rologie i - European Congress of Virology

5 th European Congress of VirologyIgG in sera from infected persons was analyzed. The results demonstratethat, although the humoral immune response to WNV in humans is heterogeneous,several dominant peptides are recognized. In addition, the dataindicate that some peptide sequences can be used for the development ofa specific serologic WNV test as they show only marginal cross reactivitywith antibodies against related flaviviruses.REF 425Comparison of workflow and data analysis for the quantificationof Cytomegalovirus (CMV) using an in house method versusQIAsymphony ® RGQ systemTim CONIBEAR, Jennifer MERRIT, Ana KUBLIK, Aysha KHANOM,Claire ATKINSONDepartment of Virology, Royal Free Hospital, London, UNITED KING-DOMObjectives: quantification of Cytomegalovirus (CMV) viral load is animportant tool in the management of CMV infections in immunocompromisedpatients. The new artus CMV QS RGQ integrated assay protocoloffers an automated solution to viral load determination. The workflowand data analysis generated with the new system was compared to an inhouse quantitative PCR method. Methods: following routine diagnostictesting, whole citrated bloods were batched into groups of 19 to facilitateoptimal testing. Eight integrated assays were performed on the QIAsymphonyaccording to the manufacturer’s instructions, utilising full barcodescanning and sample tracking. The time taken for each protocol step wasrecorded by the operator according to a designated protocol. Whole citratedblood aliquots (n=116) were tested prospectively using routine diagnosticsamples tested with the in house assay. In addition, a serial dilutionof the 1st WHO CMV international standard (NIBSC) in triplicate wastested in two separate assays. Data from all RGQ runs was analysed usingthe Rotor Gene AssayManager and compared to the laboratory in housedata. Results: the total time taken to generate a viral load result was notsignificantly reduced (average 9 minutes) however the average user handsoff time increased significantly (by 62 ± 3 minutes) using the QIAsymphonyRGQ system. There was a high level of concordance between inhouse laboratory generated data with those from the QIAsymphony RGQ.Furthermore the data generated by the new system provided results incopies/ml that were equivalent to the international standard in IU/ml (averagedeviation from the mean log10 -0.04). Conclusion: the artus CMVQS-RGQ integrated assay protocol offers an efficient and accurate alternativeto current in-house protocols for quantitation of CMV in wholeblood. The automated extraction and assay setup platform affords the usera significant increase of hands-off time which will effectively reduce thecost per test and therefore be considered during any cost benefit analysis.This study also showed good agreement and correlation of data using bothcurrent in-house protocols and the automated system. In conclusion, theQIAsymphony ® RGQ system is a versatile platform that is able to providesignificant improvements in sample handling efficiency and accuracywithin a routine virology laboratory setting.REF 426Detection of infectious viral particules of the Hepatitis A Virus (HAV)By using a pre treatment in combination with quantitative real time(RT qPCR)Coralie COUDRAY 1 , Audrey FRAISSE 1 , Sandra MARTIN LATIL 1 ,Laurent GUILLIER 2 , Sylvie PERELLE 11 Maisons Alfort Laboratory for food Safety, Food and Water Virology Unit,Maisons Alfort, FRANCE; 2 Maisons Alfort Laboratory for food Safety,Modelling of Bacterial Behaviour Unit, Maisons Alfort, FRANCEHuman enteric viruses are important agents of foodborne diseases. HepatitisA virus (HAV) is a single strand RNA virus belonging to thePicornaviridae family. HAV is responsible for hepatitis around the worldand is transmitted mainly via the faecal oral route, either by personto person contact or by ingestion of contaminated water and food.Because of the absence of a reliable cell culture method, RT qPCRis now widely used for the detection of HAV in food samples. Howeverthis approach detects virus nucleic acids of both infectious and noninfectious viruses, which limits conclusions in terms of public healthconcern. The use of photoinductible molecules like propidium monoazide(PMA) or ethidium monoazide (EMA), combined to PCR basedassays has been yet described in bacteria, parasites and fungi to distinguishviable from non viable particles. Recently, this approach has alsobeen tested in some RNA viruses. The aims of this study were to show1) EMA and PMA binding to HAV RNA under light photoactivation, 2)to test the efficacy of an associating treatment PMA/EMA and surfactantsto distinguish between infectious and non infectious HAV, 3) to usethe most promising treatment based on PMA/EMA/surfactant RT qPCRfor HAV to establish kinetic thermal inactivation curves and 4) finallyattempt to show the influence of the molecular RT qPCR models in theseassays. To conclude, we developed a molecular detection method for theHAV genome by EMA IgepalCA630 RT qPCR which provides a bettercorrelation between infectious titers and molecular titers with thermalinactivation.REF 427Viral agents in the pathogenesis of cardiomyopathyJudit DEAK 1 , Marta HÖGYE 2 , Miklos CSANÁDY 3 , GabriellaTERHES 4 , Beatrix KELE 5 , Robert SEPP 6 , Bela IVÁNYI 71 Department of Clinical Microbiology, University of Szeged, Szeged,HUNGARY; 2 2nd Department of Medicine and Cardiology Center,University of Szeged, Szeged, HUNGARY; 3 2nd Department of Medicineand Cardiology Center, University of Szeged, Szeged, HUNGARY;4 Department of Clinical Microbiology, University of Szeged, Szeged,HUNGARY; 5 Department of Clinical Microbiology, University of Szeged,Szeged, HUNGARY; 6 2nd Department of Medicine and Cardiology Center,University of Szeged, Szeged, HUNGARY; 7 Department of Pathology,University of Szeged, Szeged, HUNGARYParvoiruses are ubiquitous pathogens of humans and animals. QuantitativePCR methods are suggested for the determination of viral nucleic acids indilatative cardiomyopathies (DCMs), and some other cardiac diseases.Certain authors, have detected viral nucleic acids in autopsy samples,without cardiac diseases, at similar rates as in DCM patient samples.In our study in the past 9 years (2004-2012), 93 adult endomyocardialbiopsy samples were received from the 2nd Department of Medicine andCardiology Centre. Samples were prepared on the day of collection. Afternucleic acid isolation from the clinical samples by the Qiagen method,these were frozen or amplified with adenovirus, cytomegalovirus, EpsteinBarr virus, herpes simplex virus 1, herpes simplex virus 2, parvovirus B 19and enterovirus primers. Qualitative RT PCRs, were used; 1 5 PCRs wereperformed on the biopsy samples/patients. 406 PCRs were performed.The clinical samples contained 1 viral nucleic acid each in 20 patients; 2viral nucleic acids were positive in 2 patients, parvovirus nucleic acid in 11patients, CMV in 4 patients, and EBV, enterovirus and HSV 1 in 3 patientseach. Adenovirus and HSV 2 were not detected. Many research articleshave been published on this topic, but there is not yet a validated molecularmicrobiological method. Not only classical cardiotropic enteroviruseshave been detected in DCM, but also a high percentage of members ofthe Herpesviridae family. The possibility of antiviral therapy should betaken, into consideration in the event of DCM of HSV, EBV or CMVorigin.S238 Virologie, Vol 17, supplément 2, septembre 2013