Virological Surveillance and Antiviral Resistance of Human Influenza Virus in Argentina, 2005–2008
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Investigación original / Original research Virological surveillance and antiviral resistance of human influenza virus in Argentina, 2005–2008 Andrea Pontoriero,1 Elsa Baumeister,1 Ana M. Campos,1 and Vilma L. Savy 1 Suggested citation Pontoriero A, Baumeister E, Campos AM, Savy VL. Virological surveillance and antiviral resistance of human influenza virus in Argentina, 2005–2008. Rev Panam Salud Publica. 2011;30(6):634-40. ABSTRACT Objective. To describe the virological characteristics of the influenza strains circulating in Argentina in 2005–2008 and to assess the prevalence of antiviral resistance. Methods. On the basis of their geographical spread and prevalence, influenza A and B isolates grown in Madin–Darby canine kidney cells were selected after antigenic and genomic characterization to be analyzed for antiviral resistance by enzymatic assay and pyrosequenc- ing. Amantadine susceptibility was evaluated by pyrosequencing for known resistance mar- kers on 45 strains of influenza A. Susceptibility to oseltamivir and zanamivir was evaluated by enzymatic assay of 67 influenza A and 46 influenza B strains, some of which were further analyzed by sequencing the neuraminidase gene. Results. Resistance to amantadine was observed only on A(H3N2) strains (29/33); all of them carried the mutation S31N in their M2 sequence. Oseltamivir resistance was observed in 12 (34.3%) of the 35 A(H1N1) strains from 2008; all of them carried the mutation H275Y in their neuraminidase sequence. All these viruses remained sensitive to zanamivir. Conclusions. This study describes a high incidence of amantadine-resistant influenza A(H3N2) viruses since 2006 and an unprecedented increase in oseltamivir resistance detected only in influenza A(H1N1) viruses isolated in 2008. Influenza A and B viruses were more sensitive to oseltamivir than to zanamivir, and influenza A viruses were more sensitive to both neuraminidase inhibitors than the influenza B viruses. The national data generated and analyzed in this study may help increase knowledge about influenza antiviral drug resistance, which is a problem of global concern. Key words Influenza, human; influenza A virus; drug resistance, viral; antiviral agents; Argentina. Influenza is an infection of the upper and characterization of circulating influ- provide a valuable addition to the avail- respiratory tract that causes significant enza viruses are essential for detecting able options used to control the virus. morbidity and mortality; its economical the emergence of antigenic drift variants Two classes of specific anti-influenza impact is estimated to run into billions causing influenza epidemics and novel drugs have been developed to date: of dollars worldwide (1). Identification A subtypes with pandemic potential (2). amantadine and rimantadine, the ada- Influenza surveillance also provides a mantanes developed in the early 1960s basis for selecting the virus strains to be (5) and targeted against the M2 pro- 1 Administración Nacional de Laboratorios e Insti- included in annual formulation of influ- ton channel of influenza A viruses (6), tutos de Salud “Dr. Carlos G. Malbrán,” Instituto enza vaccines (3, 4). and zanamivir and oseltamivir, more Nacional de Enfermedades Infecciosas, De- Although vaccination provides the recently developed inhibitors of the vi- partamento Virología, Buenos Aires, Argentina. Send correspondence to: Andrea Pontoriero, primary means for protection from in- ral neuraminidase (7). The frequency of [email protected] fluenza virus infection, antiviral drugs resistance to amantadine and rimantadine 634 Rev Panam Salud Publica 30(6), 2011 Pontoriero et al • Influenza surveillance and antiviral resistance Original research among circulating seasonal influenza A in the World Health Organization Global ing neuraminidase enzyme activity in viruses has increased dramatically over Influenza Surveillance Network. the presence of oseltamivir and zanami- the past few years (8). A previous report The sampling effort of this network is vir to determine the drug sensitivity of showed the emergence of amantadine- constant year round and is part of the the viral enzyme. Sequences of the ion resistant influenza A(H3N2) viruses in influenza national surveillance system channel region of the M2 gene and the Argentina in 2006 and its circulation was of the Argentinean Ministry of Health. neuraminidase gene were also analyzed still detected in 2007 (9). The national network consists of 24 to detect mutations associated with re- A sustained antiviral susceptibility laboratories distributed throughout the duced susceptibility or drug resistance. surveillance of influenza viruses in Eu- country that routinely study 24 000– rope revealed the emergence of seasonal 31 000 respiratory samples per year for Viral strains tested for neuraminidase influenza A(H1N1) viruses naturally re- diagnostic testing. Nasopharyngeal as- susceptibility. From the total influenza sistant to the neuraminidase inhibitor os- pirates and throat or nasal swabs from A(H1N1) (478), influenza A(H3N2) (609), eltamivir (10), which is the most widely pediatric and adult outpatients and in- and influenza B (342) viruses isolated used antiviral drug for influenza and patients with acute respiratory infection between 2005 and 2008, a subset of is a potent inhibitor of influenza virus are collected and examined by immu- 113 were selected for analysis of neur- neuraminidase activity (11). The viruses nofluorescence assay for diagnosis of aminidase susceptibility: 35 influenza carried a specific histidine-to-tyrosine influenza A, influenza B, and other re- A(H1N1), 32 influenza A(H3N2), and 46 mutation at position 274 (H274Y; H275Y spiratory viruses, including respiratory influenza B viruses. Strains selected for in the N1 numbering system) in the syncytial virus, adenovirus, and para- this study were collected throughout the neuraminidase protein that confers a influenza virus. Between 800 and 1 000 study period and represent the types and high-level resistance to oseltamivir (12). influenza A- or B-positive samples per subtypes circulating in each particular Data produced by the EISS/VIRGIL net- year are submitted to the National Influ- season in different parts of the country: work until February 2009 showed that enza Center for further characterization. Catamarca, Chaco, Salta, Tucuman, Santa 97% of the European isolates tested were Fe, Mendoza, Buenos Aires, Chubut, and resistant to oseltamivir but retained sen- Virus propagation, subtype Neuquen provinces. The total number of sitivity to zanamivir, amantadine, and determination, and antigenic isolates evaluated for antiviral resistance rimantadine (13). Before emergence of characterization per year based on influenza type and the novel H1N1 swine-origin influenza subtype is shown in Table 1. A virus, neuraminidase inhibitors were Madin–Darby canine kidney cells sporadically used to treat seasonal in- were used to isolate viruses from clinical Antiviral drugs. Zanamivir was pro- fluenza in Argentina. Available data re- samples. Specimens for isolation were vided by GlaxoSmithKline (London, lated to resistance to neuraminidase in- selected taking into account the collect- United Kingdom). Oseltamivir carboxyl- hibitors in South America are poor. The ing date, the specimen preservation con- ate (GS4071), the active compound of the aims of this study were to describe the ditions, and the geographical location in ethyl ester prodrug oseltamivir phosphate virological characteristics of a subset of order to be able to obtain isolates with (GS4104), was supplied by Roche Prod- influenza A and B strains circulating in different characteristics throughout the ucts Ltd. (Burgess Hill, United Kingdom). Argentina in the period 2005–2008 and study period. Virus isolates were sero- to monitor the susceptibility to antiviral typed by hemagglutination inhibition Neuraminidase inhibition assays. To drugs. assay or indirect immunofluorescence study neuraminidase susceptibility, assay using the World Health Organi- enzymatic assays were performed at MATERIALS AND METHODS zation influenza reagent kit provided the Health Protection Agency (London, annually by the Centers for Disease United Kingdom) to determine the drug This work consists of an observa- Control and Prevention (Atlanta, Geor- concentration that provides 50% inhibi- tional study based on influenza virologi- gia, United States of America). Antigens tion (IC50) using the fluorescent substrate cal strain surveillance data carried out were characterized by hemagglutination methylumbelliferyl N-acetylneuraminic by antigenic analyses of the circulating inhibition test with postinfection ferret acid (Sigma Chemical Co., St. Louis, Mis- strains integrated with the monitoring of antisera and turkey red blood cells. souri, United States) according to previ- susceptibility to antiviral drugs. Antiviral resistance tests Specimen collection TABLE 1. Influenza A and B isolates tested for Unlike other countries, such as the oseltamivir resistance for each influenza type Weekly influenza surveillance is con- United States and European countries, and subtype, Argentina, 2005–2008 ducted routinely by Instituto Nacional routine surveillance for detection of an- de Enfermedades Infecciosas, Adminis- tiviral resistance in influenza viruses Influenza A tración Nacional de Laboratorios e Insti- has not been locally established in South Year H3N2 H1N1 Influenza B Total tutos de Salud “Dr. Carlos G. Malbran,” American countries. For a better ap- a Pan American