Recovery of a Multidrug‐Resistant Strain of Pandemic Influenza A 2009 (H1N1) Virus Carrying a Dual H275Y/I223R Mutation from a Child after Prolonged Treatment with Oseltamivir

To the Editor—Resistance to oseltamivir in strains of the pandemic influenza A 2009 (H1N1) virus, although rare, has been reported worldwide [1]. The oseltamivir resistance is caused by a single mutation, H275Y, in the neuraminidase, which does not affect susceptibility to zanamivir. Here we report an influenza A 2009 (H1N1) virus strain with 2 neuraminidase mutations (H275Y and I223R) with laboratory evidence for multidrug resistance. The patient, a 14-year-old girl with systemic lupus erythematosus, systemic vasculitis, and chronic pancreatitis who was receiving immune-suppressing medications, was hospitalized with respiratory failure and tested positive for influenza A 2009 (H1N1) on 13 October 2009. Oseltamivir was administered in dosages of 60 mg twice daily (13–16 October) and 150 mg twice daily (16–23 October) and was restarted at a dosage of 120 mg twice daily (1–14 November) for fever. During the period 23–28 November, she was treated with intravenous zanamivir (420 mg twice daily) because of persistent influenza A 2009 (H1N1) detection and suspicion of oseltamivir resistance. She died of complications on 26 December. Influenza A 2009 (H1N1)—positive bronchoalveolar lavage samples from 14 October and 10 November and a nasopharyngeal swab sample obtained 1 November were tested for the presence of known markers of drug resistance by means of pyrosequencing [2] (Table 1). H275Y was identified in the neuraminidase of the 1 and 10 November specimens. A virus strain cultured in Madin-Darby canine kidney cells from the 10 November sample was assessed for its susceptibility to oseltamivir, zanamivir, and peramivir with use of chemiluminescent and fluorescent neuraminidase inhibition assays [3]. The oseltamivir 50% inhibitory concentration (IC50) was highly elevated in both assays, compared with wild-type and H275Y control virus strains. IC50 values were also high to peramivir, causing ∼90-fold (chemiluminescent) and ∼30-fold (fluorescent) further reduction, compared with the H275Y variant. Zanamivir IC50 values were elevated ∼20-fold compared with the wild type strain and ∼10-fold compared with the H275Y mutant strain; however, the absolute IC50 values remained low. Conventional sequencing of the isolate and pyrosequencing of clinical specimens from 1 and 10 November detected H275Y/I223R mutations in the neuraminidase, which indicated shedding of a dual H275Y/I223R mutant strain for at least 10 days. An influenza A 2009 (H1N1) virus carrying a single copy of I223K isolated from an unrelated patient during routine surveillance exerted slightly elevated IC50 values (<40-fold to oseltamivir and ∼5-fold to peramivir and zanamivir). Thus, the susceptibility of the dual mutant I223R/H275Y strain was additionally reduced, compared with that of single H275Y or I223K mutant strains. Susceptibility of Pandemic Influenza A 2009 (H1N1) Virus Strains to Neuraminidase Inhibitors at Chemiluminescent (CL) or Fluorescent (FL) Neuraminidase Inhibition Assay Residue 223 is regarded as one of the key markers to monitor because of its ability to increase drug resistance by combining with other mutations [4]. To our knowledge, this is the third detection of a change at residue 223 in the neuraminidase of influenza A 2009 (H1N1) virus strains after oseltamivir exposure [5]. Dual H275Y/I223V mutants were detected in respiratory specimens from 2 campers in North Carolina; no virus was recovered. The recovery of the dual influenza A 2009 (H1N1) mutant here confirmed the functional importance of the I223R mutation when present in combination with H275Y. Because H275Y mutation alone does not affect susceptibility to zanamivir, the reduction in zanamivir susceptibility of the dual influenza A 2009 (H1N1) mutant strain, although less pronounced, is concerning. Compared with a single 275Y mutant, the dual influenza A 2009 (H1N1) mutant showed highly elevated IC50 values for peramivir, suggestive of clinically important change; however, IC50 values were still below the peak of peramivir plasma concentrations (∼10,000 nM). The potential for emergence of viruses resistant to multiple neuraminidase inhibitors reinforces efforts to develop drugs with a different mechanism of action. We thank Tiffany Sheu, Vasiliy Mishin, and Katrina Sleeman from the Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, for technical assistance and the Delaware Public Health laboratory and the Influenza Center in Santiago, Chile, for valuable contributions to this work. Financial support. Support for H.T.N for this study was provided by the Atlanta Research and Education Foundation (AREF). Potential conflicts of interest. All authors: no conflicts.