title: Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory.
abstract: Pandemic 2009 H1N1 is normally susceptible to oseltamivir, but variants harboring the H275Y (CAC → TAC) mutation exhibit resistance. We describe the use of a combined reverse-transcription polymerase chain reaction (RT-PCR)/pyrosequencing approach to identify the H275 residue. A total of 223 specimens were tested with this method: 216 randomly selected clinical specimens positive for 2009 H1N1 and 7 cell-culture supernatants from the Centers for Disease Control and Prevention (CDC; 4 resistant, 3 susceptible 2009 H1N1 strains). The assay detected H275Y in 1 clinical respiratory sample (0.5%) and all 4 oseltamivir-resistant strains from the CDC; the remaining 215 clinical and 3 susceptible CDC specimens were wild-type. Sanger sequencing confirmed the results for 50 of 50 selected isolates. The RT-PCR/pyrosequencing method was highly specific, producing no amplicons or valid sequences from samples containing non-H1N1 viruses or bacteria. Our findings suggest that this method provides a rapid tool for H275Y detection, with high sensitivity and potential benefit for patient care.
authors: Quest Diagnostics Nichols Institute, Chantilly, VA 20151, USA. jian.r.bao@questdiagnostics.com
authors: Jian R
authors: JR
authors: Bao
authors: Thomas K
authors: TK
authors: Huard
authors: Arelis E
authors: AE
authors: Piscitelli
authors: Praveena R
authors: PR
authors: Tummala
authors: Virginia E
authors: VE
authors: Aleemi
authors: Stephanie L
authors: SL
authors: Coon
authors: Ronald N
authors: RN
authors: Master
authors: Michael A
authors: MA
authors: Lewinski
authors: Richard B
authors: RB
authors: Clark
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D009439:Neuraminidase / Q000235:genetics*; D053139:Oseltamivir / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D014764:Viral Proteins; D053139:Oseltamivir; C487630:NA protein, influenza A virus; D009439:Neuraminidase