Detection of Oseltamivir Resistance during Treatment of 2009 H1N1 Influenza Virus Infection in Immunocompromised Patients: Utility of Cycle Threshold Values of Qualitative Real-Time Reverse Transcriptase PCR

Abstract

Two immunocompromised patients with 2009 H1N1 influenza pneumonia had viral shedding for over 5 weeks despite therapy with oseltamivir. Declining or persistently low cycle threshold values noted on serial qualitative real-time reverse transcriptase PCR (rRT-PCR) of respiratory specimens implied increasing viral load and probable drug resistance. Oseltamivir resistance was later confirmed by pyrosequencing. CASE REPORTSCase report 1. A 52-year-old male with myelodysplastic syndrome underwent an allogeneic hematopoietic stem cell transplantation (HSCT) complicated by graft-versus-host disease (GVHD) and bronchiolitis obliterans of his lungs. On day 864 post-HSCT, he developed cough and pleuritic chest pain. Nodular cavitary infiltrates were identified by computed tomography scan (CT) of his thorax. Aspergillus fumigatus was isolated from bronchoalveolar lavage (BAL) fluid, and the BAL fluid Aspergillus galactomannan assay was positive. Voriconazole therapy was initiated for probable pulmonary aspergillosis. Rhinovirus was detected on PCR assay of BAL fluid. Mycobacterium avium complex was also isolated from BAL fluid and was considered to represent colonization.On day 990 post-HSCT, in November 2009, he was hospitalized with fever, progressive dyspnea, and nonproductive cough. In addition to voriconazole, he was receiving methylprednisone, tacrolimus, sirolimus, imatinib mesylate, and extracorporeal photopheresis as therapy for GVHD. His oxygen saturation was 83% when he was breathing ambient air, and he had crackles at both lung bases. A computed tomography (CT) scan of the thorax demonstrated new bilateral lung infiltrates. The rapid influenza diagnostic test (RIDT) of the nasal wash was positive for influenza A virus, which was also isolated on viral culture. Oseltamivir (75 mg twice daily [BID] for 10 days) as well as empirical broad-spectrum antibiotic therapy was initiated. His symptoms improved within 7 days, and he was discharged to a rehabilitation facility. The patient was not retested for influenza virus at time of discharge.One week later he was admitted to the intensive care unit (ICU) with acute shortness of breath. A CT scan revealed interval improvement of the prior lung infiltrates. Empirical therapy with meropenem and vancomycin was begun for possible nosocomial lower respiratory tract infection. The methylprednisone dose was increased to 2 mg/kg/day for suspected worsening of bronchiolitis obliterans. On ICU day 8, he required mechanical ventilation. Bronchoscopy was performed; the BAL fluid tested positive for influenza A virus on RIDT, the novel 2009 H1N1 influenza virus (H1N1) was detected by real-time reverse transcriptase PCR (rRT-PCR), the cycle threshold (C T ) was 29, and influenza A virus was isolated from viral culture. Therapy with high-dose oseltamavir (150 mg BID) was initiated, the steroid dose was tapered, and antibiotics were discontinued. Therapy was switched to intravenous (i.v.) peramivir on day 10. Aerosolized zanamivir could not be administered, as the patient was being mech...

Prolonged Shedding of Human Coronavirus in Hematopoietic Cell Transplant Recipients: Risk Factors and Viral Genome Evolution

“…Such variability might have important implications in viral disease pathogenesis, and the study of viral genome evolution within a host can provide vital information important in developing and assessing antiviral agents [26]. For example, development of antiviral drug resistance during individualized therapy that is associated with poor outcome has been described extensively with influenza virus [27][28][29]. Grad et al reported intrahost genome evolution of respiratory syncytial virus over time in an infant with severe combined immune deficiency who underwent a bone marrow transplant [30].…”

Estimation of the Relative Sensitivity of qPCR Analysis Using Pooled Samples

“…The samples with a high number of initial copies of target nucleic acid, are detected sooner and therefore they will have low values (usually around 20–25). However, those samples with very low numbers of copies are later detected, and the values are above 30–35 [5]. The sample is defined as positive when the analyzed by the qPCR technique is less than the established (threshold value).…”

Molecular Diagnosis of Viral Respiratory Infections

“…Rapid detection of oseltamivir resistance in severe pandemic H1N1 infections is essential for patients to receive maximum therapeutic benefit, and is useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. Recent studies have reported the use of novel molecular methods to screen for the presence of the H275Y resistance mutation in the neuraminidase gene [58][59][60][61]. In addition to accurate detection of the H275Y oseltamivirresistance allele, samples collected at multiple time points even showed a switch from wild-type oseltamivir-sensitive 275 H to oseltamivir-resistant 275Y population after 9 days of treatment.…”