Vilnius International Summit on Communicable Diseases 26 June - 1 July 2016, Vilnius Abstract Book Photo by Saulius Gudeliauskas

W W W . V I L N I U S - S U M M I T . E U

Užkrečiamųjų ligų ir AIDS centras Centre for Communicable Diseases and AIDS Editor-in-chief: Saulius Caplinskas M.D., Ph.D Mykolas Romeris University, Centre for Communicable Diseases and AIDS Editor-in-chief: Saulius Caplinskas M.D., Ph.D Mykolas Romeris University, Centre for Communicable Diseases and AIDS ISBN 978-9986-720-52-2 ISBN 978-9986-720-52-2 The Minister of Health of the Republic of Lithuania Lietuvos Respublikos Juras Požela sveikatos apsaugos ministras Juras Požela Dear Colleagues, We invite you to the unique event – the Vilnius Communicable Diseases Week. The programme Gerbiami kolegos, consists of Annual Conference of the Baltic Network Against Life-threatening Viral Infections, Kviečiame Jus dalyvauti unikaliame renginyje - Vilniaus užkrečiamųjų ligų savaitėje. Lecture Course and International Conference of Communicable Diseases, which will be held in the Renginio programoje - metinė Baltijos tinklo prieš gyvybei pavojingas virusines infekcijas Government House of the Republic of Lithuania. konferencija, paskaitų kursas ir tarptautinė užkrečiamųjų ligų konferencija, kuri vyks Lietuvos The objective of the Summit will be to present and discuss the evidence-based policies, Respublikos Vyriausybės rūmuose. recommendations, points-of view and best practices in fighting the communicable diseases. High- Tarptautinės užkrečiamųjų ligų konferencijos tikslas - pristatyti ir aptarti įrodymais pagrįstą politiką, level officials from the World Health Organisation, European Commission, ECDC, other international organisations, and outstanding experts in communicable diseases are invited as gaires, požiūrius ir geriausios praktikos pavyzdžius kovojant su užkrečiamosiomis ligomis, tarp speakers. The topics include current threats of communicable diseases in Europe and the world, pranešėjų - Pasaulio sveikatos organizacijos, Europos Komisijos, Europos ligų prevencijos ir refugees and communicable diseases, antimicrobial resistance and infection prevention, vaccine- kontrolės centro, kitų tarptautinių organizacijų atstovai, žymiausi užkrečiamųjų ligų specialistai. Bus preventable diseases among others. aptariamos aktualios šiuo metu temos - šiuolaikinės užkrečiamųjų ligų grėsmės Europoje ir pasaulyje, pabėgėliai ir užkrečiamosios ligos, atsparumas antibiotikams ir infekcijų prevencija, Many of Vilnius main tourist attractions and all the accommodation for delegates are in easy walking vakcinomis valdomos ligos ir daug kitų. distance from the Conference venue. We hope that this event will provide a unique opportunity to learn the newest scientific Daugelis dažniausiai Vilniaus turistų lankomų vietų ir visi viešbučiai, kuriuose gyvens delegatai nuo achievements and to strengthen the networking, and you will find the Conference and your stay in konferencijos vietos lengvai pasiekiami pėsčiomis. Vilnius both valuable and enjoyable. Tikimės, kad renginys suteiks unikalią galimybę susipažinti su naujausiais mokslo pasiekimais ir stiprinti bendradarbiavimą, o Jūsų viešnagė Vilniuje bus naudinga ir maloni.

Scientific Committee: Local Committee Scientific Committee: Local Committee Goda Adomonis, Netherlands Andrei Kozlov, Russian Federation Arvydas Ambrozaitis Goda Adomonis, Netherlands Andrei Kozlov, Russian Federation Arvydas Ambrozaitis Dalia Adukauskienė, Lithuania Liudmila Mosina, WHO, Denmark Danguole Avižiuvienė Dalia Adukauskienė, Lithuania Liudmila Mosina, WHO, Denmark Danguole Avižiuvienė Ali Arsalo, Finland Cristina Mussini, Italy Gintautas Barcys Ali Arsalo, Finland Cristina Mussini, Italy Gintautas Barcys Henrique Barros, Portugal Teymur Noori, ECDC, Sweden Vytautas Basys Henrique Barros, Portugal Teymur Noori, ECDC, Sweden Vytautas Basys Torsten Berglund, Sweden Jurijs Perevoscikovs, Latvia Joana Bikulčikienė Torsten Berglund, Sweden Jurijs Perevoscikovs, Latvia Joana Bikulčikienė Tatjana Nemeth Blažić, Croatia Daria Podlekareva, Denmark Matilda Bylaitė Tatjana Nemeth Blažić, Croatia Daria Podlekareva, Denmark Matilda Bylaitė Hans Blystad, Norway Andris Rubins, Latvia Saulius Čaplinskas Hans Blystad, Norway Andris Rubins, Latvia Saulius Čaplinskas Anna Marzec-Bogusławska, Poland Vinay Saldanha, UNAIDS Radvilė Jakaitienė Anna Marzec-Bogusławska, Poland Vinay Saldanha, UNAIDS Radvilė Jakaitienė Saulius Čaplinskas, Lithuania Mika Salminen, Finland Inga Juozapivičienė Saulius Čaplinskas, Lithuania Mika Salminen, Finland Inga Juozapivičienė Jordi Casabona, Spain Jon Sigfusson, Iceland Silvija Kiverytė Jordi Casabona, Spain Jon Sigfusson, Iceland Silvija Kiverytė Luis Sordo del Castillo, Spain Audra Sipaviciene, IOM, Lithuania Giedrė Maksimaitytė Luis Sordo del Castillo, Spain Audra Sipaviciene, IOM, Lithuania Giedrė Maksimaitytė Masoud Dara, WHO, Belgium Danica Stanekova, Slovakia Aukse Mickienė Masoud Dara, WHO, Belgium Danica Stanekova, Slovakia Aukse Mickienė Nikos Dedes, Greece Zaza Tsereteli,Estonia Rūta Nadišauskienė Nikos Dedes, Greece Zaza Tsereteli,Estonia Rūta Nadišauskienė Jack DeHovitz, United States Magnus Unemo, Sweden Irena Pivoriūnienė Jack DeHovitz, United States Magnus Unemo, Sweden Irena Pivoriūnienė Jan Drobeniuc, United States Tonka Varleva, Bulgaria Audrius Sčeponavičius Jan Drobeniuc, United States Tonka Varleva, Bulgaria Audrius Sčeponavičius Vladimir Eremin, Belarus Anders Widell, Sweden Aurelija Urbonienė Vladimir Eremin, Belarus Anders Widell, Sweden Aurelija Urbonienė Samvel Grigoryan, Armenia Ingrida Zurlyte, WHO, Lithuania Vytautas Usonis Samvel Grigoryan, Armenia Ingrida Zurlyte, WHO, Lithuania Vytautas Usonis Johannes Hallauer, Germany Skaidra Valiukevičienė Johannes Hallauer, Germany Skaidra Valiukevičienė Andrzej Horban, Poland Arūnas Valiulis Andrzej Horban, Poland Arūnas Valiulis Maria Issagouliantis, Sweden Leonas Valius Maria Issagouliantis, Sweden Leonas Valius Saleem Kamili, United States Astra Vitkauskienė Saleem Kamili, United States Astra Vitkauskienė Eduard Karamov, Russian Federation Vytautas Zimnickas Eduard Karamov, Russian Federation Vytautas Zimnickas Irena Klavs, Slovenia Jadvyga Zinkevičiūtė Irena Klavs, Slovenia Jadvyga Zinkevičiūtė Secon I: Emerging and re-emerging threats of Secon I: Emerging and re-emerging threats of communicable diseases communicable diseases Budkowska Agata HCV: molecular basis of treatment and vaccine escape Invited Budkowska Agata HCV: molecular basis of treatment and vaccine escape Invited Kamili Saleem Infectivity of hepatitis B virus mutants Invited Kamili Saleem Infectivity of hepatitis B virus mutants Invited Khudyakov Yuri Viral hepatitis of unknown etiology, analysis of the origins Invited Khudyakov Yuri Viral hepatitis of unknown etiology, analysis of the origins Invited Kucinskiene Vesta Relevant issues of bacterial sexually transmitted infections Invited in Lithuania Kucinskiene Vesta Relevant issues of bacterial sexually transmitted infections Invited Medstrand Patrik HIV subtypes and recombinants - implications for disease Invited in Lithuania progression Medstrand Patrik HIV subtypes and recombinants - implications for disease Invited Tangy Frédéric The nucleotide bias of lentiviruses genomes is associated to Invited progression AIDS pathogenesis Tangy Frédéric The nucleotide bias of lentiviruses genomes is associated to Invited AIDS pathogenesis Bartuliene Ausra Human trichinellosis epidemiological situation in Poster Lithuania, 2006-2015 Bartuliene Ausra Human trichinellosis epidemiological situation in Poster Belopolskaya Maria HBsAg level as a disease activity marker in patients with Poster Lithuania, 2006-2015 chronic hepatitis delta Belopolskaya Maria HBsAg level as a disease activity marker in patients with Poster Belopolskaya Maria Snapshot on hepatitis B virus population heterogeneity in Poster chronic hepatitis delta pregnant women chronically infected by HBV Belopolskaya Maria Snapshot on hepatitis B virus population heterogeneity in Poster Cuypers Lize Classification and nomenclature of Dengue: challenges of Poster pregnant women chronically infected by HBV the present and defining the future Cuypers Lize Classification and nomenclature of Dengue: challenges of Poster Esbjörnsson Joakim Increased understanding of viral spread and dynamics by Oral the present and defining the future the use of molecular epidemiology Esbjörnsson Joakim Increased understanding of viral spread and dynamics by Oral Geller Julia Detection, prevalence and genetic analysis of Puumala Poster the use of molecular epidemiology virus in bank voles and patients with confirmed HFRS in Geller Julia Detection, prevalence and genetic analysis of Puumala Poster Estonia virus in bank voles and patients with confirmed HFRS in Gheorghe Irina Virulence and resistance genes in methicillin resistant Poster Estonia Staphylococcus aureus and vancomycin resistant Gheorghe Irina Virulence and resistance genes in methicillin resistant Poster Enterococcus sp. clinical strains isolated in South Romania Staphylococcus aureus and vancomycin resistant Idrees Muhammad Occult hepatitis C virus infection: how its chance Oral Enterococcus sp. clinical strains isolated in South Romania increases? Idrees Muhammad Occult hepatitis C virus infection: how its chance Oral Kotovas Robertas Novaerus: closing the loop on infection control Poster increases? Kotovas Robertas The important role of bedding (soft inventory) in infection Poster Kotovas Robertas Novaerus: closing the loop on infection control Poster control Kotovas Robertas The important role of bedding (soft inventory) in infection Poster Krasko Anatoli Pathogenic rickettsia detected in Ixodes ticks in the Poster control republic of Belarus. Krasko Anatoli Pathogenic rickettsia detected in Ixodes ticks in the Poster Kyuregyan Karen & Neurologic manifestations of hepatitis E (anti-HEV Oral republic of Belarus. Malinnikova Elena prevalence in patients with neurological disorders and Kyuregyan Karen & Neurologic manifestations of hepatitis E (anti-HEV Oral clinical case) Malinnikova Elena prevalence in patients with neurological disorders and Ruzauskas Modestas ESBL-producing Escherichia coli around us: the Oral clinical case) prevalence study in animals, food products and Ruzauskas Modestas ESBL-producing Escherichia coli around us: the Oral environment prevalence study in animals, food products and Vizuje Greta Antimicrobial resistance of invasive isolates in Lithuanian Oral environment hospitals Vizuje Greta Antimicrobial resistance of invasive isolates in Lithuanian Oral Widell Anders Zika virus - an overview Oral hospitals Zukauskaite- An outbreak of Salmonella enteritidis associated with peas Poster Widell Anders Zika virus - an overview Oral Sarapajeviene with cracklings Zukauskaite- An outbreak of Salmonella enteritidis associated with peas Poster Simona Sarapajeviene with cracklings Zygutiene Milda Blackfly control in Lithuania, 1999-2015 Poster Simona Zygutiene Milda Blackfly control in Lithuania, 1999-2015 Poster

Editor-in-chief: Saulius Caplinskas M.D., Ph.D Mykolas Romeris University, Centre for Communicable Diseases and AIDS

ISBN 978-9986-720-52-2 3 HCV: MOLECULAR BASIS OF TREATMENT AND VACCINE ESCAPE

INVITED LECTURE Name E-mail Institution Country Institut Pasteur International BUDKOWSKA AGATA [email protected] France Network, Institut Pasteur, Paris

Hepatitis C virus (HCV) remains a major cause of chronic liver disease, cirrhosis and hepatocarcinoma. Spectacular progress has been done in therapy of HCV infection by introduction of direct-acting antivirals (DAA) targeting non-structural proteins involved in virus replication (NS3/NS4A protease, NS5B RNA polymerase and NS5A inhibitors). These regimens demonstrated excellent response rates (up to 96%), have few side effects and progressively replace interferon- based therapies. Important limitation of DAA is their low genetic barrier of resistance resulting in emergence of drug escape-variants. Alternative or complementary therapeutic approaches have been proposed to target host factors required for essential steps of HCV life cycle: cell entry, replication or virus assembly/release. Such drugs will have high genetic barrier and pan-genotypic activity. Virus interactions with lipid and lipoprotein metabolic pathways should also be taken into account in therapeutic considerations. Prophylactic vaccine is a crucial requirement to prevent populations at risk, stop global epidemics, and protect patients cured with DAA since resolution of infection does not confer immunity. HCV represents a difficult target for vaccination due to its considerable variability (7 genotypes, 67 subtypes and genetically diverse quasispecies). HCV genome is highly mutable and its continuous evolution results in substantial variations in E2 envelope glycoprotein targeted by neutralizing antibodies and allows the virus to evade humoral immunity. Prophylactic HCV vaccine should elicit T cell response and cross-neutralising antibodies, both strongly influenced by virus variability. Important obstacle remains the lack of reliable animal models for studies of vaccines. Realistic goal of vaccination seems to be prevention of chronicity after acute infection. HCV uses several mechanisms to evade the host immune responses: constant mutations, shielding of neutralising epitopes by virus-associated lipoproteins, dissemination via cell-to cell spread, production of exosomes by infected cells and extrahepatic reservoirs of HCV. These mechanisms facilitate transmission of infection and contribute to the propensity of HCV to establish chronic infection.

4 INFECTIVITY OF HEPATITIS B VIRUS MUTANTS

INVITED LECTURE Name E-mail Institution Country

KAMILI SALEEM [email protected] Division of Viral Hepatitis, Centers for United States of America Disease Control and Prevention, Atlanta

Mutant forms of hepatitis B virus (HBV) are capable of escaping neutralization by vaccination or emerge as a result of antiviral therapy leading to drug resistance. Mutations in the “a’ determinant region of hepatitis B surface antigen (HBsAg) have been identified in chronic hepatitis B patients, infants receiving pre-exposure prophylaxis, liver transplant patients receiving hepatitis B immunoglobulin and individuals who receive pre-exposure hepatitis B vaccination. Mutations associated with anti-viral drug resistance emerge in the reverse transcriptase (rt) region of the HBV polymerase (pol) after prolonged treatment of chronic hepatitis B with nucleotide analogues such as lamivudine and famciclovir. Because of the overlapping nature of reading frames encoding polymerase and envelope proteins, mutations in pol can bring about a concomitant mutation in the “a” determinant region of HBsAg. Many of these mutations have been shown to have markedly reduced binding to hepatitis B vaccine induced antibody against HBsAg (anti-HBs) posing challenges of breakthrough infections as well as evading detection by commonly used HBsAg diagnostic tests. Experimental infectivity studies conducted in naïve and vaccinated chimpanzees with several hepatitis B pol and S gene mutants have shown that these HBV mutants are infectious and have the potential to infect previously vaccinated individuals. Lack of detection of HBV mutants by standard HBsAg diagnostic assays may pose the problem of their entry into the blood supply and infect vaccinated recipients of blood products. Infectivity and pathogenicity studies conducted with a S gene mutant, sG145R and a pol gene triple mutant, rtV173L, rtL180M and rtM204V will be discussed.

VIRAL HEPATITIS OF UNKNOWN ETIOLOGY, ANALYSIS OF THE ORIGINS

INVITED LECTURE E-mail Institution Country Name KHUDYAKOV YURI [email protected] Centers for Disease Control and Prevention, Atlanta USA

Several definitions of occult or cryptic hepatitis B and C (OHB and OHC) of different stringency can be found in literature. The strictest definition identifies OHB and OHC as hepatitis caused by serologically silent infections with hepatitis B (HBV) and hepatitis C (HCV) viruses, genomes of which are only detectable in liver. In absence of liver biopsy material for molecular testing, such hepatitis cases without detectable serological and molecular markers of infection with known viral agents may be misclassified as cases of hepatitis of unknown etiology (HUE). The next-generation sequencing (NGS) technologies allow for sampling very large numbers of sequences from serum specimens of persons with acute HUE to explore the potential infectious nature of these cases. After removal of human sequences, computational analyses showed that a significant fraction of HUE cases are cryptically infected with HBV, with some of them co- infected with hepatitis E virus (HEV), suggesting contribution of cryptic infections with known viral agents to etiology of acute HUE.

5

RELEVANT ISSUES OF BACTERIAL SEXUALLY TRANSMITTED INFECTIONS IN LITHUANIA

INVITED LECTURE Name E-mail Institution Country Lithuanian University of Health Sciences, KUCINSKIENE [email protected] Department of Skin and Venereal Diseases, Lithuania VESTA Kaunas

Juseviciute Vijoleta [email protected]

Vitkauskiene Astra [email protected]

There are three notifiable bacterial sexually transmitted infections (syphilis, gonorrhoeae and chlamydial infection) in Lithuania. The incidence rates over the last 5 years (2011 – 2015) for chlamydial infection ranged from 11.3 to 14.1 cases/100000 population,gonorrhoeae – from 7.5 to 6.7 cases, syphilis – from 8.3 to 9.7 cases. Three cases of congenital syphilis were registered in 2015 (ULAC data). In Lithuania, all pregnant women are screened for syphilis (the RPR test) – during the first visit (up to the 12th week of pregnancy) and at 32 weeks of pregnancy. The cases of congenital syphilis occur in neonates born to women who fail to undergo clinical examination and adequate treatment for syphilis prior to or during pregnancy. Women whose neonates are suspected to have congenital syphilis are advised to give birth in university hospitals, which are capable of providing comprehensive care to the mother and the neonate. Bacterial STIs are diagnosed and managed following national recommendations and IUSTI guidelines which are updated every 2-3 years. If a person meets clinical, laboratory and epidemiological criteria of syphilis, gonorrhoeae or chlamydial infection, the case has to be reported. Laboratory criteria are crucial for notifiable STIs. The main routine laboratory tests such as microscopy of genital smears, serology for syphilis (RPR, TPHA) are available in the laboratories of bigger hospitals or even the primary health centers. Lab testing of complicated STIs (e.g. VDRL-CSF for neurosyphilis, TP IgM antibodies for congenital syphilis) as well as antibiotic resistance control is concentrated at the laboratories of the university hospitals and National Public Health Surveillance Laboratory (NVSPL). Different molecular tests for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomona vaginalis, Mycoplasma genitalium, ureoplasmas are introduced in university hospitals, NVSPL and private laboratories. Nucleic acid amplification tests for STIs are not covered by health insurance and the patients have to pay for them. In the last years several epidemiological projects on STIs have shown that young people actively seek for STIs testing and treatment in time. In 2013 January-June, in the Hospital of Lithuanian University of Health Sciences, 963 young people consented to participate in the cross sectional study. The first void urine (FUV) samples were analysed for the presence of 7 urogenital pathogens: Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Ureaplasma parvum by using multiplex real-time PCR assay “AnyplexTM II STI-7 Detection” (Seegene, Inc., Seoul, Korea). The results showed that the frequency of STIs was: 6.2% of chlamydial and 0.4% gonococcal infections, 2.7% – Mycoplasma genitalium and 0.5% Trichomona vaginalis infections. In the 20-30 years age group C. trachomatis was detected in 10.2 % of women, in younger than 20 years – 6.25% and in the group of 30-40 years – 4.69% of women. Chlamydia trachomatis was more common among those women who had more than 2 sexual partners in the last half-year. Most of the infected persons didn‘t want to give information about their sexually relationship with other partners.

6 HIV SUBTYPES AND RECOMBINANTS - IMPLICATIONS FOR DISEASE PROGRESSION

INVITED LECTURE Name E-mail Institution Country

MEDSTRAND Lund University, Department of Laboratory PATRIK [email protected] Sweden Medicine, Lund

Backgroud. Different characteristics of HIV type 1 (HIV-1) subtypes and circulating recombinant forms (CRF) and their interactions with the human host may influence HIV transmission and disease progression. Although the average time from infection with HIV type 1 (HIV-1) to the development of AIDS is less than 10 years, the rate of disease progression varies considerably among infected individuals. Numerous factors, including plasma HIV-1 RNA level, CD4 cell count, degree of immune activation, age, socioeconomic status, and host genetics, contribute to determining the rate of progression in the individual patient. Viral properties, such as coreceptor use and replication capacity, may also influence progression rates. There have been several prospective studies of the course of HIV-related disease in cohorts infected with various subtypes. It has been reported that individuals infected with HIV-1 subtypes B, C and D progress faster than CRF_AE, CRF_AG and G with subtype A being least aggressive of the HIV-1 subtypes studies. Many studies were performed in cohorts of individuals with seroprevalent infection. Although seroprevalent cohorts can provide important information they potentially exclude individuals with rapid progression which may bias the analysis. In addition, because the timing of HIV-1 infection is rarely known, it is difficult to control for duration of infection in analyses of seroprevalent cohorts. Thus, the use of a cohort of individuals with seroincident infection is a particular strength to determine differences in disease progression rate. Results. We determined the HIV-1 subtype/CRF of 152 seroincident individuals in an occupational cohort from Guinea-Bissau, West Africa. Rate of disease progression was measured as the time from estimated seroconversion to AIDS and AIDS-related death. The major subtypes/CRFs in Guinea-Bissau were CRF02_AG (53%), A3 (29%) and A3/02 (a recombinant of A3 and CRF02_AG) (13%). The median estimated time from seroconversion to AIDS and AIDS-related death was 5.0 and 8.0 years for A3/02, 6.2 and 9.0 years for CRF02_AG and 7.2 and 11.3 years for A3. Comparing our estimated progression rates with results of previous studies conducted in ART-naïve populations (of 14 identified studies, six included seroincident infections in Kenya, Senegal and Uganda) indicated that the two recombinant forms (A3/02 and CRF_02AG) have among the fastest progression rates to AIDS reported to date, similar to reports of subtype D by others. Moreover, subtype A3 showed similar progression rates as for subtype A of other cohorts. Conclusion. Although the current knowledge highlights the importance of viral characteristics in determining the rate of HIV-1 disease progression, additional factors such as viral replication may differ between subtypes. Moreover, time from seroconversion to antiretroviral therapy initiation may result in different rate of CD4+ T-cell decline depending on the infecting subtype. Further investigations in these areas are important not at least due to the well-described link between viral load and transmission and the rate of disease progression.

7

THE NUCLEOTIDE BIAS OF LENTIVIRUSES GENOMES IS ASSOCIATED TO AIDS PATHOGENESIS

INVITED LECTURE

Name E-mail Institution Country Viral Genomic and Vaccination Unit, TANGY FRÉDÉRIC [email protected] Institut Pasteur, CNRS UMR 3569, Paris France

Vabret Nicolas

Bailly-Bechet Marc

Najburg Valérie

Müller-Trutwin Michaela

Progression to AIDS in HIV-infected humans is characterized by chronic generalized immune activation. Conversely, most African primate species naturally infected with SIV maintain normal level of immune activation and never progress to AIDS. Addressing this issue is crucial for understanding AIDS pathogenesis. Lentiviral genomes have a particularly biased nucleotide composition compared to that of their primate hosts with high frequencies of Adenosine. We thus wondered whether viral A/C/G/T nucleotide ratio has an effect on innate immunity stimulation and AIDS pathogenesis.We compared the nucleotide composition (A/C/G/T frequencies) of pathogenic and non-pathogenic primate lentiviruses with that of their hosts for every described infection. For that we measured the nucleotide divergence by computing the Chi-2 distance between the A/C/G/T frequencies in the complete viral sequence and the coding sequences of each host organism. We found that primate lentiviruses having the most divergent nucleotide composition compared to their hosts induce AIDS, whereas less divergent lentiviruses cause non-pathogenic infections. Similarly, the relative pathogenicity of HIV-1 subtypes correlates with their nucleotide divergence to the human genome. To understand this observation at a molecular level, we investigated the ability of HIV-1 RNA fragments to stimulate in vitro the synthesis of type I interferon (IFN-I). We observed again that the nucleotide bias of RNA fragments strongly correlates with their ability to induce IFN-I. Based on these observations, we designed nucleotide-optimized SIV sequences derived from SIVmac239. We produced corresponding artificial viruses and analyzed their capacity to induce IFN-I in different cellular models. We demonstrate that a Pol-optimized synthetic virus presenting the same replicative capacity than WT virus has a reduced ability to induce IFN-I, thus confirming or observation. These data suggest for the first time a direct link between nucleotide composition of lentiviruses and AIDS pathogenicity. We also describe the first synthesis of large-scale nucleotide-modified SIV that displays an attenuated pathogenic potential.

8

HUMAN TRICHINELLOSIS EPIDEMIOLOGICAL SITUATION IN LITHUANIA, 2006-2015

POSTER Name E-mail Institution Country BARTULIENE AUSRA [email protected] ULAC Lithuania Zagrebneviene Galina [email protected] ULAC Lithuania Caplinskas Saulius [email protected] Mykolas Romeris University, Lithuania Centre for Communicable Diseases and AIDS

Background. Trichinellosis is an acute disease of humans and animals caused by Trichinella genus roundworms. People get infected by eating raw or insufficiently cooked infected meat of domestic or wild animals. In comparison with other European Union (EU) countries Lithuania had the highest incidence of trichinosis. Aims. To review epidemiological situation and trends of human trichinellosis in Lithuania. Materials & Methods. Data analysis was performed using descriptive epidemiological study design. The data was obtained from the information system of Centre for Communicable Diseases and AIDS. Results. According to the data of the Centre for Communicable Diseases and AIDS, 414 cases of human trichinellosis were reported in Lithuania during 2005-2016, including 21 sporadic cases and 37 outbreaks. During the decade the incidence of trichinellosis increased from 0.3 to 3.3 cases per 100,000 population. People of any profession and age can be affected by trichinellosis, but the highest incidence rate of infection was recorded among adults. Nevertheless, trichinellosis infection is not rare among children. During ten years (2005-2016), 66 cases (15.9 %) were reported in the age group of under eighteen years of age. According to the epidemiological outbreak investigation, human trichinellosis in Lithuania is spread by consumption of infected domestic pig and wild boar meat. During 2005-2016 period, 51.4 % of trichinellosis outbreaks occurred in family farms where pigs’ meat had not been examined, about 37.8 % by consuming infected wild boar and the source of infection in 10.8 % of cases was unknown. The incidence rate of trichinellosis among the rural population was higher than among the urban population during the period of 2005-2016. During this time of period average incidence rate of human trichinellosis was 1.8 cases per 100.000 population in rural population, in urban population – 1.1 cases per 100.000 population. Human trichinellosis infections outbreaks were mostly reported from central region of Lithuania. During 2005-2016, more than 4 trichinellosis outbreaks were reported in Kaunas regions. Conclusions. 1. Incidence rate of human trichinellosis has been remaining relatively high. 2. Incidence rate of human trichinellosis is higher in rural than in urban population. 3. Higher incidence rate of human trichinellosis is been registered among adults. 4. The main risk factor of outbreaks of human trichinellosis was infected and uninspected pork meat from small farms (51.5%) during 2005-2016. 5. Human trichinellosis outbreaks often recorded in the central region of Lithuania.

9

HBsAg LEVEL AS A DISEASE ACTIVITY MARKER IN PATIENTS WITH CHRONIC HEPATITIS DELTA

POSTER Name E-mail Institution Country BELOPOLSKAYA Botkin Infectious Disease Hospital, Russian [email protected] MARIA Institute of Experimental Medicine Federation Avrutin Viktor [email protected] University of Urbino Italy Russian Firsov Sergey [email protected] Botkin Infectious Diseases Hospital Federation Russian Kalinina Olga [email protected] Pasteur Institute Federation Russian Yakovlev Alexei [email protected] St-Petersburg State University Federation

Background and Aims. Hepatitis delta is one of the most severe forms of a chronic viral hepatitis. The level of cytolytic activity, the viral load and the rate of fibrosis progression are the essential markers of chronic hepatitis B and D disease activity. The goals of our study were to investigate the relationship between serum HBsAg level and viral load in HBV infected patients with and without HDV co-infection and to estimate the possibility of using HBsAg level as a disease activity marker. Methods. 54 HBV infected patients and 21 HBV/HDV co-infected patients were investigated. Quantitative detection of HBV DNA and HDV RNA was done using COBAS TaqMan HBV test (Roche Diagnostics) and AmpliSens HDV test (InterLabService), respectively. HBsAg level was measured by the Architect HBsAg QT (Abbott Laboratories) assay. Fibrosis stage was estimated by transient elastography (FibroScan). The statistical relationship was estimated by the Pearson correlation coefficient R. Results. The correlation between HBV DNA and HBsAg levels in HBV infected patients was statistically significant (R=0.4558, P=0.0005). In most cases, a low level of HBsAg corresponded to a low HBV DNA level, whereas a high HBsAg level was not always associated with a high viral load. By contrast, in HBV/HDV co-infected patients a similar correlation was not observed (R=-0.0689, P=0.7859). However, the correlation between HDV RNA and HBsAg levels (R=0.6636, P=0.0051), as well as between HDV RNA level and fibrosis stage (R=0.5057, P=0.0457) was significant. In this group, a high level of HBsAg was usually associated with a high HDV RNA level. The correlation between HBsAg level and fibrosis stage was less significant (R=0.4138, P=0.1812) in HBV/HDV co-infected patients and not observed (R=0.0724, P=0.7252) in HBV infected patients. Conclusions. 1. The correlation between HBsAg and HBV DNA levels is statistically significant only in HBV infected patients, while in HBV/HDV co-infected patients this correlation does not exist. 2. A severe fibrosis is usually associated with a high HDV RNA level. HBsAg level cannot be used for estimating the stage of fibrosis. 3. In clinical practice, a low HBsAg level can be used as a predictor of a low HBV DNA level for HBV infected patients. In HBV/HDV co-infected patients, HBsAg level can be used for estimating HDV viral load.

10

SNAPSHOT ON HBV POPULATION HETEROGENEITY IN PREGNANT WOMEN CHRONICALLY INFECTED BY HBV

ORAL Name E-mail Institution Country Belopolskaya Botkin Infectious Disease Hospital, Institute of Russian [email protected] Maria Experimental Medicine Federation Ostankova Julia [email protected] Saint-Petersburg Pasteur Institute RF Yakovlev Alexei [email protected] St-Petersburg State University RF Dmitriev [email protected] Institute of Experimental Medicine RF Alexander KALININA [email protected] Pasteur Institute RF OLGA

Background. Hepatitis B is a potential life-threatening liver infection occurring worldwide and resulting in significant social and economic losses. According to the report of World Health Organization (WHO), more than 240 million people are chronically infected by HBV, and at least half of them were infected prenatally or in childhood. In Russian Federation the prevalence of HBsAg in pregnant women significantly decreased during the last fifteen years (from 1.1% in 1997 to 0.6% in 2012). HBV genetic variability is one of the factors which associated with disease progression, effectiveness of the therapy, success of the vaccination, and it can affect the risk of vertical transmission. This study was conducted to investigate the genetic heterogeneity of HBV population in chronically infected pregnant women. Materials & Methods. A total of 19 pregnant women with chronic hepatitis B were enrolled in this study. One woman had a high viral load more than 108 IU/ml, the others had a low viral load (less than 103 IU/ml). In nine women, HBsAg was detected for the first time during this pregnancy. Six women had the HBV infection for more than four years. All newborns received standard prophylaxis (vaccine immunization with or without immunoglobulin) at the first 12 hours postpartum. None of the newborns were infected by HBV. HBV DNA was extracted from serum samples using the DNA/RNA RIBO-PREP kit (Amplisense, Russia). The DNA fragments including the Pre-S/S gene were amplified for 13 HBV strains obtained from 13 pregnant women. Following PCR amplification, libraries were prepared using Nextera XT DNA Sample Prep Kit (Illumina, USA), amplicons were tagged using Nextera XT Index Kit (Illumina, USA) according to the manufacturer’s specifications and sequenced at MiSeq (Illumina, USA) using MiSeq Reagent Kit v2 500 Standart FC (Illumina, USA). The obtained reads were edited and assembled with the Genomics Workbench software version 8.0. (CLCbio, USA). Multiple alignment and phylogenetic analysis were carried out with the Mega software version 6.0. Results. 13 HBV strains were genotyped by phylogenetic analysis: 2 strains belonged to subgenotype A2, 4 - to D1, 5 - to D2, 1 - to D3. One strain belonged to the cluster adjacent to the genotype D branch and formed by A/D recombinant HBV strains circulating in different regions. All studied strains regardless of genotype didn’t form any distinct cluster. They were intermixed with reference HBV strains of different origin. Based on analysis of “a” determinant in S gene, 13 strains were classified into three subtypes: 6 strains (4 - subgenotype D1, 1 - subgenotype D3, and 1 - subgenotype A2) belonged to ayw2 subtypes, 6 strains (5 - subgenotype D2 and 1 - A/D recombinant) belonged to ayw3 and one strain (subgenotype A2) - to adw2. No amino acid substitutions were detected in preS region or in major hydrophilic region of the S antigen. All strains which belonged to genotype D (ayw2 or ayw3 subtypes) and one A/D recombinant strain (ayw3 subtype) carried tyrosine at position 134 within “a” determinant (instead of phenylalanine). None of the mutations associated with resistance to nucleotide analogs (rtM204, rtL80, rtL180, rtA181, rtA194, rtT184, rtS202, rtN236, rtM250, rtI169, rtF166, rtV173) were revealed. Conclusions. High heterogeneity of HBV population circulating in pregnant women infected during last decade indicates crucial impact of different HBV variants on the persistence infection in Russia.

11

CLASSIFICATION AND NOMENCLATURE OF DENGUE: CHALLENGES

OF THE PRESENT AND DEFINING THE FUTURE

POSTER Name E-mail Institution Country CUYPERS LIZE [email protected] KU Leuven Belgium Artificial Intelligence Lab – Libin Pieter [email protected] Belgium Vrije Universiteit Brussel ArtificialIntelligenceLab – Nowé Ann [email protected] Belgium Vrije Universiteit Brussel KU Leuven-Rega Institute Vandamme Anne-Mieke [email protected] Belgium For Medical Research Centers For Disease Control Santiago Gilberto [email protected] USA And Prevention KULeuven-Rega Institute Theys Kristof [email protected] Belgium For Medical Research

Background & Aims. About 4 billion people are at risk of Dengue virus (DENV) infection, as the transmitting mosquito vectors are geographically widely distributed and are expanding globally. Increased frequencies and magnitudes of DENV outbreaks have been reported, as well as high transmission rates and viral spread in regions formerly not known to be affected. Dengue can be classified into four serotypes, each consisting of different genotypes. Differently to serotypes, the system of classification and nomenclature at the genotype level lacks consistency. The variability observed in DENV genotype taxonomy is easily prone to confusion and mistakes, and has been shown to complicate clinical and epidemiological interpretation. Current challenges exist on the level of naming and of clade classification. Nomenclature. Although DENV serotypes are systematically denoted in a numerical manner (DENV1-4), naming of genotypes differs across serotypes. Historically, geographical regions were used to refer to genotypes, but genotypes within DENV1, 3 and 4 are currently predominantly named by roman numerals. For DENV2, naming is mostly based on geographical association, although some publications report roman numbers. Furthermore, the terms serotype, genotype and subtype are used interchangeably in literature, and studies that describe similar phylogenetic clades often use different numerals. Classification. There exists no formal definition of a DENV genotype, resulting into different constellations of genotypes for the four DENV serotypes. Generally, it is believed that respectively five, six, five and four genotypes are circulating in DENV1, 2, 3 and 4, although lower numbers have been reported for each of the serotypes. Furthermore, studies have identified the existence of subclades within genotypes, again without consistently defining the concept of a subtype. Overall, DENV classification by phylogenetic analysis is based on the envelope gene that generates similar topologies to full-genome analysis. However, the use of sequence data encompassing single proteins other than the envelope gene has been reported, which are not known to have sufficiently high phylogenetic signal. The type of phylogenetic inference, nucleotide substitution model, number of strains and rooting of the tree, all greatly impact the structure of the resulting phylogenetic tree. Future. The global spatiotemporal evolution of Dengue has made the previous naming of genotypes by geographic association irrelevant. Worldwide, an increasing geographic spread of DENV is expected, which will result into the potential need to extend the current classification with new diverging lineages. An increasing number of strains, characterized by a high geographical spread and covering larger parts of the full-genome, is available and can be used to unify and even thoroughly revise the current nomenclature. Additionally, the formal definition of a DENV genotype should be considered, since this definition will help to extend nomenclature when new strains emerge. Extensive analyses are needed to evaluate the abandoning of geographic-based names, the division of current genotypes into subtypes and to which extent the current nomenclature is valid for the different viral proteins. Such analyses are urgently needed and ask the involvement of a designated working group to unite the DENV community around a common and sensible nomenclature and classification.

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INCREASED UNDERSTANDING OF VIRAL SPREAD AND DYNAMICS BY THE USE OF MOLECULAR EPIDEMIOLOGY

ORAL Name E-mail Institution Country University of Oxford, Nuffield ESBJÖRNSSON United [email protected] Department of Clinical Medicine, JOAKIM Kingdom Oxford

Problem statement. Increased knowledge about the determining events and factors involved in the development of new viral epidemics and subepidemics together with insights in the epidemiological dynamics of viral spread are critical for monitoring current epidemics and designing future prevention efforts. Methods. Viral genetic sequences were used together with patient data in maximum-likelihood and Bayesian phylogenetic frameworks to dissect the molecular epidemiology of HIV-1 in different geographical settings. Results. The current presentation will describe how to design a dataset suitable for analysis by molecular epidemiology and how such dataset can be explored by current state-of-the-art phylogenetic and phylogeographic analyses. Different datasets has been used to (1) in detail describe how epidemiologic and phylogenetic data can be combined to show how one single introduction of HIV-1 became epidemic in an isolated geographic location; (2) to show how strict phylogenetic analysis of a relatively small dataset can be used to show how and when different subtypes of HIV-1 was introduced and spread within a country; and (3) dissect the dynamics and spread of HIV-1 subtypes in different risk groups over time by the analysis of a large dataset representing >50% of all newly discovered HIV-1 infected individuals between 2000-2012 in the Scandinavian countries. Conclusion. Phylogenetic analysis can increase the understanding of epidemiological dynamics, enhance surveillance-based risk information and inform national programmes for monitoring and prevention of viral infections.

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DETECTION, PREVALENCE AND GENETIC ANALYSIS OF PUUMALAVIRUS IN BANK VOLES AND PATIENTS WITH CONFIRMED HFRS IN ESTONIA

POSTER Name E-mail Institution Country GELLER [email protected] National Institute for Health Development Estonia JULIA Vikentjeva [email protected] Tallinn University of Technology Estonia Maria Ivanova Anna [email protected] National Institute for Healt Development Estonia Katargina Uppsala University, Department of Medical Biochemistry [email protected] Sweden Olga and Microbiology, Zoonotic Science Center, Sweden Golovljova [email protected] National Institute for Health Development Estonia Irina

Background and aims. Hantaviruses are of zoonotic origin and are classified as rodent-borne viruses or roboviruses, which are transmittable by inhalation of air containing contaminated rodent excreta. Hantaviruses circulating in Europe, cause haemorrhagical fever with renal syndrome (HFRS) with varying degree of severity depending on the causative virus. Estonia is endemic area for hantaviruses with HFRS incidence rates of 1,4-2,7 per 100 000 population, that is twice as much as of Europe. Aims of the study were to: 1) investigate Puumala virus occurrence in natural reservoir hosts in connection with seasonality in five counties across Estonia, 2) describe genetic characterization and phylogenetic relationships of retrieved samples, and 3) establish usefulness of hantavirus RNA detection by Real-Time PCR method in patients with confirmer HFRS. Methods. Bank voles were captured in live traps in five counties in Estonia from 2012 to 2014, yearly from March to November. Anti-hantavirus specific IgG detection in rodents’ heart samples was performed using in-house ELISA method. ELISA-positive rodents’ lung samples were then used for the Real-Time PCR analysis to detect hantavirus RNA using Puumala-specific primers and probe. Hantavirus N-protein S- segment partial gene amplification was performed by RT-PCR and then sequenced. Human serum samples, positive for anti-hantavirus IgM by commercial ELISA system, Saaremaa and Puumala virus RNA detection in anti-hantavirus IgM-positive patients was performed by real-time PCR using Puumala or Saaremaa-specific primers and probes. Methods including in-house ELISA assay for anti-hantavirus antibody detection in rodents’ samples and real-time PCR for Puumala and Saaremaa virus RNA detection as well as specific primers and probes design were developed by NIHD working group in collaboration with Uppsala University. Results and conclusions. 447 bank voles were collected from five counties (Järvamaa, Lääne-Virumaa, Pärnumaa, Tartumaa and Saaremaa) in Estonia from 2012 to 2014, from which 96 (21,5%) samples revealed positive by ELISA IgG Puumala virus antibodies. All ELISA-positive samples were then analyzed by real-time PCR. In 67 (70%) rodents’ lung samples hantavirus RNA was detected, that occurred to be Puumala virus RNA. For genetic and phylogenetic analysis 53 Puumala virus sequences were used. Our result confirmed that Estonian Puumala virus strains cluster within local lineages, as samples’ sequences from each county are similar to each other and have a common ancestor. The sequences of Latvian and Estonian Puumala virus strains showed the high level of genetic identity (91%). In addition, Baltic and Russian strains showed to share a common ancestor, that corroborate a theory of virus introduction with rodents’ migration after the last Ice Age. Another important topic of hantavirus research in Estonia concerns the investigation of the presence of hantavirus RNA in patients with confirmed HFRS. In this study, 23 anti-hantavirus IgM-positive human sera samples were analyzed by real-time PCR. There were 9 (39%) samples positive for Puumala virus RNA. However, none of the analyzed samples revealed evidence of Saaremaa virus RNA presence, which may be caused by short viremic stage of Saaremaa hantavirus infection in humans. Future studies of hantaviruses in Estonia are needed for improvement and validation of hantavirus RNA detection in human samples.

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VIRULENCE AND RESISTANCE GENES IN METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS AND VANCOMYCIN RESISTANT ENTEROCOCCUS SP. CLINICAL STRAINS ISOLATED IN SOUTH ROMANIA

POSTER Name E-mail Institution Country Faculty of Biology University of Bucharest GHEORGHE IRINA [email protected] Romania (FB/UB) Almahdawy [email protected] FB/UB Romania Othman Thamer Popa Marcela [email protected] FB/UB Romania Porumbel Iuliana [email protected] FB/UB Romania Banu Otilia [email protected] FB/UB Romania Lazar Veronica [email protected] FB/UB Romania Chifiriuc Mariana [email protected] FB/UB Romania Carmen

Background and Aims. Present study was aimed to identify the SCCmec elements and types, the macrolides and vancomycin resistance, as well as virulence genes, among clinical S. aureus and Enterococcus sp. strains. Common genetic mechanism responsible for the emergence of S. aureus resistant to methicillin (MRSA) strains involves the mecA gene which directs synthesis of an abnormal penicillin binding protein PBP2. The mecA gene offers insertion sites containing plasmids and transposons which facilitate the acquisition of resistance genes to other classes of antibiotics. The SCCmec cassette is integrated at a specific site localized near the chromosomal replication origin of S. aureus. The polymorphism of the SCCmec cassette has lead to the emergence of 6 types of SCCmec I. VI. Materials & Methods. Study was conducted 141 isolates, 94 of S. aureus, 39 of E. faecalis and 8 of E. faecium, clinical strains isolated in the summer and autumn of 2015, out of which 82 from wound secretions, 20 from tracheal secretion, 18 from blood cultures, 18 from urine and 3 from catheters taken from patients hospitalized for cardiovascular surgery harboring infections with different localizations hospitalized in Institute Prof. ”C.C. Iliescu” Bucharest, Romania. Simplex and multiplex PCR was performed on genomic DNA from MSSA and MRSA isolates in order to identify the SCCmec central elements-ccr and mec complex and to establish the SCCmec type (type I, II, III, IVa-d, V);virulence genes in S. aureus-bone bound sialoprote (bbp), elastin-binding protein (ebpS), fibronectin-binding proteins (fnbA, fnbB), clumping factors A and B (clfA and clfB), collagen-binding protein (cna), coagulase (coag), Panton-Valentine leucocidin (luk-PV), hemolysine (hlg), toxic shock toxine (tst); and vancomycin resistance genes (Van A, B, C) in Enterococcus sp. isolates. Results. The incidence of MRSA was high among the S. aureus isolates (59.57% of the analyzed strains). The following elements of the SCCmec were identified in the MRSA strains: mecI (23.40%), CIF2 (11.70%), ccrC (12.76%), SCCmecVJ1 (15.95%), mecA and ccrB2 (5.31%), kdp (2.12%), SCCmecIIIJ1 element (1.06% of the isolates). The inducible (MLSBi) and constitutive (MLSBc) resistance was also encountered among the S. aureus strains (38.29% versus 6.38% of the isolates) and in the case of 6.38% of the MLSBi isolates was correlated with the presence of ermA gene. Only mecIVa and III of SCCmec types were encountered among the MRSA strains (8.51%). Regarding the virulence genes in S. aureus the most frequent was the tst gene (19.14%), followed by ebpS (15.95%), hlg and bbp (11.70%), coag gene (10.63%) and luk-PV gene (7.44%). In the case of Enterococcus sp. isolates was revealed in the same proportion vancomycin resistance VanB and VanA genes (27.65% of the isolates). Conclusions. The fact that the analyzed strains exhibited a variety of SCCmec cassette types its evidence to the capabilities of S. aureus to evolve and thus modify the perception of scientists regarding the involvement of different types of cassettes in a wide array of infections. The results confirm the prevalence of SCCmec IVa, usually community-acquired, among the MRSA strains isolated from patients hospitalized for cardiovascular surgery and the high prevalence of VanA and VanB genes among the Enterococcus sp. clinical isolates.

15

OCCULT HEPATITIS C VIRUS INFECTION: HOW ITS CHANCE INCREASES?

ORAL Name E-mail Institution Country IDREES MUHAMMAD [email protected] University of the Punjab Pakistan

Background. Occult Hepatitis C Infection Should Be Considered When Diagnosing Patients Without Detectable Anti-HCV And Serum HCV-RNA In Patients With Etiology Of Long-Standing Abnormal Liver-Function Tests (Lfts). Aim. The Aim Of The Present Study Was To Determine The Presence Of HCV RNA In The Liver Biopsies Of Patients With Abnormal Liver Tests But Without Detectable Serum HCV RNA And Anti- HCV Antibodies In Sera. Methods. Liver Biopsies And Whole Blood Of Total 101 Patients Negative For Anti-HCV Antibodies And With Elevated Liver Function Tests For The Detection Of HCV RNA. HCV RNA Status Of The Subjects Was Tested By Reverse-Transcription Polymerase Chain Reaction (RT-PCR) And Quantified Using Smartcycler II Real-Time PCR, In Their Liver Biopsies, Sera And Peripheral Blood Mononuclear Cells (Pbmcs). HCV Genotyping Was Carried Out In HCV RNA Positive Samples Using Type-Specific PCR. This Study Was Done At Division Of Molecular Virology Centre For Applied Molecular Biology, University Of The Punjab Pakistan From March 2012 To May 2016. Results. In This Study, HCV RNA Was Detected In Liver-Biopsy Specimens From 74 (73.26%) Of The Total 101 Patients Negative For Anti-HCV Antibodies And Undetectable Serum HCV RNA. HCV RNA Of Both Negative And Positive Polarity Was Found Simultaneously In The Livers Of Total 24 (23.768%) Patients. Analysis Of Genotypes Results Showed About 2/3rd Of The Patients Infected With HCV 3a, That Is Also The Predominant HCV Genotypes Circulating In This Region Of The World. However Genotype 1 (A &B) Were Also Present In Low Frequency. Six Patients Were Found With Untypable Genotype Even Though They Were HCV RNA Positive. In A Multivariate Logistic Regression Model, Patients Having Previous History Of Sharing Needles, Presence Of Steatosis, Elevated Liver Enzymes, History Of Previous Surgeries, Male Sex, Age Above 30 Years And With Liver Fibrosis Stage F1 Were Significantly Associated With The Presence Of Occult HCV Infection. Conclusions. Patients With Elevated Liver Enzymes And Negative HCV Antibodies And Negative Serum RNA May Have Intra-Hepatic HCV RNA. The Chance Of Occult HCV Infection Increases If The Patient Has Previous History Of Sharing Needles, Elevated Liver Enzymes, History Of Previous Surgeries, Male Sex And Above 30 Years Of Age.

16

NOVAERUS: CLOSING THE LOOP ON INFECTION CONTROL

POSTER Name E-mail Institution Country KOTOVAS ROBERTAS [email protected] Lithuania

Introduction and Objectives. Pathogens are constantly on the move. Scientific evidence shows that about one third of all nosocomial infections occur due to airborne transmission. Hand hygiene has reached an acceptance level of 50+% in hospitals, but yet infections are increasing, not decreasing. Is 100% hand hygienic compliance even achievable? Can we afford it? We spend billions every year in Europe, just training people how to wash their hands. Air is 99% of the volume of any space…yet we spend all our money on treating the 1%. We need to treat the real problem…the Air! Methods. Novaerus is the first scientifically proven system for airborne infection control. Utilising clinically proven Air Plasma Technology, Novaerus works twenty-four hours a day to eradicate all airborne viruses, bacteria, moulds, VOC and odours. Novaerus destroys even the most harmful contaminants; including MRSA, C-Diff, Norovirus and Influenza, among many others. Results. Novaerus plasma technology was tested in NASA „Ames research center“ and was found to be uniquely destructive to ALL Genetic Material. These results were further verified using the Synchrotron particle accelerator at Stanford University, which confirmed the total destruction of the microorganisms, and their constituent DNA material, after only a single pass through the Plasma Field. It works in real life environment too – many clinical trials allready done and more is on the way. Conclusion. While current hygiene protocols which focus on hand and surface hygiene are essential, adding highly efficient air sterilisation from Novaerus closes the “infection control loop” by radically reducing the number of pathogens in the air. Furthermore Novaerus leads to significantly reduced surface contamination, thus logically reducing the bio-burden on contact points. 1. This is a proven and safe technology, it saves lives. 2. It is designed to enhance current infection control practices, everyone is going to have to create an airborne infection control protocol in the future. 3. It requires no extra work and no maintenance. 4. Decline in pharmacy costs...Savings on expensive specialised HAI antibiotics lead reduced pharmacy costs of up to 20%. 5. Care About Patients...Eliminate HAIs for patients AND staff. Everyone wins.

17

THE IMPORTANT ROLE OF BEDDING (SOFT INVENTORY) IN INFECTION CONTROL

POSTER

Name E-mail Institution Country

KOTOVAS ROBERTAS [email protected] Lithuania

Introduction and objectives. Patient cross-infection and HAI’s in general pose a major problem for hospitals greatly increasing suffering and causing considerable expense. Bedding interiors colonized with bacteria, become a reservoir of infection, posing an increased risk of Hospital Acquired Infections. The World Health Organisation, in a comprehensive study, asserts that 9% of healthcare budgets are taken up with problems related to these phenomena. Standard hospital pillow designs are inherently flawed, enabling pathogen ingress through the cover fabric, side stitching, vents or a combination of all these entry points. Using latest available nano and other technologies, the time has come to solve these problems indefinitely. Methods. SleepAngel™ barrier bedding combats product contamination risks using patented and award winning PneumaPure™ Filter Technology, a microbiological filter that blocks pathogens while allowing the product to breathe. PureShield™ textile is strong, long lasting, waterproof, highly stain resistant. SafeWeld™ sews Pure sonic sealed, not sewn for protection. Easy to disinfect and clean, no need to wash. All those technologies used in one unique product. Results. SleepAngel™ products tested in Belgium “Centexbel”and Ireland Airmid healthgroup laboratories. The data genereated showed the product to be 100% effective barrier … to allergen, bacteria, fungi and viruses. PneumaPure™ Filter is proven to be resistant to penetration of MRSA, C.Diff, Norovirus, Pseudomonas. Resistant to water penetration (180mm head) after 10000 crumples. SleepAngel™ bedding is registered 1 class medical device. That is the one and only bedding in the world with such achievement. Tested not only in the lab, but in hospitals in UK, Scandinavia and others. All showing reduced rates in infections. Conclusion. SleepAngel™ - modern day healthcare facility standard; a reduction in number of pillows per bed: SleepAngel pillows are made with a much more substantial and supportive inner than cheaper alternatives. As a consequence, patients require less pillows per bed and hospital facilities can reduce their overall inventory of pillows. Replacement frequency of 36 months: materials used to make the SleepAngel pillow are proven in use to be extremely durable under normal conditions of wear and tear. In addition, because no fluids or contaminants can enter the inner of the pillow, loft and bulkiness is retained thereby extending it’s useful life. Both of the above result in significant cost savings for hospital facilities on conversion to using the SleepAngel pillow as standard. Savings arise both in initial product cost and also in ongoing laundry and maintenance costs. This is before considering any cost savings that would arise from a reduction in the incidence of HAI’s.

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PATHOGENIC RICKETTSIA DETECTED IN IXODES TICKS IN THE REPUBLIC OF BELARUS

POSTER Name E-mail Institution Country KRASKO Republican Research and Practical Center for Epidemiology [email protected] Belarus ANATOLI and Microbiology Republican Research and Practical Center for Epidemiology Kniazeva Olga [email protected] Belarus and Microbiology Timofeenko Republican Research and Practical Center for Epidemiology [email protected] Belarus Ksenia and Microbiology

The aim of our investigation was the study of the presence in Ixodes genus ticks of the pathogenic for human rickettsia circulating in the territory of the Republic of Belarus. To do this, using the method of PCR was investigated 750 ticks collected by the flag method in all regions of the country. Obtained in a PCR reaction with species-specific primers amplicons were sequenced and compared with Rickettsia spp. sequences in GenBank. As the targets for sequencing diagnostically significant regions of the rickettsia genome - fragments of genes encoding protein 17 kDa outer membrane protein, ompB and gltA proteins were chosen. Of the 750 analyzed ticks in 28.7% of cases (215 samples) the presence of DNA of Rickettsia spp. was determined. An analysis of the distribution of infected ticks for Rickettsia prevalence areas showed virtually the entire country. The highest infestation of ticks with Rickettsia spp. was identified in Minsk region - 37.8% from all collected ticks, in Grodno region - 37.5 % and in Brest region 36.7%. The sequencing of 11 DNA samples of rickettsia have revealed the presence of two samples from R.helvetica and nine samples from R.raoulti.

The obtained result suggests a significant contribution of pathogenic rickettsia in the tick-borne spread of infections and makes it relevant to the continuation and expansion of investigation.

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NEUROLOGIC MANIFESTATIONS OF HEPATITIS E (ANTI-HEV PREVALENCE IN PATIENTS WITH NEUROLOGICAL DISORDERS AND CLINICAL CASE)

ORAL Name E-mail Institution Country KYUREGYAN karen- Chumakov Institute of Poliomyelitis and Viruses Russian KAREN [email protected] Encephalitis Federation MALINNIKOVA Chumakov Institute of poliomyelitis and viral Russian [email protected] ELENA encephalitides Federation Federal Service for Supervision of Consumer Russian Polyakov Andrey [email protected] Rights Protection and Human Welfare in the Federation Belgorod region Chumakov Institute of poliomyelitis and viral Russian Mikhailov Mikhail [email protected] encephalitides Federation

Background and aim. Hepatitis E is one of the most intensively studied diseases. Of particular interest are the neurological manifestations of this infection, previously thought not possible for enteral hepatitis (hepatitis A E). The aim of the study was to determine the prevalence of HEV in patients of neurological wards of general hospitals, and a case report of hepatitis E confirmed by detection of HEV RNA, and, finally, the analysis of the data on the pathogenesis of neurological manifestations at hepatitis E. Methods. Anti-HEV (IgM and IgG) were tested in ELISA using commercial kits (“Diagnostic systems”, “Vector”, Russia). HEV RNA was tested in RT-PCR with primers to ORF2 region. HEV genotype was determined by sequencing of amplified ORF2 region of HEV genome. The diagnosis of acute hepatitis E was based on the standard criteria, taking into account following data: biochemical tests (bilirubin, ALT, AST); specific HEV markers, exception of the etiological role of viral hepatitis A, B, C, Epstein-Barr virus and cytomegalovirus, as well as the results of patient physical examination. The severity of the disease was determined by the magnitude of intoxication and jaundice. Retrospective analysis of 102 patients with acute hepatitis E was carried out. Anti-HEV was determined in patients (n=231, mean age 62.3 years) of neurological wards of hospitals from three regions of Central- European part of Russia. The control group (n=376) was composed of patients from other departments, who atched by age and sex. The demographic characteristics of control and study groups were similar. Results. Anti-HEV were detected in 41 patients out of 231 (17.7%) patients with neurological disorders. In control group anti-HEV prevalence was similar (19.4% (73/376), p= 0.67). Analysis of the medical records of 102 patients with a diagnosis of "acute hepatitis E" revealed one patient with neurological symptoms, which accounted for 0.98%. The patient, 59-year-old man, had a clinical diagnosis of acute hepatitis E confirmed by the detection of serum anti-HEV IgG and IgM, and genotype 3 HEV RNA. On day 10 of disease onset patient began to complain of pain in the large joints, numbness in the outer surface of the left forearm. Later joined by pain in the upper limbs, numbness of the hands. Poliradiculopathy signs appeared. Neurologist examination revealed the thoracic spine osteochondrosis. Symptomatic treatment was assigned. Over the next two weeks the patient's condition improved, biochemical parameters normalized, neurological disorders disappeared. At the 19th day of hospitalization the patient was discharged with recovery. Conclusions. Unlike the vast number of publications in our study the case of hepatitis E with neurological manifestations was confirmed by HEV RNA detection and sequencing. The relationship between HEV infection and the neurological symptoms is confirmed by the appearance of the latter at the beginning of the infection and its disappearance in convalescence. The high prevalence of anti-HEV in patients neurological wards does not mean that these patients are at an increased risk of HEV infection, as the similarly high anti-HEV prevalence was observed in control group. However, this fact does not exempt from the need for HEV markers testing in patients with central and peripheral nervous system disorders.

20

ESBL-PRODUCING ESCHERICHIA COLI AROUND US: THE PREVALENCE STUDY IN ANIMALS, FOOD PRODUCTS AND ENVIRONMENT

ORAL Name E-mail Institution Country RUZAUSKAS Lithuanian University of Health Sciences [email protected] Lithuania MODESTAS (LUHS) Siugzdiniene Rita [email protected] LUHS Lithuania Virgailis Marius [email protected] LUHS Lithuania Mockeliunas Raimundas [email protected] LUHS Lithuania Vaskeviciute Lina [email protected] LUHS Lithuania Klimiene Irena [email protected] LUHS Lithuania

β-lactamases confer resistance to penicillins and cephalosporins and are an emerging cause of multidrug resistance in gram-negative bacteria. In E. coli an extensive horizontal gene transfer by plasmids occurs, contributing to a noticeable genome plasticity in the species. As a result, E. coli has an ability to colonize and survive in many different locations. E. coli can easily spread in different ecosystems through animal husbandry, wildlife and food chain. β-lactams, including 3d generation cefalosporins are used for treatment of food-producing animals and pets. The aim of the study was to evaluate the prevalence of ESBL-producing E. coli in different environments, including food producing animals, pets, food products, soil and wild birds. Methods. Overall 1373 samples included clinical and pathological material of horses (n=401), slaughtered broilers (n=224), dogs (n=480), poultry products intended for human consumption obtained from retail markets (n=130), soil (n=8) and faeces of wild birds (n=130). The material was inoculated onto McConkey Agar (Thermo Fisher, UK) as well as on Colorex Chromogenic Extended Spectrum Beta-Lactamase (ESBL) medium (E&O Laboratories, Scotland) for the isolation of ESBL-producing and non ESBL- producing E. coli. Identification of the isolates was performed using biochemical testing with the following sequencing of 16S rRNA in case of uncertain identification. Susceptibility testing was performed using cefpodoxime disks as well as Sensititre plates with different antimicrobials (including cephalosporins, other β-lactams and β-lactamase inhibitors) and automated system ARIS 2X (Thermo Scientific, UK). Interpretation of results was performed according to EUCAST clinical breakpoints. Genes encoding resistance to β-lactams were detected by PCR. Results. From the 1373 samples tested E. coli was isolated in 752 cases (54.8%). Two hundred and eighty nine isolates (38.4% from all isolates of E. coli) were able to grow on Chromogenic ESBL medium and demonstrated resistance to cefpodoxime, while susceptibility to other cephalosporins varied between the isolates. The highest number of ESBL-producing E. coli was detected in clinical material of horses (48.8%), raw poultry products (47.6%) and broilers (38.0%). Sick dogs also were carriers of ESBL- producing E. coli (34.4%). No E. coli was found in soil samples, while 17 wild bird samples out of 100 have been found to be positive for ESBL-producing E. coli. The most prevalent genes encoding resistance to β-lactams were blaTEM (up to 86.7%; mostly found in dogs), blaSHV (up to 20.3%; mostly found in broilers) and CTX-M (up to 62.7%; mostly found in broilers). OXA-type β-lactamases also were found in this study (51.0% in dog isolates). Plasmid mediated AmpC type β-lactamases (cmy-2 gene) were detected in E. coli isolates from all sources but most often from poultry products (54.8%). Conclusions. ESBL-producing E. coli carrying plasmid mediated resistance genes are widely distributed outside, but in a close contact of humans: pets, domestic and wild birds, food products and food-producing animals. The resistance can be transferred between and within bacterial species as well as between different hosts. Thus, beta-lactams may become less and less effective antimicrobials both for humans and animals. Acknowledgements. The study was funded by a grant (SIT-06/2015) from the Research Council of Lithuania

21

ANTIMICROBIAL RESISTANCE OF INVASIVE ISOLATES IN LITHUANIAN HOSPITALS

ORAL Name E-mail Institution Country National Public Health Surveillance VIZUJE GRETA [email protected] Lithuania laboratory National Public Health Surveillance Miciuleviciene Jolanta [email protected] Lithuania laboratory

Aim. To evaluate bacteria - Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp. - isolated from blood resistance to antimicrobial drugs in Lithuanian acute care hospitals in 2014 and 2015. Methods. 12 microbiology laboratories in 2014 and 14 in 2015 provided information about invasive isolates antimicrobial resistance to National Public Health Surveillance Laboratory which coordinates National antimicrobial resistance surveillance network in Lithuania. Data analysis was performed using a computer program WHONET the English version. Results. S.aureus isolated from blood were resistant to meticillin/oxacillin 7,9 % (382/30) in 2014 and 9,94 % (463/46) in 2015. E.faecium resistant to vancomycin identified 4,5 % (40/2) in 2014, 21,3 % (75/16) in 2015. E.coli resistance to third-generation cephalosporins increased in 2015 (cefotaxime 17,68 % (560/99), ceftazidime 13,48 % (660/89)), as compared to 2014 (cefotaxime 8.4 % (479/40), ceftazidime 6.4 % (578/37)). Carbapenem-resistant E.coli was not found in both survied years. K.pneumoniae in 2014 and 2015 resistance to third-generation cephalosporins remained similar, respectively: cefotaxime 54.2% (118/64) and 55.4% (166/92), ceftazidime 49.7% (151/75) and 54.6% (218/119). Carbapenem-resistant K.pneumoniae 1.3% (152/2) were found in 2014. P. aeruginosa resistance decreased in 2015 to piperacillin / tazobactam (32.3% - 2014; 24.6% - 2015), ciprofloxacin (25.8% - 2014; 21.1% - 2015) and imipenem / cilastatin (30.0 % - 2014; 26,78% - 2015 ), increased to ceftazidime (16.7% - 2014; 17.5 % - 2015). Acinetobacter spp. resistance to carbapenems increased from 2014 to 2015: imipenem / cilastatin. - 72.6% (62/45) in 2014, 80.43% (92/74) in 2015 and meropenem 68.7% (64/44) in 2014, 82.0% (100/82) in 2015. Conclusions. More than four times increased E.faecium resistance to vancomycin and more than two times increased resistance of E. coli to third-generation cephalosporins. Antimicrobial resistance of other observed invasive bacteria (K.pneumoniae, Acinetobacter spp.) in the past 2 years did not change significantly and persists high. Therefore, antimicrobial resistance remains a significant problem in Lithuanian health care institutions.

Acknowledgements. We would like to acknowledge all microbiology laboratories for providing antimicrobial susceptibility data of invasive isolates to National Public Health Surveillance Laboratory.

22

ZIKA VIRUS - AN OVERVIEW

ORAL Name E-mail Institution Country ANDERS WIDELL [email protected] Lund University Sweden

Emerging viral epidemics from animals are unpredictable but often due to changed habitat of vectors, which in turn may be due to deforestation, travel and climate changes. The most recent additions on the list is Zika virus (ZIKV). Although first identified in a febrile monkey in the Zika forest in 1947, ZIKV left little epidemiological imprint in the human disease panorama for 50 years, although found in several countries of tropical Africa and Asia. In 2007 ZIKV was unexpectedly found to infect 75% of Micronesia Yap islanders by Aedes mosquito bites. Symptoms were mild with maculopapular rash, low degree fever, conjunctivitis, and joint and muscle pain but the infection could often pass without symptoms. The 10.8 kb viral genomes in several strains were soon sequenced, displaying a typical flavivirus genome, and an African and an Asian genotype were found. In 2013 ZIKV was found in epidemics spreading east with Aedes sp to many Pacific island nations including, French Polynesia, Cook Island, and Easter Island. In 2015 the ZIKV epidemic arrived in South America, infecting 1.5 million Brazilians and spreading throughout Latin America – and then to Cap Verde Islands. The ZIKV infection was considered benign until 2 severe complications were foundnot reported earlier from ZIKV-endemic countries. These two were microcephaly and/or other neurological damage in fetuses/newborns and Guillain-Barré Syndrome (GBS) in adults. This sparked a major WHO driven initiative to control ZIKV. The host range is humans and non- human primates. Several Aedes species in particular Aedes Aegypti and Albopictus serve as vectors, and ZIKV replicates to high levels in the Aedes salivary glands, infect the mosquito saliva. ZIKV can be grown in Vero cells, human skin fibroblasts, keratinocytes, immature dendritic cells, progenitor neural cells and in neurons. At least one human ZIKV receptor, the TAM receptor has been found and is shared with dengue virus. The virus has been visualized in brain tissue by EM and resolved to 3.8 Å by cryo-EM, demonstrating potential receptor binding structures in the viral envelope protein. Data are still incomplete but if a pregnant woman during trimesters I and II is infected by ZIKV there is a 1-2 % risk that maternal viremia passes placenta and infects the fetal brain and causes microcephaly and/or brain damage like tissue loss, calcifications, enlarged ventricles, agenesis of corpus callosum end cerebellum. These very severe sequelae have been observed following the outbreaks in both French Polynesia and Brazil and can be interpreted as ZIKV invasion of growing fetal neural cells. GBS in adults is thought to be immunological. Diagnosis is based ZIKV RNA detection in blood, urine and saliva by PCR during the first 7-10 days, thereafter shifting to specific IgM and IgG serological tests. No vaccine is available, but a recombinant Yellow Fever/ZIKV vaccine is explored – this principle has proven successful for dengue vaccine.

23

AN OUTBREAK OF SALMONELLA ENTERITIDIS ASSOCIATED WITH PEAS WITH CR ACKLINGS

POSTER Name E-mail Institution Country ZUKAUSKAITE- Centre for Communicable Diseases and [email protected] Lithuania SARAPAJEVIENE SIMONA AIDS (ULAC) Zagrebneviene Galina [email protected] ULAC Lithuania Svambariene Rita [email protected] ULAC Lithuania Caplinskas Saulius [email protected] Mykolas Romeris University, Lithuania Centre for Communicable Diseases and AIDS

Background. Salmonella Enteritidis is the third most commonly notified cause of gastroenteritis outbreaks in Lithuania. The outbreaks of Salmonella infection are commonly associated with chicken meet, bakery, eggs. Annually Salmonella outbreaks are registering in families, children’s educational institutions, catering establishments. In rare cases Salmonella outbreaks, associated with a mobile retailer, are registering. In 2015 one of the biggest Salmonella Enteritidis outbreaks has been registered in the city of Siauliai. This abstract describes investigations conducted during this Salmonella gastroenteritis outbreak. Objective. To describe an outbreak and identify the vehicle of transmission. Materials & Methods. The descriptive epidemiology and a case-control study were undertaken to identify the vehicle of transmission. The data was analyzed by WinPepi program. The microbiological testing was performed on faecal, food and environmental samples. Results. There were 53 gastroenteritis cases registered from 5 June to 15 June 2015. All ill people were music festival participants. Children up to 16 years old accounted for 15% of all fallen ill patients. 47% of outbreak cases were women and 53% were men. 21 patients (39,6%) were admitted to hospital. Salmonella Enteritidis was identified for 23 cases out of 29 cases tested. Salmonella Enteritidis isolates were the same susceptible to cefatoxim, norfloxacin, trimethoprim-sulphamethoxazole. The probable vehicle of transmission was traced to peas with cracklings produced by a cafe-bar and sold in a music festival. Results of the case-control study based on 53 cases and 106 controls (p<0,000, OR = 47,50, 95% confidence interval [CL] = 12,793-397,910). No other food items or risk factors were implicated. During the outbreak investigation the national food and veterinary service of Siauliai specialists identified many violations of hygiene and food safety in a cafe-bar. A cafe-bare was temporarily closed. Faecal specimens from 4 cafe-bars staff members were tested. Bacterial enteropathogens were not isolated. All cafe-bar environmental swabs were negative for Salmonella. Salmonella spp. was isolated from chicken meet found in a cafe-bar. Conclusions. 1.The study showed that the Salmonella outbreak may be due to non-animal food. 2.The cross-contamination of equipment and work surfaces, the inadequate food thermal processing, storage and transportation issues, workers’ lack of hygiene could lead to the development of the outbreak.

24

BLACKFLY CONTROL IN LITHUANIA, 1999 -2015

POSTER Name E-mail Institution Country ZYGUTIENE MILDA [email protected] Centre for Communicable Diseases and AIDS Lithuania Bernotiene Rasa [email protected] Centre for Communicable Diseases and AIDS Lithuania Caplinskas Saulius [email protected] Mykolas Romeris University, Lithuania Centre for Communicable Diseases and AIDS

Background and Aims. The outbreak of bloodsucking blackflies began in the 70s of the 20th century in the South-eastern part of Lithuania. By 1990, the biting activity of bloodsucking blackflies increased and had become a serious problem. The bloodsucking insects caused losses of cattle and birds and annoyed holiday-makers of the Druskininkai health-resort in 1998-2003. Blackflies female are aggressive biters. This blood feeders bites can itch and persist for several days. Allergic reactions and bacterial skin infections may occur from bites and scratching the bites. Reactions to blackfly bites are known as "blackfly fever" include headache, nausea, fever, and swollen lymph nodes in the neck. The last outbreak of bloodsucking blackflies in South-eastern part of Lithuania induced investigations of Simuliidae in Lithuania and the main pest species in the South-eastern part of Lithuania was determined as Simulium maculatum Mg. Larvae of S. maculatum developed in the largest river of Lithuania, the Nemunas River. Materials & Methods. The abundance of blackfly larvae was estimated on water plants. Uniform, band- shaped leaves of Glyceria maxima (Hartm.) were collected from the stream at a depth up to 1.5 m, and the abundance of blackfly larvae and pupae per 1 dm2 of the leaf surface was determined. Three samples were collected each time, and the mean density of blackfly larvae and pupae of each species and each larval instar was determined. Adult bloodsucking blackflies were collected from several persons in a 10-min period after 5 min of waiting. Results. Development of S. maculatum larvae starts usually in the end of April - beginning of May. Imagines of blackflies attacked people and cattle in June – July. The highest abundance of S. maculatum larvae were detected in the middle reaches of the Nemunas River from Grodno (North Belarus) to Birštonas and Kaunas hydro-electric power plant (centre Lithuania). Less density of S. maculatum larvae was determined in the lower reaches of the Nemunas River. S. maculatum produced two generations per year. The first generation developed in April – June and the second one developed in the August. The first generation was very abundant and the second generation was not numerous. Blackfly control with preparation VectoBac 12AS was started in Lithuania in the Nemunas River in 1999 and was used till 2010 afterward stopped for five years and was reactivated in 2016. The preparation is based on the effect of Bacillus thuringiensis var. israelensis. VectoBac 12AS was poured into the river from the bank in single point in 2000 - 2010. The efficacy of application was from 95.9% (in 25 km distance from the point of application) to 49.8% (in 164 km distance from the point of application). As a consequence the density of S. maculatum larvae was decreasing each year after the 1999. S. maculatum larvae density was 2042.8 ± 282 / 1dm2 of water plant surface in 1999 and it was reduced to 22.2±7/1dm2 of water plant surface in 2011. The highest bloodsucking activity of blacklfies was 356.7 ± 112,6 per 10 min in 1999 but during 2000 – 2011 the rate reduced to 3.9 ± 1,9 per 10 min. During five years when treatment of S. maculatum was stopped, density of larva regenerated and in 2015 it was 290 ± 183 / 1dm2 while activity of imago made 143±52 per 10 min. Conclusions. Blackfly control in Lithuania resulted a significant decrease in blackfly population during 1999- 2010 but after control was stopped S. maculatum regenerated.

25 2. Vaccine preventable diseases, vaccinology and biotechnology

Frelin Lars Improving vaccine immunogenicity and development of new Invited animal models of HCV infection Kozlov Andrei Preliminary results of phase II clinical trial of therapeutic DNA Invited vaccine against HIV/AIDS Tangy Frederic Development of live measles vaccine vector for new emerging Invited pathogens Wahren Britta Vaccine developments for viral hepatitis and HIV Invited

Baguzyte Izabele Adult risk group patients‘ vaccination guidelines adherence in Poster hematopoietic stem cell transplant recipients Banzhoff Angelika Vaccination against invasive meningococcal B disease: Oral international experience Berzina Anita Comparing efficacy of electroporation and lipofection in Poster eukaryotic cell lines using gene of fluorescent reporter Caplinskas Saulius Overview of side reactions to vaccines in Lithuania in 2015 Oral Gordeichuk Ilya Assessment of T-cellular immune responce in common marmosets Oral (Callithrix jacchus) following single vaccination with prototype vaccine against hepatitis E virus Hippe Laura Approbation of FRET system for studying CRAC ion channel Poster Isaguliants Maria Immunotherapy against drug resistance as a complement to Oral HAART Jansons Juris Immunogenicity in mice of plasmid DNA encoding HCV core and Poster alternative reading frame proteins Kainov Denis Analysis of available whole-genome sequences of influenza A Poster viruses allowed identification of novel antiviral therapeutics and selection of novel vaccine strain candidates Korabliov Pavel Improving cellular immunotherapeutic vaccines efficacy with gene Poster suppression in dendritic cells and T- lymphocytes Latanova Anastasia High immunogenicity in mice of plasmids encoding consensus Oral reverse transcriptase of HIV-1 clade A with primary drug resistance mutations Lyashenko Anna Steps towards the development of hepatitis E vaccine Oral

Moriizumi Yasuhiro Electroporation and peripheral technology: from DNA Oral immunotherapy to genome editing Simopekka Vänskä HPV vaccination and cervical cancer screening in Finland Oral

26

IMPROVING VACCINE IMMUNOGENICITY AND DEVELOPMENT OF NEW ANIMAL MODELS OF HCV INFECTION

INVITED LECTURE Name E-mail Institution Country

Division of Clinical Microbiology, Laboratory Medicine FRELIN LARS [email protected] Sweden Karolinska Institutet (LM/KI), Stockholm

Ahlén Gustaf [email protected] LM/KI, Stockholm

sepideh.levander@ki. Levander Sepideh LM/KI, Stockholm se Weiland Ola [email protected] LM/KI, Stockholm

Sällberg Matti [email protected] LM/KI, Stockholm

Background. The hepatitis C virus (HCV) is a major cause for chronic liver disease worldwide. Despite effective therapies it is estimated that only 10 % of all chronic HCV infected individuals receive any therapy. Hence, new therapies are needed to increase the overall cure rate of HCV. One suggested treatment-regimen is therapeutic vaccination. It is well known that strong T cell responses are involved in clearance of HCV infection, whereas patients that progress to chronic HCV have severely impaired immune responses. The idea of therapeutic vaccination is to reactivate the dysfunctional HCV-specific T cell responses in the chronic carrier. We have developed and improved the immunogenicity of HCV NS3/4A-based DNA vaccines in both pre-clinical and clinical studies. One major obstacle in in vivo delivery of DNA vaccines is the transfer of the material over the membranes of the cell. To overcome this problem we have developed a new delivery technology, named In vivo Intracellular injection (IVIN) device. The combination of optimized vaccine compositions and improved delivery technologies have been evaluated in different animal models. Method. We have developed a codon-optimized (co) HCV NS3/4A DNA vaccine (gt1a) expressed from the pVAX1 plasmid. The vaccine was delivered intramuscularly using a regular needle or the IVIN device in combination with in vivo electroporation. The NS3/4A vaccine was further optimized by addition of stork hepatitis B virus core gene sequences and co-expression of interleukin 12 (IL-12). Safety, protein expression and immunogenicity were evaluated in mice, rabbits, and pigs. Results. Our NS3/4A-based DNA vaccine have been evaluated in two clinical trials showing that the treatment was safe and tolerable, activated HCV-specific immune responses and had transient effects on the viral load. However, therapeutic vaccination alone did not cure the HCV infected patients. Thus, we have developed new improved HCV NS3/4A-based DNA vaccines and delivery technologies. These improvements have resulted in significantly enhanced HCV-specific immune responses compared with previously evaluated vaccines. In addition, our vaccines showed potent anti-HCV activity in an immuno- competent mouse model of HCV RNA replication. Conclusion. We have shown that our optimized HCV NS3/4A vaccine in combination with IVIN delivery significantly improves the vaccine immunogenicity. Our findings support the use of NS3/4A in HCV vaccine compositions.

27

PRELIMINARY RESULTS OF PHASE II CLINICAL TRIAL OF THERAPEUTIC DNA VACCINE AGAINST HIV/AIDS

INVITED LECTURE Name Institution Country The Biomedical Center, St.Petersburg Russian KOZLOV ANDREI Research Institute of Pure Biopreparations, Federation St.Petersburg (RF)

iPharma LLC, Moscow Murashev BV

Verevochkin Sergey

Masharsky Alexey

Akulova Ekaterina

Al-Shekhadat RI

Poddubnyy VA

Zozulya OV

Vostokova NV

Introduction. Despite the tremendous efforts HIV pandemic is not stopped. That is why the HIV vaccine is needed. The Biomedical Center in collaboration with Research Institute of Pure Biopreparations developed DNA vaccine "DNA-4". This vaccine is a mixture of four plasmids containing Nef, Gag, Pol (rt) and gp140 HIV genes. In 2011, the vaccine "DNA-4" successfully passed Phase I clinical trial, where its safety and immunogenicity were demonstrated. Todate the vaccine "DNA-4" has passed Phase II clinical trial conducted as "A multicenter double-blind placebo-controlled study of the safety, immunogenicity and selection of the optimal dosage of “DNA-4” vaccine in patients with HIV-1, receiving stable first line antiretroviral therapy" in lines with goverment contract № 13411. 1008799. 13.160 of 25/07-2013 between Biomedical Center and Ministry of Industry and Trade of RF. Objective. The objectives of the study were safety and immunogenicity assessment of different "DNA-4" vaccine dosage regimens in patients with HIV-1, receiving stable first line antiretroviral therapy (ART), and evaluation of vaccine influence on the dynamics of viral load and level of CTLs and helper T cells. Methods. In the study the safety of different "DNA-4" dosage regimens in patients with HIV-1, receiving stable first line antiretroviral therapy (ART) were evaluated. The frequency and severity of adverse effects reported during the treatment and follow-up periods was studied.To evaluate the immunogenicity the level of cytotoxic (CD3+CD8+) and helper (CD3+CD4+) T cell and viral load dynamics including the frequency of viral load increase above 50 copies/ml («blips») were measured. Results. 54 patients were randomized in the study (17 patients in the DNA-4 0.25 mg group, 17 patients in the DNA-4 0.5 mg group and 20 patients in the placebo group). Patients in each group were immunized four times at days 0, 7, 11, 15 followed by the follow-up period during 22 weeks. By February, 2016 all participants successfully completed the study with no serious adverse effects. In several vaccinated patients (2 patients from 0.25 mg group and 1 patient from 0.5 mg group) the increase in the magnitude of viral “blips” was found indicating the possible latent viral reservoirs activation in these patients. This might be caused by the induction of cell immunity (CD8+ T cell activation after immunization with DNA vaccine), and the expression of TNFα in vaccinated patients as shown by previous studies. Conclusions. DNA-vaccine against HIV-1 “DNA-4” is safe and well tolerated by HIV infected patients receiving ART. The data suggest the possible influence of the DNA-4 vaccine on viral reservoirs. This hypothesis requires further investigation and analysis all available data and clinical samples.

28

DEVELOPMENT OF LIVE MEASLES VACCINE VECTOR FOR NEW EMERGING PATHOGENS

INVITED LECTURE Name E-mail Institution Country TANGY FREDERIC Institut Pasteur, [email protected] France Viral Genomics and vaccination, Paris

Over the last two decades a number of new viral diseases such as SARS, MERS or Nipah virus infections have emerged at the animal-human interface, and infections such as Ebola and Lassa viruses, or recently Zika virus, have expanded beyond their usual limited territories. Each time, the global community failed to develop effective interventions in a timely manner. We developed the use of one of the safest and most efficacious vaccines available, the live attenuated measles vaccine, as a platform for the delivery of these new antigens. Measles vaccination has been used for more than 40 years in over 1 billion children and is 95% efficacious after one or two administration. Measles vaccine is genetically stable and reversion to pathogenicity has never been observed. Taking advantage of these characteristics, we cloned the attenuated measles Schwarz vaccine virus and developed a method to genetically manipulate this negative strand RNA virus into a versatile chimeric or recombinant vector. Proof of concept in humans for this technology has recently been demonstrated for a measles-Chikungunya vaccine. A Phase I clinical trial showed that the vaccine was well tolerated and induced a robust and functional antibody response after 1 (90%) or 2 immunizations (100%). This trial also demonstrated that pre-existing measles antibodies did not impair the immunogenicity of the heterologous antigen (Ramsauer, 2015, Lancet Inf. Dis.). Thus, pre-immunity to measles due to vaccination or infection will not restrict the use of recombinant measles vector for new vaccines. Many other antigens from HIV, DENV, CHIKV, WNV, SARS, H5N1, Ebola, Zika, and Plasmodium have been expressed in this vector and their strong immunogenicity or protective capacity has been established in preclinical animal models, also in the presence of pre-existing measles immunity, highlighting the potential of measles vector as a platform for rapid response to new pathogens.

VACCINE DEVELOPMENTS FOR VIRAL HEPATITIS AND HIV

INVITED LECTURE Name E-mail Institution Country BRITTA WAHREN [email protected]

The aim of this presentation is to make an overview of the status of HIV and HCV vaccine trials. These viruses have some replication properties in common, and for both there is now successful drug treatment (HCV) or means to convert the infection into a chronic stage (HIV). Thoughts on construction of vaccines, timing and devices for immunsations, prime-boost attempts, adjuvants for innate and adaptive immune responses, and relevant non-human primate studies will be presented, together with the possibilities to use a combination of vaccine and antiviral treatment in prophylaxis or therapy.

29

ADULT RISK GROUP PATIENTS’ VACCINATION GUIDELINES

ADHERENCE IN HEMATOPOIETIC STEM CELL TRANSPLANT RECIPIENTS

POSTER Name E-mail Institution Country BAGUZYTE [email protected] Vilnius University Faculty of Medicine Lithuania IZABEL Vilnius University Hospital Santariskiu klinikos Jancoriene Ligita [email protected] Lithuania Centre of Infectious Diseases Vilnius University Hospital Santariskiu Clinics Dieniniene Jolanta [email protected] Lithuania Centre ofI nfectious Disease Abelkis Eimantas [email protected] Vilnius University Faculty of Medicine Lithuania

Background Number of hematopoietic stem cell transplantation (HSCT) performed in Lithuania is increasing significantly. HSCT recipients are more susceptible to vaccine-preventable diseases, therefore, international guidelines for their vaccination were developed. In Lithuania, guidelines were published as a part of recommendations for vaccination of adult risk group patients. Aim of our study was to evaluate whether 1st dose of vaccines and intervals between vaccinations were compliant with vaccination guidelines. We also evaluated which sets of vaccines are used most often. Methods. Retrospective analysis included autologous and allogeneic HSCT recipients vaccinated at Vilnius University Hospital Santariskiu Clinics, Centre of Infectious disease. We analyzed medical documents of patients who received vaccines from 01/09-2013 to 05/04-2016. Vaccines received by HSCT recipients were compared to vaccination guidelines for adult risk group patients (Lithuania, 2015). Results. 31 patients were included in the study. 14(45,2%) women and 17(54,8%) men. Mean of age (years) 48,48±14,436. Time after HSCT at the moment of evaluation: 11(35,3%) patients ≥24 months, 15(48,4%) 13-23 months, 5(16,1%) ≤12 months. 16(51,6%) patients received first vaccine ≤6 months after HSCT, 11(35,5%) 7-12 months after HSCT, 4(12,9%) >12 months after HSCT. 30(96,8%) patients received first dose of PCV vaccine ≥3 months after HSCT. 26(83,9%), 24(77,4%), 21(67,7%) patients received first dose of Td, IPV, HiB vaccines, respectively, ≥6 months after HSCT. 5(16,1%) patients were vaccinated with first dose of HiB vaccine <6 month after HSCT. 5(16,1%), 7(22,6%), 5(16,1%) patients were not vaccinated with Td, IPV, HiB vaccines, respectively. MenB vaccine was inoculated to 1(3,2%) patient, 0 patients were vaccinated with MenC vaccine. 23(74,2%) patients received first dose of Hepatitis B vaccine ≥6 months after HSCT, 8(25,8%) were not vaccinated. 5(16,1%) patients were vaccinated with MMR vaccine ≥24 months after HSCT, 26(83,9%) were not vaccinated. 2(6,5%) patients received first dose of Tick-borne encephalitis vaccine ≥24 months after HSCT, 29(93,5%) were not vaccinated. 9(29,0%) patients were vaccinated with influenza vaccine at least once. Intervals between doses: I and II PCV 12(38,7%) ≥2 months; between II and III 1(3,2%) ≥6 months. Between I and II Td 14(45,2%) ≥2 months, 7(22,6%) <2 months; IPV 13(41,9%) ≥2 months, 7(22,6%) <2 months; HiB 8(25,8%) ≥2 months; between II and III Td 9(29%) ≥6 months, 2(6,5%) <6 months; IPV 9(29%) ≥6 months, 2(6,5%) <6 months. Between I and II Hepatitis B vaccine 20(64,5%) ≥1 months, between II and III Hepatitis B vaccine 14(45,2%) ≥5 months, 1(3,2%) <5 months. Conclusions. The beginning of vaccination were delayed for almost half of the HSCT recipients. It can be caused by lack of information about existing guidelines. Intervals between vaccines‘ doses were compliant with vaccination guidelines. Majority of patients were vaccinated PCV, Td, IPV, HiB, Hepatitis B vaccines, unfortunately, only few patients were vaccinated MMR, Tick-borne encephalitis, MenB vaccines. It can be caused by economical factors. Appropiate patients and medical personnel education, financial compensation for purchasing vaccines should be considered as possible measures to improve vaccination guidelines adherence in HSCT recipients.

30

VACCINATION AGAINST INVASIVE MENINGOCOCCAL B DISEASE: INTERNATIONAL EXPERIENCE

ORAL Name E-mail Institution Country BANZHOFF ANGELIKA Global Medical Affairs, GSK

Serogroup B meningococcal disease affects mainly infants, is easily misdiagnosed, can kill within 24 hours of onset, and may cause serious, lifelong disabilities despite appropriate medical treatment. Vaccination may be the best approach for prevention against an aggressive disease that leaves little time for intervention. BEXSERO is a novel meningococcal serogroup B vaccine for active immunization against invasive MenB disease. In clinical trials, BEXSERO has demonstrated a protective immune response in infants, children, adolescents, and adults with or without routine vaccines. BEXSERO has a demonstrated safety profile based on an evaluation of >67,000 subjects in clinical trials and postmarketing studies (from 2 months of age). Results from 9 European countries, Australia, the United States, Brazil, and Canada on nearly 2700 MenB strains estimate that 66%–91% of strains would potentially be covered by BEXSERO.

COMPARING EFFICACY OF ELECTROPORATION AND LIPOFECTION IN EUKARYOTIC CELL LINES USING GENE OF FLUORESCENT REPORTER

POSTER Name E-mail Institution Country Gamaleya Research Center of Epidemiology Anastasia [email protected] and Microbiology, Moscow, Russia; Russia Latanova Karolinska Institutet, Stockholm Anastasia Anastasia.dovbenko@biome Biomedical Research and Study Center, Riga Latvia Dovbenko d.lu.lv ANITA A. Kirchenstein Institute of Microbiology and [email protected] Latvia BERZINA Virology (KIMV), Riga Stradiņš University Laura Hippe [email protected] KIMV, Riga Stradiņš University Latvia Arnis Strods [email protected] Biomedical Research and Study Center, Riga Latvia

Martins Kalis [email protected] KIMV, Riga Stradiņs University Latvia

Pavel p.bankovsky@bioscienceme Bioscience Media Company, Riga Latvia Bankovsky dia.lv Yasuhiro [email protected] BEX Co., Ltd., Tokyo Latvia Moriizumi Maria KIMV, Riga Stradiņs University, and [email protected] Latvia Isaguliants Karolinska Institutet, Stockholm Juris Jansons [email protected] Biomedical Research and Study Center, Riga Latvia

31 Introduction: Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into biological, chemical, and physical. Each method has advantages and disadvantages; the choice depends on the experimental design and objectives. Electroporation (EP) is a method for the transfection by the application of the electric field which makes temporary holes in the membranes of the cells, allowing transportation of the desired substances, gene, drugs, etc. inside the cells. EP is a very useful and convenient method for transfection because it does not rely on the availability of chemicals, and their quality, and provides high reproducibility. However, it may damage target cells. The key to EP success is fine-tuning of the parameters adjusting them to the individual properties of the target cells. Aims: Compare the efficacy of gene transfection by EP to transfection with cationic lipids. Methods: Transfection experiments were done on eukaryotic cell lines HеLa and HEK293T. EP was performed using electroporator CUY21Edit II and electroporation chamber and unique electrodes with two to three stainless plates by BEX Co. Ltd. (Japan). Chemical transfection was performed using Lipofectamin LTX. Cells were transfected with plasmid DNA encoding near-infrared reporter iRFP670 (kind gift of Prof Verkhusha, Albert Einstein College of Medicine, USA). Expression of iRFP670 was monitored in cell culture monolayer using FLUOROWATCHER device, provided for testing by Bioscience Media Company (Latvia). Number of transfected and dead cell was assessed by flow cytometry using BD FACSAria II. Results: Cells were electroporated using regimes with varying voltage and length of pulses. Control cells were transfected with Lipofectamine LTX. Expression of iRFP670 was detected 24h post-transfection by experimental device FLUOROWATCHER which excitated cell culture monolayer using laser, and collected the released fluorescence signal with a CCD camera. Hour 48 post-transfection cells were harvested and subjected to analysis by flow cytometry. Both in HeLa and HEK293T cells, the highest percent of expressing cells was obtained using regimens with the voltage 225V and duration of 5 ms. Lower voltage and longer pulses were less efficient. For HeLa the electroporation was more traumatic than lipofection. Increase of voltage led to extensive cell death resulting in a higher percent of cells transfected by lipofectamine: 61% compared to 41% by EP. Interestingly, HEK293T demonstrated an opposite effect, with lipofectamine transfection and EP generating comparable results, 71% and 65%, respectively. Conclusion: Parameters of EP could be adjusted to give same efficacy of cell transfection as demonstrated by cationic lipids as lipofectamine. Acknowledgements: Authors acknowledge technical support by BEX Co., Ltd. (Japan) and Bioscience Media Company (Latvia) and financial support from the projects BALTINFECT, VACTRAIN and Swedish Institute 09272_2013

32

OVERVIEW OF SIDE REACTIONS TO VACCINES IN LITHUANIA IN 2015

ORAL Name E-mail Institution Country CAPLINSKAS SAULIUS [email protected] Mykolas Romeris University, Lithuania Centre for Communicable Diseases and AIDS Zukauskaite Kristina [email protected] Centre for Communicable Diseases and AIDS Lithuania Kriauciunaite Asta [email protected] Centre for Communicable Diseases and AIDS Lithuania Sebeliauskaite Ieva [email protected] Centre for Communicable Diseases and AIDS Lithuania Korablioviene Joana [email protected] ULAC Lithuania

Background. A side reaction to vaccine (hereinafter – SRV) is an undesirable and negative response of the organism to medical preparation manifesting during the use of the usual dose of a registered medical preparation for the prophylaxis, diagnostics or treatment of human disease or restoration, correction or modifica-tion of a physiological function or any dose of the researched medical preparation (Law on Pharmacy of the Republic of Lithuania, 22nd of June 2006.No.X-709). The World Health Organization classifies SRVs as easy, severe or severe-manifesting together with easy reactions. Severe SRVs are the following ones: fatal; dangerous to life; causing a need for treatment, hospitalization or prolongation of hospital treatment; causing constant or significant disability, working inability or innate abnormality, innate defect. Objective. To overview the situation of side reactions to vaccines in Lithuania in 2015. Materials & Methods.The registration of side reactions to vaccines is performed in Lithuania in accordance with Order No. 673 of the Minister of Health Care of the Republic of Lithuania of the 24th of December 2002 “On the approval of the order of obligatory epidemiologic registration, obligatory content of epidemiologic reg-istration objects and obligatory transfer of information” (Official Gazette, 2003, No.12-444; 2004, No.82-296; 2008, No.89-3585; 2011, No.14-604) and Order No. V-185 of the Minister of Health Care of the 20th of February 2013 “On the description of the order of submission of a notification of a health care or pharmacy specialist about a suspected side reaction (SSR), form of a notification of a health care or pharmacy specialist about a suspected side re-action (SSR) and form of a notification of a patient about a suspected side reaction (SSR) (Official Gazette,2013,No.12-444). Results. 54 severe SRVs and 11 easy SRVs were registered in 2015 and 48 severe SRVs and 32 easy SRVs – in 2014. Most registered SRVs (66,2 per cent; 43 cases) were related with a BCG vaccine that caused lym-phadenitis. The second frequent SRV was fever (26,2 per cent; 17 cases). It was recorded that the reaction had been caused by Bexser, Engerix B, Pentax-im, Synflorix and TicoVac vaccines. In 2015, the number of SRVs among men (36cases) was lower by 1,2 times than in 2014 (42 cases). In 2015, the number of SRVs among women (29 cases) was lower by 1,3 times than in 2014 (38 cases). In 2015 most SRVs were registered in the group of persons un-der one (49 cases; 75,4 per cent), more than one fifth of SRVs (14 cases; 21,5 per cent) were determined for 1-5 year-old children and the lowest number of SRVs – for 6-17 year-old children (1case; 1,5 per cent) and in the group of persons over 18 (1 case; 1,5 per cent). Conclusions. 1. The registered number of SRVs was lower in 2015 than in 2014 and the major part was caused by a BCG vaccine like in 2014. This vaccine must be in-jected into a baby’s skin, but sometimes side reactions can manifest because of incorrect percutaneous injection of the BCG vaccine. It is difficult for young doctors without sufficient experience to inject BCG correctly as per the characteristics of vaccine. 2. There were more registered side reactions to vaccines among men than among women in 2015. 3. Most side reactions to vaccines were registered in the group of persons under one in 2015.

33

ASSESSMENT OF T-CELLULAR IMMUNE RESPONCE IN COMMON MARMOSETS (CALLITHRIX JACCHUS) FOLLOWING SINGLE VACCINATION WITH PROTOTYPE VACCINE AGAINST HEPATITIS E VIRUS

ORAL Name E-mail Institution Country GORDEICHUK Chumakov Institute of Poliomyelitis and Viral Russian [email protected] ILIA Encephalitides (CIPVE) Federation Tukhvatulin Gamalya Research Center of Epidemiology and [email protected] RF Amir Microbiology Petkov Stefan [email protected] Karolinska Institutet Sweden Latvian Biomedical Research and Study Center, Jansons Juris [email protected] Latvia Riga Stradins University Sominskaya Irina [email protected] Latvian Biomedical Research and Study Centre Latvia Isaguliants Maria [email protected] CIPVE RF CIPVE RF Kyuregyan [email protected] Karen Gamalya Research Center of Epidemiology and RF

[email protected] Microbiology Logunov Denis

Mikhailov [email protected] CIPVE RF Mikhail

Background. Common marmosets are widely used in immunological and pharmacological research. They are naturally susceptible to a number of human pathogens including hepatitis E virus (HEV). Development of the methods of assessment of immune activation following vaccination is needed to facilitate applications of this animal model for preclinical evaluation of human vaccine prototypes. Methods. Four common marmosets (2 males; 2 females) were immunized intramuscularly with 20 µg of truncated capsid protein of HEV genotype 3 (aa 110-660, 60 kDa; HEV3deltaORF2) with alumni adjuvant and immunomodulator glutoxim (10 mg/ml). Two males were used as negative controls. Ten days later, marmosets were bled, and PBMCs were purified by Ficoll gradient centrifugation. PBMCs were stimulated with HEV3deltaORF2, negative controls capsid or alternative reading frame proteins of HCV, mitogen PHA, or medium, at 37C and 5% CO2 for 72 h in CFSE-test, or 20 h to assess IFN-g production. Immune activation was assessed by FACS using a panel marmoset-specific fluorescent antibodies: anti-CD45RO- PerCP/Cy5.5 (Biolegend, clone UCHL1, catalogue number #304221 482/690), anti-CD3-APC (Beckton Dickenson/BD; SP34, #557757 780), anti-CD4- PE-Cy7 (BD, L200, #560644), anti-CD8-PE (Biolegend , #250304496/578), anti-IFN-g-FITC (Mabtech, 1-D1K, #3420-7). Results. First, conditions for staining with fluorescent-labelled antibodies against marmoset CD3, CD4, CD8, IFN-g were determined in series of overnight stimulations of naïve marmoset PBMCs with PHA. In the immunized marmosets, stimulation with 2.5, 5 and 10 µg of HEV3deltaORF2, induced a mean increase in the proportion of proliferating CD4 T-cells of 0.8, 0.4 and 0.3%; and proliferating CD8 lymphocytes, of 4.7, 3.0 and 2.4 versus 0, 0.3 and 0.1% (CD4+ T-cells), and -1.4, -1.2 and -0.7% (CD8+ T-cells) in the non- immunized controls, respectively. In the optimal conditions, the mean increase in the proportion of IFN- producing CD4 T-cells of immunized marmosets after stimulation with 2.5, 5 and 10 µg of HEV3deltaORF2 reached 0.3, 0.4 and 0.1% vs. -0.5, -0.6 and -0.3% in the non-immunized controls, respectively. The mean increase in the proportion of IFN-g producing CD8 T-cells constituted 2.8, 3.7 and 3.1% compared to -0.6, -0.3 and -0.3% in negative controls, respectively. The difference of immunized animals with controls in CD8+ T-cell responses was statistically significant (p<0.05). Conclusion. Conditions for immune staining of activated CD4+ and CD8+ T-cells for subsequent flow cytometry analysis were established allowing sensitive assessment of immunogen-specific cellular responses in common marmosets. Single immunization of marmosets with candidate HEV vaccine antigen was found to induce a significant stimulation of CD8 T-cells. HEV vaccination was supported by grant of Russian Ministry of education and science №RFMEFI60414X0064; optimization and performance of immune assays of marmoset lymphocytes, by Russian Science Foundation №15-15-30039.

34

APPROBATION OF FRET SYSTEM FOR STUDYING CRAC ION CHANNELS

POSTER Name E-mail Institution Country LAURA A. Kirchenstein Institute of Microbiology [email protected] Latvia HIPPE* and Virology, Riga Stradiņš University Rainer Institute of Biophysics, JKU Life science [email protected] Austria Schindl Center Šimons A. Kirchenstein Institute of Microbiology [email protected] Latvia Svirskis and Virology, Riga Stradiņš University Mārtiņš A. Kirchenstein Institute of Microbiology [email protected] Latvia Kālis and Virology, Riga Stradiņš University

Background: Store-operated calcium channels are major pathway for calcium signaling and serve many functions in cells and tissues – gene expression, secretion and immune response. STIM proteins are located in ER, being Ca2+ sensors, and, after activation by ER Ca2+ store depletion, form ion channel with Orai proteins. These ion channels have an important role in human immune cells (T cells, mast cells, NK cells, B cells). Fluorescent Resonance Energy Transfer (FRET) method for studying interaction of molecules situated at a distance 10 nm or less was first described over 50 years ago. It has a wide application in modern science in biomedical research and drug discovery when studying protein interaction and conformation change in immune and nonimmune cells. FRET is rather complicated method requiring precise identification of the correct setup for the experiments, measurements and data interpretation. Aims: To identify the most optimal conditions for studying STIM1 and Orai1 Ca2+ channel subunit interaction using wide-field dual-detector FRET system. Materials & Methods: HEK 293 cell line was cultured on poly-L-lysine-treated microscope coverslips and used for transfection with plasmid vectors using lipofectamine. Plasmids (STIM1 with CFP fluorescent protein and Orai1 with YFP) were used for studies of ion channel formation as detected by FRET. Exposure time and approach of data analysis varied through experiments in order to optimize FRET data quality. Results:. Different technical approaches were used for FRET data quality assessment. Too intensive exposure of the fluorophores during the preparation of cells for the FRET measurement resulted in fluorophore bleaching and low FRET efficiency values. Use of neutral density (ND4 combined with ND8) optical filters improved emmited fluorescence intensity and respective FRET efficiency values. Low detected fluorescence intensity of either donor or acceptor fluorophore results in false FRET and affects FRET efficiency value. Our data analysis also confirmed that FRET pixel frequencies must conform to Gaussian distribution. Negative (non-bound acceptor and donor fluorophores) and positive controls (donor and acceptor fluorophores linked with a short peptide) were also introduced to detect the lowest and the highest FRET reference values for the FRET efficiency range. Conclusions: (1) Optimal conditions for FRET experiments using a wide-field fluorescence microscope require use of ND filters during the preparation of cells for the FRET measurement in order to avoid photobleaching; (2) FRET pixel frequency in a cell must conform Gaussian distribution; (3) Use of positive and negative control shows maximal and minimal FRET values for the selected FRET acceptor and donor fluorophore pair. Acknowledgements: Authors acknowledge financial support from EU project BALTINFECT.

35 IMMUNOTHERAPY AGAINST DRUG RESISTANCE AS A COMPLEMENT TO HAART

ORAL Name E-mail Institution Country Karolinska Institutet, Stockholm ISAGULIANTS (KI); and Gamalya Research Russian Federation [email protected] MARIA Center of Epidemiology and Sweden Microbiology (GRCEM), Moscow Chumakov Institute of Starodubova Elizaveta [email protected] Poliomyelitis and Viral Russian Federation(RF) Encephalitides (CIPVE) Petkov Stefan [email protected] KI, Stockholm Sweden Kilplelainen Athina [email protected] KI, Stockholm Sweden Krotova Olga [email protected] KI, Stockholm Sweden Jansons Juris [email protected] Riga Stradins University, Riga Latvia Latanova Anastasia [email protected] GRCEM, Moscow RF Latyshev Oleg [email protected] GRCEM, Moscow RF Warholm Per [email protected] Stockholm University Sweden Tukhvatulin Amir [email protected] GRCEM. Moscow RF Gordeychuk Ilya [email protected] CIPVE, Moscow RF Logunov Denis [email protected] GRCEM, Moscow RF Bobkova Marina [email protected] GRCEM, Moscow RF Mikhailov Mikhail [email protected] CIPVE, Moscow RF

Background & Aims Clinical trials demonstrated validity of therapeutic HIV vaccines for reducing viral load and improving patient well-being towards “functional cure”. HIV immunotherapy becomes actual in view of the latest findings of an effective broad T-cell response clearing HIV-1 from the latent reservoirs. Strong immune response against viral antigens responsible for drug resistance can create a bottle-neck to viral evolution forbidding or hindering the development of drug resistance. In HIV-1, such response can complement highly active antiretroviral treatment, and thus prolong the time for its effective application. We aimed to develop a complex approach for immunotherapy of HIV/AIDS based on HIV enzymes: reverse transcriptase/RT, protease/PR, and integrase/IN. Methods. The consensus humanized HIV enzyme genes, wild-type and with primary resistance mutations were created. Respective genes were designed, synthesized (Evrogen) and cloned into plasmids for DNA-immunization. Eukaryotic expression was confirmed by PAGE of the plasmid-transfected cells with subsequent Western blotting. Inactivation of enzymes was carried by site-mutagenesis yielding prototype DNA-immunogens. Gene immunogenicity was tested in BALB/c mice, injected with respective plasmids with subsequent electroporation employing flat or multi-needle electrodes (BEX). Immune responses were assessed by IFN-g/IL-2 Flourospot, and multiparametric FACS after stimulation of murine lymphocytes with HIV-derived peptides. In vivo challenge systems were developed, based on co-delivery of HIV-1 and reporter genes with follow-up of reporter expression using bioluminescent imaging (Spectrum CT), and on reporter-labelled murine tumor cell lines expressing HIV enzymes. Results. Synthetic HIV-1 enzyme genes were highly expressed in a variety of eukaryotic cells. In vivo delivery of plasmid DNA was optimized to support strong protein expression required for a potent immune response. Immunological tests demonstrated PR gene to be a potent Th1; IN, a moderate mixed Th1/Th2; and RT, a potent Th2 immunogen. Mice were primed with HIV-1 genes, and boosted with HIV gene(s) mixed with the reporter gene for firefly luciferase. In vivo imaging demonstrated that the resulting immune response effectively cleared HIV antigen/reporter co-expressing cells generated in the boost. This phenomenon, dubbed “antigen challenge”, was useful for testing lytic capacity of HIV specific cellular response in rodents. A cancer-cell challange model was also developed based on challenge of HIV gene immunized mice with syngenic tumor cells stably expressing HIV proteins. Eight lines of 4T1 cells expressing HIV proteins were established, and shown to form tumors in BALB/c mice. In model experiments, pre-immunization with Luc gene prevented Luc-expressing murine adenocarcinoma 4T1 cells from establishing tumors in immunocompetent mice. Model is currently used to test if immune response against HIV enzymes can protect immunized mice against a challenge with HIV enzyme expressing 4T1 cells. Conclusions. Promising immunogens inducing an immune response against HIV enzymes involved in drug resistance in HIV-1 infection were designed, and shown to be highly immunogenic in mice. Two in vivo challenge systems were established to assess the capacity of the immune response to lyse HIV enzyme expressing cells, useful for diverse immune therapy applications. Supported Russian Science Foundation nr 15_15_30039.

36 IMMUNOGENICITY IN MICE OF PLASMID DNA ENCODING

HCV CORE AND ALTERNATIVE READING FRAME PROTEINS

POSTER Name E-mail Institution Country Latvian Biomedical Research and Study Center (BMC), JANSONS JURIS [email protected] Latvia Riga Stradins University (RSU) Dovbenko [email protected] BMC Latvia Anastasija

Skrastina Dace [email protected] BMC Latvia

Petkov Stefan [email protected] Karolinska Institutet Sweden

Akopjana Inara [email protected] BMC Latvia

Stahovska Irina [email protected] BMC Latvia

Latvian Biomedical Research Akmenkalns Gatis [email protected] Latvia BMC Isaguliants Maria [email protected] RSU, and Karolinska Institutet Sweden Sominskaya Irina [email protected] BMC Latvia

Background. The nucleocapsid (core) protein of HCV represents an attractive target for an HCV vaccine, but the efforts to make it immunogenic had only limited success. We attempted to delineate the factors restricting the immunogenicity of HCV core, and improve its performance as DNA immunogen in mice. Besides the core protein, the 5' terminal part of HCV genome encodes a frameshift core+1/ARFP protein, the role of which in HCV life cycle is yet unclear. In the previous study we analyzed the immunogenicity in rabbits of core aa 1-173, 1-152, 147-191, and of main alternative reading frame product ARFP. An antibody response was srtong against both HCV core and ARFP, indicating that they may compete in the induction of immune response in DNA-immunization made with viral genes allowing the frameshift. Aims. To characterize the immunogenicity of plasmids expressing proteins encoded by the 5´terminus of HCV RNA in DNA-immunization, and define the correlates of immunogenicity. Materials & Methods. Plasmids carrying cDNA encompassing the 5´terminus of HCV 1b genome encoding HCV core and ARFP, were obtained by cloning. Plasmids were transfected into eukaryotic cells, and expression level of HCV core and ARFP was evaluated by Western blotting with anti-core and anti- ARFP rabbit antibodies. Groups of BALB/c mice (n=5) were immunized twice with 40 µg plasmids or empty vector intradermally with four days interval; injections were followed by electroporation (BEX, Japan). After 21 days, mice were sacrificed, spleens and sera were collected. Cellular responses in spenocytes were analyzed IFN-γ/IL2 Fluorospot after stimulation with proteins and antigen-derived peptides. Antibody response was assessed by ELISA. Results To elucidate the factors affecting immunogenicity of HCV core DNA a set of eucariotic expression plasmids carrind wild type, mutated and synthetic codon-optimized variants of of the 5' terminus of HCV genome ware constructed. The expression experiments in cell culture showed that the prohibition of ARFP synthesis by synonymous nucleotide mutations significantly increases the expression level of core protein. Expression of ARFP protein remains low in all cases. Plasmids were introduced into mice to assess their immunogenicity, and delineate the extent of HCV core and ARFP immune competition. Discussion. Immunization with HCV core genes induced the specific immune response. Both natural and mutated HCV core genes with prohibited frame-shift provide the same levels of specific cellular and antibody responses. Thus, a higher expression of HCV core from the mutated gene compared to the wild type sequence could not provide for its better immunogenicity. Efficacy of ARFP expression by the natural ribosome frameshift mechanism was low and obviously insufficient to induce a specific immune response in DNA-immunization. Thus, anti-ARFP immune response is not competing with that against HCV core, and cannot explain low immunogenicity of the latter in DNA-immunization performed with the virus- derived genes. The importance of the obtained results for anti-HCV vaccine design will be discussed. Acknowledgements. Authors acknowledge financial support from the projects BALTINFECT, VACTRAIN, Research Council of Latvia 532/2015, and Swedish Institute 09272_2013.

37

ANALYSIS OF AVAILABLE WHOLE-GENOME SEQUENCES OF INFLUENZA A VIRUSES ALLOWED IDENTIFICATION OF NOVEL

ANTIVIRAL THERAPEUTICS AND SELECTION OF NOVEL VACCINE STRAIN CANDIDATES

POSTER Name E-mail Institution Country KAINOV DENIS [email protected] UniversityofHelsinki Finland Belanov Sergey [email protected] UniversityofHelsinki Finland Anton Cheltsov [email protected] Q-MolLLC United States of America

Human influenza A viruses (IAVs) are considered a major health threat because they cause global pandemics and epidemics. These viruses evolve rapidly, making current prevention and treatment options often ineffective. Our analysis of whole-genome sequences of influenza A(H1N1)pdm09 and A(H3N2) viruses available in GeneBank revealed flexible and conserved regions in IAV proteins. Further analysis of the flexible regions allowed us to identify evolutionary markers, which may guide selection of vaccine strains and, thus, improve vaccine efficacy (1). Moreover, structural analysis of conserved regions allowed us to identify novel druggable sites in IAV proteins. In silico chemical library screening against these sights following by in vitro validation experiments identified several novel virus-directed agents which possessed excellent antiviral activities with minimal immuno- and metabolo-modulating effects. Altogether, our results may allow full development of anti-influenza options.

38

IMPROVING CELLULAR IMMUNOTHERAPEUTIC VACCINES EFFICACY WITH GENE SUPPRESSION IN DENDRITIC CELLS AND T- LYMPHOCYTES

POSTER

Name E-mail Institution Country

KORABLIOV State Research Institute, Centre for [email protected] Lithuania PAVEL Innovative MedicineĀ

Konstantinov Petersburg Nuclear Russian Filatov Michail fi[email protected] Physics Institute Federation

Korablioviene [email protected] ULAC Lithuania Joana

Mykolas Romeris University, Caplinskas Saulius [email protected] Lithuania Centre for Communicable Diseases and AIDS

Introduction: There is considerable interest in developing therapeutic vaccines based on immune system cells. In recent years there has been interest in the establishment of anticancer vaccines based on immune system cells. Modern immunological approaches to treat malignant tumor involve the use of immune system cells comprising tumor antigens such cells including dendritic cells (DCs) and/or activated lymphocytes. Therefore the establishment of effective drugs, vaccines and medical treatment is essential and despite notable achievements in this area more progress is needed. A number of immunotherapy treatments are currently being developed, which can potentially be less toxic and/or more effective for treatment. It is known that immune system is able to distinguish between the self and the foreign and under normal conditions an immune response against such antigens is occurring.Virus infected cells are recognized by the immune system as foreign though immune response is often not strong enough to block the development of viruses. One purpose of immunotherapy is to enhance the ability of immune system to recognize virus-infected cells and establish effective mechanisms to reduce viral load. An important issue for immunotherapy is the selection of antigens and their optimal presentation to the immune system. Aims: To prove cellular vaccine efficiency. Methods: Monocytes were obtained from blood and cultivated using standard methods. Isolation of monocytes and lymphocytes was done by centrifugation. For induction of formation of DC to the culture medium growth factors are added. Later, DCs were stimulated with antigenic materials. Results: An effective amount of cells for treating hepatitis C is an amount of dendritic cells, T cells or a combination of both, wherein after administration of a vaccine comprising the effective amount of such cells to a patient having hepatitis C. In an embodiment, dendritic cells in an immunogenic composition are present at a concentration 5x105 to 5x107 cells/mL. In an embodiment the T cells in immunogenic composition present at concentration 1x107cells/mL. 6 patients had not responded to standard treatment during the year (interferon (INF) plus ribavirin (RIB)). One patient did not respond to the vaccine. Conclusions: Cellular vaccine partially or completely eliminates virus from the patients body.

39 ANALYSIS OF PREGNANT WOMEN VACCINATION AGAINST INFLUENZA

ORAL Name E-mail Institution Country Centre for Communicable Diseases and KRIAUCIUNAITE ASTA [email protected] Lithuania AIDS Centre for Communicable Diseases and Zukauskaite Kristina [email protected] Lithuania AIDS Centre for Communicable Diseases and Sebeliauskaite Ieva [email protected] Lithuania AIDS Korablioviene Joana [email protected] ULAC Lithuania Mykolas Romeris University, Caplinskas Saulius [email protected] Lithuania Centre for Communicable Diseases and AIDS

Introduction. In order to ensure effective prevention of influenza people in Lithuania are vaccinated against flu by state funds and since 2007 The World Health Organization (WHO) and the European Commission recommended risk groups are vaccinated . Influenza is one of the most common respiratory tract infections and especially dangerous for pregnant women. Pregnant women are included at risk group which is vaccinating heavily and by WHO this group is as priority. It is necassary to increase the vaccination rates of risk groups if we want to prevent diseases and their complications for person and public. Objective.To assess the attitude to vaccination of pregnant women and compare vaccination coverage for 2011-2016. Methodology.Epidemiology surveillance of influenza and air respiratory infections is done accordance by 2012 January 30 order of Lithuanian Respublic Health of ministry number V-58 ,,For influenza and acute respiratory tract infections surveillance rules approval“. Results. The most of pregnant women (44,8 %) haven’t opinion about their vaccination against influenza. Assessing vaccination of pregnant women against influenza by education, mostly were unfavorably by respondents with higher education (34,5 %). Thinking that pregnant women should be vaccinated against influenza mostly were in younger respondents group (29-30 years old) than in older (older than35 years) respondents group. In 2015-2016 influenza season pregnant women vaccination coverage were higher than in previous season. Conclusions. 1. The most pregnant women haven’t an opinion of pregnant women vaccinating against influenza. 2. Pregnant women with high education are more critically of vaccinating against influenza than with lower education. 3. Younger respondents are more likely to be vaccinated against influenza than older respondents. 4. From 2011-2012 influenza season the vaccination against influenza is increasing.

40

INFLUENZA VACCINE EFFECTIVENESS DURING 2015-2016 INFLUENZA SEASON: A HOSPITAL-BASED CASE-CONTROL STUDY IN LITHUANIA

POSTER Name E-mail Institution Country KULIESE MONIKA [email protected] Lithuanian University of Health Sciences (LUHS) Lithuania Jancoriene Ligita [email protected] Vilnius University Lithuania Grimalauskaite Rita [email protected] LUHS Lithuania Zablockiene Birute [email protected] Vilnius University Lithuania Damuleviciene Gyte [email protected] LUHS Lithuania Velyvyte Daiva [email protected] LUHS Lithuania Lesauskaite Vita [email protected] LUHS Lithuania Ambrozaitis Arvydas [email protected] Vilnius Lithuania Mickiene Aukse [email protected] LUHS Lithuania Gefenaite Giedre [email protected] LUHS Lithuania

Objectives. In Lithuania, one quarter of all acute respiratory tract annual infections are estimated to occur due to influenza. This virus might cause severe infection and lead to serious complications. The primary tool for preventing influenza is annual vaccination. Our primary objective was to measure seasonal influenza vaccine effectiveness (SIVE) against influenza in patients admitted to hospital during the 2015/2016 influenza season. We also described the co-circulation of other respiratory pathogens. Methods. A test-negative case-control study was conducted in three departments in the university hospitals in Lithuania. Data on demographic and clinical characteristics, including influenza vaccination status were collected from the patients recommended to receive SIV. Influenza and other respiratory pathogens were confirmed by multiplex RT-PCR. Results. One-hundred-sixty-three patients were included; 92(56.4%) subjects were 65 years old or older. Of the total sample, 15(9.2%) subjects were vaccinated against influenza at least two weeks before the onset of influenza symptoms, 12 vaccinated subjects were 65 years old or older. Sixty-five patients (39.9%) tested positive for influenza A (50 of which A(H1N1)pdm09; 15 unsubtyped), 8 (4.9%) for influenza B and 1 (0.6%) had a coinfection. The unadjusted SIVE against influenza in the total sample was 57% (95%CI: - 41% to 87%), and 43% (95%CI: -134% to 85%) in the group of subjects who were 65 years and older. Other respiratory pathogens found were coronavirus 3(1.9%), adenovirus 7(4.3%), rhinovirus 6(3.7%), metapneumovirus 7(4.3%), respiratory syncytial virus 8(4.9%), parainfluenza 1(0.6%). Two subjects within the sample were co-infected with influenza A and metapneumovirus, 2 with influenza A and adenovirus, 1 with influenza A and respiratory syncytial virus and 1 with influenza A and coronavirus. Conclusions. Due to low statistical power (partly due to that the study is still ongoing), the point estimate of moderate SIVE during 2015/2016 season against influenza serves only as preliminary. The detected dominance of A(H1N1)pdm09 is in line with its circulation in the WHO European Region.

41

HIGH IMMUNOGENICITY IN MICE OF PLASMIDS ENCODING CONSENSUS REVERSE TRANSCRIPTASE OF HIV-1 CLADE A WITH PRIMARY DRUG RESISTANCE MUTATIONS

ORAL Name E-mail Institution Country LATANOVA Gamaleja Research Center of Epidemiology Russian [email protected] ANASTASIA and Microbiology (GRCEM), Moscow Federation Starodubova [email protected] Engehlardt Institute of Molecular Biology Sweden Elizaveta Petkov Stefan [email protected] Karolinska Institutet Sweden Latvian Biomedical Research and Study Jansons Juris [email protected] Latvia Center, Riga Stradins University Russian Tunitskaya Vera [email protected] Engelhardt Institute of Molecular Biology Federation Russian Ivanov Alexander [email protected] Engelhardt Institute of Molecular Biology Federation Kilplelainen Athina [email protected] Karolinska Institutet, Stockholm Sweden Russian Krawiec Mati [email protected] Karolinska Institutet, Stokholm Federation Sasinovich The Republican Research and Practical [email protected] Belarus Sviataslau Centre for Epidemiology and Microbiology Warholm Per [email protected] Stockholm University Sweden Gamalya Research Center of Epidemiology Russian Bobkova Marina [email protected] and Microbiology Federation Latvian Biomedical Research and Study Skrastina Dace [email protected] Latvia Center Isaguliants Maria [email protected] GRCEM, Moscow, Karolinska Institutet Sweden

Background and aims. The field of HIV-1 DNA vaccines is actively developing but the majority of vaccines target HIV-1 of clades B or C abundant in Europe, USA and/or Africa. Territories of the former Soviet Union face an HIV-1 epidemic caused by a highly homologous HIV-1 clade A strain FSU-A. These territories would benefit from the application of therapeutic HIV-1 vaccines targeting this strain. Our goal is to develop immunotherapeuticals which would induce an immune response limiting the evolvement and spread of drug-resistant FSU-A variants. In this study we focused on the key enzyme determining HIV-1 drug resistance (DR), reverse transcriptase (RT) aiming to create DNA-immunogens that induce potent immune response against RT of FSU-A in the experimental settings. Materials & Methods: RT was designed as consensus resulting from multiple alignment (MUSCLE) of >200 RT amino acid sequences from untreated HIV-1 patients. In the variable positions, amino acid variants were chosen that were predicted to have the highest antigenicity/immunogenicity in humans. Consensus RT was supplemented with frequent primary mutations of resistance to NRTI and NNRTI in use in the Russian Federation (RT_A_N; M184V/K65R; RT_A_NN; K103N/G190S). Consensus RT_A gene was synthesized (Evrogen) and cloned in vectors for expression in pro- and eukaryotic (pVax1) expression. RT inactivation and DR mutations were introduced by site-mutagenesis, generating pVAxRT_Ain, pVaxRT_Ain_N, and pVaxRT_Ain_NN. Gene expression was confirmed by Western blotting of lysates of transfected HeLa cells. RT_A variants were also expressed in E coli, and purified by Ni-agarose chromatography. On days 1 and 4, BALB/c mice were intradermally injected with 2x20 mcg RT-plasmids and electroporated (EP) with multi-needle (MN) and fork-plate (FP) electrodes using CUY21EditII (BEX Ltd., Japan). In vitro immune responses were evaluated on day 15 (PBMCs) and at the end-point on day 28 (splenocytes). Cells stimulated with respective

42 recombinant RT_A and RT-derived peptides (SynPeptide) were assessed for cytokine production by IFN-gamma ELISpot, and IFN-gamma/IL-2 Fluorospot (Mabtech). Sera were analyzed by indirect ELISA. Results. All RT A genes were equally well expressed. The strongest IFN-gamma production was registered in PBMCs and splenocytes of RT-gene immunized mice after stimulation with the respective RT_A immunogen and a peptide representing a predicted CTL-epitope of RT at aa 145-168. IFN-gamma response to aa 145-168 decreased in the following order: RT_Ain_N>RT_Ain_NN>RT_A>RT_A_in. FP-driven EP induced stronger IFN-gamma response than EP with MN-electrode. IL-2 and IFN-gamma/IL-2 response was weak to undetectable. Regions harboring DR-mutations contained no epitopes recognized in BALB/c mice. All groups responded with a high IgG production to RT_A variants. In mice receiving RT_Ain_N and RT_Ain_NN, titer of specific IgG exceeded 1x10(6). Conclusions. Consensus HIV-1 FSU_A RT_A genes are strong Th2-immunogens capable of inducing potent acute IFN-gamma, followed by anti-RT antibody response. The next step is evaluation of compatibility of RT-genes with DNA-immunogens for protease and integrase with the final goal of raising multi-specific anti-HIV immune response limiting HIV replication. Studies in DNA- vaccination against DR-HIV are supported by Russian Science Foundation №15_15_30039; researchers mobility Swedish Institute TP№09272_2013, and training by VACTRAIN №692293.

BELL'S PALSY FOLLOWING THE VACCINATION WITH PNEUMOCOCCAL CONJUGATE VACCINE IN HIV INFECTED PATIENT ON ART POSTER Name E-mail Institution Country LIUKPETRYTE [email protected] Lithuania EVELINA Vilnius University Hospital Santariskiu Jancoriene Ligita [email protected] Lithuania klinikos Centre of Infectious Diseases

Background. The safety and tolerability of pneumococcal conjugate vaccine (PCV) administration in HIV-infected adults are generally well tolerated. Bell's palsy is the acute onset of one-sided transitory paralysis of facial muscles, due to impaired function of the seventh cranial nerve. We present an acute right facial paralysis in 38-year-old HIV infected patient possibly being related to administration of 13-valent PCV. Case report. An 38-year-old HIV infected male with undetactable HIV-RNA and CD4 count 950 mm3 currently on treatment with Abacavir/Lamivudin, Lopinavir/Ritonavir, was vaccinated with 13-valent PCV. On the second day after the vaccination patient felt weakness and deformation of the mimic of the right side of his face. Physical examination revealed impairment of movement of the right side of the face, drooping of the brow and corner of the mouth, impaired closure of the right eye, and absence of forehead wrinkles. The rest physical examination was normal. Complete blood count and chemistry results were also normal. Head CT scan showed no focal cerebral pathology. There was no personal or family history of neurologic disorders. Neurologist confirmed the diagnosis of acute neuropathy of facial nerve. Puls therapy with prednisolone 60 mg per day for 10 days was administered. Conclusions. Although the administration of the pneumococcal vaccine to immunosuppressed patients is widely accepted, neurological post-vaccinal complications may still occur. Acute neuropathy of facial nerve in immunosuppressed patient was possibly associated with the vaccination with PCV. However, this possible adverse event does not yet outweigh the beneficial effects of the pneumococcal vaccination in HIV infected population. Our suggestion is to be aware about this possibility as well as collect further data on adverse events of vaccination with PCV in HIV infected population.

43 STEPS TOWARDS THE DEVELOPMENT OF HEPATITIS E VACCINE

ORAL Name E-mail Institution Country Chumakov Institute of Poliomyelitis LYASHENKO ANNA [email protected] Russian Federation and Viruses Encephalitis Chumakov Institute of Poliomyelitis Chumakov Alexey [email protected] Russian Federation and Viruses Encephalitis Chumakov Institute of Poliomyelitis Sorokin Alexey [email protected] Russian Federation and Viruses Encephalitis Chumakov Institute of Poliomyelitis Kyuregyan Karen [email protected] Russian Federation and Viruses Encephalitis Chumakov Institute of poliomyelitis Isaeva Olga [email protected] Russian Federation and viral encephalitides Chumakov Institute of Poliomyelitis Gordeychuk Ilya [email protected] Russian Federation and Viral Encephalitides Chumakov Institute of poliomyelitis Mikhailov Mikhail [email protected] Russian Federation and viral encephalitides

Objective. Hepatitis E virus is a major cause of human acute hepatitis, which is a significant public health problem in many areas of the world. Although the first vaccine against hepatitis E virus has been produced and licensed in China, it is not yet available globally and it is still questioned for public health and economic impact. PORF2 is the only capsid protein of HEV, having the property of immunogenicity and the ability to self-assemble into the HEV virus-like particles (VLP). The aim of our study was to produce the highly purified truncated PORF2 protein and assess its immunogenicity. Materials and methods. The nucleotide sequence of the HEV genotype 3 ORF2 gene have been used in our study, because genotype 3 is prevalent in countries with a temperate climate, including Russia. The sequence of ORF2 gene was modified. First 109 codons were replaced by oligo-His-Tag for protein expression and purification. The rest of the sequence encoded the 110-660 amino acid of PORF2 was optimized for effective protein expression and folding in bacterial cells based on CAI (codon adaptation index) approach. By means of PCR rare eukaryotic codons have been substituted by the same rare bacterial codons without changing the amino acid sequence to retain translation pause and rhythm of protein folding with a view to increase protein solubility. Thus the synthetic gene of 1719 bp, encoding a 60-kDa protein, has been created. It was subcloned into the expression vector and used for transformation in expression strain E.coli. Truncated PORF2 protein was purified by Ni2+-chelate affinity chromatography using nondenaturing conditions. Expression and purification was confirmed by SDS-PAGE and western blotting with anti-HEV ORF2 antibodies. Results. The various conditions of protein expression and purification have been tested. The highest yield of target protein up to 5 mg from 2 L of culture media was obtained by autoinduction expression. In addition to the full-length 60-kDa protein predicted from the sequence of the optimized ORF2 gene, abundant ORF2-related polypeptides with lower molecular weights were detected at the SDS-PAGE. Western blotting analysis with anti-ORF2 mouse antibody showed a single band of 60 kDa, which confirmed the presence of the target protein PORF2. To separate the target protein self-assembling into the VLP gel filtration chromatography was performed. It revealed two fractions, both of which were analyzed under electron microscopy. The protein of the first fraction was found to present as VLPs, while the second fraction was represented by heterogeneous structures. To evaluate the immunogenicity of both VLP- PORF2 and nonVLP-PORF2, mice were immunized with single dose of 2-, 5- and 10 μg protein with alum adjuvant. Additional adjuvant formulation of alum with immunomodulator drug glutoxim was used for the same PORF2 dosages. Anti-HEV testing using ELISA performed at days 14th and 28th postimmunization demonstrated the strongest antibody response in mice received 5 μg of nonVLP-PORF2. No correlation between the presence of immunomodulator drug in vaccine and the magnitude of immune response was observed. Conclusions. Both VLP and non-VLP fractions of truncated HEV ORF2 protein appeared to be immunogenic in mice. The further testing in non-human primate and swine models of HEV infection is needed to evaluate protective efficacy of this vaccine candidate modalities.

44

HCV CORE AND ITS ARFP VARIANTS: COMPARISON OF EXPRESSION

LEVELS IN E. COLI CELLS AND PROTEIN IMMUNOGENICITY

ORAL Name E-mail Institution Country

SOMINSKAYA [email protected] Latvian Biomedical Research and Latvia IRINA StudyCentre (BMC), Riga

BMC, Riga Ivars Petrovskis Anastasija BMC, Riga

Dovbenko Juris Jansons BMC, and Riga Stradins University, Riga BMC, Riga Santa Andersone BMC, Riga Ilva Lieknina BMC, Riga Irina Stahovska

Background and aimsHCV induces persistent infection and chronic liver disease. Therefore, the development of an anti-HCV vaccine is a goal of high priority. One of the attractive targets for HCV vaccine is the nucleocapsid (Core) protein. Core aa1-191 is highly conserved among diverse HCV strains isolated worldwide. Here, we studied the properties of different Cores and its alternative reading frame protein variants (ARFP) with the final goal to use them as vaccine prototypes. Solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve it. Here, we applied them to purify Core and ARFP with subsequent testing of immunogenicity. Methods and Results: We used an expressional vector pETM-11 (gift from G. Stier) for cloning, expression and subsequent purification by metal affinity chromatography (IMAC). Core fragments correspond to aa 1-173 and 1-159 and 1-145 ARF, 1-10 aa core+ 11- 145 ARF as well as 10-145 ARF were cloned into pETM-11 that encodes N-terminal 6xHis tag, a TEV protease recognition site and has a conserved multiple cloning site with NcoI recognition site. The NcoI recognition sequence has an in-frame ATG codon that can be used for the functional expression of the target protein, minimizing the number of non-native N-terminal amino acids. Plasmids were expressed in E.coli BL21 (DE3) and purified using IMAC. To optimize expression conditions, we evaluated several expression parameters such as IPTG concentration:0.2, 0.4, 0.6, 0.8 mM, and induction time: 2, 4, 6 h and overnight. Grow cells at lower temperature (25oC) did not improve protein production. Two major protein purification protocols were used: purification under denaturating and under native conditions. Purification was followed by dialysis by several protocols to improve protein purity. In case with ARF aa10-145 after purification under denaturating conditions, fractions were pooled and gradually dialyzed from 8 M to 0.5 M urea. However, higher amount of ARFP protein was in the pellet than in the supernatant, implying that the expressed protein is partly soluble. Under native conditions, protein was eluted with 250mM imidazole and was found in all elution fractions. Elution fractions with the purified protein were pooled and dialyzed against PBS and TBS. Purification of 1-173, 1-159 Core and 1-10 aa core+ 11- 145 ARF was possible onlu under denaturing conditions. By Western blotting, purified proteins were identical in expression levels to previously described HCV core and ARF. Proteins were used to immunize rabbits and BALB/c mice. Strong humoral immune response was induced against Core aa 1–173, and the most stable ARFP form, F- protein. Immunization with HCV core aa 1–173 led to a B-cell response of broad specificity targeting multiple linear epitopes. The C-terminally truncated Core 1–152 induced a weaker response directed against the N-terminus only implicating the role of the C-terminus in promoting humoral immunogenicity. Epitopes recognized in rabbits overlapped those identified in HCV infection. Conclusions: Analysis of proteins purified under native conditions indicated that ARF proteins were partly soluble which allowed to use several purification protocols. The highest ARF expression was achieved at 0.4 mM IPTG at 37oC after 3-h of induction. ARF protein 11-145 was stable under the purification. The highest Core 1-173 and 1-159, as well as 1-10 aa core+ 11- 145 ARF protein expression level was achieved at 0.2 mM IPTG at 37oC after 5-h of induction and purification was done under denaturated conditions. These are promising findings which allow a step forward in the development of the HCV core protein-based prototype HCV vaccines.

45

ELECTROPORATION AND PERIPHERAL TECHNOLOGY: FROM DNA IMMUNOTHERAPY TO GENOME EDITING

Authors Name E-mail Institution Country MORIIZUMI YASUHIRO [email protected] BexCo.,Ltd. Japan

Introduction: Electroporation (EP) is a method for the transfection by the use of electricity which, on the membrane of the cells, makes temporary holes through which the target gene, drugs, etc. are moved into the inside of the cells. EP is firstly developed to be applied to the transfection of E coli., followed by application for the cells, tissues, and animals. EP is a very useful and convenient method for transfection because EP needs neither special materials nor places, EP is theoretically applied to every sample including the animal alive, and it leads to high reproducibility. Recently, the efficient method for genome editing is developed by the use of EP. The key to success of EP, however, depends on how much voltage (current) is applied and how the electricity is generated to the sample. Aims: Development EP technology to solve the EP problem by the use of machines outputting accurate voltage and sample base electrodes. Materials and Methods: For EP to E. coli. and cells, cuvette electrodes are commonly used. A unique electrode with two or three stainless plates was used for these samples, especially the primary cells. For EP to animals, the various electrodes, such as the needle electrode and tweezer-like electrode, were used. The size and shape of the electrode were changed according to the animal and/or animal tissue such as the muscle, embryo, and liver. For genome editing, the zygotes of the mouse stayed in the electrode with the platinum-made electrode, followed by EP of Cas9 and gRNA to the zygotes. For all EP above, an electroporator in CUY21 series (BEX) was used to control the generation of the accurate voltage to avoid damage to lead to the death of sample. Results: The cuvette electrode is still useful for cells. A unique electrode with two or three stainless plates was, however, also effective. This unique electrode needs no removal of the cells from the culture dish or plate. For EP to animals, the transfection was successful by changing the shape and/or size of the electrodes. The needle electrode was used for the muscle, and the tweezer-like electrode was used for the embryo and other tissues such as the liver. For genome editing, knock-out mice were born by EP. Conclusion. BEX has been developing the technology for EP and peripheral technology. EP can be theoretically applied to every sample. However, the selection of the machine and electrode as well as the condition of the electricity is a key to success for EP.

HPV VACCINATION AND CERVICAL CANCER SCREENING IN FINLAND ORAL Name E-mail Institution Country Modeller in the National Institute of Health and Welfare Vaccination Programme unit, University of SIMOPEKKA VÄNSKÄ Finland Tampere

Finland introduced 11-12 year old girl vaccination against human papilloma virus (HPV) in universal mass vaccination program in November 2013. Almost 70 percent of the target cohort girls complete full vaccination course. Presentation will highlight factors that contributed to the success of HPV vaccination program in Finland. It will also outline cost-effectiveness of the HPV programme, vaccination strategies (girls only, or girls and boys), and streamlining HPV vaccination vs. PAPA and HPV testing once HPV vaccination programme is in place.

46 3. HIV/AIDS and associated viral hepatitis and sexually transmitted infections; challenges of drug use-related infections

Bartosch Birke HCV infection reprograms the hepatic glucose and glutamine Invited metabolism Francesca Chiodi HIV-1 induced impairment of B-cell functions Invited Drobeniuc Jan Hepatitis B and C- A Silent Epidemic Invited Khudyakov Yury Intra-host HCV evolution Invited Lukashov Vladimir Patterns of HIV-1 evolution in Europe Invited Ranjit Ray Oncogenic potential of hepatitis C virus Invited Widell Anders Hepatitis A virus, analysis of hepatitis A outbreaks in Europe Invited

Akulova Ekaterina Analysis of transmitted HIV-1 variants in real time among Oral acutely infected people who inject drugs in Sant Petersburg, Russia reveals the HIV-1 transmission bottleneck Caplinskiene Irma Trends in rates of chlamydia in Lithuania, 2011-2015 Oral Caplinskiene Irma HIV epidemic in Lithuania - surveillance results, 1988-2015 Oral Caplinskiene Irma Prevalence of hepatitis B and C and risky behaviour among Oral intravenous drug users in Lithuanian regional Low Threshold Centres (LTC) Caplinskas Saulius Comparative analysis of the 2014 and 2015 tuberculin skin test Poster Caplinskas Saulius BCG vaccination coverage by who region in baltic states and Poster Lithuania Cuypers Lize The host immune system may not be the main driving factor of Oral hepatitis C virus genetic diversity Dovbenko Expression of hepatitis C virus core and ARF protein variants in Poster Anastasija eukaryotic system Dudko Juliana Infections related to blood transfusion – is there a risk? Poster Eremin Vladimir The HIV outbreak in Minsk Oral Gasich Elena Ьolecular epidemiology HBV in belarus Poster Gheorghe Irina Virulence and resistance markers in colistin-susceptible Poster Pseudomonas aeruginosa strains isolated from cystic fibrosis patients in Bucharest, Romania Kazenaite Edita Preliminary results of efficacy and safety of direct-acting antivirals Poster Ombitasvir/Paritaprevir/Ritonavir + Dasabuvir with or without Ribavirin in patients with HCV genotype 1 in real life setting Kuznetsova Tatiana Molecular investigation of sporadic cases of HEV infection in Poster Estonia Pavydiene Viktorija Challenges of treatment of HIV infected children in Lithuania Poster Rimkevicius Arunas HPV lesion – oral condyloma acuminatum: case report Poster Sallam Malik Reconstruction of the regional transmission of HCV in Southern Oral Sweden by phylodynamics Sasinovich Characterization of HIV-1 recombinant forms in Belarus: diversity, Oral Sviataslau origin and domestic spread Simkunaite-Zazecke Trends in syphilis among pregnant women in Lithuania Oral Agne Valančienė Dovilė Multi-drug-resistant Acinetobacter spp. monobacteremia in Poster intensive care units of university hospital

47

HCV INFECTION REPROGRAMS THE HEPATIC GLUCOSE AND GLUTAMINE METABOLISM INVITED LECTURE Name E-mail Institution Country BIRKE BARTOSCH [email protected] Inserm U1052, Lyon France

Background and aims. Hepatitis C virus (HCV) is the only virus that is known to perturb hepatic glucose and lipid metabolism with important patho-physiological consequences. Chronic carriers often develop steatosis, insulin resistance and type 2 diabetes, which resolve with successful antiviral treatment. Therefore it is thought that HCV interferes directly with the lipogenic and glycolytic pathways and requires these changes for its replication. However, the exact circumstances of this metabolic reprogramming still remain vague and require further analysis. Here we investigate some fundamental changes in glucose and glutamine metabolism linked to HCV infection. Methods. RNA derived from Huh7.5 cells infected or not with JFH1 and from biopsies of chronic HCV patients were used for RT-qPCR analysis with primers targeting metabolic genes. Nutrient deprivation and biochemical and NMR-based metabolic flux analysis were performed with JFH1 infected Huh7.5 cell culture extracts. Results. We show that HCV modulates the transcript levels of some key regulators of glucose metabolism in the hepatocyte-derived cell-line Huh7.5 as well as liver biopsies of patients with chronic hepatitis C, which hints at changes to glycolytic fluxes. In addition, we found enzymes and factors regulating the glutamine metabolism (MYC, SLC1A5, SLC7A5, GLS) to be induced by HCV in vitro and in liver biopsies of chronic HCV carriers. This correlated with altered metabolite fluxes in HCV infected cells, which were quantified by NMR flux analysis. Furthermore, cell proliferation rates of HCV infected and uninfected cells in conditioned growth media showed that infected cells become dependent on glutamine and reduce their glucose dependence. We show furthermore that silencing of glutaminolytic genes as well as of MYC, an oncogene and metabolic transcription factor known to induce glutamine addiction, reduced HCV infection. Conclusions. Altogether, these data suggest that HCV reprograms the hepatocyte metabolism and establishes glutamine dependence. This HCV-induced metabolic reprogramming is similar to that commonly found in many types of tumor cells. Because these changes seem to be required for viral replication, we are currently investigating their roles in the various steps of the viral life cycle, and their impact on the pathological features associated with chronic hepatitis C.

48

HIV-1 INDUCED IMPAIRMENT OF B-CELL FUNCTIONS

INVITED LECTURE

Name E-mail Institution Country

CHIODI Department of Microbiology, Tumor and Cell [email protected] Sweden FRANCESCA Biology, Karolinska Institutet, Stockholm

Although HIV-1 does not directly infect B cells, B-cell numbers are reduced and their function is impaired during HIV-1 infection. Rare B-cell populations with an exhausted phenotype are expanded in the blood of HIV-1-infected patients and the frequency of classical memory B cells involved in maintenance of serological memory, declines during HIV-1 infection. As a reflection of B cell dysfunctions, antibody titres against antigens previously encountered through vaccination or natural infection are low in patients with HIV-1, including responses to T cell dependent and T-cell independent antigens.The life of B cells starts in the bone marrow; for the generation of acquired responses B cells need to migrate out of the bone marrow and reach secondary lymphoid tissues where they become activate, undergo negative selection and meet the antigens. This process of movement between organs is mediated by chemokines and chemokine receptors which ensure lymphocyte recirculation between the periphery and secondary lymphoid organs during immune responses. An increased frequency of immature transitional (IT) B cells can be detected in the blood of HIV-1 infected patients naïve to treatment and in a recent study (Amu S et al., AIDS 2016) we showed that IT B cells from HIV-1 infected patients displayed several changes in the expression of chemokine receptors pivotal for B cell differentiation and responded poorly to chemokines regulating important B cell functions. Intrinsic B-cell defects might be involved in the impairment of humoral immunity during HIV-1 infection. Abnormal T-cell activation may cause dysfunctional interaction between T and B cells in the germinal centres of lymphoid tissues (Lantto R, AIDS 2015). The expression of inhibitory receptors may impair B cell function as antibody producing cells (Rethi B, AIDS 2013). The impact of anti-retroviral treatment on B cell function and phenotypes will be discussed.

49

HEPATITIS B AND C - A SILENT EPIDEMIC

INVITED LECTURE Name E-mail Institution Country Centers for Disease Control and Prevention, United States of DROBENIUC JAN [email protected] Division of Viral Hepatitis, Atlanta America

Globally, an estimated 8 million new hepatitis B and C infections occur annually, and 390 million people are living with chronic hepatitis B and C; almost one million deaths are HBV-, or HCV- related. Over the last 15 years the incidence of acute hepatitis B decreased dramatically due to successful hepatitis B vaccination programs, and acute hepatitis C also occurs less often with the introduction of blood donations screening and wide awareness campaigns. Nevertheless, these two infections remain important public health issue with the majority of acute cases occurring among illicit drug users; numerous outbreaks occur in health care settings due to unsafe injections, inadequate infection control practices and narcotics diversion. Highly accurate estimates of the burden from hepatitis B and C determined by The Institute of Medicine (IOM) in its 2010 report, show that viral hepatitis is an underappreciated health concern, a silent epidemic for the United States, where 65%-75% of infected population remain unaware of their infection status. Despite the implementation of anti-HCV screening tests and other prevention strategies, every year approximately 20,000 new cases of hepatitis C occur in the United States, with 85% of them developing chronic disease. This enormous source of chronic HCV infections imposes a significant health burden. In the absence of treatment 15%-40% of persons living with viral hepatitis will develop liver cirrhosis. Rates of death related to hepatitis C in the United State rose at an average rate of 0.18 deaths per 100,000 persons per year, surpassing HIV mortality rates in 2007, when more than 15,000 people died from hepatitis. Health and Human Service (HHS) Action Plan for the Prevention, Care and Treatment of Viral Hepatitis launched in 2011, states that successful testing for HCV and better provision of care and treatment to those who are infected can decrease the burden of disease. Implementation of recommendations regarding anti-HCV screening among the 1945-1965 birth cohort- the US population with highest prevalence of HCV infection, is estimated to identify and refer to care additional 809,000 infected people, which will help prevent 120,000 deaths from hepatitis C and avert spending of $2.5 billion in medical costs. Additional disease prevention effect is expected from massive education campaigns. In 2010, World Health Assembly approved its “Resolution 63.18: Comprehensive Hepatitis Prevention and Control”, sponsored by Brazil, Columbia and Indonesia, calling WHO to develop a comprehensive approach to hepatitis prevention and control. The Global Hepatitis Framework set the priorities for global control of viral hepatitis, especially B and C that annually silently kill almost 1 million of their victims. The Framework Axes focus on (1) development of partnership and resource mobilization, and communication; (2) accumulation of reliable data for policy and action; (3) prevention of virus transmissions; and (4) screening care and treatment. Clear understanding of hepatitis B and C epidemiology through systematic disease surveillance, and accurate identification of infected persons early in the course of disease, and referring them to care and treatment are cornerstone goals for prevention of advanced stages of liver disease and cancer. Collaborations among public health, clinical care providers, laboratories payers and industry are essential and must be implemented at the national levels, in order to stop the silent epidemic of hepatitis B and C at the global scale.

50

INTRA-HOST HCV EVOLUTION

INVITED LECTURE Name E-mail Institution Country Centers for Disease Control and KHUDYAKOV United States of [email protected] Prevention, Division of Viral Hepatitis, YURY America Atlanta

Hepatitis C virus (HCV) is the leading cause of chronic liver disease and hepatocellular carcinoma. Approximately 180 million people are infected with HCV worldwide, with 3.5 million infected individuals residing in the United States, many of whom unaware they are infected. HCV causes chronic infection in ~80% of infected persons. However, molecular mechanisms of establishing chronic infection and molecular evolution of HCV during chronic infection are not well understood. Each infection is usually established from several founders transmitted to naïve hosts from the source patient. HCV rapidly evolves from the founders. Intra-host HCV evolution is under control of the strengthening with time negative selection, reflecting increased adaptation of HCV to the infected host during chronic infection. The structure of intra-host HCV population is shaped by single-point mutations and variation in density of subpopulations. Immune selection plays a key role in determining HCV genetic heterogeneity over time in infected persons. Recently, it was shown that the major neutralizing epitopes located in hypervariable region 1 (HVR1) are extensively cross-immunoreactive,indicating that the number of immunological specificities distributed across the entire HVR1 sequence space is limited. Frequent cross-immunoreactivity among genetically distant HVR1 variants significantly reduces the inverse relationship between the genetic distance and cross-immunoreactivity. Taken together with the observation of a significant convergence among HVR1 variants from different genotypes and subtypes, these findings point to ample opportunities for immune escape during chronic infection, thus facilitating intra-host HVR1 evolution, and to restrictions on inter-host evolution. Novel mathematical models considering a broad variation of cross- immunoreactivity among HVR1 variants in infected persons have been recently generated to show specific and extensive immunological interactions among intra-host HCV variants that fundamentally shape HCV evolution during chronic infection.

PATTERNS OF HIV-1 EVOLUTION IN EUROPE

INVITED LECTURE Name E-mail Institution Country LUKASHOV Academic Medical Center, University of The [email protected] VLADIMIR Amsterdam, Amsterdam Netherlands

Background and aims. To study the development of the HIV-1/AIDS epidemic(s) in Europe, The Netherlands/North Europe and the former Soviet Union (fSU) in particular. Methods. Phylogenetic and evolutionary analysis of >10,000 HIV-1 sequence samples collected since the early 1980s till present in over 20 European/fSU countries, together with detailed epidemiological information on infected individuals. Results and Conclusions. We were able to reconstruct the natural history of the HIV-1 epidemic(s) in Europe since its onset in the late 1970s till present, including the origin of the introduced viruses, their dissemination and propagation among various epidemiological networks, as well as networks’ characteristics (their definitions, stability, and interactions with other networks). Our data demonstrate that the HIV-1 epidemic in Europe in general, and even in particular countries, should be viewed not as a single epidemic, but as complexes of epidemics defined by characteristics of socio-epidemiological networks.

51

ONCOGENIC POTENTIAL OF HEPATITIS C VIRUS

INVITED LECTURE Name E-mail Institution Country

Saint Louis University, Internal United States of RAY RANJIT [email protected] Medicine, St. Louis America

Hepatitis C virus (HCV) infection of primary human hepatocytes induces epithelial mesenchymal transition (EMT) and extends hepatocyte life span. These hepatocytes display sphere formation on ultralow binding plate, and survive for more than 12 weeks. The sphere forming hepatocytes express a number of cancer stem-like cell (CSC) markers, including high level of stem cell factor receptor c-Kit. The c-Kit receptor is regarded as one of the CSC markers in hepatocellular carcinoma (HCC). Analysis for c-Kit mRNA in liver biopsies display a significant increase in chronically HCV-infected patients and HCV-associated HCC, when compared to control liver. c-Kit is highly expressed in cell culture grown HCV genotype 2a (clone JFH1) infected transformed human hepatocytes (THH) as compared to mock-treated controls, and virus core protein significantly up-regulates c-Kit at the transcriptional level. Infected THH display significant increase in the number of spheres on ultra-low binding plate, EMT markers (Snail, Slug, Twist and Vimentin), and express CSC markers (CD133, Nanog, Lin28A, Oct4A, Notch1, c-myc). In addition, sphere forming cells implanted in the flanks of immunodeficient mice display tumor growth. The sphere forming cells exhibit high sensitivity to sorafenib or static treatment. Combination of these two drugs display an additive effect on cell death. Together these results suggest that HCV infection potentiates CSC generation, and selected drugs can be targeted to inhibit cancer cell growth. Further work is in progress in understanding the role of Akt, β-catenin and Notch for growth regulatory role in HCV infected hepatocytes.

52

HEPATITIS A VIRUS, AND THE ANALYSIS OF HEPATITIS A OUTBREAKS IN EUROPE

ORAL Name E-mail Institution Country ANDERS WIDELL [email protected] Lund University Sweden

Hepatitis A virus (HAV) is a unique picornavirus, classified in a separate genus, Hepatovirus, and is very stable both physically, and over time and genetically. HAV only infects primates and can be separated into 6 genotypes, each designated by a Roman letter. Genotypes I, II and III circulate among humans, each with two subtypes A and B (e.g. IIIA) whereas genotype IV, V and VI only have been found in non-human primates. Genotype III circulates among both humans and New World monkeys. The viral capsid by cryo electron microscopy shows a typical icosahedral structure of picornaviruses. The modest nucleotide diversity corresponding to the viral capsid protein VP1 and the adjacent 2A protein genes, is commonly used for molecular genotype/subtype classification and also to analyze putative transmission chains. Acute hepatitis A is diagnosed by a simple serological test (IgM anti-HAV) whereas isolated IgG anti-HAV seroreactivity stands for previous exposure or vaccination. During the prodromal and the acute phase HAV RNA is detectable in serum/plasma by PCR, permitting sequencing and molecular epidemiology on a blood sample. HAV infection can become prolonged sometimes but never becomes chronic. Interestingly, HAV exposure is inversely correlated to allergy and atopia – possibly due to that the putative HAV cellular receptor the TIM-1 molecule is also part of the host pathway for allergy. The overall HAV epidemiology is generally known, being mainly caused by feco-oral transmission in particular in settings with lower socioeconomic standards, mainly during childhood and mainly in developing countries. In most West-European countries HAV circulation has in recent decades declined to low levels or nil and there are only few areas of endemic circulation. This is in contrast to most countries in Africa and Asia and Latin America where HAV infection is endemic in many regions. Travel to endemic countries is a classical way of importing HAV. In recent years, travel HAV import often is associated with non-immune preschool children of immigrant families who visit their ancestral countries, like the Middle East, get infected there and bring HAV back to Western countries. These children may have subclinical infection, but are contagious and the infection may spread in the children´s day care centers, with recognized symptoms first seen among adult staff members. In addition, HAV contaminated food like raw shellfish, salads and a range of berries, etc., have caused numerous food borne outbreaks. Outbreaks are often recognized in countries where HAV still circulates is poorer sections of society whereas those with higher hygienic standards have been spared until exposed to contaminated food. Finally, adults with intimate contacts, either sexually (in particular MSM) or via HAV viremic blood through such as illicit injecting drug use, have repeatedly been involved in large HAV outbreaks, sometimes transnationally. The author will describe a number of different HAV outbreaks including one recently associated with berries from Italy, spreading to several European countries and describe how they have been analyzed by both standard epidemiological methods as well as by molecular methods.

53

ANALYSIS OF TRANSMITTED HIV-1 VARIANTS IN REAL TIME AMONG ACUTELY INFECTED PEOPLE WHO INJECT DRUGS IN SANT PETERSBURG, RUSSIA REVEALS THE HIV-1 TRANSMISSION BOTTLENECK

E-mail Institution Country ORAL Name Peter the Great St. Petersburg Russian Federation AKULOVA EKATERINA [email protected] Polytechnic University (PGPPU) (RF) DUKHOVLINOVA ELENA [email protected] University of North Carolina USA Masharsky Alexey [email protected] PGPPU, St Petersburg RF

Shevchenko Andrey [email protected] The Biomedical Center RF

Verevochkin Sergey [email protected] Biomedical Center RF

Pavlov First St.Petersburg State Toussova Olga [email protected] RF Medical University

Vasileva Aleksandra [email protected] PGPPU, St Petersburg RF

Lineberger Comprehensive Cancer Zhou Shuntai [email protected] USA Center, UNC-Chapel Hill Hoffman Irving [email protected] University of North Carolina USA

Miller Bill [email protected] University of North Carolina USA

Frishman Dmitrij [email protected] PGPPU, St Petersburg RF

Duke University Medical Center, Duke Montefiori David [email protected] USA University Department of Biochemistry and Swanstrom Ronald [email protected] USA Biophysics, UNC-Chapel Hill

Kozlov Andrei [email protected] The Biomedical Center RF

54 Statement of purpose. In Saint Petersburg, the HIV-1 epidemic among people who inject drugs (PWID) is associated with subtype A virus of relatively low heterogeneity as compared to subtype B virus in men who have sex with men. The phenomenon of a genetic bottleneck, i.e. transmission of one or a few variants, has been widely studied for sexual route of transmission, however for PWID the available data are controversial. Here we report the first study of real-time detection and follow-up of individual cases of acute HIV-1 infection (AHI) and analysis of the genetic variability and phenotypic properties of their Env protein. Materials & Methods. We analyzed full-length env genes of transmitted strains and their evolution post- transmission using single genome amplification (SGA) and Bayesian Evolutionary Analysis Sampling Trees (BEAST) approach. We also implemented the PrimerID Illumina MiSeq approach for ultra-deep sampling of a fragment of the env gene to look for the presence of minor transmitted variants. We created a panel of pseudoviruses derived from the transmitted and persistent variants and tested their phenotypic properties in cell culture. Results. Among 163 PWID screened for the study 40 (25%) were sero-positive. The calculated AHI incidence in the followed-up cohort of 100 PWID was 9.3 per 100 person-years. We report the data on 7 cases of acute HIV-1 infection among active PWIDs and 8 potential sexual partners of PWID. In all cases the infection was due to subtype A viral variants. We detected 3 genetic clusters of viral transmission: two had a chronically infected individual present in the cluster, while the third was an AHI-to-AHI transmission case. We used BEAST to estimate the evolution rate of the transmitted strains (with and without CTL escape mutations) which fluctuated. Among all the cases studied by SGA and Primer ID approaches, including those individuals for whom the injection drug use was determined by the questionnaire as the most probable route of infection, we detected a homogeneous viral population likely produced from a single viral variant. We also detected one case of a secondary infection from a different donor, with the secondary variant outgrowing the initially transmitted virus. Adding to previously published data we have analyzed 19 cases of AHI subtype A in Saint Petersburg, and 14 (74%) had a homogeneous viral population confirming a strong genetic bottleneck during parenteral transmission. Phenotypic analysis of transmitted and persistent viral strains revealed one viral variant from a chronically infected individual that was able to use both CCR5 and CXCR4. The neutralization properties of pseudoviruses against HIVIG-C, sCD4, 4E10 and PG9 did not reveal significant differences between transmitted and chronic variants of HIV-1 subtype A, but rather a significant difference between PWID- derived isolates of subtype A and a collection of isolates from heterosexually transmitted subtype C virus. Conclusions. Our data confirms the presence of an HIV-1 subtype A strong genetic bottleneck among the PWID. The surveillance and prevention of the acute HIV-1 infection prior to the development of a potent immune response is necessary in Saint Petersburg, with AHI-to-AHI transmission and superinfection are features of the ongoing epidemic. We developed and implemented an approach to recruit, screen and follow- up the cohort of PWIDs in real time that could be translated to the health care sector.

55

TRENDS IN RATES OF CHLAMYDIA IN LITHUANIA, 2011-2015

ORAL Name E-mail Institution Country CAPLINSKIENE IRMA [email protected] CCDA Lithuania Centre for Communicable Diseases and Simkunaite-Zazecke Agne [email protected] Lithuania AIDS Mykolas Romeris University, Caplinskas Saulius [email protected] Centre for Communicable Diseases Lithuania and AIDS

Background. Chlamydia trachomatis is the most common reportable sexually transmitted infection (STI) in Lithuania and other European countries. More than a half of cases are reported in young people and the highest rates are observed among women in EU/EEA Member States. Methods. Chlamydia epidemic review is based on retrospective analysis of the national STI surveillance database. The aim was to find out trends in rates of chlamydia during the period of 2011-2015 in Lithuania. Results. Between 2011 and 2015, 1772 cases of Chlamydia trachomatis infection were reported in Lithuania. Males accounted for 52.3% (N=927) of all reported cases, while females presented 47.7% (N=845) of patients diagnosed with chlamydia in the last five years. The male-female ratio varied during the period: in 2011-2014 it was an average 1.2 and only in 2015, chlamydia infections were reported more often in women than in men with the ratio of 0.8. Young persons (15-29 years old) accounted for an average 55% of reported cases and the proportion has been increasing annually. Since 2011, the incidence of chlamydia increased almost a quarter. Between 2011 and 2012, the notifications per 100 000 population declined from 11.3 to 8.9, then stared to increase and peaked 15.3 in 2014, after then declined again up to 14.1 in 2015. Between 2011 and 2015, the rate among men has decreased from 14.2 to 13.8, while among women increased significantly from 8.9 to 14.4 per 100 000. The highest increase in rates was observed among young people diagnosed with chlamydia. Between 2011 and 2015, the rate among 15-29 years old residents increased from 27.5 to 41.4 per 100 000. By gender has increased respectively: in women from 27.9 to 49.6, more less in men from 27.1 to 33.2 notifications per 100 000 population. Conclusions. The incidence of Chlamydia trachomatis has increased during last five years in Lithuania, especially among women. The most affected population is young 15-29 years old people. Due to asymptomatic infections, the true incidence may still be unknown. To prevent the spread of chlamydia, the control strategy should be focused on sexual education program as well as testing policy development and surveillance system improvement.

56

HIV EPIDEMIC IN LITHUANIA - SURVEILLANCE RESULTS, 1988-2015

ORAL Name E-mail Institution Country CAPLINSKIENE IRMA [email protected] CCDA Lithuania Juciene Oksana [email protected] CCDA Lithuania Mykolas Romeris University, Caplinskas Saulius [email protected] Lithuania Centre for Communicable Diseases and AIDS

Background. The objective of the study was assessment of 28 years of the HIV epidemic in Lithuania. Methods. HIV epidemic review is based on retrospective analysis of the national HIV surveillance database (1988-2015). Statistical data analysis was performed by application of SPSS package. Results. Totally 2535 HIV cases were reported in Lithuania at the end of 2015. During the whole HIV reporting period totally 2017 HIV cases in males and 518 in females have been reported (by the mode of transmission: IDUs - 61.5% (n=1558) of cases, heterosexual contact - 20.8% (n=527) of cases, homosexual contact - 7.7% (n=195), perinatal - 0.2% (n=5), unknown - 9% (n=250) cases. In 1988-1996 52 HIV cases were reported. HIV incidence rate in this period varied from 0.03/100,000 population in 1988 up to 0.32/100,000 population in 1996. Heterosexual (50 % of all registered cases) and homosexual (38.5% of all registered cases) HIV transmission mode has dominated. During 1997-2003 793 HIV cases were registered. HIV incidence rate increased from 0.84/100,000 population in 1997 up to 3.8/100,000 population in 2003. In 2001 rapid increase in rate of HIV diagnoses was observed due to HIV outbreak in Alytus prison (HIV prevalence was 10.87/100,000 population). Drug use was recognised as the main mode of transmission - at least 674 registered cases (85 %). Heterosexual transmission occurred in 5.5 % of cases, homosexual in 4.4 % of cases. During 2004-2010, 889 HIV cases were registered. HIV incidence rate in years 2004-2008 varied slightly: 3.93/100,000 population in 2004, and 2.97/100,000 population in 2008. Since 2009 HIV incidence has increased - 5.7/100,000 population in 2009, and 4.9/100,000 population in 2010. In 2004-2010 HIV cases were predominantly reported in IDUs: 64.2% of all cases. During 2011-2015, 801 HIV cases were registered. HIV incidence rate in years 2011-2015 was stable: 5.5/100,000 population in 2011, and 5.4/100,000 population in 2015. In 2011-2015 HIV cases were predominantly reported in IDUs and via heterosexual contacts: 37.5% and 33.7 % of all cases. Conclusions. By prevailing modes of HIV transmission over 28 years, 3 phases of HIV spread may be distinquished. Up to 1997, the sexual HIV transmission mode prevailed; during 1997-2003, HIV was mainly transmitted via injecting equipment in the IDUs population; since 2004, the virus has spread mainly via injecting equipment in the IDUs population, while the mode of heterosexual transmission has been reported more and more often. In 2013 were more infected through sexual contacts than through intravenous drug use. This means that the HIV infection progresses to the next phase of the spread of HIV. Is likely to continue to actively spread of HIV to the general population through IDU sexual partners (bridging population).

57

PREVALENCE OF HEPATITIS B AND C AND RISKY BEHAVIOUR AMONG INTRAVENOUS DRUG USERS IN LITHUANIAN REGIONAL LOW THRESHOLD CENTRES (LTC)

ORAL Name E-mail Institution Country CAPLINSKIENE IRMA [email protected] CCDA Lithuania Venckevic Evelina [email protected] CCDA Lithuania

Introduction. HBV and HCV can easily be transmitted through injecting drug use due to unsafe injection practices. Routine HBV and HCV testing programs among LTC IDUs clients are not available in Lithuania. Bio-behavioural study among IDUs in three main regional LTC in Lithuania was conducted by Centre for Communicable diseases and AIDS under MOH together with Lithuanian Drug, Tobacco and Alcohol Control Department. Objective. To assess prevalence of hepatitis B and C among active IDUs and to evaluate the risky behaviour in year 2015. Methods. Survey type: cross-sectional study (bio-behavioural). Sampling method: LTC service based. Measurements: structured questionnaire (anonymous) and voluntary counselling and testing (linked to questionnaire, rapid tests (HBsAg, antiHCV)). Intensives: 5 EUR coupon. Results. Study participants: N=200(50+50+100) active IDUs in three LTC in different Lithuanian regional cities (Alytus, Visaginas and Klaipeda). Sociodemographic characteristics: men - 82,5% women - 17,5%; age group: 25 - 44 years; 60 % IDUs had imprisonment experience (1-2 imprisonments); 63% been treated for drug abuse; 7,5% participated in the program of methadone/buprenorphine replacement therapy; main psychoactive substance - heroine (90,5 %). Frequency of injection: 2-3 times per/day (27%), once per/day (19 %), 4 or more (18%). Sharing injecting paraphernalia (last 30 days): 72 % - did not share, 21,5 % - shared. 48% had no regular sexual partner in last 12 month, 32% - had. 69,2 % during the last sex did not use condoms with their regular partners, and 20,4 % indicated they did. 31 % - had casual sex partners and 55,6 % - did not use condoms in last sex with casual partners; 96% no sex for money, drugs, food, etc. More than two thirds (78,5%) of participants were not vaccinated against HBV, 7,5%-vaccinated, 14% - not answer. Prevalence HBsAg(+) in LTCs: Visaginas (12%), Alytus (26%), Klaipeda (2%); prevalence of antiHCV(+) in LTCs: Visaginas (82%), Alytus (72%), Klaipeda (77%). Conclusions. There is very high prevalence of HCV, most probably resulted from sharing of injecting paraphernalia. Anti-HCV prevalence among different LTC statistically did not differ (p>0,05). This study established a lower incidence of anti-HCV than previous studies carried out in Lithuania (2009, ENCAP report). Average of HBsAg prevalence was 13,3%. HBV vaccination coverage (self-reported) very low. Targeted and combined delivery of services, including vaccination programs against HBV, should be expanded among high risk groups.

58 COMPARATIVE ANALYSIS OF THE 2014 AND 2015 TUBERCULIN SKIN TEST

POSTER Name E-mail Institution Country Mykolas Romeris University, CAPLINSKAS SAULIUS [email protected] Lithuania Centre for Communicable Diseases and AIDS Centre for Communicable Diseases Zagrebneviene Galina [email protected] Lithuania and AIDS Korablioviene Joana [email protected] ULAC Lithuania Centre for Communicable Diseases Sebeliauskaite Ieva [email protected] Lithuania and AIDS Centre for Communicable Diseases Kriauciunaite Asta [email protected] Lithuania and AIDS

Introduction. In order to ensure effective tuberculosis (TB) prevention and control, in accordance with the 2013 December 27 Lithuanian Minister of Health Order No. V - 1249, tuberculin skin test (TST) every year must be carried out for all 7 years children's and 0 -17 years children's in the risk groups: unvaccinated children's (Bacillus Calmette–Guérin (BCG) vaccine), in close contact with someone who has TB, that often suffer from upper respiratory tract infections, children's with chronic diseases, treated with corticosteroids, infected with human immunodeficiency virus or in other immunodeficiency disorders, from social risk families, living in care homes or care home for infants with developmental disorders, migrants. Objective. To compare and analyze 2014 and 2015 results of TST. Methods. Data analysis was performed with: ibm lotus domino enterprise server; ibm lotus notes; ibm db2; ibm websphere portal; crystal reports server; microsoft excel; openoffice.org calc. Results. In 2015 (52.4%) TST coverage among 7 y children's and children's in the risk groups was 5.7% higher than in 2014 (46.7%) (Fig.1). In 2015 (13.1%) was 3.9% more positive TST out of carried out TST then in 2014 (9.2%) (Fig. 2). In 2015 TB (A15-19 ICD-10) was confirmed in 1.1% of the positive TST cases, it was less than in 2014 (2.3%) (Fig. 3). In 2015 open TB (A15.0-15.2, 15.5 ICD-10) was confirmed in 6.7% of all TB (A15-19 ICD-10) cases diagnosed out of positive TST. In 2014 open TB confirmed in 11.2% (Fig. 4). Conclusions. 1. In 2015 TST coverage among 7 y children's and children's in the risk groups was 5.7% higher than in 2014. 2. In 2015 was 3.9% more positive TST out of carried out TST then in 2014. 3. In 2015 TB was confirmed in 1.1% of the positive TST cases. 4. In 2015 open TB was confirmed in 6.7% of all TB cases diagnosed out of positive TST.

59

BCG VACCINATION COVERAGE BY WHO REGION IN BALTIC STATES AND LITHUANIA

POSTER Name E-mail Institution Country Mykolas Romeris University, CAPLINSKAS SAULIUS [email protected] Lithuania Centre for Communicable Diseases and AIDS Korablioviene Joana [email protected] ULAC Lithuania Centre for Communicable Diseases Sebeliauskaite Ieva [email protected] Lithuania and AIDS

Introduction. The Global Vaccine Action Plan (GVAP) is a roadmap to prevent millions of deaths through more equitable access to vaccines. Countries are aiming to achieve vaccination coverage of ≥90% nationally and ≥80% in every district by 2020. By 2014, 4 WHO regions had reached at least 90% coverage of Bacillus Calmette–Guérin (BCG) vaccine: Western Pacific (97%), Americas (95%), European (94 %) and South - East Asia (92%). Among the Baltic States highest vaccination coverage was in Lithuania (98%). Over the past 13 years the lowest BCG vaccination coverage registered in Lithuania in 2015 (97.2%). Objective. To review the BCG vaccination coverage by WHO region in Baltic states and Lithuania. Methods.Data analysis was performed with: ibm lotus domino enterprise server; ibm lotus notes; ibm db2; ibm websphere portal; crystal reports server; microsoft excel; openoffice.org calc. Results. In 2014 highest BCG vaccination coverage was in Western Pacific (97%) Americas (95%) and European (94 %) regions. Among the Baltic States highest vaccination coverage was in Lithuania (98%). In 2015 Lithuanian BCG vaccination coverage (97.2%) was 0.5% lower then in 2014 (97.7%). Over the past 13 years the lowest BCG vaccination coverage registered in Lithuania in 2015 (97.2%). Lower than the national BCG vaccination coverage (97.2%) was in 4 Lithuanian districts: Telšiai (95.6%), Vilniaus (96.2%), Kaunas (97%) and Klaipeda (97.1%). Conclusions. 1. In 2014 highest BCG vaccination coverage was in Western Pacific region (97%). 2. Among the Baltic States highest vaccination coverage was in Lithuania (98%). 3. Over the past 13 years the lowest BCG vaccination coverage in Lithuania was in 2015 (97.2%). 4. Lower than the national BCG vaccination coverage (97.2%) was in four Lithuanian districts: Telšiai (95.6%), Vilniaus (96.2%), Kaunas (97%) and KlaipД—da (97.1%).

60

THE HOST IMMUNE SYSTEM MAY NOT BE THE MAIN DRIVING FACTOR OF HEPATITIS C VIRUS GENETIC DIVERSITY

ORAL Name E-mail Institution Country CUYPERS LIZE [email protected] KULeuven Belgium Li Guangdi [email protected] Central South University China Neumann-Haefelin christoph.neumann-haefelin@uniklinik- University Of Freiburg Germany Christoph freiburg.de Artificial Intelligence Lab-Vrije Libin Pieter [email protected] Belgium Universiteit, Brussel KU Leuven-Rega Institute For Van Laethem Kristel [email protected] Belgium Medical Research Vandamme Anne- KU Leuven-Rega Institute For [email protected] Belgium Mieke Medical Research KU Leuven-Rega Institute For Theys Kristof [email protected] Belgium Medical Research

Background. The development of preventive vaccines and therapeutic cure is largely hampered by the high evolutionary rate of the hepatitis C virus (HCV), permitting rapid adaptation of HCV to changing environments. HCV variants can escape from the strong selective pressure exerted by the host innate and adaptive immune system. Detailed mapping of immunological constraints across the HCV genome enriches our understanding of viral evolution and how this is shaped by the host immune system. Methods. In total, 647 HCV subtype 1a and 408 HCV subtype 1b full-genome sequences sampled before the DAA era were obtained from the Los Alamos HCV sequence database. Amino acid conservation, selective pressure (using HyPhy), and immunological constraints (CD8+ and CD4+ T-cell epitopes, and B- cell epitopes) were mapped along the HCV genome. Correlation between amino acid conservation or selective pressure, and immune epitopes was identified using generalized linear regression models. Results. Among 3000 amino acid positions in HCV coding regions, only 3.12% were found to be under positive selective pressure. In general, a lower number of positions in the HCV genome was covered by CD8+ T-cell epitopes (HCV1a/1b: 17%) compared to B-cell (HCV1a: 49%, HCV1b: 67%) and CD4+ T- cell epitopes (HCV1a: 73%, HCV1b: 50%). However, all types of immune epitopes were mostly abundant in the core protein, since the highest proportions of mapped epitopes were observed in that region. General linear regression analysis showed that amino acid conservation was negatively correlated with the presence of B-cell epitopes (OR(1a):0.49 [0.40-0.60]; OR(1b):0.59 [0.46-0.76]), in contrast to CD4+ and CD8+ T- cell epitopes which were identified as independent predictors of higher amino acid conservation (OR(1a CD8+): 1.27 [1.09-1.50]). Positively selected positions were more likely to be identified in positions covered by CD8+ T-cell epitopes (HCV1a), and less likely for CD4+ T-cell epitopes (HCV1b). B-cell epitopes were identified as independent predictors of lower proportions of positions under negative selective pressure (OR(1a): 0.55 [0.49-0.61]; OR(1b): 0.47 [0.41-0.54]), as well as CD8+ T-cell epitopes (OR(1a): 0.71 [0.62-0.82]; OR(1b): 0.80 [0.69-0.93]). Additionally, for HCV1a, CD4+ T-cell epitopes were an independent predictor for amino acid positions under negative selective pressure. Conclusions. The high level of negative selective pressure and limited number of positively selected sites in the full-length genome indicate that HCV mainly evolves under the neutral model of evolution, and its diversity is mainly due to random genetic drift. This is consistent with the significant correlation between T-cell epitopes and positive selective pressure on one hand, but amino acid conservation on the other hand, which argues against immune selective pressure as the main force for diversifying evolution. Overall, our findings suggest that while the host immune system certainly does exert selective pressure, it may not be the major driving factor of HCV genetic diversity.

61

EXPRESSION OF HEPATITIS C VIRUS CORE AND ARF PROTEIN VARIANTS IN EUKARYOTIC SYSTEM

POSTER Name E-mail Institution Country DOVBENKO Latvian Biomedical Research and Study [email protected] Latvia ANASTASIJA Center Latvian Biomedical Research and Study Petrovska Ramona [email protected] Latvia Center Latvian Biomedical Research and Study Akopjana Inara [email protected] Latvia Center Latvian Biomedical Research and Study Jansons Juris [email protected] Latvia Center, Riga Stradins University Latvian Biomedical Research and Study Stahovska Irina [email protected] Latvia Center Latvian Biomedical Research and Sominskaya Irina [email protected] Latvia StudyCentre

Background and Aims: Hepatitis C virus (HCV) persists in up to 85% of infected individuals as a chronic infection characterized by liver infiltration of inflammatory cells that can lead to fibrosis, cirrhosis and hepatocellular carcinoma. There is no vaccine against HCV and available therapy is expensive and related with different side effects. HCV core protein is highly conserved among various HCV genotypes with amino acid homology exceeding 96% and also contains several well-characterized B-cell and cytotoxic T- lymphocyte epitopes. In addition to its role in viral capsid formation, the core protein has been suggested to directly interact with a number of cellular proteins and pathways that may be important in the viral lifecycle. Core protein can influence a variety of intracellular processes: apoptosis, lipid metabolism, transcription and the development of immune response. Recently another HCV protein was discovered. It is synthesized as a result of programmed ribosomal frameshift. This novel HCV protein was named F (for frameshift), ARFP (alternative frame protein) or core+1 (to indicate the position of the new open reading frame) protein. ARFP participates in HCV morphology or replication. ARFP is associated with endoplasmatic reticulum and can be important in gene regulation, cell signal transfer and apoptosis also it can affect immune response mechanisms. ARFP could have potential value in diagnostic tests and as a component for vaccines. The main aim of the study was design and characterization of plasmids containing different HCV core and ARF variants. Methods. Plasmids with approprite HCV core and ARF variants were designed according to FDA recommendations. Plasmids were created on the basis of pVax1 plasmid comprising T7 promoter, kanamycin as the resistance gene for bacterial growth, and CMV promoter for expression in eukaryotic system. Eukaryotic expression of HCV core and ARFP variants were tested in embryonic kidney cells (HEK 293T). Transfection was performed by TurboFect reagent according to manufacturer`s protocol. Cell monolayers were harvested 48h post transfection and cell lysates were analyzed by SDS-PAAG under denaturating conditions and ECL western blotting with rabbit anti-HCV core or anti-ARFP antibodies. Results. We used set of plasmids containing different HCV core/ARFP variants. We have made plasmids containing wild-type HCV core variant, plasmids with prohibited ARFP expression, plasmid expressing only ARFP. Inclusion of additional ATG codon in wild-type HCV core gene doesn’t improve expression level. One of constructs where mutation was introduced in 8-11 aa region of core protein gene showed higher protein expression level. Also we have constructs expressing synthetic HCV core with optimized codons for expression in mice. We compare full optimized HCV core with truncated variant. Plasmids one of which contain mutation that prohibit expression of HCV core protein and other where HCV core gene reading frame is shifted +1 nt (ARFP should express in both cases), however do not show any protein expression. Conclusion. ARF protein apparantly do not accumulate in sufficient quantity in cells to be detected by Western blot. Mutation in core gene which disrupts RNA hairpin structure at 8-11 aa position probably improves mRNA structure and protein expression respectively.

62

INFECTIONS RELATED TO BLOOD TRANSFUSION - IS THERE A RISK?

POSTER Name E-mail Institution Country Hospital of Lithuanian University of Health DUDKO JULIANA [email protected] Lithuania Sciences Kauno Klinikos ADUKAUSKIENE Hospital of Lithuanian University of Health [email protected] Lithuania DALIA Sciences Kauno Klinikos Hospital of Lithuanian University of Health Valanciene Dovile [email protected] Lithuania Sciences Kauno Klinikos

Introduction. Even though blood transfusion can be a life – saving procedure and is generally considered to be safe it still can be a potential source of infection. Vigilant donor screening for risk factors and donated blood testing reduces the risk of transmitting viral infection including hepatitis and HIV viruses. Still there are other types of viruses which occur due to migration and can be transmitted through blood products from asymptomatic blood donors. Bacterial contamination is also a common source of transfusion-transmitted disease. Methods. We performed a systemic review to evaluate the incidence of infection trasmission through blood and it‘s products transfusion. Results. Hepatitis B virus (HBV) carries a major risk of transfusion-transmitted viral infection. The incidence of HBV transfusion with blood products is 0.02% in high income countries. It can be detected with HBV surface antigen (HBsAg) tests or HBV nucleic acid testing (NAT). Anti-HBc screening could be useful when HbsAG titer is too low. Hepatitis B surface antigen carriers also may carry hepatitis delta virus (HDV) which can occur as coinfection or superinfection and deteriorate the course of disease. The risk of hepatitis C virus (HCV) transmission is 0,02 % in high income countires. HCV RNA and antigens are used for virus detection. The risk of transmitting human immunodeficiency virus (HIV) is 0,002% in high income countires. West Nile virus can be transmitted through transfused red cells, platelets and fresh- frozen plasma. It‘s incidence is infrequent in Europe. WNV NAT is performed at the single-donation level in areas with high infection rate. Human T cell lymphotropic viruses I and II (HTLV-I/II) are associated with adult T cell leukemia and can be transmitted through cellular components only. It‘s infectivity depends on component storage and declines after approximately 10 days. Malaria parasite can be transfused through blood and it‘s products. The risk of transmission in non – endemic countries is minimazed by donor screening and additional antimalarial immunoglobulin testing. Siphilis can be rarely transmitted through the transfusion of fresh blood components. In 2006 the incidence of T. pallidum infection among blood donors was 7 per 100,000 donations. Total Ig enzyme immunoassays (EIAs) are the most sensitive for T. pallidum detection. Trypanosoma cruzi - the cause of Chagas disease - is common in Central and South America and parts of Mexico. Chronic carriers of the parasite from endemic areas have a potential risk of transmitting it through their donated blood. Babesia microti - is most common intra – erythrocytic parasite transmitted through blood transfusion and is hazardous to immunocompromised patients. The risk of transmission is reduced by leucoreduction. Rickettsia rickettsii is also rarely found in blood products. Bacterial contamination mostly occures due to poor hygiene conditions or during storage - typically in plateletes (20–24°C), less in red blood cells (1–6°C). Conclusions. Level of transfusion therapy safety has increased due to the combination of donor screening and donated blood testing. Although there is still a risk of known and unknown agent transmission. For this reason blood transfusion should be considered carefully and individually.

63

THE HIV OUTBREAK IN MINSK

ORAL Name E-mail Institution Country EREMIN The Republican Research and Practical Centre for [email protected] Belarus VLADIMIR Epidemiology and Microbiology The Republican Research and Practical Centre for Gasich Elena [email protected] Belarus Epidemiology and Microbiology Republican center of hygiene, epidemiology and public Fisenko Elena [email protected] Belarus health The Republican Research and Practical Centre for Yurovsky Pavel [email protected] Belarus Epidemiology and Microbiology Sasinovich The Republican Research and Practical Centre for [email protected] Belarus Sviataslau Epidemiology and Microbiology Domnich Maxim [email protected] Belarus

Introduction. 19 827 HIV/AIDS cases are officially registered in Republic of Belarus for January 1, 2016. The greatest number of HIV/AIDS patients are registered in age groups 25-29 and 20-39 years. 39% of patients have been infected parenterally at joint introduction of drugs, 58.7% -heterosexually. Since the end of 2014 till present in Minsk increase in quantity of HIV-infection cases connected with psychotropic drugs intravenous administration is noted. For interpretation of this outbreak we used along with classical epidemiological methods, methods of the molecular-epidemiological analysis. Materials & Methods. From June till September, 2015 it has been collected 61 blood serum/plasma samples from patients aged from 24 till 47 years infected in 2014 - 2015. All samples have been checked for anti-HIV antibodies by the ELISA and Western blot (BioRad) tests, virus loading has been defined in all a sample. Sequencing was spent on gag (p17/p24), env (V3 a loop), pol (protease) HIV-1 genome regions, as well as on gene S (preS1/S2) HBV and core/E1 HCV on the genetic ABI Prism 3100 Avant and AB3500 analyzers. The analysis of the received DNA fragments was carried out with use of software products "Sequencing Analysis Software v.5.1.1", "BioEdit", "SeqScape v.2.6". The phylogenetic analysis of the received DNA fragments was carried out by means of the Mega 6 program (trees with a root are constructed by neighbor-joining method. Nucleotide distances counted by Kimura's method. Results. 26 samples of HIV-1 on gag gene, 38 on pol and 30 on gene env have been sequenced. On gene region core/E1 of HCV we have sequenced and analysed 18 samples. 3 samples have been checked for markers of hepatitis B virus. The conducted researches have shown that all samples sequenced on HIV-1 gag, pol and env genes are belonged to the A1 subtype, and one specimen has been referred to a subtype B. Of 18 samples of HCV sequenced on core/E1 region 3 have been carried to subgenotype 1b, 6 – to 3a and 9 – to 1a. Two HBV positive samples have been defined as D2 subtype. The phylogenetic analysis has shown that all samples were distributed by groups on 2-5 and lay separately from each other on a phylogenetic tree that can indicate different sources of their infection. The specimens sequenced on core/E1 gene region also settled down separately on a phylogenetic tree, except for 4 samples of 1a subgenotype, that can also indicate independent infection of patients with HCV-infection. Two HBV specimens sequenced on S (preS1/S2) gene region had a uniform source of an origin. Conclusion. Thus, as have shown our preliminary researches results, the Minsk outbreak is characterized by existence of a set of the sources of infection which aren't connected among themselves and heterogeneity of virus population that can be connected with use of psychotropic preparations spices for intravenous administration.

64

MOLECULAR EPIDEMIOLOGY HBV IN BELARUS

POSTER Name E-mail Institution Country The Republican Research and Practical GASICH ELENA [email protected] Centre Belarus for Epidemiology and Microbiology The Republican Research and Practical Eremin Vladimir [email protected] Centre Belarus for Epidemiology and Microbiology The Republican Research and Practical Nemira Alina [email protected] Centre Belarus for Epidemiology and Microbiology

Background and aims. During last time Belarus continues to be the country with low level of hepatitis B prevalence. Vaccination against HBV led to decrease the number of acute HBV infections in 9.2 times over the past 15 years: from 12,46/100000 in 1998 to 1,28/100000 in 2015 years. At the same time number of individuals with chronic HBV infection increased on 53% (from 5,68/100000 in 2002 to 8,7/100000 in 2015). Main risk age groups are persons between 20-30 and 40 and older years living mainly in the cities. Due to the fact that adult population largely remains none vaccinated, the relevance of HBV infection in Belarus will remain for a long time. The aim of our research was to conduct molecular epidemiological researches and to define the genotypes/subtypes of a virus of hepatitis B (HBV) circulating in the territory of Republic of Belarus. Materials & Methods. From 2004 to 2015 HBV genotyping was performed by direct sequencing 600 samples using primers for P gene fragment. The genotype/subtype of HBV was confirmed by Phylogenetic Maximum Likelihood. Results. Among HBV-infected patients genotypic diversity of isolates of HBV were as follows: 479 sample (80,0±1,6%) were classified as genotype D, 112 (18,6±1,6%) – genotype A, 5 (0,8±0,4%) – to genotype C and one sample assigned to Q4 (0,2±0,2%). Two isolates (0,4±0,2%) treated to recombinant forms of the virus (A/D) of (0.2±0.2%) and C2/D (0,2±0,2%). Genotypes A, C and B of HBV in Belarus are represented by only one each subgenotype - A2, C2 and B4, respectively. Dominant on the territory of Belarus D genotype of HBV is characterized by a large genetic diversity and phylogenetically consistent with the four subgenotypes of virus. So, 96 (16,0±1.8%) of samples of HBV DNA belonged to the D1subgenotype, 239 (39,8±2,0%) to the D2 subgenotype, 140 (23,3±1,7%) to D3 subgenotype and in 5 cases (0,8±0,4%) was represented by D4 subgenotype of virus. Conclusion. It is shown that D genotype of HBV is the dominant. The identified genotypes of HBV (C2 ,D2, and B4), endemic to South-East Asia, Australia and Papua New Guinea, which is probably connected with migration of population. These genotypes had not occurred in Belarus. Conducted molecular genetic monitoring of the distribution and circulation of HBV variants is important to control of the infection in Belarus.

65

VIRULENCE AND RESISTANCE MARKERS IN COLISTIN-SUSCEPTIBLE PSEUDOMONAS AERUGINOSA STRAINS ISOLATED FROM CYSTIC FIBROSIS PATIENTS IN BUCHAREST, ROMANIA

POSTER Name E-mail Institution Country Faculty of Biology University GHEORGHE IRINA [email protected] Romania of Bucharest 1. Central Reference Laboratory Corina Cristea Violeta [email protected] Romania Synevo, Bucharest, Romania Faculty of Biology University Popa Marcela [email protected] Romania of Bucharest Faculty of Biology University of Bucharest, Research Institute Czobor Ilda [email protected] Romania of the University of Bucharest - ICUB Chifiriuc Mariana Faculty of Biology University [email protected] Romania Carmen of Bucharest 1. Central Reference Laboratory Sandu Gabriela [email protected] Romania Synevo, Bucharest, Romania 2. University of Medicine and Popa Ioan Mircea [email protected] Pharmacy “Carol Davila”, Romania Bucharest, Romania

Background and aims. The pulmonary chronic infections in patients with Cystic Fibrosis (CF) are an important cause of morbidity and mortality, leading to bronchiectasis, cor pulmonale and death from respiratory failure. Pseudomonas aeruginosa is the most prevalent microorganism isolated from the sputum of adult CF patients. The aim of this study was to investigate the molecular virulence and antibiotic resistance features of P. aeruginosa isolated from CF patients in Bucharest, Romania. Materials & Methods. The sputum was collected from the CF patients in empty dry containers and transported refrigerated in less than 24 h. The Gram stain was performed for internal routine, as rejection criteria for sputum are not applying for these patients. The sputum samples were inoculated into Columbia blood agar with 5% sheep blood, Chocolate agar and Mac Conkey agar and incubated for 24-72 hours at 37ºC, the first two media in CO2 enriched atmosphere. All isolates were identified by mass spectrometry using MALDI Biotyper. Antimicrobial susceptibility testing was performed using NM40 MicroScan Walk Away panels and the EUCAST breakpoints for interpretation. Multiplex and simplex PCR assays were used to detect the genes encoding different cell-wall associated and extracellular virulence factors, as well as resistance genes. Results. We examined 15 sputum specimens from patients with CF, women (n=8) and men (n=7). All specimens were P. aeruginosa positive in sputum cultures. The median age of the positive patients was 57 years (range: 18-88 years). All strains were susceptibile to colistin with MIC < 2 mg/L. The tested strains exhibited a multidrug resistance phenotype, being resistant to ciprofloxacin 47%, levofloxacin 33%, amikacin, tobramycin, aztreonam, ceftazidime and cefepime, 20% each. The molecular results demonstrate that all studied P. aeruginosa strains harbored the genes for on-haemolytic phospholipase (plcN) and protease IV, 86.6% were positive for alginate, haemolytic phospholipase (plcH) and exoS and 33% for the exoU gene. The beta- lactams resistance was encoded by blaTEM, while the genetic support of quinolones was represented by the association between gyrB and parE genes in all strains. Conclusions. Our results demonstrate that all the strains are positive for the majority of the investigated virulence determinants. Despite the multidrug resistance phenotype, all strains remained susceptible to colistin, which still remains an effective drug for treatment of P. aeruginosa strains in patients with CF.

66

PRELIMINARY RESULTS OF EFFICACY AND SAFETY OF DIRECT- ACTING ANTIVIRALS OMBITASVIR / PARITAPREVIR / RITONAVIR + DASABUVIR WITH OR WITHOUT RIBAVIRIN IN PATIENTS WITH HCV GENOTYPE 1 INFECTION IN REAL LIFE SETTING

POSTER Name E-mail Institution Country Vilnius University, Faculty of Medicine; Kazenaite Edita [email protected] Vilnius University Hospital Santariskiu Lithuania Klinikos POLUBENKO [email protected] Vilnius University Faculty of Medicine Lithuania KATAZYNA Vilnius University Hospital Santariskiu Jancoriene Ligita [email protected] Lithuania klinikos Centre of Infectious Diseases Vilnius University Faculty of Jakutiene Jolita [email protected] Medicine,Vilnius University Hospital Lithuania Santariskiu Klinikos Buivydiene Arida [email protected] Vilnius University, Faculty of Medicine Lithuania Tolmane Ieva [email protected] Riga Stradins University Lithuania

Preliminary results of efficacy and safety of direct-acting antivirals Ombitasvir / Paritaprevir / Ritonavir + Dasabuvir with or without Ribavirin in patients with HCV genotype 1 infection in real life setting Katazyna Polubenko1, Ligita Jancoriene2, Edita Kazenaite3, Arida Buivydiene3, Jolita Jakutiene3, Ieva Tolmane4 1 – Vilnius University, Faculty of Medicine; 2 – Vilnius University Hospital Santariskiu klinikos Centre of Infectious Diseases; 3 - Vilnius University Hospital Santariskiu klinikos Centre of Hepatology, Gastroenterology and Dietetics; 4 - Riga Stradins University Department of Infectology and Dermatology; Infectology Center of Latvia, Department of Hepatology. Background and aims. The aim of this open label, non-randomised, multicenter, international, real-life study was to evaluate efficacy and safety of Ombitasvir/Paritaprevir/Ritonavir+Dasabuvir±Ribavirin (OBV/PTV/r+DSV±RBV) in treatment naive and treatment experienced HCV genotype 1 patients, who failed therapy with PegINF/RBV with or without protease inhibitor telaprevir or boceprevir previously. Methods. 109 patients (55 males) aged 54.2±10.8 years, infected with HCV genotype 1 (97 infected with subtype 1b) from Lithuania and Latvia were included. 28.8% were treatment-naive, 26.6% - null- responders, 14.7% - partial responders, 22% - relapsers and 8.3% discontinued previous treatment because of adverse events. 21 patients failured triple therapy with first generation protease inhibitor previously. 58.7% were cirrhotics. OBV/PTV/r+DSV±RBV treatment was scheduled for 12 weeks in 107 and 24 weeks in 2 patients. RBV was administered to 87 individuals. Sustained virologic response (SVR12) was assessed by HCV-RNA undetectability 12 weeks after treatment termination. Adverse events and selected laboratory measures were assessed during the treatment and follow-up period. Results. 106 patients completed therapy and had undetectable HCV-RNA at the end of the treatment. 67 patients with available follow-up evaluation achieved SVR12. The average reduction in alpha-fetoprotein (16.3 versus 4.9, p<0.001) and improvement of hepatic elasticity (17.3 versus 15.5, p<0.001) was demonstrated after follow-up period. RBV was reduced in 18 and discontinued in 11 patients. 9 patients experienced hemoglobin decline <10 mg/dL. The most frequent adverse events were asthenia (27.4%), fatigue (17%), itching (14.2%) and dyspepsia (12.3%). Treatment was disontinued in 3 patients. Due to severe exacerbation of psoriasis one patient disontinued treatment at 3rd day. Two other patients discontinued treatment because of depression (at week 5) and elevation of ALT and AST (at week 9). Both patients achieved virologic response at the end of treatment, one of them has already confirmed SVR12, the other is still on the follow up. Conclusions. The regimen of OBV/PTV/r+DSV±RBV achieved up to now 100% SVR12. Adverse events were mostly mild and related to RBV administration.

67

MOLECULAR INVESTIGATION OF SPORADIC CASES OF HEV INFECTION IN ESTONIA

POSTER Name E-mail Institution Country KUZNETSOVA TATIANA [email protected] NIHD Estonia National Institute for Health Reshetnjak Irina [email protected] Estonia Development Geller Julia [email protected] NIHD Estonia

Background and aims. Recently paradigm of HEV transmission to human was revised. Serotypes 1 and 2 prevalent in so called endemic areas are transmitted by anthroponosis manner whereas serotypes 3 and 4 are suggested to be pure zoonosis. Formerly we reported data about massive prevalence of HEV3 in pigs (principal reservoir host) and occurrence anti-HEV antibodies in persons contacting with the pigs in Estonia. Aims: The present study has two objectives: 1) Screening of a representative cohort of patients with nonA- nonC hepatitis for presence of HEV RNA; 2) Comparison of the found HEV sequences from the human isolates with sequences of the typical swine isolates circulating in Estonia. Evaluating role of domestic zoonotic infection in HEV transmission. Methods. 233 sera specimens of nonA-nonC hepatitis patients collected in period 2011-2016 in three largest hospitals of Tallinn and one hospital of Tartu were involved to the screening. Serum samples were analyzed for the presence of immunoglobulin G (IgG) antibodies against HEV (anti-HEV IgG) using the recomWell HEV IgG test (Mikrogen GmbH, Neuried, Germany). Positive sera were consecutively tested for anti-HEV immunoglobulin M (IgM) antibodies using the recomWell HEV IgM (Mikrogen GmbH, Neuried, Germany). For further confirmation of positive sera, recomLine HEV IgG/ IgM immunoblot assay for anti-HEV IgG and IgM was applied (Mikrogen GmbH, Germany). All serological samples positive for HEV-specific antibodies were analyzed for the presence of HEV RNA by real-time RT-PCR. PCR products of qPCR-positive samples were sequenced. The sequences were used for phylogenetic analysis. Results. 10 Ig-positive specimens (4% of the total cohort) were found. 6 from them were qPCR positive. 3 samples contained HEV serotype 1 and 3 isolates belonged to HEV serotype 3. All HEV3 isolates were clearly distinct from the domestic HEV3 isolates from pigs. All three patients infected with HEV3 were suggested to have a travelling history in a recent anamnesis. Conclusions. No cases of HEV3 transmission from pigs to human resulting in a clinically manifested infection were found in Estonia in 2011-2016. Nevertheless prevalence of HEV3 acute infection in Estonia arises a question about the way of HEV3 transmission to human in this area. Hypotheses about different infectivity of HEV3 genetic subtypes for human as well as different environmental conditions in different countries should be considered.

68

CHALLENGES OF TREATMENT OF HIV INFECTED CHILDREN IN LITHUANIA

POSTER Name E-mail Institution Country PAVYDIENE Children’s Hospital, Affiliate of Vilnius [email protected] Lithuania VIKTORIJA University Hospital Santariskiu Klinikos Children’s Hospital, Affiliate of Vilnius Ivaskeviciene Inga [email protected] Lithuania University Hospital Santariskiu Klinikos Children’s Hospital, Affiliate of Vilnius Usonis Vytautas [email protected] Lithuania University Hospital Santariskiu Klinikos

Introduction. HIV continues to be a major global public health issue, globally an estimated 36.9 million people were living with HIV in 2014, and 2.6 million of these were children. Until 2014 on January the total number of individuals with HIV in Lithuania was 2 378, of whom 20 percent (476) were women. There are six known HIV infected children in Lithuania, five with congenital infection transmitted from mother to child and one with an unknown route of infection. Since 2011 four children with HIV have been treated at the department of Infectious Diseases of the Children’s Hospital, Affiliate of Vilnius University Hospital Santariskiu Klinikos. As our experience in the treatment of HIV infected children is rather limited we make our clinical and treatment decisions in collaboration with the colleagues from Imperial College Healthcare Trust, Paediatric European Network for Treating AIDS (PENTA), London, UK. The aim. To share our experience and challenges of the treatment in HIV infected children of Lithuania. Methodology. We present four different case reports. Conclusions. The total number of HIV infected children in Lithuania is very low, only six children have been diagnosed as HIV positive in Lithuania until 2015. HIV infected children require intensive input of complex medical care and age appropriate knowledge about HIV and its management. Children’s HIV infection is a new challenge for Lithuanian physicians. Optimizing the management of HIV in childhood requires a national guideline foundation, a multidisciplinary team including social and psychological specialists, accessibility of pediatric drug formulations, and an appropriate follow-up strategy.

69

HPV LESION - ORAL CONDYLOMA ACUMINATUM: CASE REPORT

POSTER Name E-mail Institution Country Vilnius University, Institute RIMKEVICIUS ARUNAS [email protected] Lithuania of Odontology Vilnius University, Institute Puriene Alina [email protected] Lithuania of Odontology Vilnius University, Institute Kscenaviciute Vaiva [email protected] Lithuania of Odontology

Introduction. Condyloma acuminatum is a verrucous or papillary growth HPV lesion that is characteristically located in the anogenital region but may also involve the oral mucosa. Case report. A 42-year-old male applied to the ЕЅalgiris Clinic of Vilnius University Hospital. The patient complained of a group of growing lesions on the anterior part of his tongue. Lesions had been first noticed almost two years ago and never treated. No other symptoms were identified. Intervention. Quite characteristically, oral examination revealed on the tongue and buccal mucosas a group of multiple oral asymptomatic nodules, pink to red in colour, with thin feet, less broad-based, soft texture reminiscent of cauliflower, variably sized, up to 2–3 cm on the patient’s tongue. Three lesions were located at the contact/traumatic side of the tongue. The diagnostics of oral lesions was based on the clinical evaluation, followed by cytology which was further confirmed by biopsy. Such oral lesions may be an oral manifestation of HIV infection, thus additional laboratory diagnostics for HIV infection and other sexually transmitted diseases were requested. The tests for other diseases were negative. The lesions were surgically removed by excisional biopsy under local anesthesia and sutured with Vicryl 5–0 sutures. Results. The histopathological examination of the oral lesion, performed by the National Center of Pathology, revealed an exophytic papillary squamous proliferation, hyperplastic epithelium irregularly thickened by acanthosis and a concentration of parakeratosis, with identification of koilocytes, without evidence of dysplastic changes. Koilocytes, a hallmark of condyloma acuminatum and HPV infection- modified keratinocytes, were found in the upper spinous and corneal layers. Follow Up and Outcome. After surgical treatment and final healing, the patient was informed about possible recurrences which are common and presumably are related to the surrounding appearingly normal tissue that may act as an infectious agent. Clinical examination some 3 to 6 months after treatment, and regular yearly visits are recommended.

70

RECONSTRUCTION OF THE REGIONAL TRANSMISSION OF HCV IN SOUTHERN SWEDEN BY PHYLODYNAMICS

ORAL Name E-mail Institution Country SALLAM MALIK [email protected] Lund University Sweden Holmgren Birgitta G [email protected] Lund University Sweden Soderlund Strand Anna [email protected] Lund University Sweden Ozkaya Sahin Gulsen [email protected] Lund University Sweden Waldeck Mattias [email protected] Lund University Sweden Anders Widell [email protected] Lund University Sweden Medstrand Patrik [email protected] Lund University Sweden

Background. The molecular epidemiology studies of HCV are useful to gain in-depth knowledge of timing and patterns of viral spread. Analysis of laboratory testing sequence data can give insights for prevention of virus spread. The objectives of the current project were to characterize the genetic diversity and transmission dynamics of HCV in Southern Sweden, as well as identifying socio-demographic variables that are associated with onward transmissions. Methods. Partial HCV NS5B gene sequences (339 bp) were available from routine clinical testing for genotyping of clinical isolates. The sequences were collected between 2004 and 2015. Sub-genotyping was based on similarity search using Los Alamos HCV sequence database BLAST tool. Phylogenetic analysis using maximum likelihood method (ML) was done based on Swedish and reference sequences retrieved from GenBank using BLAST tool. ML trees were reconstructed for determination of transmission clusters representing domestic spread of HCV in Sweden. Analysis of the transmission clusters was done using Cluster Picker software with monophyletic Swedish clades defined as those having approximate Likelihood Ratio Test SH like (aLRT-SH) support value of ≥0.90 and containing more than 70% Swedish sequences. Transmission clusters with two sequences were defined as dyads, those containing between three and 14 sequences were defined as networks and those having more than 14 sequences were defined as large clusters. Estimation of the evolutionary rate and time to most recent common ancestors (tMRCAs) of the large Swedish clusters were determined using Bayesian approach in BEAST. Results. A total of 3912 sequences with Open Reading Frames (ORFs) were available for analysis. Genotyping revealed the following distribution: 1a (39%, n=1513), 3a (38%, n=1481), 2b (10%, n=390) and 1b (10%, n=374). For sub-genotype 1a, 550 sequences were part of 138 transmission clusters (36%). Those clusters were classified as 83 dyads, 50 networks and five large clusters. Possible geographic origins of some clades that resulted in domestic spread of sub-genotype 1a included: Cyprus, Greece, Ireland, Iran, Netherlands and USA. For sub-genotype 3a, 698 sequences were part of 142 transmission clusters (47%). Those clusters were classified as 73 dyads, 60 networks and nine large clusters. Possible geographic origins of some clades that resulted in domestic spread of sub-genotype 3a included: Canada, Spain, Brazil, Malaysia, Switzerland, China, Uzbekistan, Iran and Netherlands. For sub-genotype 2b, 178 sequences were part of transmission clusters (46%). Those clusters were divided into 36 dyads, 21 networks and one large cluster. For sub-genotype 1b, 43 sequences were part of transmission clusters (11%) and were classified into 17 dyads and two networks. The time to Most Recent Common Ancestor (tMRCA) of the oldest sub- genotype 1a cluster dated back to 1967 (95% HPD: 1959-1975). The tMRCA of the oldest sub-genotype 3a cluster dated back to 1969 (95% HPD: 1962-1976). Conclusions. Four HCV sub-genotypes were prevalent in Southern Sweden between 2004-2015, with sub- genotypes 1a and 3a dominating the infections in terms of prevalence. The phylodynamic approach unravelled patterns of viral spread and possible geographic origins of HCV in Southern Sweden. Further studies including association of socio-demographic variables to members of clusters are underway and might be helpful in implementing strategies for infection control.

71

CHARACTERIZATION OF HIV-1 RECOMBINANT FORMS IN BELARUS: DIVERSITY, ORIGIN AND DOMESTIC SPREAD

ORAL Name E-mail Institution Country SASINOVICH The Republican Research and Practical Centre [email protected] Belarus SVIATASLAU for Epidemiology and Microbiology The Republican Research and Practical Centre Eremin Vladimir [email protected] Belarus for Epidemiology and Microbiology The Republican Research and Practical Centre Gasich Elena [email protected] Belarus for Epidemiology and Microbiology

Background and aims. The HIV epidemic in Belarus is characterized by prevalence of HIV-1 subtype A1 (FSU-A variant). In addition, subtypes B, C, G as well as the circulating recombinant forms (CRFs) CRF02_AG, CRF03_AB, CRF06_cpx and unique recombinant form (URF) HIV-1 A/B have been found in the country. The aims of this study were to characterize HIV recombinant forms in Belarus, determine their origin and domestic spread. Methods. Partial HIV-1 polymerase (pol) and near full genome sequences were obtained from 303 HIV- infected individuals during 2008-2015 that were permanently residing in different regions of Belarus. HIV- 1 subtypes were identified by maximum-likelihood (ML) phylogenies and putative recombinant forms were further analyzed using the REGA subtyping tool and jpHMM. The 10-20 most similar sequences in Genbank to each of the Belarusian sequences were identified by BLAST and then used to construct phylogenetic trees. Phylogenetic cluster analysis was performed based on ML phylogenetic trees. An SH- aLRT of >0.90 were considered as a significant branch support defining a cluster of related sequences. Results. Among 303 HIV-1-infected patients, 275 (90.7%) were infected with subtype A1 variant A-FSU, nine (3.0%) with subtype B, and two (0.7%) with subtypes C and G each. HIV CRFs were found in 14 cases (4.6%) and among them, eight CRF03_AB, four CRF02_AG and two CRF06_cpx. In one case, an URF between A-FSU/B-FSU subtypes was found which was termed Mos. Phylogenetic analysis of eight CRF03_AB revealed six independent introductions into Belarus, four via sexual route and two via injection drug use. Four of the sequences formed two independent clusters with high SH-aLRT branch support indicating domestic transmissions. One of this two clusters contained sequences collected from two females with a self-reported sexual route of infection indicating the presence of at least one more, expected male, individual infected with CRF03_AB. No significant clustering between the Belarusian CRF03_AB and the reference sequences were found. CRF02_AG was found to be introduced into Belarus at least three times In two occasions they clustered with high branch support with reference sequences from West Africa, and in one case (an HIV-infected mother with her HIV-infected child) with a reference sequences from Uzbekistan. CRF06_cpx was found in two IDUs who were relatives This two sequences formed a significant cluster with an Estonian CRF06 reference sequences. The URF Mos was found in a child born to an HIV-infected mother in 2010. Genotyping reveal that the Mos genome consisted of HIV-1 subtypes A and B and differ from CRF03. Phylogenetic analysis of each subtype-specific segments with reference sequences revealed recombination between FSU-A and FSU-B. No significant clustering was found between the Mos fragments and the reference sequences. Conclusions. CRF03_AB was most likely introduced from Russia and Lithuania, CRF02_AG from West Africa and Uzbekistan, and CRF06_cpx from Estonia. Only two cases of domestic spread were found, both for CRF03_AB. Based on epidemiological data, we assume at least three additional individuals infected with recombinant forms. The mothers of two children infected with CRF02 and the URF Mos, and one male infected with CRF03_AB, related to one of two domestic transmission events. Detailed cluster analysis indicated that recombinant forms have been introduced into Belarus on a few occasions and have had limited spread in the country.

72

TRENDS IN SYPHILIS AMONG PREGNANT WOMEN IN LITHUANIA

POSTER Name E-mail Institution Country SIMKUNAITE-ZAZECKE AGNE Centre for Communicable Diseases [email protected] Lithuania and AIDS Caplinskiene Irma [email protected] CCDA Lithuania Mykolas Romeris University, Caplinskas Saulius [email protected] Lithuania Centre for Communicable Diseases and AIDS

Background. Between 2011 and 2015, more than 1300 cases of syphilis were reported in Lithuania. In 2015, the rate of syphilis was 9.7 per 100 000 population and it remained stable as compared to 2011 (9 per 100 000 population). In 2013, Lithuania reported one of the highest (9.1 per 100 000 population) rates of syphilis in EU/EEA (an overall notification rate 5.4/100 000). While comparing numbers of congenital syphilis (CS) during the last five years, an increase is observed: 0 and 3 reported cases in 2011 and 2015 respectively. Methods. Retrospective analysis of national STI databases for 2011-2015 was carried out. The aim was to find out syphilis trends among pregnant women during the period of 2011-2015. Results. In 2011-2015, 1307 cases of syphilis (CS included) were reported and 47.2% (617 cases) of them were females. Under the provisions of Lithuanian legislation all pregnant women are voluntary tested for syphilis twice during pregnancy. An average of 47 000 tests for syphilis have been performed among pregnant women annually and the number of screened women has remained almost stable during the last five years. Between 2011 and 2015, 103 syphilis cases in pregnant women were detected through antenatal screening programme. The number of reported syphilis cases among pregnant women increased from 17 to 24 cases in 2011 and 2015 respectively. An average of 40% of these cases have been diagnosed in young 18-24 aged women annually. The majority was from urban area. 25-53% of women had secondary education, while highly educated women accounted only for by 4-17% annually. On average, a third had some kind of employment. On average, every second women indicated husband or cohabitant as potential source of infection. In most of cases condom was not used at all or used rarely. On average, in 78% of cases contact tracing was started. During the last five years seven congenital syphilis cases were reported. In 2013, the number of CS cases per 100 000 live births in Lithuania was more than three times higher (6.7/100 000 live births) than an overall rate (2/100 000 live births) in EU/EEA Member States. In 2015, 3 cases of congenital syphilis were reported in Lithuania (0 of cases in 2011). Conclusions. Despite increasing in detection of syphilis among pregnant women in the past years, congenital syphilis cases are still reported annually. Moving towards elimination of CS in the country, screening program uptake should be improved by targeting those who do not apply for antenatal care services.

73

MULTI-DRUG-RESISTANT ACINETOBACTER SPP. MONOBACTEREMIA IN INTENSIVE CARE UNITS OF UNIVERSITY HOSPITAL

POSTER Name E-mail Institution Country VALANCIENE Hospital of Lithuanian University [email protected] Lithuania DOVILE of Health Sciences Kauno Klinikos ADUKAUSKIENE Hospital of Lithuanian University [email protected] Lithuania DALIA of Health Sciences Kauno Klinikos

Background and aim. Acinetobacter spp. causes many nosocomial infections (respiratory tract, bloodstream, intraabdominal, skin-soft tissue, etc.), which is difficult to treat due to its high resistance to antibiotics. The aim of study was to analyze characteristics of monobacteremia caused by multi-drug- resistant (MDR, as resistant to ≥ 3 classes of antibiotics) Acinetobacter spp. strain and associating factors with mortality in surgical and medical intensive care units. Methods. The retrospective data analysis of patients treated in surgical and medical intensive care units of University Hospital Kaunas Klinikos with positive blood culture for Acinetobacter spp. during 2005-2012 yrs was carried out. Results. There were found 77 cases of Acinetobacter spp. monobacteremia, 74 (96.1%) of them were caused by MDR strain. There was no significant difference among patients in gender, age, comorbidities, source of bacteremia, length of stay in intensive care unit for MDR Acinetobacter spp. bacteremia (P>0.05). Primary MDR Acinetobacter spp. bacteremia was associated septic shock (n=31, 53.4%, P=0.04, OR=2.528, CI95%=0.12-1.28), surgical procedures (n=23, 63.9%, P=0.019, OR=5.477, CI95%=1.18- 7.82), previous stay in other hospital departments (n=20, 55.6%, P=0.05, OR=1.02, CI95%=0.16-1.06) and with high mortality (n=34, 94.4%, P=0.04, OR 4.051, CI95%=0.04-1.12). There was estimated high resistance of MDR Acinetobacter spp. strains to separate antimicrobial drugs (P<0.05), as: cefotaxim (n=74, 100%), ampicillin (n=71, 95.9%), cefuroxim and ceftazidim (n=66, 89.2%), gentamicin (n=64, 86.5%), piperacillin (n=63, 85.1%), piperacillin with tazobactam (n=62, 83.8%), ciprofloxacin (n=59, 79.7%), amikacin (n=41, 55.4%), ampicillin with sulbactam (n=33, 44.6%). The low resistance to meropenem (n=6, 8.1%, P=0.03) and no resistance to imipenem was estimated. Overall mortality rate of MDR Acinetobacter spp. bacteremia was 86.5%. Lethal outcome was associated with mechanical ventilation (n=63, 98.4%, P=0.02, OR=2.341, CI95%=0.91-4.80), septic shock (n=58, 90.6%, P<0.001), incorrect empirical antimicrobial treatment (n=52, 82.1%, P=0.04, OR=2.077, CI95%=1.42-2.89), SOFA score ≥ 13 (n=48, 88.9%, P=0.02, OR=2.0, CI95%=0.50-7.99), comorbidities rate ≥3 (n=38, 86.4%, P=0.001, OR=1.386, CI95%=0.25-3.78), elderly (n=35, 89.7%, P=0.03, OR=1.810, CI95%=0.466-7.036), and primary bacteremia (n=34, 94.4%, P=0.04, OR 4.051, CI95%=0.04-1.12). Conclusions. Almost in all cases Acinetobacter spp. bacteremia has appeared as caused by MDR strain. Primary MDR bacteremia was associated with septic shock, surgical procedures, previous stay in other hospital departments and high mortality. There was estimated high resistance to different antimicrobial drugs, but carbapenems. MDR Acinetobacter spp. bacteremia had extremely high mortality rate, associated with mechanical ventilation, septic shock, incorrect empirical antimicrobial treatment, SOFA score ≥13, comorbidities rate ≥3, elderly, and primary bacteremia.

74 4. Opportunities of laboratory diagnostics

Kamili Saleem Challenges in Laboratory Diagnosis of HBV and HCV Invited Infections

Buivydiene Arida The UCKL-1, a protein with uridine kinase activity, is a Poster putative prognostic marker for hepatocellular carcinoma progression in patients with HCV-related cirrhosis Medstrand Patrik Identification of an informative and accurate region of the Oral HCV genome for phylogenetic analyses Pereckaitė Laura Extended spectrum β-lactamase production and temocillin Oral resistance in Escherichia coli and Klebsiella pneumoniae strains resistant to third generation cephalosporins Runov Andrei Viral myocarditis: modulation of host gene expression in Poster cardiac tissue Szewczyk Boguslaw MSSCP and NGS methods for detection and differentiation Poster of influenza A/H1N1 virus. Värv Kairi Detection of tick-borne encephalitis virus RNA in acute Oral samples of TBE patients Vonsky Maxim PBMC-expressed RNA biomarkers of viral myocarditis Oral Ziliukiene Jolanta Prevalence of intestinal parasites among patients with Poster gastrointestinal complains in Lithuania, 2009-2015

75

CHALLENGES IN LABORATORY DIAGNOSIS OF HBV AND HCV INFECTIONS

INVITED LECTURE Name E-mail Institution Country CDC, Division of Viral Hepatitis,

SALEEM KAMILI [email protected] United States of America Atlanta

Serologic assays for the diagnosis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are widely available throughout the world. The laboratory diagnostics of HBV infection is greatly facilitated by the availability of a range of serological markers which when detected alone or in combination can readily identify active infection, differentiate between acute, chronic or resolved infections as well as characterize whether the antibody responses detected is a result of vaccination or natural infection. The diagnostic landscape of HCV infection is very limited to just one antibody marker, anti-HCV IgG, which is detectable during the acute, chronic and resolved phases of infection. The diagnosis of active HCV infection is, therefore, solely based on the detection of HCV RNA by polymerase chain reaction methodologies. HCV Core antigen assay, though not widely available yet, is also used for diagnosing active HCV infection. Recent advances in molecular technologies, which include nanoparticle-based diagnostic assays, the use of aptamers as capture molecules and loop-mediated isothermal amplification assays, have produced promising new tools which have the potential for use in the development of new and improved assays for the diagnosis of HCV infection. The challenges in viral hepatitis diagnostics are posed by the many types of hepatitis viruses, all causing infections that are clinically indistinguishable and each having a multitude of viral genotypes, subtypes, and serotypes. Although well characterized and validated assays approved for clinical use by international regulatory agencies clinical use are available for markers of HBV and HCV infection, Non-approved or in- house research-use only assays are available and also being routinely used in various settings. This unregulated supply of diagnostic kits poses a fraught situation, particularly in resource-poor countries where such assays are also used for blood screening. An important step towards improving the diagnostics and also for blood safety will be to develop well characterized reference panels that can be used for evaluating the performance characteristics of both approved and non-approved diagnostic assays. Such panels can also be used for developing new and improved diagnostic assays.

76 THE UCKL-1, A PROTEIN WITH URIDINE KINASE ACTIVITY, IS A PUTATIVE PROGNOSTIC MARKER FOR HEPATOCELLULAR CARCINOMA PROGRESSION IN PATIENTS WITH HCV -RELATED CIRRHOSIS

POSTER Name E-mail Institution Country Vilnius University, Faculty

BUIVYDIENE ARIDA [email protected] Lithuania of Medicine

Liakina Valentina [email protected] Vilnius University Lithuania Vilnius University, Faculty

Valantinas Jonas [email protected] Lithuania of Medicine

Smaliukiene Romualda [email protected] National Center of Pathology Lithuania

Jokubauskiene Skirmante [email protected] VilniusUniversity Lithuania

Besusparis Justinas [email protected] VilniusUniversity Lithuania

Kashuba Elena [email protected] Karolinska Institutet Sweden

Background and aims. The current limitations of prognostic stratification in patients with HCV-related cirrhosis and hepatocellular carcinoma (HCC) are requiring the development of new prognostic markers for best treatment and followup of patients. One of the possible prognostic markers might be a new human enzyme, UCKL-1, that contributes to the increase of UTP nuclear pool to support increased cell proliferation. The aim of the present study is to assess UCKL-1 expression at the protein levels in the liver cells in patients with HCVrelated cirrhosis leading to HCC development. Materials & Methods. 45 HCC cases with HCV-related cirrhosis were analyzed in the study. Specimens were stained by UCKL-1 immunohistochemistry using polyclonal rabbit anti-UCKL-1 antibody and EnVision G2 double staining system. The UCKL-1 expression levels obtained by percentage of positive cells multiplied by intensity of staining were compared in groups with different tumor grade, HCV genotype, viremia and patients demographic data. GraphPad Prism software was used for the data analysis. Results. Cases were grouped by tumor grade (G1, G2, G3). Patients in these groups did not differ significantly concerning median age and body mass index. No correlation between UCKL-1 expression levels and viremia or HCV genotype was found. Median expression of UCKL-1 in cirrhotic liver revealed a trend for lower UCKL-1 expression with delayed cancerogenesis. We found a strong correlation between high expression levels of UCKL-1 in liver at cirrhosis and low grade of HCC (i.e the highest UCKL-1 level s correspond to G1 tumors). Probably, the high UCKL-1 expression is observed when liver cells prolif erate actively, i.e. liver can regenerate. Noteworthy, in HCC the inverted correlation was observed – levels of UCKL-1 increased with tumor progression. Moreover, the high UCKL1 expression at cirrhos is and low expression in HCC correlated with prolonged survival. Conclus ions. The higher UCKL-1 expression in liver tissue may indicate a high regenerative capacity of hepatocy tes resulting in a slower progression to HCC. On the other hand, high UCKL-1 expression in the tum or cells may be a sign for worse HCC prognosis. Additional studies are needed to investigate the spec ific role of UCKL-1 protein in regeneration of hepatocytes, HCC initiation and progression.

77

IDENTIFICATION OF AN INFORMATIVE AND ACCURATE REGION OF THE HCV GENOME FOR PHYLOGENETIC ANALYSES

ORAL Name E-mail Institution Country PATRIK Lund University, Department of Laboratory

MEDSTRAND [email protected] Sweden Medicine, Lund

Introduction: One of the goals of the Baltic Network Against Life-threatening Viral Infections is to gain insights into the joint evolutionary and epidemiological 76onorrhe of hepatitis C virus (HCV) infection (phylodynamics) in the Baltic area. By incorporation of tools from the field of phylogenetics, a number of fundamental questions regarding viral spread and epidemiological dynamics can be addressed. To facilitate collaboration between the different teams of the Network, criteria for inclusion of patient samples, selection of a suitable genomic region for analysis and a PCR amplification strategy is needed. Previous studies have shown that the commonly sequenced region of NS5B is suitable for genotyping and molecular epidemiology studies, however, other regions may also be optimal for phylogenetic analyses. Aims: The goal of this study was to 1) identify informative HCV regions that allow for accurate reconstruction of the “true” HCV phylogeny, including identification of “correct” and “statistically supported” clusters, and 2) suggest a convenient PCR and sequencing protocol suitable for the general molecular biology laboratory. Materials and methods: The study used the statistical Shimodaira-Hasegawa (SH) test to compare phylogenetic trees obtained from 7 regions (1236 bases of E1-E2, 933 bases of E2, 1455 bases of E2-NS3, 1272 bases of NS5A, 2934 bases of NS5A and NS5B, 1668 bases of NS5B and 640 bases of NS5B) to the near full-length genome alignment (9036 bases) representing the polyproteins of HCV. In this investigational study, all sequence regions were derived from a set of 143 HCV-1a genomes available in the Los Alamos HCV database. Maximum-likelihood (ML) trees and SH branch-support of internal branches (N=141) of each region were estimated and compared to the “true tree” obtained from the polyprotein region. Inconsistency of tree topologies of shorter regions was statistically compared to the “true tree”. False positive (FP) branches were defined as statistically supported branches (SH > 0.9) of trees obtained from subgenomic regions that were absent or lacked statistical branch support in the phylogeny of the polyprotein region. Results: In total, 75, 35, 28, 28, 26, 22, 18 and 8 branches of the polyprotein, NS5A-NS5B, E1-E2, NS5B, NS5A, E2-NS3, E2 and short-NS5B regions, respectively, displayed statistical support by the SH test as implemented in PhyML. The lowest FP rates were observed in the NS5A, E2-NS3 and E2 regions (15, 22 and 23%, respectively), whereas the FP rates in the NS5A-NS5B, E1-E2, NS5B and short-NS5B regions when tested by this ML-method were higher (37, 39, 46 and 65%, respectively). Among trees obtained from different regions, the NS5A and NS5A-NS5B maintained a phylogeny most similar to the full-coding polyprotein. Discussion & Conclusions: In summary, we show that the 1272-bp region of NS5A displayed the lowest FP rate among subgenomic regions investigated in this study and conformed a topology of the “true” tree. In comparison to full-length genome sequencing, the NS5A fragment represents a trade-off between phylogenetic accuracy/information content and feasibility in terms of convenient PCR and sequencing protocols. Thus, the NS5A region may represent an attractive target for phylogenetic and phylodynamic studies of HCV. We are currently underway to confirm our findings with other statistical methods and HCV subgenotypes, and to optimize broad and/or subgenotype specific PCR and sequencing protocols for different HCV subgenotypes.

78

EXTENDED SPECTRUM Β-LACTAMASE PRODUCTION AND TEMOCILLIN RESISTANCE IN ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE STRAINS RESISTANT TO THIRD GENERATION CEPHALOSPORINS

ORAL Name E-mail Institution Country

PERECKAITE LAURA [email protected] Lithuanian University of Health Sciences Lithuania

Giedraitiene Agne [email protected] Lithuanian University of Health Sciences Lithuania

Background and aims. Bacteria of the Enterobacteriaceae family are the most common hospital and community-acquired pathogens. Recently, antibacterial resistance of the microorganisms has been a major threat to public health. Resistant infections claim approximately 50000 lives every year in Europe and US alone, with hundreds of thousands more dying in other areas of the world. Accurate detection of ESBLs in Enterobacteriaceae is important for individual antibiotic therapy and infection control measures. Due to antimicrobial resistance, antibiotic treatment possibilities are reduced. Therefore, researchers’ interest in old compounds is renewed. One of them is temocillin, which is especially active against resistant strains of Enterobacteriaceae. The aim of our study was to confirm extended spectrum β-lactamase (ESBL) production and detect temocillin resistance in Escherichia coli and Klebsiella 77onorrhea strains, resistant to third generation cephalosporins and isolated in 2012-2014 at Kaunas Clinics, the Hospital of Lithuanian University of Health Sciences. Materials & Methods. 72 strains of E. coli and 10 strains of K. 77onorrhea resistant to third generation cephalosporins were randomly selected and tested against ESBL production and temocillin resistance. ESBL production was confirmed using combination disk diffusion test, double disk synergy test, and gradient test (ESBL E-test). Temocillin resistance was detected using disk diffusion method and gradient test (E-test). Results. All the tested strains were resistant to cefotaxime, while 40,0% of them were also resistant to ceftazidime. Using combination disk diffusion test, ESBL production was detected among 96,3% of the strains. 3 strains of E. coli were not ESBL producers; 2 of them were resistant to cefotaxime, ceftazidime, and cefoxitin, but susceptible to cefepime, which indicates AmpC β-lactamase production; 1 strain was resistant to all four mentioned antibiotics which increases suspicion of carbapenemase production. ESBL production was indicated in 93,9% of strains by double disk synergy test, and in 100,0% of strains by gradient test (ESBL E-test). Using disk diffusion test, temocillin resistance was detected among 7,3% of strains resistant to third generation cephalosporins. No strains resistant to temocillin were detected using gradient test (E-test); MIC of temocillin varied from 1 to 24 µg/ml. A logistic regression was performed to ascertain the effects of ESBL production on the likelihood that the strain of Enterobacteriaceae is resistant to temocillin. The logistic regression model was not statistically significant (χ2=0,246, p=0,620). ESBL producing strains resistant to third generation cephalosporins were only 0,076 times more likely to exhibit temocillin resistance than non-ESBL producers. Conclusions. 1. Using combination disk diffusion test and double disk synergy test, ESBL production was detected in more than 90% of strains resistant to third generation cephalosporins. ESBL production was confirmed in all tested strains by gradient test (ESBL E-test). 2. Using disk diffusion method, less than 10% of strains resistant to third generation cephalosporins were resistant to temocillin. No strains resistant to temocillin were detected using gradient method (E-test). 3. Temocillin is effective against ESBL producers among E. coli and K. 77onorrhea strains resistant to third generation cephalosporins.

79

VIRAL MYOCARDITIS: MODULATION OF HOST GENE EXPRESSION IN CARDIAC TISSUE

POSTER Name E-mail Institution Country RUNOV Russian

[email protected] Institute of Cytology RAS ANDREI Federation

Kurchakova Elena [email protected] Institute of Cytology RAS Russian Federation Almazov Federal Heart Centre, Saint Petersburg,

Moiseeva Olga [email protected] Russian Federation Russia Federal Almazov North-West Medical Research

Mitrofanova Lubov [email protected] Russian Federation Centre

Vonsky Maxim [email protected] Institute of Cytology RAS, Saint Petersburg, Russia Russian Federation

Introduction. Myocarditis is an inflammatory heart disease, commonly caused by viral infection. This pathology often causes heart failure, life-threatening arrhythmia and sudden death. Myocardial inflammation followed by cardiomyocyte necrosis modifies gene expression in affected cells. The aim of the study was to investigate the alterations of gene expression in cardiac tissue of patients with viral myocarditis. Materials & Methods. Twenty endomyocardial biopsy (EMB) samples were received from patients with verified myocarditis (12 males, 8 females, 37-62 years); six control samples of myocardial tissue were originated from donors of orthotopic heart transplantation (4 male, 2 female). mRNA was isolated using in- house phenol extraction protocol. 30 candidate genes were selected for the study. The mRNA content of the selected genes depends on the development of non-specific inflammatory response. The set includes genes, previously identified by means of microarray technology, and genes specific for the virus-host cell interaction and inflammatory response. Primers for qPCR were designed taking into account the exon- intron structure of target genes. Oligo(dT)18 primers were used to perform reverse transcription. qPCR was performed in duplicates with intercalating dye EvaGreen. Efficiency of amplification was calculated using serial cDNA dilutions. qPCR results were analyzed using Rest-2009 software with GAPDH and HPRT1 as a reference gene set. Results. RT-qPCR confirmed the expression in EMB samples of 24 out of 30 target genes. Expression profiles in male and female patient groups differed. Male patients with myocarditis had altered expression in EMB of 10 genes including ADCY7, FCER1G, GLIPR, IL2, ITGB2, NF-kB, NOTCH3, SEC24A, SIGLEC1, TMOD3. Six out of 10 genes were identified in the present study for the 1st time. Transcription level of 4 genes (ACOX1, IL1, IL2, SIGLEC1) matched to the disease progression, emphasizing their medical role in pathology development. Female patients had statistically significant changes (p < 0,033) in a panel of 12 genes (ACOX1, ADCY7, CD1D, CD14, CD8B1, FCER1G, HRH3, IL2, NOTCH3, SEC24A, SIGLEC1, TMOD3). Expression of six genes, FCER1G, IL2, NOTCH3, SEC24A, SIGLEC1, and TMOD3 was altered in both men and women, expression of ADCY7 gene was up-regulated in male and down- regulated in female patients. Conclusions. Viral myocarditis affects transcription of 10 genes in cardiac tissue samples in male and 12 genes in female patients. Expression of six genes was similarly regulated in male and female patients, while the expression of ADCY7 gene was gender-dependent. Thus, we have shown, that viral myocarditis is accompanied by modulation of gene expression in the cardiac tissues. The regulated pattern includes genes involved not only in inflammatory process and viral life-cycle, but also in a variety of other biological functions. Genes identified in the study may be considered as biomarkers for diagnostics of myocarditis supporting morphological and immunohistological criteria.

80

MSSCP AND NGS METHODS FOR DETECTION AND DIFFERENTIATION OF INFLUENZA A/H1N1 VIRUS

POSTER Name E-mail Institution Country

SZEWCZYK BOGUSLAW [email protected] University of Gdansk Poland

Rabalski Lukasz [email protected] University of Gdansk Poland

Krzysztof Lepek [email protected] University of Gdansk Poland

Zagminas Kestutis [email protected] Vilnius University Lithuania

Griskevicius Algirdas [email protected] NPHSV Lithuania

Influenza A virus belongs to the Orthomyxoviridae family. It is very variable human pathogen and it may undergo genetic changes by antigenic drift or antigenic shift. The variation of hemagglutinin (HA) is the main mechanism to escape the response of the host immunological system. Monitoring and control of infections are key parts of surveillance systems and epidemiological risk prevention. Influenza A viruses have a wide range of hosts and a potential of reassortment between different strains. Thus it is essential to study genetic variability of the virus circulating in our immediate surroundings. Here, we describe and compare two methods for detection and study of differentiation of influenza A/H1N1 2009-2011 pandemic strains isolated from human subjects. Next Generation Sequencing method produces more detailed data such us complete genome sequence and single nucleotide polymorphism but is more expensive than Multitemperature Single Stranded Conformational Polymorphism (MSSCP). On the other hand MSSCP method is much faster, thus more suitable for rapid preliminary analyses.

81 DETECTION OF TICK-BORNE ENCEPHALITIS VIRUS RNA IN ACUTE SAMPLES OF TBE PATIENTS

ORAL Name E-mail Institution Country

VÄRV KAIRI [email protected] National Institute for Health Development Estonia

Ivanova Anna [email protected] National Institute for Healt Development Estonia

Katargina Olga [email protected] National Institute for Healt Development Estonia

Lundkvist Åke [email protected] Uppsala University Sweden

Golovljova Irina [email protected] National Institute for Health Development Estonia

Introduction: Tick-borne encephalitis virus (TBEV) is small enveloped RNA virus of the genus Flavivirus. The infection of humans occurs via bite of an Ixodid tick and the main danger is that it affects central nervous system (CNS). Currently in Europe two subtypes of the virus are known to be pathogenic to humans, European and Siberian (TBEV-Eu and TBEV-Sib, respectively). The infection takes place in two phases. The first phase is manifested through flu-like symptoms and it occurs at the same time with viraemia. No antibodies are detectable at this stage. After an afebrile period of 2-10 days, seroconversion can be observed and detection of IgM and IgG by ELISA becomes possible. Thus the main problem of the serology based diagnostics, which is widely applied at the moment, is that the infection can be revealed only during the second phase when the symptoms are severe and CNS is already involved. Moreover since the viraemic phase is very short and it appears simultaneously with unspecific symptoms, RNA detection is difficult. Aims: The aim of the current study was to detect RNA of TBEV in early patient serum samples. Materials and Methods: Paired sera of 54 acute TBE patients from 5 Latvian hospitals were analyzed. The first sera, taken immediately after the patient came to the hospital, were screened for TBEV specific IgM, and both negative and positive samples were further analyzed by real-time RT-PCR. For all 54 patients the infection was confirmed by detection of TBEV specific IgM and IgG in the second phase serum, taken approximately 2 weeks after the first one. Results: The results showed that out of 33 IgM negative first phase sera, 24 had detectable levels of TBEV RNA. The presence of RNA was also demonstrated in 3 out of 5 samples that were on the borderline of IgM negativity and positivity. Finally no viral RNA was found in none of IgM positive samples. Out of 27 RNA positive samples 19 were successfully amplified and sequenced either in the complete E gene or partial E gene region. The phylogenetic analysis revealed that all sequenced samples belonged to TBEV-Eu subtype. Conclusions: IgM negativity might correlate with RNA positivity and in this case real-time RT-PCR can be a useful tool for early diagnostics of TBE. Moreover real-time RT-PCR analysis of samples from patients with a known tick bite history or who have been to a TBEV endemic area could be of benefit.

82

PBMC-EXPRESSED RNA BIOMARKERS OF VIRAL MYOCARDITIS

ORAL Name E-mail Institution Country VONSKY Institute of Cytology RAS, Saint Petersburg, Russian

[email protected] MAXIM Russia Federation

Runov Andrei [email protected] Institute of Cytology RAS Russian Federation

Kurchakova Elena [email protected] Institute of Cytology RAS Russian Federation Almazov Federal Heart Centre, Saint Petersburg,

Moiseeva Olga [email protected] Russian Federation Russia Federal Almazov North-West Medical Research

Mitrofanova Lubov [email protected] Russian Federation Centre

Isaguliants Maria [email protected] Karolinska Institutet Sweden

Introduction. Peripheral blood mononuclear cells (PBMC) can be regarded as liquid surrogate tissue in a proximate contact with the cardiovascular system. Model toxicological experiments demonstrated correlation of modulation of gene expression in the heart and in PBMCs (1,2). Viral infection is considered to be the main cause of myocarditis, an inflammatory heart disease. Histopathological and immunohistological analysis of endomyocardial biopsy (EMB) samples are still de facto a golden standard for complicated diagnosis of myocarditis despite the high cost and traumatizing effect of EMB. Recent studies identified RNA expression profile in EMB samples as a perspective biomarker of myocarditis development.. The aim of current study was to examine the expression of target genes in PBMC from patients with myocarditis and identify potential PBMC-expressed RNA biomarkers of viral myocarditis. Materials & Methods. PBMC samples were obtained from blood collected from patients with myocarditis (6 males, 4 females, 26-50 years), and from healthy donors (3 males, 3 females). PCR or RT-PCR analysis detected viral genomes in 41 % EMB samples. RNA was isolated from PBMC using commercial microcolumn extraction kit. mRNA integrity was assessed using denaturing PAAG electrophoresis. Target mRNA list was compiled of 29 candidate genes, including genes encoding receptors for virus attachment and entry, development of inflammatory response. Reverse transcription was performed with oligo(dT)18 primers, target gene expression was examined using qPCR run in duplicates with EvaGreen intercalating dye. qPCR results were analyzed using BestKeeper and Rest-2009 software with GAPDH and HPRT1 as a previously identified reference gene set (3). Efficiency of amplification was calculated using serial cDNA dilutions. Results. Gene expression was modulated similarly in PBMC of male and female patients with myocarditis. Expression levels of 7/29 genes were unchanged, whereas expression of 20 including NF-kB, IL2, CD8B1, HERC6, SEC24A, HRH3, RFFL, IL10, NEK3, CD1D, IL1, NOTCH2, Gb4Cer, SEC24D, ACOX1, MUC25AC, SULT4A1, DPF1 was up-regulated by 4,0 to up-to 51 times. Two genes, FCER1G and TMOD3, were down-regulated by mean factor of 0,003 and 0,272, respectively. Conclusions. We revealed statistically significant modulation of target genes expression in PBMC of patients affected by viral myocarditis. The results obtained create a basis for the development of noninvasive approach for differential diagnosis of myocarditis. Validity of the newly discovered myocarditis biomarkers demand further verification. 1. Brown H.R., Ni H., Benavides G., Yoon L., Hyder K., Giridhar J., Gardner G., Tyler R.D., Morgan K.T. Correlation of Simultaneous Differential Gene Expression in the Blood and Heart with Known Mechanisms of Adriamycin-Induced Cardiomyopathy in the Rat. Toxicol. Pathol., 30(4): 452–469, 2002 2. Gerling I.C., Ahokas R.A., Kamalov G., Zhao W., Bhattacharya S.K., Sun Y., Weber K.T. Gene Expression Profiles of Peripheral Blood Mononuclear Cells Reveal Transcriptional Signatures as Novel Biomarkers of Cardiac Remodeling in Rats With Aldosteronism and Hypertensive Heart Disease. J. Am Col. Cardiol. HF, 1(6): 469–476, 2013 3. Runov A.L., Kurchakova E.V., Khaschevskaya D.A., Moiseeva O.M., Vonskii M.S. Selection of Reference Genes for Transcription Analysis in Myocarditis. Cell and Tissue Biology, 9(4): 330–335, 2015

83

PREVALENCE OF INTESTINAL PARASITES AMONG PATIENTS WITH GASTROINTESTINAL COMPLAINS IN LITHUANIA, 2009-2015

Authors Name E-mail Institution Country National Public Health Surveillance

ZILIUKIENE JOLANTA [email protected] Lithuania laboratory

Objectives. To review distribution of intestinal parasites detected at National Public Health Surveillance laboratory among patients with 82onorrh gastrointestinal complains. Materials & Methods. This is retrospective analysis of laboratory testing results. All stool samples were tested with formalin ethyl acetate concentration Mini PARASEP kit (DiaSys Europe Ltd – Wokingham, UK), some samples analyzed with additional techniques: Kato-Katz, Entamoeba histolytica, Giardia lamblia, Cryptosporidium spp. Antigen detection, permanent stained smears by Ziehl Neelsen. Results. Between January 2009 and December 2015 stool samples from 3678 patients with gastrointestinal complains were examined for ova and parasites: 832 (22,6%) men, 1384 (37,6%) women, 1462 (39,7%) children under 17. 26,9% (n=988) patients were found to have intestinal parasites: 35,6% (n=296) men, 36,4% (n=504) women, 12,9% (n=188) children under 17. Ages ranged from 0 to 91 years. The largest group of subjects consisted of children under 6 years, 30 % of all patients studied. The youngest age group of 0-6 years were found to be infected at least – 10,2% and rate of infected persons is increasing with age proportionally (Fig 1). Patients were found to have 22 species of intestinal parasites: 11 species of protozoa and 11 species of 82onorrhea82. In 33 cases (0,9%) Entamoeba were not possible to identify with microscopy by species. 4,7 % of subjects were found to be infected with two species of parasites (55 men, 100 women, 17 children, respectively, 6,6%, 7,2%, 1,2%). 0,7% of subjects were found to be infected with three species of parasites 9 men (1,1%), 14 (1,0%) women, 1 (0,1%) child. Figure 1. Percent of patient infected with intestinal parasites by age group, (n=3678) 2009-2015, Lithuania Table 1. Intestinal parasites detected in stool samples from 3678 patients, 2009-2015, Lithuania Parasites causing intestinal disease were detected in 23,5% (n=864) of patients, 7,8% (n=287) parasites were non pathogenic. Helminthes were present in 1,0% (n=38) of subjects, protozoa – in 25,8% (n=950). Blastocystis hominis was the most common parasite encountered during these examinations. It was detected in 19,3% (n=709) cases, Entamoeba histolytica – 1,3%, Giardia lamblia and Dientamoeba fragilis – 0,8%, Cryptosporidium spp. – 0,2%, Cyclospora cayetanensis -0,1% (Table 1). The were 12 known cases of imported intestinal parasitosis. Conclusions. Prevalence of intestinal parasites in Lithuania patients with gastrointestinal complains is increasing with age, distribution by sex is equal, men/women ratio 1:1, protozoan infections predominate, most common is Blastocystis hominis, followed by pathogenic Entamoeba histolytica, Giardia lamblia and Dientamoeba fragilis. 5,3% of patient were infected with more than one species of parasites.

84 5. Health literacy and management of communicable diseases

Mikhail Mikhailov Vaccination against hepatitis B and anti-vaccination Invited propaganda

Caplinskas Saulius Information handouts – influenza vaccination during Poster pregnancy – acceptability and needs Caplinskiene Irma Lithuanian population awareness about HIV Oral Cardoso Ana Lucia EDCTP as a model for Europe-Africa partnership on Oral HIV/AIDS research – achievements and future directions Cooney Fionnuala Social media used to control gonorrhea in Dublin, Ireland Oral Kazenaite Edita Epidemiological analysis of pathogenic fungi in hospitals of Poster Vilnius Kivite Anda Parents as the main source of HIV and STI prevention among Poster young people in Latvia Macijauskiene Ausra Results of the prevention system of healthcare personnel from Poster the exposure to bloodborne pathogens at Vilnius University Hospital Santariskiu Klinikos during 2013-2015

85

VACCINATION AGAINST HEPATITIS B AND ANTI VACCINATION PROPAGANDA

INVITED LECTURE Name E-mail Institution Country

Chumakov Institute of poliomyelitis and viral MIKHAIL Russian [email protected] encephalitides, department of viral hepatitis, MIKHAILOV Federation Moscow

Introduction: Once in the middle of the eighteenth century, Edward Jenner created and applied the first vaccine against smallpox, vaccination opponents appeared. Over the next centuries the movement against vaccination was the constant companion of progress. Vaccination against hepatitis B and A is not an exception. Today anti-vaccination lobby exists all over the world including Russia. Ignorance associated with vaccination issues is reflected in television shows, in press (mainly in "tabloid" mass editions), on the Internet. The aim of this lecture: To consider possible causes of anti-vaccination movement activation, to analyze the main arguments made against hepatitis B and A vaccination, and to observe possible strategy to combat anti-vaccination movement. Results & Discussion: Convincing success of mass vaccination against hepatitis B and reduction of hepatitis A incidence have led to a sharp decrease in the number of severe (fatal) cases of these diseases, which gave rise to complacency society against these infections. Desire to be in the spotlight, as the saviors of mankind, from allegedly unscrupulous scientists, as well as lack of knowledge of the problem determines the nomination of anti-vaccination movement leaders. An important component in this case is an interest to press coverage of scandals attracting people's attention. Usually such cases are based on unverified data, often distorted isolated cases. Lack of awareness about vaccination, and often simply ignorance of the population in these areas is a good background for the spread of false information. It is suggested that the hepatitis B vaccination could lead to an increase in autism, multiple sclerosis, rheumatoid arthritis, etc. To study these assumptions numerous independent studies were held, including those led by WHO. Results of these studies deny any relationships between such disorders and vaccination against hepatitis B and A. Today it is clear that vaccination against hepatitis B and A is the most important tool to control these infections. Successful confrontation to anti-vaccination movement requires a clear program that unites healthcare workers with different social populations. Such program should be based on an integrated approach involving evidence based effective interventions.

86

INFORMATION HANDOUTS - INFLUENZA VACCINATION DURING PREGNANCY - ACCEPTABILITY AND NEEDS

POSTER Name E-mail Institution Country Mykolas Romeris University, CAPLINSKAS SAULIUS [email protected] Lithuania Centre for Communicable Diseases and AIDS Drasutiene Grazina [email protected] Lithuania

Korablioviene Joana [email protected] ULAC Lithuania Centre for Communicable Diseases Kriauciunaite Asta [email protected] Lithuania and AIDS Centre for Communicable Diseases Sebeliauskaite Ieva [email protected] Lithuania and AIDS

Introduction. Vaccination against influenza can reduce the risk for influenza-related illness among pregnant women and their infants. Less than 1% pregnant women got influenza vaccination in Lithuania in 2014-2015. Objective. In order to increase influenza vaccination coverage among pregnant women, to assess the information needs and form for pregnant women was prepared information handouts about vaccination against influenza. The goal - to assess the prepared information. Methods. The questionnaire consists of 20 questions about the spontaneous reaction to the understanding, attractiveness, relevance and acceptability of the handouts. The study population consisted of pregnant women from Obstetrics and Gynecology Center at Vilnius University Hospital Santariškiu Klinikos. The study included all pregnant women, not considering on their age, nationality, place of residence and so forth. 36 of 50 questionnaires were suitable for analysis. The data were summarized by descriptive statistics method. The data present in percentages. To count p value was used Microsoft Exel 2007 program. Significance level equals 0,05. Results. Useful information from handouts found 91.7% (n=33) of the women involved in the study. 91.7% (n=33) of information given to women was clear. 13.9% (n=5) of pregnant women information about flu shots was not acceptable, 5.6% (n=2) - insufficient. 33% (n=6) of women for more information about flu shots will turn to health care professionals, 33% (n=6) - to family members. Respondents who choose not to vaccinate assess the visual presentation of handouts (p=0,003). Conclusions. 1. Useful information from handouts found 91.7% of the women involved in the study. 2. 91.7% of information given to women was clear. 3. 13.9% of pregnant women information about flu shots was not acceptable. 4. 33% of women for more information about flu shots will turn to health care professionals, 33% - to family members.

87

LITHUANIAN POPULATION AWARENESS ABOUT HIV

ORAL Name E-mail Institution Country CAPLINSKIENE IRMA [email protected] CCDA Lithuania

Background and aims. Population awareness about HIV/AIDS can contribute to the HIV / AIDS stigma and risk behavior reduction. Sustainable, targeted HIV/AIDS communication ensures an adequate population awareness about HIV/AIDS, what is appropriate HIV prevention tool in the management of HIV infection. Objective of the study - to evaluate the general population knowledge about HIV transmission modes and identify misconceptions about HIV transmission modes. Methods. Representative cross-sectional survey of the Lithuanian population (2014). The study involved 996 respondents. Results. Almost all (96.7 percent) survey respondents have good knowledge about HIV transmission through non-sterile syringes and needles. Three-quarters (76.7 percent) of the respondents know that HIV can be transmitted through blood transfusions. 99 percent of the respondents are aware that HIV is spread through sexual intercourse. That condoms protect against HIV agree about a half (46.4 percent) of the respondents and 91 percent agree with the statement that condoms reduce the risk of HIV infection. That an HIV-infected pregnant woman can pass the infection to her baby during childbirth know 78.8 percent of the respondents, but about a half of the respondents (47.9 percent) do not know whether HIV can be spread through the breastfeeding. Most of the population does not have misconceptions and know that healthy- looking person can be infected with HIV; that HIV does not spread by airborne mode when an HIV patient is coughing or sneezing; know that sharing food with someone who is infected they can't acquire HIV; know that HIV does not spread through insect bites - respectively: 86.6 percent, 79.7 percent, 77.8 percent, 58.6 percent. Conclutions. Total indicator of population awareness about HIV (have the right knowledge about the transmission of HIV and have no misconceptions about the spread of HIV) has improved over the past decade and has grown from 70.6 to 82.2 percent. IEC should be supplemented by targeted actions to change the misconceptions.

88 EDCTP AS A MODEL FOR EUROPE-AFRICA PARTNERSHIP ON

HIV/AIDS RESEARCH – ACHIEVEMENTS AND FUTURE DIRECTIONS

ORAL Name E-mail Institution Country

EDCTP - European & Developing Countries Ana Lucia Cardoso [email protected] Netherlands Clinical Trials Partnership, The Hague GABRIELLE [email protected] BREUGELMANS

Ole Olesen [email protected]

Lara Pandya [email protected]

Hager Bassyouni [email protected]

HIV infection remains a major public health concern and poses challenges that go beyond the capacity of individual countries. More research is needed in Europe and Africa on the evaluation of therapeutic interventions for management of HIV. Long-term control of HIV and the development of products require a combination of innovation and development cooperation facilitated through transnational partnerships. 16 European Member States and the European Commission established in 2003 the European & Developing Countries Clinical Trials Partnership (EDCTP) to pool resources and provide a common platform for research cooperation for European and sub-Saharan African countries in the fight against HIV, malaria and TB. EDCTP supports clinical research and capacity buiding through Calls for Proposals to develop drugs, vaccines, microbicides and diagnostics. To date, EDCTP has awarded 246 grants (total grant value of 212€M). 52 (21%) focused on HIV research (total grant value of 62.4M€) and most of the funding (40€M) was allocated to HIV clinical trials (62% treatment, 31% vaccines and 7% microbicides) with 12 projects (4.9%) on HIV/TB coinfection. Other funding supported HIV immunology studies and diagnostics (total grant value of 22M€). Most EDCTP projects (69%) include one or more European collaborators, giving an average of 4 European researchers per grant. EDCTP-funded HIV clinical trials led to the registration of a paediatric formulation of an ARV (Pedimune) in Africa (Chintu et al.). A study by Bertilsson et al. found no need to increase the efavirenz dose during concomitant rifampicin based anti-TB therapy in patients with HIV/TB coinfection. Other EDCTP studies influenced national and international policies. EDCTP has assembled countries that had no tradition of working in collaboration with Africa, facilitated synergy and optimal use of resources. EDCTP offers opportunities to conduct research that cannot be funded or delivered by a single country alone and provides a tool for European countries to align research priorities around a common agenda. EDCTP can provide an important tool for researchers in Baltic countries to participate in large clinical research consortia and engage with research teams across Europe and sub- Saharan Africa. The partnership offers increased networking opportunities, including with major pharmaceutical companies, public and private

89

SOCIAL MEDIA USED TO CONTROL GONORRHOEA IN DUBLIN, IRELAND

ORAL Name E-mail Institution Country COONEY FIONNUALA [email protected] Health Service Executive Ireland

Background. A multidisciplinary gonorrhoea control group was convened in Dublin in response to a 33 % increase in annual gonorrhoea notifications in Ireland in 2012. The problem was centred mainly in the greater Dublin area, where the gonorrhoea notification rate was 49.2 per 100,000, almost three times greater than the national average. Investigation included retrospective enhanced surveillance on all laboratory-confirmed gonorrhoea notifications (n=197) received between January-March 2013. Analysis revealed two main risk groups: men who have sex with men (MSM) (62%) and young heterosexuals (males 24% and females 14% of total). Review of the health promotion materials available on gonorrhoea in Ireland found that the information for young heterosexuals was poor, in contrast to the high quality information available for MSM. In response, an information campaign was developed specifically for young heterosexuals with key messages informed from the results of the findings from the enhanced surveillance. Methods. An information campaign sub-group was convened involving a range of professionals experienced in digital media and working with young people. Based on evaluation of previous campaigns aimed at the same target population, it was decided that this campaign would be delivered primarily via a social media and would use eye-catching imagery, humour (when appropriate), quick and clear information and positive and non-judgemental messages. Using information from the enhanced surveillance, materials were developed focussing on the risks of unprotected sex and the importance of availing of routine STI screening following unprotected sex with new or concurrent partners. Materials were tested on a sample of young people who were also asked for the hashtag for the Twitter promotion and invited to model for the creatives/visuals. Both Twitter and Facebook were used with referral to an updated health service website: yoursexualhealth.ie. The campaign was delivered over a two month period, concluding in February 2014 with a St Valentine’s competition promotion. Results. The campaign trended on Twitter. Facebook promoted posts reached tens of thousands of young people, generated hundreds of thousands of impressions, with click-through rates and engagement rates well above average. The campaign website reported a 59% increase in visits. There was considerable media interest from newspapers, radio and television. During this time there were anecdotal reports of an increased demand for STI screening services but it was not possible to validate due to absence of central information systems. Surveillance demonstrated a halt in the rise in gonorrhoea notifications and the gonorrhoea control group was concluded four months after the conclusion of the campaign. Over the subsequent 12 month period there was no further increase in number of gonorrhoea notifications. Conclusions. This is the first instance of social media being used in STI outbreak control in Ireland. The campaign achieved good reach of the target population and therefore this paper advocates use of social media as an effective method of reaching young people. Based on this success of this campaign work, a Sexual Health Communications Working Group has been established tasked with planning and delivering information on sexual health at national level.

90

EPIDEMIOLOGICAL ANALYSIS OF PATHOGENIC FUNGI IN HOSPITALS OF VILNIUS

POSTER Name E-mail Institution Country Vilnius University, Faculty of Medicine; Vilnius Kazenaite Edita [email protected] Lithuania University Hospital Santariskiu Klinikos STANKEVIC [email protected] Vilnius University Faculty of Medicine Lithuania JOLITA Radaviciute Indre [email protected] Vilnius University Faculty of Medicine Lithuania Vilnius University Faculty of Medicine, Vilnius Kiveryte Silvija [email protected] Lithuania University Hospital Santariskiu Klinikos

Aim. To evaluate the epidemiology of invasive pathogenic fungi found in the test substance of patients treated in the largest hospitals of Vilnius in 2015, to evaluate the sensitivity of fungi to antifungal preparations and find links between risk factors and different species of fungi. Methodology. A retrospective study was conducted in hospitals of Vilnius in 2015. A test group consisted of 64 patients with grown cultures of invasive fungus. The selection criteria: test substance - bronchoalveolar lavage fluid, blood, punctates; fungi grown on Sabouraud agar, identified using a selective diagnostic media (CHROMagar Candida BLL), automatic identification system Vitek2 (bioMerieux, France). Invasive pathogenic fungi were divided into groups according to the class - yeast fungi and mold fungi. Evaluated risk factors: age, diabetes, immunosuppressive diseases, length of stay in the intensive care unit, type of diagnosis. Frequency of occurrence of species in patients treated in the intensive care unit during the investigation period was analysed. The sensitivity of different species of fungi to fluconazole, voriconazole, amphotericin B, caspofungin, anidulafungin was identified. The statistical analysis of the data was carried out using SPSS22.0, differences were considered statistically reliable when p<0.05. Results. The first group consisted of 59 families of yeast fungi, 30 (46.88 %) of which were Candida albicans, 26 (40.63 %) - Candida non-albicans (14 (21.88 %) of which were Candida glabrata) and 3 (4.69 %) - Trichosporon asahii. The second group consisted of 5 (7.81 %) mold fungi (Aspergillusflavus). Statistically significant link between the fungus and the type of risk factors (age, diabetes, immunosuppressive diseases) was not found, excluding the type of diagnosis (p<0.05). Yeast fungi were found more often in patients with surgical diagnosis (84.75%), while mold fungi were found more often in patients with therapeutic profile diagnosis (80%). Infections caused by invasive pathogenic fungi are characterized by high mortality (40%). Resistance to fluconazole was 3.33% in Candida albicans group, while in Candida non-albicans group it was 73.07%. The best sensitivity in Candida non-albicans group was determined to amphotericin B (84.62%). Conclusions. Candida albicans is the most frequently excreted invasive fungus. There is also a growing number of infections caused by fungi of Candida non-albicans group. Yeast fungi were identified more often in patients with surgical diagnosis. Currently, the first drug of choice for treatment of Candida albicans infection remains fluconazole. Most of the invasive species of pathogenic fungi (yeast fungi and mold fungi) were sensitive to amphotericin B.

91 PARENTS AS THE MAIN SOURCE OF HIV AND STI PREVENTION AMONG YOUNG PEOPLE IN LATVIA

POSTER Name E-mail Institution Country KIVITE ANDA [email protected] Riga StradinsUniversity Latvia Stokenberga Ieva [email protected] University of Latvia Latvia Latvia’s association for family planning Kelle Iveta [email protected] Latvia and sexual health, Papardes zieds

Background and aims. During the last decade there are many discussions carried out in the political space and mass media in Latvia regarding the health education in schools. In 2006 the Health education was repealed as a separate subject in schools and since then health (including sexual and reproductive health) issues are covered in a limited way in frame of the subject Social sciences. Additionally to that there amendments in the Education law have been introduced in 2015 (so called virtue amendments) and several teachers have been punished for discussing, e.g. homosexuality issues during the lessons. Initiators of the virtue amendments proclaim that parents should be the main source of sexual education in Latvia despite the fact that less than a half of the young people (according to the Reproductive health study 2011) have ever received any information on sexuality from their parents. Thus the aim of our research was to clarify the opinion of parents on whether the sexual education should be covered in schools, how confident they are about their own capacity to talk on sexuality with their children as well as to investigate the knowledge of parents about sexual health issues. Methods. In April 2016 a cross-sectional study was carried out, using face-to-face and internet-based questionnaire. Using stratified randomized sampling 614 parents of children aged 7-18 years have been recruited. SPSS 20.0 has been used for statistical analysis. Frequency and cross-tabulation analysis was carried out. Results. Largest part (66.2%) of parents strongly agrees with the statement that sexual education should be compulsory part of the educational programme in schools. Only 23.8% of parents agree with the statement that sexual education in schools may promote early sexual initiation among adolescents. 65.7% of parents agree that sexual education in schools can protect young people from HIV and STI. The poorest knowledge among parents is about reproductive health of a female – only 29.2% of parents gave correct answer to a statement on the length of a menstrual cycle; only 29.6% know correct answer on the question on most fertile days in the cycle. Still 35.5% of parents think that masturbation is a health threat. But 40.4% of respondents previously showed that they are confident that they can discuss this topic with their children. Only a half (51.5%) of parents have correct knowledge on HIV (have given correct answers to all five knowledge indicator-questions on HIV). Despite of that 66.6% of respondents are confident that they are able to educate the child on HIV and STI. Conclusions. Parents are convincingly supporting the idea that the issues of sexual education should be covered in schools. Parents are quite confident of their ability to talk with their children about sexuality, including issues related to HIV and STI. Unfortunately the knowledge of parents on the mentioned issues is insufficient.

92

RESULTS OF THE PREVENTION SYSTEM OF HEALTHCARE PERSONNEL

FROM THE EXPOSURE TO BLOODBORNE PATHOGENS AT VILNIUS UNIVERSITY HOSPITAL SANTARISKIU KLINIKOS DURING 2013-2015

POSTER Name E-mail Institution Country Vilnius university hospital Santariskiu MACIJAUSKIENE AUSRA [email protected] Lithuania klinikos Vilnius university hospital Santariskiu Gierasimovic Zita [email protected] Lithuania klinikos Vilnius university hospital Santariskiu Pakalniskyte Ilma [email protected] Lithuania klinikos Vilnius university hospital Santariskiu Siauliene Daiva [email protected] Lithuania klinikos Vilnius university hospital Santariskiu Valotkiene Zydre [email protected] Lithuania klinikos Vilnius university hospital Santariskiu Kerulyte Sandra [email protected] Lithuania klinikos

Introduction and purpose. Exposure to blood and other body fluids occurs in a wide variety of occupations. Healthcare personnel can be exposed to blood through a needle - needle stick or other sharp injuries, mucous and skin lesions. The pathogens of primary concern are the human immunodeficiency virus (HIV), the hepatitis B virus (HBV) and the hepatitis C virus (HCV). The aim of this post-exposure system is to introduce our standardized system for the registration of blood and body fluids exposure reports, to analyze them and to improve protection for healthcare personnel from occupational exposure. Materials & Methods. We analyzed the collected data from the reports of percutaneous injuries, blood and body fluid exposures, and post-exposure questionnaires from all the departments of the hospital during 2013-2015. Summary of results. We received 69 percutaneous injury reports during 2015 (80 reports were included into the study within 2014, 55 injury reports were included into the study within 2013). The mean age of medical staff injured was 39 years in 2015, 42 years in 2014, 41 year in 2013: 38 female cases (55,1 %) and 31 male cases (44,9 %) in 2015, 38 female cases (47,5 %) and 42 male cases (52,5 %) in 2014, 37 female cases (67,3 %) and 18 male cases (32,7 %) in 2013. Most of the injuries occurred in operating theatres: 48 cases (69, 6 %) in 2015, 56 cases (70 %) in 2014, 35 cases (63,7 %) in 2013. Mostly doctors were injured: 38 cases (55, 1 %) in 2015, 56 cases (70 %) in 2014, 34 cases (61,9 %) in 2013 . Most of the injuries were caused by a suture needle: 50 cases (72,6%) in 2015, 48 cases (60 %) in 2014, 39 cases (71 %) in 2013. 8 injuries (11,8 %) in 2015, 10 injuries (13 %) in 2014, and 11 injuries (20,8 %) in 2013 were in risk of Hepatitis C infection. 2 injuries (2,9 %) in 2015, 4 injuries (5,2 %) in 2014, were in risk of HIV. No cases of dangerous injuries of HIV were noticed in 2013. During 1999-2015, 1400 members of medical staff received a full course of vaccination (3 doses of the hepatitis B vaccine) and serological blood testing for anti-HBs were performed for these employees. After vaccination, immunity against HBV developed in 1229 (87, 8 %) employees. Conclusions. 1. Our data collected during 2013-2015 seem to indicate that the reports received include only a little part of all the injuries (if the danger of infection was really great). 2. There were no occupational illnesses at Vilnius University Hospital Santariskiu Klinikos during 1999-2015. 2. It is necessary to convince employees that they need to report all the needle-stick injuries. 3. We have to educate healthcare personnel about the risks associated with sharp injuries.

93 Vilnius International Summit on Communicable Diseases 26 June - 1 July 2016, Vilnius

725 W Lombard St. | Baltimore, MD 21201-1009 | 410.706.1966 Organizers www.gvn.org

Baltimore, May 9, 2016 To participants at the Vilnius International Summit on Communicable Diseases, Užkrečiamųjų ligų ir AIDS centras Centre for Communicable Diseases and AIDS Although we are unable to participate in this important meeting, on behalf of the Global Virus Network (GVN) we want to extend a warm welcome to all of you. The scientific program is comprehensive and of a very high quality, with several sessions dedicated to the threat of emerging viral diseases. The successful development of antimicrobial drugs and vaccines during the first 60 years Partners of the last century led to the belief that "it (was) time to close the book on infectious diseases, and declare the war against pestilence won." Nobel Laureate Joshua Lederberg once said that "the war between humankind and microbes was ongoing, and that no one could be sure about who will win it." Joshua Lederberg, who died in 2008, could clearly see the imminent risk that emerging and re-emerging viruses will continue posing to humankind. The medical community experienced a rude awakening when AIDS was described as a new disease in 1981. A new virus, the human immunodeficiency virus or HIV, was identified as the cause of AIDS in 1983-1984. What is sobering is to realize that HIV was introduced in West Africa, from chimpanzees to humans, in the 1920s, and that HIV circulated "silently" for 60 years before we were aware of its potential to cause human disease. Only in the last thirteen years, at least nine different viruses have emerged or re-emerged to cause human epidemics. As you meet in Vilnius, countries in the Americas are struggling to control an epidemic of the Zika virus, with the unexpected and dramatic association with microcephaly. Angola is experiencing a severe epidemic of yellow fever, and Nigeria is experiencing one of Lassa fever. We can only predict that new viral epidemics will arise in the future and we, as virologists, need to be alert and prepared to properly respond to these future viral threats. In 2011, the Global Virus Network was co-founded to harness progress in science to address and be prepared to confront emerging and re-emerging epidemics caused by a full range of pathogenic viruses, and to train the medical virologists to address this problem. We look forward to working together with medical virologists in the region as part of an extended network of the GVN Centers of Excellence.

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W W W . V I L N I U S - S U M M I T . E U ROLE OF HUMAN PAPILLOMAVIRUS IN DEVELOPMENT OF CERVICAL CARCINOMA IN SITU

INVITED LECTURE Name E-mail Institution Country JOANA CELIESIUTE [email protected] Lithuanian University of Health Lithuania Agne Vitkauskaite [email protected] Sciences, Department of Daiva Vaitkiene [email protected] Obstetrics and gynaecology, Kaunas

Our aim was to identify what HPV types actually caused high grade cervical intraepithelial neoplasia (CIN3/Cis). Analysis of 5 years old formalin fixed paraffin – embedded tissue of cervical cone with morphologicaly confirmed CIN3/Cis was done. 100 samples were analyzed for 24 HPV genotypes (15 HR HPV, 3 RHR HPV and 6 LR HPV) using polymerase chain reaction (Optilpex HPV genotyping Kit). 100 cervical cone formalin fixed paraffin – embedded histological samples were analyzed. In 77 of them HPV was detected – there was sufficient number of HPV DNR copies to make further analysis. Other 23 samples had lower number of HPV DNR copies that cannot be analyzed (this could be due to following factors: tissue coagulation, small amount of the disease in the sample, difficulties to find the lesion in the sample because of the discoloration; but no correlation with negative HPV status ant mentioned factors was observed). The mean age of women treated for CIN3/Cis at the moment of surgery was 35.85 years (SD: 7.72; min. value 22 years, max. value 58 years). HPV 16 type was the most common HPV type of all identified, it was detected in 86.8% of the samples (95%SD: 78.7 – 94.5%; p<0.05). Some trends were noticed: HPV 33 type was detected more often in 25 – 30 year group. HPV 52, 56, 58, 59 were found more often in women older than 30 year age. In 85.7 % (n = 66) of HPV positive samples only one HPV type was identified while multiple HPV types were detected in 14.4 % (n = 11). In all cases with multiple HPV infection on of the HPV was type 16. HPV 16 is most common HPV type causing high grade cervical intraepithelial neoplasia (CIN3/Cis). Our data is in good agreement with other European countries. According to our data in almost all cases vaccination could prevent high grade cervical intraepithelial neoplasia (CIN3/Cis) and cervical cancer development.

Vilnius International Summit on Communicable Diseases 26 June - 1 July 2016, Vilnius Abstract Book Photo by Saulius Gudeliauskas

W W W . V I L N I U S - S U M M I T . E U

Užkrečiamųjų ligų ir AIDS centras Centre for Communicable Diseases and AIDS