Novel Assays for Detection of Influenza Virus

Study Overview
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Brief Summary
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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.

Detailed Description
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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: Our new RT-PCR assay targeting PB2 gene Our new RT-PCR assay targeting NS gene M gene RT-PCR published by the World Health Organization 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

Official Title
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Evaluation of Novel Molecular Assays for the Detection of Influenza Virus

Conditions
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Influenza A Virus

Intervention / Treatment
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* Diagnostic Test: PB2 gene RT-PCR; NS gene RT-PCR


Participation Criteria
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Eligibility Criteria
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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
Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| NPA positive<br>NPA specimens that are tested positive for influenza A virus by commercially available assay or by the Public Health Laboratory Services Branch of Hong Kong | Diagnostic Test: PB2 gene RT-PCR; NS gene RT-PCR<br>* PB2 gene RT-PCR: RT-PCR targeting the PB2 gene of influenza A virus NS gene RT-PCR: RT-PCR targeting NS gene of influenza A virus<br>|
| NPA negative<br>NPA specimens that are tested negative for influenza A virus by commercially available assay or by the Public Health Laboratory Services Branch of Hong Kong | Diagnostic Test: PB2 gene RT-PCR; NS gene RT-PCR<br>* PB2 gene RT-PCR: RT-PCR targeting the PB2 gene of influenza A virus NS gene RT-PCR: RT-PCR targeting NS gene of influenza A virus<br>|
| Saliva positive<br>Saliva specimens that are tested positive for influenza A virus by commercially available assay or by the Public Health Laboratory Services Branch of Hong Kong | Diagnostic Test: PB2 gene RT-PCR; NS gene RT-PCR<br>* PB2 gene RT-PCR: RT-PCR targeting the PB2 gene of influenza A virus NS gene RT-PCR: RT-PCR targeting NS gene of influenza A virus<br>|
| Saliva negative<br>Saliva specimens that are tested negative for influenza A virus by commercially available assay or by the Public Health Laboratory Services Branch of Hong Kong | Diagnostic Test: PB2 gene RT-PCR; NS gene RT-PCR<br>* PB2 gene RT-PCR: RT-PCR targeting the PB2 gene of influenza A virus NS gene RT-PCR: RT-PCR targeting NS gene of influenza A virus<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| RT-PCR result | The result of RT-PCR can be positive or negative | Through study completion, an average of 2 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Cycle threshold value | The cycle threshold is a surrogate for viral load | Through study completion, an average of 2 months |