Multidrug-Resistant Pandemic (H1N1) 2009 Infection in Immunocompetent Child

Recent case reports describe multidrug-resistant influenza A pandemic (H1N1) 2009 virus infection in immunocompromised patients exposed to neuraminidase inhibitors because of an I223R neuraminidase mutation. We report a case of multidrug-resistant pandemic (H1N1) 2009 bearing the I223R mutation in an ambulatory child with no previous exposure to neuraminidase inhibitors.

Recent case reports describe multidrug-resistant infl uenza A pandemic (H1N1) 2009 virus infection in immunocompromised patients exposed to neuraminidase inhibitors because of an I223R neuraminidase mutation. We report a case of multidrug-resistant pandemic (H1N1) 2009 bearing the I223R mutation in an ambulatory child with no previous exposure to neuraminidase inhibitors.  (1). Zanamivir resistance is also rare in infl uenza viruses. A Q136K (glutamine to lysine mutation, N2 NA numbering) mutation conferring zanamivir resistance in infl uenza (H1N1) viruses has been described in an in vitro study but has not been detected in clinical specimens from patients (2). An infl uenza B strain carrying a R152K (arginine to lysine) mutation and resistant to oseltamivir and zanamivir has been reported (3). Recent case reports described multidrug-resistant pandemic (H1N1) 2009 infection in immunocompromised patients exposed to oseltamivir and zanamivir because of an I223R (isoleucine to arginine) mutation in NA (4-6). We report a case of infection by multidrug-resistant pandemic (H1N1) 2009 virus bearing the I223R mutation in an ambulatory child with no previous exposure to NAI.

The Study
On October 30, 2009, a 15-year-old girl with a history of asthma sought treatment at an emergency department in the Greater Toronto area after 3 days of cough and rhinorrhea and 1 day of chest pain. Several children at her school also had respiratory symptoms. On arrival, she was febrile to 39.6°C and mildly dehydrated; physical examination was otherwise unremarkable. Blood count and chest radiograph showed no abnormalities. The child received intravenous rehydration in the emergency department, was discharged home with a prescription for oseltamivir therapy, and recovered uneventfully. A nasopharyngeal swab was forwarded to Ontario Agency for Health Protection and Promotion (OAHPP) for infl uenza testing. Pandemic (H1N1) 2009 was detected by real-time reverse transcription PCR (7). Subsequently, the specimen was screened by a single-nucleotide polymorphism assay distributed by Canada's National Microbiology Laboratory and the World Health Organization pyrosequencing protocol for the presence of the H275Y mutation (8). Both assays confi rmed the isolate was wild type (histidine) at aa 275 of NA.
As part of pandemic surveillance, the specimen was cultured in rhesus monkey kidney cells and whole genome sequencing was performed by using a modifi ed World Health Organization protocol (9). Sequences were deposited into GenBank under accession nos. CY060619-CY060626. In comparison with A/California/7/2009 (H1N1), several nonsynonymous mutations were identifi ed: I201V and E538K in polymerase; S220T, D239E, and K465R in hemagglutinin; V100I and M316I in nucleoprotein; S99P and I123V in nonstructural protein; T16I, V106I, I223R, N248D, and N369K in NA. Apart from I201V, which is of unknown signifi cance and has not been previously documented in pandemic (H1N1) 2009, these mutations were detected in 22% to 72% of pandemic (H1N1) 2009 strains circulating in Ontario at the same time that underwent whole genome sequencing. The I223R mutation results from a 1 nucleotide substitution at codon 223 of NA. To rule out the possibility of acquisition of I223R during culture in rhesus monkey kidney cells, the NA gene of the primary sample and its fi rst passage were sequenced. Both had 100% identical nucleotide composition.
The 50% inhibitory concentration (IC 50 ) values for oseltamivir carboxylate and zanamivir, determined by chemiluminescent NAI assay (NA-Star; Applied  . Compared with a wild-type control, the I223R mutant exhibited 28-and 12-fold increases in IC 50 s for oseltamivir and zanamivir, respectively. The oseltamivir IC 50 of the I223R strain was elevated, but not as much as observed in an H275Y control, which had a 168-fold IC 50 elevation compared to the wild-type strain and was 6× higher than that of the I223R strain when tested in parallel. Similar results were obtained when the sample was retested at the National Microbiology Laboratory (Tables 1, 2). The clinical signifi cance of the I223R mutation is poorly understood because the IC 50 s for oseltamivir and zanamivir are well below achievable serum levels when administered at recommended doses. Oral oseltamivir at a dose of 75 mg 2×/d resulted in a maximum serum concentration (C max ) of 348 ng/mL (1,115 nmol). Repeated inhalation of 10 mg of the dried powder formulation of zanamivir produced a C max of 39 to 54 ng/mL (117.5-162.7 nmol) at 1 to 2 postdose, with an elimination half life of 4-5 (10). Intravenous zanamivir at a dose of 600 mg resulted in a C max of 32,000-39,000 ng/mL (96,300-117,360 nmol).
I223 is recognized as one of the framework residues responsible for stabilizing the NAI active site; typespecifi c mutations at these residues have resulted in reduced susceptibility to NAIs (11,12). Although the exact mechanism by which mutations at the framework residue alter susceptibility to particular NAIs is not clear, simulation studies suggest that the NA electrostatic potential plays a major role in the interaction and stabilization of NAIs within the NA cavity (13). Nonhomologous substitution of a nonpolar hydrophobic amino acid, isoleucine, with the positively charged (polar) hydrophilic amino acid, arginine (I223R), seems to be a key point in alteration of the NA cavity. These changes most likely result in active site endpoint interactions affecting drug binding affi nity and could disturb the proposed electrostatic binding funnel instrumental in directing NAIs into and out of binding sites on NA (14). Three independent case reports described infections caused by multidrug-resistant pandemic (H1N1) 2009 in immunocompromised patients who received prolonged treatment with oseltamivir followed by zanamivir; 2 of the infections were fatal. In 2 patients, infection developed (H275Y followed by I223R alone with simultaneous reversion to wild type at position 275) (4,5); dual H275Y/ I223R mutations developed in the third patient (6). Our patient is unique because she was immunocompetent, had no prior exposure to NAIs, and had an uneventful recovery. A similar resistance profi le was seen in the published case exhibiting I223R alone, where IC 50 s for oseltamivir, zanamivir, and peramivir were elevated by 45-, 10-, and 7-fold, respectively (4). The origin of the multiresistant isolate in this patient's case could not be established. The I223R mutation may have occurred spontaneously in our patient. Alternatively, she acquired infection in the ambulatory setting, possibly as part of a school outbreak. Resistance may have evolved following random mutation, or during NAI therapy in another patient. We could not investigate this further because no samples were submitted from contacts. Using reverse genetics, it has been recently shown that an I223V NA change increased oseltamivir and peramivir resistance in pandemic (H1N1) 2009 and also restored NA substrate affi nity and replication fi tness in vitro (15).

Conclusions
Although the I223 residue is highly conserved across pandemic (H1N1) 2009 strains, the global distribution of pandemic (H1N1) 2009 was made possible by the virus adapting for stable circulation through genetic changes contributing to fi tness and facilitating transmissibility from person to person. This report of community acquisition of a multidrug-resistant strain of pandemic (H1N1) 2009 reinforces the need to continue close monitoring for the emergence of resistant viruses and incorporation of screening for newly discovered resistance mutations into clinical diagnostics.
Phenotypic resistance testing was partially funded by a research grant provided to Ontario Agency for Health Protection and Promotion (J.B.G. and D.E.L.) by GlaxoSmithKline Inc.
Dr Eshaghi is a research technologist in the Molecular Research department at The Public Health Laboratories, Ontario Agency for Health Protection and Promotion. His research interests focus on molecular evolution and characterization of respiratory viruses, including emergence of resistance in infl uenza viruses.