Evaluation of Novel Molecular Assays for the Detection of Influenza Virus
Conditions: Influenza A Virus
Sponsor: The University of Hong Kong

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Protocol Title 
 
Evaluation of novel molecular assays for the detection of 
influenza virus 
 
 
Principal Investigator 
Dr. Kelvin To 
MBBS, FRCPath, FHKCPath, FRCP (Edin), MD 
 
Date: 10th January 2019 
Protocol number: FluA_20190110 

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1. PROTOCOL SYNOPSIS 
 
Protocol title 
 
Evaluation of novel molecular assays for the detection of influenza virus 
Hypothesis 
Our novel molecular assays for the detection of influenza A virus will be 
non-inferior to current molecular assays published by the World Health 
Organization and the US Centers for Disease Control and Prevention 
Primary objective 
 
To evaluate the sensitivity and specificity of different molecular assays 
Clinical specimens 
 
Nasopharyngeal and saliva specimens previously tested for influenza A 
virus 
Study design 
We 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.  
 
Primary outcome 
 
Results of different RT-PCR assays 
 
Secondary outcome 
Cycle threshold (Ct) value of different RT-PCR assays 
 
Data analysis 
Sensitivity, specificity, positive predictive value and negative predictive 
value will be determined 
 
 
2. 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% [1-3]. Swine influenza viruses from 
pigs have also infected humans [4]. 
 
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 [5, 6]. 
 
However, recent studies have shown that mutations in the M gene have led to a reduced 
sensitivity of RT-PCR assay targeting this gene [7, 8]. Therefore, it is important to use 

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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.  
 
3. STUDY OBJECTIVES 
 
1. To evaluate the sensitivity and specificity of different molecular assays 
 
 
4. STUDY DESIGN 
 
4.1. Overall study design 
 
We 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 and specificity will be determined as we described previously [9]. 
 
4.2. Outcome measurements 
 
4.2.1. Primary outcome measure 
1. Results of different RT-PCR assays 
 
4.2.2 Secondary outcome measure:  
1. Cycle threshold (Ct) value of different RT-PCR assays 
 
4.3. Confidentiality of data 
 
The specimens will be tested in an anonymous manner.  
 
4.4. 
Archive of data 
 
The investigator will retain all study documentation pertaining to the conduct of the study at the 
study site for a period of at least 5 years. 
 
4.5. 
Ethical endorsement 
 
The use of archived clinical specimen for microbiological testing has been approved by the 
Institutional Review Board (IRB) of the University of Hong Kong and Hospital Authority. 

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5.  
SELECTION OF CLINICAL SPECIMENS FOR TESTING 
 
5.1.  
Inclusion criteria 
 
1. Nasopharyngeal or saliva specimens of patients in Queen Mary Hospital of Hong Kong 
2. Tested for influenza A virus using a commercially available assay or by the Public Health 
Laboratory Services Branch in Hong Kong 
 
5.2  
Exclusion criteria 
 
1. Insufficient specimen volume 
 
 
6. 
STUDY PROCEDURES 
 
6.1 
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) as we described previously [10]. 
Briefly, 250 μL of each specimen will be mixed with lysing buffer. After extraction, the nucleic 
acids will be recovered using 55 μL of elution buffer.  
 
Monoplex real-time RT-PCR assays for influenza A virus will be performed using QuantiNova 
Probe RT-PCR Kit (QIAGEN, Hilden, Germany). The reagent mixture (20 μL) will contain 1x 
QuantiNova Probe RT-PCR Master Mix, 1x QN Probe RT-Mix, 0.8 μM of each forward and 
reverse primer, 0.2 μM of probe and 5 μL of TNA as the template. The thermal cycling 
conditions will be 10 min at 45 °C for reverse transcription, 5 min at 95 °C for PCR initial 
activation, and 50 cycles of 5 s at 95 °C and 30 s at 55 °C. All reactions will be performed using 
the LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland). The primers and 
probes for the M gene RT-PCR have been published by the WHO and the US CDC [5, 6]. 
 
7. 
STATISTICAL METHODS 
 
7.1 
Sample size 
 
 
We 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 

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- 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 
 
The sample size is based on feasibility of the study 
 
7.2 
Analysis of the study 
 
Sensitivity, specificity, positive predictive value and negative predictive value will be calculated 
for  
1. All specimens 
2. Nasopharyngeal specimens only 
3. Saliva specimens only 
 
 
8. 
REFERENCES 
 
1 
To KK, Chan JF, Chen H, Li L, Yuen KY. The emergence of influenza a h7n9 in human 
beings 16 years after influenza a h5n1: A tale of two cities. Lancet Infect Dis. 2013; 13: 
809-821. 
2 
To KK, Tsang AK, Chan JF, Cheng VC, Chen H, Yuen KY. Emergence in china of 
human disease due to avian influenza a(h10n8)--cause for concern? J Infect. 2014; 68: 
205-215. 
3 
To KK, Ng KH, Que TL, et al. Avian influenza a h5n1 virus: A continuous threat to 
humans. Emerg Microbes Infect. 2012; 1: e25. 
4 
Freidl GS, Meijer A, de Bruin E, et al. Influenza at the animal-human interface: A review 
of the literature for virological evidence of human infection with swine or avian influenza 
viruses other than a(h5n1). Euro Surveill. 2014; 19. 
5 
World Health Organization. Who information for the molecular detection of influenza 
viruses. Available at 
http://www.Who.Int/influenza/gisrs_laboratory/who_information_for_the_molecular_det
ection_of_influenza_viruses_20171023_final.Pdf. Accessed on august 15, 2018. 2017. 
6 
Selvaraju SB, Selvarangan R. Evaluation of three influenza a and b real-time reverse 
transcription-pcr assays and a new 2009 h1n1 assay for detection of influenza viruses. J 
Clin Microbiol. 2010; 48: 3870-3875. 
7 
Stellrecht KA. History of matrix genes mutations within pcr target regions among 
circulating influenza h3n2 clades over ten-plus-years. J Clin Virol. 2018; 107: 11-18. 
8 
Yang JR, Kuo CY, Huang HY, et al. Newly emerging mutations in the matrix genes of 
the human influenza a(h1n1)pdm09 and a(h3n2) viruses reduce the detection sensitivity 
of real-time reverse transcription-pcr. J Clin Microbiol. 2014; 52: 76-82. 
9 
To KK, Yip CC, Lai CY, et al. Saliva as a diagnostic specimen for testing respiratory 
virus by a point-of-care molecular assay: A diagnostic validity study. Clin Microbiol 
Infect. 2018. 

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10 
To KK, Lu L, Yip CC, et al. Additional molecular testing of saliva specimens improves 
the detection of respiratory viruses. Emerg Microbes Infect. 2017; 6: e49.