Detection of the rapid emergence of the H275Y mutation associated with oseltamivir resistance in severe pandemic influenza virus A/H1N1 09 infections

In 2009 a new swine-origin influenza virus A/H1N1 (A/H1N1 09) emerged, causing the century's first pandemic. Most isolates of the new A/H1N1 09 virus are susceptible to neuraminidase inhibitors, but the H275Y mutation in the neuraminidase gene region associated with high-level oseltamivir resistance has been detected. Using rolling circle amplification (RCA) technology, 96 A/H1N1 09-specific RT-PCR positive clinical samples collected from 80 oseltamivir-treated and untreated patients were screened for the presence of the H275Y mutation. Samples positive for 275Y mutation by RCA were cloned and sequenced for confirmation. From 25 patients who had been treated with oseltamivir and remained A/H1N1 09 RT-PCR positive, we identified three (12%) individuals with the H275Y mutation: one immuno-suppressed adult, one immuno-competent adult and one child. Samples collected at multiple time points from the two adults showed a switch from wild-type oseltamivir-sensitive 275H to oseltamivir-resistant 275Y population after 9 days of treatment. The child had the 275Y mutation detected after being persistently A/H1N1 09 RT-PCR positive while receiving oseltamivir treatment. Resistance was not detected in 17 pre-treatment samples and 54 A/H1N1 09 RT-PCR positive outpatients. RCA demonstrates the rapid emergence of the H275Y resistance mutation in individuals with severe A/H1N1 09 infection receiving neuraminidase inhibitors. Rapid detection of oseltamivir resistance in severe infection is essential for patients to receive maximum therapeutic benefit. In the light of emerging resistance, close monitoring and understanding of the nature and dynamics of resistance mutations in newly emerging strains should be a priority.