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Four cases of oseltamivir-resistant influenza A(H1N1)pdm09 virus infection were detected among inhabitants of a border detention center in Texas, USA. Hemagglutinin of these viruses belongs to 6B.1A5A-156K subclade, which may enable viral escape from preexisting immunity. Our finding highlights the necessity to monitor both drug resistance and antigenic drift of circulating viruses.

We report characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 viruses and patients infected with these viruses in the United States. During 2013–14, fifty-nine (1.2%) of 4,968 analyzed US influenza A(H1N1)pdm09 viruses had the H275Y oseltamivir resistance–conferring neuraminidase substitution. Our results emphasize the need for local surveillance for neuraminidase inhibitor susceptibility among circulating influenza viruses.

The recent increase in zoonotic avian influenza A(H7N9) disease in China is a cause of public health concern. Most of the A(H7N9) viruses previously reported have been of low pathogenicity. We report the fatal case of a patient in China who was infected with an A(H7N9) virus having a polybasic amino acid sequence at its hemagglutinin cleavage site (PEVPKRKRTAR/GL), a sequence suggestive of high pathogenicity in birds. Its neuraminidase also had R292K, an amino acid change known to be associated with neuraminidase inhibitor resistance. Both of these molecular features might have contributed to the patient's adverse clinical outcome. The patient had a history of exposure to sick and dying poultry, and his close contacts had no evidence of A(H7N9) disease, suggesting human-to-human transmission did not occur. Enhanced surveillance is needed to determine whether this highly pathogenic avian influenza A(H7N9) virus will continue to spread.

Stockpiling drugs to prevent and treat influenza would be economically effective., We compared strategies for stockpiling neuraminidase inhibitors to treat and prevent influenza in Singapore. Cost-benefit and cost-effectiveness analyses, with Monte Carlo simulations, were used to determine economic outcomes. A pandemic in a population of 4.2 million would result in an estimated 525–1,775 deaths, 10,700–38,600 hospitalization days, and economic costs of $0.7 to $2.2 billion Singapore dollars. The treatment-only strategy had optimal economic benefits: stockpiles of antiviral agents for 40% of the population would save an estimated 418 lives and $414 million, at a cost of $52.6 million per shelf-life cycle of the stockpile. Prophylaxis was economically beneficial in high-risk subpopulations, which account for 78% of deaths, and in pandemics in which the death rate was >0.6%. Prophylaxis for pandemics with a 5% case-fatality rate would save 50,000 lives and $81 billion. These models can help policymakers weigh the options for pandemic planning.

Analysis of mutations I117V and I117M in the neuraminidase of influenza A pandemic (H1N1) 2009 viruses showed that I117V confers a mild reduction in oseltamivir sensitivity and has a synergistic effect of further increasing resistance when combined with H275Y. Contrary to recent reports, the I117M mutation does not alter oseltamivir sensitivity. Published Version

Oseltamivir is 1 of 2 antiviral medications available for the treatment of influenza B virus infections. We describe and characterize a cluster of influenza B viruses circulating in North Carolina with a mutation in the neuraminidase active site that may reduce susceptibility to oseltamivir and the investigational drug peramivir but not to zanamivir.

To the Editor: Oseltamivir is a neuraminidase inhibitor approved for treatment and prevention of influenza virus infection. Oseltamivir resistance caused by a single amino acid substitution from histidine (H) to tyrosine (Y) at position 274 of the neuraminidase active site has been reported in persons infected both experimentally and naturally with influenza A virus subtype H5N1 (1,2). Evidence suggests that using lower doses of oseltamivir or shorter-than-recommended treatment periods can contribute to emergence of viral resistance (1,3) Currently, oseltamivir is being used in several countries that may be affected by epidemics of H5N1. Therefore, monitoring for oseltamivir-resistant strains of H5N1 during oseltamivir administration is essential for outbreak management and prevention. Although real-time PCR or pyrosequencing is more rapid than high-throughput assays for mutation detection (4,5), the conventional PCR technique can be applied to detect drug-resistant mutation (6) in areas lacking real-time PCR or pyrosequencing capabilities. Therefore, to discriminate between oseltamivir-sensitive and oseltamivir-resistant strains, we developed a simple method, based on PCR, which takes advantage of the H274Y substitution. The forward primer was designed from the conserved region common to both wild-type and mutant strains; the reverse primers were designed specifically for wild-type and mutant strains, respectively, derived from the 3´ terminal base of each primer. The primers consisted of a forward primer N1f (nt 517-534: 5´-GGGGCTGTGGCTGTATTG-3´) and reverse primer H274r (nt 759-784: 5´-GGATAACAGGAGCAYTCCTCATAGTG -3´) for wild-type strain detection or Y274r (nt 759-784: 5´-GGATAACAGGAGCAYTCCTCATAGTA-3´) for mutant strain detection. Both strains yielded products of ≈267 bp; hence, the assay consisted of 2 separate reactions for detecting wild-type and mutant strains, respectively. For each reaction, 1.0 μL cDNA was combined with a reaction mixture that contained 10 μL 2.5× MasterMix (Eppendorf, Hamburg, Germany), forward and reverse primers at a final concentration of 0.15 μM, and nuclease-free water to a final volume of 20 μL. Thermocycling conditions comprised initial denaturation at 94°C for 3 min and 35 cycles of amplification including denaturation (94°C, 30 s), annealing (65°C, 50 s), extension (72°C, 45 s), and final extension (72°C, 7 min). Subsequently, 10 μL of the amplified products was analyzed by using 2% agarose gel electrophoresis. To optimize the assay, we performed PCR-based H274Y mutagenesis of the N1 fragment of the H5N1 virus (primers on request). The resulting mutagenic and wild-type products were cloned into the pGEM-T Easy Vectors (Promega, Madison, WI, USA), confirmed by direct sequencing, and then used as positive controls. Preliminary results showed that the wild-type primer was specific for the oseltamivir-sensitive strain, whereas the mutant primer can be used to detect the oseltamivir-resistant strain exclusively because no significant cross-amplification had been observed. To establish sensitivity, serial 10-fold dilutions of the standard N1 plasmids (wild-type and mutant) ranging from 109 to 101 copies/μL were used as a template. The threshold concentrations for detection of wild-types and mutants were 103 copies/μL. To provide semiquantitative data to detect subpopulations of the resistant variants, the 2 control plasmids were mixed at wild-type:variant ratios of 108:102, 107:103, 106:104, 105:105, 104:106, 103:107, and 102:108. The result showed that the density of the expected bands depended on the amount of DNA templates (Figure, B). However, the mixing experiments indicated that the predominant mixtures of wild-type:resistant variant were 80:20, which is the lowest ratio of resistant variants that the assay can reliably detect (data not shown). To assess specificity, human DNA and viral cDNA extracted from other subtypes of influenza A virus (N2–N9) were subjected to this assay. No cross-reaction occurred with human DNA or other subtypes of influenza A virus. Figure A) Representative result from conventional PCR that used H274r primer for oseltamivir-sensitive and Y274r primer for oseltamivir-resistant detection in samples isolated from human plasma (P), tiger (T), and Vietnamese patient (V). Plasmids containing ... We further validated the assay by testing 3 specimens from hosts treated with oseltamivir and 17 specimens from untreated hosts; infection with H5N1 was detected by using multiplex real-time PCR (7). The specimens from oseltamivir recipients were isolated from a Vietnamese patient on the third day after he received a prophylactic dose (1) and from 2 tigers (CU-T7 and KU-11) (8). The specimens from untreated hosts were isolated from the plasma of an H5N1-infected human (9) and from virus-containing allantoic fluid isolated from infected chickens, ducks, and cats (n = 16) during a 2005 outbreak in Thailand. The specimen isolated from the Vietnamese patient yielded detectable bands after amplification by wild-type and mutant primer sets, which indicates that this specimen contains mixed populations of wild-type and resistant strains (Figure, A). The result was confirmed by cloning the amplicon of the Vietnamese strain into the pGEM-T EASY vector (Promega). Ten colonies were randomly picked and sequenced; 9 clones were mutant, and 1 was wild type. The other specimens produced a visible positive band only on amplification using the wild-type primer set, which indicates that samples containing these strains were oseltamivir sensitive. The assay described here provides an accurate, cost-effective, and highly efficient approach to detecting oseltamivir-sensitive and oseltamivir-resistant H5N1 strains; it is based on conventional PCR and takes advantage of the H274Y substitution within the neuraminidase gene. This simple technique can be applied in all laboratories that lack more advanced and expensive instruments; thus, it provides a valuable tool for managing and preventing influenza A H5N1 outbreaks. Concerning the spread of mutant viruses, however, the fitness of the viruses needs further investigation.

To the Editor: In the Netherlands, the outbreak of pandemic influenza A (H1N1) 2009 led to a 100-fold increase from 2008 in prescriptions for the antiviral neuraminidase inhibitor oseltamivir (1). The guidelines for prescribing oseltamivir during the 2009 pandemic were adapted throughout the year. After August 7, prescribers were advised to restrict prescriptions to patients with influenza symptoms plus 1 additional risk factor (2) (Table). Table Reported risk factors of patients with and without influenza symptoms who were dispensed a prescription for oseltamivir, the Netherlands, 2009–10 Community pharmacists dispensed oseltamivir as a 5-day course of sachets produced exclusively for the Dutch government program and documented all prescriptions. Our objective was to assess whether oseltamivir dispensed through community pharmacies was prescribed according to the national guideline for the pandemic virus and to investigate how patients used oseltamivir. The Institutional Review Board of the Division of Pharmacoepidemiology and Clinical Pharmacology of Utrecht University approved the study. Pharmacists in 19 pharmacies belonging to the Utrecht Pharmacy Practice Network for Education and Research (UPPER) selected all patients who had filled a prescription for oseltamivir during May 1, 2009–February 8, 2010. These patients were contacted by phone and, after giving consent, completed a structured questionnaire. The questionnaire contained questions about potential risk factors, the reason for receiving the oseltamivir prescription (influenza symptoms or other reasons), and whether the oseltamivir course was started and completed. Of the 630 patients eligible for contact, 361 (57.3%) completed the questionnaire. To assess whether the current guidelines were adhered to, because of the changes in policy throughout the year, we analyzed only the 300 respondents who had filled the oseltamivir prescription at the height of the pandemic, i.e., after August 7, 2009. A total of 156 (52.0%) participants were female patients; most participants were 18–59 years of age. Of the 212 patients >18 years of age, education level was available for 195; of these, 55 (28.2%) had a low education level, 94 (48.2%) a middle education level, and 46 (23.6%) a high education level. Of the 300 respondents, 111 (37.0%) received a prescription while they did not meet guideline criteria (Table). They had risk factors but did not experience influenza symptoms (67 [22.3%] of all respondents); had influenza symptoms but not risk factors (34 [11.3%]); or had neither influenza symptoms nor any risk factors (10 [3.3%]). Compared with respondents who had a low education level, respondents >18 years of age who had a middle or high education level were 2× more likely to receive an oseltamivir prescription that was not in accordance with guideline criteria (odds ratio 2.20; 95% CI 1.12–4.32). Sex and age were not associated with the likelihood of receiving off-guideline oseltamivir. Of the 189 respondents who received oseltamivir in accordance with guideline criteria, 184 (97.4%) started treatment and 167 (90.8%) completed the oseltamivir course. Of the 111 respondents who received a prescription for oseltamivir that was not in accordance with guideline criteria, 62 (55.9%) started treatment, and 56 (90.3%) completed the course. We showed that during the pandemic the guideline criteria were not met by nearly one third of patients who received an oseltamivir prescription. Patients with a higher education level more often received a prescription, suggesting that they are more informed or empowered than patients with a lower education level to request a prescription. Another explanation for the inadequate adherence to guideline criteria is that prescribers themselves were not immediately aware of the current criteria, possibly because of changes throughout the year. In addition, in nearly half of instances in which guideline criteria were not met but in which oseltamivir was prescribed, the patients did not start the oseltamivir course. These prescriptions could have been used for stockpiling, which also occurred during the influenza A (H5N1) outbreak in 2005 (3). In the Netherlands, stockpiling did not lead to drug shortages, but in countries where oseltamivir is not reimbursed by the government, stockpiling might lead to problems with availability for patients truly in need of antiviral therapy but without the necessary means to acquire it. The limited effect of oseltamivir on reducing disease duration, usually only shortening the duration by 1 day in healthy persons (4), the possibility of serious side effects (5), the possibility of the virus developing resistance to neuraminidase inhibitors (6,7), and the cost to health care of unnecessary prescriptions are reasons to strive for better adherence to prescribing guidelines. Prescribers need to be properly informed about current guidelines to reduce overprescribing caused by lack of knowledge. Furthermore, improving communication between prescribers and patients might help relieve patients’ concerns and increase awareness about the limited benefits of oseltamivir treatment in healthy persons.

To the Editor: In March 2009, a new strain of influenza A (H1N1) virus of swine origin emerged; the virus had crossed the species barrier to humans and acquired the capability of human-to-human transmission. Soon after, the World Health Organization raised the worldwide pandemic alert to level 6 (www.who.int/en), declaring the first influenza pandemic in the past 42 years. The virus was named influenza A pandemic (H1N1) 2009 virus. The illness caused by this virus is particularly dangerous for pregnant women and for patients with chronic diseases (1). The preferred treatment is a neuraminidase inhibitor, zanamivir or oseltamivir (2). Around the world, several dozen cases of resistance to oseltamivir in persons with or without exposure to the drug have been reported (3). However, only limited information is available with regard to initial infections with oseltamivir-resistant viruses (4). We report a case of possible human-to-human transmission of pandemic (H1N1) 2009 virus in Israel. After the recent discovery of oseltamivir-resistant strains, we conducted a retrospective study of oseltamivir-resistance mutations in viral RNA amplified from specimens from patients hospitalized >1 week with pandemic (H1N1) 2009. All samples were first tested for the H275Y mutation by using an in-house real-time reverse transcription–PCR (RT-PCR) assay developed at the Central Virology Laboratory of Chaim Sheba Medical Center; positive results were confirmed by sequencing. The histidine-to-tyrosine mutation at the 275 position of the neuraminidase protein results in reduced binding of oseltamivir. During June–August 2009, ≈80 children in an institution for disabled children were suspected of being infected with pandemic (H1N1) 2009 virus. The children had influenza-like signs and symptoms, and at that time the only influenza virus circulating in Israel was pandemic (H1N1) virus. Of these 80 patients, 10 were hospitalized because of the severity of their clinical signs or disease complications, and for 7, RNA of the pandemic (H1N1) 2009 virus was detected in throat and nasal swabs by real-time RT-PCR. Patient 1 was a 13-year-old boy with cerebral palsy and partial blindness, who was treated with oseltamivir (60 mg twice a day) for 5 days (July 27–31, 2009). After some improvement, his condition worsened, and he was hospitalized on August 13 for breathing difficulty and high fever. Real-time RT-PCR indicated infection with pandemic (H1N1) 2009 virus. During our survey, we found that patient 1 was infected with a virus carrying the H275Y mutation, suggesting that the mutation evolved during oseltamivir treatment. Patient 2 was a 10-year-old girl who had lived in the room next to patient 1 and who also had cerebral palsy. Her signs and symptoms started on August 13, 2009, and she was hospitalized on August 15. She was treated in the hospital for 5 consecutive days with oseltamivir, steroids, and augmentin; she was discharged on August 21. Her diagnosis was made early in the clinical course of her infection, and she was infected with pandemic (H1N1) 2009 virus carrying the H275Y mutation. In contrast, none of the other 8 children who were hospitalized for pandemic (H1N1) 2009 carried the mutation. Although we cannot rule out the possibility that the virus was transmitted by a third person, we suggest that the virus carrying the resistance mutation was probably transmitted from patient 1 to patient 2. This transmission is probable because these 2 patients lived in adjacent rooms, they were in contact with each other, clinical onset of patient 1 preceded that of patient 2 by a few days, and patient 2 had the mutation at the beginning of her disease. Fortunately, despite the conditions that favor virus spread in such institutions, this virus did not seem to spread further; the other 8 hospitalized children from this institution were infected with the wild-type virus. Nevertheless, the potential for spread of pandemic (H1N1) 2009 virus carrying the oseltamivir resistance mutation exists, thereby emphasizing the urgent need for a vaccination to prevent illness and for alternative drugs to treat infected patients.

To the Editor: Each year, approximately 2 million Muslims travel from all over the world to participate in hajj. Approximately 22,000 pilgrims travel from the United Kingdom to Makkah, Saudi Arabia; of those, approximately 1,000 pilgrims reside in the east end of London. In the past, infectious diseases research conducted during these pilgrimages focused on meningococcal disease because of outbreaks associated with the hajj. Since 2000, the dates of the hajj have been moved back into the winter season; this time change could lead to a seasonal increase in outbreaks of respiratory infections caused by influenza and other viruses. From 1991 to 1992, influenza A was a common cause of respiratory infection in pilgrims tested in Makkah (1). However, the incidence rate of influenza among pilgrims from Europe is not well-known. A previous study of influenzalike illness among pilgrims from Pakistan reported rates of 36% in influenza-vaccinated pilgrims and 62% in influenza-nonvaccinated pilgrims; these results were based on clinical endpoints without microbiologic confirmation (2). We assessed the risk for influenza infection among a cohort of pilgrims from the east end of London who participated in the hajj in 2003. From December 2002 to January 2003, we enrolled 115 participants who planned to take part in hajj in 2003. The study was approved by the North London Multicentre Research Ethics Committee and the Trustees of East London Mosque. Informed consent was obtained through appropriate translators. All participants attended the East London Mosque, Whitechapel, London; 30 were vaccinated with influenza vaccine (A/New Caledonia/20/99 [H1N1]-like strain, A /Moscow/10/99 [H3N2]-like strain, B/Sichuan/379/99-like strain). Venous blood samples were collected, and questionnaires were completed before the participants departed for the hajj and within 2–3 weeks of their return in February to March 2003. Tests for influenza A and B were conducted by using hemagglutination inhibition against the following influenza antigens: A/NewCalidonia/20/99, A/Wuhan/371/91, A/Sydney/5/97, A/Panama/2007/99, B/Sichuan/379/99, and B/Harbin/7/94. A diagnosis of influenza was made based on seroconversion with at least a fourfold rise in antibody titer. Based on seroconversion, the influenza attack rate among all pilgrims was 38% (44/115). The attack rate was 30% among the vaccinated and 41% among the nonvaccinated participants (Table) (odds ratio for influenza in vaccinees = 0.61, p = 0.28). Of the 44 patients, 42 (37%) were infected with influenza A H3N2; 1 had influenza A H1N1, and 1 had influenza B infection. Six influenza A H3N2 patients were dually infected; two patients seroconverted to A H1N1, and four patients seroconverted to influenza B. Nearly half (21/44) of the patients with influenza received a course of antimicrobial drugs while on the hajj compared with 38% (27/71) of those who did not seroconvert. The attack rate in the vaccinated patients was lower than the rate in nonvaccinated patients, which is consistent with some protective effect of the influenza vaccine. Table Seroconversion and respiratory symptoms due to influenza infection and vaccination status among U.K. pilgrims Even though blood was collected from five convalescing patients within 3 weeks of their return from the hajj, some of the patients may have acquired influenza B infection immediately after their return to the United Kingdom, as it was the main strain circulating in the United Kingdom in late February to March 2003. Many pilgrims from throughout the world, some of whom may carry H3N2 drift variants, mingle closely during the hajj. This type of exposure increases the risk for worldwide spread of new drift variants and other contagious respiratory diseases (3). Given the potential for the high influenza attack rate documented in this study, all pilgrims, regardless of age, should be offered influenza vaccination before they travel on the hajj during winter months. On-site testing for influenza should be available to medical services in Makkah (and countries of origin), and treatment with a neuraminidase inhibitor should be offered to persons who test positive and have been symptomatic for