Novel Assays for Detection of Influenza Virus

Evaluation of Novel Molecular Assays for the Detection of Influenza 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.

Study Overview

• Status: Withdrawn
• Conditions: Influenza A Virus
• Intervention / Treatment: Diagnostic test: PB2 gene RT-PCR; NS gene RT-PCR

Detailed Description

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

• Study Type: Observational

Contacts and Locations

Study Locations

• Hong Kong
  • Hong Kong, Hong Kong
    • Queen Mary Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult; Older Adult
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Non-Probability Sample

Study Population

These archived nasopharyngeal and saliva specimens are collected from patients in Queen Mary Hospital

Description

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

Study Plan

How is the study designed?

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
NPA positive; 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; 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
NPA negative; 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; 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
Saliva positive; 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; 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
Saliva negative; 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; 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

What is the study measuring?

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

Collaborators and Investigators

• Sponsor: The University of Hong Kong
• Investigators: Principal Investigator: Kelvin To, MD, The University of Hong Kong

Study record dates

Study Major Dates

• Study Start (Anticipated): April 18, 2019
• Primary Completion (Anticipated): July 30, 2019
• Study Completion (Anticipated): July 30, 2019

Study Registration Dates

• First Submitted: January 11, 2019
• First Submitted That Met QC Criteria: April 18, 2019
• First Posted (Actual): April 23, 2019

Study Record Updates

• Last Update Posted (Actual): April 25, 2019
• Last Update Submitted That Met QC Criteria: April 23, 2019
• Last Verified: April 1, 2019

More Information

Terms related to this study

• Additional Relevant MeSH Terms: RNA Virus Infections; Virus Diseases; Infections; Respiratory Tract Infections; Respiratory Tract Diseases; Orthomyxoviridae Infections; Influenza, Human
• Other Study ID Numbers: FluA_20190110

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No