rologie i - European Congress of Virology

5 th European Congress of Virologyversatile and compact LHS allows automation of PCR set up, improvesthroughput and reliability. It can easily be integrated in any laboratoryworkflow to link extraction and amplification steps.REF 455Transmission electron microscopic quantification of viruses grown incell culturesHana MALENOVSKÁVeterinary Research Institute, Brno, CZECH REPUBLICInfectious titration of viruses is laborious and alternative protocols forquantification of viruses are needed. In this study, suspensions of Canineadenovirus 1 (CAdV 1), Feline calicivirus (FCV) and Bovine herpesvirus1 (BoHV 1) were quantified comparatively by transmission electronmicroscopy (TEM) and TCID50 to investigate their correlation. Each ofthe viruses was grown in five replicates until complete cytopathology wasrecorded (time 0), then frozen. In order to evaluate the influence of timingon virus harvest, they were left for additional periods of 16, 32 and 48 hoursat 37 ◦ C. At each time point, the infectious ability was characterised byTCID50 and the number of virions quantified by TEM. The virus particlecount determined by TEM did not change for any of the viruses throughoutthe experiment. The relationship between virus particle counts withTCID50 at time 0 showed good linearity response; their ratio was almostconstant. The proportion of non infectious particles did not change throughoutthe experiment for either CAdV 1 or BoHV 1. However, a decreasein virus infectious ability disclosed by TCID50 indicated that the fractionof non infectious particles in FCV increased 300 000 times when time0 and 48 hours were compared. In TEM quantification account must betaken of harvesting time as virus counts need not necessarily correlate withvirus infectious ability.Supported by the Ministry of Agriculture and the Ministry of Education,Youth and Sports of the Czech Republic (Grant no. MZe 0002716202,AdmireVet; Grant No. CZ.1.05/2.1.00/01.0006 ED 0006/01/01).REF 456A portable point-of-entrance system for the detection of lumpy skindisease virus using recombinase polymerase amplification assayAyman EL DEEB 3 , Ahmed ABD EL WAHED 1 , Mohamed ELTHOLOTH 2 , Mohamed SHALABY 3 , Frank HUFERT 1 , ManfredWEIDMANN 11 Institute of Virology, University Medical Center, Goettingen, GER-MANY; 2 Virology Department, Mansoura University, Mansoura, EGYPT;3 Virology Department, Cairo University, Giza, EGYPTLumpy skin disease is viral disease of cattle characterized by fever, followedshortly by the development of nodular lesions in the skin thatsubsequently undergo necrosis. Early diagnosis of its infectious agentshelps to diminish their impact by adequate outbreak management. Samplescollected from animals in the field or at quarantine stations are sent longdistances to the laboratory for PCR analysis because portable PCR isneither available nor suitable for on-site screening. The recombinase polymeraseamplifications (RPA) assay is an isothermal DNA amplification anddetection technology. In contrast to PCR, RPA is performed at a single temperature(42 ◦ C) and yield a result after only 5-15 minutes. In this study,we describe the development of a real-time RPA assay for the detection oflumpy skin virus (LSDV). Molecular DNA standards representing a part ofthe GPCR gene of LSDV were prepared. The assay sensitivity was determinedby probit analysis of eight assay runs of serial dilutions of the molecularstandard. The assay specificity was evaluated against a panel of virusesconsidered for differential diagnosis with LSDV. The assays were validatedusing clinical samples from 22 LSDV-infected cattle. The LSDV wasrapid (15 minutes) and showed an analytical sensitivity of 179 moleculesdetected. No cross reactivity with other viruses causing similar clinicalpictures were observed. In comparison to real-time PCR, LSDV RPAssensitivity was 100%. In conclusion, LSDV RPA yielded similar resultsas the corresponding PCR assays, but RPA were quicker and much easierto handle in the field. Thus RPA could be easily implemented to performdiagnostics at quarantine stations or farms for rapid on-site viral detection.REF 457Using EQA results to better understand the observed variation withinquantitative molecular testing and support standardisationCaterina DI LORENZO 1 , Paul S. WALLACE 1 , Hubert G.M.NIESTERS 2 , Anton M. VAN LOON 31 QCMD, Glasgow, UNITED KINGDOM; 2 University Medical Centre,Groningen, THE NETHERLANDS; 3 University Medical Centre, Utrecht,THE NETHERLANDSMolecular technologies have been the mainstay of infectious disease diagnosticsfor over twenty years. In addition, over the last decade, quantitationand the determination of pathogen load has become a core diagnosticparameter. For some pathogens such as HIV 1 and HCV, there is a clearrelationship between disease status and pathogen load. In addition, thedevelopment of international standards and the improved 2nd and 3rdgeneration commercial molecular assays available in this area have helpedconsiderably in improving test reproducibility within the laboratory,which in turn helps facilitate comparison of results across laboratories. Incontrast, for many other pathogens now routinely tested, the relationshipbetween pathogen load and disease status still remains unclear. For thesepathogens there are often not many commercial assays available. There area wide range of in house developed assays utilising different extraction andamplification methods and there is also a lack of suitable reference materialor international standards. This makes it difficult to compare quantitativeresults from laboratory to laboratory. Using the EQA data obtained over thelast decade, analysis of the overall variation observed within the reportingof quantitative EQA data from a range of EQA programmes will be made.A comparison between the variation observed within programmes wherean International Standard in known to be available, where a Standard hasonly just been introduced and also where no International Standards arepresent will be performed.REF 458Using proficiency testing programmes as a tool to assess and improvediagnostic methods for infectious diseasesCaterina DI LORENZO 1 , Paul S. WALLACE 1 , HubertG.M. NIESTERS 2 , Anton M. VAN LOON 31 QCMD, Glasgow, UNITED KINGDOM; 2 University Medical Centre,Groningen, THE NETHERLANDS; 3 University Medical Centre, Utrecht,THE NETHERLANDSProficiency testing is an essential tool used by laboratories to evaluate theperformance of existing and evolving molecular diagnostic technologies.The rapid evolution of molecular based diagnostic techniques and theever increasing number of clinically relevant pathogens, pose significantchallenges for proficiency testing providers as testing programmes mustcontinually evolve to meet the requirements of today’s laboratories.Quality Control for Molecular Diagnostics (QCMD) is responsible forthe coordination of international proficiency testing (PT) programmesand has kept at the forefront by adding new PT programmes eachyear covering a wide range of emerging and re emerging pathogens.Data from over 1500 laboratories in more than 100 countries using awide range of molecular diagnostics methods were collected and theresults were analysed to determine trends in the use and performanceof molecular diagnostics for a wide range of human pathogens and toevaluate laboratory performance over time. Results on QCMD PT panelsshowed that laboratories have improved precision of their (quantitative)assays in recent years. Furthermore the percentage of false positive resultsS246 Virologie, Vol 17, supplément 2, septembre 2013