To control influenza A virus infections, the US Food and Drug Administration has approved the use of matrix 2 (M2) blockers, amantadine and rimantadine, and the neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir (5). However, for pandemic (H1N1) 2009, therapeutic options are limited to the NAIs because this virus has a swine virus–origin M2 gene, which contains a mutation associated with resistance to adamantanes (6). NAI resistance in pandemic (H1N1) 2009 viruses has been rare; nevertheless, 285 oseltamivir-resistant cases were reported worldwide as of April 14, 2010 (7). All oseltamivir-resistant viruses have the H275Y substitution that confers resistance to oseltamivir but not to zanamivir. Spread of oseltamivir-resistant seasonal influenza A virus (H1N1) was first detected in 2007, and this virus has now become the predominant lineage of influenza A virus (H1N1) in humans (8,9). This finding raises strong concerns that the H275Y mutation could become dominant in pandemic (H1N1) 2009 as well. We report oseltamivir-resistant pandemic (H1N1) 2009 detected through virologic surveillance in Mexico.

Samples were collected from patients in all Mexican states. Patients did not differ significantly by sex, and persons 10–29 years of age were most commonly affected, similar to the number of incident cases of acute respiratory infection in Mexico (Table 1). Viral RNA was extracted by using either MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics, Rotkreuz, Switzerland) or QIAmp Viral RNA Mini Kit (QIAGEN, Hilden, Germany).

An endpoint RT-PCR was performed for all 692 samples screened for the H275Y molecular marker by using the Superscript III RT-PCR system (Invitrogen, Carlsbad, CA, USA) and FLUAN1–721F and FLUAN1–924R primers spanning position 275 of the NA gene (Table 2). Direct sequencing of these PCR products was performed by using a sequencing primer (FLUAN1–904R, Table 2) and the BigDye Terminator version 3.1 cycle sequencing reaction kit on an ABI PRISM 3130xl DNA analyzer (Applied Biosystems, Foster City, CA, USA).

All the sequences obtained indicated that the H→Y mutation at the NA 275 residue was absent, except in 1 NPS from an 8-month-old girl (A/Mexico/InDRE797/2010). This patient received oseltamivir treatment from the evening of January 22 through January 27. She had no known history of travel, contact with a person treated with this drug, or diagnosed immunodeficiency. Her clinical record mentioned 2 respiratory events in the 2 months before the influenza diagnosis: broncholitis, which required hospitalization, and readmission to the hospital 2 weeks after discharge because of influenza-like illness and severe pneumonia. In addition to oseltamivir, the child required other antimicrobial drugs (ceftriaxone and vancomycin) and mechanical ventilation. She slowly recovered.

The clinical specimen was collected, and pandemic (H1N1) 2009 was laboratory confirmed on January 27, 2010. In this sample and in isolating the virus we further analyzed, we discarded any of the 9 previously described mutations (5,10) associated with resistance to NAIs (V116, I117, E119, Q136, K150, D151, D198, I223, and N295 [N2 numbering]) by the full sequence of the NA gene by using 2 overlapping RT-PCR products (Figure 1, Table 2). The complete NA sequence of the A/Mexico/InDRE797/2009 virus obtained from NPS and MDCK isolate (GenBank accession no. CY057074) confirmed the H275Y substitution and showed no other NA mutations known to be associated with NAI resistance (Figure 2, panel A). Pyrosequencing analysis performed on the clinical specimen from the 8-month-old patient showed oseltamivir-resistant H275Y and wild-type H275 virus variants (Figure 2, panel B). The A/Mexico/InDRE797/2009 virus was tested at the Centers for Disease Control and Prevention by using an NA inhibition assay (NA-Star kit, Applied BioSystems) and showed an ≈120-fold increase in oseltamivir 50% inhibitory concentration over that of a sensitive control (27.3 nmol/L vs. 0.23 nmol/L; this result was consistent with H275Y and H275 variants. No change in zanamivir susceptibility was detected (0.32 nmol/L vs. 0.30 nmol/L), which was in accord with the NA sequencing analysis. We also sequenced the M2 gene and confirmed the S31N substitution that confers M2 blocker resistance. Sequencing of the HA gene showed that the substitution D222G, potentially associated with severe clinical outcome (12), was not present in this isolate (data not shown).

In addition, 24 virus isolates collected during December 2009–January 2010 were tested in the NA inhibition assay. All were sensitive to both NAIs, with 50% inhibitory concentrations of 0.12–0.29 nmol/L and 0.17–0.36 nmol/L for oseltamivir and zanamivir, respectively.