rologie i - European Congress of Virology

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5 th European Congress of VirologyConclusion: Our method is very specific and allows detection of co infectionswhile commercial methods are more sensitive but failed to detect coinfections. The accuracy of our method was confirmed by the identificationof expected HBV genotypes (A,D: most frequent genotypes in Europe) inpatient and in QCMD samples.REF 473An exercise in instrument consolidation and redundancy in atertiary teaching hospital: Comparative evaluation of Diasorin Liaison/LiaisonXL Hepatitis B Serology AssaysCraig LEEMAN, Elizabeth MARLAND, Elizabeth GALVIN, SpirosREPOUSIS, Jane CORNWALLSydPath St Vincent’s Hospital, Darlinghurst AUSTRALIASt Vincent’s Hospital is one of Australia’s leading tertiary teaching hospitalsand recognised as a centre for excellence in clinical care, research andtraining with specialty areas including transplantation and HIV/AIDs care.The need to streamline pathology services by centralisation or consolidationaffects laboratories of all sizes. From 2011 to 2013 a project has beenundertaken within SydPath to reduce test platforms and subsequent costswhile remaining compliant with ISO and cGMP requirements for redundancyfor provision of donor services. The solution sought was to increasethroughput and capacity while eliminating restrictive batch testing of traditionalELISA technology without compromising result quality and patientcare. Stage one realized automation and consolidation of EBV, HSV, Parvo,MMV, Toxo and Hep A testing to LIAISON/LIAISON XL. Stage twoprovides consolidation and redundancy for Hepatitis B testing. Aim: Toevaluate LIAISON/LIAISON XL murex Hepatitis B CLIA assays vs currentAbbott Architect CMIA and BioMerieux VIDAS ELFA. Method: 150characterised retrospective and prospective samples tested for HBsAg, sAgneut, HBeAg/Ab, HBc IgM to determine concordance/serostatus; PelispyT38 & T17, Bio Rad Virotrol III & Viroclear assessed limit of detection,linearity and precision; HBsAg mutant panel challenged mutant detectioncapability. Results: Excellent concordance. Superior HBsAg mutantdetection capability demonstrated by LIAISON XL murex HBsAg Quantassay. Throughput, capacity and consolidation were achieved improvingpatient care and clinical outcomes.REF 474Reconsidering primary HPV testing in cervical cancer screeningCarlo LIVERANI 1,21 Fondazione IRCCS, Ospedale Maggiore Policlinico, Milano, ITALY;2 University of Milan, Milano, ITALYInappropriate testing for HPV types on healthy subjects increases costswithout benefit and potentially results in overtreatment (1). HPV testingalso has a negative psychosocial impact on women, increasing anxiety,stress, and concerns on sexual relationships (2,3). Giving the fact thatrecently HPV testing has been shown to have similar sensitivity but moreoverdiagnosis than cytology (4), and also giving the fact that false negativeresults may be higher than previously suspected (5), primary screeningwith HPV tests in European countries should be reconsidered. Resourcessaved in molecular testing may well be addressed in implementing vaccinationstrategies which are still underused, and may possibly includemales as well as women (6,7).References1. Solomon D, et al. Statement on HPV DNA Test Utilization. Acta Cytol2009; 53 (3): 247-8.2. Rosen NO, et al. The impact of intolerance of uncertainty on anxietyafter receiving an informational intervention about HPV: a randomisedcontrolled study. Psychol Health 2010; 25 (6): 651-68.3. Kwan TT, et al. Psychological burden of testing positive for highrisk human papillomavirus on women with atypical cervical cytology:a prospective study. Acta Obstet Gynecol Scand 2011; 90 (5):445-51.4. Malila N, et al. The HPV test has similar sensitivity but more overdiagnosisthan the Pap test A randomised health services study on cervicalcancer screening in Finland. Int J Cancer 2012 Sep 18. doi: 10.1002/ijc.2785.5. Liverani CA, et al. High risk HPV DNA subtypes and E6/E7 mRNAexpression in a cohort of colposcopy patients from Northern Italy withhigh grade histologically verified cervical lesions. Am J Transl Res 2012;4 (4): 452-7.6. Oscarsson MG, et al. Young women’s decision making process for HPVvaccination. Sex Reprod Healthc 2012; 3 (4): 141-6.7. Jin XW, et al. Human papillomavirus vaccine: Safe, effective, underused.Cleve Clin J Med 2013; 80 (1): 49-60.REF 475Development of a new diagnostic tool for the detection and quantificationof Parvovirus B19 by Real Time PCRPatricia MARECHAL, Gwendoline FOUCAUD, Stéphane MAGRO,Come BARRANGERBioMérieux, Verniolle, FRANCEObjectives: Parvovirus B19 is an important human pathogen causing avariety of diseases with outcomes ranging from asymptomatic to severe,such as chronic anemia immunocompromised patients or fetal hydrops anddeath after maternal infection. The bioMérieux Parvovirus B19 R gene ®real time PCR assays allows to quantify the three B19 genotypes on themajor extraction and real time PCR platforms. The results expressed incopies/mL can be converted to international units by using a conversionfactor determined on the basis of the 2nd WHO International Standard forParvovirus B19.Methods: Viral DNA was extracted from 200 L of whole blood orplasma on NucliSENS ® easyMAG TM and eluted in 50 L. 10 L ofpurified nucleic acids were added to 15 L of ready to use amplificationpremix. An Internal control added before extraction step was usedas extraction/inhibition control. Parvovirus B19 and internal control wererespectively detected at 530 nm and 560 nm on ABI 7500 Fast.Results: On the Parvovirus B19 QCMD Panel 2012, 100% (8/8) of thesamples were correctly identified and quantified. Analytical sensitivitystudies performed in whole blood and plasma specimens showed a highlevel of sensitivity.Coefficient of variations below 3% were observed for the intra/inter assayreproducibility studies carried out on the 2nd WHO IS Parvovirus B19diluted in whole blood negative samples.Conclusion: The high quality associated with its compatibility with themajor extraction and real time PCR platforms allows immediate integrationof this tool into most routine diagnostic laboratories.REF 476Does the HCV INNO LIPA 2.0 assay correctly assigns genotype 4?Camelia MOKHTARI 1 , Arielle ROSENBERG 2 , Stephanie HAIMBOUKOBZA 1 , Eric MARCHADIER 1 , Anne Marie ROQUE AFONSO 11 Virologie,AP HP Hôpital Paul Brousse, Villejuif, 94800, France;2 Virologie,AP HP Hôpital Cochin, Paris, 75006, FRANCEThe strategy used to treat HCV infection depends on the viral genotype particularlywith the upcoming direct antiviral agents. For therapeutic trials,European medicine authorities recommend the use of NS5B sequencingor the reverse hybridization assay Inno Lipa 2.0, based on the simultaneousdetection of 5’UTR and Core regions. However, one of our patients,Virologie, Vol 17, supplément 2, septembre 2013S251
5 th European Congress of Virologyinfected by HCV genotype 4r, as determined by NS5B sequencing, wasdenied inclusion in a genotype 4 dedicated trial because the centralizedscreening method (InnoLipa) gave an undetermined result. To study theability of the Inno Lipa assay to correctly assign genotype 4 for this particularsubtype, we tested retrospectively all genotype 4r samples identifiedat our center. From 2008 to 2012, 20 patients (2%) infected by genotype 4rwere identified. InnoLipa assigned genotype 4e in 10 samples, as determinedby the band pattern 1,2,5,16,23. An undetermined result was obtainedfor the remaining 10 samples: a pattern showing an additional band on line3 was observed in 8 cases, an additional reactivity on line 9 was observedin one case and the missing of line 16 was observed in one case. In conclusion,although Versant HCV genotype 2.0 assay is easiest to implement inroutine genotyping, NS5B sequencing must remain the reference method,particularly for therapeutic trials.REF 477Evaluation of a rapid test device for the detection of hepatitis C virusinfection at the point of first contactSandeep RAMALINGAM 1 , Mina OHARA 2 , Jacky SHAW 2 , PeterMCCULLOCH 1 , Clifford LEEN 31 Department of Microbiology, Royal Infirmary of Edinburgh, Edinburgh,SCOTLAND; 2 Substance Misuse Directorate, NHS Lothian, Edijnburgh,SCOTLAND; 3 Department of Infectious Diseases, Western General Hospital,Edinburgh, SCOTLANDIntroduction: Diagnosis of hepatitis C virus (HCV) infection is primarilylaboratory based. In high risk groups, a proportion would not return forresults, thereby reducing the effectiveness of intervention projects.Objectives: To determine if a rapid test device for HCV could be used in theclinic and if its performance characteristics allowed its safe introductioninto clinical practice.Methods: Individuals attending the Substance Misuse Directorate forHCV screening were consented. Samples were tested by ImmunoFlowHCV (IFHCV) prior to being sent to the laboratory for routine testing.Difficulty in processing specimen or reading results were noted alongwith whether participants wished to know the result immediately if thetest was introduced.Results: All 135 individuals tested expressed their desire for knowingresults immediately. The sensitivity, specificity, positive and negative predictivevalues for diagnosing antibody status and active HCV infectionswere 75%,99%,96%,93%; and 100%,96%,86%, 100% respectively. Therewere three acute HCV infections, all of whom were detected by IFHCV.Conclusions: IFHCV was cheap and easy to use in the clinic.ThoughIFHCV was less sensitive in detecting HCV antibody, the assay had goodperformance characteristics in identifying active HCV infections. IFHCVcould hence be used to give a preliminary result and organize furtherappointments at the first clinic visit whilst waiting for confirmation ofresults from the laboratory.REF 478Towards standardization of HDV RNA quantificationRoland SAHLI 1 , Christopher DOERIG 2 , Christine ESTRADE 1 , AmalioTELENTI 1 , Darius MORADPOUR 21 Institute of Microbiology, Centre Hospitalier Universitaire Vaudois andUniversity of Lausanne, Lausanne, SWITZERLAND; 2 Division of Gastroenterologyand Hepatology, Centre Hospitalier Universitaire Vaudoisand University of Lausanne, Lausanne, SWITZERLANDChronic infection by hepatitis D virus (HDV) is a severe complication inpatients with hepatitis B. HDV RNA quantification in plasma is importantto confirm the diagnosis of chronic hepatitis D and to monitor the responseto treatment. HDV RNA quantification has been difficult to standardize.We explored the contribution of RNA extraction, cDNA synthesis andPCR assays to viral load determination. Two RNA extraction methods, 3RNA melting temperatures prior to cDNA synthesis, and 3 PCR assays(HDVL, Le Gal et al., 2005; HDVR, in house multiplex assay; HDVG,Ferns et al., 2012) were assessed in clinical samples and international qualitycontrol samples. HDV RNA quantification was influenced mainly byviral sequences and PCR assay. HDVL underestimated loads by 5 foldcompared to HDVR. However, HDVR was susceptible to sequence polymorphismsin a low percentage of genotype 1 strains downstream the 3’ endof the forward primers binding site, resulting in 100 fold lower viral loadestimates relative to HDVL or HDVG. HDVG offered the best compromisebetween viral load estimates and independence to viral sequence variation.In addition, fast thermocycling diminished the PCR assay dependent readoutand an endogenous extraction control could correct for inhibitors.Selecting PCR assays with fast thermocycling and with primers havingtheir 3’ end near loop structures of the cDNA target may allow for optimalstandardization of HDV RNA quantification. Definition of an internationalstandard would be required to allow for comparison of HDV RNA resultsobtained in different laboratories.REF 479Comparison of two immunoblot assays for the confirmation of hepatitisC antibodiesHelvi Holm SAMDAL 1 , Vethanayaki SRIRANGANATHAN 1 , RagnhildVassbø EID 1 , Bente HAGH 2 , Svein Arne NORDBØ 21 Department of Microbiology, Oslo University Hospital, Ullevål, Oslo,NORWAY; 2 Department of Medical Microbiology, Trondheim UniversityHospital, Trondheim, NORWAYObjective: to compare recomLine HCV IgG (Mikrogen) and Chiron ®RIBA ® HCV 3.0 SIA (Novartis) in verifying repeatedly reactive resultsobtained in primary HCV antibody testing. Methods: Sera from 13 blooddonors and 78 patients, reactive in Abbott Architect Anti HCV test, wereanalyzed by Chiron ® RIBA ® SIA HCV 3.0 containing NS5 and c33crecombinant proteins and c100p, 511pandc22p synthetic peptides, andrecomLine HCV IgG containing Core1, Core2, Helicase, NS3, NS4 andNS5 recombinant proteins. Interpretation was done according to manufacturersguidelines. Samples from all blood donors and 51 patients were alsoanalyzed by PCR (In house and CAP/CTM (Roche). Results: the resultswere in accordance for 59 sera. Seven sera were RIBA positive, but indeterminateor negative in recomLine. Follow up gave no indication of HCVinfection, including negative PCR results. Three sera were RIBA indeterminateand recomLine positive. PCR analyses were positive in two of thesera, which reacted strongly against RIBA c22p. The difference is due todifferent antigen representation and interpretation criteria. Seventeen sera,indeterminate in RIBA (mainly reactive against c33c), were negative inrecomLine and PCR analyses. Five sera, negative in RIBA and PCR, wereindeterminate in recomLine. Conclusion: in our study, recomLine reducedthe number of indeterminate results compared to RIBA. For three patientsrecomLine was more sensitive in detecting HCV infection. RecomLineseems well suited for confirmation testing for HCV antibodies.REF 480Evaluation of a novel real time PCR assay for Parvovirus B19 (B19 V)genome detection and quantificationAurélie SCHNURIGER 1,2 , Kenda SALOUM 1 , Yanne MICHEL 1 ,Valérie MARINHO 1 , Isabelle ANSART 1 , Antoine GARBARGCHENON 1,21 APHP Trousseau hospital, Virology Department, Paris, FRANCE; 2 ERLINSERM U1057/UMR7203, UPMC University Paris 6, Paris, FRANCES252 Virologie, Vol 17, supplément 2, septembre 2013
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Contents5 th European Congress of V
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i r o l o g i eList of Keynote Spea
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i r o l o g i eList of Chairpersons
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i r o l o g i eWelcomeWelcome to th
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i r o l o g i eExhibitorsThe indust
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i r o l o g i eFacilities for the 5
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i r o l o g i eGeneral Information
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PROGRAMME AT A GLANCEWednesday 11th
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