title: 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.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Liverpool Specialist Virology Centre, Royal Liverpool University Hospital, Liverpool, UK. m.hopkins@liv.ac.uk
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Hopkins
authors.forename: Jay F
authors.initials: JF
authors.lastname: Moorcroft
authors.forename: Jailson B
authors.initials: JB
authors.lastname: Correia
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Hart
mesh_terms: D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D058873:Pandemics; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D012680:Sensitivity and Specificity