents a set of minimal and acceptable properties of the product obtained from the applied medico-pharmaceutical research activity, related to the respective disease and which will lead to a competitive advantage in the profile market [1]. TPP is perfectable: as the product develops and clinical trials lead to a better understanding of product safety and efficacy, the experimental data is compared to the TPP attributes previously established, and TPP becomes QTPP (Quality Target Product Profile).

Objective: The main objective was to develop a TPP in order to start the initial research phase, so as to develop a new rabies vaccine based on the cultivation of the fixed rabies virus in cell cultures.

Methods: In accordance with the WHO recommendations [2], the current scientific data [3], the medical practice in Romania, and the practical possibilities of research and development at the institutional level, a TPP-type profile was established which presents the minimum and desirable characteristics for the development of a new rabies vaccine for human use.

Results: The TPP profile states the following characteristics that a rabies vaccine for human use must include: a) indications for use; b) the target population; c) safety/reactogenicity; d) measures of efficacy; e) duration of protection; f) route of administration; g) valence (covering the etiological specificity); h) dose regimen; i) product stability and storage; j) co-administration with other vaccines; k) presentation form for administration; l) substrate for preparation; m) registration.

Conclusion: The data included in the TPP will be considered for the development of a new type of rabies vaccine for human use, prepared on cell cultures, by creating an initial model of preclinical/non-clinical research (early-stage model).

Acknowledgements: the funding source – The Romanian Ministry of Research and Innovation (MCI), Project Nucleu PN 19 14 01 01.

REFERENCES1. Lee BY, Burke DS. Constructing target product profiles (TPPs) to help vaccines overcome post-approval

obstacles. Vaccine 2010;28(16):2806-09.2. WHO-World Health Organization. Annex 2. Recommendations for inactivated rabies vaccine for human

use produced in cell substrates and embryonated eggs. WHO Technical Report Series No 941, 2007, p. 132.3. Rupprecht CE, Nagarajan T, Ertl H. Rabies vaccines. In: Plotkin SA, Orenstein WA, Offit PA, Edwards KM,

editors. Plotkin’s vaccines. 7Th Edition, Philadelphia (PA): Elsevier Inc; 2018. p. 918-942.e12.


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pROFILUL pRODUSULUI ÞINTÃ (Tpp - TARGET pRODUCT pROFILE) CA INSTRUMENT CENTRAL DE pLANIFICARE pENTRU CERCETAREA BIOFARMACEUTICÃ: ApLICARE pENTRU DEZVOLTAREA UNUI NOU

VACCIN ANTIRABIC DE UZ UMAN

Radu Iulian Tãnasã*, Paulina Podgoreanu, Ilinca-Mihaela MarandiucInstitutul Naþional de Cercetare-Dezvoltare Medico-Militarã „Cantacuzino”, Bucureşti, România

Introducere: În esenţă, TPP reprezintă un set de proprietăți minimale și aceptabile pe care trebuie să le aibă produsul obţinut din activitatea de cercetare aplicată medico-farmaceutică, raportat la afecțiunea/boala respectivă şi care va conduce la un avantaj competitiv pe piaţa de profil [1]. TPP este perfectibil: pe măsură ce produsul se dezvoltă şi studiile clinice conduc la o mai bună înțelegere legată de siguranţa şi eficacitatea produsului, datele experimentale sunt comparate cu atributele TPP stabilite anterior, iar TPP devine QTPP (Quality Target Product Profile – Profilul de Calitate al Produsului Ţintă).

Obiectiv: S-a avut ca obiectiv principal elaborarea unui TPP în vederea demarării etapei iniţiale de cercetare, în scopul dezvoltării unui nou vaccin rabic bazat pe cultivarea virusului rabic fix în culturi celulare.

Metode: În acord cu recomandările OMS [2], datele ştiintifice actuale [3], practica medicală din România şi posibilitățile practice de cercetare-dezvoltare la nivel instituţional, s-a alcătuit un profil tip TPP care prezintă caracteristicile minimale şi dezirabile pentru dezvoltarea unui nou vaccin rabic de uz uman, de generatie contemporană.

Rezultate: Profilul tip TPP precizează următoarele caracteristici pe care trebuie să le includă un vaccin rabic de uz uman: a) indicațiile de utilizare; b) populația țintă; c) siguranța/reactogenitatea; d) eficacitatea; e) durata protecției; f) mod de administrare; g) valența (acoperirea specificităţii etiologice); h) dozajul; i) stabilitate şi conditii de păstrare; j) co-administrare cu alte vaccinuri; k) forma de prezentare pentru administrare; l) substrat pentru preparare; m) înregistrare.

Concluzie: Datele cuprinse în TPP vor fi luate în considerare pentru dezvoltarea unui nou tip de vaccin rabic de uz uman, preparat pe culturi celulare, prin crearea unui model iniţial de cercetare preclinică/non-clinică (early-stage model).

Mulțumiri: sursa de finanţare – Ministerul Cercetarii și Inovării (MCI), Proiect Nucleu PN 19 14 01 01.

REFERENCES1. Lee BY, Burke DS. Constructing target product profiles (TPPs) to help vaccines overcome post-approval

obstacles. Vaccine 2010;28(16):2806-09.2. WHO-World Health Organization. Annex 2. Recommendations for inactivated rabies vaccine for human

use produced in cell substrates and embryonated eggs. WHO Technical Report Series No 941, 2007, p. 132.3. Rupprecht CE, Nagarajan T, Ertl H. Rabies vaccines. In: Plotkin SA, Orenstein WA, Offit PA, Edwards KM,

editors. Plotkin’s vaccines. 7Th Edition, Philadelphia (PA): Elsevier Inc; 2018. p. 918-942.e12.


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20. THE MAIN OBJECTIVES IN THE pRODUCTION OF VACCINES/ADJUVANTS AND THEIR pLACING/MAINTAINING ON THE MARKET.

REGULATIONS

Yuksel Rasit1, Cristina Dumitrache1, Mariana Vãduva2

1National Chemico-Pharmaceutical Institute for Research and Development – ICCF, Bucharest, Romania2Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

ABSTRACTVaccination is the primary tool for primary disease prevention and one of the most cost-effective

measures in terms of public health. Immunization through vaccination is the best defense we have in place to fight serious, preventable and sometimes deadly diseases.

The development and production of the vaccine/adjuvant comprises the following stages:- Non-clinical (preclinical) studies - Necessary to determine the safety profile of the vaccine,

adapting the manufacturing technologies and selecting the antigen/adjuvant, performing in vitro and in vivo tests. All information is essential for the initiation of clinical trials.

- Clinical studies:• Phase I - testing on volunteers to evaluate the safety, dosage range and side effects of the

vaccine.• Phase II - In the case of the convenient results obtained in Phase I, the potential vaccine is

administered to a consistent group of healthy volunteers to complete the safety data, immunogenicity assessment, correct dose determination and administration schedule.

• Phase III - performed on thousands of subjects, evaluating the safety and efficacy on the target population of the vaccine and the co-administration of other vaccines.

• Phase IV - Pharmacovigilance studies, after commercialization, for the strict surveillance of the safety of the vaccine, by acquiring data on possible adverse reactions after immunization and to obtain evidence that the protection offered by the vaccine is long-lasting.

The regulations on vaccine production can be found in “Guidelines on the nonclinical evaluation of vaccines adjuvants and adjuvanted vaccines - WHO 2013” and in “Guidelines on clinical evaluation of vaccines: regulatory expectations - WHO Technical Report Series, No. 1004, 2017.”


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pRINCIpALELE OBIECTIVE ÎN pRODUCEREA DE VACCINURI/ADJUVANÞI ŞI INTRODUCEREA/MENÞINEREA LOR pE pIAÞÃ. REGLEMENTÃRI

Yuksel Rasit1, Cristina Dumitrache1, Mariana Vãduva2

1Institutul National de Cercetare-Dezvoltare Chimico-Farmaceutica – ICCF, Bucureşti, România2Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã Cantacuzino, Bucureşti, România

REZUMATVaccinarea este principalul instrument de prevenire primară a bolilor și una dintre măsurile cele

mai eficiente din punct de vedere al costurilor în materie de sănătate publică. Imunizarea prin vaccinare este cea mai bună cale de apărare de care dispunem pentru a lupta

împotriva bolilor grave, care pot fi prevenite și împotriva bolilor contagioase, uneori mortale.Dezvoltarea și producerea de vaccin/adjuvant cuprinde următoarele etape:- Studii non-clinice (preclinice) - Necesare pentru a determina profilul de siguranță al vaccinului,

adaptând tehnologiile de fabricație și selectând antigenul/adjuvantul, efectuând teste în vitro și în vivo. Toate informațiile sunt esențiale pentru inițierea studiilor clinice.

- Studii clinice: • Faza I - testare pe voluntari pentru a evalua siguranța, intervalul de dozare și efectele

secundare ale vaccinului.• Faza II - în cazul rezultatelor convenabile obținute în faza I, vaccinul potențial este administrat

unui grup consistent de voluntari sănătoși pentru a completa datele de siguranță, evaluarea imunogenicității, stabilirea corectă a dozei și programul de administrare.

• Faza III - efectuate pe mii de subiecți, evaluând siguranța și eficacitatea asupra populației țintă a vaccinului și co-administrarea altor vaccinuri.

• Faza IV - Studii de farmacovigilență, după comercializare, pentru supravegherea strictă a siguranței vaccinului, prin achiziționarea de date despre posibilele reacții adverse după imunizare și pentru a obține dovezi că protecția oferită de vaccin este de lungă durată.

Reglementările privind producerea de vaccinuri se regasesc în ”Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines - WHO 2013” și în ”Guidelines on clinical evaluation of vaccines: regulatory expectations - WHO Technical Report Series, No. 1004, 2017.”


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21. BIOBANKING - TREND OR NECESSITY?

Andreea-Roxana Lupu, Alexandra-Maria NãscuþiuCantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

Population ageing is associated with changes in the profile of certain diseases and the predominance of chronic, slowly progressive diseases as against acute diseases. More and more cases of complex diseases are being also registered, with multiple co-morbidities, while re-emergent infectious diseases need new approaches with respect to diagnostic and specific prevention.

Identification of genetic or molecular markers involved in the predisposition to the onset and development of a certain pathology can contribute to the development of highly effective therapeutic compounds, with reduced side-effects. All these can lead to the improvement of the health status of individual patients, as well as the population’s life quality. In this frame, the development of personalised and translational medicine are tightly linked to complex studies that use several types of biological samples.

Biobanks have evolved from simple repositories of biological samples to complex units that collect, process and store biological samples as well as associated data, according to the regulations established through European conventions, protocols, directives and rules, as well as through national legislations. The diversity of samples, of available testing methodologies and of aims for use of samples and data have led to a rapid development of this field and to the creation of national and international biobank networks.

As common regulations do not manage to cover all the aspects, most of the countries observe mainly the national regulations, which leads to differences in the processing of biological samples and their associated data, and to the reduced availability of certain biospecimens for vast (international) studies with respect to the pathologies of interest. Hence the necesity of harmonizing the procedures regarding samples and associated data, and of establishing protocols for transfer between biobanks and use of research results.

Biological samples in the biobank can offer support for experimental models in biomedical research, for epidemiological studies, and for manufacturing processes (biological products like vaccines or therapeutical proteins, reagents for in vitro diagnostic).

The biological samples from pacients with different infectious and non-infectious pathologies can be used in Reference laboratories for national and international studies, in order to evaluate the competencies of the users in the field (laboratories for medical analyses) and for validation of testing methods.

Through method standardization and development of new methodologies (like experimental models based on 3D cultures or co-cultures), biobanks can be involved in both research-development and learning, ensuring the training of the staff from research laboratories, technological development laboratories, as well as medical analyses laboratories.

Besides, the use of samples from the biobanks contributes to cost decrease in the pharmaceutical industry, in vitro diagnostic devices industry (through investment decrease for obtaining of new therapeutical and diagnostic compounds, increase of quality, safety and users’ trust in biotechnology).

Through the involvement in research-development and innovation fields, biobanking activity contributes to the development of knowledge in the biomedical field, with major social and economic implications, becoming a necessity for research-development-innovation institutes and companies.

This work was financed by the PN 19 14 01 02 project.


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BIOBANCA - MODÃ SAU NECESITATE?

Andreea-Roxana Lupu, Alexandra-Maria NãscuþiuInstitutul Naþional de Cercetare-Dezvoltare Medico-Militarã „Cantacuzino”, Bucureşti, România

Îmbătrânirea populației este asociată cu modificarea profilului anumitor boli și preponderența bolilor cronice și lent progresive față de cele acute. De asemenea, se înregistrează tot mai multe cazuri de boli complexe, cu multiple co-morbidități, iar bolile infecțioase reemergente necesită noi abordări pe linie de diagnostic și prevenție specifică.

Identificarea unor markeri genetici sau moleculari care predispun la instalarea și dezvoltarea unei anumite patologii poate contribui la dezvoltarea unor compuși terapeutici cu eficacitate crescută și efecte adverse reduse. Toate acestea au drept rezultat îmbunătățirea stării de sănătate a pacienților în particular și a calității vieții populației în general. În acest context, dezvoltarea medicinei personalizate și a medicinei translaționale este strâns legată de efectuarea de studii complexe utilizând diverse tipuri de probe biologice.

Biobăncile au evoluat de la simple depozite de probe biologice la unități complexe care colectează, procesează și depozitează atât probe biologice, cât și date asociate acestora, conform cadrului normativ stabilit prin convenții, protocoale, directive și regulamente europene, precum și prin acte normative la nivel național. Diversitatea probelor, a metodologiilor de testare disponibile și a scopurilor în care sunt utilizate probele și datele, a dus la o dezvoltare rapidă a domeniului și la crearea de rețele naționale și internaționale de biobănci.

Regulamentele comune nereuşind să acopere toate aspectele, majoritatea țărilor respectă în primul rând legislațiile naționale, ceea ce duce la diferențe în procesarea probelor biologice și a datelor asociate acestora și la disponibilitatea scăzută a anumitor biospecimene pentru studii ample (internaționale) privind anumite patologii de interes. De aici necesitatea armonizării procedurilor privind probele și datele asociate acestora, stabilirea condiţiilor transferului între biobănci și utilizării rezultatelor cercetării.

Probele biologice din cadrul biobăncii pot oferi suport pentru modele experimentale în cercetarea biomedicală, pentru studii epidemiologice şi pentru procesele de fabricație (produse biologice de tipul vaccinurilor sau proteinelor terapeutice, reactivi de diagnostic in vitro).

Materialul biologic provenit de la pacienți cu diferite patologii infecțioase sau non-infecțioase poate fi utilizat de laboratoarele de referință pentru studii la nivel național sau internațional având ca scop evaluarea nivelului de competență a utilizatorilor din domeniu (laboratoare de analize medicale) și pentru validarea unor metode de testare.

Prin standardizarea de metode şi dezvoltarea de metodologii noi (de exemplu modele experimentale având la bază culturi 3D sau co-culturi), biobanca poate fi implicată atât în cercetare-dezvoltare, cât și în învățământ, putând asigura instruirea personalului din laboratoarele de cercetare, dezvoltare tehnologică și laboratoarele de analize medicale.

În plus, utilizarea probelor din biobănci contribuie la scăderea costurilor din industria farmaceutică, industria dispozitivelor de diagnostic in vitro (prin scăderea investițiilor pentru obținerea unor noi compuși terapeutici și de diagnostic, creșterea calității, a siguranței și a încrederii utilizatorilor în biotehnologie).

Prin implicarea în domeniile de cercetare-dezvoltare și inovare, activitatea de biobanking contribuie la dezvoltarea cunoașterii în domeniul biomedical, cu implicaţii socio-economice majore, devenind o necesitate pentru institutele și companiile de cercetare-dezvoltare-inovare.

Elaborarea acestei lucrări a fost finanţată din proiectul PN 19 14 01 02.


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22. DIACHRONIC MODULATION OF ANTIBODY-ANTIGEN AFFINITY THROUGH TEMpERATURE MODULATION

Rãzvan Stan1,2

1Department of Proteomics and Structural Biology, Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

2Department of Immunology, University of São Paulo, São Paulo, Brazil

ABSTRACTIn order to optimize energy use and anticipate events in their immediate surroundings, proteins

must be able to reversibly alternate between at least one active conformational state back to an inactive conformer. This property can be invoked by single or judiciously repeated applications of a physical (eg. temperature) or chemical stimulus.

I review and present evidence that monoclonal antibody affinity for antigens can be enhanced through:

1. thermal equilibration of both immune partners at relevant temperatures, prior to assays 2. adjusting the temperature of the assays according to the disease model 3. cyclic thermal stimulations. Time-dependent features of conformational memory in antibodies

will also be discussed.

REFERENCES1. Stan et al. Cellular Adaptation Relies on Regulatory Proteins Having Episodic Memory, BioEssays.

2020;41(1).2. Stan et al. Increased grp78 transcription is correlated to reduced tlr4 transcription in patients surviving

sepsis. Clinical & Experimental Immunology. 2019;198(2):273-280.3. Stan et al. Febrile temperatures increase in vitro antibody affinity for malarial and dengue antigens, PLOS

Neglected Tropical Diseases. 2019;13(4): e0007239.4. Stan et al. Memory of Periodic Thermal Stimulation in an Immune Complex, ChemistrySelect,

2019;4(12):3325-3328.

MODULAREA DIACRONICÃ CU TEMpERATURA A AFINITÃÞII ANTICORpILOR FAÞÃ DE ANTIGENE

Rãzvan Stan1,2

1Departamentul de Proteomicã şi Biologie Structuralã, Institutul Naþional de Cercetare-Dezvoltare Medico-Militarã “Cantacuzino”, Bucureşti, România

2 Departamentul de Imunologie, Universitatea São Paulo, São Paulo, Brazilia

REZUMATPentru a optimiza resursele energetice și pentru a anticipa evenimente din mediul înconjurător,

proteinele trebuie să fie capabile să alterneze între diferite conformații. Această proprietate poate fi indusă printr-o singură aplicare, sau prin aplicări repetate ale unui stimul fizic (ex. temperatura) sau chimic (ex. liganzi).

Afinitatea anticorpilor monoclonali contra antigenelor poate fi îmbunătățită prin: 1. echilibrarea termică a ambilor parteneri imuni înainte de formarea complexului imun 2. ajustarea temperaturii măsurătorilor în funcție de modelul de boală cercetat 3. stimulare termică ciclică. Sunt prezentate caracteristici ale dependenței în timp a memoriei

conformaționale a anticorpilor.


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BIBLIOGRAFIE1. Stan et al. Cellular Adaptation Relies on Regulatory Proteins Having Episodic Memory, BioEssays.

2020;41(1).2. Stan et al. Increased grp78 transcription is correlated to reduced tlr4 transcription in patients surviving

sepsis. Clinical & Experimental Immunology. 2019;198(2):273-280.3. Stan et al. Febrile temperatures increase in vitro antibody affinity for malarial and dengue antigens, PLOS

Neglected Tropical Diseases. 2019;13(4): e0007239.4. Stan et al. Memory of Periodic Thermal Stimulation in an Immune Complex, Chemistry Select,

2019;4(12):3325-3328.


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CURRENT pROGRESS AND pERSpECTIVES OF MALDI-TOF MASS SpECTROMETRY AppLICATIONS FOR THE MEDICAL MICROBIOLOGY

FIELD: A MINI-REVIEW

Andreia Amzuþa1*, Alina Lenghel2, Elena Diana Giol3, Mihaela Palela1*1Biotehnology Development and Pilot Studies Microstructure, Cantacuzino National Medico-Military Institute for Research

and Development, Bucharest, Romania2Proteomics and Structural Biology Laboratory, Cantacuzino National Medico-Military Institute for Research and

Development, Bucharest, Romania3Pharmaceutical Development and Methods Validation Microstructure, Cantacuzino National Medico-Military Institute for

Research and Development, Bucharest, Romania

* Corresponding author: Andreia Amzuþa: [email protected], 0745025586; Mihaela palela: [email protected], 0748704833

ABSTRACTMatrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry or MALDI-TOF MS is

a high-throughput method of biomolecules analysis based on the incorporation of a sample into a matrix composed of an energy-absorbing organic compound that will subsequently allow its desorption by laser light. When used for microbiological purposes, this method can identify bacterial genera, species, and subspecies, while when applied in proteomics it can provide important information regarding the molecular structure of proteins. To date, a large number of studies have highlighted the accuracy of the MALDI-TOF MS technique in identifying a wide range of microorganisms including Gram positive and Gram negative glucose-fermentative and non-fermentative bacteria, aerobic/anaerobic bacteria as well as fungi. The present review focuses on the microbiological applications of MALDI-TOF MS technique, offering a mini-review of the current state of the art.

Keywords: MALDI-TOF, mass spectrometry, microbiological applications

REZUMATMALDI–TOF MS (en. Matrix Assisted Laser Desorption Ionisation, Time of Flight Mass Spectrometry)

este o metodă de procesare a biomoleculelor, bazată pe încorporarea unei probe într-o matrice reprezentată de un compus organic absorbant de energie care va permite ulterior desorbția prin lumina laser. În domeniul microbiologiei, această metodă poate identifica genul, specia și subspecia unui microorganism, în timp ce, atunci când este utilizată în proteomică, oferă informații importante cu privire la structura moleculară a proteinelor. Până în prezent, studiile au evidențiat acuratețea tehnicii MALDI-TOF MS pentru identificarea unui spectru larg de microorganisme, incluzând bacterii Gram pozitive, bacterii Gram negative glucozo-fermentative și non-fermentative, bacterii aerobe/anaerobe, precum și fungi. Acest minireview prezintă aplicațiile microbiologice ale tehnicii MALDI-TOF MS.

Cuvinte-cheie: MALDI-TOF, spectrometrie de masă, aplicații microbiologice

INTRODUCTION

Bacterial infections represent a worldwide issue that can still be responsible for high mor-tality rates. In hospitals, or other healthcare facilities, they represent a burden for public healthcare systems by severely increasing the costs and hospitalization time of the patients

[1]. The straightforward and correct identifi-cation of bacteria contributes to reducing the morbidity and mortality rates. Generally, the bacterial identification and characterization methods have been grouped into phenotypic and genotypic methods. Through the pheno-typic assays, information about morphology


250

(cells and colonies shape/size), biochemical (cells metabolism evidenced by conventional individual tests, Analytical Profile Index-API, VITEK®, BD Phoenix™ tests and other auto-mated systems) and physiological (suscepti-bility to antimicrobial agents/phages/bacteri-ocins) features are provided [1-2]. However, these methods are laborious, time-consuming and less efficient and present some limitations in terms of sensitivity and specificity [3].

Conversely, genotypic methods rely on molecular biology principles and include more specialized techniques such as hybridization methods, plasmid profiling and analysis of plasmid polymorphism, all based on molecu-lar typing techniques: enzymes digestion and separation by pulsed-field gel electrophoresis (PFGE), ribotyping, polymerase chain-reac-tion (PCR) or ligase chain reaction PCR (LCR), respectively, and their variations. Transcrip-tion-Based Amplification System (TAS), Multi-locus Sequence Typing (MLST), Spoligotyping and MIRUS-VNTR, GenoType Mycobacterium CM systems are among the most often used typing techniques, and have been applied to isolated pathogens etc. These methods are best to be applied to identify bacterial strains isolated from diverse samples and to improve research in the context of molecular epidemiol-ogy. The main advantages of these techniques are their straightforwardness, improved sen-sitivity and diagnostic specificity with respect to other detection techniques, including cul-ture, in some cases even allowing simultane-ous detection of several microbial agents from the same sample, as well as their resistance genes. These analyses are laborious and expen-sive to be used for routine investigations [3]. The current trend in both research and diag-nostic microbiology is based on using rapid analysis techniques, such as establishing the protein profiles directly from the bacterial col-onies or directly from the biological samples. MALDI-TOF MS is an emerging technique that proved to be a versatile diagnostic tool with great potential for microorganism identifica-tion (Gram positive, Gram negative bacteria and/or fungi). The present review article pro-vides a brief summary of the principles behind

the MALDI-TOF MS technique and its direct input in the microbiological field [4].

Principles and procedural description of MALDI-TOF MS

Since its development in the 1980’s, mass spectrometry (MS) has been applied as an ana-lytical investigation method. The principle be-hind it relies on analyzing with great accuracy the composition of different chemical elements by allowing the measurement of ions derived from molecules and separating them in func-tion of their mass/charge (m/z) ratio, thus de-termining the so-called “chemical trace” of a compound [5]. Recently this method has been applied in the microbiological diagnosis labo-ratories, propelling it as a fast and reliable al-ternative method for microbial identification. In parallel, MALDI is an ionization technology in which a matrix absorbs energy from a UV laser source to create ions from large mole-cules with minimal cellular fragmentation that has been successfully used for decades for the identification of bacteria [6-8]. The combina-tion of both techniques offers a fast, reliable and in depth investigation of microorganisms, highlighting the best characteristics of these techniques, namely the “fingerprint chemical trace” of the MS and the straightforwardness and quickness of the MALDI-TOF.

Several standard operating procedures are applied during MALDI-TOF investigations. In general, microorganisms can be examined directly without pretreatment. This is possi-ble due to the fact that most of the vegetative forms of bacteria are bound by exposure to water, organic solvents and/or strong acids on the MALDI matrix. In the case of more resist-ant microorganisms, as for example bacterial spores and fungal cells, a pretreatment with organic acids and/or alcohols is required be-fore proper analysis. For Actinomyces species, protein extraction procedures are used [9].

One of the most used procedures is the di-rect transfer (DT) method, where in a first step, a small amount of the investigated single colo-ny is applied directly onto a spot on a Ground Steel MALDI target plate. Next, a matrix is overlaid on top of the sample, allowed to dry

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at room temperature and then introduced into the MALDI-TOF equipment for measurement. When the matrix crystallizes during drying, the sample from the matrix co-crystallizes. It is ionized in an automated manner, with a la-ser beam, and ionization with the laser beam generates protonated ions of the analytes in the sample. The protonated ions are then acceler-ated and separated from each other based on their mass-to-charge ratio (m/z). For microbio-logical applications, mainly TOF mass analyz-ers are used. During the MALDI-TOF analysis, the m/z ratio of an ion is measured by deter-mining the time required to travel along the flight tube length. Based on the TOF informa-tion, a characteristic spectrum is generated for each analyzed sample.

One of the most used matrices in microbio-logical applications is α-Cyano-4-hydroxycin-namic acid (HCCA), but others such as 2.5-di-hydroxybenzoic acid have been proposed. A brief matrix preparation procedure is based on the mixing of HCCA with standard solvent (50% acetonitrile, 47.5% ultra-pure water, 2.5% trifluoroacetic acid), to obtain a high-yield matrix for identification and classification of microorganisms, measuring peptides and pro-teins in the range of 0.7 – 20 kDa. The cells are lysed by the aprotic acetonitrile solvent in acid media, resulting in the release of proteins. The samples are then protonated by trifluoroacet-ic acid that facilitates energy absorption of the matrix and ionization of the proteins [10-12]. As a variation of the direct method, the ex-tended Direct Transfer (EDT) method can be applied by using the formic acid before the HCCA matrix, or a formic acid extraction (EX) method, applied for the identification of diffi-cult samples, where an initial extraction in eth-anol and centrifugation, is applied before the formic acid and acetonitrile (pre)treatment of the sample [13].

The cultivation and sample preparation for filamentous fungi require a different pro-cedure. In order to obtain enough biomass in liquid media, cultivation tubes are inoculated and incubated by horizontally rotating in a ro-tator device under the optimal environmental conditions. Part of the culture is centrifuged

(13,000 rpm/2 minutes) and the obtained pellet (biomass) is washed several times with deion-ized water, re-suspended in ethanol, dried and finally treated with formic acid and acetoni-trile, before being placed on a MALDI Ground Steel Target plate.

For rigorous, easy and fast bacterial iden-tification, current MALDI-TOF MS systems have a fully automated analysis flow, with a large upgradable database [6-8, 14]. It is well known that the sequence and size of ribosomal proteins are specific to each bacterial species and, therefore, this feature is used to identify individual types of bacteria. Individual mass peaks are used to identify microorganisms and to provide valuable information about the “fin-gerprint” of the analyzed bacterium. One of the main softwares used for MALDI-TOF MS devices is MBT Compass. The acquired mass spectrum is processed, resulting in a peak pat-tern that is compared to the Reference Library. A report is generated, showing the matching hints and their score values. The higher the score, the higher is the level of confidence. A score above 2 offers a high-confidence identi-fication, while usually a score between 1 and 2 shows a low-confidence identification (and should be correlated with other identification methods). A score lower than 1 indicates no possible matching of the recorded spectrum with database ones. If no identification is pos-sible, the sample should be spotted again on a target plate, considering using another method (such as formic acid extraction instead of direct transfer) while being cautious that no errors during sample preparation are made [15].

Advances in the state of the art of MALDI-TOF MS applications

Until now, a large number of studies have highlighted the accuracy of the MALDI-TOF MS system for identifying a wide spectrum of Gram positive/negative, fermentative/non-fer-mentative, aerobic/anaerobic bacteria [16-20]. Dupont et al. compared the results obtained by MALDI-TOF MS analysis with those obtained from the biochemical tests used in the current microbiology practices [16]. The study showed a correlation of 99.1% between the results of

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the analyzed samples. Regarding the coagu-lase-negative staphylococci, a 93.2% correla-tion with the current investigation methods has been observed. A similar study conduct-ed in 2011 by Spânu et al. has demonstrated a 99.3% accuracy of the MALDI-TOF results ob-tained on different staphylococcal species [17]. A correct identification of 94.9% and 83.4% was observed at the level of bacterial genera (39 genera) and bacterial species (102 species), respectively [18]. Moreover, MALDI-TOF MS was shown to allow the identification of patho-gens with major impact on public health, such as mycobacteria [21]. Out of the 190 species of Mycobacterium genus, M. tuberculosis is the most important for human health. However, studies have shown that the non-tuberculosis mycobacterium (NTM) species may also cause numerous infections, especially in immuno-suppressed patients [22, 23].

In a recent study, Samli et al. compared and evaluated three methods of identification of M. tuberculosis and NTM species: MALDI -TOF MS, nucleic acid hybridization and a specific antigen test (cfr. MPT64 - major culture filtrate protein) [24]. These methods help to differentiate the complex of M. tuberculosis from mycobacteria other than tuberculosis (MOTT).The identification of the M. tuberculosis strains using MALDI TOF was correct but for non–tuberculosis strains the ratio was 38.5%. In conclusion, this technique is suitable for the differentiation of TB and non-tuberculosis species, giving the possibility to initiate therapy protocols, to stewardship the control and prevention methods. The study clearly concludes that the MALDI-TOF MS technique may be used as a sensitive, fast and efficient method, not only for the “usual” bacteria identification, but also for the characterization of mycobacteria. The time required for an analysis using this system is about 6 minutes and the costs are 70-80% lower compared to conventional methods of identification, where a sample processing can last between 7 and 21 days, depending on the species and the breeding period of the colonies [25].

The MALDI – TOF MS system has also been extended to foodborne pathogens of pub-

lic health interest. For example, Dieckmann et al. described the identification of Salmonella at the level of genus, species, subspecies and se-rovar, based on the ribosomal proteins profile [26]. The study confirmed 12 serotypes with 100% specificity and sensitivity for S. enteri-ca subspecies. Studies confirm that whole-cell MALDI-TOF mass spectrometry can be a rap-id method for prescreening S. enterica subsp. enterica isolates to identify epidemiologically important serovars and to reduce sample num-bers that have to be subsequently analyzed using conventional serotyping methods such as slide agglutination techniques. This kind of identification using MALDI-TOF relies on certain biomarker identification rather than on pattern recognition. Similarly, by using the appropriate interpretation module, the sero-types of L. monocytogenes from dairy products could be identified [27, 28]. In another study, the anaerobic Clostridium difficile was inves-tigated through MALDI-TOF MS and PCR ribotyping methods [29]. Proteins with high molecular weight were isolated and analyzed from 500 samples and showed a 89% correla-tion between the recorded data, revealing that MALDI-TOF MS technique can be used for the screening of isolates of Clostridium difficile in-fection. Similar results were gathered for res-piratory pathogens such as Legionella [30, 31]

and Streptococcus pneumoniae [32, 33]. A correct identification of 97.7% for Enterobacteriaceae, 92% for glucose non-fermentative Gram ne-gative bacteria, 94.3% for staphylococci, 84.8% for streptococci and 84% for bacteria in the HACCEK group (Haemophilus, Actinobacillus, Cardiobacterium, Capnocytophaga, Eikenella, and Kingella) were obtained using the MALDI-TOF MS system [34].

However, despite the multiple advantages of the MALDI-TOF MS technique, there are limitations in the detection of microorganisms from polymicrobial infections, where only one pathogen could be identified. Its use for subspecies identification requires technological improvements in terms of the sensitivity of the equipment, the quality of the database, post-test analysis methods [35-37].

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CONCLUSIONS

MALDI-TOF MS is an efficient, fast, accurate, and easy to use technique that could become a very precise instrument for high-throughput microbial identification and successfully replace not only conventional bio-chemical tests, but also even molecular typing systems.

Disclaimer: The views expressed in this review article do not express an official position of the institution or funder.

Source of support: The authors would like to acknowledge the support from Cantacuzino National Medico-Military Institute for Research and Development.

Conflict of interests: No conflict of interests to declare.

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EXpERIMENTAL BIOFILM MODELS: A REVIEW

Ştefania Mãdãlina Negrea1, Raluca Elena Iurea2, Costin Ştefan Caracoti1, Brînduşa Elena Lixandru1, Elena Carmina Drãgulescu1, Cristin Coman1, Irina Codiþã1,2*

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

* Corresponding author: Irina Codiþã, Cantacuzino National Medico-Military Institute for Research and Development, Splaiul Independenþei 103, 050096, Bucharest, Romania, E-mail: [email protected]; Tel. 004 021 3069226

ABSTRACTNowadays, one of the greatest concerns of scientists all over the world is antibiotic resistance of bacteria

as such, and mostly of those able to develop biofilms. Single- or polyspecies biofilms, formed in different en-vironments, starting with the aquatic ones, up to hospital environment, on both biotic and abiotic surfaces, are characterized by higher resistance to antimicrobials than planktonic bacterial cells; bacteria’s structure and physiology are modified, making them able to withstand minimal survival conditions. To understand how bacteria interact with themselves and different surfaces to develop a biofilm and the changes that bacteria suf-fer during that process, many methods and models were designed to reproduce, in experimental conditions, a microbial biofilm.

This article comprises a critical review on several models of experimental biofilm development described in the scientific literature.

Keywords: biofilm, resistance to antimicrobials, experimental models

REZUMATÎn zilele noastre, una dintre cele mai mari îngrijorări ale oamenilor de ştiinţă din întreaga lume este re-

zistenţa la antibiotice a bacteriilor ca atare, dar, mai cu seamă, a celor care au capacitatea de a dezvolta bio-filme. Biofilmele mono- sau multispecifice, formate în diferite medii, începând cu cele acvatice şi până la cel spitalicesc, atât pe suprafeţe biotice, cât şi pe cele abiotice, sunt caracterizate printr-o rezistenţă mai mare la substanţe antimicrobiene faţă de celulele bacteriene planctonice; structura şi fiziologia bacteriilor sunt modifi-cate, ceea ce le face apte să reziste în condiţii minime de supravieţuire. Pentru a înţelege cum interacţionează bacteriile, atât între ele, cât şi cu suprafaţa la care aderă, precum şi modificările pe care bacteriile le suferă în cadrul acestui proces, au fost dezvoltate diferite metode şi modele, cu scopul de a reproduce biofilmul în condiţii experimentale.

Acest articol cuprinde o revizuire critică a diferitelor modele de dezvoltare a biofilmului publicate în literatura de specialitate.

Cuvinte-cheie: biofilm, rezistenţă la substanţe antimicrobiene, modele experimentale


INTRODUCTION

Most laboratory observations in micro-biology, historically and until today, have been made on planktonic cells, although, as they exist in nature, bacteria and fungi grow pre-dominantly as complex communities named biofilms. This form of existence was discovered since the beginnings of microbiology; Antonie van Leeuwenhoek was the one that, using the most simple microscopes, described for the

first time this phenomenon, characteristic of bacteria, of being able to develop and adhere to basically any exposed surface [1].

A biofilm can be defined as an aggregate of microorganisms in which the cells are embedded within a self-produced matrix of extracellular polymeric substance (EPS) and adhered to each other and/or to a surface. The matrix is also referred to as slime and is a polymeric conglomeration composed of

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extracellular biopolymers in various structural forms [2-3]. It is considered that biofilms are a primitive form of cellular differentiation, being metabolically more efficient, protected and resistant to antimicrobials than the bacteria themselves. Microbial biofilms present in different environments, in natural, industrial and hospital settings influence many aspects of our lives. While in a natural environment, the biofilm can be beneficial, in the medical field the biofilm formation is harmful for the human body, as the cells can attach to medical devices, generating chronic infections, difficult to treat [4]. The microorganisms involved in these infections are characterized by higher resistance to different antimicrobials, based on the combination between genetic mechanisms and induced factors. The genetic mechanisms can be classified in two classes: innate resistance factors and induced resistance factors, resulted from induction by antimicrobial administration. As a result of the environment in which the biofilm is developed, there are three main categories of infections: biofilm organ-related infections, biofilm implant-related infections and biofilm related waterborne diseases [3].

Biofilm formationStudies on biofilm formation revealed that

even if the most common biofilms are made by multiple bacterial species, the single-species ones should not be disregarded, as they exist mostly on medical devices and are involved in several infections. Pseudomonas aeruginosa is the most studied Gram negative bacteria, in-volved in single-species biofilm development,

followed by Escherichia coli and Pseudomonas fluorescens. On the other hand, Staphylococcus aureus and Staphylococcus epidermidis are the most studied Gram positive microorganisms that are responsible of biofilm formation on bi-otic and abiotic surfaces [5].

According to the scope of the investiga-tion, three main categories of models have been designed throughout the years to develop a microbial biofilm: in vitro, ex vivo and in vivo models.

Several simplified in vitro models ad-dressed basic questions about biofilm forma-tion and physiology. These models, conceived either as static models, microcosms or open models, offer a number of advantages, as well as disadvantages [6]. A list of different biofilm models is provided in Table 1.

1. In vitro modelsClosed or static models, such as microtiter

plates, Calgary device and biofilm ring test, are characterized by limited nutrients and aera-tion, enabling direct quantification of biofilm mass or viable cells by using assays like XTT (2, 3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) reduction or staining with different dyes as crystal violet, safranin or Congo red. In contrast, the princi-ple of open or dynamic systems is similar to the one of continuous cultures, characterized by constantly replacing of spent culture (wastes, metabolic products, dead cells) with fresh medium. The most common open models are represented by flow chamber, Robbins device and modified Robbins device, rotary biofilm

Static methods Dynamic methods Microcosms Ex vivo models In vivo modelsMicrotiter plate Flow chamber Reconstituted human

epithelia (RHE)Root canal biofilms Mammalian in

vivo modelsCalgary biofilm device

Robbins device and modified Robbins device

Zürich burn biofilm model

Cardiac valve ex vivo model

Non-mammalian in vivo models

Biofilm ring test Rotary biofilm reactor Zürich oral biofilm model

Microvascular endothelial vessels

Colony biofilm model

Microfluidics simple or co-culture models

Endothelial cells under flow model

Vaginal mucosa for Candidiasis

Drip flow biofilm reactor

Airway epithelial cell model

Table 1. In vitro, ex vivo and in vivo biofilm models

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NEGREA et al.

reactor, microfluidics and drip flow biofilm reactors. These methods also allow control of some environmental parameters. Scientists tend to use one of these methods to study a microbial biofilm, even though they often de-mand specialized equipment [6].

Microcosms represent more specifically models designed to replicate in situ conditions, often including several microbial species and materials from different environments, such as addition of hydroxyapatite and saliva to dental biofilm models [6, 8] or covering abiotic surfaces with human cells to mimic an in vivo situation [6, 9].

These models include several environ-mental parameters, taking into consideration the complexity of natural settings [6]. There are several models of microcosms and here we mention the most common and used ones:

reconstituted human epithelia (RHE), Zürich oral and burn biofilm models, endothelial cells under flow model and airway epithelial cell model [6].

2. Ex vivo modelsIn this kind of models, the tissue/organ

used are extracted from an experimental animal host (porcine or murine – in vivo) and placed in an artificial environment (in vitro) for further investigations. It is more useful to use one of these models for imaging or analysing the progression of biofilm development [6]. Examples of these models are represented by root canal biofilms, cardiac valve or microvascular endothelial vessels ex vivo model [10], vaginal mucosa for candidiasis etc., which are characterized in Table 4, together with the in vivo models.

Table 2. Advantages, disadvantages and use of static methods – adapted after Azeredo et al., 2017

Method Advantages Disadvantages Use References

Microtiter plate

InexpensiveNo need for advanced equipment (except plate reader)Allows running a range of tests in parallelAllows testing many samples at the same timeAllows noninvasive imagingSuitable for molecular tests

Sensitive to sedimentation (can be detached during washing steps)Exhaustion of nutrientsUsually short term experimentsPoor reproducibilityNot suitable for investigating the early stages of biofilm developmentDistinction between viable/ non-viable cells and EPS is hard to make

Testing the effect of different compounds on bacterial capacity of adhesionScreening for biofilm formation capacity

[3, 6, 11, 19]

Calgary biofilm device

InexpensiveNo need for advanced equipment Allows testing many samples at the same timePossibility to change growth conditionsLess sensitive to sedimentation

Direct inspection difficultDetachment methods may not remove firmly attached cells or may interfere with physiological characteristicsInadequate for investigating initial steps of biofilm developmentPossible contamination resulting from manipulation

Screening for biofilm formation capacityTest for biofilm minimal inhibitory concentration

[11, 13]

Biofilm ring test

Allows testing many samples at the same timeSimple method (no need of washing, fixing or staining procedures)Not dependent on the person or the laboratory

Requires expensive machines (magnetic device and scanners)

Screening for biofilm formation capacityTesting antimicrobial substances

[11, 13]

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Experimental biofilm models: a review

3. In vivo modelsDuring infections, several interactions are

evolving between the host and the bacteria and also between bacteria themselves. Scien-tists use in vitro models to mimic the natural environment in the human host, in order to de-cipher these interactions and to find a way to break them and, of sound importance, in order to validate in vitro results.

In the past years, more and more models have been designed. Non-mammalian in vivo models have been designed to overcome the practical and ethical issues associated with the use of mammalian models [6].

Other models, used nowadays, are tissue associated biofilm models and in vivo models associated with medical devices (Table 4).

We describe in detail below the most used models in each category mentioned above.

1. In vitro models1.1. Closed or static models

a) Microtiter plates is certainly the most used method, developed in 1977 by Madilyn Fletcher to investigate bacterial attachment [6]. The principle of this method consists in growing bacterial cells in the wells of the microtiter plate, in a nutritive medium. At different time points, the wells are emptied and washed to remove planktonic cells and then the biomass attached to the surface of the wells is fixed by using a chemical method or heat and stained with different dyes. The biomass can also be quantified after detachment and subsequent plating [11].

b) Calgary device was developed in 1999 [12] to overcome the artifact that may appear when using a microtiter plate, more precisely

Table 3. Advantages, disadvantages and use of dinamic methods – adapted after Azeredo et al., 2017

Method Advantages Disadvantages Use ReferencesFlow Chamber

Allows single cell visualizationHigh quality images

Requires special equipmentDoes not allow direct access to the biofilm cells

Evaluation of biofilm formationTesting the efficiency of antimicrobial products

[6, 11]

Robbins device and modified Robbins device

Sustain continued biofilm growthThe sampling plugs can be used aseptically

Does not allow direct observation of biofilm developmentExpensive

Application in biomedical industry (mimic throat conditions and evaluate the efficiency of different products)

[6, 11, 21]

Rotary biofilm reactor

Can be used very long periods of time without interventionThe coupons can be removed and used to further investigate biofilm development

ExpensiveRequires flow systemsDoes not allow in situ observations

Study biofilm formation in different ecosystemsEvaluate interspecies interactions in multispecies biofilmsInvestigate the activity of high-dose vancomycin and moxifloxacin against Staphylococcus aureus biofilms

[11, 22, 27, 33]

Microfluidicdevices

Can be custom made for specific purposesAllows single cells analysis

Require expensive and special equipment

Provide in situ mixing of reagents

[11]

Drip Flow biofilm reactor

Allows both solid-liquid and solid-air biofilm developmentThe possibility to analyze samples noninvasively

Heterogeneity of biofilm formation on the coupons

Visualization and quantification of biofilm developmentEvaluation of several disinfectants efficiency

[6, 11]

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the biomass may not be solely formed as the result of the biofilm formation. By using this method, the biofilm is formed at the coverlid, composed of pegs that fit into the microtiter plate’s wells. The biomass that is attached to these pegs is a result of microbial sessile deve-lopment [11]. The pegs are specifically designed so that they can be removed without opening the vessel, thus possible contamination being not allowed [12]. The bacteria are inoculated in the microtiter plate’s wells filled with a nutri-tive broth; the plate is incubated with or with-out shaking, allowing the biofilm to be formed around the pegs and the planktonic bacteria to remain in the wells [13]. Once formed, the bio-mass can be quantified, usually starting with detachment of the bacteria through, for exam-ple, sonication. This method of detachment has limitations, as only between 5 to 90% of the sessile community is recovered, and also the

cells that are detached can suffer physiological changes during the process [11]. To facilitate the growth of bacteria, the pegs are usually coated with different materials [13], such as L-lysine or hydroxyapatite, which helps to in-vestigate or promote the biofilm formation on different biotic and abiotic supports [11].

c) Biofilm ring test is a method created specifically for evaluating the first steps of bio-film development. This device has been vali-dated on four bacterial strains: Listeria monocy-togenes [14], Escherichia coli [15], Staphylococcus xylosus [16] and Staphylococcus carnosus [17]. This method is based on bacteria’s capacity to immobilize microbeads in the extracellular matrix during biofilm development, when for-ming a biofilm at the surface. Blocked beads thus cannot be mobilized when a magnetic field is near [11]. Measurement of this microbead

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Table 4. Advantages, disadvantages and use of ex vivo and in vivo models – adapted after Azeredo et al., 2017

Model Method Advantages Disadvantages Use References

Ex vivo

Root canal biofilms

Irrigation of dental surfaces ExpensiveNeed to have teeth extracted from pacients

Remove dental biofilms

[38]

Cardiac valve ex vivo model

Allows imagingAllows, after a valve replacement surgery, studying the initial interactions between bacteria and the valve replaced

ExpensiveNeed of special equipment

Evaluate progression of endocarditis

[41]

Candidiasis in vaginal mucosa

Allows microscopic investigation, such as confocal and scanning microscopy

Need of rabbits to perform the biofilm developmentSpecial equipment

Evaluate candidiasis

[46]

In vivo

Non-mammalian in vivo models

Allow studying the interactions between the host and the bacteria

ExpensiveNeed of different organisms, like Drosophila melanogaster or Danio rerio (the most used models)

Overcome the difficulties associated with using mammalian models

[6]

Mammalian tissue-or device-associated biofilm models

Mimic precisely the environment of biofilm development in superior organisms

Expensive, In order to validate the results, there is a needed to experiment on different animals, such as rats, hamster, cats, pigs, rabbits etc.

Understanding the process of biofilm development

[6]

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immobilization by adherent cells of a biofilm allows the assessment of the biofilm’s forma-tion kinetics, which may provide information with practical impact, facilitating the thera-peutical decision to administrate the right an-tibiotics to prevent biofilm formation [18]. The advantages and disadvantages of these models are represented in Table 2.

1.2. Open or dynamic systemsa) Flow chamber model is suitable for

growing microbial biofilms with continuous supply of fresh medium. The system consists of a double trap that captures air bubbles in the growth medium and the flow chamber. On top of the chamber is attached a microscope coverslip; only one channel is mounted with silicone tubing [11]. The growth medium flows through the channel; depending on the geometry of the chambers, the flow may be laminar or turbulent, influencing the distribution of nutrients from the medium and dismissal of waste [11, 20]. The bubble traps were developed in response to the need to remove stray bubbles that influence the biofilm development [11].

b) Robbins device and modified Robbins device

The Robbins device consists of a pipe with numerous threaded holes where are mounted coupons, on the end of screws placed into the liquid stream. The original device was developed to monitor biofilm formation under various fluid velocities in a simulated drinking water facility [21]. The device has been later adapted and the modified Robbins device (MRD) was developed; it can be constructed using a stainless steel or a plastic, with a number of separate ports in a linear array through a channel of rectangular section. The microbial suspension is later filled in the MRD and the device is reversed to enhance the adhesion of microbial planktonic cell to the discs; the tubing is fixed off and the remaining microbial suspension is flushed out through the bypass [11, 22]. The system can operate under various hydrodynamic conditions, from laminar to turbulent flow [11, 23].

c) Rotary biofilm reactorThere are 3 types of rotary biofilm reactors:

rotary annular, rotary disk and the concentric cylinder reactor [11]. The rotary annular reac-tor, developed in 1968 [24] consists in a statio-nary outer cylinder and a rotating inner one. A motor controls the rotation frequency of the internal cylinder, so that constant shear stress fields are obtained [25]. The coupons used as the surface for biofilm development are re-trievable, thus allowing chemical, biochemical and microscopy observation of the biofilm [11]. It has been used to simulate microbial biofilms that develop in drinking water systems and to assess the efficiency and the effect of antimi-crobial agents [11, 26]. The rotary disk reactor was designed in a specific way, to hold several coupons. It is attached to a magnet that pro-vides rotational speed when placed on top of a magnetic stirrer [11, 27]. There is a version of this device, called CDC rotary biofilm reactor, developed and used by the Center of Disease Control in the USA, in the standard methods E2196-12 and E2562-12 for quantification of Pseudomonas aeruginosa biofilms [11, 28, 29]. The CDC biofilm reactor consists in a glass vessel with a polyethylene lid that supports 8 removable polypropylene rods; each rod can grasp 3 removable coupons [22].

d) Microfluidic devices provide a closed system where microbial biofilms interact with hydrodynamic environments. The channels of the device were designed to elucidate the effects of several factors on biofilm formation [11, 30]. These devices can be fabricated from a range of different materials [31]. The devices are designed for special purposes, such as mimicking air-liquid interfaces. Microfluidic devices are not usually used as a standard method in biofilm formation, but they are a very promising approach.

e) Drip flow biofilm reactor was designed in 2009 [31] and it consists in a device with four parallel chambers; each chamber has a coupon where the biofilm will develop [11]. The micro-bial suspension and the medium enter in each chamber through a gauge needle; during this


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operation, the reactor will tilt 10º from horizon-tal and the fluid will pass through the length of the coupons [11]. This kind of device is used to evaluate of disinfectant efficiencies and study biofilm heterogeneity [6].

1.3. Microcosmsa) Reconstituted Human Epithelia (RHE)In this method, the microbial biofilm will

form on top of the human keratinocytes from buccal mucosa [34]; it is used to study oral bio-films and human cells – bacterial biofilm inter-actions [6, 35]. One of the advantages of this method is that it offers the possibility of taking into account some of the local host factors, such as receptor specificity [6].

b) Zürich oral and burn biofilm modelsThe oral model of biofilm development

is used nowadays, to study antimicrobial resistance in different bacterial populations at the same time. In one of the most used models the biofilm is developing on a 24-well microtiter plate lined with hydroxyapatite [35]. The Zürich burn biofilm model allows the development of microbial biofilms and studying its structure, being suitable to assess antimicrobial efficiencies. In contrast to the oral biofilm model, the 24-well microtiter plate is lined with a proteic pellicle that allows polymicrobial growth [36].

c) Endothelial cells under flow modelThe main characteristic of this model is that

the biofilm develops on human microvascular endothelial cells that are attached to a microscope slide, perfused with medium [6]. The biofilm development can be imaged using an inverted fluorescent microscope, which allows tracking the cells concomitantly.

d) Airway epithelial cell model is used as a model to study chronic rhinosinusitis, cystic fibrosis and many other biofilm-associated pul-monary infections, allowing air-liquid inter-face biofilm development. The most common-ly used tool for the growth and development of the microbial biofilm is a membrane that is collagen–coated and has disposed airway epi-thelial cells, to mimic in vivo model [37].

2. Ex vivo modelsa) Root canal biofilmsWhen a periapical lesion appears, it

suggests that the root canal system is infected with bacteria, usually developed in a biofilm; moreover, the presence of bacteria in the apical segment interferes with treatment [38]. It was demonstrated in a study in 1990 [39] that by chemo-mechanical debridement and obturation, the bacterial load in the root canal is significantly reduced, enabling periapical healing in almost 80% of the cases taken into consideration for the study. Using mechanical instruments is not enough to access the entire root canal system, in order to irrigate it for further treatment. This disadvantage was firstly overcome by using simulated canals in plastic blocks, later by extraction of the problematic teeth and treatment with different substances. Nowadays, scientists try to find novel methods to reduce infection of the root canal systems, and they infect the teeth of different animals, in vivo, then those teeth are extracted, coated in silicone putty and irrigated in vitro, with different substances [40].

b) Cardiac valve ex vivo modelMost likely, endocarditis is initiated with

the adherence of bacteria to the valve surface when bacteria infect the bloodstream. The most used model for studies on the early stages is porcine heart extracted from pigs in which endocarditis was induced with different pathological agents. In order to establish how endocarditis appears and how we can prevent it, this method is suitable to study initial interactions between bacteria and valve tissue, in vitro, using, for example, field emission scanning microscopy [41].

c) Vaginal mucosa for candidiasisEx vivo murine or rabbit vaginitis models

were designed in order to examine biofilm formation of Candida spp. on the vaginal mu-cosa and the implication in various diseases. A 2010 study regarding biofilm formation on murine vaginal mucosa indicated high fungal burden, highlighted by colony forming units count and microscopic analysis. The mice used for the experiment were initially treated with

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hormones, several days prior to the vaginal harvest; the tissue was removed after the mice were euthanized and cultured with medium, antibiotic and fungal inoculum. After several days in the incubator, the tissue was examined and the biofilm formation was confirmed [42].

3. In vivo modelsA study from 2013, regarding in vivo bio-

films, concluded that there are two differenc-es between in vitro and in vivo biofilms: in vivo biofilms do not have the same ”mushroom like” structure as the ones formed in vitro and they appear smaller [40]. In order to improve the understanding of biofilms, it is important that these models resemble in vivo conditions.

3.1. Non-mammalian animal modelsThe most common non-mammalian

ani mals used as in vivo models for biofilm de velopment and to study host-microbe interactions were Drosophila melanogaster and Danio rerio. The biggest advantage of using these models is short generation time due to rapid deve lopment. The fact that their genome is fully sequenced offers greater possibility to study the genetic changes that they undergo during biofilm development. As any model used, this one has its limitations due to the short lifespan, when studies of immune system responses in chronic infections caused, most of the time, by biofilm formation are performed [6].

3.2. Mammalian animal modelsIn the past years, animal models have been

associated with various chronic diseases, such as lung infections, otitis media, wound infec-tions, rhinosinusitis, endocarditis, osteomyeli-tis etc. [40]. Animals are one of the best models, in these cases, as they can integrate not only their immune system, but also different other factors, such as flow conditions, nutrients or surface of adherence [43]. There is also a big disadvantage in using these models regarding the long-term inflammatory response that is hard to mimic. In order to overcome this draw-back, different research groups used ingenious solutions for mimicking specific tissue or de-vice-related infections.

a) Tissue-associated modelsThe most widely used animal for urinary

tract infection modelling is the rat; in order to develop bladder stones and understand the kinetics of stone formation and, most of all, the role of bacterial biofilms in this process, foreign bodies like zinc or chalk discs are placed in the bladder of the animal [6, 44].

Another model, that is useful nowadays, as the prevalence of this stomatological condition is increasing, is the denture model. It was studied for a long time, the early models including monkeys and rats with acrylic custom plates, useful for describing the inflammatory process of the adjancent oral mucosa and the host response to the biofilm formation. These studies included the use of different substances, such as clorhexidine and miconazole, to treat the acrylic custom plates, the results being promising, as prevention of mucosal lesions development was highlighted [43].

In the recent years, a very useful non animal model has been created for replacing tissue associated animal eperiments. For mimicking chronic lung infections with Pseudomonas aeruginosa, the bacteria is embedded in seaweed-derived agar or native alginate recovered from the bacteria, with only one to three weeks lifetime [40].

b) Device-associated modelsThe vascular catheter is possibly the most

used model adapted to rat, rabbit, mouse, and most likely to be inserted in the jugular vein. The biofilm formed on the catheter surface is exposed to proteins, immune components and other host conditions, the quantification and visualization of the biofilm being done by mi-croscopy methods [43].

In order to investigate catheter-associated urinary tract infections, a murine model was developed by Wang et al. [45]. The procedure consists in a guided wire inserted in the ure-thra of a female mouse and a catheter segment threaded over the wire and into the bladder, being secured by suture. After a couple of days, the mouse is infected with different bacteria of medical interest. This model is the most used


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one, as it was described that the immune sys-tem of the mouse is similar to the human one, with the clarification that only the wildtype of immunocompromised animals should be used in these types of studies [43].

Animal testing ethics and regulationsThough cell culture and ex vivo models may

be used as preliminary methods, many studies are focused nowadays on using in vivo mo dels, as they are the ones which offer the best con-ditions, most similar to the human body, in respect of biofilm formation and dynamic. It has to be highlighted that whenever an animal model is used for research purposes, Europe-an and national regulations on ethical aspects, regarding both humans and laboratory ani-mals, as well as recommendations of Helsinki Declaration and its revisions have to be taken into consideration. Research projects involving animals have to respect the 3Rs principles, i.e. making every effort to replace, reduce, and re-fine experiments. To proceed with the experi-mental activities in vivo, research institutions involved in preclinical activities will present the experimental study to the Ethics Committee to be approved and to the Veterinary Authori-ty to be authorized. All experiments involving animals have to be performed following the national and international laws regarding ani-mal testing, in particular, the European Direc-tive 2010/63/EU. In Romania, this directive was translated by Romanian Parliament into Law 43/2014 concerning the protection of animals used for scientific purposes. The animal facili-ties have to implement appropriate procedures and to address the requirement to continuous-ly refine experiments. Animal facilities have to ensure a safe working environment, looking to precautions taken when working with any allergens, minimizing the risks of injury by hazardous medical equipment/reagents etc. Securing animals safely helps to avoid sprains, slip and fall accidents, and other physical inju-ries. All requirements addressing health, safety and environment (HSE) issues have to be addressed.

CONCLUSION

Increasing interest of scientists involved both in applied and fundamental research on bacterial biofilms resulted in an impressive number of proposed biofilm models. However, biofilms are still not exhaustively studied and an ideal method has not been developed, in order to exactly quantify bacterial biofilm formation and dynamics. Therefore, using different methods should be very carefully adapted to the purpose of research and there is often need to compare the results obtained by several methods.

Novel methods are still awaited to solve specific questions in the current practice. Knowledge based on previous experience urg-es us to keep up with the bacteria, as they al-ways were and will be a step in front of us.

Conflict of interests: None of the authors of this paper has declared any conflict of interests.

The present study was supported by the National Authority for Research and Innova-tion, Project PN-III-P1-1.2-PCCDI-2017-0010, “Emerging molecular technologies based on micro- and nanostructured systems with bio-medical applications”.

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ULTRA-DEEp SEQUENCING ASSESSMENT OF GENETIC VARIABILITY IN HEpATITIS C VIRUS INFECTING ROMANIAN pATIENTS

Sonia Spandole-Dinu1*#, Eugen Radu2#, Sorin Dinu3#, Georgeta Cardoş1, Graþiela Þârdei4, Petre Iacob Calistru4, Emanoil Ceauşu4, Laurenþiu Micu5, Simona Ruþã6, 7, Camelia Sultana6, 7, Gabriela Oprişan3, 8

1Personal Genetics Laboratory, Bucharest, Romania2Molecular Biology and Pathology – Molimagex, Emergency University Hospital Bucharest, Romania

3Molecular Epidemiology Laboratory, Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

4Dr Victor Babeş Clinical Hospital for Infectious and Tropical Diseases, Bucharest, Romania5Department of Nephrology and Internal Medicine, Fundeni Clinical Institute, Bucharest, Romania

6Emergent Diseases Department, Stefan S. Nicolau Institute of Virology, Bucharest, Romania7Virology Discipline, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

8Faculty of Pharmacy, Titu Maiorescu University, Bucharest, Romania

* Corresponding author: Sonia Spandole-Dinu, personal Genetics – Medical Genetics Center, 4 Frumoasã Street, 1st District, 010987, Bucharest; email: [email protected]; phone: 0040 764 824 281

#Authors that contributed equally to this work

ABSTRACTIntroduction: Hepatitis C virus (HCV) infection is one of the leading causes of hepatic disease worldwide. HCV

quasispecies are the substrate for selective pressure exerted by the immune system or antiviral drugs. Objectives: The aim of our study was to identify by next-generation sequencing polymorphic HCV genomic

regions associated with treatment resistance, and to estimate the viral intra-host diversity of HCV subtype 1b infecting naïve patients selected for treatment with interferon and ribavirin.

Methods: Sera from eleven patients infected with HCV subtype 1b were collected before interferon and ribavirin treatment and purified viral RNA was used to obtain a 454 Roche sequencing library. Sequence data were analyzed using a software package developed by the Broad Institute.

Results: Core 91M and 70Q/H mutations have been identified in 100% of reads in eight, respectively three patients. The existence of a viral population consisting of quasispecies with both mutant variants of core 70 was found in two patients. NS3 drug resistance mutations with low frequency that most likely would have been missed using Sanger sequencing were also identified.

Conclusion: Next-generation sequencing is a sensitive tool for assessing viral intra-host diversity in HCV infected patients. This method can identify polymorphisms in HCV genome potentially predicting treatment outcome.

Keywords: ultra-deep sequencing, HCV, intra-host diversity, haplotypes

REZUMATIntroducere: Infecţia cu virusul hepatitei C (HCV) este una dintre cauzele principale ale bolii hepatice, la nivel

mondial. Cvasispeciile HCV reprezintă substratul asupra căruia acţionează presiunea selectivă exercitată de sistemul imunitar sau de terapeuticele antivirale.

Obiective: Scopul studiului nostru a fost identificarea prin secvenţierea de nouă generaţie a regiunilor polimorfice din genomul HCV, asociate cu rezistenţa la tratament şi estimarea diversităţii virale intra-gazdă la pacienţi naivi, infectaţi cu HCV subtip 1b şi selectaţi pentru terapia cu interferon şi ribavirină.

Metode: Au fost colectate seruri de la unsprezece pacienţi infectaţi cu HCV subtipul 1b, înainte de iniţierea tratamentului cu interferon şi ribavirină, iar ARN viral extras şi purificat din seruri a fost utilizat pentru a obţine biblioteci ADN 454 Roche. Datele tip secvenţă au fost analizate cu un pachet de programe dezvoltat de Broad Institute.

Rezultate: Mutaţiile 91M şi 70Q/H în regiunea core au fost identificate în 100% din secvenţele obţinute de la opt pacienţi, respectiv trei pacienţi. Existenţa unei populaţii virale constând în cvasispecii cu ambele mutaţii în poziţia 70 din regiunea core a fost pusă în evidenţă la doi pacienţi. Mutaţii de rezistenţă la tratament în regiunea NS3, având o frecvenţă mică şi care, cel mai probabil, nu ar fi fost detectate prin secvenţierea Sanger, au fost puse, de asemenea, în evidenţă.

Concluzie: Secvenţierea de nouă generaţie este o tehnică sensibilă pentru evaluarea diversităţii virale intra-gazdă la pacienţii infectaţi cu HCV. Această metodă poate identifica polimorfismele din genomul HCV, care ar putea prezice evoluţia bolii sub tratament.

Cuvinte-cheie: secvenţiere cu randament înalt, HCV, diversitate intra-gazdă, haplotipuri


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SPANDOLE-DINU et al.

INTRODUCTION

Next generation sequencing (NGS) tech-niques allow reading a very high number of relatively short DNA fragments, thus revolu-tionizing metagenomics and virology in the last decade [1]. The large volume of sequence information obtained permits the evaluation of a wide spectrum of quasispecies including minor variants, discovery of rare viral variants, and identification of a large number of muta-tions [2-4].

In particular, the use of these techniques in hepatitis C virus (HCV) infection study enabled exploring viral populations diversi-ty during their evolution [5, 6], detecting and evaluating the persistence of therapy-resistant variants [7-10] and analysis of viral diversity during various treatments [11-15].

HCV is an enveloped ssRNA virus, ap-proximately 9600 nucleotides long [16, 17]. The virus encodes a single precursor poly-protein that is cleaved into ten structural and non-structural proteins (core - E1 - E2 - p7 - NS2 - NS3 - NS4A - NS4B - NS5A - NS5B, in 5’ to 3’ order) [18]. The high rate of mutation in the HCV replication, along with other mecha-nisms, causes the occurrence of highly related groups of mutant viruses – quasispecies – rep-resenting the target of evolutionary phenome-na. Quasispecies analysis can lead to biologi-cally relevant information, but also exhibits ex-perimental and data analysis problems (e.g. the

need for amplification reactions, the length of sequenced fragments, and number of reads of the same regions) [19]. Due to its longer reads, the 454/Roche pyrosequencing platform facili-tates assembling complete genomes, even if the total number of nucleotide reads is lower [20].

Sustained virological response (SVR, i.e. undetectable HCV RNA six months post-treat-ment) is achieved in only 40-50% of interfer-on-naïve patients infected with HCV subtype 1b – the most common subtype in Romania [21]. Along with a relatively low SVR rate, combined pegylated interferon and ribavirin (PEG-IFN/RBV) are accompanied by severe side effects and entail significant costs. Hence, the patients who will not achieve SVR need to be identified prior to therapy, in order to avoid unnecessary side effects and high costs.

In order to estimate the viral diversity, as well as to identify polymorphic genomic re-gions associated with treatment resistance, deep sequencing of some PEG-IFN/RBV sensi-tive or resistant HCV genomes was performed using the 454/Roche platform.

MATERIALS AND METHODS

PatientsEleven HCV subtype 1b-infected, previ-

ously untreated patients, with detectable HCV viral load (> 1000 IU/ml) were selected for this study based on their response to PEG-IFN/RBV therapy (Table 1). The patients were recruited

Patient code Age (years) Gender (M/F) Viral subtypea Viral load (IU/mL)b Treatment responseSVB003 29 M 1b 3,090,000 SVRSVB011 32 M 1b 4,010,000 SVRSVB016 55 f 1b 2,220,000 SVRSVB019 37 M 1b 5,070,000 RelapseSVB028 50 f 1b 2,600,801 NRSVB054 48 M 1b 1,010,000 SVRSVB072 41 M 1b 279,000 SVR

R04 34 f 1b 105,000 SVRR06 58 M 1b 8,160,000 SVRR14 58 f 1b 970,000 NRR16 62 f 1b 1,090,000 Relapse

Table 1. Patients included in the study, their initial viral load and treatment response

a Genotype identified using a commercial reverse hybridization line probe assay (VERSANT™ HCV Genotype 2.0 Assay, Siemens, Germany) and confirmed by Sanger sequencing as described in [21]

bViral load was tested before treatment by RT-PCR (Cobas Amplicor HCV Monitor, vers 2.0, Roche, Germany)

269

Ultra-deep sequencing of HCV infecting Romanian patients

in the “Dr Victor Babeş” Clinical Hospital for Infectious and Tropical Diseases from Bucha-rest. The study was approved by the Bioethics Committee of research institutions involved, in accordance with the Helsinki Declaration, and written informed consents was obtained from all enrolled patients.

NGS library preparation and runThe strategy used for library preparation

was to reverse-transcribe, then amplify through nested-PCR four overlapping fragments span-ning almost the entire HCV genome.

Viral RNA isolated from serum samples collected prior to PEG-IFN/RBV treatment was reverse-transcribed using specific primers [22] and M-MLV Reverse Transcriptase (Promega Corporation, Madison, WI). Complementary DNA was then used in nested-PCR reactions (FastStart High Fidelity PCR System, Roche, Germany) as described elsewhere [22]. Amplification products were tested for quantity and quality using fluorometric assays (Qubit dsDNA HS Assay, Life Technologies) and, respectively, microcapillary electrophoresis (2100 Bioanalyzer, Agilent). The correct-length amplicon for 3’ end of the genome, coding for NS5 and 3’ UTR, was not obtained due to technical difficulties related to efficient reverse transcription.

In order to directly estimate the error rate of the whole experimental procedure (includ-ing PCR amplification and pyrosequencing), a plasmid (pFK-Con1) containing the wild-type HCV Con1 isolate genome (GenBank accession number AJ238799) was sequenced within the same run [23]. The plasmid sample was a gen-erous gift from Ralf Bartenschlager, Heidel-berg University. The resulting amplicons were equimollarly mixed for each patient and con-trol, and random-fragmented through nebuli-zation, according to Roche instructions. After purification, the Roche Rapid Library Prepara-tion Kit (Roche Diagnostics GmbH, Germany) was used to attach MID adaptors to fragment-ed DNA. Library quantification and dilution were performed according to the manufactur-er-supplied protocol. Optimal target molecules per bead ratio was derived from a small-vol-

ume emulsion (em) PCR titration experiment and subsequently used for the large-volume emPCR amplification, according to Roche protocols. Finally, the libraries were mixed in equal quantities and sequenced on two large regions of a PTP device using XLR70 Titani-um Sequencing reagents (Roche Diagnostics GmbH, Germany).

Data analysisSequencing data analysisImage acquisition and primary processing

was performed on the GS FLX (Roche 454 Sequencing) system, and a Roche Titanium Cluster computer was used for shot-gun data processing. Sequences extracted for each sample were then filtered using mothur software [24], to exclude reads with ambiguities (homopolimers longer than eight bases and an average quality score lower than 35 in any 50 bp window). Contigs were assembled using Vicuna software, an overlap-layout-consensus de novo assembler tuned for highly diverse viral populations [25]. Finishing and reorienting of contigs, as well as annotation (HCV 1b isolate Con1 genome was used as a reference) and frameshift corrections were performed using V-FAT (http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/v-fat). Pyrosequencing-specific error correction was made using RC454 software [26] on the unfiltered reads. Contigs were manually curated to correct an instance of homopolymer error that initially led to erroneous frameshift changes.

Sequence variations (single nucleotide polymorphisms and length polymorphisms) were called using V-Phaser 2 Software [27]. Core mutations with frequency over 20% were confirmed by Sanger sequencing [21]. ShoRAH software [28] was used for reconstructing viral haplotypes.

Statistical analysisAll data were expressed as mean ± standard

deviation or absolute values and statistical analysis was conducted using SPSS 20.0 for Windows (SPSS Inc., Chicago, IL, USA). The Shapiro-Wilk test was performed to assess the

270

normality of data distribution. Student’s t-test was used to compare normally distributed variables and Mann-Whitney U test was used for variables with non-gaussian distribution among groups. Instances with low statistical resolution (e.g. low number of samples to be analyzed) were eliminated from analysis. For all statistical tests P < 0.05 was considered significant.

Genetic variabilityGenetic variability of HCV genome regions

encoding for proteins involved in shaping the response to treatment was assessed by estimating number and distribution of non-synonymous nucleotide substitutions.

Phylogenetic analysisPhylogenetic analysis was performed

on viral reconstructed haplotype sequences (7220 nucleotides corresponding to positions 125-7339 in HCV Con1b reference genome, GenBank accession number AJ238799) obtained for each patient with ShoRAH. The nucleotide sequences were aligned using the online software Clustal Omega [29]. MEGA v6.0 [30] was employed to infer the evolutionary history using the Neighbor-Joining method with Tajima-Nei substitution model. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the analyzed taxa.

RESULTS

Following the quality filtering of raw results, 1,069,542 reads totaling 424,886,322 bases were obtained. Reads had a mean length of 450 bases and an average quality score of 30.81. The contig obtained for Con1 control had a length of 7837 nucleotides with an average depth of 3500x. The HCV genomic fragment with lowest depth (~1400x) included 43,772 reads totaling 16,899,914 nucleotides, while the highest depth was approximately 5000x.

The genomic regions encoding for core, p7, NS2, NS3, NS4A and NS5A proteins (~75% of the polyprotein) were analyzed. Distribution of the non-synonymous substitutions was

compared in terms of frequency for each protein (Table 2).

For each selected region, the non-synony-mous substitutions were identified and listed in supplementary table.

Core 70H/Q mutations were found in five and core 91M mutation in eight samples, with 100% frequency in three, respectively eight samples. All mutations with frequency over 20% were confirmed by Sanger sequencing.

A number of resistance mutations to protease inhibitors, which target NS3 protein, have been identified with different frequencies in samples from our patients (Table 3).

No significant association was found between the numbers of non-synonymous substitutions in core, NS2 and NS3 proteins and response to treatment.

Ten Interferon Sensitivity-Determining Region (ISDR) amino acid substitutions (Table 4) compared with HCV Con1 b reference (GenBank AJ238799) were found; three of them in 100% of reads. Five of these substitutions were previously found in HCV with mutant type ISDR in a cohort of 334 patients [31].

None of these mutations was common in more than one patient. ISDR amino acid sequence was identical to the reference for two patients, both of which were relapsers. Inter-host variability estimate was achieved by constructing phylogenetic trees using genomic sequences of viral haplotypes obtained for each patient. The highest number of haplotypes with a frequency above 1% was recorded for patient R04, while at the opposite pole patient SVB019 harbored a single viral type. No haplotype was common to more patients (Fig. 1).

DISCUSSION

Due to technical restraints related to the reverse transcription step preceding library preparation, the 3’ extremity of HCV genome containing NS5A and NS5B was sequenced partially or not at all. Complete NS5A region was sequenced in five out of eleven samples.

Pairwise alignment of Con1b sequence obtained experimentally and the database reference sequence identified six nucleotide miss-matches, resulting in a sequencing error


271

ProteinNon-synonymous substitutions (mean ±sd) P

frequency <1% frequency ≥ 1%core (191 aa) 4.27 ±5.8 2.18 ±1.8 0.89 (Mann-Whitney)

p7 (63 aa) 2.45 ±2 2.18 ±1.6 0.72 (T-test)

NS2 (217 aa) 8 ±7.6 9 ±4.5 0.27 (Mann-Whitney)NS3 (621 aa) 14.9 ±8.9 8.36 ±3.9 0.04 (T-test)

NS4A (54 aa) 1.27 ±1.8 1.27 ±1.6 0.91 (Mann-Whitney)

NS5A* (447 aa) 36.8 ±26.3 16 ±13.3 0.15 (Mann-Whitney)*Five patients with complete NS5A sequence.

Table 2. Distribution of non-synonymous substitutions in the tested proteins

Table 4. Amino-acid substitution in the interferon sensitivity-Determining region (ISDR) found in samples from tested patients

Amino acid position in

NS5A protein

Amino acid position in polyprotein

Reference (Gen Bank accession number AJ238799.1)

SVB003 SVB011 SVB019 SVB054 R16*

240 2212 K - - - R 0.74%

-244 2216 T - A

1.35%- - -

248 2220 D - - - N 100%

-

249 2221 S P 0.7%

- - - -

255 2227 I - - - T 100%

-

256 2228 E - - - D 11.5%

-

263 2235 Q - - - P 1.88%

-

268 2240 N - D 8%

- S 0.35%

-

274 2246 S - - - L 100%

-

276 2248 N - - - D 2.18%

-

MutationPatients

SVB 003

SVB 011

SVB 016

SVB 019

SVB 028

SVB 054

SVB 072

R04 R06 R14 R16

Q80L - - - - L (100%)

- - - - - -

R117H - - - H (7.22%)

- - H (100%)

- - - -

I132V V (63.74%)

-V

(100%)- V

(99.53%)- - V

(100%)V

(3.66%)V

(100%)V

(100%)

I153V - - - - - - - - V (0.5%)

- -

D168N - - - - - - N (1.64%)

- - - -

Table 3. Resistance mutations to viral protease inhibitors targeting NS3 found in samples from tested patients


272

rate of 0.076%, a value that is concordant to previously reported error rates for this type of experiment [12].

For each selected region, the number of substitutions was recorded. The non-synonymous substitutions recorded for each analyzed region were divided into two subtypes: with frequency below 1% and equal or exceeding 1%, respectively. Viral variability

is reflected in the number of non-synonymous substitutions in the proteins encoded by the HCV genome. The large number of non-synonymous substitutions with low frequency (e.g. <1%) may suggest an evolving viral genome due to the combined effect of adaptability and host factors or sequencing artifacts.

A higher number of substitutions with low frequency were observed preponderantly, with

Fig. 1. Bootstrap consensus tree inferred from 1000 replicates using genomic sequences obtained for haplotypes with frequency >1% from tested patients. Mega6, Neighbourg-joining,

Tajima-Nei substitution model. Sample SVB054 was excluded from phylogenetic analysis due to no viable haplotypes with cut-off frequency


273

no statistical significance. For the NS3 protein, a significantly higher number of low frequency substitutions (p=0.04) were detected, indicat-ing that NS3 may be under selective pressure, as was suggested before [32, 33]. A high degree of genetic variability among all HCV geno-types in protease inhibitors-naïve patients was reported [34], and this finding that is also sup-ported by our study.

Core 70H/Q and 91M mutations found with frequency over 20% were confirmed by Sanger sequencing. However, two patients were found to carry simultaneously both var-iants of core 70 mutation with different fre-quencies (one patient displayed variant 70Q in 1.38% of reads, and the other one harbored 70H in 3.08% of reads) only by NGS (Supplemen-tary table). A similar finding was encountered for NS3 resistance mutations (e.g. sample from a patient harbored both NS3 D168N variants, with a 168N dominance of 98.36% of reads) un-derlining the sensitivity of NGS analysis, im-portant for the detection of genotypes with low frequency and potential resistance mutations that can be selected after therapy.

A series of previously reported resistance mutations to protease inhibitors, which target NS3 protein, was found in samples from our study (Table 3). Furthermore, Q80L polymor-phism associated with 1b subtype HCV strains circulating in Asia and conferring resistance to faldaprevir and paritaprevir [35, 36] was found in one non- responder patient in 100% of reads. Given the fact that patients in this study have not been treated with protease inhibitors, this suggests that NS3 resistance mutations pre-exist in viral populations infecting naïve patients. Indeed, in the past years, data have also demonstrated the presence of a naturally occurring resistant variant in treatment-naïve subjects [7, 37-39]. Thus, studies suggested the need for screening of resistance-associated variants not only in patient candidates for pro-tease inhibitors therapy, but also in the overall HCV-infected population [40].

To test the association between genetic variability and therapy response, patients were classified as sustained virologic response (SVR),

non-response and relapse after the evaluation performed at six months after completing the therapy. Subsequently, for each individual protein, the association of non-synonymous substitutions and the type of therapy response was tested. Since p7 and NS4A proteins have a low number of amino acids, statistical tests cannot be applied successfully in these cases and were excluded from further analysis. Nevertheless, we identified the p7 L20F resistance mutation to adamantane [42] in three patients and confirmed the high conservation degree of H17 [43], G39 [44] and P49 residues [45]. G46, along with other four glycine residues, is believed to be of extreme importance for helix to helix interaction in p7 porine [46]. G46S mutation (i.e. substitution of the non-polar amino acid residue glycine with the polar un-charged serine) with unknown biological significance in this context was found in two patients in this study.

The region encoding NS5A was sequenced in only five patients, three responders and two relapsers.

NS2 protein is a transmembrane protein essential for completion of the viral replication cycle [47, 48]. The C-terminal residues of NS2 play an important role in NS2/3 auto cleavage. The domain required for this cleavage was mapped between amino acids 827 and 1207 of the polyprotein at the C-terminus of NS2 and start of NS3 [49]. The association between the numbers of amino acid substitutions in the 827-1026 residues range of HCV polyprotein (belonging to NS2) from our patients and treatment failure was tested with borderline statistical significance. To better depict the difference in amino acid sequence variability of NS2 between patients, fig. 2 shows a heatmap of NS2 variation in samples from responder and non-responder patients. However, on closer analysis in both responder and nonresponder patients, most amino acid substitutions from C-terminal portion of NS2 scored positive or slightly below zero in the BLOSUM62 matrix for amino acid substitutions [50], thus being unlikely to modify protein structure. This observation may indicate that data


274

from our patients is too scarce to imply an association between amino acid substitutions and treatment response and an increase of the power of the study by analyzing more patients may be required.

Genetic variability of NS5A and particularly ISDR-V3 region was previously associated with good prognostic in PEG-IFN/RBV [51]. NS5A along with ISDR was completely sequenced in five of eleven patients (three responders and two relapsers).

The phylogenetic analysis grouped haplotypes from each patient into a distinct clade in 100% of bootstrap replications (Fig. 1). Considering similarity of HCV nucleotide sequence between patients is ~90%, haplotype distribution suggests that once reached the

host, viral isolates may evolve differently depending on host factors.

High throughput sequencing techniques represent rapid and reliable tools that greatly improved our understanding of virus infection. NGS allows a more effective characterization of complex viral populations and their evolution within a host. Our NGS-based study aimed to identify polymorphic HCV genomic regions associated with treatment resistance, and to estimate the viral diversity of HCV subtype 1b in naïve patients. The same approach is now used with the third-generation long-read sequencers for evaluation of the virus resistance variants to the new direct-acting antiviral (DAAs) treatment [52, 53]. Even though DAAs resolves HCV infection in over 90% of treated

Fig. 2. Heatmap of amino acid variability for NS2 protein in samples from responder (top row) versus non-responder (bottom row) patients. Colored spots represent polymorphic regions; warmer color

denotes higher frequency of variants. The heatmap was constructed using V-Phaser 2


275

patients, it can lead to adverse reactions, viral resistance, drug interactions and low efficacy for some HCV genotypes [53].

CONCLUSIONS

NGS analysis of variability across core and NS3 regions allowed the detection of low-frequency mutations previously associated with drug resistance that might have been missed with Sanger sequencing, underlining the effectiveness of deep sequencing in identifying these types of variants in a clinical context.

Our findings showed that this method can identify polymorphism in HCV genome potentially predicting treatment outcome. Minor viral variants harboring treatment resistance mutations that may have been

missed by Sanger sequencing were detected in naïve patients. As previously reported, NS5A variability appears higher in responding patients. Despite the great variability of HCV genome, haplotype analysis suggested that once reached the host, viral isolates may evolve differently, and this is probably due to host factors.

AcknowledgementsFunding: This work was supported by a

grant of the Romanian National Authority for Scientific Research, CNDI–UEFISCDI, project number 88/2011.

Conflict of interests: No conflict of interests to declare.


276

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277

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V (2

.14%

) V

(99.

44%

)

V

867I

V

-

- -

- -

- -

- -

I (10

0.00

%)

-

T8

78M

/V/A

T

M (6

2.90

%)

- -

- V

(97.

71%

) A

(100

.00%

) -

V (1

00.0

0%)

- -

A (0

.72%

)


278

Prot

ein

Mut

atio

n

Patie

nts

A

880V

A

V

(100

.00%

) V

(100

.00%

) -

- V

(0.9

2%)

- -

- -

- -

I8

81V

/A/F

/T

I V

(100

.00%

) V

(96.

85%

); A

(3.1

5%)

V (1

00.0

0%)

F (1

00.0

0%)

V (9

8.17

%);

A

(1.0

2%)

V (1

00.0

0%)

A (1

00.0

0%)

V (1

00.0

0%)

V (5

4.08

%);

I (21

.93%

);

A (1

1.36

%);

F (1

1.30

%)

A (1

00.0

0%)

I (98

.93%

);

V (1

.07%

)

H

882Y

H

-

- -

Y (1

00.0

0%)

- Y

(100

.00%

) Y

(100

.00%

) -

Y (5

.46%

) Y

(99.

00%

) -

P8

83S

P -

- -

- -

- -

- -

- S

(80.

68%

)

L8

85P

L -

- -

- -

P (1

3.67

%)

- -

- -

-

I8

86V

/T

I V

(100

.00%

) V

(24.

84%

) -

- -

- -

T (1

00.0

0%)

V (0

.41%

) -

-

T8

88D

/E

T D

(100

.00%

) E

(100

.00%

) E

(100

.00%

) D

(100

.00%

) D

(100

.00%

) E

(100

.00%

) D

(100

.00%

) D

(100

.00%

) E

(100

.00%

) E

(100

.00%

) D

(100

.00%

)

I8

92L/

F I

L (1

00.0

0%)

- -

L (1

00.0

0%)

L (1

00.0

0%)

- L

(100

.00%

) -

- -

F (9

7.51

%)

I8

96V

/M/L

I

- V

(100

.00%

) -

- L

(0.7

7%)

- M

(95.

99%

); L

(4.0

1%)

M (0

.48%

) V

(100

.00%

) -

L (9

9.33

%)

L8

97F

L -

- F

(100

.00%

) -

- -

- -

F (0

.62%

) -

-

L9

00P

L P

100.

00%

) -

P (2

.86%

) -

- -

- -

- -

-

M

901N

/T

M

- T

(100

.00%

) -

- -

- -

- N

(65.

01%

); T

(34.

47%

) T

(99.

39%

) N

(8.6

7%)

G

906A

/S

G

- A

(100

.00%

) A

(100

.00%

) -

- S

(100

.00%

) -

- S

(100

.00%

) -

S (1

00.0

0%)

I9

07M

/L

I -

M (9

6.70

%);

L (3

.30%

) L

(100

.00%

) L

(1.3

5%)

- -

- -

- M

(100

.00%

) -

T9

08I/V

T

- I (

100.

00%

) V

(100

.00%

) -

I (96

.55%

) I (

100.

00%

) -

- I (

100.

00%

) -

V (1

00.0

0%)

K

909R

K

R

(100

.00%

) -

- -

R (1

00.0

0%)

R (1

00.0

0%)

R (1

00.0

0%)

R (1

00.0

0%)

R (1

00.0

0%)

R (0

.46%

) -

Y

912F

Y

-

- -

- -

- -

- F

(100

%)

- -

V

914T

V

-

T (1

00.0

0%)

T (1

00.0

0%)

T

(100

.00%

) T

(100

.00%

) -

- T

(100

.00%

) T

(100

.00%

) -

H

917Q

H

-

- Q

(98.

80%

) Q

(100

.00%

) Q

(23.

21%

) Q

(100

.00%

) Q

(100

.00%

) -

Q (1

00.0

0%)

- Q

(100

.00%

)

A

922M

/I A

M

(100

.00%

) -

- I (

100.

00%

) -

- -

- -

- M

(97.

63%

)


279

Prot

ein

Mut

atio

n

Patie

nts

M

924T

/V

M

- -

- -

T (2

2.05

%)

- V

(98.

00%

) -

- -

-

V

929I

/A

V

- -

- -

I (92

.67%

) -

- A

(100

.00%

) -

- I (

25.1

0%)

V

935I

V

-

- -

- -

- -

I (10

0.00

%)

- -

I (81

.57%

)

L9

39F

L -

F (9

6.54

%)(

- F

(96.

25%

) F

(2.4

0%)

F (1

00.0

0%)

- -

F (1

.19%

) F

(76.

74%

) -

M

940V

M

-

- -

- V

(1.7

0%)

- -

- -

- V

(19.

06%

)

K

941R

K

-

R (0

.81%

) R

(100

.00%

) -

R (6

.88%

) -

R (2

.21%

) -

- -

R (9

9.92

%)

A

944S

A

-

- S

(100

.00%

) -

- -

- -

- -

-

T9

55S

T -

- -

- -

- -

- S

(45.

50%

) -

-

R

958Q

/K

R

- Q

(100

.00%

) -

K (1

00.0

0%)

- Q

(100

.00%

) -

Q (1

00.0

0%)

Q (0

.39%

) Q

(100

.00%

) -

A

963S

/T

A

S (1

00.0

0%)

- T

(99.

03%

) S

(100

.00%

) -

- S

(100

.00%

) -

T (0

.59%

) S

(100

.00%

) S

(100

.00%

)

G

964S

G

-

- -

S (1

00.0

0%)

- -

S (1

00.0

0%)

- -

- -

V

976I

V

I (

100.

00%

) -

I (10

0.00

%)

- -

- -

I (10

0.00

%)

- -

I (1.

87%

)

T9

82I

T -

- -

- -

- -

- -

- I (

100.

00%

)

V

984I

V

-

I (97

.72%

) I (

100.

00%

) -

I (10

0.00

%)

I (10

0.00

%)

I (10

0.00

%)

I (10

0.00

%)

I (10

0.00

%)

I (10

0.00

%)

-

L9

99S/

N/A

L

S (1

00.0

0%)

S (1

00.0

0%)

S (1

00.0

0%)

S (0

.62%

) -

S (1

00.0

0%)

S (5

.16%

) S

(100

.00%

) S

(1.2

7%)

S (9

9.13

%)

A (7

0.14

%);

N (2

0.07

%)

R

1007

K/E

R

-

- -

- K

(96.

01%

) -

- -

K (0

.20%

) K

(94.

08%

) E

(100

.00%

)

R

1009

K

R

K (0

.14%

) K

(0.0

2%)

- -

- K

(100

.00%

) -

- -

K (2

.18%

) -

I1

011V

I

- -

- -

- V

(77.

52%

) -

- -

- -

S1

018G

/R

S G

(100

.00%

) G

(100

.00%

) G

(1.1

6%)

- R

(100

.00%

) -

- -

G (3

.66%

) G

(100

.00%

) -

L1

019F

L

F (1

00.0

0%)

F (1

00.0

0%)

- -

- F

(77.

08%

) F

(100

.00%

) -

F (9

8.82

%)

- -

E1

020R

/K

E -

R (1

00.0

0%)

K (1

00.0

0%)

- -

R (1

00.0

0%)

- -

K (1

00.0

0%)

K (1

00.0

0%)

-

G

1021

E G

E

(100

.00%

) -

E (1

.09%

) -

- -

E (9

0.12

%)

E (1

00.0

0%)

E (1

00.0

0%)

E (9

8.69

%)

-

Q

1022

R

Q

R (9

9.78

%)

- R

(97.

76%

) -

- -

- -

- -

-

NS3


280

Prot

ein

Mut

atio

n

Patie

nts

S1

033A

S

- -

- A

(100

.00%

) A

(98.

74%

) -

- -

A (1

00.0

0%)

- A

(99.

82%

)

R

1052

K

R

K (1

00.0

0%)

K (1

00.0

0%)

K (1

00.0

0%)

K (9

9.62

%)

K (9

9.37

%)

- K

(100

.00%

) K

(100

.00%

) K

(100

.00%

) K

(100

.00%

) K

(100

.00%

)

T1

072S

T

- -

- -

S (1

00.0

0%)

- -

- -

- S

(26%

)

V

1074

I V

-

I (99

.39%

) -

I (10

0%)

I (10

0%)

- I (

100%

) I (

100%

) -

I (10

0%)

I (10

0%)

Y

1082

F Y

-

F (1

00.0

0%)

F (1

00.0

0%)

- F

(100

.00%

) F

(100

.00%

) -

- -

- -

S1

087T

/A

S T

(100

.00%

) -

- T

(93.

59%

); A

(6

.41%

) -

- -

A (1

00.0

0%)

T (2

.86%

) -

-

I1

097V

I

- V

(13.

96%

) -

- V

(99.

1%)

- -

- V

(0.6

9%)

- -

T1

098I

T

I (10

0.00

%)

I (99

.05%

) -

- -

- I (

0.64

%)

- -

- -

Q

1106

L Q

-

- -

- L

(100

.00%

) -

- -

- -

-

Q

1112

P/L

Q

- P

(100

.00%

) P

(100

.00%

) -

- P

(100

.00%

) L

(100

.00%

) -

- -

-

P1

115S

/A

P -

A (1

00.0

0%)

- -

- -

- S

(100

.00%

) -

S (1

00.0

0%)

-

L1

120M

L

- -

- -

- -

- -

- -

M (1

00.0

0%)

S1

127G

S

- -

- G

(63.

46%

) -

- -

- -

- -

R

1143

H/C

R

-

- C

(100

.00%

) H

(7.2

2%)

C (1

00.0

0%)

- H

(100

.00%

) -

- -

-

S1

148N

/T

S -

T (1

.84%

) -

N (1

00.0

0%)

T (9

6.86

%)

- T

(4.3

2%)

- -

- -

R

1149

K

R

- -

- -

- -

- K

(60.

57%

) -

- -

V

1158

I V

I (

36.2

6%)(A

TC)

I (10

0.00

%)

- I (

100.

00%

) I (

0.47

%)

I (10

0.00

%)

I (10

0.00

%)

- I (

96.3

4%)

- -

S1

173L

S

- -

- -

L (9

8.8%

)

F1

210Y

F

- -

- -

Y (1

00.0

0%)

- -

- -

- -

I1

274V

I

- -

- -

- V

(100

%)

V (1

00%

) V

(100

%)

V (1

00%

) V

(100

%)

V (1

00%

)

D

1275

E D

-

- -

E (1

00.0

0%)

- -

- -

- -

-

V

1282

A

V

- -

- -

- -

- -

- A

(27.

86%

) -

R

1283

K

R

K (1

00.0

0%)

- -

- -

- -

- -

- -

P1

290S

P

- -

- -

S (1

00.0

0%)

- -

- S

(12.

59%

) -

-


281

Prot

ein

Mut

atio

n

Patie

nts

I1

314M

I

- M

(41.

03%

) -

- -

M (1

.97%

) -

- M

(48.

98%

) -

-

A

1339

S A

-

- -

- -

- -

S (2

6.78

%)

- -

-

S1

354L

S

- -

- -

- -

- -

- L

(20.

78%

) -

V

1355

I V

-

- -

- -

- -

- I (

15.5

%)

I (1.

08%

) -

P1

360S

P

S (9

9.70

%)

S (1

00%

) -

S (1

00%

) S

(100

.00%

) -

- -

- -

-

N

1361

S N

-

S (1

0.94

%)

- -

- -

- -

- -

-

T1

370I

T

- -

I (1.

42%

) -

I (10

0.00

%)

- -

- -

- I (

10.3

2%)

I1

380L

I

- -

- -

- -

- -

- -

L (1

00.0

0%)

I1

382L

L (1

00.0

0%)

- L

(100

.00%

) L (1

00.0

0%)(C

TC)

L (1

00.0

0%)

L (100

.00%

)(CTC

) -

L (1

00.0

0%)

- -

L (1

00.0

0%)

T1

384A

/V/I

T -

A (9

8.90

%)

V (9

7.68

%)

A (1

00.0

0%)

V (7

9.02

%);

A

(19.

56%

) -

A (1

00.0

0%)

I (99

.38%

) -

A (

100.

00%

) A

(97.

63%

);

V (1

.27%

)

K

1397

R

K

R (1

00.0

0%)

- -

- -

- -

- -

- -

S1

408L

/Q

S -

- -

- -

L (1

00.0

0%

- Q

(100

.00%

) -

- -

G

1409

S/A

G

-

S (1

00%

) S

(100

.00%

) A

(100

.00%

) -

S (1

00.0

0%)

- -

- S

(98.

81%

) -

L1

412I

/V

L -

I (0.

67%

) V

(100

%)

- V

(0.3

8%)

V (1

00%

) I (

92.7

2%);

V

(7.2

8%)

- -

I (10

0%)

I (0.

06%

)

V

1425

A/I

V

A (4

3.07

%)

- -

- -

I (1.

19%

) A

(3.8

7%)

- -

- -

T1

428A

T

- -

- A

(2.8

5%)

- A

(21.

93%

) -

- -

A (

100.

00%

) A

(0.2

0%)

D

1431

N

D

- -

- N

(100

.00%

) -

- -

- N

(99.

64%

) N

(0.2

5%)

N (9

9.78

%)

V

1432

A

V

- -

- -

- -

- -

- A

(99.

66%

) A

(0.3

%)

F1

444Y

F

Y (1

00%

) Y

(100

%)

Y (1

00%

) -

- -

- -

- -

Y (9

9.93

%)

M

1496

R/L

/A

/N

M

L (1

00.0

0%)

- R

(100

.00%

) R

(100

.00%

) R

(100

.00%

) R

(100

.00%

) R

(100

.00%

) A

(100

.00%

) R

(98.

4%)

N (0

.19%

) N

(99.

8%)

T1

503A

T

A (0

.57%

) -

- -

- A

(18.

85%

) A

(6.9

4%)

- A

(0.6

8%)

- -

A

1523

S A

-

S (1

0.82

%)

- -

- -

- S

(100

.00%

) -

- -

S1

536T

S

T (1

00.0

0%)

- T

(100

.00%

) T

(100

.00%

) T

(100

.00%

) -

- T

(100

.00%

) -

- T

(99.

90%

)

A

1570

S A

-

- -

- -

- -

- -

- S

(100

.00%

)


282


Prot

ein

Mut

atio

n

Patie

nts

D

1581

E D

E

(100

.00%

) -

- E

(100

.00%

) -

- -

- -

- E

(100

.00%

)

F1

583L

/Y

F -

- L

(100

.00%

) -

- Y

(100

.00%

) -

- L

(99.

80%

) -

L (0

.80%

)

V

1587

T V

-

T (1

00%

) -

- -

- -

- T

(100

%)

- -

I1

612T

/A

I -

T (6

5.50

%);

A (3

3%)

- -

- T

(100

.00%

) -

- -

- T

(0.7

6%)

Q

1632

S Q

-

- -

S (1

00.0

0%)

- -

- -

- -

-

E1

634D

E

D (1

00.0

0%)

- -

- -

- -

- -

- -

T1

636N

/I T

- -

- -

- -

N (1

00.0

0%)

- -

I (98

.99%

) -

I1

641V

/M

I M

(99.

78%

) -

- V

(79.

65%

) -

- -

- V

(91.

87%

) -

V (1

00.0

0%)

A

1647

T A

T

(99.

61%

) -

T (1

00.0

0%)

T (1

00.0

0%)

T (1

00.0

0%)

- -

- T

(0.3

0%)

- T

(100

.00%

)

NS4

A

S1

664G

G

G

(100

.00%

) G

(100

.00%

) G

(100

.00%

) G

(100

.00%

) G

(100

.00%

) G

(100

.00%

) G

(100

.00%

) G

(100

.00%

) G

(100

.00%

) G

(100

.00%

) G

(100

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283


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286

EVALUATION Of THE IMMUNOgENICITY Of REVERSE-VACCINOLOgY DERIVED STAPHYLOCOCCUS AUREUS PROTEINS ON EXPERIMENTAL ENDOCARDITIS MODEL – Mihaela Oprea, Bogdan Cordoș, Monica Străuț, Adriana Costache, Felicia Antohe, Adrian Onu 5

CURRENT EXPERIMENTAL METHODS fOR ANALYZINg PROTEIN-PROTEIN INTERACTIONS – Darshak K. Bhatt, Elena D. Giol, Adriana Costache, Răzvan C. Stan .............. 17

IMPORTANCE OF SPECIFIC IgM SEROLOGY IN DIAGNOSIS OF PERINATAL INFECTIONS – Adrian Băncescu, Dan Ionescu ................................................................................................................... 30

ETIOLOGY AND INCIDENCE OF INFECTIOUS DISEASES OF THE HEART. AN UPDATE – Irina Alina Cucu, Mariana Carmen Chifiriuc .......................................................................................... 45

A REVIEW OF ZIKA VIRUS – Daniela Bădescu, Ani-Ioana Cotar, Cornelia-Svetlana Ceianu ..... 62

STUDY Of bILE SALT HYDROLASE IN LYSINIBACILLUS SPHAERICUS: A POTENT fISH PRObIOTIC AND ITS IN SILICO STRUCTURE PREDICTION FOR CATALYTIC INTERACTION – Arindam Ganguly, Amrita Banerjee, Asish Mandal and Pradeep Kumar Das Mohapatra ............ 81

PREVALENCE Of DERMATOPHYTOSIS AND ASSESSMENT Of ANTIfUNgAL SUSCEPTIBILITY IN PATIENTS REFERRED TO A MYCOLOGY CLINIC IN TEHRAN – Shadi Pakroo, Armin Tarrah, Ayatollah Nasrollahi Omran .................................................................. 91

DENDRIMERS – AS NEW PROMISING TOOLS IN THE MANAGEMENT OF MULTI-DRUG RESISTANT BACTERIAL INFECTIONS – Cristina Iulia Mitran, Mădălina Irina Mitran, Costin Ștefan Caracoti, Irina Codiță ........................................................................................................... 98

THE WEST NILE VIRUS (WNV) MOLECULAR DETECTION IN VECTORS: FROM THE BENCHTOP TO THE FIELD - DEPLOYABLE LABORATORY – Alexandru Filip Vladimirescu, Valeria Ciulacu-Purcărea, Liviu Florian Prioteasa, Elena Fălcuță ....................................................... 106

EVALUATION Of CLINICAL AND LAbORATORY EffECTS IN THE REPRODUCTION Of OSTEOMYELITIS IN RABBITS USING A HUMAN MRSA STRAIN – Diana Larisa Ancuța, Jasmina Manolescu, Claudiu Gal, Alexandru Muntean, Petronica Gheorghiu, Andrei Constantin Stoian, Cristin Coman .................................................................................................................................. 112

2018/19 INFLUENZA VACCINE EFFECTIVENESS IN ROMANIA IN A SEASON WITH INFLUENZA A SUBTYPES CIRCULATING – Mihaela Lazăr, Maria Elena Mihai, Carmen Maria Cherciu, Cătălina Pascu, Elena Stoian ...................................................................................................... 124

UPDATINg THE ROUTINE SPECIATION Of ROMANIAN CAMPYLOBACTER STRAINS – Mădălina Militaru, Marilena Sorokin, Simona Adriana Ciontea, Daniela Cristea, Codruța-Romanița Usein, Ileana Stoica ................................................................................................... 145

SUBJECT INDEX


287


PRO-AND ANTITUMOR ROLE OF THE INTERLEUKINS 1 TO 41– Ovidiu Farc, Victor Cristea 14

CIRCULATING TUMOR CELLS: FASCINATING INSIGHTS IN THE CLINICAL RELEVANCE – Iulia I. Niță, Valentin Vasile, Laura Zaharia, Vlad Tofan, Cătălin Țucureanu, Ramona Caraghergheopol, Aurora Sălăgeanu, Iuliana Caraș .............................................................................. 163

CYTOTOXICITY EVALUATION Of fE-bASED STAbILIZED SUSPENSIONS fOR bIOMEDICAL APPLICATIONS – Dora Domnica Baciu, Iulia Ioana Lungu, Andrei-Mihai Dumitrașcu, Gabriel Prodan, Aurora Sălăgeanu and Florin Dumitrache ................................................................................ 168

A PRObLEM-SOLVINg INTEgRATIVE APPROACH Of ESCHERICHIA COLI O157:H7 SEROTYPE DIAGNOSIS – Codruța-Romanița Usein, Mădălina Militaru, Mihaela Oprea, Sorin Dinu, Daniela Cristea, Adriana Simona Ciontea ................................................................................... 177

VIRULENCE AND RESISTANCE PROfILES Of SHIGELLA STRAINS ISOLATED IN ROMANIA FROM 2016 TO 2018 – Daniela Cristea, Adriana Simona Ciontea, Melania Mihaela Andrei, Andrei Popa, Mădălina Zamfir, Lavinia Zota, Maria Nica, Codruța-Romanița Usein .................... 182

EOSINOPHILIA AND PARASITIC DISEASES – Dan Steriu ................................................................. 189

CANTACUZINO NATIONAL MEDICO-MILITARY INSTITUTE fOR RESEARCH AND DEVELOPMENT SYMPOSIUM – Abstracts .............................................................................................. 209

CURRENT PROgRESS AND PERSPECTIVES Of MALDI-TOf MASS SPECTROMETRY APPLICATIONS fOR THE MEDICAL MICRObIOLOgY fIELD: A MINI-REVIEw – Andreia Amzuța, Alina Lenghel, Elena Diana Giol, Mihaela Palela ................................................................. 249

EXPERIMENTAL bIOfILM MODELS: A REVIEw – Ştefania Mădălina Negrea, Raluca Elena Iurea, Costin Ştefan Caracoti, Brînduşa Elena Lixandru, Elena Carmina Drăgulescu, Cristin Coman, Irina Codiţă .................................................................................................................................... 256

ULTRA-DEEP SEQUENCINg ASSESSMENT Of gENETIC VARIAbILITY IN HEPATITIS C VIRUS INFECTING ROMANIAN PATIENTS – Sonia Spandole-Dinu, Eugen Radu, Sorin Dinu, Georgeta Cardoș, Grațiela Țârdei, Petre Iacob Calistru, Emanoil Ceaușu, Laurențiu Micu, Simona Ruță, Camelia Sultana, Gabriela Oprișan ................................................................................. 267

288


AUTHOR INDEX

AAmzuța Andreia 249Ancuța Diana Larisa 112Andrei Melania Mihaela 182Antohe Felicia 5

BBaciu Dora Domnica 168Banerjee Amrita 81Bădescu Daniela 62Băncescu Adrian 30Bhatt Darshak K. 17

CCalistru Petre Iacob 267Caracoti Costin Ștefan 98, 256Caraghergheopol Ramona 163Caraş Iuliana 163Cardoș Georgeta 267Ceauşu Emanoil 267Ceianu Cornelia-Svetlana 62Cherciu Carmen Maria 124Chifiriuc Mariana Carmen 45Ciontea Adriana Simona 145, 177, 182Ciulacu-Purcărea Valeria 106Codiță Irina 98, 256Coman Cristin 112, 256Cordoş Bogdan 5Costache Adriana 5, 17Cotar Ani-Ioana 62Cristea Daniela 145, 177, 182Cristea Victor 149Cucu Irina Alina 45

DDas Mohapatra Pradeep Kumar 81Dinu Sorin 177, 267Drăgulescu Elena Carmina 256Dumitrache Florin 168Dumitraşcu Andrei-Mihai 168

FFarc Ovidiu 149Fălcuță Elena 106

GGal Claudiu 112Ganguly Arindam 81Gheorghiu Petronica 112Giol Elena Diana 17, 249

IIonescu Dan 30Iurea Raluca Elena 256

LLazăr Mihaela 124Lenghel Alina 249Lixandru Brînduşa Elena 256Lungu Iulia Ioana 168

MMandal Asish 81Manolescu Jasmina 112Micu Laurențiu 267Mihai Maria Elena 124Militaru Mădălina 145, 177Mitran Cristina Iulia 98Mitran Mădălina Irina 98Muntean Alexandru 112

NNegrea Ştefania Mădălina 256Nica Maria 182Niță Iulia I. 163

OOmran Ayatollah Nasrollahi 91Onu Adrian 5Oprea Mihaela 5, 177Oprişan Gabriela 267

289


PPalela Mihaela 249Pascu Cătălina 124Popa Andrei 182Prioteasa Liviu Florian 106Prodan Gabriel 168

RRadu Eugen 267Ruţă Simona 267

SSălăgeanu Aurora 163, 168Shadi Pakroo 91Sorokin Marilena 145Spandole-Dinu Sonia 267Stan Răzvan C. 17Steriu Dan 189Stoian Andrei Constantin 112Stoian Elena 124Stoica Ileana 145Străuţ Monica 5Sultana Camelia 267

TTarrah Armin 91Tofan Vlad 163

ȚŢârdei Graţiela 267Țucureanu Cătălin 163

UUsein Codruța-Romanița 145, 177, 182

VVasile Valentin 163Vladimirescu Alexandru Filip 106

ZZaharia Laura 163Zamfir Mădălina 182Zota Lavinia 182

290

CATEGORIES OF MANUSCRIPTSThe following types of articles are accepted for

publication in Romanian Archives of Microbiology and Immunology:

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PREPARATION OF MANUSCRIPTSManuscripts should be divided into the following

sections and order: Title page, Abstract and keywords, Introduction, Materials and Methods, Results, Discussion, Conclusions, Acknowledgements, References, Tables, Figure Legends and Figures.

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The same product names should be used throughout the text with the manufacturer’s name written in parentheses (at the first use).

5. Results should be clear and concise and be presented in a logical sequence in the text.

6. Discussion section should start by briefly summarizing the main findings.

It should enrich and not repeat previous sections (3 or 5).

7. Conclusions. In this section, the author(s) can summarize the paper’s findings and generalize their importance. It is also where the writer can raise questions, discuss ambiguous data, and recommend places for further research. Conclusions often occur in a section titled “Discussion.” This writing is an extension of a conversation(s) with colleagues.https://writingcenter.gmu.edu/guides/introductions-and-conclusions-for-scientific-papers

8. Acknowledgements (if applicable) of technical help and of financial material support.

9. References should be recent, with 70% of the titles dating from the past 5 years. They should be numbered consecutively in the order in which they are first mentioned in the text. Identify references in text, tables, and legends by Arabic numerals in square brackets (e.g. [1], [2-6], etc.). Authors are responsible for the accuracy and completeness of all references.

For the style and format of the References, authors should follow the standards promoted by the NLM’s International Committee of Medical Journal Editors ICMJE (www.nlm.nih.gov/bsd/uniform_requirements.html)

Journals:Hallal AH, Amortegui JD, Jeroukhimov IM,

Casillas J, Schulman CI, Manning RJ, et al. Magnetic resonance cholangiopancreatography accurately detects common bile duct stones in resolving gallstone pancreatitis. J Am Coll Surg. 2005;200(6):869-75.

Books:Carlson BM. Human embryology and develop-

mental biology. 4th ed. St. Louis: Mosby; 2009. 541 p.Book chapters:Blaxter PS, Farnsworth TP. Social health and class

inequalities. In: Carter C, Peel JR, editors. Equalities and inequalities in health. 2nd ed. London: Academic Press; 1976. p. 165-78.

Web pages:Diabetes Australia. Diabetes globally [Internet].

Canberra ACT: Diabetes Australia; 2012 [updated

292

Guidelines for authors

2012 June 15; cited 2012 Nov 5]. Available from: http://www.diabetesaustralia.com.au/en/ Understanding-Diabetes/DiabetesGlobally/

10. Tables with suitable captions at the top and numbered with Arabic numerals should be collected at the end of the text on separate sheets (one page per Table). Each column should be given a short or an abbreviated heading. Footnotes to tables should be marked with a) b) c) etc and *, **, *** should be reserved for p values. Each table must be understood independently of the text. All tables must be cited in the text.

11. Figures (illustrations) Figures should be submitted on separate pages at the end of the article (new page for each complete figure). They should be numbered in the order of their appearance with Arabic numerals. Figures should be submitted as TIFF files at a proper resolution as follows: Graphs at 800-1200 dpi; Photos at 400-800 DPI; Color 300-400 DPI. Text in figures should be 8-10 point in size. Each figure must have a separate legend. The legends should not appear under the figures, but be gathered in a separate section (Figure legends). Color figures can only be printed if the author is prepared to pay the cost incurred.

12. Figure legends should be supplied at the end of the manuscript, double spaced, with relevant figure numbers, labeling symbol and explanation.

Units of measurement, Symbols and abbreviationsSymbols for physical units should be those of the

Système Internationale (SI) Units.Alternative or non-SI units may be used, but these

must be defined at their first occurrence in the text.Nomenclature of MicroorganismsBinary names, consisting of a generic name and a

specific epithet (e.g., Escherichia coli), must be used for all microorganisms.

Genetic NomenclatureTo facilitate accurate communication, it is

important that standard genetic nomenclature be used whenever possible and that deviations or proposals for new naming systems be endorsed by an appropriate authoritative body.

Peer-Review Submitted manuscripts judged by the Editor-in-

Chief to be of potential interest to the biomedical scientific community are sent for formal review and critical assessment to expert reviewers (at least two/manuscript).

Reviewer selection is of utmost importance and the Editor-in-Chief bases his choice on such factors as expertise, scientific reputation, specific

recommendations etc. The work, effort and time spent by the reviewers on evaluating articles submitted for publication in Romanian Archives of Microbiology and Immunology are gratefully acknowledged and highly appreciated.

To the extent to which manuscripts are authors’ private property and authors may be harmed by premature disclosure of any or all of a manuscript’s details, reviewers should keep manuscripts and the information therein strictly confidential. Also, reviewers should declare their conflicts of interest and recuse themselves from the peer-review process if a conflict exists.

In a critical yet constructive manner, reviewers are expected to evaluate submitted manuscripts and comment on such aspects as:

- relevance of the manuscript to the journal- novelty and originality- clarity- technical quality- importance of the subject matter related to state-

of-the-art in the respective field- satisfactory presentation of data and conclusions

that are clearly supported and derived from presented data

When reviewers accept to assess a paper, we understand that they implicitly agree to review all subsequent revisions, in case these are necessary.

After reading the manuscripts, the reviewers may make the following recommendations to the editor:

- accept the manuscript, with or without minor/major revision

- reject the manuscript, but indicate to the authors that further work might improve the paper and justify resubmission

- reject the manuscript outright on lack of novelty of the information included in the paper, outdated references, major technical/interpretational problems etc.

The reviewers are kindly expected to write some comments about the manuscript in support of their recommendations, besides filling out the peer-review form.

In case one reviewer opposes publication and the other/others does/do not, the Editor-in-Chief may bring in additional reviewers to resolve the dispute.

The Editorial Team informs the corresponding author of the manuscript within 90 week days after submission that the paper is accepted for publication in the journal, needs minor/major revision or is rejected. Revised manuscripts should be resubmitted as soon as possible but not later than 14 week days.

Manuscripts revised by the authors according to reviewers’ observations and recommendations are revised by the reviewers/Editor-in-Chief.

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