Oseltamivir-Resistant Influenza Viruses A (H1N1), Norway, 2007–08

Resistance was not associated with oseltamivir use or more severe disease.

S easonal infl uenza, caused by infl uenza A subtypes H3N2 and H1N1 and infl uenza B viruses, occurs as annual epidemics. Although vaccination remains the primary measure for prevention, antiviral drugs are available for prevention and treatment of infl uenza. The infl uenza virus neuraminidase inhibitors zanamivir and oseltamivir were introduced into clinical practice in various parts of the world from 1999 through 2002 (1). Oseltamivir limits replication of both infl uenza A and B viruses (1). In most European countries, neuraminidase inhibitors are not widely used to treat seasonal infl uenza, but they are being stockpiled in many countries as part of their pandemic infl uenza preparedness. In Norway, oseltamivir is registered for prophylactic and therapeutic use in persons >1 year of age; however, it is not available without a prescription and it is rarely prescribed (2).
Until 2007, resistance against neuraminidase inhibitors was rarely observed (1,3,4). Nevertheless, to better understand the potential for development of resistance against neuraminidase inhibitors, surveillance of antiviral susceptibility in infl uenza virus in Europe has been ongoing since 2004 (5). As part of the World Health Organization (WHO) Global Infl uenza Surveillance Network, the national infl uenza centers in Europe submit infl uenza viruses to the WHO Infl uenza Collaborating Centre in the United Kingdom each infl uenza season. Within the framework of the European Surveillance Network for Vigilance against Viral Resistance (VIRGIL), these viruses are also tested for drug susceptibility at the Health Protection Agency in London.
In mid-January 2008, antiviral susceptibility testing (enzyme inhibition assays) of the fi rst shipment of infl uenza viruses from Norway for the 2007-08 season showed an unusually large proportion (5/7) of infl uenza viruses A (H1N1) with high-level resistance to oseltamivir. In subsequent days, testing of additional viruses from Norway at the Norwegian national infl uenza center and at the Health Protection Agency confi rmed the high proportion of oseltamivir resistance. This unexpected and unprecedented discovery had possible public health implications of international concern. On January 25, 2008, the Norwegian Institute of Public Health notifi ed WHO of these fi ndings under the International Health Regulations (6) and notifi ed the European Commission through the Early Warning and Response System. The Institute also informed hospitals and physicians in Norway about a possible lack of therapeutic effect when treating patients with oseltamivir. By the end of January, oseltamivir-resistant viruses had been reported from several European countries (7).
The oseltamivir-resistance trait is caused by a previously described point mutation in the virus neuraminidase gene (histidine to tyrosine at position 275 of the N1 neuramini-dase, commonly referred to as H274Y in N2 numbering), which is known to confer high-level resistance to oseltamivir while retaining susceptibility to zanamivir (8). Infl uenza viruses A (H1N1) carrying the H274Y mutation have reduced ability to replicate and transmit effi ciently when compared with parental, susceptible virus, but the clinical implications of infection with these viruses have been largely unknown (9). Consequently, we undertook studies to determine whether the emergence and spread of the resistant viruses were associated with exposure to oseltamivir, whether resistant viruses would continue to circulate in similar proportions into the epidemic phase of the season, and whether the new resistant viruses differed from their susceptible counterparts in their ability to cause disease. To do so, we tested all infl uenza viruses A (H1N1) available from the 2007-08 outbreak for oseltamivir susceptibility. We furthermore enhanced surveillance by collecting an extended set of data regarding clinical symptoms, complications, and prior exposure to oseltamivir for all laboratoryverifi ed cases of infl uenza viruses A (H1N1) infection.

Methods
The infl uenza viruses A (H1N1) included in this study were obtained from the sentinel and nonsentinel collaborators as part of routine national virologic infl uenza surveillance. From all 19 counties, 71 sentinel practices collect samples from patients with infl uenza-like illness and send them to the national infl uenza center for diagnostic testing. From all parts of the country, 15 medical microbiology laboratories submit materials containing infl uenza A or B materials (original specimens, nucleic acid preparations from original specimens, or viral isolates) to the national infl uenza center for further characterization. Most of these samples originate from primary care clinics; the rest, from hospitals.
Viruses confi rmed as infl uenza A (H1), by either reverse transcription-PCR (RT-PCR) or virus isolation in MDCK cells and subsequent subtyping by immunofl uorescence, were included in the study. In-country susceptibility testing was performed by detecting the H274Y mutation by sequence analysis, through either a pyrosequencing assay targeting the single relevant point mutation (10) or through full-or partial-length cycle sequencing of the coding region for the viral neuraminidase. These analyses were mostly performed on RNA prepared from the original patient specimen. A large proportion of the isolated viruses were sent to the WHO Collaborative Centre for Infl uenza Research and Reference in the National Institute of Medical Research, Mill Hill, UK, for further characterization. Within the framework of VIRGIL, these viruses were forwarded to the Health Protection Agency for phenotypic antiviral susceptibility testing and more extensive genotypic analyses. To determine neuraminidase susceptibility, assays to determine the drug concentration that provides 50% inhibition (IC 50 ) were performed by using the fl uorescent substrate methylumbelliferyl N-acetylneuraminic acid based on the method described by Wetherall et al. (11) with minor modifi cations.
Relative quantitative data on virus shedding, i.e., virus RNA content in the patient specimens, were obtained through a real-time RT-PCR targeting a conserved part of the matrix protein (M1) gene of infl uenza A virus. Two microliters of nucleic acid prepared from specimens (MagNApure LC Total Nucleic Acid Isolation Kit; Roche Diagnostics, Mannheim, Germany) was added to a 23-μL reaction mixture containing 0.3 μM forward primer M52c (5′-CTT CTA ACC GAG GTC GAA ACG-3′); 0.3 μM reverse primer M149r (5′-CTT GTC TTT AGC CAT TCC ATG AG-3′); 0.15 μM probe M93c (FAM-5′ CCG TCA GGC CCC CTC AAA GCC GA 3′-Black Hole Quencher 1); and 5× QIAGEN OneStep RT-PCR buffer, dNTP mixture, and enzyme mixture according to the manufacturer's instructions (QIAGEN OneStep RT PCR Kit; QIAGEN, Hilden, Germany). Forward primer and probe sequences were as described by Fouchier et al. (12), and the reverse primer was designed by Tom Øystein Jonassen (Akershus University Hospital, Lørenskog, Norway). Reactions were run in a Corbett Rotorgene RG-3000 or RG-6000 thermocycler (Corbett Research Pty Ltd, Sydney, New South Wales, Australia) with the following cycling conditions: reverse trancription for 30 min at 50°C, then 15 min at 95°C, followed by 50 cycles at 95°C for 10 sec, 54°C for 30 sec, and 72°C for 20 sec.

Participants and Study Design
We included all patients with a diagnosis of infl uenza virus A (H1N1) infection made by national infl uenza center during the 2007-08 infl uenza season. For the 72 patients who received this diagnosis before the end of January 2008, data were collected retrospectively. From February on, data were collected as soon as possible after laboratory confi rmation of infl uenza virus A (H1N1) infection. Structured questionnaires returned from consulting physicians provided auxiliary information about clinical signs and symptoms, complications, predisposing diseases for severe outcome of infl uenza (diabetes, cardiac disease, lung disease, and immunodefi ciency), use of oseltamivir, and infl uenza vaccination status. If the questionnaire was not returned by mail within 3 weeks, a reminder call was made. When available, relevant clinical information on the original referral sample form was used to supplement the data from the written questionnaire. The consulting physician, usually the primary care physician, was not informed about the result of the susceptibility testing when the information was collected. Information for the fi rst 12 patients infected with a resistant virus was collected from the consulting physicians by telephone.

Statistical Analysis
Data from the questionnaires and selected laboratory testing outcomes were merged, checked for quality, and analyzed by using Stata version 9.0 (StataCorp LP, College Station, TX, USA). We used the Fisher exact test to compare the proportions of possible confounders among those infected with a resistant and a susceptible virus. To estimate the association between exposure (resistant virus infection) and outcome (subsequent clinical fi ndings and complications), we calculated crude risk ratios (RRs) and 95% confi dence intervals (CIs). We used binomial regression to calculate RRs adjusted for possible confounders. For each variable, we used the number of respondents as the denominator, except for predisposing disease, for which missing values were coded as "no."

Results
The overall infl uenza activity in Norway was low in 2007-08 compared with that of previous years. Virologic surveillance showed most infl uenza virus A and 95% of subtyped viruses to be subtype H1N1 (13). From the sentinel practices, the national infl uenza center received 229 specimens for infl uenza testing. Of the 108 that were positive for infl uenza virus, 61 were type A, subtype H1N1, and most of the rest were type B. In total, 297 patients had an infl uenza virus A (H1N1) infection confi rmed by the national infl uenza center in Norway from week 47 in 2007 until the end of week 20 in 2008. We obtained a resistance profi le for 272 of the 297 viruses. We could not determine the resistance profi le for the remaining 25 because of low virus content and consequently excluded them from analysis.
A total of 196 viral isolates were available (133 carried the resistance mutation); of these, 113 (79 with the resistant genotype) were reference tested by the VIRGIL laboratory. Phenotypic and genotypic reference analysis results agreed completely with the in-country genotypic testing results; all mutant viruses showed large reductions in susceptibility to oseltamivir when compared with non-H274Y viruses (IC 50 260-2,161 nM, mean 673 nM, for the 274Y mutant and 0.4-5.6 nM, mean 2.6 nM, for the nonmutant viruses). No evidence of mixed genotype or phenotype was observed. In phylogenetic analysis of the H1 gene, all viruses tested grouped together in subclade 2B (Figure 1). In the phylogenetic tree, the resistant viruses from Norway all formed a single branch that was distinct, but closely related, to the susceptible viruses from Norway.
Of the 272 infl uenza viruses A (H1N1), 183 (67.3%) were oseltamivir resistant ( Table 1). The proportion of resistant viruses did not differ between samples from sentinel 67.9% (38/56) and nonsentinel 67.1% (145/216) practices and persisted throughout the season (Figure 2). No difference in virus shedding, as quantifi ed by real-time RT-PCR of available patient specimens, was observed between susceptible and resistant viruses (Figure 3). From the original sample form, we obtained demographic information for all 272 patients. Returned questionnaires provided information for 265 patients (97.4%), but the response rate on individual questions varied. Of the 272 patients infected with infl uenza viruses A (H1N1), 132 (48.5%) were male (Table 1), and slightly more than half (50.7%) were 29-64 years of age (median 27 years, range 2 months-71 years); median ages of those infected with a resistant and a susceptible virus were 31 and 21 years, respectively. The highest proportion of resistant virus infection was found for those 25-59 years of age (102/138, 73.9%) and differed significantly from the proportion for only those 5-14 years of age (25/45, 55.6%) (Fisher exact p = 0.03). We obtained infl uenza viruses A (H1N1) from 18/19 counties (Figure 4).The oseltamivir resistance proportion was >80% in 8 counties in southern Norway, compared with 63.5% in the rest of the country (Fisher exact p = 0.001).
Information about use of antiviral drugs was obtained for 237 patients. No patients had received antiviral treatment in the 14 days before the onset of symptoms, and none had been in close contact with others known to have used antiviral drugs. Oseltamivir was received after sampling by 7 patients, 5 of whom were infected with an oseltamivirresistant virus. Of 225 patients, 9 had traveled abroad in the week before symptom onset; 4 were infected with a resistant virus. Of all 272 patients, 2 had been vaccinated against infl uenza and were both infected with a resistant virus.
We received information about predisposing disease for 213 patients. Having a predisposing disease more than doubled the risk for complications (RR 2.5, 95% CI 1.2-5.4) but was not clearly associated with being infected with a resistant virus (RR 1.4, 95% CI 0.6-3.2). Information about clinical symptoms was obtained for 252/272 patients; most frequently reported were fever (229/242) and dry cough (182/218). Resistant virus infection was not associated with any particular symptom ( Table 2). Of 241 patients, 58 (24.1%) had >1 complications recorded, but no difference was observed between those infected with a resistant virus and those infected with a susceptible virus (Table 2). Bronchitis and pneumonia were the most frequent complications, reported for 22 and 17 patients, respectively. The age of the 17 patients who had pneumonia ranged from 8 months to 65 years (mean 29 years): 2 (12.5%) were 0-4 years of age, 5 (31.3%) were 5-14 years of age, 2 (12.5%) were 15-24 years of age, 4 (25.0%) were 25-59 years of age, and 3 (18.8%) were >59 years of age. Of the 17 patients with pneumonia, 15 were infected with a resistant virus. The attack rates of pneumonia and of sinusitis were higher for those infected with a resistant virus than for those infected with a susceptible virus, although the risk ratios were not statistically signifi cant after adjusting for age, gender, and predisposing disease (pneumonia RR 3.2, 95% CI 0.7-13.7; sinusitis RR 1.7, 95% CI 0.4-7.5) ( Table  2). Of 264 patients, 45 had been hospitalized, 28 and 17 in-  fected with a resistant and a susceptible virus, respectively. No deaths were reported for patients included in the study.

Discussion
During the 2007-08 infl uenza season in the Northern Hemisphere, widespread circulation of oseltamivir-resistant infl uenza viruses A (H1N1) was observed. Percentage of resistant viruses circulating in different countries varied markedly; the highest proportion reported worldwide (67%) was in Norway (17,18).
Our study did not show any association between oseltamivir use in Norway and emergence of the oseltamivir-resistant infl uenza viruses A (H1N1). Because only a minority of infl uenza cases are laboratory confi rmed, oseltamivir use in nonsampled persons could have contributed to the development of resistance. However, for this suggestion to be plausible, use of oseltamivir would have to be widespread to exert substantial selective pressure on the viruses. Sales of oseltamivir in Norway have been low:  (2). In countries where oseltamivir use has been high, e.g., Japan, the proportion of oseltamivirresistant infl uenza viruses A (H1N1) reported during the 2007-08 season was low (18). Because infl uenza strains from Norway were genetically similar to resistant viruses that appeared just as early in several other European countries (A. Hay, pers. comm.), we consider it unlikely that the resistant variant originated in Norway. Conceivably, the initial emergence of a resistant virus could be associated with oseltamivir use elsewhere. Our data indicate that the viruses carrying this resistance mutation are fully capable of persistence and spread in the absence of selective pressure.
In Norway, the initially high proportion of resistant infl uenza viruses A (H1N1) was maintained throughout the entire 2007-08 infl uenza season; countrywide, 2 of 3 viruses  were resistant. The reason for this exceptionally high resistance proportion is unknown. However, it likely refl ects the proportion of resistant viruses introduced into Norway in the fall of 2007. Globally, the proportion of resistant infl uenza viruses A (H1N1) reported is highly variable between the different countries for which data are available (18). This large variation, apparently in the absence of oseltamivir selective pressure, suggests that a high level of randomness determined the frequency of resistance. In temperate countries, the infl uenza viruses are reintroduced each autumn after the absence of infl uenza during the summer. If only a limited number of viruses are reintroduced into each country and initiate virus circulation and outbreaks, the result will be considerable random variation in virus variant proportions between the different countries. Consistent with this result, almost all the characterized infl uenza viruses A (H1N1) in Norway could be assembled into a small number of genetically discernible groups (Figure 1). We propose that such randomness in virus introductions may be suffi cient to explain the differences in the proportions of resistant viruses between the countries. Conceivably, a difference in the antigenic characteristics of the resistant and susceptible viruses could have fa-vored one virus over the other in the face of host population immunity. Such differences might contribute to different relative effect of the 2 viruses in different populations (e.g., countries) or subpopulations (e.g., age groups). However, the resistant and susceptible viruses were closely related and were not distinguishable in hemagglutination inhibition tests (19).
Overall, the observed clinical manifestations associated with infl uenza viruses A (H1N1) in this study were as expected for seasonal infl uenza. No differences were noted for virus shedding, primary symptoms, or overall complication and hospitalization rates caused by oseltamivir-resistant and -susceptible viruses. We did fi nd, although not a statistically signifi cant fi nding, that patients infected with a resistant virus appeared to be more likely than those infected with a susceptible virus to have pneumonia or sinusitis. Patients with more severe illness may be more likely to be sampled; however, the resistance pattern of the virus was not known by the physician at the time of sampling and reporting. We therefore believe that these fi ndings are not infl uenced by selection bias. Adjusting for possible confounders (age, sex, and predisposing disease) did not change the results. Because of our limited sample size, the precision of our estimates is low, but they do indicate fi ndings that warrant further investigation. Our data will also be analyzed with data from other European countries, and the fi ndings may strengthen the conclusions about the clinical implications of oseltamivir-resistant infl uenza viruses A (H1N1) .
The future effect of resistant infl uenza viruses A (H1N1) is unpredictable. In Europe, the H1N1 subtype was predominant during the 2007-08 infl uenza season and, according to historical patterns, is unlikely to predominate during the 2008-09 infl uenza season. In the following 2008-09 season in the Northern Hemisphere, infl uenza viruses A (H1N1) may well predominate in areas where they had not recently been present in large numbers. Early reporting from the Southern Hemisphere 2008 infl uenza season indicates that detection of infl uenza virus A (H1N1) is low (20). However, in South Africa, oseltamivir resistance has been detected in 100% of infl uenza viruses A (H1N1) tested (21).
Whether oseltamivir-resistant viruses will persist beyond 2008 depends on several factors. First, their persistence will depend on the prevalence of resistant viruses in the populations that are the source of global infl uenza spread. Countries in East and Southeast Asia have been proposed as the most likely source for global dissemination of new infl uenza virus variants (22). The prevalence of resistant infl uenza viruses A (H1N1) in this region may therefore be more likely to infl uence future occurrence of these viruses than the prevalence in Europe; resistance monitoring thus needs to be global. Second, changes in re- cent infl uenza viruses A (H1N1) may have provided a genetic background that permits H274Y mutants to replicate and transmit. Previous studies have concluded that resistant viruses are less pathogenic and less transmissible than their susceptible counterparts (9,23). In contrast, however, reverse genetics-derived mutants (A/WSN/33 or PR8 backbone) had the same phenotype as wild type viruses in vitro and in vivo (24,25). A recent study on the enzymatic properties of the N1 neuraminidase of the resistant viruses from the 2007-08 season suggested some genetic background changes that could potentially be involved (26).
As long as such a postulated permissive genetic background is common, resistant mutants may arise anew in purely oseltamivir-susceptible infl uenza virus A (H1N1) populations. Identifi cation of such predisposing genetic traits and monitoring of their occurrence in infl uenza viruses A (H1N1) and other infl uenza viruses should continue.
Similar resistance can arise in viruses other than the current human infl uenza viruses A (H1N1). Resistance in a more virulent infl uenza virus can have serious public health implications because of fewer therapeutic and prophylactic options, which may result in more persons being affected by infl uenza and more severe illness and death in those who become infected. Oseltamivir is a prime option for infl uenza treatment and prophylaxis and forms a substantial part of pandemic preparedness in many countries. The prevalence of oseltamivir-resistant viruses reported in Europe throughout the 2007-08 infl uenza season clearly shows that this resistant mutation is stable and that these viruses sustain their fi tness and ability to spread among persons. These fi ndings should be taken into consideration when shaping future strategies for treating and preventing seasonal and pandemic infl uenza.