Novel Assays for Detection of Influenza Virus

Influenza A Virus

Seasonal influenza virus causes an estimated 0.3-0.6 million deaths per year. Avian influenza virus H5N1, H7N9 and H5N6 has fatality rate of over 30%. Swine influenza viruses from pigs have also infected humans. Molecular assays are now used routinely in the detection of influenza viruses. The M gene is often used as the target for all influenza A viruses because the nucleotide sequence of this gene is relatively conserved among all the influenza A viruses. The World Health Organization and the US Centers for Disease Control and Prevention (CDC) have published protocols for molecular detection of influenza A virus M gene. However, recent studies have shown that mutations in the M gene have led to a reduced sensitivity of RT-PCR assay targeting this gene. Therefore, it is important to use alternative conserved genes as the target of RT-PCR. In this study, our aim is to evaluate two new RT-PCR assays that are based on PB2 and NS gene segment.

I. Background * Seasonal influenza virus causes an estimated 0.3-0.6 million deaths per year. Avian influenza virus H5N1, H7N9 and H5N6 has fatality rate of over 30%. Swine influenza viruses from pigs have also infected humans. * Molecular assays are now used routinely in the detection of influenza viruses. The M gene is often used as the target for all influenza A viruses because the nucleotide sequence of this gene is relatively conserved among all the influenza A viruses. The World Health Organization and the US Centers for Disease Control and Prevention (CDC) have published protocols for molecular detection of influenza A virus M gene. * However, recent studies have shown that mutations in the M gene have led to a reduced sensitivity of RT-PCR assay targeting this gene. Therefore, it is important to use alternative conserved genes as the target of RT-PCR. In this study, our aim is to evaluate two new RT-PCR assays that are based on PB2 and NS gene segment II. Study objective -To evaluate the sensitivity and specificity of 2 new RT-PCR assays III. Overall study design * The investigators will randomly retrieve archived nasopharyngeal and saliva specimens that were previously tested for influenza A virus using commercially available assays in our laboratory, tested for influenza A virus at the Public Health Laboratory Service Branch in Hong Kong. These specimens will be tested for influenza A virus by 4 different RT-PCR assays as listed below: 1. Our new RT-PCR assay targeting PB2 gene 2. Our new RT-PCR assay targeting NS gene 3. M gene RT-PCR published by the World Health Organization 4. M gene RT-PCR published by the US CDC Sensitivity, specificity, positive predictive value and negative predictive value will be determined. IV. Nucleic acid extraction and real-time reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus * Saliva and nasopharyngeal specimens will be subjected to total nucleic acid (TNA) extraction by NucliSENS easyMAG (BioMerieux, Boxtel, Netherlands). * Monoplex real-time RT-PCR assays for influenza A virus will be performed. The primers and probes for the M gene RT-PCR have been published by the WHO and the US CDC. V. Sample size: * The investigators will perform all 4 RT-PCR assays on a total of 320 specimens, including * 80 nasopharyngeal specimens which tested positive for influenza A by commercially-available molecular assays or by testing performed at the Public Health Laboratory Services Branch in Hong Kong * 80 nasopharyngeal specimens which tested negative for influenza A by commercially-available molecular assays or by testing performed at the Public Health Laboratory Services Branch in Hong Kong * 80 saliva specimens which tested positive for influenza A by commercially-available molecular assays * 80 saliva specimens which tested negative for influenza A by commercially-available molecular assays

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