© 2008 International Medical Press 1359-6535 809 Antiviral Therapy 13: 809–820 Background: Emerging resistance of influenza viruses to neuraminidase inhibitors is a concern, both in surveillance of global circulating strains and in treatment of individual patients. Current methodologies to detect resistance rely on the use of cultured virus, thus taking time to com- plete or lacking the sensitivity to detect mutations in viral quasispecies. Methodology for rapid detection of clini- cally meaningful resistance is needed to assist individual patient management and to track the transmission of resistant viruses in the community. Methods: We have developed a pyrosequencing method- ology to detect and quantitate influenza neuraminidase inhibitor resistance mutations in cultured virus and directly in clinical material. Our assays target polymorphisms asso- ciated with drug resistance in the neuraminidase genes of human influenza A H1N1 as well as human and avian H5N1 viruses. Quantitation can be achieved using viral RNA extracted directly from respiratory or tissue samples, thus eliminating the need for virus culture and allowing the assay of highly pathogenic viruses such as H5N1 without high containment laboratory facilities. Results: Antiviral-resistant quasispecies are detected and quantitated accurately when present in the total virus population at levels as low as 10%. Pyrosequencing is a real-time assay; therefore, results can be obtained within a clinically relevant timeframe and provide informa- tion capable of informing individual patient or outbreak management. Conclusions: Pyrosequencing is ideally suited for early identification of emerging antiviral resistance in human and avian influenza infection and is a useful tool for laboratory surveillance and pandemic preparedness. The neuraminidase inhibitor (NI) class of drugs (oseltamivir and zanamivir) was introduced in 1999/2000. Since this time, there has been a gradual increase in global use of these drugs, particularly osel- tamivir, to treat seasonal influenza A and B infections, with the highest per capita use in Japan followed by the United States. Limited therapeutic options and lack of vaccines for zoonotic H5N1 infection means that osel- tamivir is the drug of choice in the treatment of severe or life-threatening H5N1 infection [1]. Anti-influenza drugs will be one of the first lines of defence in the event of an influenza pandemic. Circulation of amantadine- resistant human H3N2 and avian H5N1 strains is already widely recognized, meaning that the use of the M2 blocker class of drugs alone is not advised [2,3]. Many countries have already acquired large stockpiles of oseltamivir during pre-pandemic planning; therefore, the emergence of NI-resistant strains with a capacity to spread is of concern. Oseltamivir resistance following treatment of seasonal and avian influenza infection has been documented and low levels of phenotypic NI resis- tance were found (0.3%) during several untargeted sur- veillance studies for drug susceptibility [4–13]. Oseltamivir-resistant human H1N1 virus emerged globally during the 2007–2008 Northern Hemisphere winter season with no evidence of drug exposure [14]. The resistant variants harboured a mutation in the N1 gene (H275Y; N1 nomenclature is used throughout this text because we only refer to N1-containing viruses. This position corresponds to the 274 position when using N2 numbering) and was detected early in the season (by the surveillance systems already in place) using a combina- tion of phenotypic and genotypic assays that included the pyrosequencing methodology described in this paper. The most commonly used method for NI suscepti- bility surveillance is an enzyme assay measuring 50% Original article Rapid quantitation of neuraminidase inhibitor drug resistance in influenza virus quasispecies Angie Lackenby 1 *, Jane Democratis 1,2 , Marilda M Siqueira 3 and Maria C Zambon 1 1 Health Protection Agency, Centre for Infection, London, UK 2 Department of Infection and Tropical Medicine, University Hospitals Leicester NHS Trust, Leicester Royal Infirmary, Leicester, UK 3 Instituto Oswaldo Cruz, FIOCRUZ, Department of Virology, Rio de Janeiro, Brazil *Corresponding author: E-mail: [email protected] Introduction
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