Using the full spectral capacity (six channels) of a real‐time PCR instrument can simplify diagnostic laboratory screening and typing protocols for pandemic H1N1 influenza

Please cite this paper as: Hopkins et al. (2011) Using the full spectral capacity (six channels) of a real‐time PCR instrument can simplify diagnostic laboratory screening and typing protocols for pandemic H1N1 influenza. Influenza and Other Respiratory Viruses 5(2), 110–114. Background Timely reporting of influenza A virus subtype affects patient management. Real‐time PCR is a rapid and sensitive method routinely used to characterise viral nucleic acid, but the full spectral capability of the instruments is not employed. Objectives To evaluate a hexaplex real‐time PCR assay (Flu‐6plx assay) capable of detecting influenza A and B, hMPV, respiratory syncytial virus (RSV) and distinguishing 2008 ‘human’ influenza A/H1 from 2009 pandemic A/H1 subtypes. Methods Respiratory specimens (n = 213) were tested using the Flu‐6plx assay and a further four monoplex PCRs targeting hMPV, RSV, influenza A and B. The FDA‐approved ProFlu ST test was used to validate the Flu‐6plx PCR influenza A/H1 subtyping components. Discrepant 2009 pandemic A/H1 results were further tested using the CDC swine H1 assay. Results The Flu‐6plx assay had excellent sensitivity identifying 106/106 influenza A RNA–positive samples. The ProFlu ST test was a less sensitive subtyping test, and discrepant analysis could not confirm A/H1 status for four samples resulting in Flu‐6plx PCR specificities of 98% and 95% for human A/H1 and 2009 pandemic A/H1, respectively. Co‐infection affected the sensitivity of the Flu‐6plx PCR hMPV component whereby low‐level hMPV RNA could be masked by much higher concentrations of influenza A virus RNA. Conclusions The Flu‐6plx assay is a sensitive and specific test for the universal detection of influenza A infection and determination of A/H1 subtype. Concomitant detection of influenza B, hMPV and RSV demonstrates the utility of hexaplex real‐time PCRs in viral diagnostics.