A comparison of pyrosequencing and neuraminidase inhibition assays for the detection of oseltamivir-resistant pandemic influenza A(H1N1) 2009 viruses

Currently most pandemic influenza A(H1N1) 2009 (H1N1pdm) viruses are sensitive to oseltamivir, but a single point mutation (H275Y) in the neuraminidase (NA) gene of H1N1pdm can lead to resistance and such viruses have been reported from several countries. In this study we compare the performance of a pyrosequencing-based method for the detection of the H275Y mutation in H1N1pdm viruses with a conventional NA inhibition assay. Pyrosequencing could detect as little as 5% H275Y mutants in a mixed viral population, while mixtures with 25% or greater mutant virus were required before a significant increase in IC50 value could be detected. However, the sensitivity of the NA inhibition assay could be enhanced by using a more sophisticated curve-fitting analysis to generate similar results to the pyrosequencing assay. Of 181 H1N1pdm clinical samples examined by pyrosequencing, nine samples from five patients were found to contain H275Y mutant viruses, four of whom were under oseltamivir treatment. Changes in the ratio of H275Y mutant to wild-type viruses were observed in serial clinical specimens from two patients over the duration of their treatment. This study highlights the need for close monitoring of the H275Y mutation in clinical samples, in particular from severely ill patients infected with H1N1pdm. The use of pyrosequencing and the NA inhibition assay provide powerful tools for the rapid detection and quantitation of resistant influenza viruses in mixed populations.