STAPHYLOCOCCUS AUREUS STRAINS Isolated from INFECTIVE ENDOCARDITIS Mihaela Oprea, David Sebastian Patriche, Monica Str Ãuþ, Felicia Antohe

ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY

Founded by PROFESSOR ION CANTACUZINO in 1928

VOLUME 73 - Issue 3-4 July - December 2014

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MICROBIOLOGY Editorial Board: AND Viorel Alexandrescu (Cantacuzino NIRDMI, Bucharest, Romania) Gabriela Anton (Şt. S. Nicolau Virology Institute, Bucharest, Romania) IMMUNOLOGY Antonios Antoniadis (Aristotelian University of Thessaloniki, Greece) Daniela Bădescu (Cantacuzino NIRDMI, Bucharest, Romania) Print ISSN 1222-3891 Adrian Băncescu (Carol Davila UMPh, Bucharest, Romania) INDEXED IN MEDLINE Gabriela Băncescu (Carol Davila UMPh, Bucharest, Romania) Coralia Bleotu (University of Bucharest, Faculty of Biology, Romania) Jean-MarcCavaillon (Pasteur Institute, Paris, France) Ana Călugăru (Cantacuzino NIRDMI, Bucharest, Romania) Cornelia Ceianu (Cantacuzino NIRDMI, Bucharest, Romania) Carmen Chifiriuc (Cantacuzino NIRDMI, Bucharest, Romania) Radu Cojocaru (Ministry of Health, National Center of Public Health, Republic of Moldova) Nicolae Corcionivoschi (National Children Research Centre, Dublin, Republic of Ireland) Irina Codiţă (Cantacuzino NIRDMI, Bucharest, Romania) Lidia Cremer (Cantacuzino NIRDMI, Bucharest, Romania) Ionica Deliu (University of Piteşti, Romania) Adina Daniela Iancu (Cantacuzino NIRDMI, Bucharest, Romania) Angel Galabov (Bulgarian Academy of Sciences, Sofia, Bulgaria) Steliana Huhulescu (Austrian Agency for Health and Food Safety, Vienna, Austria) Luminiţa Smaranda Iancu (G r. T. Popa UMPh, Iaşi, Romania) Gabriel Ionescu (Cantacuzino NIRDMI, Bucharest, Romania) Anca Israil (Cantacuzino NIRDMI, Bucharest, Romania) Veronica Lazăr (University of Bucharest, Faculty of Biology, Bucharest, Romania) Emilia Lupulescu (Cantacuzino NIRDMI, Bucharest, Romania) Gina Manda (Victor Babeş NIRD, Bucharest, Romania) Grigore Mihăescu (University of Bucharest, Faculty of Biology, Bucharest, Romania) Roxana Moldovan (Victor Babeş UMPh, Timişoara, Romania) Geza Molnar (Ministry of Health, Bucharest, Romania) Alexandra Maria Năşcuţiu (Cantacuzino NIRDMI, Bucharest, Romania) Monica Neagu (Victor Babeş NIRD, Bucharest, Romania) Marian Neguţ (Carol Davila UMPh., Bucharest, Romania) Maria Nica (Carol Davila UMPh, Bucharest, Romania) Norica Nichita (Institute of Biochemistry, Romanian Academy, Bucharest, Romania) Adrian Onu (Cantacuzino NIRDMI, Bucharest, Romania) Marina Pană (Cantacuzino NIRDMI, Bucharest, Romania) Hervé Pelloux (Centre Hospitalier Universitaire, Grenoble, France) Mircea Ioan Popa (Carol Davila UMPh, Bucharest, Romania) Graţiela Pîrcălăbioru (University College Dublin, Republic of Ireland) Cristina Purcărea (Institute of Biology, Romanian Academy, Bucharest, Romania) Alexandru Rafila (Carol Davila UMPh, Bucharest, Romania) Anca Roşeanu (Institute of Biochemistry, Romanian Academy, Bucharest, Romania) Lila Shundi (Institute of Public Health, Tirana, Albania) Constantin Spânu (National Public Health Center, Chişinău, Republic of Moldova) Demetrios Spandidos (Medical School, University of Crete, Greece) Crina Stăvaru (Cantacuzino NIRDMI, Bucharest, Romania) Dan Steriu (Carol Davila UMPh, Bucharest, Romania) Cătălina-Suzana Stîngu (University of Medicine, Leipzig, Germany) Edit Székely (UMPh Târgu-Mureş, Romania) Subscription orders: Radu Iulian Tănasă (Cantacuzino NIRDMI, Bucharest, Romania) Orders can be placed directly Galina Tseneva (Pasteur Institute, Sankt Petersburg, Russia) with the publisher: Vasilica Ungureanu (Cantacuzino NIRDMI, Bucharest, Romania) „Cantacuzino“ National Codruţa-Romaniţa Usein (Cantacuzino NIRDMI, Bucharest, Romania) Institute of Research-Development Alexandru Filip Vladimirescu (Cantacuzino NIRDMI, Bucharest, Romania) Hans Wolf (Regensburg University, Germany) for Microbiology and Immunology Imola Zigoneanu (University of North Carolina at Chapel Hill, USA) C.P. 1-525, Splaiul Independentei 103, 050096, Bucureºti, România Editorial Team: Felicia RAPILAT , Monica TRăISTARU Fax: (40.21)306.93.07 TOTAL PUBLISHING HOUSE E-mail: [email protected] www.roami.ro Copyright © 2014 CANTACUZINO INSTITUTE B ucharest ROMANIAN ARCHIVES OF MICROBIOlOgy AND IMMUNOlOgy

CONTENTS

MICROBIOlOgy

65 IS THERE A DIFFERENCE IN THE INCIDENCE OF HELICOBACTER PYLORI INFECTION IN PATIENTS WITH SOME CHRONIC DISEASES? Cristian Adrian Constantinescu, Elena Mihaela Constantinescu

69 SERUM ALPHA FETOPROTEIN, A SURROGATE MARKER FOR LIVER DISEASE PROGRESSION IN CHRONIC HEPATITIS C Camelia Sultana, Claudia Diþã, Alina Botescu, Camelia grancea, Simona Ruþã

74 MOlECUlAR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS STRAINS ISOlATED FROM INFECTIVE ENDOCARDITIS Mihaela Oprea, David Sebastian Patriche, Monica Str ãuþ, Felicia Antohe

84 MICRORNA BIOgENESIS AND ITS ROlE IN HIV-1 INFECTION Adelina Roşca, gabriela Anton, Simona Ruþã

IMMUNOlOgy

92 MECHANISMS OF THROMBOSIS IN SySTEMIC lUPUS ERyTHEMATOSUS Adriana Metoni, Cristina Mambet, laura g. Necula, Ioana M. Aldea, Ana I. Neagu, lilia Matei, Denisa Dragu, Coralia Bleotu, Daniela A. Ion, Carmen C. Diaconu

REVIEW

99 ROMANIAN SCIENTISTS IN THE NOMINATION DATABASE FOR THE NOBEl PRIZE IN PHySIOlOgy OR MEDICINE, 1901-1951 Costin Cernescu

105 ROlE OF SALmOnELLA SEROlOgy A CENTURy AFTER THE WIDAl ERA Alexandra-Maria Nãşcuþiu

119 SUBJECT INDEX 121 AUTHOR INDEX

VOlUME 73 ISSUE 3- 4 JUly - DECEMBER 2014

63 INSTRUCTIONS TO AUTHORS

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64 IS THERE A DIFFERENCE IN THE INCIDENCE OF HELICOBACTER PYLORI INFECTION IN PATIENTS WITH SOME CHRONIC DISEASES?

Cristian Adrian Constantinescu, Elena Mihaela Constantinescu * Transilvania University of Braşov, Faculty of medicine, Braşov, România

ABSTRACT REZUMAT Globally, Helicobacter pylori infection is esti - La nivel global, se estimează că infecţia cu Heli - mated to be present in two-thirds of the population. cobacter pylori se întâlneşte la două treimi din This longitudinal, observational, comparative case- populaţie. Studiul longitudinal, observaţional, com - control study conducted in the county of Braşov on parativ, de tip caz-control efectuat în judeţul Braşov 763 subjects split into two groups: one with Heli - pe 763 de persoane care au fost împărţite în două lo - cobacter pylori infection (412 people) and the other turi, unul cu infecţie cu Helicobacter pylori (412 per - without infection (351 people) allowed us to com - soane) şi celălalt fără infecţie (351 persoane) a condus pare our results with other international epidemio - la obţinerea unor rezultate care au fost comparate cu logical studies. Thus, statistical interpretation of cele ale altor studii epidemiologice internaţionale. results indicates diabetes as an indifferent factor in Astfel, rezultatele interpretării statistice indică dia - the interrelation with Helicobacter pylori infection, betul ca factor indiferent în interrelaţia cu infecţia cu while asthma and hypertension might be considered Helicobacter pylori, astmul bronşic şi hipertensiunea as protective factors. arterială ca posibili factori de protecţie.

Keywords : Helicobacter pylori infection, diabetes, asthma, hypertension

IntroductIon The observation sample consisted of two groups of subjects: one group with Helicobacter pylori in - CDC (Center for Disease Control) states that ap - fection (n = 412) and the other group without infec - proximately 2/3 of the world population is infected tion (n = 351). with Helicobacter pylori , being in a situation of Separation between the two groups was done by chronic pain, for months, years, or decades, with using the Helicobacter pylori stool antigen test. The high social, family and economical implications. first group contained adult patients with positive He - Basically, Helicobacter pylori infection is a licobacter pylori fecal antigen test and the control public health problem because of the need for group included adults with negative test. Inclusion repeated consultations and laboratory examinations, criteria for the case group were: age over 18, men - the high cost of therapy, and high indirect costs tally healthy, informed consent of the respondent, the [1, 2]. presence of Helicobacter pylori in feces. Exclusion Because the infection with Helicobacter pylori criteria for case group: persons aged under 18, with is a public health problem, the implementation of impaired mental faculties, recent treatment with pro - good prophylaxis, consisting in a population screen - ton pump inhibitors (2 months prior to completing ing using noninvasive means, is necessary [3, 4, 5]. medical advice sheet type), antibiotics (2 months prior to completing medical advice sheet type), ab - sence of Helicobacter pylori in feces. MATERIALS AND METHODS A self-administered typical medical consultation The study was an observational, analytical, lon - chart was used, consisting in closed and open ques - gitudinal, case-control one, carried out from tions that people belonging to the two groups were 10.01.2012 to 30.01.2014, and enabled us to study kindly asked to answer. Chronic diseases of patients the possibility of an association between Helicobac - included in the study were documented based on ter pylori infection and the presence of chronic dis - records of personal consultation sheets from the gen - eases such as diabetes, asthma or hypertension. eral practitioners offices.

*Corresponding author: Elena Mihaela Constantinescu, Transilvania University of Braşov, Faculty of Medicine, Braşov, Romania, e-mail: [email protected]

65 CONSTANTINESCU and CONSTANTINESCU

All subjects voluntarily participated. The groups [7]. Therefore, it is tenable that the relationship be - were heterogeneous in terms of occupational status, tween Helicobacter pylori infection and asthma is level of education, including basic patient education, unclear and poorly supported or unsupported by ev - secondary and higher education, residing in both idence [8, 9, 10, 11]. rural and urban areas. The results of the study conducted on the popu - lation of Braşov county, demonstrates that there is a Statistical Protocol description statistically significant negative association between Descriptive presentation of data was done by them (OR = 0.72 and p = 0.03), supported by the fol - recording the data in tables which synthetically sum - lowing findings: 11.40% of people uninfected with marized the measured values. Statistical indicators Helicobacter pylori have asthma, while the percent - describing distributions of studied cases in separate age of people infected who have asthma is of only groups were calculated. 8.50%; of all subjects with asthma 46.67% are in -

The distinction of variables from the point of fected vs 53.33% individuals with asthma who are view of distribution was done by using the Kol - uninfected (Table 1). mogorov - Smirnov normality test. Application conditions for each statistica l te st ta ble 1. Stati stic al an alysis of the re lationship betw een were checked. the status infecte d/no n-in fected wi th Helicoba cter Analysis of qualitative variables (binary or pylori and the presence of asthma nominal) was performed by using the Chi-square ASTHMA test, taking into account the same significance level Ag HP absent present Total α = 0.05. in stool Interpretation of statistical results included eval - uation of the p value returned by applying the infer - absent 311 40 351 ence test. Basically, this value indicates the Line % 88.60% 11.40% 100.00% significance of the difference between the studied variables, by comparing the value of α with the value Column % 45.20% 53.33% 46.00% of p. The following was stated in statistical terms: if present 377 35 412 p > α ( p > 0.05) there is no statistical significance, but if p ≤ α ( p ≤ 0.05 ) there is statistical significance. Line % 91.50% 8.50% 100.00% Starting from the assumption that statistical sig - Column % 54.80% 46.67% 54.00% nificance does not necessarily require scientific sig - nificance and vice versa, the boundary results p <=> TOTAL 688 75 763 α were discussed by using the scientific experience Line % 94.48% 5.52% 100.00% of the author. Column % 100.00% 100.00% 100.00% All statistical analysis was performed by using

EpiInfo utility, a free software available from CD C Legend: Ag HP = Helicobacter pylori antigen Atlanta.

The resu lts r egarding the in cidenc e of Hel i - RESULTS AND DISCUSSION cobact er p ylori infection a nd m etabolic contro l of diabetes are co ntra dictor y. Some stud ies could no t Based on current knowledge, it can be said that identify significant differences in the incidence of there is not a clear relationship established between Helicobacter pylori infection in diabetics versus

Helicobacter pylori infection and asthma and we non-diabetics [12, 10]. Others have revealed a high cannot find a correlation between the presence of incidence of Helicobacter pylori infection in sub - Helicobacter pylori antigen, spirometric changes jec ts with insu lin-dependen t d iabe tes, a s well as in and duration of respiratory disease [6]. On t he other people w ith type 2 diab etes [13 , 14 ]. hand, the continuing decline in the frequenc y of The p otentially proposed bi olo gica l mecha - Helicobacter pylori infection in developin g coun - ni sms w hic h could exp lain this co nnection inv olve tries is in a direct relationship with increase d cases me tabol ic hom eo s tasis of th e host bacteri a by inter - of asthma and allergic phenomena through a m ech - f er ing wit h the secr etion of g hrelin, r espectively lep - anism of immune imbalance between Th1 and Th 2 tin, or t he delay o f g astric empty ing, wh ich lymphocytes function because of modern life style c ontributes to local bacterial colonization or an over -

66

Is there a difference in the incidence of Helicobacter pylori infection in patients with some chronic diseases? growth of bacteria in the gastrointestinal tract, as a Helicobacter pylori ; the latter act on the hyper reac - result of autonomic neuropathy [15, 16]. tive artery wall, especially in the smooth muscle Our results confirm that there is a positive as - fibers. This explains the decrease in blood pressure sociation, not statistically significant (OR = 2.38 and values, and especially the diastolic pressure, after p = 0.11), between the two groups su pported by Helicobacter pylori is eradicated. (Table 2) Hypertension morbidity in Braşov county pop - - the proportion of 1.14% of noninfected people ulation is significantly higher for the Helicobacter who have diabetes mellitus pylori uninfected group (p = 0.03), versus the group - percentage of 2.67% of infected people with of Helicobacter pylori infected people. diabetes The study conducted in Braşov county indicated - of all people with diabetes, 73.33% are in - a statistically significant negative association be - fected persons vs 26.67% uninfected. tween the presence of Helicobacter pylori infection

and hypertension (OR = 0.57, p = 0.03). The results table 2. S tatistic al an alys is of the rel ati onship bet ween showed us the following (Table 3): status infected/uninfected H. pylori and the p resence - persons belonging to the uninfected group - of diabetes the pre sence of hyp erte nsio n was found i n a perc ent - DIABETES age of 15.10 % - persons belo ngin g to the infecte d group - th e Ag HP absent present Total in stool presence of hypertension was found in a proportion of 9. 22% absent 347 4 351 - o f all subjects with h ypertensi on, 58.24% were Line% 98.86%1.14%100.00%non-infected subjects and 41.76% were infected

Column% 46.39%26.67%46.00% ta ble 3. Stat isti cal a nalysis of the relationsh ip betw een status infecte d/un infec ted H. pyl ori and the presen ce present 401 11 412 of hypertension Line% 97.33%2.67%100.00% HYPERTENSION Column% 53.61%73.33%54.00% Ag HP in stool absent Present Total TOTAL 748 15 763 Absent 298 53 351 Linie% 97.96%2.04%100.00%Line % 84.90% 15.10% 100.00% Column% 100.00%100.00%100.00% Column % 44.35% 58.24% 46.00% Legend: Ag HP = Helicobacter pylori antigen present 374 38 412

Line % 90.78 % 9.22% 100.00% A number of epidemiological studies conducted Column % 55.65% 41.76% 54.00% in the 200 1-2004 p eriod co ncluded t hat the inc idence of Hel icobac ter pylor i in fection in pati ent s wi th hy - TOTAL 672 91 763 p ertension is be tween 55-85% and in co ntrol sub - jects between 50-66% [17]. The results of another Line % 88.05% 11.95% 100.00% study showed a higher value of the systolic compo - Column % 100.00% 100.00% 100.00% nent of blood pressure in Helicobacter pylori in - Legend: Ag HP = Helicobacter pylori antigen fected patients [18]. The growth of blo od press ure va lues as a conse quence of He licoba cte r p y l o r i i n - fection has mechanisms which are difficult to ex - CONCLUSION pl ain. One o f th e hypothes es th at attemp t t o explain this mec han ism is the molec ular m imic ry between The size of the study group (763 subjects) al - He licobacter pyl ori and th e c ompon ents of the arte - l owed us, af ter statistical processing, to suggest an rial w all (e ndothelial cell s, smo oth mu scle fib ers). interrelation between some chronic diseases and in - Othe r hypotheses me ntion the fact that v asocon stric - fect ion wit h Helico bacter pylori . Thu s, chronic dis - tion appears and re sults in the g rowth o f a rte rial pre s - eas es c o ul d be ranked as protec tive, r isk or sur e by re leasing the cytokines induced b y indifferent fa ctors as follow s: diabete s can be con -

67

CONSTANTINESCU and CONSTANTINESCU sidered as an indifferent factor unrelated with Heli - 8. Asbjoernsdottir H, Sigurjonsd rB, Sveinsd SV, et cobacter pylori infection, asthma and hypertension al . Foodborne infections in Iceland: relationship to al - might be considered as “protective” factors for in - lergy and lung function. Laeknabladid. 2006. 92 :437- fection with Helicobacter pylori . 444. The evaluation of the various chronic diseases 9. Jarvis d, Luczynska c, chinn S, et al . The associa - tion of hepatitis A and Helicobacter pylori with sensi - as risk, protective, or indifferent factors, was done tization to common allergens, asthma and hay fever in when their presence in the total group of subjects of a population of young British adults. Allergy . the study had no evidently elevated values. The level 2004. 59 :1063-1067. of these values is due to the random selection of par - 10. Mccune A, Lane A, Murray L, et al . Reduced risk ticipants included in the study, most of them not pos - of atopic disorders in adults with Helicobacter pylori sessing comorbidities of the discussed kind. infection. Eur J Gastroenterol Hepatol . 2003. 15 :637- The involvement of Helicobacter pylori in the 640. pathogenesis of some gastric and extragastric dis - 11. Pessi t, Virta M, Adjers K, et al . Genetic and envi - eases has paved the way for research aimed at clari - ronmental factors in the immunopathogenesis of fying the still unknown pathogenic potential of this atopy: interaction of Helicobacter pylori infection and bacterium. The results of the survey conducted in IL4 genetics. Int Arch Allergy Immunol. Braşov county represent an additional database for 2005. 137 :282-283. 12. Bener A, Micallef r, Afifi M, et al . Association be - further research. tween Type 2 diabetes mellitus and Helicobacter py - lori infection. Turk J Gastroenterol . 2007. 18 :225-229. 13. 10. devrajani Br, Shah SZA, Soomro AA, devra - rEFErEncES jani t . Type 2 diabetes mellitus: A risk factor for He - licobacter pylori infection: A hospital based 1. Everhart JE, Kruszon-Moran d, Perez-Perez, GI., case-control study. Int J Diabetes Dev Ctries. 2010. tralka tS, McQuillan G . Seroprevalence and ethnic 30 (1):22–26. differences in Helicobacter pylori infection among 14. Bener A, Micallef r, Afifi M, et al. Association be - adults in the United States. J Infect Dis. 2000. tween Type 2 diabetes mellitus and Helicobacter py - 181 (4):1359-1363. lori infection. Turk J Gastroenterol. 2007. 18 :225. 2. Kuipers EJ, Pena AS, G. van Kamp, uyterlinde AM, 15. candelli M, rigante d, Atlanta G, et al . Helicobac - Pals G, Pels nF, Kurz-Pohlmann E, Meuwissen SG . ter pylori, gastrointestinal and metabolic control in Seroconversion for Helicobacter pylori. Lancet . 1993, young type 1 diabetes mellitus patients. Pediatrics . 342 :328-331 2003. 111 (4): 800-803. 3. osawa HG, et al . Helicobacter pylori infection. World 16. Yamagata H, Kiyohara Y, nakamura S, et al . Im - J Gastroenterol. 2008. 14 :6327–6333. pact of fasting plasma glucose levels on gastric cancer 4. Parsonnet J : The incidence of Helicobacter pylori in - incidence in a general Japanese population. The fection. Aliment Pharmacol 1995, Ther . 9 (Suppl. Hisayama Study. Diabetes Care. 2005. 28 :789-794. 2):45-51. 17. Migneco A, ojetti V, Specchia L. Eradication of H. 5. Perez-Perez GI, rothenbacher d, Brenner H . Epi - pylori infection Let.al. Inproves blood pressure values demiology of Helicobacter pylori infection. Helicobac - in patients affected by hypertension Helicobacter. ter 2004. 9 (Suppl. 1): 623-627. 2003. 8: 585-589. 6. tsang KW, Lam WK, chan Kn. et al . Helicobacter 18. Harvey r, Lane A, Murray L, et al . Helicobacter pylori seroprevalence.Asthma Respiratory Medicine pylori infection Effect of blood pressure on a commu - 2000. 94 : 756-759. nity based cross reational study. BMJ 2001. 323 : 264- 7. chen Y, Blaser MJ, et al . Inverse associations of He - 265. licobacter pylori with asthma and allergy. Arch Intern Med. 2007. 167 :821–827.

68 SERUM ALPHA FETOPROTEIN, A SURROGATE MARKER FOR LIVER DISEASE PROGRESSION IN CHRONIC HEPATITIS C

Camelia Sultana 1,2 , Claudia Diþã 2, Alina Botescu 1, Camelia Grancea 2, Simona Ruþã 1,2 * ¹Carol Davila University of medicine and Pharmacy, Bucharest, Romania; ²Ştefan S. nicolau Institute of Virology, Bucharest, Romania

ABSTRACT REZUMAT Serum alpha fetoprotein (AFP) is a com - Determinarea nivelului seric al alfafetoproteinei monly used marker in the screening for hepatocel - (AFP) este utilizată în mod curent ca test de screening lular carcinoma. This study aims to evaluate the pentru carcinomul hepatocelular (HCC). Studiul prezent value of AFP as an early predictor of the evolution şi-a propus evaluarea AFP ca marker timpuriu de pre - of chronic hepatitis C. In a retrospective study on dicţie a evoluţiei hepatitei C cronice. 116 HCV-infected patients (62.9% females, mean Într- un studiu retrospectiv pe 116 pacienţi cu hepa - age 49.13 ±1.73 years), increased levels of serum tită cronică C (62.9% femei, vârstă medie - 49.13±1.73 AFP (> 7 ng/mL) w ere found in 39.7% of cases. ani), valori crescute ale AFP serice (peste 7 ng/ml) au High serum AFP levels were more frequently de - fost detectate în 39.7% din cazuri. Un nivel crescut de tected in older patients and in those with severe AFP se înregistrează mai frecvent la pacienţii cu vârste fibrosis and cirrhosis (62.2% and 76.9% respecti - peste 40 de ani şi la cei cu fibroză hepatică avansată sau vely vs. 11.6% in those without significant fibro - ciroză (62.2% , respectiv 76.9% vs. 11.6% la cei fără fi - sis, p=0.0001). Increased AFP levels were broză semnificativă, p=0.0001). Se înregistrează corela - significantly associated with markers of hepatic ţii semnificative între nivelul AFP şi cel al markerilor de cytolysis ( ALT- r=0.245, p=0.009 and AST citoliză hepatică (ALT-r=0.245, p=0.009; AST -r=0.441, r=0.441, p=0.0001) and cholestasis ( GGT level- p=0,0001) şi de colestază (GGT-r=0.947, p=0.0001), in - r=0.947, p=0.0001), but not with HCV viral load. dependent de nivelul replicării virale. A predictive model based on AFP level and Un model predictiv al evoluţiei hepatitei C, bazat routinely monitored biochemical markers of liver pe corelaţia dintre nivelul seric al AFP şi markerii de fi - fibrosis and necroinflammatory activity can be a broză şi citoliză hepatică, poate fi extrem de util în prac - useful clinical tool in chronic HCV infection. tica clinică.

Keywords : chronic hepatitis C, alpha fetoprotein, aspartate aminotransferase (AST), alanin aminotransferase (AlT), gamma-glutamyl transpeptidase (ggT), liver fibrosis

INTRODUCTION [4], such as viral factors (baseline viral load, geno - type, diversity of quasispecies) or host variables Chronic hepatitis C is complicated by liver cir - (polymorphisms in the IL28B gene, age, race, co - rhosis and hepatocellular carcinoma (HCC), with morbidities and the strength of the immune re - high morbidity and mortality. Alpha fetoprotein sponse). (AFP) is a glycoprotein produced by the fetal liver, The aim of this study was to evaluate the poten - routinely used as an early screening marker for tial use of AFP as an early predictor for the progres - HCC. However, increased values of AFP (above 20 sion of chronic hepatitis C and, therefore, for the µg/L) have been also found in patients with ad - decision to treat and the likelihood of treatment re - vanced chronic hepatitis C, even in the absence of sponse. HCC [1], and its value as a solely tumor marker is debated [2, 3]. There is scarce information regarding MATERIALS AND METHODS the significance of AFP levels during the natural course of hepatitis C virus (HCV) infection and its This is a retrospective study on 116 HCV-in - association with other well-established predictors fected patients, diagnosed in Ş tefan S. Nicolau In -

*Corresponding author: Simona Ruþã, Address: Ştefan S Nicolau Institute of Virology, 285, Şos. Mihai Bravu, 030304, Bucharest, Romania, Telephone/fax:+40213242590 ; e-mail address; [email protected]

69 SULTANA et al. stitute of Virology. Informed consent was obtained (AST) was 50 IU/L in men and 35 IU/L in women, from all the patients, and the study was approved by for alanin-aminotransferase (ALT): 50 IU/L in both the Bioethic Committee of the Institute. Baseline men and women; and for gamma-glutamyl transpep - HCV viral load was measured using Cobas Ampli - tidase (GGT): 61 IU/L in men and 36 IU/L in cor TM HCV Monitor, vers 2.0, Roche, with a linear women. range of HCV RNA quantification between 600 - Serum AFP levels were measured using electro - 700 000 IU/mL, and a low detection limit of 600 chemiluminescence (ECLIA, Roche Diagnostics), IU/mL. HCV Genotyping was performed using a with a low detection limit of 0,24 ng/mL, and normal commercial Line Probe Assay (VERSANT™ HCV values defined as less than 7.0 ng/mL, according to Genotype 2.0 Assay), according to manufacturer’s the manufacturer’s instructions . instruction. Statistical analysis was performed using SPSS Liver fibrosis was appreciated using FIB-4, a 20 and GraphPad InStat 3; continuous variables were biochemical composite index calculated according compared using T student test, and categorical vari - to the Sterling’s formula [5]; using age, aspartate ables - using Chi-square test; statistical significance aminotransferase (AST), alanine aminotransferase was accepted at p value <0.05. (ALT) and platelet count: a FIB-4 value less than 1.45 shows no or minimal fibrosis (F0/F1) ; values between 1.45 - 3.25 (F2/F3) reflect severe fibrosis, RESULTS while values >3.25 indicate cirrhosis (F4) . The aminotransferases, alkaline phosphatase Virologic and biochemical markers of HcV and birubine levels were measured with the spec - infection. 116 HCV infected patients (62.9% fe - trophotometric standardized methods of the Interna - males) with a mean age of 49.13 ± 1.73 years, tional Federation for Clinical Chemistry. The upper mostly from urban areas ( 80.2%), were included in normal limit (ULN) for aspartataminotransferase the study (Table I). All patients were infected with

Fig. 1. Positive corelation between serum AFP levels and the degree of liver fibrosis (expressed by FIB-4 index) in 116 HcV infected study subjects .

70

Serum alpha fetoprotein, a surrogate marker for liver disease progression in chronic hepatitis C

Table I. General characteristics of the study patients Parameter Mean Limits % CI Age (years) 49.1 ± 11.73 20-72 46.9 – 51.3 HCV- RNA (IU/mL) 1.2 x10 6 ± 1.35 x10 5 0-6.8 x106 9.6 x105 - 1.5 x106 ALT (IU/L) 84.03 ± 56.9 17-609 68 – 100.1 AST (IU/L) 62.58 ± 61.9 16-345 51.2 – 73.4 GGT (IU/L) 72.76 ± 77.31 6-464 34.4 – 95.4 Alkaline phosphatase (IU/L) 72.37 ± 20.19 44-136 34.4 – 95.2 Total bilirubin (mg/dL) 0.65 ±0.34 0.11-136 0.51 – 0.72 Direct bilirubin (mg/dL) 0.20 ±0.12 0.01-0.83 0.17 – 0.23 Alpha fetoprotein (ng/mL) 9.3 ± 22.8 1.01-212 5.1 – 13.6

HCV genotype 1b, m ore than half ( 52.6%) had high of liver fibrosis: 11.6% of those without significant baseline HCV viral load (>600.000 IU/mL), and a fibrosis vs. 62.2% and 76.9% of those with severe significant percentage presented hepatic cytolysis fibrosis and cirrhosis, respectively; p=0.0001. (with increased levels of ALT and AST in 56% and Increasing AFP levels were significantly asso - 67.2% of the study subjects, respectively) and cho - ciated with markers of liver cytolysis ( ALT and AST lestasis (elevated level of GGT in 73.3% of the level higher than the upper normal limit) and cases). A minimal degree of liver fibrosis (defined cholestasis ( GGT values higher than the upper nor - by FIB-4 values lower than 1.45) was present in 50% mal limit). The highest levels of AFP (> 20 ng/mL – of the study subjects, while 38.4% had advanced he - considered as the cut-off in HCC screening) were patic fibrosis and 11.6% liver cirrhosis. A direct cor - constantly present in patients with severe fibrosis relation between FIB-4 values and GGT levels was and increased necroinflammatory activity. Although observed ( 70.6% of the patients with high GGT level a high HCV viral load (> 600 000 IU/mL) was more had an advanced degree of liver fibrosis vs 32.3% of frequently detected in patients with high levels of those with normal GGT levels , p=0.026). AFP, the association did not reach statistical signifi - cance. correlation between AFP levels and other predictors of liver disease progression. Increased AFP serum values (> 7 ng/mL) were found in 39.7% DISCUSSION of the patients. Higher levels of AFP were more fre - quently present in older patients (in 72.4% of those In this study, an increased AFP level was ob - aged 41-60 years vs 18 % of those younger than 40 served in patients with chronic hepatitis C, dirrectly years , p=0.04). Mean values of serum AFP were sig - correlated with the degree of liver fibrosis, one of nificantly higher in patients with severe liver fibrosis the main predictors of liver disease progression. Fi - (Fig. 1). The percentage of patients with elevated brosis is a conservative way of response to chronic AFP l evel increased proportionally with the degree hepatocyte injury, the result of abnormal increase in

Table II. correlation between serum AFP levels and fibrosis/necroinflammatory markers Serum AFP Percentage of patients with <7 ng/mL 7 -20 ng/mL > 20 ng/mL P value (n = 70) (n = 30) (n = 16) High AST levels 50 90 100 0.001 High ALT levels 42.9 73.3 81.3 0.009 High GGT levels 55.7 86.6 87.5 0.03 Severe liver fibrosis 28.6 56.6 72.7 0.01 (FIB4= 1.45 - 3.25) Cirrhosis (FIB-4> 3.25) 2.9 23.3 25 0.001 P<0.05 was considered significant

71

SULTANA et al. the synthesis of collagen and other extracellular ma - AcKnoWLEdGEMEntS trix components. Progressive fibrosis leads to cirrho - This study was partially supported by the Ro - sis, that will develop slowly over a 20 year period in manian Academy Programme for the Institute of Vi - 10-20% of patients with chronic hepatitis C [6], con - rology, and by Grant No. 5P30 AI036211-19 from ducting to the disruption of normal liver architecture, NIH, through Baylor International Pediatric AIDS appearence of fibrotic bands, parenchymal nodules Initiative. and vascular distortion; 1-4% of these cases will pro - gress every year to hepatocellular carcinoma. Mo - reover, there are studies indicating a small REFERENCES percentage of malignant transformation even in pa - tients with non-cirrhotic liver fibrosis [7], as such, 1. di Bisceglie A, Sterling r, chung r, Everhart J, the AFP level can constitute a simple and affordable dienstag J. Serum alpha-fetoprotein levels in patients marker for the early prognosis of the disease. with advanced hepatitis C: Results from the HALT-C A significant association between increased lev - Trial. Journal of Hepatology 2005 4(3) : 434–441. els of AFP and markers of hepatic cytolysis and 2. Bruix, J, Sherman M . Management of hepatocellular cholestasis was also observed; similar data have carcinoma: an update. Hepatology 2011 53 : 1020– been previously reported for HCV infected patients 1022. with liver fibrosis, but not for those with hepatocel - 3. Volk ML, Hernandez, Jc, Su GL . Risk factors for he - patocellular carcinoma may impair the performance of lular carcinoma [8, 9]. In logistic regression models, biomarkers: a comparison of AFP, DCP, and AFP-L3. elevated levels of transaminases, especially ALT and Cancer Biomark 2007. 3: 79–87. GGT, were associated both with advanced stages of 4. cernescu c, ruta S, Gheorghe L, Iacob S , Popescu liver fibrosis and high necro-inflammatory activity I, Wanless rS . “The Flying Publisher Guide to Hepa - [10], indicating a high risk of progression. Due to titis C Treatment”, Flying Publisher & Kamps, 2011, the association of AFP with both necroinflamatory http://www.flyingpublisher.com/pdf/FPG_004_Hepati - and fibrosis markers, future studies might test a tisCTreatment2011.pdf, ISBN: 978-3-942687-04-1 mathematical model based on the AFP value ad - 5. Sterling rK, Lissen E, clumeck n, Sola r . Devel - justed according to the transaminases levels, as a opment of a simple noninvasive index to predict sig - nificant fibrosis in patients with HIV/HCV coinfection. predictor of malignant transformation risk. Hepatology 2006. 43(6): 1317-25 The direct correlation between the AFP level 6. chen S and Morgan t. The Natural History of Hepa - and the degree of liver fibrosis observed in the cur - titis C Virus (HCV) Infection. Int J Med Sci 2006. 3(2): rent study might also have therapeutic applications. 47-52 An increased level of fibrosis is a marker of poor re - 7. Lok AS, Seeff LB, Morgan tr, di Bisceglie AM, sponse with the currently used terapeutic regimens. Sterling rK, curto tM . Incidence of hepatocellular The interferon-based treatment regimens have a carcinoma and associated risk factors in hepatitis C-re - modest impact on the progression of advanced lated advanced liver disease. Gastroenterology 2009. chronic hepatitis C towards HCC, with a very small 136: 138–148. 8. richardson P, duan Z, Kramer J, davila J, tyson proportion of patients with cirrhosis achieving a sus - G, El-Serag H. Determinants of serum alpha-fetopro - tained virologic response [11-14]. The best available tein levels in hepatitis C–infected patients. Clinical therapeutical option for non-responders is repre - Gastroenterology and Hepatology 2012. 10 (4) : 428– sented by the new associations of direct acting an - 433 tivirals (DAA). Combinations of two DAA - an 9. Hu KQ, Kyulo n, Lim n, Elhazin B, Hillebrand d, NS5B polymerase inhibitor (sofosbuvir) and an Bock t . Clinical significance of elevated alpha-feto - NS5A inhibitor (ledipasvir) [15, 16], or between protein (AFP) in patients with chronic hepatitis C, but three DAA - an HCV protease inhibitor (ABT-450, not hepatocellular carcinoma. The American Journal of boosted with ritonavir) plus an NS5A inhibitor (om - Gastroenterology 2004. 99: 860-865 bitasvir) and a non-nucleoside polymerase inhibitor 10. Bisceglie AM, Sterling rK, chung rt, Everhart JE. Serum alpha-fetoprotein levels in patients with (dasabuvir) [17], proved to be extremely efficient, advanced hepatitis C: Results from the HALT-C Trial. with sustained virologic response achieved in 82 - Journal of Hepatology 2005. 43: 434–441 100% of the cases. As such, patients with increased 11. Yoshida H, Shiratori Y, Moriyama M. Interferon levels of AFP can represent the most appropriate therapy reduces the risk for hepatocellular carcinoma: candidates for an interferon free - first line therapy. national surveillance program of cirrhotic and noncir -

72 Serum alpha fetoprotein, a surrogate marker for liver disease progression in chronic hepatitis C

rhotic patients with chronic hepatitis C in Japan. IHIT 14. nishiguchi S, Kuroki t, nakatani S. Randomised Study Group. Inhibition of Hepatocarcinogenesis by trial of effects of interferon-alpha on incidence of he - Interferon Therapy. Ann Intern Med 1999. patocellular carcinoma in chronic active hepatitis C 131(3): 174–81. with cirrhosis. Lancet 1995. 346(8982 ):1051–1055. 12. Imai Y, Kawata S, tamura S . Relation of interferon 15. Afdhal n, reddy Kr, nelson dr. Ledipasvir and therapy and hepatocellular carcinoma in patients with Sofosbuvir for Previously Treated HCV Genotype 1 chronic hepatitis C. Osaka Hepatocellular Carcinoma Infection. N Engl J Med 2014. 370: 1483-1493 Prevention Study Group. Ann Intern Med 1998. 16. Afdhal n, Zeuzem S, Kwo P . Ledipasvir and Sofo s- 129(2): 94–99. buvir for Untreated HCV Genotype 1 Infection. N 13. Mazzella G, Accogli E, Sottili S . Alpha interferon Engl J Med 2014. 370 :1889-1898 treatment may prevent hepatocellular carcinoma in 17. Ferenci P, Bernstein d, Lalezari J. ABT-450/r–Om - HCV-related liver cirrhosis. J Hepatol 1996. bitasvir and Dasabuvir with or without Ribavirin for 24(2): 141–147. HCV. N Engl J Med 2014. 370 :1983-1992

73 MOlECUlAR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS STRAINS ISOlATED FROM INFECTIVE ENDOCARDITIS

Mihaela Oprea 1*, David Sebastian Patriche 2, Monica Str ãuþ 1, Felicia Antohe 3

¹Cantacuzino national Institute of Research-Development for microbiology and Immunology, 103 Splaiul Independenþei, 050096, Bucharest, Romania; 2Cantacuzino national Institute of Research-Development for microbiology and Immunology, 103 Splaiul Independenþei, 050096, Bucharest, Romania (internship); 3nicolae Simionescu Institute of Cellular Biology and Pathology, 8 BP Haşdeu, 050568, Bucharest, Romania

ABSTRACT REZUMAT Infective endocarditis (IE) is an infection of the Endocardita infecţioasă (EI) reprezintă o heart endothelium and valves and is frequently a infecţie a endoteliului inimii şi a valvelor şi apare, consequence of a sanguine flow turbulence and in - frecvent, ca o consecinţă a turbulenţei fluxului san - jury of endocardium. Recent studies revealed an in - guin şi a traumelor endocardului. Studiile recente au crease of Staphylococcus aureus strains involved in arătat o creştere a numărului tulpinilor de Staphylo - IE, but no evident correlations between the genetic coccus aureus implicate în EI, dar încă nu s-au background of this bacterium and IE involvement of evidenţiat corelaţii între genomul acestei specii şi certain strains have been found yet. In this study we implicarea anumitor tulpini în EI. În acest studiu, am analyzed the virulence profile, including adhesins, analizat profilul de virulenţă, incluzând adezinele, exotoxinele, superantigenele şi determinanţii de exotoxins, superantigens and biofilm determinants, biofilme, alături de detectarea tipului agr , pentru along with agr type detection, for S. aureus strains tulpini de S. aureus izolate din EI, comparativ cu isolated from IE, versus non-IE originating strains. tulpini izolate în alte tipuri de bacteriemii. De aseme - We performed also bacterial typing (SCC mec typing, nea, am utilizat tehnici de tipizare moleculară spa -typing and MLST typing), in order to compare (tipizare SCC mec , spa şi MLST) pentru a compara our strains with international databases repositories. tulpinile noastre cu cele aflate în bazele de date Although the study was carried out on a reduced internaţionale. Chiar dacă acest studiu a fost realizat number of isolates, our observations confirm the pre - pe un număr redus de tulpini, observaţiile noastre vious works, showing that no major differences were confirmă studiile anterioare, nefiind semnalate observed between the genetic backgrounds of the diferenţe majore între informaţia genetică a two groups of strains analyzed. Notably, the added tulpinilor din cele două grupuri studiate. Ca un câştig value of this study was optimization of two new mul - al acestui studiu, au fost optimizate doua noi proto - tiplex PCR protocols, and the enrichment of interna - coale PCR multiplex şi au fost îmbogăţite bazele de tional databases with three new spa -types, three new date internaţionale cu trei tipuri spa noi, trei noi al - MLST alleles and four new MLST sequence types. lele MLST şi patru tipuri noi MLST.

Keywords : S. aureus, molecular characterization, endocarditis

IntroductIon Coagulase-negative staphylococci and viridans streptococci were considered the predominant cause Infective endocarditis (IE) is an infection of the of IE, but recent studies revealed an increasing num - heart endothelium and valves and is frequently a ber of Staphylococcus aureus strains involved in IE. consequence of a sanguine turbulence and an injury A study conducted by “International Collaboration of endocardium. Initially, a sterile vegetation can on Endocarditis” between 2000 and 2003 revealed occur, but it can be infected by bacteria accidentally that 31.4% of 1779 IE cases were due to S. aureus entering the bloodstream [1]. [2]. Another work, published in 2012, studying cases

*Corresponding author: Mihaela Oprea, Cantacuzino National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independen ţei, 050096, Bucharest, Romania, e-mail [email protected], tel. +40213069223

74 Molecular characterization of Staphylococcus aureus strains isolated from infective endocarditis from 1999 to 2008 reported a proportion of 46% out biofilm determinants detection, along with agr type of 87300 cases [3] as well. This may be due to a detection for S. aureus strains isolated from IE, ver - changing epidemiology of IE, with an increase of sus non-IE derived strains. Also, the methicillin sen - medical care involvement (prosthetic devices, sitivity was determined, as this issue is very gastrointestinal surgery, dental medicine, etc.), as important for treatment options. We performed also S. aureus is an important nosocomial agent. bacterial typing, in order to compare our strains with Immuno compromised subjects, with a predisposition the strains from international database repositories. for IE, can be easily infected with nosocomial S. au - reus isolates, sometimes with a multidrug resistance phenotype. Also, S. aureus is the predominant MATERIAL AND METHOD species found in narcotic consumers with IE. But S. aureus may be also community-acquired, entering Strains Collection the bloodstream following a superficial lesion and We characterized the genetic background of producing IE, especially in those patients with an af - seven S. aureus strains isolated from patients with IE, fected endocardium [1]. The IE produced by S. au - along with nine isolates from other blood infections reus is often very severe due to characteristics of this and one reference S. aureus strain, namely S. aureus versatile bacterium. MW2 (GenBank accession no. BA000033.2). A number of studies were conducted in order to The IE S. aureus strains were isolated from pa - reveal if there is any difference between the isolates tients with definite endocarditis from three different producing IE and the other isolates circulating in the hospitals and were labeled from 1 to 7. The nine non- bloodstream but not producing IE. The results did IE strains were labeled from 8 to 16 and were col - not demonstrate any striking genetic or phenotypic lected from patients with other bloodstream characteristics of IE strains analyzed so far [4-6]. In infections than IE and hospitalized in a single unit, addition, when the IE strains belonged predomi - at the same time period as IE isolates. The reference nantly to some clonal complexes (CC), the results strain S. aureus MW2 was labeled in this study with from different studies did not overlap. Nienaber et no. 17. al . [7] suggested that CC30 is frequently associated with IE comparing with other staphylococcal infec - DNA Extraction tions. Instead, Nethercott et al . [8] showed that CC12 DNA extraction was performed with Nucleo- and CC20 are predominantly associated with IE, Spin Tissue kit (Macheray Nagel, Germany), while CC45 could not be detected in this disease. following producer’s instructions. The latter also reported a t160 predominant spa- type for IE strains. On the contrary, other researchers did Detection of Virulence Factors Genes not find any evident relation between IE strains and The presence of some virulence factors genes spa -types [4-5]. was determined by simplex or multiplex PCR reac - The involvement of some adhesins in IE progres - tions, using 1.5 U of GoTaq Polymerase (Promega sion was already described. It is now known that Corporation, Madison, WI, USA), 200 µM each clumping factor A and B (ClfA and ClfB), which are dNTPs (Promega Corporation, Madison, WI, USA) adhesion proteins for fibrinogen, fibronectin binding and GeneAmp PCR system 2700 thermocycler proteins (FnbPA and FnbPB), serine-rich adhesin for (Applied Biosystems CA, USA). platelets (SraP) or collagen adhesion (Cna) are impor - l Triplex PCR for nuc , lukF/S and mecA genes am - tant factors in the development of IE vegetations. plification These proteins are present in the majority of S. aureus An already published PCR protocol was used to strains. However, FnbPB, ClfB and Cna seem to be amplify in a single reaction three genes: nuc (coding associated more frequently to IE strains [7, 9-10]. for thermonuclease, as internal positive control), Some studies also reported correlations between lukF/S (coding for Panton-Valentine leukocidin, a IE and superantigens bearing strains or producing potent virulence factor) and mecA gene (the main de - extracellular fibrinogen binding protein (Efb) and a terminant of methicillin resistance) [11]. Primers subunit compound of leukocidin/hemolysin family used are listed in Table 1 and the amplification pro - [6]. gram was: 95ºC – 4 min., 35 cycles with 94ºC – 1 In this study, we analyzed the virulence profile, min., annealing at 55ºC – 1 min., extension at 72ºC including adhesins, exotoxins, superantigens and – 1,5 min., and final extension at 72ºC – 4 min.

75 OPREA et al.

table 1. Primers for nuc, lukS/F and mecA amplification

table 2. Primers for efb , clfA and clfB genes amplification

table 3. Primers for cna, eno and ebpS genes amplification

l Triplex PCR for efb, clfA and clfB genes amplifi - Several simplex PCR were performed in order cation to identify the corresponding genes. The amplifica - Fragments of genes coding for proteins binding tion reaction conditions for previous triplex reactions to fibrinogen (Efb, ClfA, ClfB) were amplified in a runs were used with slight modifications: 0.2 µM single reaction, optimized in this study. For a 50 µl was used for each primer and the annealing temper - reaction, 1.5 units of enzyme were used. Primers are ature was decreased to 50ºC. listed in Table 2 and concentrations used were: 0.14 Primers used for these reactions are listed in µM for efb and 0.08 µM for clfA and clfB . The same Table 4. In this study, primers for sraP were designed amplification program as above was used. with GeneRunner software (www.generunner.net/). l Triplex PCR for cna, eno and ebpS genes amplifi - l PCR for amplification of icaA gene, a member of cation ica locus For amplification of cna (coding for adhesin to For identification of icaA gene (coding for collagen), eno (coding for enolase, a protein binding N-acetylglucosamine transferase, responsible for to laminin) and ebpS (coding for protein binding to polysaccharide intercellular adhesin synthesis), elastin) a triplex PCR was optimized, using 0.08 µM primers ica3F and ica4R (Table 5) were used, and primers for cna , 0.04 µM primers for eno and amplification program was: 94ºC – 5min., 25 cycles 0.26 µM primers for ebpS (Table 3), 1.5 units of with 94ºC – 1min., 52ºC – 45 sec., 72ºC – 1min. and enzyme, in a volume of 50 μl and the same amplifi - final extension 2 min. at 72ºC [20]. cation program as above was used. agr Type Identification l PCR for amplification of genes coding for adhesins agr locus, the main and the most studied system (fnbA, fnbB, sraP - gene coding for serine-rich ad - involved in the control of virulence factors expres - hesin for platelets , map/eap – coding for MHC sion, was identified by PCR, using 5 primers: a uni - class II analog protein ), enterotoxins A, B, C, D, versal primer, specific for all five agr types and one H, I, J, M (sea, seb, sec, sed, seh, sei, sej, sem), ex - primer for each distinct agr type (Table 6). The pa - foliatins A, B, D (eta, etb, etd) and toxic shock rameters of the reaction were described by Holtfreter syndrome toxin (tst). et al . [18].

76 Molecular characterization of Staphylococcus aureus strains isolated from infective endocarditis

table 4. Primers for simplex Pcrs

table 5. Primers for icaA gene amplification

Detection of SCC mec Type for mecA Positive spa -Typing Strains For amplification of a fragment spanning spa Detection of SCCmec type can be used as a gene, SeqNet protocol was followed (http://www.se - rapid typing method and also can give information qnet.org/downloads.html; [25]). For better results, about strains traceability [21]. Until now, 11 SC - FastStart High Fidelity PCR system, dNTPack kit Cmec types are known, but SCC mec I to V are the (Roche) was used. Amplicons were purified with predominant types (http://www.sccmec.org/Pages/ NucleoSpin Extract II (Macheray Nagel, Germany), SCC_ClassificationEN.html). directly from the amplification products. The same In this study types I to V were identified in a 25 SeqNet protocol was followed for sequencing, using µl reaction with parameters described by Boye et al ., ABI Prism BigDye Terminator v3.1 Ready Reaction 2007 [22]. Primers are listed in Table 7. Cycle Sequencing Kit (Applied Biosystems CA, The results were interpreted as presented in USA). Reaction products were purified with DyeEx Table 8. 2.0 Spin Kit (Qiagen) and ABI 3130 Avant Genetic

77 OPREA et al.

Analyzer (Applied Biosystems CA, USA) was used RESULTS for sequencing. Methicillin Resistance Distribution The resulting sequences were analyzed with Only for two S. aureus IE isolates mecA gene Ridom StaphType software (Ridom GmbH, Ger - PCR amplification yielded a positive result (strains many) [25] and synchronized with Ridom spa server 6 and 7). Also, for three out of nine S. aureus non- (http://www.spaserver.ridom.de/). IE isolates a specific 532 bp amplicon was obtained (strains 9, 13 and 16). The internal control was pos - MLST Typing itive for all the reactions and the MW2 strain methi - MLST typing consists of sequencing fragments cillin resistance was also confirmed (Fig. 1). spanning seven housekeeping genes and the recom - mended protocol and primers were published by En - Virulence Factors Distribution right et al ., 2000 [26]. DNA sequences obtained with Only two non-IE isolates harbored gene for Pan - ABI 3130 Avant Genetic Analyzer, were edited with ton-Valentine leukocidin, along with the reference BioEdit Sequence Alignment Editor v7.0.9 software strain (Fig. 1). All the tested isolates were positive (http://www.mbio.ncsu.edu/bioedit/bioedit.html) for genes coding for proteins binding to elastin and [27] and analyzed on http://saureus.mlst.net/ site for laminin. Two out of 7 IE isolates and one out of 9 identification of every single allele and, finally, se - non-IE isolates do not bear cna gene (Fig. 2). quence type (ST) for each strain.

table 6. Primers for agr type identification

table 7. Primers for Scc mec typing

table 8. Interpretation panel for Scc mec typing

78 Molecular characterization of Staphylococcus aureus strains isolated from infective endocarditis

table 9. s pa -types distribution

Amplification of clfA and clfB genes was posi - Regarding the enterotoxin genes presence, three tive for all strains. Three IE and two non-IE isolates IE isolates were positive for enterotoxin M, whereas did not exhibit gene efb (Fig. 3). zero was positive in non-IE group. No isolates were Performing PCR for other adhesins and secreted found positive for enterotoxin B (bioterrorism agent) virulence factors revealed that some genes were and enterotoxin J. All the isolates were positive for present in all the isolates ( fnbPA, sraP and eap /map ), icaA gene. and other genes ( eta, etb and etd ) in none of the iso - A complete distribution of tested virulence fac - lates. tst gene was present only in two IE isolates. tors is presented in Fig. 4. Three IE isolates and two non-IE isolates did not harbor fnbPB gene. agr Type Identification In IE group, four isolates were agr 3, two were agr 2 and one was agr 4. In non-IE group, four iso - lates were agr type 1, four isolates were agr 3 and one isolate was agr 4 (Fig. 5).

SCCmec Typing Both methicillin resistant IE isolates harbored mecA gene in a type IV SCC mec element. Also, one non-IE methicillin resistant strain harbored a SCC mec IV type and other three non-IE isolates revealed the presence of SCC mec type III. For MW2 strain, type IV SCC mec was confirmed.

Fig. 1. triplex Pcr for nuc, lukS/F and mecA genes spa -Typing Legend: 1 – isolate no. 8, 2 – isolate no. 3, 3 - isolate Submitting the sequences obtained for spa gene no, 4 – molecular weight (MW) marker 100 bp, 5 – isolate hyper-variable region to the Ridom spa server, spa no. 14, 6 – S. aureus MW2, 7 – negative control types were assigned, as follows:

Fig. 2. triplex Pcr for cna , eno and ebpS genes Fig. 3. triplex Pcr for efb , clfA and clfB genes Legend: 1 – MW marker 100 bp, 2 – isolate no. 1, 3 – iso - Legend: 1 – isolate no. 8, 2 - MW marker 100 bp, 3 – iso - late no 2., 4 - isolate no 4., 5 – isolate no.8, 6 – isolate no. late no. 9, 4 - isolate no. 10, 5 – negative control 11, 7 – MW marker 100 bp

79 OPREA et al.

Fig. 4. distribution of virulence factors genes: a) adhesins; b) exoenzymes; c) enterotoxins

Fig. 5. a gr type identification Legend: 1 – isolate no. 9, 2 – isolate no. 10, 3 - isolate no. 8, 4 – isolate no. 13, 5 – isolate no. 14, 6 – isolate no. 11, 7 – MW marker100 bp, 8 – isolate no. 12, 9 – isolate no. 1, 10 – isolate no. 16, 11 – negative control.

80 Molecular characterization of Staphylococcus aureus strains isolated from infective endocarditis

table 10. St distribution

spa- types t5891, t5890 and t9057, detected in IE isolates where the corresponding genes were not IE isolates, were considered, at the time of synchro - detected. Toxic shock syndrome protein and entero - nization with Ridom spa server, new types, and a spa toxin M are potent superantigens, enhancing the vir - type number was assigned for each allele. Also, the ulence potential of these strains. strains with these new spa alleles were entered into Jarraud et al ., [18] showed that agr 1 type pres - the server database. ence could be positively correlated with IE. In con - trast with these findings, we identified mostly types MLST Typing 2 and 3 in IE isolates and agr types 1 and 4 in non- Following the sequencing and questioning of S. IE isolates. aureus MLST database, we identified a new arrange - The spa and MLST types identified in this study ment of the seven alleles and the new ST was desig - revealed a great diversity of isolates, without an epi - nated as ST 2959. Also, three new alleles were found demiological or causal correlation. and a unique number was assigned by the database Notably, we identified three new spa -types for curator for each allele, respectively each new ST: tpi IE isolates, methicillin-sensitive, suggesting that gene – allele 320 (ST 2958), aroE gene – allele 435 these strains are not bearing virulence determinants (ST2980) and tpi gene – allele 323 (ST2981). ST dis - that can give the status of successful clones. tribution in the two groups is shown in Table 10. Some authors suggested that particular CC are prevalent in IE [7-8], but those findings seem to be related to circulating strains characteristics for the DISCUSSION respective area or to the study design, because the results do not overlap entirely. The four new ST No major differences were observed between IE identified in this study lead us to the same conclu - and non-IE isolates in terms of genetic background. sion, despite the limited statistical data about the cir - The methicillin resistant IE isolates, harboring culating ST in Romania. Maybe a better knowledge SCC mec IV cassette, seems to be a community ac - of local MLST types is required in the future. quisition, as this type of cassette is predominantly Following the molecular analysis of IE versus found in community strains [28]. It is not surprising non-IE isolates, we cannot point out major differ - that gene for Panton-Valentine leukocidin was not ences between the two groups or that the genetic detected in IE strains, since this virulence factor was background alone can influence the course of IE. reported to be rarely present in bloodstream isolated However, the IE isolates tested in this study seem strains [29]. However, two non-IE strains were more likely to be community acquired, entered the positive for this gene, but the medical history of pa - bloodstream as a result of quotidian activities, than tients suggested a nosocomial acquisition. nosocomial strains, as a results of medical proce - Adhesins distribution was uniform between the dures. examined groups, therefore we cannot conclude that Despite the fact that the study was performed some of the adhesins are favoring the attachment of on a reduced number of isolates, our observations bacteria to vegetation. confirm the previous studies [4-6]. Taken together, We can remark two strains with potential for these data support the conclusion that there is not an toxic shock syndrome, and also four strains entero - evident correlation between pathogenic potential of toxin M producing in IE group, in contrast to non- particular strains of S. aureus involvement in IE.

81 OPREA et al.

However, local and micro-environmental favoring 9. Hienz S, et al. Collagen binding of Staphylococcus au - conditions should be taken into consideration when reus is a virulence factor in experimental endocarditis. evaluating the causal-effect relation between J Infect Dis 1996. 174: 83-88. S. aureus and IE co-existence. 10. Heying r, van de Gevel J, Que YA, Moreillon P, Regardless of the number of studied strains, as Beekhuizen H. Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experi - a gain of this study, we optimized two new multiplex mental endocarditis: FnBPA is sufficient to activate PCRs protocols that can reduce the time and the human endothelial cells. Thromb Haemost 2007. costs for further studies. Also, we identified three 97(4): 617-626. new spa -types, three new MLST alleles and four 11. drăgulescu Ec, oprea M, Străuţ M, Irina codiţă . new ST and, thus, we contributed to the enrichment Detectarea rapidă a tulpinilor de Staphylococcus au - of available databases repositories. reus comunitar meticilino-rezistent folosind tehnica PCR multiplex, A XI-a Reuniune Anuală de Microbi - ologie, 24-26 mai 2007, Mamaia, Romania - poster; AcKnoWLEdGEMEntS : This study was 12. Strommenger B, Kettlitz c, Werner G, Witte W. financially supported by Ministry of National Multiplex PCR assay for simultaneous detection of Education - PARTENERIATE 42-119/2008 and nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol 2003. PN 09-22 01 01. 41(9):4089-4094 13. Lina G, Boutite F, tristan A, Bes M, Etienne J, Vandenesch F . Bacterial competition for human nasal rEFErEncES cavity colonization: role of staphylococcal agr alleles. Appl Environ Microbiol 2003. 69(1): 18-23. 1. Prendergast Bd. The changing face of infective endo - 14. Brakstad oG, Aasbakk K, Maeland JA . Detection carditis. Heart 2006, 92: 879-885. of Staphylococcus aureus by polymerase chain reac - 2. Fowler VG, et al. Staphylococcus aureus endocarditis: tion amplification of the nuc gene . J Clin Microbiol a consequence of medical progress. JAMA 2005. 293 1992. 30(7): 1654-1660. (24): 3012-3021. 15. tristan A, Ying L, Bes M, Etienne J, Vandenesch, 3. Federspiel JJ, Stearns Sc, Peppercorn AF, chu VH, Lina G . Use of multiplex PCR to identify Staphylo - Fowler VG . Increasing US rates of endocarditis with coccus aureus adhesins involved in human hematoge - Staphylococcus aureus : 1999-2008. Arch Intern Med nous infections. J Clin Microbiol 2003. 2012. 172(4): 363 – 365. 41(9):4465-4467. 4. chi cY, Wang SM, Lin cc, Liu cc. Microbiological 16. Mongodin E, Bajolet o, cutrona J, Bonnet n, characteristics of community-associated Staphylococ - dupuit F, Puchelle E, de Bentzmann S. Fibronectin- cus aureus causing uncomplicated bacteremia and in - binding proteins of Staphylococcus aureus are in - fective endocarditis. J Clin Microbiol 2010. volved in adherence to human airway epithelium. 48:292-294. Infect Immun 2002. 70(2): 620-630. 5. Feil EJ, cooper JE, Grundmann H, robinson dA, 17. Peacock SJ, Moore cE, Justice A, Kantzanou M, Enright Mc, Berendt t, et al., How clonal is Staphy - Story L, Mackie K, o’neill G, day nP. Virulent lococcus aureus ? J Bacteriol 2003. 185:3307-3316. combinations of adhesins and toxins genes in natural 6. tristan A, rasigade J-P, ruisendaal E, Laurent F, populations of Staphylococcus aureus . Infect Immun Bes M, Meugnier H, et al. Rise of CC398 lineage of 2002. 70(9): 4987-4996. Staphylococcus aureus among infective endocarditis 18. Holtfreter S, Grumann d, Schmudde M, nguyen isolates revealed by two consecutive population-based Htt, Eichler P, Strommenger B, Kopron K, Ko - studies in France 2012, PloS One 7:e51172.doi: lata J, Giedrys-Kalemba S, Steinmetz I, Witte W, 10.1371/journal.pone.0051172. Bröker BM . Clonal distribution of superantigen 7. nienaber JJc, Sharma Kuinkel BK, clarke-Pear - genes in clinical Staphylococcus aureus isolates. J son M, Lamlertthon S, Park L, rude tH, et al. Me - Clin Microbiol 2007. 45(8): 2669-2680. thicillin susceptible Staphylococcus aureus 19. Jarraud S, Mougel c, thioulouse J, Lina G, Meug - endocarditis isolates are associated with clonal complex nier H, Forey F, nesme X, Etienne J, Vandenesch F . 30 genotype and a distinct repertoire of enterotoxin and Relationships between Staphylococcus aureus genetic adhesins. J Infect Dis 2011. 204: 704-713. background, virulence factors, agr groups (alleles), and 8. nethercott c, Mabbett An, totsika M, Peters P, human disease. Infect Immun 2002. 70(2):631-641. ortiz Jc, nimmo Gr, coombs GW, Walker MJ, 20. chokr A, Watier d, Eleaume H, Pangon B, Ghnas - Schembri MA. Molecular characterization of endo - sia Jc, Mack d, Jabbouri S. Correlation between carditis-associated Staphylococcus aureus . J Clin Mi - biofilm formation and production of polysaccharide crobiol 2013. 51(7): 2131-2138. intercellular adhesin in clinical isolates of coagulase-

82 Molecular characterization of Staphylococcus aureus strains isolated from infective endocarditis

negative staphylococci. Int J Med Microbiol 2006. tant Staphylococcus aureus in a university hospital 296(6):381-388. setting using a novel software for spa -repeat determi - 21. Stephens AJ, Huygens F, Giffard PM. Systematic nation and database management. J Clin Microbiol derivation of marker sets for staphylococcal cassette 2003. 41: 5442-5448. chromosome mec typing. Antimicrob Agents 26. Enright Mc, day nPJ, davies cE, Peacock SJ, Chemother 2007. 51:2954-2964. Spratt BG . Multilocus sequence typing characteriza - 22. Boye K, Bartels Md, Andersen IS, Møller JA, tion of methicillin-resistant and methicilin–susceptible Westh H. A new multiplex PCR for easy screening of clones of Staphylococcus aureus . J Clin Microbiol methicillin-resistant Staphylococcus aureus SCC mec 2000. 38 (3): 1008-1015. types I–V. Clin Microbiol Infect 2007. 13: 725–727. 27. Hall tA. BioEdit: a user-friendly biological sequence 23. oliveira dc, de Lencastre H. Multiplex PCR strat - alignment editor and analysis program for Windows egy for rapid identification of structural types and 95/98/NT. Nucl Acids Symp Ser 1999. 41:95-98. variants of the mec element in methicillin-resistant 28. Vandenesch F, naimi t, Enright Mc, Lina G, Staphylococcus aureus. Antimicrob Agents nimmo Gr, Heffernan H, Liassine n, Bes M, Chemother 2002. 46(7): 2155–2161. Greenland t, reverdy ME, Etienne J. Community- 24. Hanssen AM, Kjeldsen G, Sollid Ju. Local variants acquired methicillin- resistant Staphylococcus aureus of staphylococcal cassette chromosome mec in spo - carrying Panton-Valentine leukocidin genes: World - radic methicillin-resistant Staphylococcus aureus and wide emergence. Emerg Infect Dis 2003. 9(8):978- methicillin-resistant coagulase-negative staphylo - 984. cocci: evidence of horizontal gene transfer? Antimi - 29. Loffler B, et al. Staphylococcus aureus Panton-Valen - crob Agents Chemother 2004. 48(1):285-296. tine leukocidin is a very potent cytotoxic factor for 25. Harmsen d, claus, Witte W, rothgänger J, claus human neutrophils. PLoS Patog 2010, H, turnwald d,Vogel u. Typing of methicillin-resis - 6:e1000715.doi:10.1371/ journal.ppat.1000715.

83 MICRORNA BIOgENESIS AND ITS ROlE IN HIV-1 INFECTION

Adelina Roşca 1, Gabriela Anton 2*, Simona Ruþã 1,2 1Carol Davila University of medicine and Pharmacy, Bucharest; 2Stefan S nicolau Institute of Virology, Bucharest, Romania

ABSTRACT REZUMAT MicroRNA (miRNA) are small- 19-24 nu - MicroARN (miRNA) sunt molecule ARN mici, cleotides, non-coding RNA molecules that regulate necodificante, de aproximativ 19-24 nucleotide, ca - translational and post-translational processes pabile să intervină în reglarea post-translaţională a through mRNA degradation and protein translation ARN, prin degradarea ARNm sau prin reprimarea repression, or sometimes through heterochromatin traducerii proteice, sau mai rar inducând formarea formation or activation of protein translation. de heterocromatină sau activarea sintezei proteice. Lately, miRNA are investigated as predictive În ultima vreme, a fost investigat rolul miRNA biomarkers for the evolution and prognosis of viral în bolile infecţioase. Rezultate promiţătoare au fost diseases, as well as therapeutic targets. obţinute folosind miRNA ca biomarkeri pentru Although the role of non-coding RNA mole - predicţia evoluţiei şi prognosticului bolilor virale şi cules during HIV infection is not yet fully eluci - chiar ca ţinte terapeutice. dated, several studies have reported strong Deşi în infecţia HIV-1, rolul moleculelor ARN correlations between cellular and viral miRNA ex - necodificator nu este încă pe deplin elucidat, studiile pression and the immunologic and virological status au raportat existenţa unor corelaţii puternice între ex - of infected patients. presia miRNA şi caracteristicile imunologice şi viru - Some studies have proven the existence of host sologice ale pacienţilor. cellular miRNA able to influence all important steps Studii importante au evidenţiat existenţa unor in HIV replicative cycle and to interfere with the es - miRNA celulari capabili sa influenţeze toate etapele tablishment of latent infection in CD4+ cells. ciclului replicativ viral şi chiar stabilirea latenţei vi - Although the function and existence of viral en - rale în limfocitele CD4+. coded miRNA remains controversial, new studies Deşi funcţia şi chiar existenţa miRNA codificaţi have shown their potential in modulating the host de virusul HIV-1 rămân controversate, studii recente cell response or the efficiency of viral replication. au arătat potenţialul acestora în a modela răspunsul This review aims to summarize the current level celulei gazdă şi chiar producţia virală. of knowledge in the interaction between miRNA and Acest review sintetizează cunoştinţele actuale HIV-1 and to describe new therapeutic strategies en - asupra interacţiei dintre miRNA şi HIV-1 şi descrie tailing miRNAs as new and potent players in con - posibile noi strategii terapeutice ce utilizează trolling viral infectivity, replication and latency. miRNA ca factori în controlul infectivităţii, replicării şi latenţei virale.

Keywords : microRNA, HIV-1, post-transcriptional regulation, Human Immunodeficiency Virus

Recent studies have shown that the molecular translation and post-translational processes regula - basis of pathological processes that occur in eukary - tion. MicroRNA (miRNA) are part of the small non - ote cells can be explained through the RNA molec - coding RNA family, along with at least two other ular biology. The focus is on the small noncoding RNA species: si-RNA (short interfering RNA mole - RNA molecules acting as key regulators of gene ex - cules able to influence gene expression through both pression [1], which play an important role in various mechanisms) and pi-RNA (Piwi - interacting RNA cellular pathways [2] through their intervention in molecules involved in spermatogenesis) [3].

*Corresponding author: gabriela Anton, Address: Ştefan S Nicolau Institute of Virology, 285, Şos. Mihai Bravu, 030304, Bucharest, Romania, Tel/Fax: +4021-324.25.90; e-mail: [email protected]

84 Micro RNA biogenesis and its role in HIV -1 infection

The term miRNA designates a species of small plications: cirrhosis or hepatocellular carcinoma noncoding, single-stranded RNA molecules 19-24 [19]. Moreover, a new drug, Miravirsen, an oligonu - nucleotides long. MiRNA transcripts are generated cleotide that forms stable bonds with mature miR- from endogenous hairpin structures, usually being 122, thus inhibiting miRNA function and preventing transcribed from noncoding DNA regions or intronic HCV replication, is already in phase 2a clinical trials DNA (about 25% of the miRNA) [4]. The genes that for patients infected with HCV genotype 1 [20]. encode miRNA molecules are found on all chromo - somes (except the Y chromosome), mostly in clus - ters of 2-7 genes [5]. Many miRNA are expressed MIRNA BIOGENESIS together with genes that encode proteins, being MiRNA biogenesis (Fig. 1) begins in the nu - likely to indicate a common mechanism involved in cleus, where RNA polymerase II transcribes pri- translation control [6]. miRNA precursors, consisting of hundreds of Usually, miRNA bind to the target messenger nitrogenous bases [21]. The first step in their matu - RNA (mRNA) in the 3’ untranslated region (3’ ration is the pri-miRNA processing in hairpin struc - UTR), inhibiting its translation, but in some cases it tures of about 60-70 nucleotides, called pre-miRNA can induce heterochromatin formation or activation [22], performed by the nuclear RNase III Drosha to - of protein translation. MiRNA binds to mRNA using gether with a cofactor called DiGeorge syndrome an imperfect sequence homology. As such, a single critical region 8 (DGCR8) [23]. The dsRNA pre- miRNA molecule can interact with up to 300 differ - miRNA structure is recognized by exportin-5 which ent mRNA sequences and a single mRNA sequence transfers them in the cytoplasm. Here pre-miRNA is allows a large number of miRNA to interact with it. processed in a duplex with a length of 18-23 bases So far, more than 2000 mature miRNA (mirbase.org) by Dicer [24], a cytoplasmic, highly conserved en - have been described in the human genome. These zyme. One of the miRNA duplex strands is loaded molecules modulate the translation of mRNA into onto an Ago protein to generate the RISC effector proteins and control up to 60% of the RNA transla - complex (RNA-induced silencing complex), and the tion [7]. Although not all miRNA functions are iden - other is degraded. The binding of miRNA with com - tified, there is evidence suggesting their involvement plementary nucleotides in the 3’ UTR of mRNA in key cellular processes, in immune response mod - transcripts activates the RISC complex [7]. ulation, but also in the progression of many patho - logical processes. Recent studies have reported the involvement of miRNA in the pathogenesis of viral CELLULAR MICRORNA WITH ANTIVIRAL diseases (hepatitis C) [8], Epstein-Barr virus infec - ACTION tion [9]), in the development of various cancers [10, 11] or, neurological diseases (multiple sclerosis [12], MiRNA play an important role both in nonspe - Alzheimer disease [13]), as well as in the individual cific and specific immunity [25]. Small RNA mole - susceptibility to cognitive decline, including mem - cules are able to influence the signaling cascades that ory impairment in the elderly population by control - mediate activation of the immune response and mod - ling the synaptic protein translation, along with other ulate the expression of immune cells specific pro - genetic and epigenetic factors [14]. It has been sug - teins. Production of viral miRNA that are able to gested that microRNA are responsible for altered target vital components of their own genome reduce neuronal gene expression and involved in the devel - viral production inside the host cell, a mechanism opment and progression of neuropsychiatric disor - used by a series of human DNA viruses to evade the ders consecutive to HIV-1 infection [15]. host immune response [26]. The use of RNA interference as an antiviral treatment was evaluated for more than a decade [16]. the role of cellular mirnA in HIV-1 Infection Currently, one of the most studied micromolecules Using an in silico approach, a number of cellu - is miR-122, specifically found in hepatocytes. It has lar miRNA able to target HIV regulatory and matu - already been proven that the presence of miR-122 is ration genes have been identified: miR-29a and required for the replication of hepatitis C virus miR-29b (targeting nef gene - important in survival (HCV) [17] and several studies used miR-122 as a of infected lymphocytes and apoptosis of uninfected biomarker for the necroinflammatory lesions caused ones), miR-149 targeting vpr (accessory gene facili - by HCV infection [18], as well as for its related com - tating the proviral DNA transport to the nucleus),

85 ROŞCA et al.

Fig. 1. MirnA Biogenesis Fig. 1. RNA polymerase II transcribes pri-miRNA precur - sors that are cleved by RNase III Drosha together with Di - George syndrome critical region 8 (DGCR8) forming pre-miRNA which is exported to the cytoplasm, bound by Argonaute 2 protein and incorporated in the RNA induced silencing complex (RISC) to silence diferent mRNA targets. miR-378 targeting env and vpu genes and miR-324- Different miRNA levels (Table 1) in monocytes 5p targeting vif (a maturation gene important for viral and macrophages were shown to correlate with per - infectivity, that blocks the activity of APOBEC 3G - missiveness to HIV-1 infection [32], although the a cellular factor that opposes viral replication) [27]. significance of these miRNA in modulating viral in - Although these microRNA are expressed in T fection was later questioned [33]. lymphocytes, it is unknown if they are expressed Four miRNA (miR-28, miR-125b, miR-150, mainly in HIV-1 target cells - the CD4+ cells. A miR-382) have been shown to have a reduced ex - comparative study on the miRNA profile in HeLa pression in PBMC isolated from patients addicted to cells transfected with an HIV-1 clone (NL4-3) vs na - heroin [34]. It was also suggested that overexpres - tive HeLa cells, revealed significant expression dif - sion of these miRNA in PBMCs could confer an in - ferences, possibly determined by the regulatory viral creased resistance to HIV infection [35]. Changes in protein tat [28]. the level of a neuronal miRNA (miR-9) were demon - The expression of different miRNAs could be strated in frontal cortex autopsy specimens from used as predictive biomarkers for disease HIV-positive addicts and in subsequent experimental progression. A distinct profile of the cellular miRNA models (SH-SY5Y cell line) [36]. expression was found in HIV-1 infected patients vs Other noncoding RNA molecules (miR-29a and healthy controls, as well as in HIV infected patients miR-29b) appear to have an antiviral effect in HIV- with high viral loads vs elite controllers (subjects 1 infection by targeting the HIV nef gene [37]. naturally controlling HIV infection, maintaining a Several miRNA that indirectly inhibit HIV-1 low viral load in the absence of treatment) and HIV replication by either targeting cofactors of the viral uninfected subjects, despite multiple exposures [29, protein tat, by binding the gag gene and interfering 30]. MiR-29 family, miR-125, miR-150, miR-31 and with viral proteins assembly or by reducing the level miR-31* were proposed as markers that can of proteins important for HIV-1 DNA nuclear distinguish healthy controls and non-progressors translo cation, were identified (Table 2) [38-41]. from HIV-1 infected patients with active viral Cellular miRNA can block virus production not replication [31]. only through gene silencing, but also by preventing

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Micro RNA biogenesis and its role in HIV -1 infection

table 1. cellular mirnA with direct antiviral action miRNA Target Mechanism of action Examined cells Study miR-28, miR-125b, 3’UTR Role in establishing viral latency, CD4+ cells, [35] miR-150, miR-223, inhibition of protein translation peripheral blood [62] miR-382 and virus production monocytes [34] miR-29a, miR-29b Nef Binds nef protein, inhibition of HEK293T [37] 3LTR/UTR viral replication and infectivity virus assembly at the cell membrane, a process ing necessary for miRNA synthesis would result in stimulated by Argonaute-2 protein depletion and degradation of the entire viral genome [45] inhi bited by target mRNA. This inhibition of However, retroviruses reverse transcript their progeny virions production, mediated by the mixture genome into a double-stranded DNA that is inte - of R ISC and mRNA unbound miRNA, might be grated into the host cell genome. As such, retro - even more important than the activity of an viruses, including HIV, can access both cytoplasmic individual miRNA [42]. components and RNA interference nuclear complex, similar to human miRNA. An in silico study [46] identified five potential miRNA precursors encoded VIRUS-ENCODED M iR NA by the HIV virus.

Shortly after the identification of the first human the role of HIV-1 Encoded mirnA in Infection miRNA, DNA viruses-encoded miRNAs have also Progression bee n described [43, 44]. Meanwhile, the existence A nef derived miRNA, called miR-N367, was of miRNA, encoded by RNA viruses remains con - identified in an in vitro infection model [47], al - troversial. though further research failed to validate its presence Various explanations for the apparent inability [48]. HIV1 encoded miR-H1 seems able to influence of RNA viruses to synthesize their own miRNA were cell apoptosis, by targeting different cellular proteins formulated. For most RNA viruses, the replication (c-myc, Par-4, Bcl-2 or Dicer) and to inhibit a cellu - cycle takes place in the cytoplasm, a fact that ex - lar miRNA (miR-149) that targets the vpr protein. cludes contact with the nuclear protein, Drosha and Overexpression of another HIV-1 encoded miRNA TRB P, indispensable for microRNA processing. In -miR-H3-3p- located at the reverse transcriptase addition, it was suggested that even for RNA viruses gene level, leads to increased viral replication, while that replic ate in the nu cleus, the enzy matic pro cess - table 2. cellular mirnA with indirect antiviral action miRNA Target Mechanism of action Examined cells Study miR-17-5p, PCAF Targets PCAF and indirectly inhibits HIV-1 infected [38] miR-20a HIV-1 PBMC miR-9 K channel - Affects the release of SH-SY5Y, [36] KCNMA1 neurotransmitters in dopaminergic Frontal cortex neurons (HAND) tissue miR-198, Cyclin T1 Reduces cyclin T1 expression Monocytes in [39] miR-27b, primary culture [41] miR-29b, Resting CD4+ miR-223, cells miR-150 miR-15a, Pur-Į Reduces Pur-Į protein expression Infected CD4+ T [40] miR-15b, cells miR16, miR- 20a, miR-93, miR-106b miR-155 (LEDGF)/p75 Inhibits viral cDNA integration into Primary [63] ADAM10 the host cell genome macrophages NUP153

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ROŞCA et al.

table 3. Viral mirnA miRNA Coding Role Examined cells Study region mir-N367 nef Nef degradation and decreased Jurkat T cells and MT-4 T [47] viral replication cells miR-H3-3p RT gene Increases viral replication CD4+ T-lymphocytes [49] miR-TAR-5p, TAR Inhibits transcription 293T cells [57] miR-TAR-3p Anti-apoptosis NL4- Replication and persistence 3-infected primary human [53] CD4+ T lymphocytes hiv1-miR-H1 3’ UTR Targets a transcription factor Mononuclear cells [64] that oposes apoptosis; inhibits cellular proteins c- myc, Par-4, Bcl-2 and Dicer; Reduces the activity of miR- 149 mutations in its sequence result in decreased viral PERSPECTIVES replication [49]. A complex interaction between HIV-1 and RNA Although microRNA are commonly found in micromolecules has been described. Cellular noncoding regions of the viral genome, a recent microRNAs are able to alter viral gene expression study [50] proposed the env and gag-pol regions as by interfering with virtually all replicative steps, as a source of miRNA-like molecules. well as with the establishment of viral latency and Several studies focused on a TAR element as a with the host cell response to viral invasion. The miRNA source. TAR is a stable, highly conserved mechanism by which miRNA allow alteration of [51] hairpin RNA structure located at the 5’ end of gene expression without a strict complementarity HIV-1 transcripts. A second copy of TAR is present with the target mRNA is both an advantage and an near the 3’ end of the viral genome. Two separate obstacle for the discovery of functional antiviral studies [52, 53] observed that TAR is cleaved in vitro molecules. A single miRNA can bias hundreds of by Dicer protein and described two functional viral RNA sequences and one RNA sequence can be the miRNA (Table 3), miR-TAR-5p and miR-TAR-3p, target of many miRNA. This lack of specificity can derived from its processing. These miRNAs were be overcome by an excellent knowledge of the suggested to be important in maintaining viral la - functions and mechanisms of action for microRNA tency by remodeling the LTR promoter chromatin and other RNA interference mediators. [54]. Furthermore HIV-1 TAR was shown to inhibit Although the existence and the function of viral the expression of ERCC1 and IER3, two cellular encoded small RNA molecules remain controversial, genes involved in apoptosis regulation [55]. These one should not ignore their potential in modulating results were confirmed by several other studies, the host cell response, the efficient viral production using deep sequencing techniques [56, 57]. Recently, or the setting of viral latency. Many of these ques - TAR-derived miRNA were detected in HIV-1 in - tions may be answered by the advent of new deep fected cells exomes, suggesting that HIV can influ - sequencing techniques. ence the neighboring uninfected cells through small RNA interference is emerging as a highly prom - non-coding RNA expression [58]. ising antiviral mechanism. Post-transcriptional HIV-1 encoded miRNAs expressed at very low mRNA cleavage, mediated by microRNA, followed levels were identified using selection and com - by viral or human genes silencing may become an pounds enrichment techniques [59]. Nevertheless important therapeutic strategy. Given the role of other experimental studies argue against the exis - miRNA in HIV latency is possible that these micro - tence of HIV encoded microRNA [48,60]. These dis - molecules can be used as therapeutic targets to avoid crepancies can be explained either by a miRNA the establishment of viral reservoirs, the most im - profile restricted to certain viral strains or by the portant obstacle for viral clearance. technical difficulties in miRNA detection using con - MiRNA might become important biomarkers ven tional cloning strategies [61]. for the evolution of viral infection. The development

88 Micro RNA biogenesis and its role in HIV -1 infection of Miravirsen - the first drug that uses a miRNA as 13. cui L, Li Y, Ma G, Wang Y, cai Y, Liu S, chen Y, therapeutic target in hepatitis C, is paving the way to et al. A functional polymorphism in the promoter re - very promising, yet unexploited, antiviral treatments. gion of microRNA-146a is associated with the risk of Alzheimer disease and the rate of cognitive decline AcKnoWLEdGEMEntS in patients. PLoS One 2014. 9: e89019. 14. tatro Et, risbrough V, Soontornniyomkij B, This work received financial support through Young J, Shumaker-Armstrong S, Jeste d V, the project entitled “CERO – Career profile: Achim cL . Short-term recognition memory corre - Romanian Researcher”, grant number POSDRU/ lates with regional CNS expression of microRNA- 159/1.5/S/135760, cofinanced by the European 138 in mice. Am J Geriatr Psychiatry 2013. 21: Social Fund for Sectoral Operational Programme 461–73. Human Resources Development 2007-2013 15. tatro Et, Scott Er, nguyen tB, Salaria S, Baner - jee S, Moore dJ, Masliah E, et al. Evidence for Al - teration of Gene Regulatory Networks through REFERENCES MicroRNAs of the HIV-infected brain: novel analysis of retrospective cases. PLoS One 2010. 5: e10337. 1. carthew rW, Sontheimer EJ . Origins and Mecha - 16. cernescu c, ruta S . Interferenţa ARN - un nou nisms of miRNAs and siRNAs. Cell 2009. 136: 642– mecanism antiviral? In: Medicamente antivirale. Ed - 55. itura Universitară “Carol Davila,” Bucuresti 2003, pp 2. Khraiwesh B, Arif MA, Seumel GI, ossowski S, 220. Weigel d, reski r, Frank W . Transcriptional Control 17. Jangra rK, Yi M, Lemon SM . Regulation of hepa - of Gene Expression by MicroRNAs. Cell 2010. 140: titis C virus translation and infectious virus produc - 111–22. tion by the microRNA miR-122. J Virol 2010. 84: 3. Siomi Mc, Sato K, Pezic d, Aravin AA . PIWI-inter - 6615–25. acting small RNAs: the vanguard of genome defence. 18. trebicka J, Anadol E, Elfimova n, Strack I, Nat Rev Mol Cell Biol 2011. 12: 246–58. roggendorf M, Viazov S, Wedemeyer I, et al. He - 4. Bartel dP . MicroRNAs: Genomics, Biogenesis, Mech - patic and serum levels of miR-122 after chronic anism, and Function. Cell 2004. 116: 281–97. HCV-induced fibrosis. J Hepatol 2013. 58: 234–9. 5. tanzer A, Stadler PF . Evolution of microRNAs. 19. Xu J, Wu c, che X, Wang L, Yu d, Zhang t, Methods Mol Biol 2006. 342: 335–50. Huang L, et al. Circulating microRNAs, miR-21, 6. Morlando M, Ballarino M, Gromak n, Pagano F, miR-122, and miR-223, in patients with hepatocellu - Bozzoni I, Proudfoot nJ . Primary microRNA tran - lar carcinoma or chronic hepatitis. Mol Carcinog scripts are processed co-transcriptionally. Nat Struct 2011. 50: 136–42. Mol Biol 2008. 15: 902–09. 20. Janssen HLA, reesink HW, Lawitz EJ, Zeuzem S, 7. Bartel dP . MicroRNAs: Target Recognition and Reg - rodriguez-torres M, Patel K, van der Meer AJ, et ulatory Functions. Cell 2009. 136: 215–233. al. Treatment of HCV infection by targeting mi - 8. Sedano cd, Sarnow P . Hepatitis C Virus Subverts croRNA. N Engl J Med 2013. 368: 1685–94. Liver-Specific miR-122 to Protect the Viral Genome 21. Lee Y, Kim M, Han J, Yeom K-H, Lee S, Baek SH, from Exoribonuclease Xrn2. Cell Host Microbe 2014. Kim Vn . MicroRNA genes are transcribed by RNA 16: 257–64. polymerase II. EMBO J 2004. 23: 4051–60. 9. Ma L, deng X, Wu M, Zhang G, Huang J . Down- 22. Borchert GM, Lanier W, davidson BL . RNA poly - regulation of miRNA-204 by LMP-1 enhances CDC42 merase III transcribes human microRNAs. Nat Struct activity and facilitates invasion of EBV-associated na - Mol Biol 2006. 13: 1097–01. sopharyngeal carcinoma cells. FEBS Lett 2014. 588: 23. Han J, Lee Y, Yeom K-H, Kim Y-K, Jin H, Kim Vn . 1562–70. The Drosha-DGCR8 complex in primary microRNA 10. Fukuda-Yuzawa Y, tanabe M, tsuji S, Sasakawa processing. Genes Dev 2004. 18: 3016–27. c, Fukao t, Kiga K . Comprehensive Silencing of 24. Bernstein E, caudy AA, Hammond SM, Hannon Target-Sharing microRNAs is a Mechanism for GJ . Role for a bidentate ribonuclease in the initiation SIRT1 Overexpression in Cancer. RNA Biol 2014. step of RNA interference. Nature 2001. 409: 363–66. 11. 25. o’connell rM, rao dS, chaudhuri AA, Baltimore 11. Mei Q, Li X, Guo M, Fu X, Han W . The miRNA d. Physiological and pathological roles for microR - network: micro-regulator of cell signaling in cancer. NAs in the immune system. Nat Rev Immunol 2010. Expert Rev Anticancer Ther 2014: 1–13. 10: 111–22. 12. Ma X, Zhou J, Zhong Y, Jiang L, Mu P, Li Y, Singh 26. Gottwein E, cullen Br . Viral and Cellular MicroR - n, et al. Expression, Regulation and Function of Mi - NAs as Determinants of Viral Pathogenesis and Im - croRNAs in Multiple Sclerosis. Int J Med Sci 2014. munity. Cell Host Microbe 2008. 3: 375–87. 11: 810–18.

89 ROŞCA et al.

27. Hariharan M, Scaria V, Pillai B, Brahmachari SK . 40. Shen c-J, Jia Y-H, tian r-r, ding M, Zhang c, Targets for human encoded microRNAs in HIV Wang J-H . Translation of Pur- α is targeted by cellu - genes. Biochem Biophys Res Commun 2005. 337: lar miRNAs to modulate the differentiation-depen - 1214–8. dent susceptibility of monocytes to HIV-1 infection. 28. Yeung ML, Bennasser Y, Myers tG, Jiang G, FASEB J 2012. 26: 4755–64. Benkirane M, Jeang K-t . Changes in microRNA 41. chiang K, Sung t-L, rice AP . Regulation of cyclin expression profiles in HIV-1-transfected human cells. T1 and HIV-1 Replication by microRNAs in resting Retrovirology 2005. 2: 81. CD4+ T lymphocytes. J Virol 2012. 86: 3244–52. 29. Houzet L, Yeung ML, de Lame V, desai d, Smith 42. chen AK, Sengupta P, Waki K, Van Engelenburg SM, Jeang K-t . MicroRNA profile changes in SB, ochiya t, Ablan Sd, Freed Eo, et al. Mi - human immunodeficiency virus type 1 (HIV-1) croRNA binding to the HIV-1 Gag protein inhibits seropositive individuals. Retrovirology 2008. 5: 118. Gag assembly and virus production. Proc Natl Acad 30. Bignami F, Pilotti E, Bertoncelli L, ronzi P, Gulli Sci U S A 2014. M, Marmiroli n, Magnani G, et al. Stable changes 43. Kincaid rP, Sullivan cS . Virus-encoded microR - in CD4+ T lymphocyte miRNA expression after ex - NAs: an overview and a look to the future. PLoS posure to HIV-1. Blood 2012. 119: 6259–67. Pathog 2012. 8: e1003018. 31. Witwer KW, Watson AK, Blankson Jn, clements 44. chen cJ, Kincaid rP, Seo GJ, Bennett Md, Sulli - JE . Relationships of PBMC microRNA expression, van cS . Insights into Polyomaviridae microRNA plasma viral load, and CD4+ T-cell count in HIV-1- function derived from study of the bandicoot papil - infected elite suppressors and viremic patients. Retro - lomatosis carcinomatosis viruses. J Virol 2011. 85: virology 2012. 9: 5. 4487–500. 32. Wang X, Ye L, Hou W, Zhou Y, Wang Y-J, Metzger 45. cullen Br . Five questions about viruses and microR - dS, Ho W-Z . Cellular microRNA expression corre - NAs. PLoS Pathog 2010. 6: e1000787. lates with susceptibility of monocytes/macrophages 46. Bennasser Y, Le S-Y, Yeung ML, Jeang K-t . HIV- to HIV-1 infection. Blood 2009. 113: 671–4. 1 encoded candidate micro-RNAs and their cellular 33. Swaminathan S, Zaunders J, Wilkinson J, Suzuki targets. Retrovirology 2004. 1: 43. K, Kelleher Ad . Does the presence of anti-HIV miR - 47. omoto S, Ito M, tsutsumi Y, Ichikawa Y, okuyama NAs in monocytes explain their resistance to HIV-1 H, Brisibe EA, Saksena nK, et al. HIV-1 nef sup - infection? Blood 2009. 113: 5029–30; author reply pression by virally encoded microRNA. Retrovirol - 5030–1. ogy 2004. 1: 44. 34. Wang X, Ye L, Zhou Y, Liu M-Q, Zhou d-J, Ho W- 48. Lin J, cullen Br . Analysis of the interaction of pri - Z. Inhibition of anti-HIV microRNA expression: a mate retroviruses with the human RNA interference mechanism for opioid-mediated enhancement of HIV machinery. J Virol 2007. 81: 12218–26. infection of monocytes. Am J Pathol 2011. 178: 41–7. 49. Zhang Y, Fan M, Geng G, Liu B, Huang Z, Luo H, 35. Huang J, Wang F, Argyris E, chen K, Liang Z, Zhou J, et al. A novel HIV-1-encoded microRNA en - tian H, Huang W, et al. Cellular microRNAs con - hances its viral replication by targeting the TATA box tribute to HIV-1 latency in resting primary CD4+ T region. Retrovirology 2014. 11: 23. lymphocytes. Nat Med 2007. 13: 1241–7. 50. Holland B, Wong J, Li M, rasheed S . Identification 36. tatro Et, Hefler S, Shumaker-Armstrong S, Soon - of human microRNA-like sequences embedded tornniyomkij B, Yang M, Yermanos A, Wren n, et within the protein-encoding genes of the human im - al. Modulation of BK channel by MicroRNA-9 in munodeficiency virus. PLoS One 2013. 8: e58586. neurons after exposure to HIV and methampheta - 51. Leonard Jn, Shah PS, Burnett Jc, Schaffer d V . mine. J Neuroimmune Pharmacol 2013. 8: 1210–23. HIV evades RNA interference directed at TAR by an 37. Ahluwalia JK, Khan SZ, Soni K, rawat P, Gupta indirect compensatory mechanism. Cell Host Mi - A, Hariharan M, Scaria V, et al. Human cellular mi - crobe 2008. 4: 484–94. croRNA hsa-miR-29a interferes with viral nef protein 52. Klase Z, Kale P, Winograd r, Gupta M V, Heydar - expression and HIV-1 replication. Retrovirology ian M, Berro r, Mccaffrey t, et al. HIV-1 TAR el - 2008. 5: 117. ement is processed by Dicer to yield a viral 38. triboulet r, Mari B, Lin Y-L, chable-Bessia c, micro-RNA involved in chromatin remodeling of the Bennasser Y, Lebrigand K, cardinaud B, et al. viral LTR. BMC Mol Biol 2007. 8: 63. Suppression of microRNA-silencing pathway by 53. ouellet dL, Plante I, Landry P, Barat c, Janelle HIV-1 during virus replication. Science 2007. 315: M-E, Flamand L, tremblay MJ, et al. Identification 1579–82. of functional microRNAs released through asymmet - 39. Sung t-L, rice AP . miR-198 inhibits HIV-1 gene ex - rical processing of HIV-1 TAR element. Nucleic pression and replication in monocytes and its mech - Acids Res 2008. 36: 2353–65. anism of action appears to involve repression of 54. ouellet dL, Vigneault-Edwards J, Létourneau K, cyclin T1. PLoS Pathog 2009. 5: e1000263. Gobeil L-A, Plante I, Burnett Jc, rossi JJ, et al.

90 Micro RNA biogenesis and its role in HIV -1 infection

Regulation of host gene expression by HIV-1 TAR 60. Whisnant AW, Bogerd HP, Flores o, Ho P, Powers microRNAs. Retrovirology 2013. 10: 86. JG, Sharova n, Stevenson M, et al. In-depth analy - 55. Klase Z, Winograd r, davis J, carpio L, Hildreth sis of the interaction of HIV-1 with cellular mi - r, Heydarian M, Fu S, et al. HIV-1 TAR miRNA croRNA biogenesis and effector mechanisms. MBio protects against apoptosis by altering cellular gene 2013. 4: e000193. expression. Retrovirology 2009. 6: 18. 61. Ebhardt HA, thi EP, Wang M-B, unrau PJ . Ex - 56. Schopman nct, Willemsen M, Liu YP, Bradley t, tensive 3’ modification of plant small RNAs is mod - van Kampen A, Baas F, Berkhout B, et al. Deep se - ulated by helper component-proteinase expression. quencing of virus-infected cells reveals HIV-encoded Proc Natl Acad Sci U S A 2005. 102: 13398–403. small RNAs. Nucleic Acids Res 2012. 40: 414–27. 62. Wang X, Ye L, Hou W, Zhou Y, Wang Y-J, Metzger 57. Harwig A, Berkhout B, das A . Characterization of dS, Ho W-Z . Cellular microRNA expression corre - microRNAs derived from the HIV-1 TAR RNA hair - lates with susceptibility of monocytes/macrophages pin. Retrovirology 2013. 10: P24. to HIV-1 infection. Blood 2009. 113: 671–4. 58. narayanan A, Iordanskiy S, das r, Van duyne r, 63. Swaminathan G, rossi F, Sierra L-J, Gupta A, Santos S, Jaworski E, Guendel I, et al. Exosomes navas-Martín S, Martín-García J . A role for mi - derived from HIV-1-infected cells contain trans-acti - croRNA-155 modulation in the anti-HIV-1 effects of vation response element RNA. J Biol Chem 2013. Toll-like receptor 3 stimulation in macrophages. 288: 20014–33. PLoS Pathog 2012. 8: e1002937. 59. Althaus cF, Vongrad V, niederöst B, Joos B, di Gi - 64. Kaul d, Ahlawat A, Gupta Sd . HIV-1 genome-en - allonardo F, rieder P, Pavlovic J, et al. Tailored en - coded hiv1-mir-H1 impairs cellular responses to in - richment strategy detects low abundant small fection. Mol Cell Biochem 2009. 323: 143–8. noncoding RNAs in HIV-1 infected cells. Retrovirol - ogy 2012. 9: 27.

91 MECHANISMS OF THROMBOSIS IN SySTEMIC lUPUS ERyTHEMATOSUS

Adriana Metoni 1# , Cristina Mambet 2# , Laura G. Necula 2, Ioana M. Aldea 2, Ana I. Neagu 2, Lilia Matei 2, Denisa Dragu 2, Coralia Bleotu 2, Daniela A. Ion 1,3 , Carmen C. Diaconu 2* 1Carol Davila University of medicine and Pharmacy, Bucharest, Romania; 2Ştefan S. nicolau Institute of Virology, Bucharest, Romania; 3Prof. Dr. matei Balş national Institute of Infectious Diseases, European HIV/AIDS Academy, Bucharest, Romania

ABSTRACT REZUMAT Thrombotic events are highly prevalent in sys - Evenimentele trombotice au o prevalenţă temic lupus erythematosus (SLE). Antiphospholipid crescută în lupusul eritematos sistemic (LES). antibodies play an essential role in promoting throm - Anticorpii antifosfolipidici deţin un rol esenţial bosis by activating several intracellular signaling în inducerea trombozei prin activarea unor meca - pathways (TLR4, p38MAPK, NFkB) in platelets, nisme de semnalizare intracelulară (TLR4, monocytes and endothelial cells. New therapeutic p38MAPK, NFkB) în trombocite, monocite şi celule opportunities might be offered by addressing these endoteliale. Noi opţiuni terapeutice ar putea fi create molecular targets. prin influenţarea acestor ţinte moleculare. Chronic inflammatory status, the degree of dis - Inflamaţia cronică, gradul de activitate a bolii şi ease activity and accelerated atherosclerosis are also ateroscleroza accelerată sunt, de asemenea, respon - responsible for the thrombotic phenotype in patients sabile pentru fenotipul trombotic al pacienţilor cu with SLE. LES. The aim of this review is to highlight thrombo - Scopul acestui articol este de a evidenţia meca - sis mechanisms and to look for possible connection nismele trombozei şi de a explora posibile conexiuni between SLE, antiphospholipid antibodies and can - între LES, anticorpii antifosfolipidici şi cancer, în cer, especially myeloproliferative neoplasms. special neoplasmele mieloproliferative.

Keywords : antiphospholipid antibodies, b2gPI , premature atherosclerosis, SlE

INTRODUCTION diagnosis, involvement of both arterial and venous systems and polymorphic clinical manifes - Systemic lupus erythematosus (SLE) is a tations [4, 5]. chronic systemic autoimmune disease triggered by Pathogenesis of thrombosis in SLE is multifac - a combination of genetic, hormonal and environ - torial. Conventional risk factors are involved as well mental factors and characterized by the inflamma - as disease-specific factors, such as antiphospholipid tion of several tissues and overproduction of antibodies (aPL), high level of inflammation and dis - autoantibodies against nuclear constituents [1-3]. ease activity, other thrombophilic risk factors and Thrombosis is a major complication of SLE, ac - drugs used for therapeutic purpose [6, 7]. counting for a significant patient morbidity and mor - tality. According to various studies, the prevalence Antiphospholipid antibodies - a main pro - of thrombotic events in SLE exceeds 10% and may thrombotic factor in SLE be over 50% in high-risk patients. aPL are heterogenous autoantibodies that inter - Thrombosis in SLE patients has several charac - act primarily with phospholipid-binding plasma pro - teristics: onset at a younger age than in general teins, β2- glycoprotein I ( β2 GPI) and prothrombin population, increased incidence in the first year from (PT) being the main antigenic targets [8]. Antibodies

*Corresponding author: Carmen C. Diaconu, Ştefan S. Nicolau Institute of Virology, Bucharest, Romania, e-mail: [email protected]; [email protected] # Authors that contributed equally to this paper

92 Mechanisms of thrombosis in systemic Lupus erythematosus against β2 GPI-cardiolipin complexes are detected One mechanism involved in aPL-mediated hy - with currently anti-cardiolipin (aCL) immunoassays, percoagulability is increased expression of tissue while anti- β2 GPI and anti-PT antibodies account for factor (TF) - the initiator of extrinsic coagulation the most of lupus anticoagulant (LAC) positive find - cascade and a major risk factor of thrombosis - on ings [9]. In SLE patients, aPL antibodies may be as - monocytes through activation of toll-like receptors sociated or not with the antiphospholipid syndrome (TLR), p38 mitogen activated protein kinase (APS) – a condition characterized by venous, arte - (MAPK) and nuclear factor (NF) κB pathways. It rial, or microcirculation thrombosis and/or preg - was shown that the fraction of polyclonal IgG that nancy poor outcome [10, 11]. bound β2 GPI derived from patients with aPL and ve - aCL antibodies, anti- β2 GPI antibodies and nous thrombosis induced phosphorylation of LAC are the main aPL tested in the clinical practice p38MAPK, NF κB and up-regulation of TF on and are also included in the laboratory diagnostic cri - healthy monocytes and human promonocytic (U937) teria of APS [12]. cell line. TF expression was diminished by inhibitors aPL antibodies were commonly found in of p38MAPK, NFkB, and TLR4 suggesting that this Hopkins’ Lupus Cohort patients: 47% had aCL, category of aPL antibodies might preferentially ac - 32.5% anti- β2 GPI and 26% LAC. In case of LAC tivate TLR4 on the monocyte surface [19]. The re - presence at the onset of SLE, a 50% risk of a deep sults of further studies indicated that the TLR4 and venous thrombosis/pulmonary embolism in the next NF κB signaling pathways were involved in the anti- 20 years was predicted. Among aPL antibodies, LAC β2 GPI/ β2 GPI-induced expression of TF together was most strongly associated with arterial and ve - with some proinflammatory molecules (IL-6, IL-8 nous thrombosis and uniquely associated with my - and TNF- α) in human acute monocytic leukaemia ocardial infarction [13]. THP-1 cells and these might represent pathogenic Moreover, positive LAC in recently diagnosed mechanisms of APS [20-22] . SLE patients confers an increased risk of intracranial In a study performed by Vega-Ostertag et al. it thrombosis [14]. was shown that aPL induced increased TF transcrip - Renal manifestations of primary or SLE-related tion, function and expression as well as IL-6, IL-8 APS include renal artery stenosis or thrombosis, renal and inducible nitric oxide synthase (iNOS) expres - infarction, renal vein thrombosis and antiphospholipid sion on human umbilical vein endothelial cells (HU - antibody (aPL)-associated nephropathy defined by a VECs). Phosphorylation of p38 MAPK and thrombotic disorder affecting small vessels [15]. activation of NF-kappaB were important steps in Genetic mechanisms might contribute to the de - velopment of thrombosis in patients with SLE. Re - these processes [23]. lated to this aspect, it was recently demonstrated that Furthermore, it was demonstrated that NF- κB– HLA-DRB1*04 and HLA-DRB1*13 alleles con - dependent activation of endothelial cells by anti ferred a genetic predisposition for vascular disease β2 GPI antibodies is mediated by a signaling com - in SLE patients, being also associated with aPL an - plex on the cell surface composed of annexin A2, tibodies [16]. The study performed by Liang W et al TLR4, calreticulin, and nucleolin [24]. analyzed the polymorphism at position 247 of the β2 Another mechanism that might be responsible -glycoprotein I ( β2 GPI) gene in a Chinese lot of for prothrombotic tendency in APS is platelet acti - SLE patients and showed that VV genotype and the vation by the anti-beta2GPI antibody-beta2GPI V-encoding allele at position 247 of the β2 GPI gene complex mediated by von Willebrand factor receptor was strongly correlated with thrombotic events and glycoprotein Ib (GPIb) alpha and apolipoprotein E the production of the anti- β2 GPI antibodies [17]. receptor 2’ [25]. Despite many accumulated experimental data, p38 MAPK signaling pathway is also involved the precise pathogenesis of aPL-mediated thrombo - in platelet activation and aggregation generated by sis is not fully elucidated. Several mechanisms were aPL antibodies [26]. proposed referring to the activation of endothelial However, the concept that anti- β2 GP1 autoan - cells, monocytes, and platelets, and/or alteration of tibody/ β2 GP1 complex activates both endothelial natural hemostatic balance by antiphospholipid an - cells and platelets has been very recently challenged. tibodies [18]. In a mouse thrombosis model of APS, Proulle et al . a. Monocyte, endothelial cell and platelet acti - showed that this complex firstly activates platelets vation through interaction of circulating aPL with leading to subsequent increased endothelium activa - membrane bound β2 GPI tion and fibrin generation [27].

93 METONI et al.

The complement system might also contribute roles of inflammation, disease activity and prema - to the aPL-mediated thrombosis. Thus, it was sug - ture atherosclerosis in SLE-related thrombosis gested that in SLE, aPL antibodies could increase There is a accumulated evidence supporting the C4d deposition on platelets by enhancing platelet ac - fact that atherosclerosis has a higher prevalence and tivation and providing complement-fixing antibod - an early occurrence in SLE. However, its mecha - ies. In its turn, complement deposition on platelets nisms are only partially explained by traditional risk was associated with disease activity and venous, but factors, SLE specific factors and corticosteroid ther - not arterial, thrombosis [28]. apy. b. Alteration of natural hemostatic balance by SLE patients have a relative risk of myocardial aPL antibodies infarction 5-8 times higher than that of general Alteration of anticoagulant and fibrinolytic sys - population and this may even exceed 50 in 35-44 tems by aPL antibodies may represent another mech - year-old women [34]. anism of thrombosis in SLE patients. A significant percentage of patients with SLE Related to this, several effects of aPL antibodies and APS demonstrates a β2 GPI-specific T cell reac - on the anticoagulant protein C pathways were de - tivity, which is associated with subclinical athero - scribed: decreasing protein C and/or S plasma levels, sclerosis [35]. inducing of an acquired activated protein C resist - Due to the fact that atherosclerosis is considered ance (APC-R) and disrupting protein C activation as a chronic inflammatory disease, it has been postu - a result of reduced thrombin levels [29]. The study lated that persistent inflammatory status in patients performed by Nojima et al . suggested that acquired with SLE is the key mechanism of accelerated APC-R in SLE patients was mainly due to the anti- atherogenesis. Thus, disease activity and especially protein S antibodies interfering with APC pathway renal involvement dictate the severity of this process. [30]. Autoantibodies in SLE might contribute to athero - A significantly reduced annexin A5 anticoagu - genesis by causing endothelium injury and altering lant associated with the presence of LAC and anti - the lipoprotein metabolism [36]. bodies to the domain I portion of β2- glycoprotein I Endothelial dysfunction is a major pathogenic (anti-DI antibodies) was found in children with SLE factor for premature atherosclerosis. Circulating im - but further studies are needed to demonstrate the mune complexes and anti-endothelial cell antibodies clinical relevance of this finding [31]. [36], as well as increased neutrophil extracellular Regarding alteration of fibrinolytic system, it trap (NET) formation in SLE can induce endothelial was shown that the tightness of fibrin clots generated damage. In the latter case, the activation of matrix in plasma from APS patients was increased com - metalloproteinase 2 (MMP-2) by matrix metallopro - pared with healthy subjects and warfarin treated non- teinase 9 (MMP-9) contained in NETs promotes en - APS thrombosis controls [32]. dothelial dysfunction [37]. Proposed mechanisms of impaired fibrinolysis Another mechanism that may promote prema - in APS include: increased plasminogen activator in - ture atherosclerosis is altered lipid metabolism. Lipid hibitor type 1 (PAI-1) activity, presence of antibodies abnormalities are a common finding in SLE patients that target tissue plasminogen activator (tPA), plas - and include increased triglycerides and reduced minogen, plasmin and annexin 2, high levels of HDL-cholesterol with proinflammatory HDL pro - lipoprotein (a) that is structurally similar to plas - duction [34]. SLE patients with persistent inflamma - minogen and may interfere with plasminogen/plas - tory activity have increased titers of antibodies that min binding of fibrin, anti- β2 GP1 autoantibodies target high-density lipoprotein (HDL) and apoli - affecting the interaction of β2 GP1 with tPA [33]. poprotein A1 and are inversely correlated with ac - As a conclusion of the presented data, aPL tivity of paraoxonase, an enzyme responsible for the antibodies represent an important player in the anti-oxidant properties of HDL [36]. pathogenesis of SLE associated thrombosis. Type I interferons (IFNs) might also exert neg - However, not all patients with aPL antibodies will ative effects on the vascular system. Using mouse develop thrombosis and conversely, thrombosis is models, Thacker SG et al . have shown that exposure detected in approximately 40% of adults with SLE to IFN α led to accelerated thrombosis and platelet who are negative for aPL antibodies, suggesting that activation [38]. other factors are required to trigger the thrombotic In a very recent study, gene expression profiling process [5]. performed in monocytes obtained from patients with

94 Mechanisms of thrombosis in systemic Lupus erythematosus

APS, APS plus SLE and SLE, as well as healthy respectively 13% of cases. For symptomatic carriers donors, demonstrated specific atherosclerosis/car - the presence of aPL antibodies increased the throm - diovascular disease/inflammation-related gene sig - botic risk 4.4-fold. The authors concluded that it nature that differentiated patients with these linked might be clinically relevant to test for aPL antibodies autoimmune conditions. Moreover, IgG aCL titers in this category of patients [41]. were correlated with mRNA levels of certain inflam - The 4G/5G polymorphism of plasminogen ac - matory molecules in monocytes of APS plus SLE tivator inhibitor type 1 (PAI-1) gene together with patients being an independent predictive factor for PAI-1 levels was investigated in patients with pri - both atherosclerosis and thrombosis [39]. mary and SLE-associated APS. Patients with SLE- Corticosteroids are commonly used in SLE due associated APS that were carriers of 4G allele had to their potent anti-inflammatory properties. How - higher PAI-1 levels than patients with primary APS. ever, they are associated with many side effects in - As a conclusion, measuring the levels of plasmino - cluding an increased risk of thrombosis. On the gen and PAI-1 as well as the 4G/5G polymorphism contrary, the antimalarial agent hydroxychloroquine of PAI-1 gene may have utility for identification of displays a beneficial antithrombotic effect explained APS patients that have a high risk of thrombosis by inhibition of platelet aggregation and adhesion, [42]. and arachidonic acid release from activated platelets. In addition, it decreases the thrombus size and the comments and perspectives time of thrombus generation and has also choles - Thrombosis in SLE is multifactorial and there terol-lowering effects [5, 36]. are many unknown molecular pathways that remain to be elucidated. the contribution of thrombophilic factors to However, in vitro and in vivo studies have SLE-associated thrombosis demonstrated the existence of a characteristic in - The incidence of thrombosis in general popula - flammatory and prothrombotic fenotype that corre - tion as well as in SLE patients has large ethnic vari - lates with clinical manifestations of SLE. ations and it was assumed that genetic factors, such TLR4, p38 MAPK and NF κB signal transduc - as known thrombophilic defects and polymorphism tion pathways mediate the pathogenic effects of aPL of genes involved in coagulation pathways, might on different cell types and may represent potential explain these differences. therapeutic targets in patients with primary or SLE- The study conducted by Kaiser et al. have geno - associated antiphospholipid syndrome. typed 33 single-nucleotide polymorphisms (SNPs) Patients with SLE have an increased risk of can - that were previously correlated with increased pre - cer, especially of lymphoma probably due to im - disposition to thrombotic events (thrombophilia), in mune dysregulation, persistent inflammatory milieu two ethnically diverse SLE cohorts. Subsequently, [43] and immunosuppressive treatment [44]. Other the SNPs were tested for their association with hematological malignancies were also reported, such thrombosis using complex statistical methods. Fac - as leukemia and multiple myeloma [45]. tor V Leiden ( FVL ) rs6025 and methylenetetrahy - In a large population-study it was shown that the drofolate reductase ( MTHFR ) rs1801133 showed a risk of myeloproliferative neoplasms (MPNs) was correlation with the overall thrombotic risk in Euro - statistically significant in patients with previously pean Americans and fibrinogen gamma ( FGG ) diagnosed autoimmune diseases, and this could rs2066865 in Hispanic Americans. SNPs associated imply common genetic predisposition [46]. In addi - with venous thrombosis were MTHFR rs1801131, tion to this, an increased prevalence of aPL antibod - MTHFR rs1801133, FVL rs6025 and FGG ies was found in essential thrombocythemia (ET) rs2066865 in European Americans. FGG rs2066865 [47], the most common BCR-ABL negative MPN. was the only polymorphism related to arterial throm - These data suggest that there could be a possible link bosis in Hispanics [40]. between primary or SLE-associated APS and MPN, In a retrospective case-control study that in - especially ET, concerning thrombosis mechanism, cluded carriers of FV Leiden and prothrombin which needs further investigation. G20210A gene mutation it was shown that postop - erative state and the presence of aPL antibodies rep - AcKnoWLEdGEMEntS resented risk factors for developing thromboembolic This paper is supported by the Sectoral Opera - events and adverse pregnancy outcomes in 6% and tional Programme Human Resources Development

95 METONI et al.

(SOP HRD), financed from the European Social Koike t, Meroni PL, reber G, Shoenfeld Y, tin - Fund and by the Romanian Government under the cani A, Vlachoyiannopoulos PG, Krilis SA. Inter - contract number POSDRU/159/1.5/S/137390/ and national consensus statement on an update of the contract number POSDRU /159/1.5/S/135760. classification criteria for definite antiphospholipid syndrome (APS). Journal of Thrombosis and Haemostasis : JTH 2006. 4(2): 295-306. 13. Petri M. Update on anti-phospholipid antibodies in SLE: the Hopkins’ Lupus Cohort. Lupus 2010. 19 (4): REFERENCES 419-423. 1. Mathian A, Arnaud L, Amoura Z. Etiopathogenesis 14. Hanly JG, urowitz MB, Su L, Bae Sc, Gordon c, of systemic lupus erythematosus: a 2014 update. La clarke A, Bernatsky S, Vasudevan A, Isenberg d, Revue de Medecine Interne / fondee par la Societe Na - rahman A, Wallace dJ, Fortin Pr, Gladman d, tionale Francaise de Medecine Interne 2014. 35 (8): romero-diaz J, Sanchez-Guerrero J, dooley MA, 503-511. Bruce I, Steinsson K, Khamashta M, Manzi S, 2. Lisnevskaia L, Murphy G, Isenberg d. Systemic ramsey-Goldman r, Sturfelt G, nived o, van Vol - lupus erythematosus. Lancet 2014. 384 (9957): 1878- lenhoven r, ramos-casals M, Aranow c, Mackay 1888. M, Kalunian K, Alarcon GS, Fessler BJ, ruiz- 3. Franklyn K, Hoi A, nikpour M, Morand EF. The Irastorza G, Petri M, Lim S, Kamen d, Peschken need to define treatment goals for systemic lupus ery - c, Farewell V, thompson K, theriault c, Merrill thematosus. Nature Reviews Rheumatology 2014. Jt. Autoantibodies as biomarkers for the prediction 10 (9): 567-571. of neuropsychiatric events in systemic lupus erythe - 4. chang Er, Pineau cA, Bernatsky S, neville c, matosus. Annals of the Rheumatic Diseases 2011. clarke AE, Fortin Pr. Risk for incident arterial or ve - 70 (10): 1726-1732. nous vascular events varies over the course of systemic 15. tektonidou MG. Identification and treatment of APS lupus erythematosus. The Journal of Rheumatology renal involvement. Lupus 2014. 23 (12): 1276-1278. 2006. 33 (9): 1780-1784. 16. Lundstrom E, Gustafsson Jt, Jonsen A, Leonard 5. Al-Homood IA. Thrombosis in systemic lupus erythe - d, Zickert A, Elvin K, Sturfelt G, nordmark G, matosus: a review article. ISRN Rheumatology 2012. Bengtsson AA, Sundin u, Kallberg H, Sandling 2012: 428269. JK, Syvanen Ac, Klareskog L, Gunnarsson I, 6. Burgos PI, Alarcon GS. Thrombosis in systemic lupus ronnblom L, Padyukov L, Svenungsson E. HLA- erythematosus: risk and protection. Expert Review of DRB1*04/*13 alleles are associated with vascular Cardiovascular Therapy 2009. 7(12): 1541-1549. disease and antiphospholipid antibodies in systemic 7. Burgos PI, McGwin G, Jr., reveille Jd, Vila LM, lupus erythematosus. Annals of the Rheumatic Dis - Alarcon GS. Factors predictive of thrombotic events in eases 2013. 72 (6): 1018-1025. LUMINA, a multi-ethnic cohort of SLE patients 17. Liang W, Zhou J, Zhou S, Wang t, Yang L, Xu d, (LXXII). Rheumatology (Oxford) 2010. 49 (9): 1720- deng G, Huang d, Mei c, He Y, Zhang Z. 1725. Val/Leu247 polymorphism of β2 - glycoprotein I and 8. nunez-Alvarez cA, cabiedes J. Pathogenic mecha - thrombosis in Chinese patients with SLE. Int J Lab nisms of the anti-phospholipid antibodies. Reumatolo - Hematol 2014. [Epub ahead of print]. gia Clinica 2011. 7(1): 72-76. 18. chaturvedi S, Mccrae Kr. Recent advances in the 9. Yang YH, chang cJ, chuang YH, Hsu HY, chen antiphospholipid antibody syndrome. Current Opin - PP, chiang BL. Identification of anti-prothrombin an - ion in Hematology 2014. 21 (5): 371-379. tibodies in the anti-phospholipid syndrome that display 19. Lambrianides A, carroll cJ, Pierangeli SS, Peri - the prothrombinase activity. Rheumatology (Oxford) cleous c, Branch W, rice J, Latchman dS, 2010. 49 (1): 34-42. townsend P, Isenberg dA, rahman A, Giles IP . 10. radic M, Martinovic Kaliterna d, radic J. Vascu - Effects of polyclonal IgG derived from patients with lar manifestations of systemic lupus erythematosis. different clinical types of the antiphospholipid syn - The Netherlands Journal of Medicine 2013. 71 (1): drome on monocyte signaling pathways. J Immunol 10-16. 2010. 184 (12): 6622-6628. 11. Emmi G, Silvestri E, Squatrito d, ciucciarelli L, 20. Zhou H, Yan Y, Xu G, Zhou B, Wen H, Guo d, cameli AM, denas G, d’Elios MM, Pengo V, Zhou F, Wang H . Toll-like receptor (TLR)-4 medi - Emmi L, Prisco d. An Approach to Differential Di - ates anti-beta2GPI/beta2GPI-induced tissue factor ex - agnosis of Antiphospholipid Antibody Syndrome and pression in THP-1 cells. Clinical and Experimental Related Conditions. The Scientific World Journa l Immunology 2011. 163 (2): 189-198. 2014. 2014: 341342. 21. Xie H, Zhou H, Wang H, chen d, Xia L, Wang t, 12. Miyakis S, Lockshin Md, Atsumi t, Branch dW, Yan J. Anti-beta(2)GPI/beta(2)GPI induced TF and Brey rL, cervera r, derksen rH, PG dEG, TNF-alpha expression in monocytes involving both

96 Mechanisms of thrombosis in systemic Lupus erythematosus

TLR4/MyD88 and TLR4/TRIF signaling pathways. 33. Krone KA, Allen KL, Mccrae Kr. Impaired fibri - Molecular Immunology 2013. 53 (3): 246-254. nolysis in the antiphospholipid syndrome. Current 22. Zhou H, Sheng L, Wang H, Xie H, Mu Y, Wang t, Rheumatology Reports 2010. 12 (1): 53-57. Yan J. Anti-beta2GPI/beta2GPI stimulates activation 34. tazi Mezalek Z, Harmouche H, Ammouri W, Maa - of THP-1 cells through TLR4/MD-2/MyD88 and NF- mar M, Adnaoui M, cacoub P. Atherosclerosis in kappaB signaling pathways. Thrombosis Research systemic lupus erythematosus. Presse Med 2014. 2013. 132 (6): 742-749. 43 (10 Pt 1): 1034-1047. 23. Vega-ostertag M, casper K, Swerlick r, Ferrara 35. conti F, Spinelli Fr, Alessandri c, Pacelli M, cec - d, Harris En, Pierangeli SS . Involvement of p38 carelli F, Marocchi E, Montali A, capozzi A, But - MAPK in the up-regulation of tissue factor on en - tari B, Profumo E, Sorice M, Arca M, Valesini G, dothelial cells by antiphospholipid antibodies. Arthri - rigano r. Subclinical atherosclerosis in systemic tis and Rheumatism 2005. 52 (5): 1545-1554. lupus erythematosus and antiphospholipid syndrome: 24. Allen KL, Fonseca FV, Betapudi V, Willard B, focus on beta2GPI-specific T cell response. Arte - Zhang J, Mccrae Kr. A novel pathway for human riosclerosis, Thrombosis, and Vascular Biology 2014. endothelial cell activation by antiphospholipid/anti- 34 (3): 661-668. beta2 glycoprotein I antibodies. Blood 2012. 119 (3): 36. narshi cB, Giles IP, rahman A. The endothelium: 884-893. an interface between autoimmunity and atherosclero - 25. urbanus rt, Pennings Mt, derksen rH, de Groot sis in systemic lupus erythematosus? Lupus 2011. PG. Platelet activation by dimeric beta2-glycoprotein 20 (1): 5-13. I requires signaling via both glycoprotein Ibalpha and 37. carmona-rivera c, Zhao W, Yalavarthi S, Kaplan apolipoprotein E receptor 2’. Journal of Thrombosis MJ. Neutrophil extracellular traps induce endothelial and Haemostasis : JTH 2008. 6(8): 1405-1412. dysfunction in systemic lupus erythematosus through 26. Krulik r, Zvolsky P. The effect of lithium on the me - the activation of matrix metalloproteinase-2. Annals tabolism of experimental animals. Activitas Nervosa of the Rheumatic Diseases 2014. [Epub ahead of Superior 1970. 12 (3): 279-283. print]. 27. Proulle V, Furie rA, Merrill-Skoloff G, Furie Bc, 38. thacker SG, Zhao W, Smith cK, Luo W, Wang H, Furie B. Platelets are required for enhanced activa - Vivekanandan-Giri A, rabquer BJ, Koch AE, tion of the endothelium and fibrinogen in a mouse Pennathur S, davidson A, Eitzman dt, Kaplan thrombosis model of APS. Blood 2014. 124 (4): 611- MJ. Type I interferons modulate vascular function, 622. repair, thrombosis, and plaque progression in murine 28. Lood c, tyden H, Gullstrand B, Sturfelt G, Jonsen models of lupus and atherosclerosis. Arthritis and A, truedsson L, Bengtsson AA. Platelet activation Rheumatism 2012. 64 (9): 2975-2985. and anti-phospholipid antibodies collaborate in the 39. Perez-Sanchez c, Barbarroja n, Messineo S, ruiz- activation of the complement system on platelets in Limon P, rodriguez-Ariza A, Jimenez-Gomez Y, systemic lupus erythematosus. PloS One 2014. 9(6): Khamashta MA, collantes-Estevez E, cuadrado e99386. MJ, Aguirre MA, Lopez-Pedrera c. Gene profiling 29. urbanus rt, de Laat B. Antiphospholipid antibodies reveals specific molecular pathways in the pathogen - and the protein C pathway. Lupus 2010. 19 (4): 394- esis of atherosclerosis and cardiovascular disease in 399. antiphospholipid syndrome, systemic lupus erythe - 30. nojima J, Iwatani Y, Ichihara K, tsuneoka H, matosus and antiphospholipid syndrome with lupus. Ishikawa t, Yanagihara M, takano t, Hidaka Y. Annals of the Rheumatic Diseases 2014. [Epub ahead Acquired activated protein C resistance is associated of print]. with IgG antibodies to protein S in patients with sys - 40. Kaiser r, Li Y, chang M, catanese J, Begovich AB, temic lupus erythematosus. Thrombosis Research Brown EE, Edberg Jc, McGwin G, Jr., Alarcon 2009. 124 (1): 127-131. GS, ramsey-Goldman r, reveille Jd, Vila LM, 31. Wahezi dM, Ilowite nt, Wu XX, Pelkmans L, Laat Petri MA, Kimberly rP, taylor KE, criswell LA. B, Schanberg LE, rand JH . Annexin A5 anticoag - Genetic risk factors for thrombosis in systemic lupus ulant activity in children with systemic lupus erythe - erythematosus. The Journal of Rheumatology 2012. matosus and the association with antibodies to 39 (8): 1603-1610. domain I of beta2-glycoprotein I. Lupus 2013; 22 (7): 41. deSancho Mt, Berlus n, christos PJ, rand J. Risk 702-711. factors for clinical manifestations in carriers of Factor 32. Vikerfors A, Svenungsson E, Agren A, Mobarrez V Leiden and prothrombin gene mutations. Blood F, Bremme K, Holmstrom M, Eelde A, Bruzelius Coagulation & Fibrinolysis : An International Jour - M, Elgue G, Wallen H, Antovic A. Studies of fibrin nal in Haemostasis and Thrombosis 2010. 21 (1): 11- formation and fibrinolytic function in patients with 15. the antiphospholipid syndrome. Thrombosis Research 42. Aisina rB, Mukhametova LI, ostriakova EV, Seredavkina nV, Patrushev LI, Patrusheva nL, 2014. 133 (5): 936-944.

97 METONI et al.

reshetniak tM, Gulin dA, Gershkovich KB, na - 45. Apor E, o’Brien J, Stephen M, castillo JJ. Sys - sonov EL, Varfolomeev Sd . Polymorphism of the temic lupus erythematosus is associated with in - plasminogen activator inhibitor type 1 gene, plas - creased incidence of hematologic malignancies: a minogen level and thrombosis in patients with an - meta-analysis of prospective cohort studies. tiphospholipid syndrome. Biomeditsinskaia Khimiia Leukemia Research 2014. 38 (9): 1067-1071. 2014. 60 (1): 72-93. 46. Kristinsson SY, Landgren o, Samuelsson J, Bjork - 43. Mészáros I, Krausz tL, Fischer EF, Major ZZ, holm M, Goldin Lr. Autoimmunity and the risk of Popescu o. Expression of immunomodulatory genes myeloproliferative neoplasms. Haematologica 2010; in inflammatory and autoimmune diseases. Romanian 95 (7): 1216-1220. Biotechnological Letters 2010. 15 (6): 5729-5737. 47. Harrison cn, donohoe S, carr P, dave M, Mackie 44. cloutier Bt, clarke AE, ramsey-Goldman r, Gor - I, Machin SJ. Patients with essential thrombo - don c, Hansen JE, Bernatsky S. Systemic lupus cythaemia have an increased prevalence of antiphos - erythematosus and malignancies: a review article. pholipid antibodies which may be associated with Rheumatic Diseases Clinics of North America 2014. thrombosis. Thrombosis and Haemostasis 2002. 40 (3): 497-506. 87 (5): 802-807.

98 ROMANIAN SCIENTISTS IN THE NOMINATION DATABASE FOR THE NOBEl PRIZE IN PHySIOlOgy OR MEDICINE, 1901-1951

Costin Cernescu* Ştefan S. nicolau Institute of Virology, Bucharest, Romania

ABSTRACT REZUMAT Nobelprize.org site is the most reliable and Site-ul www.nobelprize.org este sursa cea mai complete resource of information on the Nobel completă şi de încredere privind laureaţii Premiului Prize and the Nobel Laureates. The nomination Nobel. Accesând baza de date privind nominalizările database for Physiology or Medicine, 1901-1951, la Premiul Nobel pentru Medicină sau Fiziologie 1901- offers exciting facts about the Romanian Schools 1950 găsim fapte incitante despre Facultăţile de of Medicine from Bucharest, Iaşi and Cluj. Be - Medicină din Bucureşti, Iaşi şi Cluj. În intervalul 1920- tween 1920-1950, four Romanian scientists were 1950, patru savanţi români au fost propuşi pentru nominated for the Nobel Prize: Victor Babeş atribuirea prestigiosului premiu: Victor Babeş (1854- (1854-1926), Ion Cantacuzino (1863-1934), 1926), Ion Cantacuzino (1863-1934), Thoma Ionescu Thoma Ionescu (1860-1926) and Constantin Leva - (1860-1926) şi Constantin Levaditi (1874-1953). În diti (1874-1953). This paper discusses these nom - acest articol detaliem numele persoanelor care au făcut inees, the nominators and the motivations, as well aceste propuneri, motivaţiile lor şi numele evaluato - as the specific publications that endorse the candi - rilor. Din alte surse, am ales publicaţiile care justifică dates’ scientific activity. Recommendations made propunerile. De asemenea, sunt menţionate recoman - by Romanian professors for foreign researchers to dările făcute de profesori din România pentru cerce - receive the Nobel Prize are also included. tători din străinătate pentru decernarea premiului.

Keywords : Victor Babeş, Ion Cantacuzino, Thoma Ionescu, Constantin levaditi, Nobel Prize

INTRODUCTION monumental work “Les principes fondamentaux de l’histoire” (‘The fundamental principles of history’). Nobelprize.org site is the conclusive source of While accessing the nomination database, one information on the Nobel Prize and the Nobel can find exciting evidence about the scientific activ - Laureates [1]. Information concerning three fields ar e ity of the Romanian Schools of Medicine [2]. Be - currently posted: Literature, Peace and Physiology tween 1920-1950, four Romanian scientists were or Medicine. The Nomination Database for Physio - nominated for the Nobel Prize: Victor Babeş (1854- logy or Medicine encompasses all candidates nomi - 1926), Ion Cantacuzino (1863-1934), Thoma nated for the Nobel Prize from 1901 (when the prize Ionesco (1860-1926) and Constantin Levaditi (1874- was instituted) until 1950. That limitation, namely 1953). This paper discusses these nominees, the inclusion of material older than 50 years, follows the nominators and the motivations, as well as the spe - clause stipulated by the statute of the Nobel Foun - cific publications that endorse the candidates’ scien - dation: “all evaluations are archived in written form tific activity. Also, included are applications made and open to historic research only after 50 years”. by Romanian professors for foreign researchers to On nine occasions, during the World Wars years: in receive the Nobel Prize. 1915, 1916, 1917, 1918, 1921, 1925, 1940, 1941 and 1942 the Prize was not awarded. SEARCH STRATEGy AND In its introductory year, the Nobel Committee SELECTION CRITERIA received 136 nominations for the Prize for Peace, 83 for Physiology or Medicine and 37 for Literature. Data for this review were identified by search Among the latest is the name of the great Romanian of the nomination databases posted on www.nobel - hystorian Alexandru D. Xenopol, designated for his prize.org [2]. Currently there are three sections, each

*Corresponding author: Costin Cernescu, Ştefan S. Nicolau Institute of Virology, 285, Mihai Bravu Str, Bucharest 030304, Romania, E-mail: [email protected], phone: 00.40.740176984

99 COSTIN CERNESCU dedicated to specific domains: Physiology or Medi - the Nobel prize was motivated by his contributions cine, Peace and Literature. Using “Romania” as a related to the etiology and pathology of pellagra primary search term, all the Romanian nominations (Victor Babeş and V. Sion „Die Lesionen des Nerven and nominees in the Database for Physiology or System in der Pellagra”, Münich, 1899). Pelagra is Medicine were identified. In the second step, sup - caused by a chronic deficiency of niacin (PP vitamin plementary search terms were introduced, such as - “pellagra-preventive factor”). Babeş’s interest in the names of the nominees, their home universities this vitamin deficiency disease was stimulated by the or institutes and specific key words from the moti - endemic prevalence of pellagra skin lesions (pelle vations. As all Romanian nominees were educated agra - pelle = skin; agra = sour) in the Romanian or worked for long time periods in France, especially rural population, in which a maize-dependent diet in the Pasteur Institute, the search was extended to was common. In addition, Babeş illustrated the neu - the Paris Pasteur Institute site. The OLDMEDLINE rological lesions in pellagra [4]. Further studies have subset in PubMed ®, that contains journal article ci - demonstrated that the slaked-lime treatment of corn tations from two print indexes: Cumulated Index makes niacin nutritionally available and reduces the Medicus (CIM) and the Current List of Medical Lit - risk of developing pellagra. However, the work on erature (CLML) was also used. Approximately pellagra was only marginally relevant for Babeş 2,010,000 article citations from international bio - curriculum, and as such, his nominations were not medical journals that cover the fields of medicine, evaluated [5]. On the other hand, the interest in the preclinical sciences and allied health sciences dating role of vitamins was yet to come, as the Nobel Com - before 1965 can be found here. OLDMEDLINE mittee awarded the prize for the discovery of vita - records, most of which are included in the mins five years later, in 1929, to Christian Eijkman MEDLINE ® database, can be searched via PubMed. (The Nederlands) and Sir Frederick Hopkins (Great Britain). Furthermore, in 1924, two shared nomina - RESULTS tions for Victor Babeş and for Thoma Ionescu were signed by Professor Ernest Juvara (clinical surgery) The first nomination for Victor Babeş (professor and D. Manolescu (ophthalmology) - entries 43-1 of bacteriology and pathology at Bucharest School and 57.2, respectively. The motivation for Babeş’s of Medicine) was made in 1924 for “work on the nomination was again for his “work on the patholo - pathological anatomy, symptomatology, prophylaxis gical anatomy, symptomatology, prophylaxis and and treatment of pellagra”. The nominator was treatment of pellagra” and the one for thoma Gheorghe Marinesco, professor of neurology (entry Ionescu for his: “work on the anatomy, physiology 31 in Database). In the same year other nominations and surgery of neck and chest sympathicus” [6]. Ad - for Babeş (with the same motivation) were made by ditional nominators were Professors: I. Bălăcescu distinguished professors of Bucharest School of (orthopedics), A. Jianu (urology), N. Gheorghiu (ob - Medicine: C. Daniel, (ginecology; entry 32), M. stetrics), D. Manolescu (ophthalmiatrics), N.C. Manicatide (obstetrics), I. Nanu Muscel (clinical Paulescu (physiology), all from Bucharest. In total, medicine), Mina Minovici (forensic medicine), M. Babeş collected 13 nominations in Nobel Database Meţianu (otolaryngology), A. Obregia (neuropsychi - 1924. In that year 78 nominations were registered, atry), A. Teohari (clinical therapy) and D. Gerota including nominees like: Maurice Arthus (work on (clinical surgery) – entries 33 to 39 in the Database. anaphylaxis and snake venoms), T.H. Morgan (work Victor Babeş was trained in Budapest and Vienna on the physiology of embryonic development) or and was appointed professor of pathology and bac - Hermann Sahli (work on tuberculosis therapy and teriology in Bucharest in 1887, after a remarkable immunity). The 1924 Nobel Prize was awarded to scientific career in Rudolf Virchow’s and Robert Willem Einthoven for his discovery of the mecha - Koch’s laboratories in Berlin. In addition, he published nism of the electrocardiogram. with Cornil the first European textbook of bacteriology In 1924, Dimitrie Ionescu (professor of medi - [3] (Cornil A.V. and Babeş V. “Les Bacteries”, Felix cine, Bucharest) made a nomination for Thoma Alcan ed. Paris 1885). An excelent digital photoprint Ionescu (professor of topographical anatomy), a sur - of Victor Babeş can be found in OldMedline geon of world wide reputation, with the same above (http://ihm.nlm.nih.gov/images/B01315). mentioned motivation. Additional nominators came Although Babeş’s list of publications encom - from Bucharest Medical School professors: I. pass many important investigations in the fields of Bălăcescu (orthopedics), A. Jianu (urology), E. Ju - tuberculosis and bacteriology, his nominations for vara (clinical surgery), N. Gheorghiu (obstetrics), D.

100 Romanian scientists in the nomination database for the Nobel Prize in Physiology or Medicine, 1901-1951

Manolescu (ophthalmiatrics), N.C. Paulescu (phys - In the Nobel Prize Database, Cantacuzino is men - iology) (entries 30-3). Thoma Ionescu, a surgeon tioned for the first time in 1922 as a nominator for with excelent skills for innovative procedures was Émile Roux, director of Pasteur Institute, with the known for the introduction of high rachianesthesia, following incentive: “The discovery of toxins, the as a practice for the thoracic surgery [7]. Rachianes - use of antidiphtheria serum on human and isolation thesia was introduced in the medical practice by J.L. of the first known filterable virus”. Corning (1855-1923), who injected cocaine between Cantacuzino’s international reputation was fur - the spinous processes of the lower lumbar vertebrae. ther strenghtened by his support of the Bucharest An initial controversy was centered on whether Medical School objectives of excellence in research Corning’s injection was a spinal or an epidural neu - and education. Driven by the pursuit of quality pub - raxial blokade. Corning also published the principle lishing and maximum dissemination of new public of using spinal anaesthesia as local medication of the health accomplishments, he founded the journal cord. The high rachianesthesia was proposed by “Archives roumaines de pathologie expérimentale et Thoma Ionescu as a therapeutic technique for angina de microbiologie ” in 1928 (in 1991, the title was pectoris [8]. Other Romanian surgeons are cited in changed to Romanian Archives of Microbiology and the history of intrathecal and epidural anaesthesia: Immunology). Here he published several articles, in - Nicolae Racoviceanu-Piteşti (1860-1942) was the cluding the review that was cited for his nomination first to use opioids for intrathecal analgesia and the in 1928 [11]. All nominations for Cantacuzino were obstetrician E. Aburel (1899-1975) described caudal assessed by Upsalla Professor Alfred Pettersson. In epidural injection as an analgesic procedure during 1933, 74 nominations were registered in the Data - delivery. base involving clasic names in bacteriology like: The Nomination Database for the Nobel Prize Calmette and Guerin (work on tuberculosis), in Physiology or Medicine contains four nomina - d´Hérelle (work on bacteriophagy), Ramon (work on tions for Ion cantacuzino - professor of experimen - anatoxins), Uhlenhuth (work on precipitation reac - tal medicine at Bucharest School of Medicine - made tions) etc. The Nobel Prize winner in 1933 was T.H. by Reims professors: J. Bouillot (biochemistry) – Morgan for his discoveries concerning the role entry 47; J.E. Magou (bacteriology) – entry 36; J. played by the chromosome in heredity. Malassez (medical physics) – entry 62, and E. The first nomination for constantin Levaditi Techoueyres (histology) – entry 35. The motivation was made in 1922 by Professor H.R. Bruynoghe in all nominations was: “for his work on immunity (professor of Hygiene, Louvain) for his “research on reaction on invertebrates and contact immunity”. Ion polioviruses, influenza viruses and sleeping-sick - Cantacuzino studied at the Medical School in Paris ness” [12, 13]. In 1924, professor J.A. Pupo (profes - between 1887-1893, after graduating (in 1895) from sor of therapy, Sao Paolo) made a new nomination “Faculte de Lettre et Phylosophie de Paris”. In 1893, for Levaditi for his ˝work on experimental syphilis˝ as an intern, he was accepted in Metcinikoff’s La- [14]. The same motivation can be found in the nom - boratory at Pasteur Institute. His PhD thesis: inations made in the following years by Professors: “Recherches sur le mode de la déstruction du vibrion G. Wirgin (Upsalla, 1927), E. Hoffmann (Bonn, cholerique dans l´organisme. Contribution à l’ étude 1930), Carol A. Kling (Stockholm, Karolinska Uni - du probleme de l’immunité”, Ed Steinheil Paris versity, 1930), Jean Lepine (Lyon, 1930), Emile 1884, was conducted by Metcinikoff [9]. While in Roux (Paris, Institut Pasteur, 1930), and again, G. France, Cantacuzino began an intense research ac - Wirgin (Upsalla, 1930). Scientists from different tivity at the Roscoff Institute for Marine Biology. His countries simultaneously signed Levaditi’s nomina - experimental results were recorded in a series of im - tions, all based on his contributions to infectious dis - portant articles and publications. A review of his eases and therapy. Although he was engaged in many contributions was published in Annales de l’Institut areas of the rapidly growing field of immunology, Pasteur in 1920 in a jubilee volume dedicated to his name was associated mainly with research in Metcinikoff: “La pathogénie du choléra et la vacci - syphilis and poliomyelitis [15]. nation anticholérique” [10]. Owing to his fame and Constantin Levaditi, who was professor of Bac - his selfless devotion to medical research, Can - teriology at Cluj Medical Faculty between 1926-27, tacuzino was appointed professor of Bacteriology at is considered one of the most significant Pasteurian Bucharest Medical School in 1901 and as Head of scientist in immunology and virology. Two of his Laboratory of Experimental Medicine (later “Can - nominations for Nobel Prize were signed by Profes - tucuzino Institute for Sera and Vaccines”) in 1904. sors of Cluj Faculty of Medicine in 1931: Nicolae

101 COSTIN CERNESCU

Minovici (forensic medicine) and C. Grigoriu (ob - contributions. We must observe the emphasis on stetrics) (entry 58-0 and 58-1 in the database). Most works of Pasteurian researchers such as Émile Roux, of the Cluj Faculty professors are cosignatories: J. Albert Calmette and Camille Guérin on vaccines Guiart (medicine history), Ion Haţieganu (clinical against diphtheria or tuberculosis. Sometimes, rele - medicine), P. Thomas (biochemistry), Ion Moldovan vant proposals made by Romanian professors con - (hygiene), Ion Iacobovici (clinical surgery), C. Ure - ducted to the Nobel Prize award: Willem Einthoven chia (psychiatry), Ion Minea (neurology), T. Gane (1924 – for electrocardiograpy), Sir Charles Scott (pediatrics), T. Vasiliu (pathological anatomy), Ion Sherrington (1932 – for neuronal physiology), I. Niţescu (physiology), D. Mihail (ophthalmology), George Whipple (1934 – for anti anemia therapy) D. Negru (radiology), V. Papilian (anatomy). and Wendell Stanley (1946 – Nobel for Chemistry The scientific work of Levaditi was multilateral. for works on tabacco mosaic virus). In collaboration with numerous famous scientists of his era, such as Landsteiner, Metchnikoff and Lepine DISCUSSION he studied the etiology of poliomyelitis, herpes en - cephalitis, neuroectodermoses; introduced new sero - Between the two World Wars, in all Romanian logical techniques for the diagnosis of syphilis; and Medical Schools the amount of new information that applied bismuth as treatment for this disease. Heavy was gathered in biomedicine was well known. Med - metals - first mercurial and later arsenic and bismuth icine was not only clinical, it also covered cell biol - preparations - were commonly used to treat syphilis ogy, biochemistry, physiology, pathology, since 1930. For his contributions in bismuthother - neuro biology and more. Even nowadays, medicine apy, Levaditi was nominated for Nobel Prize in 1931 extends from the organism to the molecule. It is un - by Professors Holmgreen and Kling (Karolinska), G. fortunate to observe the absence of a homeland nom - Wirgin (Upsalla), George W. Raiziss (Philadelphia, ination for Nicolae Paulescu for the discovery of chimiotherapy) and W. Scholtz (Kőningsberg, der - ˝pancreine˝ (later named insulin). The prize awarded matology and syphyligraphy). The same motivation to Banting and Macleod for the discovery of insulin can by found in prestigious proposals made in 1932 (1923) was questioned right from the time it was an - by Karl Landsteiner (director at Rockfeller Institute nounced. Paulescu’s original publication on insulin for Medical Research), Nobel Prize winner in 1930, appeared in 1921, two years before the Canadian sci - and also by Johan Almkvist, Carol A. Kling and entists were awarded the prize. Israel Holmgreen (all from Karolinska University). The method used by Paulescu to prepare the Other nominations for Levaditi were registered pancreatic extract was published in the Archives In - between 1934-39: L. Spillman (Nancy, 1934), H. ternationales de Physiologie (Liege, Belgium) in Au - Gougerot (Paris, 1935), Ştefan S. Nicolau (Paris, gust 1921 [17]. Fortunately, Paulescu was mentioned 1938), R. Le Blaye, M. Roblin and H. Vincent (all in the Presentation Speech made by Professor J. from Poitiers, 1939). Altogether, Levaditi received Sjöquist, member of the Nobel Committee for Phys - 27 nominations, being among the most cited nomi - iology or Medicine on December 10, 1923, in Stock - nees who did not received the Prize (http://nobel - holm, during the Awarding Ceremony[18]. prize.org/nobel_prizes/medicine/nomination). At the end of the First World War, Romania had Levaditi’s research papers (cited by nominators as a vibrant research environment with world-class ex - motivations for their proposals) were evaluated by perts. Working in that environment was extremely Upsalla University Professors: Johan Almkvist and stimulating and many disciples of the four Romanian Carl G. Santesson in 1929 or A. Pettersson and Einar nominees for the Nobel Prize became scientific fig - Hammarsten în 1933. ures with worldwide recognition. Noteworthy, three Many Nobel Prize proposals registered in the of Nobel nominees were from different domains of Data Base were made by Romanian professors, act - Microbiology. Babeş, Cantacuzino and Levaditi im - ing as nominators for foreign scientists (Table 1). plemented their scientific knowledge on potential Some of them were not sustained by appropriate ar - novel treatments to raise relevant public health ques - gumentation and were not evaluated. The Nobel tions and succeeded to bring therapeutic agents or Prizes are awarded to scientists who bring novelty procedures in practice as a response to major infec - to the pathogeny and treatment of diseases. The qua - tious diseases. lity and strength of the discovery are important, not the number of partial discoveries. One clear disco - very is a requirement, rather than several stepwise

102 Romanian scientists in the nomination database for the Nobel Prize in Physiology or Medicine, 1901-1951

table 1. romanian Scientists recorded as nominators in nobel Prize for Physiolo gy or Medicine database Nominator and Year Nominee Motivation university E. Riegler Highly eminent and intelligent researchers (in 1907 E. Salkowski (1854-1929), Iaúi fact discoverer of JDVWULFWXEH) C. Levaditi E. Roux 1910-1914 Work on serum therapy and diphtheria toxin. (Pasteur Institute) (1853-1933) The discovery of toxins, the use of I. Cantacuzino 1924 E. Roux antidiphtheria serum on human and isolation of (Bucharest) the first known filterable virus. E. Riegler Sir LT Brunton 1913 No motivation given. (Iaúi) (1844-1916) E. Haekel No motivation given. 1913 C.Thiron (Iaúi) (1834-1919) Ontogeny recapitulates phylogeny theory D. Danielopolu The invention and design of the string 1924 *W. Einthoven (1884-1955) galvanometer. D. Danielopolu *Ch. S. Sherrington 1926 Works on physiology of neurons. (Bucharest) (1857-1952) C.I. Parhon The preparation of the active hormone of the B.J. Collip 1928 (1874-1969) parathyroid gland and demonstration of its (1892-1965) (Iaúi) mode of action and uses. A. Slătineanu A. Calmette (1873-1939), (1863-1933) Discovery of a vaccine against tuberculosis 1928 M. Ciucă C. Guérin (BCG) with Guérin; sera against snake venom. (1883-1968) (1872-1961) (Bucharest) The discovery that regeneration of red blood I. Enescu cells and formation of haemoglobin in dogs *G.H. Whipple 1934 (1884-1972) made anaemic by haemorrhage could be (1878-1976) (Iaúi) greatly accelerated by providing liver or kidney tissue in the diet Gr. T. Popa G. Elliot Smith 1934 (1892-1948) Studies on neopallium (1871-1973) (Iaúi) 1936 Gr.T. Popa L. Lapicque Work on chronaxy. G. Popoviciu P. György Work on vitamins, especially vitamins B2 and 1940 (Iaúi) (1893-1976) B6. I. Bâlteanu (1888-1968) R. Leriche 1943 A. Cosăcescu Vascular surgery, especially arteriectomy. (1879-1955) (1888-1951) (Bucharest) *W. Stanley Isolation of virus proteins. Studies of tobacco 1943 Gr. T. Popa (1904-1971) mosaic virus. Work on corneal transplantation and on tissue G. Năstase V.P. Filatov 1950 transplantations in other ocular and some (Iassy) (1875-1956) dermal diseases. *6cientistsSURSRVHGto win the Nobel Prize

103 COSTIN CERNESCU

REFERENCES 11. cantacuzino I - Recherches sur le reactions d’immu - nite chez les invertebres. Arch. Roum. Pathol. Exp. 1. “Nomination Database - Credits”. Nobelprize.org. Microbiol. 1928, 1, 7-80. Nobel Media AB 2014. http://www.nobelprize.org/ 12. Landsteiner K, Levaditi c - La transmission de la nomination/archive/medicine/credits.html paralysie infantile aux singes. C. R. Soc. Biol. 2. “Manual for the Nomination Database for the Nobel 1909:592–594. Prize in Physiology or Medicine, 1901-1951”. Nobel - 13. Levaditi c and Lépine P - Le virus poliomyélitique. prize.org. Nobel Media AB 2014. http://www.nobel - In ˝Les ultravirus des maladies humaines˝. Eds. Leva - prize.org/nomination/archive/medicine/manual.html diti C, Lépine P (Librairie Maloine, Paris, France, 3. cornil AV et Babeş V - Les Bacteries (Felix Alcan ed. 1938), p 572. Paris 1885). 14. Levaditi c and Sazerac r - Action de certain derivés 4. Babeş V and Sion V - “Die Lesionen des Nerven Sys - phénoliques du bismuth sur la syphilis. C.R. de la So - tem in der Pellagra˝, München, 1899. ciété de Biologie. Paris, May, 1922. 5. Babeş V and Sion V - “Die Pellagra” in Spezielle 15. Levaditi c - “Le Bismuth dans le traitement de la Pathologie und Therapie (red. K.W. Nothnagel) vol 24, syphilis ˝, Paris 1924. fasc 2. Holder Verlag, Wien 1901. 16. Iftimovici r - “Istoria Universală a Medicinei şi Far - 6. Ionescu t - Le sympathique cervico-thoracique. Bull maciei” (The Universal History of Medicine and Acad Nat Med, Paris 1923, 87, 76. Pharmacy). Ed. Academiei Române, Bucureşti,2008, 7. Ionescu t - La rachianestésie générale, Paris, 1919. ISBN 978-973-27-1708-0. 8. Ionescu t - Angine de poitrine guérie par la resection 17. Paulescu nc - Recherche sur le rôle du pancréas dans du sympathique cervico-thoracique. Bull Acad Nat l’assimilation nutritive, Archives Internationales de Med , Paris, 1920, 84, 93. Physiologie 1921, August 31, 17: 85–103. 9. cantacuzino I - Recherches sur le mode de la déstruc - 18. “Physiology or Medicine 1923 - Presentation Speech”. tion du vibrion holerique dans l´organisme. Contribu - Nobelprize.org. Nobel Media AB http://www.nobel - tion a l’ étude du probleme de l’immunité. Ed Steinheil prize.org/nobel_prizes/medicine/laureates/1923/ Paris 1884. press.html 10. ciufecu c, neguţ M, (editors) - “Profesorul Can - tacuzino, 150 ani de la naştere”, Total Publishing, Bucharest 2014, ISBN: 978-606-8003-28-3.

104 ROlE OF SALmOnELLA SEROlOgy A CENTURy AFTER THE WIDAl ERA

Alexandra-Maria Nãşcuþiu* Cantacuzino national Institute of Research-Development for microbiology and Immunology, Bucharest, Romania Carol Davila University of medicine and Pharmacy Bucharest, Romania

ABSTRACT REZUMAT Typhoid fever remains an important health Febra tifoidă rămâne o importantă problemă de burden in the developing world, whereas non-ty - sănătate publică în ţările în curs de dezvoltare, în vreme phoid salmonelloses are one of the most common ce salmonelozele non-tifoidice se numără printre cele food-borne illnesses throughout the world and can mai frecvente boli transmise prin alimente în lumea be subjected to extra-intestinal complications. întreagă, putând antrena şi complicaţii extraintestinale. Culture is the gold standard for diagnosing a Sal - Cultivarea reprezintă standardul de aur pentru diagnos - monella infection. Serology can also provide evi - ticarea infecţiei cu Salmonella . Serologia poate oferi de dence of infection. Serological methods for the asemenea dovezi ale infecţiei. Există variate metode diagnosis of Salmonella infections in humans and serologice pentru diagnosticarea infecţiilor cu Salmo - animals vary widely and can as well be used in nella la om şi animale, ele fiind folosite şi în studiile epi - epidemiologic studies to detect carriers, to assess demiologice pentru detectarea purtătorilor, pentru infection rates, disease burden and vaccine re - evaluarea ratei infecţiilor şi a răspunsurilor vaccinale. sponses. Ca orice test serologic, şi detectarea titrului anticor - As with all serology, detecting antibody titers pilor anti- Salmonella îşi are limitele, acestea ţinând în in Salmonella infections has its limits, mainly re - principal de sensibilitatea şi specificitatea scăzută, de lated to low sensitivity and specificity, high run - costurile ridicate şi de cinetica şi particularităţile anti - ning costs, and antibody kinetics and peculiarities. corpilor. Sunt disponibile comercial teste rapide, mai Fast and more or less reliable immunoassays mult sau mai puţin fiabile, care detectează Salmonella which detect Salmonella enterica serovar Typhi enterica serovar Typhi. Sectorul veterinar şi cel de con - are commercially available. Veterinary and food trol al alimentelor sunt dotate cu teste comerciale pentru sectors are well-provided with commercially tests detectarea salmonelelor non-tifoidice antigene şi anti - for non-typhoid salmonellosis, while most im - corpi, în vreme ce majoritatea testelor imunologice pen - munoassays for non-typhoid human Salmonella tru diagnosticarea infecţiilor umane cu Salmonella sunt diagnosis are developed and used in-house mainly dezvoltate şi utilizate in-house , în principal în scop de for research or surveillance purposes. So far, there cercetare şi supraveghere. Până în prezent nu există un is no international consensus for the development consens internaţional pentru dezvoltarea unor asemenea of such serological tests for routine diagnostics. teste serologice pentru diagnosticul de rutină. 119 years after the observations made by La 119 ani după observaţiile făcute de George Fer - George Fernand Isidore Widal, this work intends nand Isidore Widal, acest referat intenţionează să treacă to review and analyze the present state of facts and în revistă şi să analizeze realitatea şi controversele în controversies in the field of Salmonella serology. domeniul serologiei pentru Salmonella .

Keywords : Salmonella ser. Typhi, non-typhoid salmonellae, serology, limits and perspectives

Salmonella serology has a very long story in the be of use for diagnostic and epidemiological pur - even longer history of immunology. The purpose of poses and to whether research in this field is worth this review is to list and analyze the present state of being done. facts and controversies in the field of Salmonella Salmonella is one of the leading causes of food- serology and to give a possible answer to whether borne outbreaks worldwide, with continuously in - Salmonella antibodies are still capable nowadays to creasing incidence of reported cases, as well as the

*Corresponding author: Alexandra-Maria Nãşcuþiu, Cantacuzino Institute, 103, Splaiul Independenþei, Bucharest, Romania, e-mail: [email protected] 105 ALEXANDRA-MARIA NÃŞCUÞIU agent of a major health problem for developing than one week should be considered typhoid until countries: typhoid (enteric) fever. Due to the severity proved otherwise [5]. of the illness and to its health impact, there has been For either typhoid or non-typhoid salmonellosis more insight to the diagnostic of typhoid fever which the gold standard for diagnostic is bacteriological is a condition more common in children and young culture. Identification is a step-wise process that can adults and is most prevalent in impoverished, over - be labor- and time-consuming, especially in geo - crowded areas that have poor access to sanitation. graphical areas that lack resources for automated Non-epidemic incidence estimates suggest that equipment. Thus, an alternative means of diagnostic South-Central and Southeast Asia, as well as south - is serological diagnostic. Furthermore, in the ab - ern Africa are regions with high incidence of Salmo - sence of viable bacteria (which can be present some - nella ser. Typhi infection (more than 100 cases per times only for a few days), antibody tests can give 100,000 person-years), while other regions of Asia evidence of infection provided that suitable im - and Africa, Latin America, the Caribbean, and Ocea - munoassays, based on well-characterized antigens nia have a medium incidence of 10 to 100 cases per are used and that sufficient time has elapsed between 100,000 person-years. However, these estimates are the onset of symptoms and serum collection (Fig. 1). not 100% realistic, being based on extrapolation of Detection of serum antibodies might prove very use - data across regions and age groups and they are lim - ful in the case of post-infectious complications such ited by lack of consistent reporting from all areas of as reactive arthritis, a form of arthritis with chronic the world [1]. progression. This was showed to be associated In Europe and the USA, salmonellosis is the mostly with Salmonella ser. Typhimurium and, oc - second enteric bacterial infection as incidence, casionally, Enteritidis infections (group B and D sal - following Campylobacter infection. Worldwide, monellae, respectively). By the time of onset of these there are an estimated 93.8 million cases of arthritic complications, the microbe might no longer gastroenteritis due to non-typhoid Salmonella be culturable. infection each year, resulting in approximately The usefulness of serology is obvious in clinical 155,000 deaths [2]. Annual 1.4 millions of cases of suspicion of typhoid fever, when sampling for cul - salmonellosis are esti mated only in the USA, 95% tural purposes is performed, followed by antibiotic of them being food-borne infections. About 220 out administration. If failure of isolation previous to an - of 1000 cases are hospitalized, 8 of those being fatal. tibiotic ingestion, further sampling most probably 31% of the deaths related to food consumption are will result negative as well, so diagnostic can not be due to Salmonella spp. infections [3]. Enteric properly performed otherwise than by serological salmonelloses still present an increased rate of means (Fig. 2). Furthermore, in areas where typhoid notification in EU countries (29.75/100.000). Value s fever has a high incidence, culture is not always are nevertheless encouraging, lately the tendency available, so that serological assays become highly being to continuous decrease, which might be due, desirable as facilities [6]. at least partly, to the control measures implemented The use of serology is not solely restricted to in zootechny (poultry farming) [3, 4] . diagnosis. Serology is important for the detection of The diagnostic of typhoid fever is rather diffi - carriers (convalescence and chronic faecal carriage), cult to perform, the presenting symptoms being di - the assessment of infection rates, of disease burden verse and similar to those encountered in other in a community in order to determine the need for febrile illnesses (malaria, amebic liver abscess, vis - vaccination programs, the assessment of vaccine ceral leishmaniasis, toxoplasmosis, influenza, responses, and for other epidemiological purposes. dengue and other arboviroses, infectious mononu - Each specific role may warrant a dedicated test, cleosis, tuberculosis, leptospirosis, brucellosis, ty - using different samples, targets and methods to serve phus or other rickettsial diseases, encephalitis, their respective purpose [7]. endocarditis, deep abscesses, lymphoproliferative 119 years ago, in 1896, George Fernand Isidore disease, and connective-tissue diseases), clinical di - Widal was the first to think of a test that could agnosis being thus considered unreliable. For pa - emphasize the presence of Salmonella ser. Typhi tients in countries where typhoid is not endemic, a antibodies. Widal test - the first test to be conceived travel history is crucial. Though clinical algorithms and performed in this field - is still widely used, with have been developed, they have not generally been several brands available. It is an agglutination test validated, so it is usually stipulated that in endemic that can be performed on slide or in tubes, areas a fever without evident cause that lasts more demonstrating the presence of antibodies directed

106

role of Salmonella serology a century after the Widal era

Direct diagnosis – CULTURE Serological GOLD STANDARD diagnosis

Salmonella non-Typhi ser. Typhi salmonellae

can be labor- and time-intensive Bone marrow, in areas of the world Blood culture, Stools that lack resources Stools, Urine for automated equipment.

Isolation

Identification - Cultural aspects - Biochemical tests - Antigenic identification - Phage Typing / PFGE

Fig. 1 – diagnosis of Salmonella infections

Clinical suspicion of typhoid fever

Sampling for culture purposes (blood, stools, urine, Antibiotic administration other body fluids)

Failure of isolation Further sampling

Serology for defining diagnosis Failure of isolation

Fig. 2 – usefulness of serology for the diagnosis of Salmonella infections against somatic (O) and flagellar (H) antigens of A positive agglutination during the acute and Salmonela ser. Typhi. convalescent phases of the disease increases the sus - It is still the test of choice in endemic develop - picion for the presence of typhoid fever. It was the - ing countries, as it is unanimously accepted that the oretically stated that a four fold increase in antibody Widal test is easy and inexpensive (main reason for titer in dual serum is diagnostic, with serum taken 7 its popularity) and that it can definitely be of diag - to 10 days apart (acute- and convalescent-phase nostic value when blood cultures are not available serum, respectively) [8, 9], but this does not always or practical. happen in the field, as sometimes antibody titers rise

107 ALEXANDRA-MARIA NÃŞCUÞIU even before the clinical onset of the disease [10]. In showed those marked discrepancies at different cut- blood-culture-confirmed cases one or two fold rise off values [21]. The results of the study have been of antibody titer is the rule nowadays. This can be previously supported by Hoffman et al. and by explained either by the presence of high levels of Olopenia and King, respectively. The former stated background antibodies in endemic regions, or by that results from a single Widal test are virtually non- early treatment of cases of fever with antibiotics interpretable, unless the performances of the test are (blunted antibody response), or by the delay in ob - well known (in terms of laboratory experience and taining acute phase samples in the natural history of antibody level titer in the specified population) [22]. diseases, or by a possible lack of antibody response The later stated that the value of Widal test depends in immunosuppressed patients [9, 11]. As on the upon standardization and maintenance of the anti - field not few are the cases when only one serum gens to produce consistent results [20]. sample is available from the patient, some studies Regarding the type of antibodies in the blood of agreed that, with a judicious interpretation, Widal the patients that could be more reliable for diagnos - test can sometimes be valuable even as single test tic, there have been several trends. The earliest sero - performed in the early stage of disease [12, 13, 14, logical response in acute typhoid infection is the rise 15]. Yet, other authors have proved in their studies of anti-O antibody titer, the anti-H antibody titer ris - that a single Widal test is not sufficient to sustain the ing more slowly, but persisting longer [23]. In 1962, diagnosis of typhoid fever [16, 17, 18, 19]. The main Huckstep has claimed that only O titer is helpful, as reasons that were stated as arguments were that pa - the level of H antibodies remains elevated a longer tients in endemic regions are either repeatedly ex - period than the O level following an acute episode posed to Salmonella ser. Typhi or to other of typhoid fever. Furthermore, H antibodies were Salmonella spp. that express type 9 or type 12 so - found by Huckstep to also rise in response to other matic antigens (cross-reactivity in group B or D sal - infections [24]. Recent studies supported the obser - monellae), or they are previously vaccinated. In vation that the level of O antibodies is more helpful endemic areas, sera collected from patients in the than the H level: for instance a study performed in the first week of illness may present high titers of H and endemic area of Lahore, Pakistan [9] and a study per - O antibodies, which is attributable to a hyperimmune formed in Iraqi population [25]. Yet, throughout the or immunologically sensitized population continu - years, evidences that state that H antibodies are at least ally exposed to Salmonella infection [12]. as useful as O antibodies (if not even more) were ob - For the Widal test, over 100 years of faithful tained by several other researchers in field studies per - services also meant over 100 years of controversies formed in the Transvaal [26], South-East Asia that featured, besides the interpretability of single (Malaysia) [12] and again in South Africa [27]. Any - Widal test, the quality of antigens used, the useful - how, the baseline O and H titers in the local popula - ness of O versus H titer, or the difficulties of inter - tion must be acknowledged when interpreting results. preting results in endemic regions and the The antibody titer detectable in populations of dependency upon standardization for producing con - different areas varies considerably, depending also sistent results [16, 20, 21]. It is well recognized that on the level of infection due to other salmonellae agglutination tests have serious shortcomings. Dis - with cross-reacting antigens, which is an additional crepancies in results between laboratories have been argument to the fact that evaluation of Widal test reported. These might show up even within the same should be performed in the geographical area in laboratory, for instance when preparations of the which it is to be used [9, 28, 29]. Low titers on a sin - antigens have come from different sources or when gle specimen which are considered significant in the technique has been slightly modified [19]. What non-endemic areas are of no diagnostic significance else can be reproached to Widal test? Its inability to in areas where Salmonella ser. Typhi is endemic. discriminate between different antibody classes or In order to keep Widal test alive, standardization between current infection and previous infection or is a must. Different brands should give consistent re - vaccination, its possible cross-reactivity with other sults. Until this goal is achieved, research must be Salmonella spp., its poor sensitivity, especially when done for the improvement of sensitivity and speci - using increased cut-off values, the fact the test is ficity of the available tests. Dual use of Widal tests time-consuming when performed in dynamics [20]. is now recommended, that is screening sera by A lot of Widal test variants are commercially highly sensitive variants of Widal tests and later trac - available, but they unfortunately show great discrep - ing false positive by testing those positive sera in the ancies. A 2011 study on 4 different Widal brands first testing by a highly specific variant of Widal test.

108 role of Salmonella serology a century after the Widal era

If more than one Widal brand not available or afford - naturally acquired typhoid fever in endemic areas. able, than the cut-off value of the single test used Some studies showed the Vi reaction is very helpful must be determined for the specific population (de - for the detection of carriers in the context of out - pending on the background level of typhoid fever break investigations (being non-equaled in terms of and the level of vaccination). Titers of 1/160 or more widespread acceptance for this purpose, as stated by are generally thought to be consistent with acute ty - Malaysian scientists) [61], but less helpful for the phoid [21]. general population. Initially performed with a crude The WHO has shown reticence in using this test extract of Citrobacter freundii (Ballerup O group in the future, strongly insisting on the use of cultures, 29), the test was later developed into a passive haem - whenever possible. Besides, concerning serology, magglutination assay using purified, highly poly - the recommendations issued by the WHO are clear merised Vi antigen [62]. A similar Vi reaction has and very rigorous: “the method used as the gold been used to screen for S. enterica ser. Typhi carriers standard for the laboratory diagnosis of typhoid in Chile, with encouraging results in terms of sensi - should approach 100% each for sensitivity, speci - tivity and specificity for a highly endemic region ficity, and positive and negative predictive values” [32]. Yet, sera used in passive haemagglutination [30]. Whether this is a realistic, achievable goal has assay need to be pre-absorbed with sheep erythro - been a topic of debates in the last decades [31]. cytes before being used and this may not be conven - The difficulties in the interpretation of the Widal ient for screening of large populations. test, together with the need for a rapid and inexpen - The usefulness of haemagglutination tests for de - sive laboratory test for early and accurate diagnostic tection of anti-LPS (lipopolysaccharide) antibodies have lead, in time, to the exploration of many vari - was evaluated in different endemic areas, results de - eties of serologic detection methods including noting it is a good replacement for Widal test [27, 33]. haemagglutination, counter-immunoelectrophoresis, radial counter immunoelectrophoresis (cIE) radioimmunoassay, dot immunoassays, and enzyme- using cellulose acetate membranes was tried more linked immunosorbent assays (ELISAs). In Table 1 than 30 years ago, proving to be a rapid and useful and Table 2 we tried to gather information from sev - tool especially for screening large number of serum eral databases listing studies performed worldwide samples from patients suspected of typhoid fever and point out the performances of the tests in terms [63]. In spite of some promising results when using of sensitivity and specificity. Yet, these studies are veronal buffer extract as antigen [64], the method not comparable as such because they use different was abandoned, as several studies showed no corre - protocols applied on different population groups, lation of CIE antibody with Widal antibody titers from regions with different degree of endemicity of [65]. Even if the sensitivity of some CIE for anti - the disease (when testing for Salmonella ser. Typhi body detection rose from first to second week of ill - antibodies) and with different sampling timing with ness, it still remained below 25% [66]. Yet, Sharma respect to number of days since onset of the disease. et al. recommended a panel of antigens (somatic O, Newer, rapid antibody tests are commercially flagellar H and capsular polysaccharide Vi antigens available at present and comparative studies of their of Salmonella ser Typhi) for the rapid diagnosis of performances have been already available. Tests typhoid fever by CIE [67]. such as the colorimetric TUBEX® test, the dot en - An important approach to the rapid diagnosis of zyme immunoassays Typhidot®, immunochromato - infectious diseases is the detection of specific anti - graphic assays or variants of ELISAs are in place. bodies (IgM, IgG and IgA). The presence of IgM is All of these tests have been evaluated in highly en - usually indicative of a recent or ongoing infection, demic regions for typhoid, mostly not giving satis - from the first week onwards. factory enough results regarding performance. That A solid-phase radioimmunoassay (rIA) was is probably the reason why the WHO has not issued used to assess antibody response to the LPS of Sal - recommendations on the use of rapid tests for sero - monella ser. Typhi during enteric fever. All three logical diagnosis of typhoid fever yet. types of serum antibodies (IgM, IgA and IgG) were As diagnostic method using agglutination, de - found, with higher levels than the intestinal antibody tection of antibodies directed against the very special levels, but no significant correlation was noted be - Vi capsular antigen of Salmonella ser. Typhi must tween serum antibodies and antibodies in intestinal not be forgotten. There are very few published data secretions [68]. Typhoid patients showed a progres - on the antibody response to this Vi capsular polysac - sive increase in IgA, IgG and IgM levels which ex - charide (Vi-CPS) of Salmonella ser. Typhi during tended for at least 8 weeks [69]. Even if RIA has

109 ALEXAND RA-M ARIA NÃŞC UÞIU

Table 1. S ensitivity and s pecific ity of several types of serological reactions for Salmonella ser. typhi antibodies detection for different sample populations belonging to different geographical areas Test Location, year * Sensitivity Specificity Reference Haemagglutination Chile, 1983 75% 92% Lanata et al. [32] (Vi) Haemagglutination South Africa, 1986 80% 98.4% Coovadia et al. [27] (LPS) Haemagglutination India, 1997 60% 98.2% Shukla et al. [33] (LPS) TUBEX® Vietnam, 2001 87% 76% House et al. [34] TUBEX® Malaesia, 2002 78% 80% Gopalakrishnan et al. [35] TUBEX M® Vietnam, 2004 78% 94% Olsen et al. [36] TUBEX® Bangladesh, 2007 91% 82% Rahman et al. [37] TUBEX® Philippines, 2007 95% 80% Kawano et al [38] Sub-Saharan Africa, TUBEX® 73% 69% Heddy et al [39] 2007-2009 TUBEX® Bangladesh, 2008 60% 58% Naheed et al. [40] TUBEX® Egypt, 2011 75% 87% Fadeel et al. [41] Papua New Guinea, TUBEX® 51.1% 88.3% Siba et al. [42] 2012 IgM 69.0% IgM 79.3% ICT (IgM, IgG) Philippines, 2007 Kawano et al. [38] IgG 70.7% IgG 76.1% ICT (IgM, IgG) Bangladesh, 2013 90.3% 92.5% Hassan et al [43] Lateral flow IgM Indonesia, 2007 41.2-89% 97.8% Abdoel et al. [44] Lateral flow IgM Egypt, 2010 80% 71.4% Nakhla et al. [45] Typhidot® Malaesia, 2002 98% 76.6% Gopalakrishnan et al. [35] Typhidot® Pakistan, 2006 85% 77% Bhutta et al. [46] Typhidot® India, 2006 92% 98% Jesudason et al. [47] Sub-Saharan Africa Typhidot IgM® 75% 60.7% Heddy et al. [39] 2007-2009 Sub-Saharan Africa Typhidot IgG® 69.2% 70.4% Heddy et al. [39] 2007-2009 Typhidot® Bangladesh, 2008 67% 54% Naheed et al. [40] Typhidot M® India, 2010 92.6% 37.5% Narayanappa et al. [6] Typhidot M® India, 2010 90% 100% Beig et al. [48] Typhidot® Egypt, 2011 63% 92% Fadeel et al. [41] Papua New Guinea, Typhidot M® 70% 80.1% Siba et al. [42] 2012 Dipstick IgM Vietnam, 2001 77% 95% House et al. [34] Dipstick IgM Indonesia, 2002 76.9-83.1% Up to 100% Hatta et al. [49] Dipstick IgM Kenya, 2002 73.7% 84.2% Hatta et al. [49] Dipstick IgG Vietnam, 2004 89% 50% Olsen et al. [36] ELISA Chile, 1992 94% 92% Quiroga et al. [50] IgM 75% ELISA LPS Vietnam, 2001 IgG 55% >= 93% House et al. [34] IgA 52% ELISA flagellum Vietnam, 2001 IgG 28% >=93% House et al. [34] IgM 90% 49% ELISA Philippines, 2007 Kawano et al. [38] IgG 96% 39% ELISA IgM India, 2010-2011 89.47% 96.87% Anagha et al [51] * year when study was performed or, if not stated, year when data were published

110 role of Salmon ella serology a centur y a fter the Widal era

Table 2. Sensitivity and specificity of several types of serological reactions for non-typhoidal Salmonella serovars fo r human use (modified after Kuhn, ref. 52) Antigen Location, Test Sensitivity Specificity Reference (serovar) year* TUBEX® LPS (S. Ent) Poland, 2003 92.6% 94.8% Oracz et al. [53] LPS (S. Ent, ELISA Sweden, 1979 92% NR Svenungsson et al. [54] S. Tm) LPS (S. Typhi, ELISA Germany, 1983 100% 90% Hirschl et al. [55] S. Tm) LPS Cross-reactivity S. Tm 92% ELISA (S. Tm, Finland, 1989 51% Isomaki et al. [56] S. Ent 85% S. Ent)** Y. enterocolitica LPS group B Sweden / ELISA group C South-East 78% 94% Jertborn et al. [57] S. Newport Asia, 1990 LPS S. Tm, S. Ent 70% NR Cross- ELISA Denmark, 2005 Dalby et al. [58] S. Ent** S. Tm 77% reactivity LPS (S. Tm, S. Ent 95% S. Ent 97% ELISA Denmark, 2007 Strid et al. [59] S. Ent) S. Tm 89% S. Tm 94% Indirect mixed LPS S. Tm 86% S. Tm 95% ELISA Denmark, 2013 Falkenhorst et al. [60] (S. Tm, S. Ent) S. Ent 92% S. Ent 95% (IgM, IgA, IgG) Legend: NR = not reported * year when study performed or, if not stated, year when data were published ** commercially available S. Ent = Salmonella ser. Enteritidis, S. Tm = Salmonella ser. Typhimurium p roved its superiority to Widal test for the diagnosis reliable compared to Widal test (field studies, per - of typhoid fever, it involves the use of expensive in - formed mainly in the endemic area of Bangladesh struments and hazardous radioactive reagents, thus showed figures of more than 90% for sensitivity, it is not suitable for routine use [69]. specificity, positive and negative predictive values) tubex ® (IDL, Biotech) is a test that detects [43]. ICT do not require any specialized laboratory anti- Salmonella O9 IgM antibodies. It uses magnetic or highly skilled personnel. Commercial kits are al - particles coated with Salmonella ser. Typhi LPS. Re - ready available (Typhifast®, AB Diagnopath Man - sults are rapidly read, being semi-quantitatively ex - ufacturing Ltd.). pressed according to a score. Research in the field Lateral flow rapid tests are designed for the has shown some inconveniences of this test, that is detection and differentiation of anti- Salmonella it gives interferences between group D salmonellae, Typhi and Paratyphi IgG and IgM in human serum, mainly between serovars Typhi and Enteritidis [70]. plasma or whole blood. These tests have high levels What seemed at first to be a disadvantage, turned out of reproducibility and results are obtained within 15 to become an advantage, as the test might be used in minutes. They are recommended as screening tests. non-endemic regions for typhoid fever for the sero - Reactive specimen must be however confirmed with logical diagnostic of food-borne group D Salmonella alternative methods. The fact that the test kits can be infections, even of reactive arthritis [53, 71, 72]. For used and stored in a wide range of temperatures this use, most probably, false-positive reactions due make them suitable in remote tropical endemic re - to previous Salmonella ser. Typhi infections are rare. gions that lack facilities. Field testing yielded satis - The results obtained with Tubex® are, overall, en - factory results in the endemic typhoid region of couraging. South Sulawesi, Indonesia, [44], but have proved Immunochromatographic assays (Ict) less specific in Egypt [45]. which can detect IgM and IgG, separately or com - More recent tests include a dot enzyme im - bined, with IgM detection as early as 4 days after munoassay (typhidot ®, Malaysian Biodiagnostic fever onset, appear to be easier to perform and more Research ) detecting either both IgM and IgG or

111 ALEXANDRA-MARIA NÃŞCUÞIU solely IgM antibodies directed against a 50kDa and rather cheap for use in human medicine. Further - outer-membrane protein (OMP). It is an easy to per - more, result interpretation is superior for ELISA, as form test, but nevertheless it requires additional differentiation between positive and negative reac - steps, laboratory equipment and preparation of con - tion is performed using preset cut-off values and do sumables. Yet, it is more specific when compared not require subjective evaluations for colour inten - with other available tests. Performed very early in sities of spots or bands produced (as is the case with the disease evolution, Typhidot M® (Malaysian Bio - Typhidot® and Tubex®) [41]. Yet, the knowledge diagnostic Research) showed a sensitivity of 90% about the current availability and application of such and a specificity of 100% in blood culture proved tests is scarce and most of them lack standardization. cases from Indian settings [48]. In another study per - There were attempts to use either the outer- formed in India as well, some blood-culture con - membrane LPS or the flagellar antigens. In the vet - firmed cases yielded negative results for Typhidot erinary field, in the search for quick and easy M®, which might in part be explained by the de - serological methods for screening poultry or for de - creasing levels of IgM against the OMP of the bac - veloping herd profiles of exposure to Salmonella fol - terial cell wall and masking of IgM by IgG in the lowing outbreaks of diarrhea or abortion, ELISA second week of the disease [6]. Being a simpler test immunoassays have initially focused on Salmonella than blood culture or even Widal, Typhidot M® is a LPS as the detecting antigen, as it induces strong hu - reliable, more suitable test for use in settings lacking moral response directed mostly to the O antigen facilities [48]. Besides, the high negative predictive component. Good results were obtained with this value of the test suggested its usefulness in highly type of method for instance in the attempt to eradi - endemic areas. cate Salmonella ser. Dublin in diaries [73]. As for dipstick tests are simplified versions of ELISA poultry screening many cross reactions were noted, with very good specificity that can detect IgM anti - especially with other Gram-negative organisms, bodies as soon as 5-7 days after fever onset. Their studies have moved towards the use of flagellin as sensitivity increases when testing paired samples, antigen. With this respect, opinions and study results which makes them useful in culture-negative pa - are divergent. Dalby et al . noted a lower sensitivity tients showing clinical signs consistent with enteric and specificity of these tests and a higher cross-re - fever. Results are rapidly obtained, stability of the activity [58]. On the contrary, Minicozzi et al . ob - reagents has been proved, and there is no need for tained good results with purified Salmonella special laboratory equipment, so dipsticks are ideal flagellin which is easy to clone and express [4]. for use in places that lack laboratory facilities [49]. Whatever the results obtained in veterinary sectors, An alternative that enables detection of all classes of these tests cannot be directly applied to human sam - serum antibodies, not solely agglutinating antibodies ples. For the detection of Salmonella ser. Typhi an - is immunofluorescence, which is neither rapid nor tibodies by means of ELISA there has been a lot of easy to perform, but is more specific [54]. development performed, especially in South-East ELISA s are nowadays the optimal candidates Asia. Attempts to standardize such antibody assays for serological investigation of Salmonella infec - for the diagnosis of typhoid fever were done. These tions, the veterinary and food sectors across Europe kits yet cannot be used for detecting zoonotic Sal - and in the USA using them as routine tools. Though monella species. Very few papers have been pub - diagnostic tests such as Widal, TUBEX-TF® and lished on the subject. Most immunoassays for Typhidot® are more suitable and convenient than non-typhoid human Salmonella diagnosis are devel - ELISA for bedside testing and in small-scale, inad - oped and used in-house for research or for regional equately equipped or field laboratories, ELISA is su - surveillance purposes. Serology for the diagnosis of perior in routine surveillance studies or outbreak Salmonella -associated complications is used only by investigations [41]. Another application is etiolog- a few laboratories. Time and studies decided that for ical diagnosis in patients with suspected post-infec - detection of Salmonella antibodies in human sera O tious complications such as reactive arthritis, even antigen is the most suitable candidate for standard - when faecal shedding of salmonella has ceased. We ized, commercially available tests. have assisted to over 35 years of ELISAs in the field Different types of ELISAs for antibody detec - of Salmonella , with different performances. A lot of tion in humans have been already tested: sandwich research on the field of ELISAs was done in both ELISAs (Enterocheck WB® for serovar Typhi IgM veterinary and human medicine, ELISAs proving to detection, Zephyr Biomedicals) [51], indirect IgG have a promising potential, being reliable, accurate and IgM and capture IgM ELISAs [74].

112 role of Salmonella serology a century after the Widal era

ELISAs that estimate the level of Salmonella phimurium and Enteritidis LPS would cover 70–90 ser. Typhi antibodies use either the somatic or the % of all reported Salmonella isolates in Europe, for flagellar antigen and can detect IgM, IgG or IgA, de - the USA the kit should also contain LPS from pending on the conjugate used. Differences of sen - serovar Newport (group C) which is responsible for sitivity were obtained, due to the detection limit of almost 10 % of reported salmonelloses in the region. each assay. An Egyptian study pointed out that Other group B salmonellae which are frequently iso - ELISA may be sensitive to antibody concentrations lated are expected to be captured by the Ty - as low as 0.05ng, while agglutination tests may re - phimurium antigens [78]. quire concentrations greater than 500 ng [75]. An - The levels of IgG, IgM and IgA antibodies were other Egyptian study showed that among the assessed, with IgG persisting for several months indi vidual isotype ELISAs, the most sensitive after infection for a considerable proportion of pa - proved to be IgM and the least sensitive IgA. The tients, and IgM and IgA antibodies declining 3 to 4 total Ig ELISA showed strong correlation with cul - months after the onset [59], making thus of IgM and ture isolation. Besides ELISAs are fast and cost-ef - IgA better sensitive diagnostic parameters for recent fective, with excellent throughput, which infection and of IgG a tool allowing a retrospective recommend them for epidemiological survey studies diagnosis. or outbreak events [41]. A literature review identified only one commer - Preliminary attempt of building up ELISA as - cially available antibody assay for detection of non- says revealed that the Vi antigen showed poor bind - typhoid Salmonella in human sera (the ing to the microtiter plates, except when IMTEC-Salmonella-Antibodies Screen tyra mi nated. House et al. used an indirect ELISA (IgG/IgA/IgM)/Antibodies IgA®, Human-IMTEC) with a tyraminated, purified Vi-CPS to assay anti- based on pooled antigens from serovars Enteritidis Vi-CPS antibodies from typhoid fever cases and con - and Typhimurium and using high IgA titers as a trols living in urban and rural regions of Viet Nam. marker for the diagnosis of reactive arthritis. Even Findings showed significantly higher antibody re - if the sensitivity of this commercially available test sponses to Vi-CPS in typhoid patients who have was reported by the manufacturers as encouraging, been ill for more than two weeks than in those in the no further evaluating studies were performed [78]. first two weeks of illness. Unfortunately, these anti - When testing for the presence of non-typhoid bodies were also detected in the sera of non-typhoid antibodies, one must have in mind the differences in patients, with a frequency increasing with age, prob - the quality of the immune response (amplitude and ably reflecting increased exposure to Salmonella ser. duration of elevated antibody concentrations) be - Typhi [76]. Hence the limits of the test as a general tween serovars. Salmonella ser. Enteritidis was population screening test for carriage. Carrier detec - shown to induce a stronger IgA and IgG (but not tion is not only important for public health issues, IgM) response than serovar Typhimurium. This but, in the light of recent developments which have might be explained in part by the means of transmis - shown an increased risk of gallbladder carcinoma sion of the disease. For serovar Typhimurium trans - among typhoid carriers, it should be of use in pro - mission is often foodborne (consumption of raw or moting an effective management of such complica - undercooked pork, beef or poultry meat), but contact tions [77]. with animals and/or animal faeces in the environ - Serological diagnosis of non-typhoid salmonel - ment also plays a role in transmission and may lead losis was developed targeting mostly the antibodies to frequent low-dose exposures [79, 80]. Infection directed against the LPS of a single serovar, either with serovar Enteritidis is predominantly foodborne, Enteritidis or Typhimurium. More recently, mixed raw eggs being the most important transmission ve - LPS ELISAs have been developed (incorporating hicle. Meals prepared with raw eggs allow for mul - equal amounts of serovars Enteritidis and Ty - tiplication of bacteria, the average size of the phimurium LPS as antigen). There has been a lot of inoculum being larger for serovar Enteritidis. Some cross-reactivity, especially between Gram-negative researchers yet believe that serovar Enteritidis might bacteria (diarrheagenic E. coli , Y. enterocolytica , be more immunogenic per se [60]. Campylobacter spp., Helicobacter pylori ). The ELISAs determining antibodies against H anti - choice of LPS must reflect the circulation of serovars gens were not considered useful for diagnosis of in the human population, thus commercial ELISA non-typhoid salmonellosis, as their level decreases kits will have to depend on geographical zone of in - very rapidly [58]. tended use. If an ELISA based on serovars Ty - The most recent published study (2013) is that

113 ALEXANDRA-MARIA NÃŞCUÞIU on an indirect mixed ELISA, which determines three [81]. In a study dated 2011, Parry et al . stated that classes of antibodies directed against the two pre - “The antigenic cross-reactivity of Gram-negative mi - dominant serovars involved in gastroenteritis [60]. croorganisms means that developing new methods The mixed ELISA is a useful additional tool for the is a considerable challenge and must be conceived diagnosis of acute salmonella gastroenteritis, as it al - in physiological reality ” [7]. This reality is best re - lows rapid analysis of multiple samples. Anti-LPS vealed by Henk L Smits in a key paper evaluation in IgA detection is particularly important when trying March 2013: “ Validation of an index test requires to establish the etiological diagnostic of a reactive good understanding of the diagnostic performance arthritis, a form of arthritis with chronic progression and assay characteristics of the reference test, and most commonly linked to group B or D salmonellae. criteria and principles for study design and report - Screening tests are already available, high classes of ing outlined by the Quality Assessment of Diagnostic IgA titers being considered highly sensitive markers Accuracy Studies and the Standards for Reporting for the diagnosis of reactive arthritis. Studies have Studies of Diagnostic Accuracy should be followed ” been performed, demonstrating the correlation be - [82]. For gaining credibility and become widely tween the presence of these IgA antibodies and the used, serological tests for typhoid fever will require severity and duration of the disease [72]. Commer - thorough evaluation in endemic regions with a rig - cial ELISA kits are already available for this pur - orous selection of patients and careful attention to a pose, using lipopolysaccharides specific for gold standard reference [7]. Typhimurium and Enteritidis serovars. What is still to be done when considering this Literature search has proved that so far there is type of serological tests for diagnostic is acquiring no international consensus for an established ELISA an extensive and in-depth knowledge about antibody method for the serological diagnosis of non-ty - kinetics profiles after the onset of infection, identi - phoidal Salmonella infections, and we still lack fying new antibodies targeting for other than the guidelines for the choice of antigens, the cut-off val - classical Salmonella antigens and trying to reduce ues for antibody titers and the overall definition of cross-reactivity. Novel antigen candidates - which seropositivity. Yet, results obtained so far express the have to be conserved and highly immunogenic - likelihood that an LPS-based ELISA which detects might be identified by bioinformatics and used as IgG, IgM and IgA responses could prove specific, basis for the development of new tests [31]. sensitive, fast, easy, and reliable for routine analyses With this “physiological reality” in mind, one of human sera [78]. Besides, very recently, Kuhn and thing is obvious: there is still much to be done in the his team have suggested a protocol for a standard - field of Salmonella serology, for both patient care ized ELISA for non-typhoid salmonellae antibody and surveillance, and the road is open ahead. detection in Europe, based on their experience at the Statens Serum Institut, Denmark. The proposal has REFERENCES been already accepted by the European Centre for Disease Control and Prevention (ECDC) [78]. 1. crump JA, Luby SP, Mintz Ed . The global burden of typhoid fever. Bull World Health Organ 2004; 82: In spite of the huge development of serological 346. methods and of the constant attempts to improve di - 2. Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk agnosis we have to admit that the field of Salmonella M, o’Brien SJ Jones tF, Fazil A, Hoekstra rM, In - serology can be concisely characterized by the term ternational collaboration on enteric disease “Bur - “stagnation”. This is best reflected by two assertions, den of Illness” studies. The global burden of separated by 60 years: in 1950, Felix, in the Bulletin nontyphoidal Salmonella gastroenteritis. Clin Infect of the World Health Organization, noted that “ Some Dis Off Publ Infect Dis Soc Am, 2010, 50: 882–889. years before the Second World War, the Health Or - 3. năşcuţiu A-M . [Phenotypic and molecular markers for ganization of the League of the Nations started an the diagnostic and epidemiology of food-borne infec - international inquiry aiming at the standardization tions determined by Enterobacteria belonging to genus Salmonella ]. PhD thesis, “Carol Davila” University of of the serum diagnosis of enteric fevers. A consider - Medicine and Pharmacy, 2011. able amount of work was done. […] serological tests 4. Minicozzi J, Sanchez S, Lee Md, Holt PS, Hofacre are indispensable for the successful conduct of epi - cL, Maurer JJ. Development of Recombinant Flagel - demiological field work […]. The need is empha - lar Antigens for Serological Detection of Salmonella sized for taking action without delay to standardize enterica Serotypes Enteritidis, Hadar, Heidelberg, and the diagnostic agglutination tests for enteric fevers ” Typhimurium in Poultry. Agriculture 2013, 3, 381-397.

114 role of Salmonella serology a century after the Widal era

5. Parry cM, Hien MB, dougan Md, White nJ, Far - 22. Hoffman SL, Flanigan tP, Klancke d . The Widal rar Mt . Typhoid fever. N Engl J Med , 2002, 347: slide agglutination test, a valuable rapid diagnostic 1770-1782. test in typhoid fever patients at the infectious disease 6. narayanappa d, Sripathi r, Jagdishkumar K, ra - hospital in Jakarta. Am J Epidemiol, 1986, 123: 869- jani HS. Comparative study of dot enzyme immunoas - 875. say (Typhidot-M) and Widal test in the diagnosis of 23. Shanti J, usha rani r, Balagurunathan r . A brief typhoid fever. Indian Pediatr, 2010, 47: 331-333. study of diagnosis and frequency of typhoid fever in - 7. Parry cM, Wijedoru L, Arjyal A, Baker S . The util - cidence by Widal test. Annals Biol Res , 2010, 3(4): ity of diagnostic tests for enteric fever in endemic lo - 1847-1851. cations. Expert Rev Anti Infect Ther , 2011, 9(6): 24. Huckstep rL . Typhoid fever and other Salmonella 711-725. infections. Livingstone, London, 1962, pp. 87. 8. Sansone P, Saslaw MS, Hennekens cH . High titer 25 . Mussa A . Reassessment of Widal test in the Diagnosis Widal reaction. JAMA , 1992, 220: 1615-1616. of Typhoid Fever. Diyala J Med , 2011, 1(2):13-25. 9. Aftab r, Khurshid r . Widal agglutination titre: a 26. Sommerville Pc, Lewis M, Kornhof HJ, Alberts rapid serological diagnosis of typhoid fever in devel - M, Alberts HW, raymond r . The Widal test in the oping countries. Pak J Physiol , 2009, 5(1): 65-67. diagnostic of typhoid fever in the Transvaal. S Afr 10. House d, chinh nt, diep S Parry cM, Wain J, Med J , 1981, 58(Suppl 24): 851-854. dougan G, White nJ, Hien tt, Farrar JJ. Use of 27. coovadia YM, Singh V, Bhana rH, Moodley n. paired samples for serodiagnosis of typhoid fever. J Comparison of passive haemagglutination test with Clin Microbiol , 2005, 43: 4889-4990. Widal agglutination test for serological diagnosis of 11. Schroeder SA . Interpretation of serological test for typhoid fever in an endemic area. J Clin Pathol , 1986, typhoid fever. JAMA , 1968, 206: 839-840. 39: 680-683. 12 . Pang t, Puthucheary S. Significance of Widal test 28. Saha SK, ruhulamin M, Hanif M Islam M, Khan in the diagnosis of typhoid fever in endemic areas. WA. Interpretation of the Widal test in the diagnosis J Clin Pathol , 1983, 36: 471-475. of typhoid fever in Bangladeshi children. Ann Trop 13 . choo KE, razif Ar, oppenheimer SJ, Ariffin WA, Paediatr 1996, 16(1): 75-78. Lau J, Abrahan t. Usefulness of Widal test in diag - 29. Senewiratne B, Senewiratne K . Reassesment of the nosing childhood typhoid fever in endemic areas. J Widal test in the diagnosic of typhoid. Gastroenterol - Paediatr Child Health 1993, 29: 36-9. ogy , 1977, 73(2): 233-236. 14. Wicks AB, cruickshank JG, Museeve n . Observa - 30. *** WHo . The diagnosis, treatment and prevention tions on the diagnosis of typhoid fever in an endemic of typhoid fever. Communicable Disease Surveillance area. S Afr Med J , 1974, 48: 1368-70. and Response Vaccine and Biologicals , 2003, pp. 7- 15. Saleh MMS, Mahmud SA . Frequency of Salmonella 18. antibodies among febrile patients with suspected en - 31. Baker S, Favorov M, dougan G . Searching for the teric fever. Al Taqani , 2013, 26(3):46-55. elusive typhoid diagnostic. BMC Infectious Diseases 16. reynolds dW, carpenter rL, Simon WH . Diagnos - 2010, 10: 45. tic specificity of Widal’s reaction for typhoid fever. 32 . Lanata cF, ristori c, Jimenez L, Garcia J, Levine JAMA , 1970, 214: 2192-3. M, Black rE, Salcedo M, Sotomayor V . Vi serol - 17. olopenia L, oyewole F, onafowokan r et al . Widal ogy in detection of chronic Salmonella typhi carriers agglutination in malaria infection in Nigeria. Clin Im - in an endemic area. Lancet 1983, 322 (8347): 441- munol Immunopathol , 1995, 75(3): 314. 443. 18. Aquino rL, Lansong MAd, Quimpo VS, Sam - 33 . Shukla S, chitnis dS . Haemagglutination system for brero Lt, Saniel Mc . Evaluation of a single Widal the simultaneous detection of LPS and anti LPS anti - test in the diagnosis of enteric fever. SE Asian J Trop bodies of S. typhi . India J Med Scien , 1997, 51: 265- Med Public Health , 1991, 22: 375-9. 9. 19. Levine MM, Grados o, Gilman rH, Woodward 34. House d, Wain J, Ho VA, diep tS, chinh nt, Bay WE, Solis-Plaza r, Waldman S. Diagnostic value BV, Vinh H, duc M, Parry cM, dougan G, White of Widal test in areas endemic for typhoid fever. Am nJ, Hien tt, Farrar JJ . Serology of Typhoid Fever J Trop Med Hyg 1978, 27: 795-800. in an Area of Endemicity and Its Relevance to Diag - 20. olopoenia LA, King AL . Widal agglutination test - nosis. J Clin Microbiol , 2001, 39(3): 1002-1007. 100 years later: still plagued by controversy. Postgrad 35 . Gopalakrishnan V, Sekhar WY, Soo EH, Vinsent Med J , 2000, 76: 80-84. rA, devi S. Typhoid fever in Kuala Lumpur and a 21 . Bakr WMK, El Attar LA, Ashrour MS, El toukhy comparative evaluation of two commercial diagnostic AM . The dilemma of Widal test – which brand to kits for the detection of antibodies to Salmonella use? A study of four different Widal brands: a cross Typhi . Singapore Med J , 2002, 43: 354–358. sectional comparative study. Ann Clin Microbiol An - 36. olsen SJ, Pruckler J, Bibb W, nguyen tM, tran timicrob , 2011, 10: 7. Mt, nguyen tM, Sivapalasingam S, Gupta A,

115 ALEXANDRA-MARIA NÃŞCUÞIU

Phan tP, nguyen tc, nguyen Vc, Phung dc, in a resource poor setting. Braz J Infect Dis , 2010, Mintz Ed . Evaluation of rapid diagnostic tests for 14(6): 589-593. typhoid fever. J Clin Microbiol, 2004, 42: 1885-1889. 49. Hatta M, Goris MGA, Heerkens E, Gooskens J, 37. rahman M, Siddique AK, tam Fc, Sharmin S, Smits HL . Simple dipstick assay for the detection of rashid H, Iqbal A, Ahmed S, nair GB, chaignat Salmonella Typhi -Specific IgM antibodies and the cL, Lim PL . Rapid detection of early typhoid fever evolution of the immune response in patients with ty - in endemic community children by the TUBEX-TF phoid fever. Am J Trop Med Hyg , 2002, 66(4): 416– O9-antibody test. Diagn Microbiol Infect Dis , 2007, 421. 58: 275-281. 50 . Quiroga t, Goycoolea M, tagle r, Gonzalez F, ro - 38 . Kawano rL, Leano SA, Agdamag dM . Comparison driguez L, Villarroel L . Diagnosis of typhoid fever of serological test kits for diagnosis of typhoid fever by two serologic methods. Enzyme-linked im - in the Philippines. J Clin Microbiol , 2007, 45: 246- munosorbent assay of antilipopolysaccharide of Sal - 247. monella typhi antibodies and Widal test. Diagn 39 . Heddy KH, Sooka A, Letsoalo ME, Hoyland G, Microbiol Infect Dis, 1992, 15(8): 651-6. chaignat cL, Morrissey AB, crump JA. Sensitiv - 51 . Anagha K, deepika B, Shahriar r, Sanjeev K. The ity and specificity of typhoid fever rapid antibody Easy and Early Diagnosis of Typhoid Fever. J Clin tests for laboratory diagnosis at two sub-Saharan Diagn Res , 2012, 6(2): 196-199. African sites. Bull WHO , 2011, 89: 640-647. 52. Kuhn KG, Falkenhorst G, ceper tH, dalby t, 40 . naheed A, ram PK, Brooks WA, Mintz Ed, Hos - Ethelberg S, Mølbak K, Krogfelt KA. Detecting sain MA, Parsons MM, Luby SP, Breiman rF . non-typhoid Salmonella in humans by ELISAs: a lit - Clinical value of TUBEX-TF and Typhidot rapid di - erature review. J Med Microbiol , 2012, 61: 1-7. agnostic tests for typhoid fever in an urban commu - 53 . oracz G, Feleszko W, Golicka d, Maksymiuk J, nity clinic in Bangladesh. Diagn Microbiol Infect Dis, Klonowska A, Szajewska H . Rapid diagnosis of 2008, 61: 381-386. acute Salmonella gastrointestinal infection. Clin In - 41. Fadeel MA, House BL, Wasfy MM, et al . Evaluation fect Dis , 2003, 36: 112-115. of a newly developed ELISA against Widal, 54. Svenungsson B, Jörbeck H, Lindberg AA . Diagno - TUBEX-TF and Typhidot for typhoid fever surveil - sis of Salmonella infections: specificity of indirect lance. J Infect Dev Ctries, 2011, 5(3): 169-175. immunofluorescence for rapid identification of Sal - 42. Siba V, Horwood PF, Vanuga K, Wapling J, Sehuko monella enteritidis and usefulness of enzyme-linked r, Siba PM, Greenhill Ar . Evaluation of Serologi - immunodorbent assay. J Infect Dis, 1979, 140: 927- cal Diagnostic Tests for Typhoid Fever in Papua New 936. Guinea Using a Composite Reference Standard. Clin 55. Hirschl A, Stanek G, rotter M, niemetz AH, diridl Vaccine Immunol, 2012, 19(11): 1833–1837. G. [Comparison of the ELISA (lipopolysaccharide) 43 . Hasan B, nahar SG, Shamsuzzaman AKM, Aftab and Widal reactions (O antigen) in the diagnosis of S, Yusuf A . Detection of anti- Salmonella antibodies Salmonella infections]. Zentralbl Bakteriol Mikrobiol by immunochromatographic assay at Rajshahi Med - Hyg [A] , 1983, 255: 247-257. ical College, Bangladesh. J Microbiol Antimicrob , 56. Isomäki o, Vuento r, Granfors K . Serological di - 2013, 5(11): 119-123. agnosis of Salmonella infections by enzyme im - 44 . Abdoel tH, Pastoor r, Smits HL, Hatta M. Labo - munoassay. The Lancet , 1989, 333(8652): 1411-1414. ratory evaluation of a simple and rapid latex aggluti - 57. Jertborn M, Ahrén c, Perlman dM, Kaijser B, nation assay for the serodiagnosis of typhoid fever. Svennerholm AM . Serum antibody responses to bac - Trans Roy Soc Trop Med Hyg , 2007, 101(10): 1032– terial enteropathogens in Swedish travelers to south- 1038. east Asia. Scand J Infect Dis , 1990, 22: 699-704. 45 . nakhla I, El Mohammady H, Mansour A, Klena 58 . dalby t, Strid MA, Beyer nH, Blom J, Mølbak K, Jd, Hassan K, Sultan Y, Pastoor r, Abdoel tH, Krogfelt KA. Rapid decay of Salmonella flagella an - Smits H. Validation of the Dri-Dot Latex agglutina - tibodies during human gastroenteritis: a follow up tion and IgM lateral flow assays for the diagnosis of study. J Microbiol Methods , 2005, 62: 233–243. typhoid fever in an Egyptian population. Diag Micro - 59 . Strid MA, dalby t, Mølbak K, Krogfelt KA . Ki - biol Infect Dis, 2011, 70(4): 435–441. netics of the Human Antibody Response against Sal - 46 . Bhutta ZA, dewraj HL. Current concepts in the di - monella enterica Serovars Enteritidis and agnosis and treatment of typhoid fever. BMJ , 2006, Typhimurium Determined by Lipopoplysaccharide 333: 78-82. Enzyme-Linked Immunosorbent Assay. Clin Vaccine 47 . Jesudason MV, Sivakumar S. Prospective evaluation Immunol , 2007, 14(6): 741-747. of a rapid diagnostic test Typhidot for typhoid fever. 60 . Falkenhorst G, ceper tH, Strid MA, Mølbak K, Indian J Med Res , 2006, 123: 513-516. Krogfelt KA. Serological follow-up after non-ty - 48 . Beig FA, Ahmad F, Ekram M, Shukla I . Typhidot phoid salmonella infection in humans using a mixed M and Diazo test vis-à-vis blood culture and Widal lipopolysaccharide ELISA. Int J Med Microbiol, test in the early diagnosis of typhoid fever in children 2013, 303(8): 533-8.

116 role of Salmonella serology a century after the Widal era

61 . Ismail A. New Advances in the Diagnosis of Typhoid Indirect immunoglobulin G (IgG) and IgM enzyme- and Detection of Typhoid Carriers Malays J Med Sci, linked immunosorbent assays (ELISAs) and IgM cap - 2000, 7(2): 3–8. ture ELISA for detection of antibodies to 62 . nolan cM, Feeley Jc, White Pc Jr, Hambie EA et lipopolysaccharide in adult typhoid fever patients in al. Evaluation of a new assay for Vi antibody in Pakistan. J Clin Microbiol, 1989, 27(6): 1298-302. chronic carriers of Salmonella typhi. J Clin Micro - 75. el Fattah MM, Hassan EM, Shaheen H, Awad rA, biol. 1980, 12(1): 22-6. Girgis n . Evaluation of enzyme-linked immunosor - 63. Gupta AK, rao KM . Simultaneous detection of Sal - bent assay for detection of Salmonella Typhi antigen monella Typhi antigen and antibody in serum by and antibodies. J Egypt Public Health Assoc , 1991, counter-immunoelectrophoresis for an early and rapid 66: 145-157. diagnosis. J Immunol Methods , 1979, 30: 349-353. 76. House d, Ho VA, diep tS, chinh nt et al. Anti - 64. tsang rSW, chau PY . Serological diagnostic of ty - bodies to the Vi capsule of Salmonella Typhi in the phoid fever by counterimmuno-electrophoresis. Br serum of typhoid patients and healthy control subjects Med J 1981, 282: 1505-1507. from a typhoid endemic region. J Infect Dev Ctries , 65. Sundararaj t, Ilango B, Subramanian S. A study 2008, 2(4): 308-12. on the usefulness of counter immuno-electrophoresis 77. dutta u, Garg PK, Kumar r, tandon rK . Typhoid for the detection of Salmonella typhi antigen in the carriers among patients with gallstones are at in - sera of suspected cases of enteric fever. Trans R Soc creased risk for carcinoma of the gallbladder. Am J Trop Med Hyg , 1983, 77(2): 194-7. Gastroenterol , 2000, 95:784-787. 66. Mishra M, thakar YS, chande c, tankhiwale nS, 78. Kuhn KG, Emborg H-d, Krogfelt KA, Mølbak K . Saoji AM . Rapid detection of enteric fever by coag - Detecting Non-typhoid Salmonella in Humans by En - glutination and countercurrent immunoelectrophore - zyme- Linked Immunosorbent Assays (ELISAs): sis. Indian J Pathol Microbiol, 1998, 41(4): 391-6. Practical and Epidemiological Aspects. Meth Mol 67. Sharma M, datta u, roy P, Verma S, Sehgal S . Biol , 2013, 1225: 117-126. Low sensitivity of counter-current immuno-elec - 79. doorduyn Y, van den Brandhof WE, Van duyn - trophoresis for serodiagnosis of typhoid fever. J Med hoven Yt, Wannet WJ, Van Pelt W. Risk factors Microbiol , 1997, 46: 1039-42. for Salmonella Enteritidis and Typhimurium (DT104 68. chan PY, tsang rS, Lam SK, La Brooy Jt, row - and non-DT104) infections in The Netherlands: pre - ley d. Antibody response to the lipopolysaccharide dominant roles for raw eggs in Enteritidis and sand - and protein antigens of Salmonella Typhi during ty - boxes in Typhimurium infections. Epidemiol. Infect , phoid infection. II. Measurement of intestinal anti - 2006, 134: 617-626. bodies by radioimmunoassay. Clin Exp Immunol , 80. Wegener Hc, Hald t, Lo Fo Wong d, Madsen M, 1981, 46(3): 515-520. Korsgaard H, Bager F, Gerner-Smidt P, Mølbak 69. tsang rS, chan PY, Lam SK, La Brooy Jt, row - K. Salmonella control programs in Denmark. Emerg ley d . Antibody response to the lipopolysaccharide Infect Dis , 2003, 9: 774-780. and protein antigens of Salmonella Typhi during ty - 81 . Felix A . Standardization of diagnostic agglutination phoid infection. I. Measurement of serum antibodies tests. Typhoid and Paratyphoid A and B fevers. Bull by radioimmunoassay. Clin Exp Immunol , 1981, WHO, 1950, 39(2):643-647. 46(3): 508-514. 82. Smits KL . Limitations of typhoid fever diagnostics 70. Lim P-L, tam FcH, cheong Y-M, Jegathesan M. and the need for prevention. Expert Rev Mol Diagn , One-Step 2-Minute Test To Detect Typhoid-Specific 2013, 13(2): 147-149. Antibodies Based on Particle Separation in Tubes. J Clin Microbiol , 1998, 36(8): 2271–2278. 71. Ham FcH, Ling tKW, Wong Kt, Leung dtM, chan rcY, Lim PL . The TUBEX test detects not only typhoid-specific antibodies but also soluble anti - gens and whole bacteria. J Med Microbiol , 2008, 57(3): 316-323. 72. thomson Gt, derubeis dA, Hodge MA et al. Post- Salmonella reactive arthritis: late clinical sequelae in a point source cohort. Am J Med, 1995, 98(1): 13-21. 73. Hoorfar J, Feld nc, Schirmer AL et al. Serodiag - nosis of Salmonella dublin infection in Danish diary herds using O-antigen based enzyme-linked im - munosorbent assay. Can J Vet Res , 1994, 58: 268- 274. 74. Sippel J, Bukhtiari n, Awan MB, Krieg r, duncan JF, Karamat KA, Malik IA, Igbal LM, Legters L.

117 ROMANIAN ARCHIVES OF MICROBIOlOgy AND IMMUNOlOgy

118 ROMANIAN ARCHIVES OF MICROBIOlOgy AND IMMUNOlOgy

SUBJECT INDEX

MIcroBIoLoGY

ESCHERICHIA COLI ST131 CAUSING INVASIVE INFECTIONS IN ROMANIAN PATIENTS – A THREAT WE CAN NO LONGER IGNORE Codruţa-Romaniţa Usein, Maria Condei, Dana Cristea, Simona Adriana Ciontea, Dorina Tatu-Chiţoiu, Violeta Cristea, Monica Străuţ ...... 5

MOLECULAR DETECTION OF NINE ANTIBIOTIC RESISTANCE GENES IN METHICILLIN RESISTANT STAPHYLOCOCCuS AuREuS ISOLATES Ghaleb Adwan, Kamel Adwan, Naser Jarrar, Alaa Amleh ...... 9

SEROPREVALENCE STUDY OF ANTI DIPHTHERIA ANTIBODIES IN TWO AGE-GROUPS OF ROMANIAN ADULTS Cristiana Cerasella Dragomirescu, Ileana-Luminiţa Coldea, Anamaria Ilie, Aurora Stănescu, Vasilica Ungureanu, Mircea Ioan Popa ...... 18

DIFFERENTIATION OF INFLUENZA B LINEAGES FROM CLINICAL SAMPLES BY ONE-STEP REAL-TIME PCR Carmen Maria Cherciu, Gheorghe Necula, Maria Elena Mihai, Alina Elena Ivanciuc, Mihaela Lazăr, Cristina Ţecu, Daniela Piţigoi, Emilia Lupulescu ...... 25

HEMATOLOGY REFERENCES FOR THREE LABORATORY MICE STRAINS Bogdan Marinescu, Gheorghiţa Isvoranu, Laurenţiu Anghelache, Florina Cionca ...... 30

ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF ALCOHOLIC EXTRACTS OBTAINED FROM VEGETATIVE ORGANS OF A. RETROFLExuS Ioana Cristina Marinaş, Carmen Chifiriuc, Eliza Oprea, Veronica Lazăr ...... 35

IS THERE A DIFFERENCE IN THE INCIDENCE OF HELICOBACTER PYLORI INFECTION IN PATIENTS WITH SOME CHRONIC DISEASES? Cristian Adrian Constantinescu, Elena Mihaela Constantinescu ...... 65

SERUM ALPHA FETOPROTEIN, A SURROGATE MARKER FOR LIVER DISEASE PROGRESSION IN CHRONIC HEPATITIS C Camelia Sultana, Claudia Diţă, Alina Botescu, Camelia Grancea and Simona Ruţă ...... 69

MOLECULAR CHARACTERIZATION OF STAPHYLOCOCCuS AuREuS STRAINS ISOLATED FROM INFECTIVE ENDOCARDITIS Mihaela Oprea, David Sebastian Patriche, Monica Străuţ, Felicia Antohe ...... 74

MICRORNA BIOGENESIS AND ITS ROLE IN HIV-1 INFECTION Adelina Roşca, Gabriela Anton, Simona Ruţă ...... 84

119 ROMANIAN ARCHIVES OF MICROBIOlOgy AND IMMUNOlOgy

IMMunoLoGY

KIR GENOTYPING IN THE SELECTED POPULATION IN ANDHRA PRADESH (INDIA) G. Vaishnav, S. Krupanidhi, C.B. Sanjeevi ...... 43

IMMUNOLOGICAL INVESTIGATIONS IN PROSTATIC PATHOLOGY – A PROSPECTIVE STUDY Dan Spînu, Dan Mischianu, Mihaela Surcel, Radu Huică, Adriana Munteanu, Ioana Pîrvu, Dan Ciotaru, Ovidiu Bratu, Cătălin Farcaş, Ştefan Manache, Cornel Ursaciuc ...... 51

MECHANISMS OF THROMOBOSIS IN SYSTEMIC LUPUS ERYTHEMATOSUS Adriana Metoni, Cristina Mambet, Laura G. Necula, Ioana M. Aldea, Ana I. Neagu, Lilia Matei, Denisa Dragu, Coralia Bleotu, Daniela A. Ion, Carmen C. Diaconu ...... 92

SHort coMMunIcAtIon

STATUS OF IMMUNITY AGAINST POLIOMYELITIS IN THE ACUTE FLACCID PARALYSIS (AFP) CASES IN ROMANIA BETWEEN 2009-2012 Anda Băicuş ...... 56

rEVIEW

ROMANIAN SCIENTISTS IN THE NOMINATION DATABASE FOR THE NOBEL PRIZE IN PHYSIOLOGY OR MEDICINE, 1901-1951 Costin Cernescu ...... 99

ROLE OF SALMONELLA SEROLOGY A CENTURY AFTER THE WIDAL ERA Alexandra-Maria Năşcuţiu ...... 105

120 ROMANIAN ARCHIVES OF MICROBIOlOgy AND IMMUNOlOgy

AUTHOR INDEX

A H Huică Radu 51 Adwan Ghaleb 9 Adwan Kamel 9 Aldea Ioana M. 92 I Amleh Alaa 9 Anghelache Laurenţiu 30 Ilie Anamaria 18 Antohe Felicia 74 Ion Daniela A. 92 Anton Gabriela 84 Isvoranu Gheorghiţa 30 Ivanciuc Alina Elena 25

B J Băicuş Anda 56 Bleotu Coralia 92 Jarrar Naser 9 Botescu Alina 69 Bratu Ovidiu 51 K

c Krupanidhi S. 43

Cernescu Costin 99 Cherciu Carmen Maria 25 L Chifiriuc Carmen 35 Cionca Florina 30 Lazăr Mihaela 25 Ciontea Simona Adriana 5 Lazăr Veronica 35 Ciotaru Dan 51 Lupulescu Emilia 25 Coldea Ileana-Luminiţa 18 Condei Maria 5 Constantinescu Cristian Adrian 65 M Constantinescu Elena Mihaela 65 Cristea Dana 5 Mambet Cristina 92 Cristea Violeta 5 Manache Ştefan 51 Marinaş Ioana Cristina 35 Marinescu Bogdan 30 d Matei Lilia 92 Metoni Adriana 92 Diaconu Carmen C. 92 Mihai Maria Elena 25 Diţă Claudia 69 Mischianu Dan 51 Dragomirescu Cristiana Cerasella 18 Munteanu Adriana 51 Dragu Denisa 92

n F Năşcuţiu Alexandra-Maria 105 Farcaş Cătălin 51 Neagu Ana I. 92 Necula Gheorghe 25 Necula Laura G. 92 G

Grancea Camelia 69

121 ROMANIAN ARCHIVES OF MICROBIOlOgy AND IMMUNOlOgy

o t

Oprea Eliza 35 Tatu-Chiţoiu Dorina 5 Oprea Mihaela 74

Ţ P Ţecu Cristina 25 Patriche David Sebastian 74 Piţigoi Daniela 25 Pîrvu Ioana 51 u Popa Mircea Ioan 18 Ungureanu Vasilica 18 Ursaciuc Cornel 51 r Usein Codruţa-Romaniţa 5

Roşca Adelina 84 Ruţă Simona 69, 84 V

Vaishnav G. 43 S

Sanjeevi C.B. 43 Spînu Dan 51 Stănescu Aurora 18 Străuţ Monica 5, 74 Sultana Camelia 69 Surcel Mihaela 51

122