Monitoring of oseltamivir resistance in influenza A H1N1 strains in Western Australia using a rapid real-time PCR

Session VII – Harnett, Gerald

Monitoring of oseltamivir resistance in influenza A H1N1 strains in Western Australia using a rapid real-time PCR

Harnett GB1, Pratt EJ1, Ryan NK1, Al-Mousa G1,2, Hurt A3, Smith DW1,2

1Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA, Nedlands, Australia; 2Discipline of Microbiology and Immunology, University of Western Australia, Nedlands, Australia; 3WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia.

Oseltamivir is a neuraminidase inhibitor that is used for the treatment and prevention of influenza. Resistance to this drug has been uncommon until the appearance of resistance in influenza A/H1N1 virus strains was reported in several European countries in late 2007 and since then that clade has been found throughout the world, including Australia. These particular strains have a histidine-to-tyrosine mutation at position 274 in the neuraminidase protein gene that confers resistance to oseltamivir. In order to rapidly test for this mutation in H1N1 strains, we developed one-step TaqMan real-time assays that targeted the wild-type and the mutant sequences and applied them to 150 Western Australian H1N1 strains from 2007 and 2008. A nested PCR was also performed, which allowed DNA sequencing across the mutation site. All H1N1 cultured strains were also referred to the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne for phenotypic testing, with results currently available for 38 of the 150 samples tested by TaqMan.  The earliest detection of a mutant was in August 2008 and this was the predominant H1N1 strain throughout the 2008 season. In some cases the TaqMan assays identified strains where sequencing was unsuccessful, reflecting the higher sensitivity of the TaqMan assays. Sequencing of the haemagglutinin gene was also performed to determine whether the 2007 and 2008 strains were antigenically different.