Unit EPIDEMIOLOGy of INFECTIous diseases

Scientific seminar on infectious diseases Brussels, 19 MAY 2016

Spring Symposium BVIKM/SBIMC

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Scientific Institute of Public Health (WIV-ISP) Operational directorate Public health and surveillance Unit Epidemiology of Infectious Diseases Rue Juliette Wytsmanstraat 14 | 1050 Brussels | Belgium Belgian Society of Infectiology and Clinical Microbiology Route de Lennik 808 | 1070 Brussels | Belgium Scientific Seminar on Infectious Diseases | 19 May 2016 | Brussels, Belgium PHS Report 2016-013 Legal deposit: D/2016/2505/11

Table of Contents Partners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Programme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Sponsors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Scientific committee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Abstracts of presentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Dr. Tinne Lernout & Prof. Dr. Veroniek Saegeman Surveillance strategies for Lyme borreliosis in Belgium and expectations and limitations of diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Dr. Sophie Bertrand Recent trends of Listeriosis in Belgium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Prof. Dr. Greet Ieven Vancomycin-resistant Enterococci (VRE) epidemiology . . . . . . . . . . . . . . . . . . . . 19 Prof. Dr. Elizaveta Padalko & Dr. Béatrice Swennen HPV diagnosis and prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Dr. Chantal Reusken MERS-coronavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Prof. Dr. Steven Callens Clinical approach of uncommon emerging and traveling causes of encephalitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Prof. Dr. Steven Van Gucht Bats as reservoirs of emerging pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Dr. Amaya Leunda Biorisk management: current state of play . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Dr. Franz Karcher The new EU framework on health security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Dr. Rémy Demeester Refugees, asylum seekers, migrants and infectious diseases . . . . . . . . . . . . . . . . 37 Dr. Marjan Van Esbroeck Zika virus in Belgium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Abstracts of posters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

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Partners

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Programme 8:30

Registration with walking breakfast / Visit of stands Session 1 Dr Olivier Denis (ULB) & Dr Bart Gordts (ZNA)

09:15

Welcome address Prof. Dr Herman Van Oyen (WIV-ISP) & Dr Denis Piérard (BVIKM-SBIMC)

09:30 Surveillance strategies for Lyme borreliosis in Belgium and expectations and limitations of diagnostics Dr Tinne Lernout (WIV-ISP) & Prof. Dr Veroniek Saegeman (UZ Leuven) 10:00

Recent trends of Listeriosis in Belgium Dr Sophie Bertrand (WIV-ISP)

10:25

Vancomycin-resistant Enterococci (VRE) epidemiology Prof. Dr Greet Ieven (UZA)

10:50

HPV diagnosis and prevention Prof. Dr Elizaveta Padalko (UZ Gent) & Dr Béatrice Swennen (ULB)

11:20

Coffee break / Visit of stands Session 2 Dr Yves Van Laethem (CHU St Pierre) & Prof. Dr Elizaveta Padalko (UZ Gent)

11:50 MERS-coronavirus Dr Chantal Reusken (Erasmus MC Rotterdam) 12:20

Clinical approach of uncommon emerging and traveling causes of encephalitis Prof. Dr Steven Callens (UZ Gent)

12:45

Bats as reservoirs of emerging pathogens Dr Steven Van Gucht (WIV-ISP)

13:10

Walking lunch / Visit of stands OR General assembly of BVIKM-SBIMC (members only) followed by walking lunch Session 3 Dr Ruud Mak (ZG) & Dr Marc Vekemans (HIS-IZZ)

14:25

Biorisk management: current state of play Dr Amaya Leunda (WIV-ISP)

14:50 The new EU framework on health security Dr Franz Karcher (EC- DG SANTE) 15:15

Refugees, asylum seekers, migrants and infectious diseases Dr Rémy Demeester (CHU de Charleroi)

15:40

Hot Topic: Zika virus in Belgium Dr Marjan Van Esbroeck (ITG)

16:00

Closing address and end of seminar

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Sponsors

altona DIAGNOSTICS

ELITECH BENELUX

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Scientific committee Boudewijn Catry Head of unit Healthcare-associated infections and antimicrobial resistance, Scientific Institute Public Health, Brussels [email protected] Georges Daube Professor Food Microbiology, Université de Liège [email protected] Greet Ieven Head of unit Clinical Microbiology, AntwerpUniversity Hospital Professor Microbiology, University of Antwerp [email protected] Tinne Lernout Chair of SsID scientific committee Scientific Institute Public Health, Brussels [email protected] Ruud Mak Zorg en Gezonheid Coordination of infectious diseases control [email protected] Pierrette Melin Head of unit Clinical Microbiology, CHU de Liège Professor Microbiology, Université de Liège. [email protected] Marcella Mori Head of Unit Co- ordination Veterinary Diagnose Epidemiology and Risk Analysis CODA-CERVA, Brussel m.mori@ coda-cerva.be Elizaveta Padalko Head of Clinical Virology, University Hospital Ghent [email protected] Denis Piérard Head unit Microbiology and hospital hygiene UZ Brussels [email protected] Catherine Potvliege Microbiologist, Head of Laboratory CHU Tivoli [email protected] Sophie Quoilin Head of unit Epidemiology Infectious Diseases Scientific Institute Public Health, Brussels [email protected]

Hector Rodriguez-Villalobos Microbiologist at Cliniques universitaires Saint-Luc [email protected] Carole Schirvel Head of Infectious disease surveillance unit Fédération Wallonie Bruxelles [email protected] Jean‐Marie Trémérie Health inspector at Commission Communautaire Commune – Gemeenschappelijke Gemeenschapscommissie, Brussels [email protected] Viviane Van Casteren Head of unit Healthcare services research Scientific Institute Public Health, Brussels [email protected] Steven Van Gucht Head of unit Viral diseases Scientific Institute Public Health, Brussels [email protected] Yves Van Laethem Head of the Clinical Dept. Infectious Diseases CHU St-Pierre [email protected] Herman Van Oyen Operational director, Scientific Institute Public Health, Brussels Professor of epidemiology, University Ghent [email protected] Jan Verhaegen Clinical microbiologist at the University Hospital Gasthuisberg [email protected] Kris Vernelen Quality of Medical Laboratories, Scientific Institute of Public Health, Brussels [email protected]

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Abstracts of presentations

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Dr. Tinne Lernout Scientific Institute of Public Health [email protected]

Biography Tinne Lernout is a MD with a specialization in tropical medicine and a master’s degree in Public Health. She has 13 years of experience working for (public health) institutions in Belgium (WIV-ISP, University Antwerp) and in France (InVS), in the area of infectious diseases, surveillance, field epidemiological investigations and surveys. She is currently employed as a scientific researcher at the WIV-ISP, where she is in charge of the coordination of the cell Environment-related Infectious Diseases.

Surveillance strategies for LYME BORRELIOSIS in Belgium and expectations and limitations of diagnostics (1) Monitoring of vector-borne diseases needs to integrate different strategies, in which surveillance of both the disease and the vector are important. In Belgium three sources contribute to surveillance of Lyme disease. Laboratory surveillance is done by a network of sentinel laboratories and by the National Reference Center (NRC: UCL – UZ Leuven) which both report the number of positive laboratory tests for Borrelia burgdorferi. The yearly number of persons hospitalized for Lyme disease in Belgium is monitored through data extracted from the hospitals’ minimum clinical (RMC) datasets. Finally, the incidence of erythema migrans is estimated based on repeated studies carried out by a sentinel network of general practitioners. Up to 2015, none of these sources identified a significant increase in the incidence of Lyme disease in Belgium. Surveillance of the vector of Lyme disease (mainly Ixodes ricinus ticks) in Belgium was initiated in June 2015, with the launch of an online citizen-based platform for the reporting of tick bites (tekennet.be). In addition to the activities in vector and disease surveillance, the WIV-ISP is also involved in a number of research projects on Lyme borreliosis. In 2013-2014, WIVISP collected 3200 serum samples representative of the Belgian population. The NRC tested these samples for IgG antibody reactivity against B. burgdorferi sl. in 2015. We estimated the overall seroprevalence of Borrelia IgG at 1.06% (95% CI: 0.67-1.67%). In May 2016, a study on the burden and cost of Lyme disease and the occurrence of Post Treatment Lyme Disease Syndrome (PTLDS) will start. In this study, a cohort of approximately 800 patients with Lyme disease will be followed up over time (6 to 24 months), to collect information on acute and persistent symptoms, quality of life and costs related to the diagnosis and treatment of their disease.

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References 1. Bleyenheuft C, Lernout T, Berger N, Rebolledo J, Leroy M, Robert A, et al. Epidemiological situation of Lyme borreliosis in Belgium, 2003 to 2012. Arch Public Health. 2015 Jul 3;73(1):33 2. Rebolledo J, Lernout T, Litzroth A, Van Beckhoven D, et al. Zoonoses and vectorborne diseases. Epidemiological surveillance in Belgium, 2013 and 2014. Report in Dutch and in French. Available online at https://epidemio.wiv-isp.be/ID/reports/Zoönosen%20en%20vectoroverdraagbare%20 ziekten.%20Jaarrapport%202013%20en%202014.pdf

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Prof. Dr. Veroniek Saegeman UZ Leuven [email protected]

Biography Veroniek Saegeman graduated in Medicine in 2003 at the KULeuven. Afterwards, she specialized in Clinical Biology at KULeuven. Her special interest goes to Infectious Diseases and Infection Control. She defended her PhD thesis in Biomedical Sciences entitled ‘Microbiological contamination and decontamination of human tissue allografts’ in 2010. Related with this topic, she is secretary of the ESCMID Study Group for Forensic and Postmortem Microbiology. Since 2011, she is adjunct clinical head in Microbiology at the Department of Laboratory Medicine and at the Department of Hospital Epidemiology and Infection Control of University Hospitals Leuven, Belgium. In consortium with the Université Catholique de Louvain, she is responsible for the National Reference Center of Lyme disease.

Surveillance strategies for Lyme borreliosis in Belgium and expectations and limitations of diagnostics (2) Lyme disease is the most common tickborne infection in the US and in Europe. it is caused by Borrelia burgdorferi, a bacterium that is transmitted by the tick Ixodes ricinus (in Europe). Clinical manifestations most often involve the skin, joints, nervous system and heart. Diagnostic testing performed in laboratories is recommended for confirmation of extracutaneous Lyme disease. As diagnostic tests, one can distinguish either direct methods detecting the causal agent (culture or PCR) and indirect methods detecting the immune response against the agent (antibodies). Serological assays are compliant with the EUCALB criteria and currently follow the two-tiered testing system. Some tips in Lyme serology will be discussed. As a new biomarker in Lyme neuroborreliosis, CXCL13 will be presented. This chemokine is increased in acute neuroborreliosis.

Reference 1. Wormser G, Dattwyler R, Shapiro E et al. IDSA guidelines: The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the infectious Diseases Society of America. CID 2006;43:1089 2. Schmidt C, PLate A, Angele A et al. A prospective study on the role of CXCL13 in Lyme neuroborreliosis. Neurology 2011;76:1051

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Dr. Sophie Bertrand Scientific Institute of Public Health [email protected]

Biography Head of Service Bacterial Diseases, Institute of Public Health In Belgium. Her main area of expertise is in the field of microbiology and molecular biology. She is responsible of three National Reference Centres in Belgium (NRC Salmonella and Shigella, NRC Listeria and NRC Neisseria meningitidis). Her research interests cover the surveillance of these pathogens but also the study of virulence mechanisms and mechanism of antibiotic resistance of the strains. She participates actively to national and international networks like this of ECDC, i.e. IBD (invasive bacterial Diseases) and FWD (Foodborne and Water Diseases).

Recent trends of Listeriosis in Belgium Listeriosis is a rare but severe disease mainly caused by Listeria monocytogenes. This study shows the results of the laboratory-based surveillance of Listeriosis in Belgium over the period 1985-2014. Besides the incidence and some demographic data we present also more detailed microbiological and molecular characteristics of human strains isolated since 2000. The strains from the latter period were also compared to food and animal strains from the same period. Our study has also shown that different alimentary products were commonly contaminated with L. monocytogenes presenting the same PFGE profile as in patient’s isolates. Since 1985 we observed a significant decrease in incidence of the Materno-Neonatal cases (from 0.15 to 0.04 cases /100,000 inhabitants) probably to be attributed to active prevention campaigns targeting pregnant women. Nevertheless, despite the strengthening of different control measures by the food industry, the incidence of non-Materno-Neonatal listeriosis increased (from 0.3 to 0.7 cases /100,000 inhabitants) in Belgium probably largely due to the rise of highly susceptible patients (cancers, immunosuppressive treatments, transplantation….) in an aging population. Moreover the risk population is susceptible to low level of contamination of the food stressing the need of prevention campaigns specifically targeting these persons.

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Prof. Dr. Greet Ieven Head of unit Clinical Microbiology, Antwerp [email protected]

Biography Greet Ieven, head of laboratory of medical microbiology at University hospital Antwerp and professor at the faculty of medicine, is responsible for the microbiological diagnosis performed in the clinical laboratory and is involved in a number of national and international projects. She has long standing experience with the development and implementation of conventional and molecular diagnostic tests in the clinical microbiology laboratory and has published her research in over 150 full papers in peer-reviewed journals. The Laboratory of Medical Microbiology, University of Antwerp has a broad expertise in studying antibiotic use and resistance, as well as on the development of molecular diagnostic tests for respiratory tract infections and is currently involved in several EU projects. Based on its expertise, the lab also houses the reference centers for enterococci, group A streptococci and respiratory pathogens.

Vancomycin-resistant Enterococci (VRE) epidemiology E. faecalis and E. faecium are common gastrointestinal commensal organisms acquiring resistance through the transfer of plasmids and transposons and recombination or mutation events. Infection with vancomycin-resistant enterococci (VRE) is a growing problem. Currently, there are not many data available on the epidemiology of Enterococci and VRE in Belgium. A survey was initiated by Belgian Reference Centre for Enterocci (NRC) to assess the epidemiology of enterococci received from hospital laboratories between 01/01/2009 and 31/03/2016 both on strains isolated from an infection as well as on outbreak strains. Since 2009, the NRC receives increasing numbers of enterococcal strains from hospital laboratories: n=32, 27, 81, 131, 178, 300, 578 and 72 in respectively 2009, 2010, 2011, 2012, 2013, 2014, 2015 and till March 31 2016. The E. faecium % increased from 56.2% to 88.0%. The VRE% increased from 56.3% to 83.3%, in the same period. VanA increased from 27.8% to 98.4%, vanB decreased from 61.1% to 0.8%. All outbreaks except 1 outbreak were caused by E. faecium, with an increase in number between 2009 and 2015; the majority of the outbreak strains were vanA positive. Overall, the most prevalent STs were 80, 117 and 203, each representing more than 1 PFGE-type.

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The majority of VRE outbreaks is caused by E. faecium and E. faecalis, whereas vancomycin-resistant organisms of other species appear only sporadically. Only a few studies described the clonal spread of VRE E. raffinosus so far. In 2015, the Belgian NRC received 2 vanA positive E. raffinosus strains from the same region. Further investigation indicated that both patients resided in the same nursing home. Screening from most exposed contacts was performed and leaded to the identification of an additional case. PFGE-typing indicated clonal spread of a vanA positive E. raffinosus clone. A low level of LZ resistance was detected over the last 2.5 years (2.3%, MIC 8-64) in both E. faecium and E. faecalis. None of the LZ R E. faecalis strains were VRE, 9/11 E. faecium were VRE. All, except 1 E. faecium and 1 E. faecalis strain (MIC 4.0), were sensitive to tigecycline. Rare species are also more often submitted for confirmation eg. E. avium, E. gilvus, E. mundtii, E. raffinosus. In conclusion, since Belgian laboratories for clinical microbiology are not legally bound to submit their VRE strains to the NRC, it is difficult to conclude on the exact epidemiology of VRE and on the number of outbreaks. However, based on the strain submissions in previous years, the NRC received increasing numbers of E. faecium isolates. ST80, 117 and 203 are most frequently identified. E. raffinosus may be an important reservoir of van-genes and may contribute to the dissemination of VRE. Therefore, E. raffinosus should be taken into consideration when implementing control procedures for enterococcal infections. Furthermore, such strains should be submitted to the NRCs for confirmation and surveillance.

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Prof. Dr. Elizaveta Padalko Head of Clinical Virology, University Hospital Ghent [email protected]

Biography Elizaveta Padalko, MD, PhD, is a Head of the Laboratories of Infectious Serology and Molecular Microbiology at Ghent University Hospital. She received medical and doctoral degrees and fulfilled her interneship in Clinical Biology at Catholic University of Leuven and University Hospitals of Leuven. As a part of her academic training, she followed research fellowship at Johns Hopkins Medical Institutions, School of Medicine in Baltimore, USA. She is a member of several national and international organisations active in the field of Clinical Virology. She is a Lecturer in Virology at Faculties of Medicine and Biomedical Sciences at Hasselt and Ghent Universities. Since her PhD degree in the field of antiviral chemotherapy of enteroviral infections, she is mostly interested in the evaluation of methods for diagnostic laboratories with accents on the Women’sHealth including role of infections in assisted reproduction, sexually transmitted infections, viral-based screening methods fo cervical cancer, congenital and neonatal infections.

hpv diagnosis and prevention (1) Approximately 530,000 women worldwide develop cervical cancer and ~275,000 women die from it annually making cervical cancer the 3rd most common cancer in women worldwide; in Belgium is cervical cancer the 8th most frequent cancer in women and the 3rd most frequent gynaecological tumour. Infection with oncogenic (high-risk) human papillomavirus (hrHPV) is a necessary but insufficient event in the aetiology of cervical cancer. Even if we take into account only currently available commercial HPV tests, we’ll find unusually excessive variety of them making HPV the most interesting microbial target for molecular diagnostic companies. Nevetheless there is an enormous choice of diagnostic asssays, HPV tests are one of the least regulated on the market as only one third of them do have documented analytical and/or clinical performance data. There is definitely a positive trend as there is clear improvement in availability of scientific literature in comparison with last 3 years: in 2012 still 75% of tests did not have a single publication in peer-reviewed literature; in 2015 there were 43% of tests fulfilling this description. Particular feature of the molecular HPV testing making it different from the majority of the molecular targets in medical microbiology is that analytical sensitivity for the detection of HPV is not the prime driver of test performance. The majority of currenly available HPV molecular tests have a high analytical

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sensitivity without established clinical cut-offs leading to a substantial yield of clinically insignificant positives with as results a) unneccesary referrals for colposcopy and biopsy; b) decreased correlation with histology and c) increased anxiety in screened women with treatment applied to healthy ones. Methodologically we can differentiate hrHPV DNA tests providing no genotyping, limited/partial genotyping, type- or group-specific genotyping or full genotyping. mRNA hrHPV tests are based on the use of the most relevant transcripts for diganostic purposed encoidng viral oncogenes E6 and E7 and are disigned in order to try to achieve higher specificity that DNA-based testing. Special attention is paid to the self-sampling of cervico-vaginal material for hrHPV as one of the possible solutions for increase in participation rate in the currently available organised screening programs.

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Dr. Béatrice Swennen Hôpital Universitaire des Enfants Reine Fabiola [email protected]

Biography Béatrice Swennen received her MD from the Université Libre de Bruxelles and her M.P.H from the Johns Hopkins University (USA). Since 1989, she is in charge of PROVAC (inter-university group UBL-UCL-Ulg) working for the Vaccination programme of the Fédération Wallonie-Bruxelles (FWB). From 1996 to 2006 she worked as scientific secretary for the Vaccination group of the Superior Health Council and is member of it since 2007. At the Federal level, she is member of the National Certification Committee for the Eradication of Poliomyelitis, Belgium and of the Committee for the elimination of Measles and Rubella. She is also member of the European Society for Paediatric Infectious Diseases (ESPID). She has made contribution in the field of vaccination and public health politics publishing over 40 articles in peer-reviewed and general journals.

HPV diagnosis and prevention (2) HPV vaccines are non-infectious virus like particles vaccines (L1-VLP). Since 2007, two vaccines are licensed in Belgium: bivalent vaccine (bHPV) directed against HPV16 and 18 and quadrivalent vaccine (qHPV) against HPV6,11,16 and 18.both are licensed for women. Only qHPV is also licensed for men. In 2007, the Superior Health Council of Belgium recommended a general immunization programme for adolescent girls between 10 and 13 years. The first goal of the pre-exposure prophylaxis of young women was to reduce incidence and mortality by cervical cancer. From 2007, HPV vaccine were reimbursed by the social security on an individualized base. HPV-vaccination programme for adolescent girls started in September 2010 in Flanders and one year later, in September 2011, in Wallonia (Fédération Wallonie-Bruxelles). Those programmes were based on free of charge delivery system of vaccine to all vaccinators included the scholar medicine. The high efficacy of HPV vaccination reported by RCT was confirmed by population based effectiveness studies up to 4 years after the implementation of HPV vaccination programmes in different settings. In countries with high female vaccination coverage (> 50%) reduction around 60% was described for HPV 16 and 18 and HPV 6 and 11 as well. Cross-protection and herd effects with reductions of HPV 31, 33 and 45 infection in girls younger than 20 years of age and reduction of

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anogenital warts in men and older women.(1). No such effects were described in countries with low coverage. The longest follow-up of an HPV vaccine has been 9,5 years for bHPV and 8 years for qHPV and the protection is confirmed for almost 10 years. Epidemiological data collected during the last ten years have increased the awareness of the burden of HPV diseases in both genders. Specific oncogenic HPV type is associated with nearly all cervical cancers and in many vulvar, vaginal, penile, anal and oropharyngeal cancers. More than 90% of ano-genital warts are associated with HPV6 and 11. (2) In 2015, EMA authorized a nine valent vaccine (9HPV) which should be availabe in Belgium at the end of this year. 9HPV contains HPV 6,11,16,18 and HPV 31,33,45,52 and 53 VLPs and is licensed for women and men. 90% of all cervical cancer may be avoided be 9HPV. It is now time for a paradigm change in the prevention of HPV infection and related disease. HPV vaccination should be broadened to universal vaccination in adolescents (girls and boys) and catch-up programs for women up to 26 years of age and men up to 21y.

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Dr. Chantal Reusken Erasmus MC Rotterdam [email protected]

Biography Dr. Chantal Reusken currently works as senior scientist at the Viroscience department of Erasmus MC. She worked for 9 years at the Centre for Infectious Disease Control of the RIVM, the last three years as head of the unit for rare and emerging viral diseases and laboratory response. Her public health and research activities at the RIVM and Erasmus MC focus on viral zoonoses, in particular rodent-borne and arboviruses. Her research at the Viroscience department mainly involves activities/research focusing on emerging disease preparedness and response and as such she works at the researchdiagnostics interphase. Earlier she worked in the field of Virology at the LUMC, CVI, Leiden University and SUNY Stony Brook on plant viruses, HCV, poliovirus and CSFV.

MERS-coronavirus No abstract available

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Prof. Dr. Steven Callens Universitair Ziekenhuis Gent [email protected]

Biography Prof. Dr. Steven Callens is an internist and infectious disease specialist at the Ghent University Hospital. He started his career as a district medical officer in Homa Bay on the shores of Lake Victoria in Kenya in the late nineties. He specialzed at the University of Leuven and conducted his doctoral research assistant at the Antwerp University on the treatment of children with HIV in Kinshasa, Democratic Republic of Congo. They main interests are tuberculosis, import pathology, travel medicine and vaccinations, as well as general internal medicine clinic and diagnostics.

Clinical approach of uncommon emerging and traveling causes of encephalitis Increased mobitility of both men and vector has increased the changes of rare imported causes of encephalitis in Europe. Furthermore, vectors have profited from global warming and reduced verctor control measures to spread in areas previously free. Invasive mosquito species, as well as disease transmitting ticks have changed the epidemiology of Japanese encephalitis and tick born encephalitis. In addition, some tourist diregard environmental risk, and expose themselves to rodent born diseases, such as lyssa virus and leptospirosis. In this talk, the approach of uncommon, emerging and traveling causes of encephalitis will be explored from a clinical point of view.

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Prof. Dr. Steven Van Gucht Scientific Institute of Public Health, Brussels [email protected]

Biography Steven Van Gucht, Viral Diseases, WIV-ISP

Bats as reservoirs of emerging pathogens Bats are increasingly recognized as important reservoirs of emerging and reemerging viruses, which are highly pathogenic for humans or other mammals. Typical examples are rabies virus (Lyssavirus), Ebola virus and other filoviruses, MERS and SARS coronaviruses and certain types of paramyxoviruses (Hendra, Nipah viruses). Despite the fierce course of infection in other mammals, these viruses tend to cause only mild or subclinical infections in bats. Clearly, for species which come into competition with bats in Africa (Ebola, Marburg), South-East Asia (Nipah, Hendra) or South- and Middle-America (Rabies), contact with bats can have devastating consequences. It is tempting to speculate that the co-evolution of these viruses with bats, offers an evolutionary advantage to bats in the battle between species for food and resources. At least, FAO now recommends not to hunt for bats anymore or consume their meat. So the apparent alliance between bats and their viruses seems to work at the benefit of the reservoir host. In contrast to other mammals, bats seems to be able to harbour these dangerous viruses without developing severe disease. This might be explained by simple co-evolution of viruses and their preferred reservoir host, but some researchers believe that bats are somehow better suited to serve as a reservoir than other species. There are a number of social and behavioural characteristics which facilitate pathogen maintenance between bats. Bats live in dense colonies and maintain close contacts between each other, for example by mutual grooming or sharing food. Some bats migrate over long distances and regularly mix with colonies of different bat species. Still, similar characteristics can also be found in other typical reservoir species, such a rodents and birds. Bats have also acquired some unique physiological and immunological properties, associated with the trait of flight, which may render them more suited to support viral infections, without developing severe disease. Recently, it was discovered that different bat species constitutively express interferon-alpha in their tissues, whereas this typical antiviral cytokine needs to be induced first by virus infection in other mammals. Remarkably, this constitutive expression does

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not induce a hyperinflammatory state in bats, as would be expected in other mammals. With the advent of open genetic detection techniques and the new-found interest in bats, new virus species are being discovered in bats on a regular basis. Most often, there is no reason to assume that these viruses pose a threat to humans. It is not the seemingly high diversity of viruses, this can also be found in other types of colony-forming animals, but the exceptionally pathogenic nature of some of the bat viruses, which is worrying. It is important to point out that each of the important zoonotic events can be narrowed down to one or a few of the more than 1240 bat species, each time under specific circumstances and in specific regions of the world. Until now, in Europe, the role of bats as a source of zoonotic infections seems to be limited to lyssaviruses, with 4 proven cases of bat rabies in humans since the seventies. Considering the useful role of European bats as insect eaters, one bat can eat almost one third of its body weight in insects per night, and their protected status as vulnerable species, the potential risk of bats should not be overemphasized, especially in Europe.

References 1. Backhed F, Ding H, Wang T, et al. The gut microbiota as an environmental factor that regulates fat storage. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(44):15718-23 2. Amar J, Serino M, Lange C, et al. Involvement of tissue bacteria in the onset of diabetes in humans: evidence for a concept. Diabetologia. 2011;54(12):3055-61 3. Vrieze A, Van Nood E, Holleman F, et al. Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome. Gastroenterology. 2012;143(4):913-6 e7

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Dr. Amaya Leunda Scientific Institute of Public Health, Brussels [email protected]

Biography I obtained a PhD in Biomedical Sciences from the Université Catholique de Louvain. Alter working at the University as an Research fellow, I integrated the Biosafety and Biotechnology Unit at the Scientific Institute of Public Health in 2004. First I contributed to the implementation of the National Reference Laboratory for the detection of genetically modified organisms (GMOs) in food and feed in Belgium. Then and until now, I am working as an adviser for the authorities for scientific and technical matters concerning the biosafety in facilities where GMOs and /or pathogens are manipulated.

Biorisk management: current state of play Biorisk is defined by the WHO as being the probability or chance that a particular adverse event, possibly leading to harm, will occur, the hazard being a biological agent. Both biosafety and biosecurity focus on risks related to this hazard, however the perspectives are different. Biosafety (in the context of the contained use of GMOs and/or pathogens) aims at protecting public health and the environment from an accidental exposure to GMOs and pathogens. Biosecurity aims at preventing misuse through loss, theft, diversion or intentional release of pathogens, toxins and any other biological materials. Biosafety is well-defined and regulated in Belgium since 1997 with the implementation of biosafety European Directives at the federal and regional levels. In contrast with some neighbouring EU member states, biosecurity is not yet regulated in Belgium. This communication aims at explaining the current situation of biorisk management in Belgium. Both biosafety and biosecurity will be defined and distinguished by an overview of their emergence and evolution in the course of time.

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Dr. Franz Karcher European Commission, DG Sante

Biography Based in Luxembourg Franz Karcher works for the European Commission, currently on developing an EU health information system aggregating available country knowledge data in support of designing, reviewing and evaluating feasible EU health policies. In his previous assignment he worked on esablishementreview of the EU health security framework that addresses serious cross border threats to health as a principal administrator in charge of generic and pandemic preparedness. He started his work in the Commission with auditing how the EU Member States and third countries implement the EU acquis on animal health and related fields. A graduate of the University of Munich Mr Karcher holds a degree in veterinary medicine. Following post-graduate field work in the Philippines in protozoology he holds a doctorate with a special interest in comparative tropical veterinary medicine. Mr Karcher has worked as a veterinary surgeon and in national veterinary administrations in Germany and overseas before joining the Commission

The new EU framework on health security With the adoption of Decision 1082/2013 /EU on serious cross-border threats to health the EU framework on health security has undergone a major review. Main changes concern: • the enlarged scope which now, apart from biological threats, includes environmental and chemical threats; • enhanced coordination of response through a strengthened mandate of the Health Security Committee (HSC) which has become a high-level body for consultation. Recent changes of the epidemiological situation have underlined the added EU value of the Commission’s role in timely organising and chairing numerous ad hoc HSC audio meetings, in addition to the regular bi-annual plenaries as a driver in assisting Member States in their consultations on coordinated risk management; • a legal basis on preparedness and response planning which obliges Member States to consult within the HSC and to report where they are with national planning, particularly on the collaboration between key and critical sectors, on business continuity and the implementation of the International Health Regulations (IHR, 2005). The Commission works

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with Member States on the basis of means and tools, such as scenario exercises, provided for by the health programme, or country assessments, now under the global governance of the WHO as the recognized hub for the Joint External Evaluation Tool (JEE), an important outcome of the late Lyon conference on global health security. • invited by the Commission 24 Member States now work together on voluntary joint procurement for medical countermeasures; • surveillance has been reinforced and work with the European Centre for Disease Prevention and Control (ECDC) is in progress; progressive surveillance now covers 47 communicable diseases and two special health issues with case definitions in place and further communicable diseases in the pipeline; the Centre compiles data provided by Member States for annual reports. • the Early Warning and Response System (EWRS) has become a confidential and lively tool for notifying relevant changes of the epidemiological situation, interlinking Member States EWRS contact points and the Commission in a timely manner, for example on outbreaks of Zika virus or recent Lassa transmission in the EU, etc. The ECDC’s role in providing rapid risk assessments, not least on Ebola, is increasingly recognised by the EU public health community; • so far the Commission has not made use of the option to recognise a situation of public health emergency for the sake of accelerating marketing authorisation of medical countermeasures in need, as provided for by Article 12 of the Decision in the context of major health events such as Ebola and Zika, since WHO has declared these outbreaks a Public Health Emergency of International Concern (PHEIC).

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In summary, the Commission’s work on implementing the health threats Decision and strengthening the reviewed EU framework on health security is on track, results have been shared with and debated in the European Parliament. Targets are nevertheless moving with new developments since November 2013, when the Decision was adopted. Ebola has been a true test case for the EU Framework, also in the context of reports of delays in response and assistance provided by the International Community. The Commission has, in collaboration with the Council, undertaken an in depth review of the lessons learned in public health on Ebola. In conclusion, there is added value in coordinating risk management but less so in exchange of information within the HSC. Current audit work already provides for ongoing scrutiny which may entail ideas or orientations for further critical reviews, potentially also in the context of the Commission’s work on supporting the Global Health Security Framework.

Dr. Rémy Demeester CHU Charleroi Médecin spécialiste en Médecine Interne, Maladies Infectieuses et Médecine Tropicale [email protected]

Biography Rémy Demeester graduated as Medical Doctor in 2001 and as specialist in Internal Medicine at the Université Libre de Bruxelles in 2006. He followed the post-graduate course of Tropical Medicine in Antwerp in 2002-2003 and thereafter practiced 6 months in Rwanda in an AIDS project of the Luxemburg cooperation. He worked in the hospitalisation ward of the Tropical Institute in the University Hospital of Antwerp from 2006 until 2008 and in the department of Infectious Diseases of the University Hospital St Pierre in Brussels from 2008 until 2010. He was recognized specialist in Infectious diseases in 2009 at the Université Libre de Bruxelles. He works since 2010 in the AIDS reference centre, travel clinic and department of infectious diseases of the University Hospital of Charleroi. He is member of the Management Board of Médecins du Monde Belgium since 2015.

Refugees, asylum seekers, migrants and infectious diseases Europe is experiencing the most severe migrant crisis since World War II. Migrants may be subject to specific risks of infectious diseases in relation to their country of origin, the countries visited during their journey and their conditions of living in Europe (1). The risk for the EU countries of outbreaks of infectious diseases as a consequence of the current influx of migrants is extremely low. But poor living conditions and overcrowding can facilitate the transmission of several infections that may represent a potential threat for the migrants’ health. Therefore screening for tuberculosis and vaccinations are recommended (2). Access to medical care must be guaranteed to migrants whatever their social status (3). Public health objectives, respect of the fundamental rights of the migrants and respect of the medical deontology must be guaranteed (4). This investment would be the best way to ensure a peaceful common future.

References 1. www.ecdc.europa.eu : migrant health: infectious diseases of specific relevance to newly-arrived migrants in the EU/EEA (19 November 2015) 2. www.wiv-isp.be : prise en charge des maladies infectieuses chez les demandeurs d’asile (22 December 2015)

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3. www.assembly.coe.int : Council of Europe: Parliamentary Assembly: Resolution 1946 (2013): Equal access to healthcare 4. www.fra.europa.eu : European Union Agency for Fundamental Rights: monthly data collection on the current migration in the EU: April 2016: thematic focus: healthcare

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Dr. Marjan Van Esbroeck Instituut Tropische Geneeskunde [email protected]

Biography Marjan Van Esbroeck works as a clinical biologist at the Institute of Tropical Medicine. The Institute houses several reference laboratories and national reference centers among which the National Reference Center for arboviruses.

Zika virus in Belgium After its discovery in Uganda in 1947 serosurveys showed that the zika virus (ZIKV) is distributed in Africa and parts of Asia. A first outbreak occurred in the State of Yap (Micronesia) in 2007 followed by outbreaks in French Polynesia and some other Pacific islands in 2013 and 2014 (1). The virus arrived in Brazil in February 2015 from where it spread rapidly over part of the American continent. Species distribution modelling has shown environmental suitability for ZIKV in a large portion of tropical and sub-tropical regions (2). Transmission occurs mainly through the vector Aedes aegypti but Aedes albopictus has the intrinsic ability to transmit the disease as well. Other routes of transmission are from mother to child during pregnancy and delivery and transmission via sexual contact. Transmission via blood transfusion has not been described but is likely to occur (1). The infection goes unnoticed in 80% of the people. If symptoms are present they are usually mild. The most common clinical feature is rash (90%). Fever is not always present (65%). Other common symptoms are arthralgia (65%), non-purulent conjunctivitis (55%) and myalgia (48%) (3). Laboratory diagnosis is, as for other arboviruses, based on RT-PCR in the early viremic phase and serology in the convalescent phase of the disease (4). PCR on serum is only useful in the first 3-5 days although the viremia may be longer in pregnant women (5). PCR on urine allows to extend the diagnostic window for PCR to 10-14 days. IgM antibodies appear 5-7 days after the start of the symptoms, IgG antibodies after 10-14 days. The ELISA currently used for the diagnosis of ZIKV is specific and cross-reactions with other flaviviruses do not seem to pose a big problem (6). In case of doubt a virus neutralization test is available.

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References 1. Petersen LR, Jamieson DJ, Powers AM, Honein MA. Zika Virus. N Engl J Med. 2016 2. Messina JP, Kraemer MU, Brady OJ, Pigott DM, Shearer FM, Weiss DJ, Golding N, Ruktanonchai CW, Gething PW, Cohn E, Brownstein JS, Khan K, Tatem AJ, Jaenisch T, Murray CJ, Marinho F, Scott TW, Hay SI. Mapping global environmental suitability for Zika virus. Elife 2016. 19 (5) 3. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS et al.. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med. 2009;360(24):2536-43 4. http://ecdc.europa.eu/en/publications/Publications/zika-virus-guidance-healthcare-providersand-laboratory-diagnosis.pdf 5. Driggers R, Ho CY, Korhonen EM, Kuivanen S, Jääskeläinen AJ, Smura T et al. Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain Abnormalities. NEJM. 30 March 2016 6. Huzly D, Hanselmann I, Schmidt-Chanasit J, Panning M. High specificity of a novel Zika virus ELISA in European patients after exposure to different flaviviruses. Eurosurveillance. 21 (16). 21 April 2016

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1. Herbrecht R, et al. N Engl J Med. 2002;347(6):408-15. 2. SmPC Vfend® 3. Walsh TJ, et al. Clin Infect Dis. 2008;46(3):327-60. 4. Herbrecht R, et al. Antifungal therapy in leukemia patients. Update ECIL 4. September 6, 2011 slidedeck 5. Pfaller MA, et al. J Clin Microbiol. 2010;48(4):1366-77. 6. Nivoix Y, et al. Clin Infect Dis. 2008;47(9):1176-84. * H.U. : Hospital Use Please consult the included SmPC for information related to the safety of this product

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Abstracts of posters

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HPV DNA DETECTION IN URINE: EFFECT OF A FIRST-VOID URINE COLLECTION DEVICE AND TIME OF COLLECTION Alex Vorsters1, Severien Van Keer1, Samantha Biesmans1, Vanessa Vankerckhoven1,2, Manon de Koeijer2, Koen Beyers2, Margareta Ieven1,3, Pierre Van Damme1 1. University of Antwerp, Vaxinfectio, Belgium 2. Novosanis, Wijnegem, Belgium 3. Antwerp University Hospital, Belgium

Objectives To evaluate the use of a prototype first-void (FV) urine collection device (ColliPee™, Novosanis) and assess the effect of collection time on detection of human and HPV DNA in women. Methods Participants with a self-reported HPV infection were asked to provide eight FV urine samples (four FV first of the day urine samples and four FV evening urine samples) over a period of four days. Two FV urine collection methods were alternated, i.e. the Colli-Pee™ device and a collection set with a pipet for transfer of urine. Both used a preservative pre-filled vial. Human DNA quantification as well as HPV DNA detection, genotyping and quantification were performed on all samples. Results Complete and correctly labelled sample sets from 22 women were analysed. All samples tested positive for 14 women; all eight samples were negative for 4 women. Two women had one out of eight samples negative and one women had six out of eight samples negative. Significant more copies of human DNA were detected in Colli-Pee™ collected urine. Although not significant, a similar effect was observed for HPV DNA. We did find a significant correlation between the amount of HPV DNA and amount of human DNA. No significant differences between first urine of the day samples and samples collected later on the day was observed. Conclusions A FV urine collection device may help to enhance HPV DNA detection in urine, independent whether the FV is taken from first urine of the day or from urine provided later in the day.

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The NRC Norovirus activities since 2011 Nadine Botteldoorn, Sarah Denayer, Katelijne Dierick Scientific Institute of Public Health, Unit Food-borne pathogens, Brussels, Belgium

Keywords Norovirus, Q-PCR detection, Noronet, Norovirus variants Since 2011 the National reference centre of Norovirus at WIV-ISP has collected all available data on Norovirus outbreaks in Belgium by analysing and confirmation of stool and/or food samples collected during outbreaks in healthcare facilities (e.g. hospitals, elderly homes) and Foodborne Outbreaks (FBO’s). The diagnosis of Norovirus is done using a Real-time PCR based method for which an accreditation ISO15189 was obtained in 2012. Epidemiological data collection and analysis are performed on (1) food borne suspected Norovirus outbreaks and on (2) suspected NoV outbreaks reported by the health inspectors and the clinical lab’s. Clinical lab’s performing an antigen based detection of Norovirus, or having samples of an acute gastroenteritis with clinical symptoms of Norovirus send the samples to the NRC for the confirmation of the case by real time PCR detection. The diagnosis helps the general practitioners to have an idea of the transmission routes and to implement specific measures to prevent further spread of the infection. Since the designation as NRC Norovirus our laboratory received more and more samples especially during outbreaks in institutional settings and hospitals. The most important transmission route is from human to human especially during outbreaks in healthcare facilities. During foodborne outbreaks the transmission route is more from a food handler who contaminates the food during preparation or distribution.

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A comparison and evaluation of different real time PCR detection kits was performed. From every positive Norovirus outbreak at least one sample is used for the variant determination of Norovirus. The characterisation (genogroup, genotype and variant) of the strains is done using sequencing of the ORF1 (polymerase region) and ORF2 (capside region) and this allows to exchange information with Noronet. This enables to follow the genetic evolution of noroviruses circulating in Belgium and to compare them with those circulating in Europe. The shift of Norovirus GII.4 New Orleans to GII.4 Sydney was observed in 2013 and in 2015 the new variant GII.17 was also detected in the population.

Development of a pentaplex immunoassay for a seroprevalence study of Diphtheria, Tetanus and Pertussis Raissa Nadège Caboré, Jean-Marc Collard, Kris Huygen Scientific Institute of Public Health, Unit Immunology, Brussels, Belgium

Background Seroprevalence studies can detect subgroups at risk and can therefore deliver information that is often lacking in routine surveillance and vaccination coverage studies. In order to monitor the protection of the general population against Diphtheria, Tetanus and Bordetella pertussis infection in a seroprevalence study in Belgium, we developed a magnetic bead based pentaplex immunoassay (MIA) for the quantification of IgG antibodies against Pertussis Toxin, Filamentous Hemagglutinin, Pertactin, Diphteria toxin and Tetanus toxin in a single well. As compared to classical ELISA, the simultaneous detection of 5 analytes reduces significantly the price of the tests, the volume of serum needed and the workload for the technicians. Methods The five protein antigens were covalently coupled to carboxylated magnetic microspheres at an optimized antigen concentration to obtain five monovalent bead sets. Using mixed beads sets and detection antibodies conjugated to phycoerythrin, we assessed the performance of pentaplex MIA on 37 serum samples and compared the results to those of five commercially available ELISA. The possible interference between the different bead sets was investigated by comparing IgG concentrations generated from the monoplex to the pentaplex MIA. The inhouse reference standard sample was calibrated against the international pertussis (NIBSC code 06-140) in a triplex MIA and against the international Diptheria (NIBSC code 10-262) and Tetanus (NIBSC code TE-3) reference standard in an individual monoplex. Results The pentaplex MIA was reproducible, with good intra and inter-assay coefficient of variation (CV) for all antigens and shown to correlate with the five single ELISA. No evidence for bead interference with various antigen coupled bead regions was found between monoplex and pentaplex MIA. The pentaplex mean fluorescence intensities were comparable to those of monoplex MIA and good correlation between IgG concentrations obtained of both methods for all antigens tested were shown. The recovery of reference standard observed values was between 80 to 120% compared to expected values. A good linearity was demonstrated between the international reference serum and the in-house reference serum. Conclusion The pentaplex immunoassay is validated and used for testing a new serumbank of more than 3000 samples collected from July 2013 to December 2014 through the Belgian sentinel laboratory network.

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Oral fluid for diagnosis of viral infections – application in public health Veronik Hutse, Steven Van Gucht Scientific Institute of Public Health, Unit Viral Diseases, Brussels, Belgium

Classically, the diagnosis of viral diseases occurs by the detection of antibodies/ antigens and nucleic-acids in serum and nasopharyngeal secretions. Oral fluid as a diagnostic sample can however open perspectives for the diagnosis and surveillance of viral infections. Indeed, compared to traditional venepuncture or nasopharyngeal swabbing, the collection of oral fluid is not invasive, less painful, less expensive and safer. The immunoglobulin classes and specificities of antibody found in plasma are similar to those in the gingival crevicular fluid (GCF). GCF is the fraction of oral fluid which transudes from the capillary bed situated beneath the margin between teeth and gum, and it contains the highest concentrations of IgA (1/10), IgM (1/400) and IgG (1/800) relative to serum. The Oracol collection device (Malvern Medical Development, UK) is specifically designed to target the gingival crevice and absorb GCF, providing an optimal sample quality for antibody testing. Although reliable tests to detect anti-HAV, HBsAg and anti-HCV in serum are widely used, assays for testing oral fluid are not commercially available and have to be developed in-house by adapting commercial kits. The past years we have validated and applied several oral fluid assays for the serological and/or molecular detection of measles, mumps, hepatitis A, C and HBsAg. Compared to the gold standard PCR assay on nasopharyngeal secretion, the PCR assay on oral fluid had a sensitivity and specificity of 100% for detection of measles and mumps RNA. Compared to the serum ELISAs, the oral fluid ELISAs for measles IgM, HBsAg, anti-HAV and anti-HCV, had a sensitivity of 92%, 91%, 85% and 89% respectively. A specificity of 100% was obtained for all ELISAs. We have applied oral fluid assays in numerous studies and surveillance programs, leading to at least 9 peer-reviewed publications. The overall good sensitivity and specificity of these oral fluid assays allow oral fluid assays to be used for epidemiological monitoring, prevalence studies for anti-HAV, HBsAg and anti-HCV, screening of possible measles and mumps cases and monitoring outbreaks of measles, mumps or HAV. Nevertheless serum tests remain the gold standard for laboratory diagnosis in individual patients. 48

Time for a change in blood culture sampling: from multiple to unique venipuncture in the emergency department? Marie-Astrid van Dievoet, Caroline Pieters, Anne-Marie Van den Abeele, Dieter Devriese, Jos Van Acker AZ Sint-Lucas, Gent, Belgium

Background The sensitivity of blood cultures (BC) in diagnosing blood stream infections (BSI) increases when collecting a larger volume of blood. The standard method of multiple blood cultures (MBC, 2x2 culture bottles; 2x20 mL) has the drawback of a second venipuncture. Due to time restraints, second venipuncture is sometimes omitted, resulting in a smaller volume of blood taken. Moreover, the threshold to execute two instead of one venipuncture is higher and could consequently result in less blood cultures drawn. We implemented unique blood culture (UBC, 1x4 culture bottles; 1x40mL) venipuncture in the emergency department (ED). In addition, to minimize the BC contamination rate, initial specimen diversion technique (ISDT) was adopted. This technique is based on the hypothesis that skin fragments detached by the needle can be a cause of contamination. Material/methods In October 2014 we implemented both UBC and ISDT in the ED: two BC pairs (BACTEC Plus Aerobic and BACTEC Lytic/10 Anaerobic, Becton-Dickinson, USA) were filled after collection of a serum tube (BD Vacutainer SSTTM, 6 mL, BectonDickinson, USA). All BC bottles were incubated on BD BACTEC FX. We compared a nine-month period before (January-September 2014) and after (JanuarySeptember 2015) implementation. Analysis of differences between proportions in these two periods were studied with the Chi-square test. Results Neither in positive BC ratio (positive BC pairs/total number of BC pairs, before 12.3%, after 11.4%, p=0.43) nor in contamination ratio (contaminated BC pairs/ total numbers of BC pairs, before 4,0%, after 3.1%, p=0.14) significant differences were observed between the two time periods. Contrarily, a significant increase was observed in the number of BC pairs drawn/ED admission (before 15.7%, after 22.0%, p