Emergence of Oseltamivir- Resistant Pandemic (H1N1) 2009 Virus within 48 Hours Masafumi Inoue, Timothy Barkham, Yee-Sin Leo, Kwai-Peng Chan, Angela Chow, Christopher W. Wong, Raphael Tze-Chuen Lee, Sebastian Maurer-Stroh, Raymond Lin, and Cui Lin An oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus evolved and emerged from zero to 52% of de- tectable virus within 48 hours of a patient’s exposure to oseltamivir. Phylogenetic analysis and data gathered by pyrosequencing and cloning directly on clinical samples suggest that the mutant emerged de novo. E arly descriptions of emergence of H275Y mutants in pandemic (H1N1) 2009 virus showed resistance after 11 and 23 days of therapy in immunosuppressed patients (1). Also in previous reports, transmission of mutant vi- ruses occurred in immunosuppressed patients (2), although a cluster among healthy persons demonstrated that H275Y mutants could replicate and cause disease in the absence of drug pressure (3). Additional reports noted decreasing times to detection of resistance, from 14 to 4 days after therapy (4–6). We report development of oseltamivir-resis- tant pandemic (H1N1) 2009 virus in an infected woman in Singapore within 48 hours of drug treatment. The Study Pandemic (H1N1) 2009 virus was first detected in Singapore in May 2009. Infected patients were placed in isolation and offered oseltamivir, and respiratory samples were collected for screening for H275Y, the principal mu- tation associated with oseltamivir resistance in influenza A N1 viruses. H275Y was detected in a pandemic (H1N1) 2009 virus isolated from a sample from a 28-year-old fe- male patient on the sixth day of illness within 48 hours of her exposure to oseltamivir. (Written patient consent was obtained under Review Board approval no. E09-230.) A sore throat, myalgia, redness of the right eye, and a mild fever with a productive cough had developed on the day she returned to Singapore from Hawaii. Eleven close con- tacts, exposed before emergence of the mutant, were given oseltamivir prophylaxis on the patient’s fourth day of treat- ment, and they remained well. By performing sequencing directly on 6 of her respiratory samples and on their viral isolates (online Technical Appendix, www.cdc.gov/EID/ content/16/10/1633-Techapp.pdf), we investigated the ori- gin of this H275Y mutant (the second earliest sample of this mutation to be deposited in GenBank). Only wild-type sequences were detected in samples collected on the day before, the day of, and 14 hours af- ter initiation of oseltamivir therapy (online Technical Ap- pendix Table 1). Similarly, only wild-type sequences were detected in 192 clones generated from a sample collected a few hours before initiation of oseltamivir. Pyrosequenc- ing directly on clinical samples collected 38 and 45 hours after initiation of therapy showed 24% and 52% mutant se- quences, respectively (Figure). The relative amount of vi- rus detected, as determined by the strength of PCR results (online Technical Appendix Table 1), increased from days 3 to 5 of illness by ≈1,000-fold. Oseltamivir treatment was initiated on day 4 of illness. On the same day, her maximum body temperature (38.8°C) was recorded, although no other signs or symptoms of clinical deterioration were observed. Her fever resolved on day 5 of illness, and she was allowed out of isolation on day 7 of illness. When we compared the mutant drug-resistant isolate GN285 with the wild-type drug-sensitive isolate ON129, we found only 1 aa difference, the H275Y resistance-caus- ing mutation in the neuraminidase gene, whereas a com- parison of GN285 and ON129 with the reference strain A/ Texas/05/2009(H1N1) showed several mutations (online Technical Appendix Table 2). Mutation PB1 I435V, shared between GN285 and ON129, did not occur in any of the other 7 drug-resistant strains included in the analysis. The whole genome maximum likelihood tree (online Technical Appendix Figure 1) showed that the wild-type and resistant viruses isolated from this patient were more closely related to each other than to any other virus in the analysis. Nota- bly, GN285 and ON129 clustered together in 376 of the 500 bootstrap tests. Conclusions Our data indicate that oseltamivir resistance developed within 2 days. This time is similar to the interval for de- velopment of resistance to adamantanes in subtype H3N2 viruses when 30% of treated patients shed resistant, trans- Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 10, October 2010 1633 Author affiliations: Agency for Science, Research and Technology, Singapore (M. Inoue, C.W. Wong, R.T.-C. Lee, S. Maurer-Stroh); Tan Tock Seng Hospital, Singapore (T. Barkham, A. Chow); Com- municable Disease Center, Singapore (Y.-S. Leo); Singapore Gen- eral Hospital, Singapore (K.-P. Chan); and Ministry of Health, Sin- gapore (R. Lin, L. Cui) DOI: 10.3201/eid1610.100688