Comparison of the novel ResPlex III assay and exis

Authors: M Eggers B Roth B Schweiger M Schmid JP Gregersen M Enders

Publish Date: 2011/10/20

Volume: 31, Issue: 6, Pages: 1257-1265

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Abstract

Influenza virus is a major cause of disease worldwide The accurate detection and further subtyping of influenza A viruses are important for epidemiologic surveillance and subsequent comprehensive characterization of circulating influenza viruses is essential for the selection of an optimal vaccine composition ResPlex III is a new multiplex reverse transcriptase polymerase chain reaction RTPCRbased method for detecting typing and subtyping influenza virus in clinical specimens The ResPlex III assay was compared with other methods with respect to sensitivity and accuracy using 450 clinical specimens obtained from subjects throughout Germany during the 2006–2007 influenza season Samples were analyzed for the presence of influenza virus in MadinDarby canine kidney MDCK cells by rapid cell culture using peroxidase staining and conventional cell culture confirmed by hemagglutination inhibition assay a rapid diagnostic assay Directigen Flu A+B test BD Diagnostic Systems Heidelberg Germany inhouse realtime RTPCR RRTPCR and ResPlex III Qiagen Hilden Germany ResPlex III had the highest sensitivity for detecting influenza virus in clinical specimens followed by inhouse RRTPCR 96 compared with ResPlex III Conventional cell culture in MDCK cells rapid culture and quick test assays were substantially less sensitive 55 72 and 39 respectively Virus subtyping results were identical using ResPlex III and the standard virological subtyping method hemagglutination inhibition ResPlex III is a quick accurate and sensitive assay for detecting and typing influenza A and B viruses and subtyping influenza A viruses in clinical specimens and might be considered for a supplemental role in worldwide seasonal and pandemic influenza surveillanceSeasonal influenza is responsible for a high disease burden worldwide Up to 5–15 of the global population is affected by influenza annually 21 Influenza epidemics result in an average of 294000 hospitalizations and 36000 deaths each year in the US alone 15 16 The best way to prevent influenza is yearly vaccination with seasonal vaccines that are designed to include antigens from three strains recommended by the World Health Organization WHO Virological surveillance to identify predominant circulating influenza virus strains is essential to determine the optimal strains as seasonal influenza vaccine components and to provide information about the possible emergence of new virus strains with pandemic potential 6 If vaccine strains do not closely match the strains causing disease in a given year vaccine efficacy may be reduced with a potentially significant impact on public health 2Reverse transcriptase polymerase chain reaction RTPCRbased methods have replaced viral culture as the reference standard for the detection of influenza viruses 10 12 Results are obtained in 2–4 h 3 and compared to viral culture methods the specificity is improved and the sensitivity is 2–13 higher 22 Due to its speed sensitivity and specificity realtime RTPCR RRTPCR has been recognized as an attractive method of detecting influenza viruses However singleplex realtime onestep RTPCR cannot distinguish type A and B viruses or allow further subtyping of type A viruses Thus different assays have to be used or combined into multiplex PCR assays New technologies that provide a quicker method of typing and subtyping could enhance global surveillance programs and more rapid identification of potential pandemic strains would allow more time to prepare for severe outbreaks of disease These new technologies would need to be at least as sensitive and accurate as existing methods to ensure the continued precision of global surveillance programs Therefore the new ResPlex III assay which detects HA/NA subtypes ie H1 seasonal H2 H3 H5 H7 H9 N1 and N2 and two influenza A or B generic targets of the nonstructural protein NS gene was compared with current diagnostic assays and conventional subtyping by using specific immune sera Molecular assays are valuable tools for the rapid detection typing and subtyping of influenza viruses However global influenza surveillance also requires the isolation of virus in culture to allow for a comprehensive analysis of the antigenic profile of circulating influenza viruses which is important for an optimal vaccine compositionThroat or nasal swabs were obtained from 450 outpatients with acute respiratory symptoms during the 2006–2007 influenza season by officebased physicians mainly pediatricians and general practitioners in the southern region of Germany Samples were collected during calendar weeks 4 through 14 of 2007 and stored in virus transport media for routine diagnosticsRapid test conventional and rapid cell culture with adherent MadinDarby canine kidney MDCK cells nucleic acid extraction and RTPCR were performed at the Laboratory Prof Enders Partner Stuttgart Germany Aliquots of the extracted nucleic acid were sent to Novartis Vaccines Diagnostics GmbH for ResPlex III testing Conventional cell culture isolates were submitted to the National Influenza Reference Center for Influenza for typing/subtyping by hemagglutination inhibition HI In addition RRTPCRnegative but ResPlex IIIpositive clinical specimens were used for the isolation of influenza viruses in MDCK33016PF suspension culture developed by Novartis Vaccines Diagnostics GmbH 9

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