Hematology–oncology patients who were infected with pandemic (H1N1) 2009 virus and received care at adult and pediatric Seattle Cancer Care Alliance (Seattle, WA, USA) units or clinics during May 1, 2009–April 30, 2010, were identified by using infection control data and laboratory databases. All samples with pandemic (H1N1) 2009 virus detected by in-house real-time reverse transcription–PCR targeting the matrix, and hemagglutinin genes were retrospectively tested by our allele-specific real-time PCR (ASPCR) for H275Y genotype (9,10). ASPCR uses 2 allele-specific forward primers (wild-type and mutant) and a common reverse primer and probe. Wild-type and mutant genotypes were defined by the difference in PCR cycle threshold values (∆cycle threshold_{mutant –wild type}) between the mutant primer and the wild-type primer amplification curves for the same sample. The ASPCR directed toward the H275Y mutation only was designed and validated in our laboratory, with good correlation demonstrated by pyrosequencing (9). ASPCR also can provide an accurate quantitative result of mutant percentage in a mixed population, as described (11). Samples were not tested for adamantanes resistance because pandemic (H1N1) 2009 virus was considered to be uniformly resistant to adamantanes.

Specimens collected from inpatients or outpatients were either nasal wash (NW) samples or bronchoalveolar lavage (BAL) samples. NW samples were tested by using multiplex real-time reverse transcription–PCR for respiratory syncytial virus, parainfluenza virus 1–4, human metapneumovirus, adenovirus, bocavirus, coronavirus (OC43, 229E, HKU1, and NL63), and rhinovirus at the same time as influenza A subtyping and influenza B. BAL samples were tested for the same respiratory viruses and for bacterial, mycobacterial, viral, and fungal cultures, fungal PCR, galactomanan, cytomegalovirus by shell vial, respiratory syncytial virus by shell vial, respiratory virus direct immunofluorescence assay, and Pneumocystis spp. direct immunofluorescence assay. NW samples were collected by instilling 5 mL of normal saline in each nare and having the patient blow his or her nose directly into a sterile cup. For younger children, suction was used to collect nasal secretions. BAL samples were obtained according to a standardized protocol. Samples were refrigerated within 4 h after collection and transported to the molecular virology laboratory. All available samples were kept frozen at –80°C for up to 8 months after initial clinical testing. Total nucleic acid was extracted as described (12). Institutional review board approval was obtained from our institutional committee. A chart review of all patients with mutant H275Y virus was retrospectively performed by using standardized case record forms.

During the pandemic (H1N1) 2009 outbreak, neuraminidase-resistant viruses emerged rapidly in immunocompromised patients. Our retrospective analysis suggests a high rate of oseltamivir resistance conferred by the H275Y mutation in treated immunocompromised patients. The rate of mutation development ranged from 8 (16%) of 49 patients, with all infected patients as denominator, to 7 (58%) of 12 on the basis of samples obtained after antiviral drug therapy began. Two other studies have reported similar rates. One study from Scotland reported a 50% rate (5/10) of H275Y mutation in immunocompromised patients with samples available after oseltamivir therapy began (14). The other study, in Australia, reported a 13.3% rate (4/30) of H275Y mutation in treated immunocompromised patients (not all of them tested) or 57% rate (4/7) in only treated patients for whom samples were available after treatment began (15).

The 8 patients in our study who had H275Y mutant virus demonstrated a wide range of clinical disease, from benign upper respiratory tract symptoms to severe and fatal respiratory insufficiency. Whether the H275Y mutation is associated with higher rates of death or severe disease is unclear. The small number of immunocompromised patients infected with resistant pandemic (H1N1) 2009 virus did not enable us to compare them with patients infected with sensitive pandemic (H1N1) 2009 virus. However, our case series highlights the rapid emergence of resistant viruses in the context of mild to severe influenza disease. In 7 of the 8 cases in our study, resistance was not associated with long-term consequences and was not even suspected in 4 of the 7 cases. This observation could suggest that resistant viruses are not more virulent than wild-type viruses.

Influenza resistance kinetics as provided in this study by the percentage of H275Y mutants enable a better determination of the timing of emergence of resistant virus and the possible subsequent clearance of this resistant viral population. Our results suggest that elevated viral loads seen early in disease might be associated with more rapid emergence of resistance, but more complete and regular testing after the initial sample is necessary to confirm that hypothesis.

In this case series, some patients conserved the resistant viral population after antiviral pressure removal, whereas another patient (patient 1) cleared his resistant viral population to recover a fully wild-type virus. Similarly, the H275Y mutation described in another case report disappeared after antiviral drug therapy was completed, while others showed conserved resistant viral population (14,16). Those 2 different evolutions of mutant viral population suggest the possibility of quasispecies with different fitness. The H275Y neuraminidase mutation had affected in vitro viral fitness when incorporated in seasonal (H1N1) virus, but data looking at 2008–09 seasonal (H1N1) virus showed that the circulating H275Y mutant strain had recovered its fitness, possibly explaining its sustainable transmission (17). Recently, permissive secondary mutations in the neuraminidase gene leading to fitness recovery have been proposed in seasonal (H1N1) viruses (18). Substitutions V234M and R222Q buffer deficiencies in neuraminidase folding or stability caused by H275Y and simultaneously allow the virus to keep its neuraminidase/hemagglutinin balance, which is implicated in viral fitness (19). Further molecular and in vitro analysis of viral strains from patients who cleared and did not clear the mutant viral population might provide more input about permissive secondary mutations in pandemic (H1N1) 2009 virus.

We were not able to provide information about whether an H275Y mutant viral population can develop in the upper respiratory tract and not in the lower respiratory tract or the converse scenario. The limited data we described on paired NW and BAL samples suggest that the upper and lower respiratory tracts are likely to be infected with the same viral H275Y genotype. Others have shown that quasispecies harboring the D222G hemagglutinin mutation that confers improved pneumocyte receptors binding could be found specifically or in larger proportions in endotracheal aspirates than in paired nasopharyngeal aspirates (20). Analysis of D222G mutant virus has not yet been reported in immunocompromised patients, a condition in which mutations develop more readily.

We have demonstrated that the H275Y mutation can develop in immunocompromised patients under different antiviral drug treatment regimens. Oseltamivir is the antiviral agent most associated with the emergence of H275Y. Peramivir selects for the H275Y mutation in seasonal (H1N1) by successive passages in vitro, but data have not clearly confirmed this observation in vivo (21). One patient in our study had received oseltamivir and peramivir before resistance detection. We believe that if peramivir was not the drug that selected the initial mutation, it did not prevent establishment of dominant H275Y virus population (13). Because peramivir is rarely used as frontline therapy, data to support this hypothesis are likely to be difficult to obtain. Combination therapy with oseltamivir and rimantadine did not prevent development of H275Y mutation in 2 of the patients in our study. The issue of antiviral drug resistance in immunocompromised patients makes determination of optimal initial therapy necessary. Inhaled zanamivir is often contraindicated because the patients may have respiratory failure or underlying lung disease, and novel agents (e.g., DAS181) are not yet available. Triple-combination therapy (i.e., oseltamivir, rimantadine, and oral ribavarin) potentially could reduce emergence of resistance, but more data are needed to support this hypothesis (22,23).

This case series has some limitations. It does not provide a totally accurate incidence of resistance in immunocompromised patients. Some cases of pandemic (H1N1) 2009 might have been missed, particularly if symptoms were mild or if the patient was not identified by infection control surveillance. Also, repeat testing was performed in only 17 patients, in whom 5 viral loads were too low to genotype. Systematic testing of infected patients at 5 or 6 days after initial pandemic (H1N1) 2009 diagnosis might have caught even more minor mutant viral populations. Our ASPCR was aimed only at detection of the H275Y mutation. Presence of other rare mutations as the I223R could have influenced our results (24,25). However, our data highlight the need for surveillance and clinical testing for resistant mutations in immunocompromised patients by using sensitive and rapid molecular diagnostic tests.