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Short communication
Neuraminidase-inhibitor resistance testing for pandemic influenza A (H1N1) 2009 in Ontario, Canada Jean Longtin a,b,∗ , Samir Patel a , Alireza Eshaghi a , Ernesto Lombos a , Rachel Higgins a , David Alexander a,d , Romy Olsha a , John Doyle e , Dat Tran e , Alicia Sarabia f , Christine Lee g , Nathalie Bastien c , Yan Li c , Donald Low a,d , Guy Boivin b , Jonathan Gubbay a,d,e a
Ontario Agency for Health Protection and Promotion, Public Health Laboratories, 81 Resources Road, Toronto M9P 3T1, Canada Infectious Diseases Research Center, CHUQ, Québec, Québec, Canada c Influenza and Respiratory Viruses Section, National Microbiology Laboratory, Public Health Agency of Canada, Canada d Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada e The Hospital for Sick Children, Toronto, Ontario, Canada f Credit Valley Hospital, Mississauga, Ontario, Canada g Medical Microbiology, St Joseph’s Healthcare, Hamilton, Ontario, Canada b
a r t i c l e
i n f o
Article history: Received 19 October 2010 Received in revised form 2 December 2010 Accepted 12 December 2010 Keywords: Influenza A Neuraminidase-inhibitors Resistance
a b s t r a c t Background: Oseltamivir resistance-associated H275Y mutation in the neuraminidase (NA) gene of pandemic influenza A (H1N1) 2009 was occasionally reported worldwide during the 2009–2010 influenza season. A significant proportion of those were found in immunocompromised or severely ill persons. This phenomenon remains infrequent and clear recommendations for resistance testing are lacking. Objectives: Present the suggested clinical selection criteria for antiviral susceptibility testing for influenza in Canada and to describe the Ontarian experience during the 2009–2010 influenza season. Study design: Using a defined algorithm, we prospectively screened for OsR with pyrosequencing and phenotypic testing during the 2009–2010 influenza season. Zanamivir resistance was screened using phenotypic and sequencing technique on selected occasions. Clinical data was gathered for the resistant cases. Results: A total of 804 clinical H1N1 (2009) positive samples from Ontario were screened for oseltamivir resistance between June 2009 and March 2010. We identified oseltamivir resistance in 5 (0.6%) distinct patients aged 9–62 years. All the resistant strains bore the H275Y mutation. Susceptibility to zanamivir was maintained in all of them. Three patients harboring oseltamivir resistant strain were intensive care unit patients and four were immunocompromised. All were tested for susceptibility because of a repeat positive result for influenza A PCR. Conclusion: Oseltamivir resistance was not frequent during the 2009–2010 influenza season but was identified with a systematic and prospective approach to resistance testing. In order to be as sensitive as possible in the detection of those few cases, we report the suggested indications for antiviral susceptibility testing in Canada. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Background Influenza viruses can cause significant morbidity and mortality in certain populations and oseltamivir is advocated as first-line treatment for pandemic H1N1 (2009) virus. Surveillance
Abbreviations: H275Y, histidine to tyrosine mutation of residue 275 of the neuraminidase gene; IC50, 50% inhibitory concentrations; NA, neuraminidase; NAI, neuraminidase inhibitor; OAHPP, Ontario Agency for Health Protection on Promotion; H1N1 (2009), pandemic influenza A (H1N1) 2009. ∗ Corresponding author at: Infectious Disease Research Center, Laval University, 2705 Laurier blvd, Room RC-709, Quebec City, Quebec, Canada G1V 4G2. Tel.: +1 418 656 4141x47882; fax: +1 418 654 2194. E-mail address:
[email protected] (J. Longtin).
for antiviral resistance in Canadian H1N1 (2009) isolates revealed generalized resistance to adamantanes by a S31N mutation in the matrix gene.1,2 Sporadic resistance to oseltamivir by a histidine to tyrosine mutation of residue 275 (H275Y) of the neuraminidase (NA) gene was reported in 319 cases of H1N1 (2009) around the world since it emerged.3 The first Canadian case of oseltamivir resistance was reported from Québec in June 2009 and a total of 12 cases were reported in Canada during the 2009–2010 influenza season.1,4 2. Objectives We discuss the different approaches to screen, confirm and characterize antiviral resistance that were put in place for the first and
1386-6532/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2010.12.007
Please cite this article in press as: Longtin J, et al. Neuraminidase-inhibitor resistance testing for pandemic influenza A (H1N1) 2009 in Ontario, Canada. J Clin Virol (2011), doi:10.1016/j.jcv.2010.12.007
Healthcare setting
Underlying conditions setting
Date of illness
Details of NAI therapy onset
Influenza A/subtyping/OsR testing Test date
1
25 years, MICU
Os, August 10–20 August 9
September 1 Os, August 25–September 18 September 7 September 9
Outcome
Other clinical information
24 days intermittent therapy
None
16 days
Recovered
Parainfluenza coinfection was detected in August 17 and 24 samples. Discharged, readmitted 2 weeks later with fatal EBV infection. Family member had respiratory illness prior to onset of illness in patient. September 7 sample with H275Y mutation was first detected in early October when screened at Ontario PHL as part of surveillance testing
18 days
Zanamivir
18 days
Recovered
October 22 sample tested by rapid test only. Admitted with pneumonia. On steroids for SLE; had concomitant Salmonella Group B bacteremia. Patient made a complete recovery. No reports of respiratory illness in contacts
Result H1N1 2009/H275
FluA H1N1 2009/H275Y
H1N1 2009/H275Y H1N1 2009/H275Y FluA not detected
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August 17 August 24
Duration of shedding
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August 9, 2009 Haematological malignancy, bone marrow transplantation
Duration Os Change therapy prior in therapy to onset OsR due to OsR
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PatientAge, sex
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Table 1 Detailed clinical information for 3 patients with OsR influenza A pandemic (H1N1) 2009.
Patient Age, sex
9 years, F
ICU
Oncology ward
Lupus, asthma, October 22, hypertension 2009
Haematological June 13, 2009 malignancy
Details of NAI therapy onset
Os, September 25–27 Os October 22–November 10
Influenza A/subtyping/OsR testing Test date
Result
September 24
FluA not detected
October 22
FluA
October 28 November 9 November 16 and 17 November 20
H1N1 2009/H275 H1N1 2009/H275Y FluA not detected FluA not detected
Os, June 14–19 June 13
Os, June 30–July 5 Os, August 4–14
H1N1 2009/H275
July 30
H1N1 2009/H275Y
August 26
H1N1 2009/H275Y
September 8 December 1
H1N1 2009/H275Y H1N1 2009/H275Y
Duration Os therapy prior to onset OsR
Change in therapy due to OsR
Duration of shedding
Outcome
Other clinical information
10 days
No antivirals given
122 days
Recovered
Resistance first detected when samples from December 1, 2009 were tested due to persistent shedding of virus at the treating clinician’s request. Well at time of detection of OsR strain. Subsequently earlier samples were forwarded to Ontario PHL and resistance was confirmed on all samples since first detected on July 30, 2009. No reports of respiratory illness in contacts
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3
33 years, F
Date of illness
M: male; F: female; ICU: intensive care unit; Os: oseltamivir; OsR: oseltamivir resistance; FluA: influenza A virus; SLE: systemic lupus erythematosus.
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Healthcare Underlying setting conditions setting
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Table 1 (Continued)
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Table 2 Clinical selection criteria for antiviral susceptibility testing for influenza in Canada.15 1 2 3 4 5 6
Failed therapy (ex ICU patient, 10 days post-treatment) Positive test with influenza-like illness while receiving or after receiving prophylaxis Positive test for Influenza A in a traveler returning from area where resistance is endemic Persistent infection in immunocompromised host Nosocomial transmission in clinical areas with immunocompromised hosts Positive test from a case in contact with immunocompromised case
second waves of the 2009 influenza pandemic in the province of Ontario and present the clinical selection criteria for antiviral susceptibility testing for influenza in Canada that will be in use for the 2010–2011 influenza season. 3. Study design The vast majority of influenza testing in Ontario is done at the Ontario Agency for Health Protection on Promotion (OAHPP) Public Health Laboratories using influenza A PCR. Samples were collected by flocked nasopharyngeal swab, nasopharyngeal aspiration, or bronchoalveolar lavage. Positive samples are subjected to resistance testing upon physician requests for patients who developed influenza after oseltamivir or zanamivir prophylaxis, are severelyill, have persistent viral shedding (defined as a repeat PCR test positive after 7 days or more of treatment), immunocompromised patients and those non-responding to antiviral therapy.5 We also tested random specimens from the community for surveillance purposes and on special request.5 A pyrosequencing method using the WHO published protocols was established as a preliminary screening tool for the H275Y mutation.6 The result was further confirmed by Sanger sequencing of a 550 bp segment of the NA gene.7 Phenotypic testing for the detection of neuraminidase inhibitor (NAI) resistance was measured using a fluorometric method. NAIs were obtained from GlaxoSmithKline Inc. (Zanamivir) and Roche Ltd. (Oseltamivir); concentration used ranged from 0.03 to 1000 nM. We gathered clinical information on resistant cases after obtaining written patient consent under Review Board approval. 4. Results During the 2009–2010 influenza season, we screened 804 influenza A positive clinical samples for NAI resistance using our clinical and surveillance indications. Patient setting was available for 191 samples tested: most were from intensive care units (66%) and other inpatients (23%). Ambulatory patients and long-term care residents represented 6% and 5%, respectively. Risk factor information was available for 132 samples – 59% and 30% were from persons below age 5 years and over 65 years, respectively, and 12% of samples screened were from persons with chronic lung disease. 5% of samples were from patients in whom H1N1 2009 was detected in 2 or more respiratory specimens collected at different times. Two-thirds were tested through the epidemiological surveillance program, chosen randomly from all our positive samples. Resistance to oseltamivir was detected in 5 patients, all bearing the H275Y mutation and was confirmed at Canada’s National Microbiology Laboratory (NML) by sequencing. Phenotypic resistance assay was also conducted when virus could be isolated in culture. No mutations conferring resistance to zanamivir were detected. Mean (±SD) oseltamivir 50% inhibitory concentrations (IC50) were 0.52 (±0.37) nM for susceptible (H275 wt) isolates, and 78.09 (±16.12) nM for isolates from 2 patients with H275Y mutation (Table 1). Mean zanamivir IC50 was 0.75 (±0.54) for oseltamivir susceptible isolates; 6 oseltamivir-resistant isolates (3 strains each from patients 1 and 5) had a mean zanamivir IC50 of 1.13 nM (±0.72), consistent with zanamivir susceptibility. Patients harboring the resistant strains were aged 9–62 years old.
Detailed clinical information for the 3 patients for which consent was obtained is presented in Table 1. All were tested for resistance based upon the clinical criteria of persistence of a positive influenza A PCR result. All 4 of the patients for which clinical information was known were immunocompromised and developed oseltamivir resistance while on oseltamivir therapy; 3 were in ICU during the course of their infection. No patients were epidemiologically linked and no resistant strains were detected in the general population through our surveillance program. 5. Discussion Oseltamivir, zanamivir and peramivir bind in different manners to the NA catalytic site, resulting in different profiles of resistance.8,9 In N1-containing viruses the most usual mutation conferring high-level resistance to oseltamivir is H275Y (H274Y in N2 numbering).10,11 For H3N2 influenza viruses, the predominant NA mutation observed in clinical trials of oseltamivir was R292K with a lower incidence of E119V.9 There is a published clinical occurrence of influenza B zanamivir resistance emerging under zanamivir treatment due to a R152K mutation.12 In vitro studies also identified Q136K as possibly conferring resistance to zanamivir in seasonal influenza A (H1N1) viruses.13 An oseltamivirresistant strain carrying the H275Y mutation remains sensitive to zanamivir while the Q136K zanamivir resistant strain remains sensitive to oseltamivir. However R152K influenza B is resistant to both oseltamivir and zanamivir.12 Resistance in H1N1 (2009) has been caused by the H275Y mutation in all but one case. A recent European report warned of a novel I223R mutation in H1N1 (2009) that conferred resistance to oseltamivir, zanamivir, and peramivir.14 This array of possible resistance mechanism is why we must perform phenotypic testing in addition to the targeted genotypic screening of H275Y. Nonetheless oseltamivir resistant strains were very infrequent in Ontario and Canada during the 2009–2010 influenza season. The Canadian experience reflects what was seen globally at the same time, with only rare reports of oseltamivir resistance.3 However, the same rarity was observed for seasonal influenza prior to 2007 when resistance was restricted to countries with high-levels of oseltamivir use such as Japan.8 A rapid increase in oseltamivir resistance was observed during the 2007–2008 season and by the following year, almost all characterized influenza A/Brisbane/59/2007(H1N1)-like strains from North America and Europe were reported to be resistant to oseltamivir, carrying the H275Y mutation. The emergence of resistance was no longer linked to oseltamivir use suggesting that the NA mutation was no longer affecting viral fitness and transmissibility.8 This phenomenon can occur anytime with the new influenza A/H1N1 2009 pandemic strain and prospective resistance screening may help to minimize the spread of resistant viruses. In our experience no resistant strains were found during routine screening but 5 were identified using defined high-risk criteria. Likelihood of developing resistance increases during prolonged infections and the Canadian Public Health Laboratory Network has put forward suggested indications for targeted susceptibility testing in any type of influenza (Table 2).15 One of our resistant cases had prolonged shedding of at least 4 months and immunocompromised patients have been reported to shed influenza A virus for up to one year.16 Most cases
Please cite this article in press as: Longtin J, et al. Neuraminidase-inhibitor resistance testing for pandemic influenza A (H1N1) 2009 in Ontario, Canada. J Clin Virol (2011), doi:10.1016/j.jcv.2010.12.007
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of oseltamivir resistance reported to WHO to date have occurred in immunocompromised persons (30% of cases) or associated with treatment (32% of cases), which is similar to our observation.3 In conclusion oseltamivir resistance was not frequent in the 2009–2010 influenza season but a sensitive detection algorithm is necessary to optimize patient care. Clinicians should be aware of the potential emergence of NAI resistance during treatment, especially in those patients with prolonged shedding, such as immunocompromised patients. To optimize resistance screening, we report the suggested indications for antiviral susceptibility testing recommended by the Canadian Public Health Laboratory Network. In addition to specific clinical indications, surveillance programs for influenza resistance should be conducted to rapidly identify clusters involving transmission of drug-resistant viruses. Conflicts of interest Jean Longtin, Samir N. Patel, Reza Eshaghi, Ernesto Lombos, Rachel Higgins, David C. Alexander, Romy Olsha, Dat Tran, John Doyle, Alicia Sarabia, Christine Lee, Nathalie Bastien and Yan Li: no conflicts of interest. Donald E. Low participated in advisory board committee meetings for GlaxoSmithKline Inc. and Hoffman-La Roche. He has also received research funding from both companies. Guy Boivin has received research grants from Hoffmann La Roche to work on resistance to neuraminidase inhibitors. Jonathan B. Gubbay has received a research grant from GlaxoSmthKline Inc. to work on resistance to neuraminidase inhibitors. General. In June 2010, OAHPP received a research grant from GlaxoSmithKline to study phenotypic resistance in influenza virus. Acknowledgments None.
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