Monitoring the susceptibility to oseltamivir of Influenza A(H1N1) 2009 virus by nested-PCR and pyrosequencing during the pandemic and in the season 2010-2011.

For the early detection of the H275Y mutation as a marker of oseltamivir resistance in A(H1N1) pandemic strains, a sensitive and specific pyrosequencing assay was developed. This assay analyses a region 99nts long, encompassing the H275Y site, amplified by a nested PCR. Seventy-five respiratory specimens, obtained from 62 patients during the pandemic and in the 2010-2011 influenza season, in Tuscany, were tested. Resistant strains were demonstrated in 10 patients. In three other patients, resistant and sensitive variants were found. This pyrosequencing assay may be a useful method for monitoring the spread of resistant influenza H1N1 2009 strains.