title: Ultrasensitive detection of drug-resistant pandemic 2009 (H1N1) influenza A virus by rare-variant-sensitive high-resolution melting-curve analysis.
abstract: Oseltamivir (Tamiflu), an oral neuraminidase inhibitor, has been widely used to treat pandemic 2009 (H1N1) influenza A. Although a majority of 2009 (H1N1) influenza A virus remains oseltamivir susceptible, the threat of resistance due to the His275Tyr mutation is highlighted by the limitations of alternative therapies and the potential for rapid, global fixation of this mutation in the circulating influenza A virus population. In order to better understand the emergence of resistance, we developed a rare-variant-sensitive high-resolution melting-curve analysis method (RVS-HRM) that is able to detect the His275Tyr oseltamivir resistance mutation to 0.5% in a background of susceptible virus. We applied RVS-HRM to clinical specimens from patients who developed oseltamivir resistance and demonstrated the ultrasensitive detection of influenza A virus N1 neuraminidase quasispecies. Interestingly, we were unable to detect the oseltamivir resistance mutation in pretreatment samples, suggesting that resistant virus does not reach even this very low detection threshold until exposed to selective drug pressure. Thus, patients naive to oseltamivir are most likely to be susceptible when this drug is used as a first-line treatment modality.
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Neng
authors.initials: N
authors.lastname: Chen
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Pinsky
authors.forename: Betty P
authors.initials: BP
authors.lastname: Lee
authors.forename: Min
authors.initials: M
authors.lastname: Lin
authors.forename: Iris
authors.initials: I
authors.lastname: Schrijver
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D024882:Drug Resistance, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology*; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D053139:Oseltamivir / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D053139:Oseltamivir