novel assays for detection of influenza virus

Conditions: Influenza A Virus

Summary: 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

Eligibility criteria:
inclusion criteria: * nasopharyngeal or saliva specimens of patients in queen mary hospital of hong kong * tested for influenza a virus using a commercially available assay or by the public health laboratory services branch in hong kong exclusion criteria: * insufficient specimen volume