JCM Accepts, published online ahead of print on 4 November 2009

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J. Clin. Microbiol. doi:10.1128/JCM.00698-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Rapid and specific detection of amantadine-resistant Ser31Asn mutated influenza A viruses by cycling probe method

Yasushi Suzuki^*, Reiko Saito, Hassan Zaraket, Clyde Dapat, Isolde Caperig-Dapat, and Hiroshi Suzuki

Department of Public Health, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan

^* To whom correspondence should be addressed. Email: yasshi{at}med.niigata-u.ac.jp.

Amantadine is one of the antiviral agents against influenza A viruses, but resistant strains have widely emerged worldwide. In this study, we developed a novel method to detect amantadine-resistant strains harboring Ser31Asn mutation in the M2 gene based on cycling probe method using real time PCR, and we studied amantadine-resistant rate in the 2007-2008 season in Japan. Two different primers and cycling probes sets were designed respectively for A/H1N1 and A/H3N2 to detect a single nucleotide polymorphism corresponding to Ser/Asn at 31 residue of the M2 protein. Using nasopharyngeal swabs from patients with influenza-like and other respiratory illnesses, and virus isolates, the specificity and sensitivity of the cycling probe method were evaluated. High frequencies of amantadine-resistant viruses were detected among A/H1N1 (411/663, 62%) and A/H3N2 (56/56, 100%) collected from six prefectures in Japan in the 2007-2008 influenza season. We confirmed that the cycling probe method is suitable for screening of amantadine-resistant strains from both nasopharyngeal swabs and influenza isolates, and showed that the incidence of amantadine-resistant viruses remained high for both A/H1N1 and A/H3N2 in Japan during the 2007-2008 season.