We characterized 28 seasonal influenza A(H1N1) viruses from 2008 to 2010 that were dually resistant to oseltamivir and adamantanes from 5 countries: the United States, Canada, China, Kenya, and Vietnam. These viruses retained susceptibility to only 1 licensed influenza antiviral agent, inhaled zanamivir. Seven of the dual-resistant viruses were collected during the 2009–2010 influenza season, 21 were from the previous season (2008–2009), and 1 was detected during the 2007–2008 season. Although dual-resistant viruses are still rare, the increase in prevalence among seasonal influenza A(H1N1) viruses was notable during the last 3 seasons, .06% (1 of 1753 tested in 2007–2008), 1.5% (21 of 1426 in 2008–2009), and 28% (7 of 25 in 2009–2010) (χ2; P<.001). Although few seasonal A(H1N1) viruses circulated during 2009-2010 and it is uncertain whether seasonal and pandemic A(H1N1) viruses will cocirculate in future seasons, the detection of dual-resistant seasonal A(H1N1) viruses from 5 countries warrants concern because of the limited treatment options currently available for dual-resistant influenza A viruses.
The acquisition of a mutation in an influenza A virus that confers resistance to an antiviral agent may occur as a result of drug selection, spontaneous mutation, or through genetic reassortment with another drug-resistant influenza A virus. The development of resistance to oseltamivir while on therapy was documented to occur in 1%–18% of patients, including immunocompromised patients, depending on age, treatment regimens, and other differences [11
]. The emergence and widespread detection of oseltamivir-resistant seasonal influenza A(H1N1) viruses in the 2008–2009 season is not well understood; however, some evidence suggests that the H275Y mutation in the NA may have occurred spontaneously, without apparent drug pressure or reassortment [12
]. In contrast, adamantane resistance has been reported in 30%–80% of isolates from patients treated in clinical studies or during outbreak investigations [3
]. In addition, current seasonal influenza A(H1N1) viruses from clade 2C are typically oseltamivir-susceptible and carry the S31N marker of adamantane resistance in the M2 protein, whereas recent clade 2B seasonal influenza A(H1N1) viruses are primarily adamantane-susceptible and carry the oseltamivir resistance-conferring mutation H275Y in the NA gene.
In this study, the dual-resistant viruses belonged to 4 genetic backgrounds on the basis of full genome sequence analysis. Of the US viruses of genotype 1, all genes similar to clade 2B viruses, 2 were identified in severely immunocompromised patients after adamantane treatment. The third US genotype 1 virus was isolated from a patient that did not have a severe immunocompromising condition and received no antiviral agents, suggesting that the virus causing this infection either spontaneously acquired the S31N mutation in the M2 protein or was possibly transmitted from a person treated with adamantane. All 3 of these cases would be consistent with an oseltamivir-resistant clade 2B virus acquiring adamantane resistance through treatment or transmission.
The fourth US case was a genotype 2 virus from a patient with an influenza infection unassociated with adamantane treatment. As evidenced by a 2B backbone and a 2C M gene (genotype 2), adamantane resistance in this virus was likely the result of a reassortment between clade 2B and 2C viruses. An additional 21 dual-resistant viruses in this report were similar reassortants belonging to genotype 2. The predominance of genotype 2 dual-resistant viruses may suggest that the presence of the 2C M gene provides either a fitness advantage over viruses with clade 2B M gene or could be the result of the continued use of adamantanes in some populations. The S31N mutation is also the marker detected in nearly all recent adamantane-resistant seasonal A(H3N2) viruses and 2009 pandemic A(H1N1) viruses (http://www.cdc.gov/h1n1flu/
]. However, adamantane resistance in A(H3N2) viruses and 2009 pandemic A(H1N1) viruses were acquired independently (http://www.cdc.gov/h1n1flu/
Interestingly, 3 of the 5 viruses from North America, including the Canadian virus, A/Alberta/RV2859/2009 (H1N1), contained the less common V27A change in the M2 protein, whereas the other 2 viruses contained the most commonly detected mutation: S31N. Of note, one dual-resistant virus, A/Kenya/1720/2009 (H1N1), also contained another less common adamantane resistance marker: L26F. At this time it is unclear whether these uncommon changes (L26F and V27A) in the M2 protein, all genotype 1 viruses, emerged due to treatment as seen in the 2 US cases, A/Texas/38/2009 (H1N1) and A/Kentucky/08/2009 (H1N1), or if adamantine-resistant viruses with 2B M genes with those mutations circulate regionally.
Genotype 2 and genotype 3 dual-resistant viruses from Asia appear to be genetically similar to previously reported dual-resistant viruses from Hong Kong SAR [8
]. The genotype 4 virus, A/Liaoning-Huanggu/1144/2009 (H1N1), was the only dual-resistant virus with a nearly complete 2C genome. Oseltamivir resistance in the A/Liaoning-Huanggu/1144/2009 (H1N1) virus appears to be the result of a reassortment between 2B and 2C viruses, as revealed by the presence of the oseltamivir-resistant clade 2B NA gene. Although the exact mechanisms are unknown, it is likely that the development of dual resistance in reassortant viruses, genotypes 2–4, arose from coinfection with a clade 2B oseltamivir-resistant virus and a clade 2C adamantane-resistant virus. Such intrasubtype reassortment of cocirculating strains has been observed in influenza A viruses [16
The detection of influenza A(H1N1) viruses that are resistant to both adamantanes and oseltamivir warrants close monitoring. If circulation of viruses with dual resistance to both oseltamivir and adamantanes becomes more widespread among any of the predominant circulating influenza A viruses (eg, 2009 pandemic H1N1, H5N1, etc), treatment options would be extremely limited, especially in young children and for severely ill patients. New antiviral agents and strategies for antiviral therapy are likely to be necessary in the future.