Detection of influenza A H1N1 and H3N2 mutations conferring resistance to oseltamivir using rolling circle amplification.
In the event of an influenza pandemic, the use of oseltamivir (OTV) will undoubtedly increase and therefore it is more likely that OTV-resistant influenza strains will also arise. OTV-resistance genotyping using sequence-based testing on viruses isolated in cell culture is time consuming and less likely to detect the low-level presence of drug-resistant virus populations. We have developed a novel rolling circle amplification (RCA) method to achieve the sensitive detection of OTV-resistant viruses from clinical specimens. Using artificially created templates, RCA could detect the presence of OTV-resistant mutations (N2: 119V, 292K, N1: 274Y) even if the population carrying the mutations was <1% of the total. By applying RCA to clinical samples, we identified the emergence of the 274Y mutation in one OTV-treated patient, as well as in seven individuals who were treatment-naïve (confirming community transmission of 274Y-containing resistant influenza A H1N1). These results were further confirmed by neuraminidase region sequencing. In conclusion, RCA technology can provide rapid (<24 h), high-throughput diagnosis of OTV resistance mutations with a high specificity and sensitivity.
Steain
Megan C
MC
Department of Infectious Diseases and Immunology, University of Sydney, NSW 2006, Australia.
Dwyer
Dominic E
DE
Hurt
Aeron C
AC
Kol
Chenda
C
Saksena
Nitin K
NK
Cunningham
Anthony L
AL
Wang
Bin
B
D000935:Antifungal Agents / Q000627:therapeutic use*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D024882:Drug Resistance, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000235:genetics*; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects / Q000235:genetics*; D007251:Influenza, Human / Q000175:diagnosis / Q000188:drug therapy* / Q000821:virology; D008969:Molecular Sequence Data; D009154:Mutation; D053139:Oseltamivir / Q000627:therapeutic use*
D000935:Antifungal Agents; D053139:Oseltamivir