Diagnostic accuracy of an allele-specific reverse transcriptase-PCR assay targeting the H275Y oseltamivir resistant mutation in 2009 pandemic influenza A/H1N1 virus

AbstactBackgroundOseltamivir resistant 2009 pandemic influenza A/H1N1 viruses (pH1N1) are emerging and rapid molecular assays identifying these strains are needed for clinical management.ObjectiveDevelopment and evaluation of an allele-specific, real-time reverse transcriptase-PCR assay (ASPCR) targeting the H275Y oseltamivir resistant mutation in pH1N1 virus.Study designASPCR uses two allele-specific forward primers (wild-type and mutant) and a common reverse primer and probe. Wild-type and mutant genotypes were defined by the difference in PCR Ct values (ΔCtmut–wt) between the mutant primer and wild-type primer amplification curves for the same sample. Mixtures of wild-type and mutant genotypes were analyzed to evaluate sensitivity and determine assay cut-off values. ASPCR results were confirmed using an allelic discrimination assay (AD) and pyrosequencing.ResultsMixtures containing 5–95% mutant genotype could be detected. A ΔCtmut–wt ≥ 3.5 identified wild-type genotype (<10% mutant); between 3.5 and −3.5 identified mixed genotypes (10–90% mutant); and ≤−3.5 identified fully mutant genotype (>90% mutant). Among 264 clinical samples, 171 were wild-type, 10 were mixed, and 29 were fully mutant. The 39 samples with mixed or mutant results were from 11 patients. Of 107 samples with sufficient volume tested by ASPCR and AD, 12 were indeterminate by AD due to low viral load, 86 were wild-type by both assays, and 9 were mutant by both assays. Thirteen samples were confirmed by pyrosequencing and one discrepant sample was mixed by ASPCR and fully mutant by pyrosequencing.ConclusionsASPCR is sensitive, quantitative and specific for H275Y mutation analysis and provides an accurate approach for detecting pH1N1 oseltamivir resistance in clinical samples.