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Pandemic 2009 H1N1 isolates containing the neuraminidase inhibitor resistance mutation H275Y have been reported. We describe rapid selection for the H275Y resistance mutation during therapy in two immunocompromised individuals in 9 and 14 days respectively, and the first described case of clinically significant resistance to peramivir.
The 2009 H1N1 influenza virus pandemic has highlighted the limited armamentarium of antivirals available to treat influenza A, especially for those at high risk of severe disease and complications of influenza. Oseltamivir, one of the neuraminidase inhibitors (NAIs), has been the drug of choice for treatment. Early in the pandemic, reports of resistance to oseltamivir were limited, but recently a number of isolates have been detected that contain the H275Y mutation in the neuraminidase gene (NA) conferring significant reduction in sensitivity to oseltamivir[1-4].
In addition to oseltamivir, two other antivirals are available: zanamavir, an FDA approved inhaled NAI, and peramivir, an intravenous (IV) NAI released under an emergency use authorization. In vitro laboratory testing of seasonal H1N1 isolates containing the H275Y mutation have shown an increase in IC50 to peramivir [6, 7], but the demonstration of clinically significant resistance to peramivir in an individual patient has not yet been described.
Selection for resistance mutations in immuncompromised individuals infected with influenza A viruses has recently been reported[2, 8-10]. In this report, we describe two cases of influenza in immunocompromised hosts infected with the 2009 pandemic H1N1 virus treated with an extended course of NAIs.
Patient A is a transfusion dependant 24-year-old woman who was treated for myelodysplastic syndrome with a matched unrelated peripheral blood stem cell transplantation in December 2008 complicated by post transplant lymphoproliferative disorder and graft versus host disease. She recently received a stem cell boost and courses of corticosteroids, rituximab, and tacrolimus in the two months prior to infection with influenza.
The patient presented on October 24th, 2009 with fever of 38.5°C, coryza, myalgias, productive cough with clear sputum, and loose stool. She had course breath sounds bilaterally, heart rate was 120 beats per minute, and she was tachypnic with an oxygen saturation of 89%. Chest radiography demonstrated a right-sided infiltrate and complete blood count showed slight lymphopenia. The patient was treated with oxygen, empiric oseltamivir 75mg twice daily, and piperacillin/tazobactam. Rapid test for influenza A was positive and all bacterial cultures negative. The patient required oxygen for approximately 8 days, and chest CT performed on day 6 showed bilateral patchy infiltrates. The patient was treated for 30 days continuously with oseltamivir and remained symptomatic, but respiratory symptoms began to improve after day 21. All repeat nasopharyngeal washes and a bronchioalveolar lavage remained positive for influenza A by viral culture until 44 days after initial diagnosis (Table 1A).
Patient B is a 49-year-old male who underwent matched unrelated donor peripheral blood stem cell transplantation in March 2009 to treat recurrence of diffuse large B-cell lymphoma. The patient was recently treated for cytomegalovirus reactivation and graft versus host disease, and medications included sirolimus and prednisone. On October 22, 2009, this patient presented with mild upper respiratory symptoms. He was afebrile and breathing comfortably. Chest radiography showed no evidence of disease and complete blood count revealed lymphopenia. The respiratory virus culture was positive for influenza A at 24 hours and the patient was subsequently treated with 75mg oseltamivir twice daily.
After 14 days of oseltamivir therapy the patient was admitted with worsening fatigue, cough, sinus pressure, and significant lower extremity edema. Radiographs and chest CT demonstrated extensive bilateral patchy infiltrates. Cultures from both a nasal wash and bronchoscopy were positive solely for influenza A. Oseltamivir therapy was continued and levofloxacin was added empirically.
After 24 days of continuous oseltamivir therapy he developed respiratory distress and was admitted to the ICU for treatment with noninvasive positive pressure ventilation. A second bronchoscopy was performed that revealed positive influenza cultures. Further empiric antibiotics were added and 10 days of peramivir IV was administered. Thirty one days after diagnosis the patient’s severe symptoms had stabilized, but the patient remained symptomatic and nasopharyngeal wash samples remained positive for influenza A on day 32 and day 40. The patient then received 10 days of inhaled zanamivir therapy and by day 46 a negative nasopharyngeal wash was obtained with overall improvement of symptoms (Table 1A).
Samples were collected with consent of participants under an NIAID IRB approved protocol “Influenza in the Normal and Immunocompromised Host” (IRB number 07-I-0229).
Viral isolates were obtained using shell vial culture technique. Viruses were passed 1-2 times in Madin-Darby canine kidney cells as previously described.
Reverse-transcription polymerase chain reaction (RT-PCR) and sequencing was performed on the primary viral isolates as previously described. NA sequences determined have been deposited in GenBank (GenBank accession numbers GU571152-GU571156).
Antiviral susceptibility and NA activity was measured using methyl-umbelliferyl-N-acetyl neuraminic acid substrate as previously described[10, 13], and 50% inhibitory concentration (IC50) was determined by regression analysis (Prism, GraphPad Software Inc., La Jolla, CA). A/California/07/2009 (H1N1) was used as control.
Viral isolates collected from Patient A on day 0 and 5 contained the wild-type histidine at position 275 of the NA gene. All other isolates after day 9 contained the H275Y mutation (Table 1A). Viral isolates collected on day 0 from Patient B contained the wild-type NA, but all further isolates contained the H275Y mutation. No other changes were observed in the NA gene (Table 1A).
IC50 values for viral isolates collected containing the wild-type NA and H275Y mutation are shown in Table 1B. The H275Y-containing isolates from both patients show a greater than 200-fold increase in IC50 to oseltamivir and 50-fold increase to peramivir, indicating a significant loss in sensitivity to both antivirals. No significant change in IC50 to zanamivir was observed.
Selection during therapy for the H275Y mutation in pandemic H1N1 infections of two immunocompromised hosts with prolonged illness was previously demonstrated early in the pandemic, but only after more than 24 days of continuous therapy. The two cases described here are the first to demonstrate a rapid selection of the H275Y mutation, in less than 9 and 14 days of oseltamivir therapy respectively. Oseltamivir treatment failed clinically in both cases and peramivir failed to reduce shedding in one of these individuals. This is the first described case of clinically significant resistance to peramivir correlating to the measured IC50 in vitro.
The IC50 that signifies NAI resistance may differ between viruses and drugs tested, but a change in IC50 of 10-fold or greater between a single virus pre- and post-treatment is commonly considered the hallmark of resistance. Isolates collected from these patients containing the H275Y mutation in the NA gene clearly meet this definition, demonstrating a significant increase in IC50 to both oseltamivir and peramivir.
The clinical failure of therapy in both Patient A and B further strengthens the importance of this change in IC50. Patient A showed signs of illness and viral shedding for 45 days and Patient B progressed to a more severe respiratory illness with signs of viral pneumonia despite continuous oseltamivir therapy for the first 30 and 24 days respectively. Patient B then received a 10-day course of IV peramivir that failed to reduce viral shedding.
In both cases these individuals were significantly immunocompromised at the time of infection which likely contributed to the failure of the antivirals. These two cases were observed during a two-month period in which just 7 other cases of pandemic influenza in immunocompromised individuals were enrolled at the NIH Clinical Center. This high frequency of resistance development is concerning, as it might suggest that the selection for multi-drug resistant viruses in immunocompromised hosts may be more common than previously believed.
It has become fairly common for immunocompromised patients at higher risk of severe complications of influenza infection to receive a longer course of oseltamivir therapy than the recommended 5 days if they remain ill or continue to shed. These two cases, along with the reports of similar resistance selection in immunocompromised patients[2, 8-10], suggest that we may need to reevaluate our usage of NAIs. Demonstration of rapid selection of resistance during therapy, in as fast as 9-14 days in these cases or 5 days in seasonal influenza, suggests that prolonged therapy with a single agent may provide conditions that are optimal for the development of drug resistance mutations. Therefore, discontinuation of oseltamivir therapy or switching to zanamivir if possible after the recommended 5 days may be an appropriate strategy to prevent these mutations.
The rapid selection of the H275Y mutation leading to clinical failure of peramivir to reduce shedding in one of these cases is a significant finding, and suggests that we need to investigate further the effectiveness of peramivir as a treatment for patients who fail oseltamivir therapy. The rapid emergence of oseltamivir and peramivir resistance in already amantadine resistant pandemic H1N1 viruses highlights the difficulties faced in treating influenza, especially in those patients who are susceptible to prolonged infection such as immunocompromised individuals. These results indicate that further investigation of drug resistance and the development of new classes of antivirals is imperative in order to reduce the adverse impact current and future influenza pandemics can have on human health.
The authors would like to acknowledge Jocelyn Voell, R.N., M.S., Charles Fiorentino, R.N., M.Ed., Richard Davey, M.D., Jeffrey Cohen, M.D., Daniel Fedorko, Ph.D., and the Clinical Microbiology Laboratory in the Division of Laboratory Medicine for their support of this study. This research was supported by the Intramural Research Program of the NIH and the NIAID.
All authors certify that they have no potential conflicts of interest to disclose.