Development of a novel bead-based multiplex PCR assay for combined subtyping and oseltamivir resistance genotyping (H275Y) of seasonal and pandemic H1N1 influenza A viruses

BACKGROUND: The identification of influenza A virus subtypes in clinical specimens is becoming increasingly important for clinical laboratories since seasonal H1N1, H3N2 and pandemic H1N1 influenza A viruses can have defined antiviral resistance patterns and subtyping can be used as a surrogate for antiviral resistance testing. OBJECTIVES: To develop a novel multiplex PCR (M-PCR) assay for the combined identification of influenza A subtype and oseltamivir resistance (H275Y) genotype in a combined assay format using Luminex xMAP™ technology. STUDY DESIGN: The M-PCR assay employed five degenerate primers to amplify the hemagglutinin (HA) and neuraminidase (NA) genes and eight tagged primers in a target specific primer extension reaction (TSPE). Products were analysed using xTAG™ beads containing specific anti-tag oligonucleotides. RESULTS: M-PCR correctly identified the subtype for 54/54 specimens that were influenza A positive, including 13/13 seasonal H3N2, 17/17 seasonal H1N1 and 24/24 pandemic H1N1 for both HA and NA genes. For oseltamivir resistance the M-PCR assay correctly identified 41/41 H1N1 viruses as oseltamivir sensitive (H275) or resistant (H275Y). Analysis of sequential specimens from two immunocompromised patients revealed the appearance of the H275Y allele at earlier time points after infection compared with Sanger sequencing. CONCLUSIONS: The combined M-PCR assay correctly subtyped seasonal and pandemic influenza A viruses and accurately detected the H275Y oseltamivir resistance allele. This assay should provide useful information to clinicians for appropriate patient management.