Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates

During the 2007-2008 influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza A H1N1 isolates. Although oseltamivir resistance rates vary from region to region, 16% of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase gene of influenza A. In order to implement effective resistance testing locally a novel real-time reverse-transcriptase PCR (RT-PCR) assay was developed for the detection of the H275Y mutation. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. In comparison to Sanger sequencing, the sensitivity and specificity of the H275Y RT-PCR assay were 100% (40/40) and 100% (61/61) respectively, while the sensitivity and specificity of pyrosequencing were 100% (40/40) and 97.5% (60/61) respectively. Although all three methods were effective in detecting the H275Y mutation associated with oseltamivir resistance, the H275Y RT-PCR assay was the most rapid and could easily be incorporated into an influenza subtyping protocol.