Marys Medicine



Original Research Oseltamivir resistance among infl uenza viruses: surveillance in northern Viet Nam, 2009–2012Hoang Vu Mai-Phuong,a Nguyen Co Thach,a Nguyen Le Khanh Hang,a Nguyen Thi Kim Phuongb and Le Quynh MaiaCorrespondence to Hoang Vu Mai-Phuong (e-mail: [email protected] or [email protected]).
Introduction: Antiviral resistance has been reported in seasonal influenza A viruses and avian influenza A(H5N1) viruses
in Viet Nam, raising concerns about the efficacy of treatment.
Methods: We analysed specimens from two sources during the period 2009–2012: influenza-positive samples from
influenza-like illness patients at sentinel clinics in northern Viet Nam and isolates from patients with confirmed A(H5N1)
infections. Pyrosequencing was used to detect mutations: H275Y [for A(H1N1) and A(H5N1)], E119V [for A(H3N2)]
and I117V [for A(H5N1)]. A neuraminidase inhibition assay was used to determine the Inhibitory Concentration 50 (IC )
values for all influenza A and B isolates.
Results: There were 341 influenza A positive samples identified; influenza A(H1N1)pdm09 was identified most frequently
(n = 215). In 2009, oseltamivir resistance was observed in 100% (19 of 19) of seasonal A(H1N1) isolates and 1.4%
(3/215) of A(H1N1)pdm09 isolates. This H275Y mutation was not found in influenza subtypes A(H5N1) or A(H3N2)
Discussion: In Viet Nam, seasonal and A(H5N1) influenza vaccines are not currently available; thus, effective treatment is
required. The presence of oseltamivir-resistant viruses is therefore a concern. Active surveillance for oseltamivir resistance
among influenza viruses circulating in Viet Nam should be continued.
Influenza infection causes annual epidemics throughout for the treatment of influenza infections,4,5 yet antiviral
the world. There are two common types of influenza resistance to influenza A viruses is increasingly being
viruses that cause human infection – influenza A and reported.6,7 Oseltamivir is currently recommended as the
influenza B. Influenza A viruses caused several influenza first-line option by the Viet Nam Ministry of Health for
pandemics in the 20th century, and a pandemic treating suspected infections of A(H5N1) and A(H1N1)
caused by the influenza A(H1N1)pdm09 virus occurred pdm09. The emergence of oseltamivir resistance
in 2009.1 National influenza surveillance was initiated of clinical isolates of influenza A virus has been
in Viet Nam in 2006, and the data collected so far associated with substitution at residue V116, I117,
have shown that influenza viruses circulate year-round E119, Q136, K150, D151, D199, I223, H275 and
with similar peaks and subtypes observed across N295 in the neuraminidase active site.8 For influenza
all surveillance regions.2 Between 2003 and 2012, B there have been two main substitutions: residues
123 human cases of A(H5N1) infection were confirmed R152 and D198.8,9 In Viet Nam, oseltamivir-resistant
from 40 of the 63 provinces in Viet Nam, with 81 cases strains harboring mutations at positions I117V, H275Y
(66%) from northern Viet Nam.3 Although influenza and N295S were reported for A(H5N1) in 2005,6
vaccines that protect against A(H1N1)pdm09 or A(H1N1) in 200710 and A(H1N1)pdm09 in 2009.7,11
influenza A(H5N1) are being developed in Viet Nam, The limitations of other antiviral drugs, as well as the risk
they are currently only available through private market of oseltamivir resistance, have raised concerns about the
efficacy of oseltamivir for influenza infection treatment. We report here on a pilot study for the establishment of The neuraminidase inhibitors oseltamivir and a routine antiviral resistance surveillance programme in zanamivir are the primary antiviral agents recommended northern Viet Nam.
National Infl uenza Center, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam.
b Military Hospital 108, Hanoi, Vietnam.
Submitted: 21 March 2013; Published: 26 June 2013doi: 10.5365/wpsar.2013.4.1.010 WPSAR Vol 4, No 2, 2013 doi: 10.5365/wpsar.2013.4.1.010 Influenza virus oseltamivir resistance, northern Viet Nam, 2009–2012 MATERIALS AND METHODS
was conducted using the Qiagen PyroMark PCR kit
with specific primer sets for A(H1N1)pdm09, seasonal
A(H1N1), A(H3N2) and A(H5N1).12,13 Three sets of
As an initial step in establishing a surveillance programme RT–PCR primers were used to generate corresponding for antiviral resistance in northern Viet Nam, genetic amplicons of the neuraminidase gene segment covering analysis was conducted for both clinical specimens and the sequences encoding the target residues 117, isolates collected through sentinel sites and isolates 119 and 275 according to procedures described of influenza A(H5N1). Neuraminidase activity was previously.14
measured using a phenotypic method for viral isolates of influenza A and B. Pyrosequencing assays were then applied to detect the common mutations related The pyrosequencing reactions and data analysis to reducing susceptibility or resistance of influenza A were performed using a PyroMark Q24 ID Platform viruses to oseltamivir – I117V, E119V and H275Y. (Qiagen, USA). Briefly, biotinylated PCR products Data from the National Influenza Surveillance System in were washed through a series of buffers, and single-Viet Nam were also analysed for the period 2009–2012.
stranded DNA was generated and used as a template for hybridization to residue-specific sequencing primers, Source of samples
which were used at a concentration of 100 μM.11,15
Throat swabs were collected as part of the sentinel Neuraminidase inhibition (NAI) assay
surveillance system for influenza-like illness in
northern Viet Nam between 2009 and 2012 and were Oseltamivir carboxylate (GS4071) and its active form
screened using standard methodology (conventional (GS4104) were provided by Roche Laboratories, Inc,
reverse transcriptase polymerase chain reaction, Basel, Switzerland. The reference influenza viruses
RT–PCR) at the National Influenza Center of the National were provided by the World Health Organization (WHO)
Institute of Hygiene and Epidemiology in Hanoi.2 Collaborating Centre for Reference and Research on
Influenza isolates were then cultured from throat swabs Influenza, Melbourne, Australia. Influenza A(H5N1)
positive for influenza A and B.2,6 In addition, isolates isolates were inactivated by 1% formalin for 24 hours
from throat swabs, pharyngeal swabs or tracheal swabs before the NAI assay. The NAI assay was performed
collected from A(H5N1)-infected patients admitted according to procedures described previously.8
to intensive care units of general hospitals in northern
Viet Nam between 2009 and 2012 were obtained.
Madin-Darby canine kidney (MDCK) cells, obtained During the period 2009–2012, there was year- from the American Type Culture Collection, were used to round circulation of seasonal influenza viruses with
culture viruses. Swabs positive for influenza A(H5N1) frequent co-circulation of influenza A and influenza B
by RT–PCR were inoculated biosafety level III culture (Figure 1). A total of 341 influenza A positive samples
facilities. Viruses were harvested and stored at −80 ˚C were identified by RT–PCR (Table 1). Of these, influenza
for further analysis. All influenza isolates with a minimum A(H1N1)pdm09 was identified most frequently
of eight haemagglutination units by haemagglutination (n = 215) throughout the whole study period; 100 were
inhibition assay were selected for neuraminidase A(H3N2) and seven were A(H5N1). Seasonal A(H1N1)
inhibition assay.6,8,10,11
was isolated only in 2009 (n = 19).
Oseltamivir resistance, as determined by detection of H275Y in the neuraminidase gene, was observed in Pyrosequencing assays were used to further characterize 100% (19 of 19) of seasonal A(H1N1) isolates in 2009
all RT–PCR influenza-positive samples (n = 341) and and was identified in 1.4% (3/215) of A(H1N1)pdm09
influenza isolates (n = 67). Viral RNA was extracted isolates collected in 2009. This H275Y mutation was
directly from clinical specimens or from supernatants not found in influenza subtypes A(H5N1) or A(H3N2)
of isolates propagated in MDCK cells by using a viral isolates. I117V was not observed in any of the isolates of
RNA extraction kit (Qiagen, USA) according to the subtypes A(H1N1), A(H5N1) or A(H1N1)pdm09; I119V
manufacturer's instructions. RT–PCR amplification also was not found in any A(H3N2) isolates (Table 1).
2 WPSAR Vol 4, No 2, 2013 doi: 10.5365/wpsar.2013.4.1.010 Influenza virus oseltamivir resistance, northern Viet Nam, 2009–2012 Figure 1. Percentage of influenza viruses by subtype, northern Viet Nam, 2009 to 2012
There were 67 isolates that underwent NAI assay; Table 1. Influenza A subtypes and resistance mutations
six seasonal A(H1N1), 14 A(H3N2), seven A(H5N1), identified by year, northern Viet Nam,
27 A(H1N1)pdm09 and 13 influenza B. All of the 2009 to 2010
seasonal A(H1N1) isolates (n = 6) had Inhibitory Concentration 50 (IC ) values that ranged from Infl uenza A subtypes
541.61 to 703.48 nM higher than the reference virus (A/Mississippi/3/2001, oseltamivir-resistant) and reached 1000-fold higher than the reference wild-type virus (A/Mississippi/3/2001, oseltamivir-sensitive). Among the 27 viruses of A(H1N1)pdm09, IC values ranged from 118.59 to 127.91 nM and reached 250-fold higher than the reference wild type virus. The IC values obtained from non-mutant viruses [A(H1N1)pdm09, A(H3N2), A(H5N1) or influenza B] had median IC ranging from 1.07 nM [A(H1N1)pdm09] to 8.56 nM [A(H5N1)] and 24.79 nM (B), i.e. sensitive to oseltamivir (Table 2).
The A(H1N1)pdm09 virus was first detected in June 2009 in Viet Nam and was the predominant virus Our study of oseltamivir-resistant influenza viruses during the 2009 and 2011 influenza seasons. However, in northern Viet Nam shows that seasonal A(H1N1) in this study, the oseltamivir-resistent mutation (H275Y) isolates circulating in 2009 were oseltamivir-resistant by was identified in only two specimens in 2009 and one virtue of having the H275Y mutation and IC values in 2011, a rate of 1.5% in 2009. A separate cluster indicative of resistance. This finding is consistent with of seven cases of oseltamivir-resistant A(H1N1)pdm09
reports from Japan, the United States of America and was also reported from Viet Nam in July 2009.5
Europe of high rates of resistance (100%) during the The rate from this study is consistent with that collected
2008 to 2009 season.12,13,15 WHO also reported the through the WHO Global Influenza Surveillance and
spread of resistant A(H1N1) strains worldwide during Response System of 1.7% frequency of resistance of
that time.
A(H1N1)pdm09 in the first two years of the pandemic WPSAR Vol 4, No 2, 2013 doi: 10.5365/wpsar.2013.4.1.010 Influenza virus oseltamivir resistance, northern Viet Nam, 2009–2012 Table 2. The IC of oseltamivir carboxylate of non-mutant and mutant influenza viruses (H275Y) by subtype,
northern Viet Nam, 2009 to 2012
Infl uenza subtype
IC median (nM)
A(H1N1) (n = 6)
A/Viet Nam/32036/2009 A/Viet Nam/ELI197/2009 A/Viet Nam/Q271/2009 A/Viet Nam/31808/2009 A/Viet Nam/34381/2009 A/Viet Nam/N116/2009 A(H1N1)pdm09 (n = 27)
A/Viet Nam/33419/2009 A/Viet Nam/36530/2011 A/Viet Nam/DN42/2009 A/H3N2 (n = 14)
A(H5N1) (n = 7)
Infl uenza B
IC – Inhibitory Concentration 50 (2009–2010).16,17 Similar data reported elsewhere influenza A(H3N2) and A(H1N1)pdm09 and the quality
ranged from 0.5% in the United States of America of samples caused a reduced number of viral isolates
to 0.8% in the United Kingdom and 1.1% in the to be tested. Also, it should be noted that phenotypic
data (and sequencing) can only indicate that a virus is resistant: a direct relationship between IC values In our study, A(H5N1), A(H3N2) and B viruses and actual clinical resistance is yet to be proven. were determined to be oseltamivir-sensitive by genotypic Our data represent northern Viet Nam only and might and phenotypic testing. These results are reassuring for not provide an accurate picture of the prevalence of future treatment of A(H5N1) infections in Viet Nam with oseltamivir-resistant viruses in the whole country. oseltamivir, as the A(H5N1) influenza vaccine is not The data collected from the national influenza available. However, an oseltamivir-resistant A(H5N1) surveillance system did not report periodic antiviral use, virus was reported in human isolates in 2005, and and therefore it is difficult to assume that any resistance the emergence of mutations associated with reducing found in our study was due to transmitted resistance. susceptibility (I117V) to oseltamivir was also determined Active surveillance in the future should be expanded to among A(H5N1) isolates from both human and poultry include data on oseltamivir use in hospitals and private in 2009–2010. Thus, continuing oseltamivir-resistance clinics.
surveillance is critical for public health as oseltamivir is the most widely used antiviral medication for H5N1 In conclusion, phenotypic and sequencing data indicated that oseltamivir resistance was present in seasonal A(H1N1) and A(H1N1)pdm09 subtypes in This study is subject to several limitations. Viet Nam during the period 2009–2012. An increase The main limitation is the sample size of both biological of antiviral-resistant influenza viruses might occur in and viral isolates, as we experienced difficulty in growing Viet Nam in the future. Enhancing active surveillance by 4 WPSAR Vol 4, No 2, 2013 doi: 10.5365/wpsar.2013.4.1.010 Influenza virus oseltamivir resistance, northern Viet Nam, 2009–2012 expanding this pilot study to different regions, monitoring 8. Gubareva LV, Webster RG, Hayden FG. Detection of influenza virus resistance to neuraminidase inhibitors by an enzyme inhibition the use of oseltamivir, analysing more specimens assay. Antiviral Research, 2002, 53:47–61. doi:10.1016/ and reviewing more epidemiological information is recommended for Viet Nam.
9. Sheu TG et al. Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008. Antimicrobial Agents and Chemotherapy, Confl icts of interest 2008, 52:3284–3292. doi:10.1128/AAC.00555-08 pmid:18625765 None of declared.
10. Nguyen, LKH. Study on influenza infections in northern Viet Nam during the early years of 2000s [in Vietnamese]. Viet Nam, 2010, 66–68. 11. Hurt AC et al. Increased detection in Australia and Singapore of a novel influenza A(H1N1)2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation. This styudy was supported by a grant from the Korea Euro Surveillance: European Communicable Disease Bulletin, Center for Diseases Control.
2011, 16(23):19884. pmid: 21679678 12. Hurt AC et al. Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa.Antiviral Research, 2009, 83:90–93. doi:10.1016/j.
antiviral.2009.03.003 pmid:19501261 We are grateful the National Institute of Hygiene and 13. Meijer A et al.; European Influenza Surveillance Scheme. Epidemiology for their continued support for our works, Oseltamivir-resistant influenza virus A (H1N1), Europe, 2007–08 season. Emerging Infectious Diseases, 2009, 15:552–60. we would like to thank technical support from WHO Viet Nam and WHO Collaborating Center, Melbourne, 14. Lackenby A et al. Continued emergence and changing epidemiology of oseltamivir-resistant influenza A(H1N1) 2009 virus, United Kingdom, winter 2010/11. Euro Surveillance: European Communicable Disease Bulletin, 2011, 16(5):19784. 1. Dawood FS et al. Novel Swine-Origin Influenza A (H1N1) Virus 15. Deyde VM et al.Detection of molecular markers of drug resistance Investigation Team. Emergence of a novel swine-origin influenza in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing. A (H1N1) virus in humans. The New England Journal of Medicine, Antimicrobial Agents and Chemotherapy, 2010, 54:1102–1110. 2009, 360:2605–2615. doi:10.1056/NEJMoa0903810 16. McKimm-Breschkin JL et al.Reduced sensitivity of influenza 2. Nguyen HT et al.; Vietnam National Influenza Surveillance and A (H5N1) to oseltamivir. Emerging Infectious Diseases, Evaluation Team. National influenza surveillance in Vietnam, 2007, 13:1354–1357. doi:10.3201/eid1309.07-0164 2006–2007. Vaccine, 2009, 28:398–402. doi:10.1016/j.
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A. Les McDonald, Founding CNAC Executive Director (Editor's Note: The following article appeared in the Globe & Mail "Lives Lived" section on April 3, 2008. It waswritten by Les' good friend, Ron Jette. I am pleased to share it with you. It's hard for me to believe it has already beensix months. Ron really captured Les' spirit and made me hear my good friend's laugh one more time.Thanks Ron. – Cheryl)