Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory

Pandemic 2009 H1N1 is normally susceptible to oseltamivir, but variants harboring the H275Y (CAC → TAC) mutation exhibit resistance. We describe the use of a combined reverse-transcription polymerase chain reaction (RT-PCR)/pyrosequencing approach to identify the H275 residue. A total of 223 specimens were tested with this method: 216 randomly selected clinical specimens positive for 2009 H1N1 and 7 cell-culture supernatants from the Centers for Disease Control and Prevention (CDC; 4 resistant, 3 susceptible 2009 H1N1 strains). The assay detected H275Y in 1 clinical respiratory sample (0.5%) and all 4 oseltamivir-resistant strains from the CDC; the remaining 215 clinical and 3 susceptible CDC specimens were wild-type. Sanger sequencing confirmed the results for 50 of 50 selected isolates. The RT-PCR/pyrosequencing method was highly specific, producing no amplicons or valid sequences from samples containing non-H1N1 viruses or bacteria. Our findings suggest that this method provides a rapid tool for H275Y detection, with high sensitivity and potential benefit for patient care.