The new england journal of medicinen engl j med 353;25 www.nejm.org december 22, 20052667brief reportOseltamivir Resistance during Treatment of Influenza A (H5N1) InfectionMenno D. de Jong, M.D., Ph.D., Tran Tan Thanh, M.Sc., Truong Huu Khanh, M.D., Vo Minh Hien, M.D., Gavin J.D. Smith, Ph.D., Nguyen Vinh Chau, M.D., Bach Van Cam, M.D., Phan Tu Qui, M.D., Do Quang Ha, M.D., Ph.D., Yi Guan, M.D., Ph.D., J.S. Malik Peiris, D.Phil., M.D., Tran Tinh Hien, M.D., Ph.D., and Jeremy Farrar, D.Phil., F.R.C.P.From the Oxford University Clinical Re-search Unit, Hospital for Tropical Dis-eases (M.D.J., T.T.T., D.Q.H., J.F.), Pediat-ric Hospital Number One (T.H.K., B.V.C., P.T.Q.), and the Hospital for Tropical Dis-eases (V.M.H., N.V.C., T.T.H.)— all in Ho Chi Minh City, Vietnam; and the Depart-ment of Microbiology, University of Hong Kong, Queen Mary Hospital, Hong Kong Special Administrative Region, China (G.J.D.S., Y.G., J.S.M.P.). Address reprint requests to Dr. de Jong at the Oxford Uni-versity Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, District 5, Ho Chi Minh City, Vietnam, or at [email protected] Engl J Med 2005;353:2667-72.Copyright © 2005 Massachusetts Medical Society. summaryInfluenza A (H5N1) virus with an amino acid substitution in neuraminidase con-ferring high-level resistance to oseltamivir was isolated from two of eight Vietnam-ese patients during oseltamivir treatment. Both patients died of influenza A (H5N1) virus infection, despite early initiation of treatment in one patient. Surviving pa-tients had rapid declines in the viral load to undetectable levels during treatment. These observations suggest that resistance can emerge during the currently recom-mended regimen of oseltamivir therapy and may be associated with clinical deteriora-tion and that the strategy for the treatment of influenza A (H5N1) virus infection should include additional antiviral agents.Influenza A (h5n1) virus causes severe disease in humans and poses an unprecedented pandemic threat.1-3 The neuraminidase inhibitor oseltamivir constitutes an important treatment option, and stockpiling of this drug is part of pandemic-preparedness plans.4 However, data on the efficacy and development of drug resistance in human influenza A (H5N1) virus are scarce. We report the isola-tion of oseltamivir-resistant influenza A (H5N1) variants from two patients who died of the infection, in one case despite the early initiation of treatment. Further-more, we provide evidence suggesting that the presence of detectable virus after the completion of treatment is associated with a poor outcome. These observations have implications for the treatment of influenza A (H5N1) virus infection.case reportA previously healthy 13-year-old Vietnamese girl weighing 28 kg (Patient 1 in Table 1) presented to a hospital in Dong Thap Province on January 22, 2005, with a one-day history of fever and cough. The day before, her mother (Patient 2 in Table 1) had died of influenza A (H5N1) virus infection after one day of oseltamivir treatment. Virus isolated from the mother did not reveal oseltamivir-resistance mutations. Be-cause influenza A (H5N1) virus infection was suspected in the child at presentation, she received an initial 75-mg dose of oseltamivir and was transferred to a pediatric referral hospital. On admission, she had a temperature of 40.3°C, a pulse of 106 beats per minute, a respiratory rate of 36 breaths per minute, and normal blood pressure. Results of physical examination and routine biochemical measurements were unre-Copyright © 2005 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org on September 24, 2006 . For personal use only. No other uses without permission.The new england journal of medicinen engl j med 353;25 www.nejm.org december 22, 20052668markable. Hematologic measurements showed a white-cell count of 4800 cells per cubic millimeter (normal range, 5500 to 15,500), with 12 percent lymphocytes, and a platelet count of 183,000 cells per cubic millimeter (normal range, 250,000 to 550,000). A blood culture showed no growth. A chest radiograph revealed a small focal pulmo-nary infiltrate in the right middle lobe (Fig. 1A). The patient received a second 75-mg dose of oseltamivir within 6 hours after the first, fol-lowed by a third dose within the first 24 hours after admission. Treatment was then continued for four days at the standard dose of 75 mg twice daily. Antibiotic treatment with ceftriaxone and amikacin was also given. During the first three days after admission, the patient remained in stable condition and did not require supplemen-tal oxygen. A chest radiograph obtained on Janu-ary 24 showed minimal progression of the infil-trate (Fig. 1B). At that time, her white-cell count was 3100 cells per cubic millimeter, with 26 per-cent lymphocytes. On January 25 (the fourth day of oseltamivir treatment), the child’s respiratory condition wors-ened and supplemental high-dose oxygen was giv-en, initially by nasal cannula and later by continu-ous positive airway pressure. Antibiotic treatment was switched to vancomycin, ciprofloxacin, and amikacin. At this time, the pneumonia involved most of the right middle zone (Fig. 1C). A chest radiograph obtained on January 26 showed fur-ther progression of the infiltrate (Fig. 1D). Hema-tologic and biochemical measurements revealed a white-cell count of 1800 cells per cubic millime-ter, with 41 percent lymphocytes; a platelet count of 97,000 cells per cubic millimeter; and increased serum alanine aminotransferase and aspartate aminotransferase levels (144 and 279 U per liter, respectively; normal range, less than 55 and less than 50, respectively). Her respiratory condition continued to worsen, and she was intubated and ventilation was begun on January 27. A chest ra-diograph obtained on January 28 showed pneu-monia involving the entire right lung and exten-sion to the left lung (Fig. 1E). She died the same day. No autopsy was performed.methodspatients and clinical specimensWe examined sequential pharyngeal swabs from Patient 1 and seven additional patients with in-fluenza A (H5N1) infection from whom at least one pharyngeal swab obtained before treatment and during treatment with oseltamivir was avail-able. The swabs were collected in viral-transport medium and stored at −80°C.virologic investigationsVirus isolation was performed in Madin–Darby canine-kidney cells in biosafety level III culture