Emergence of oseltamivir resistant influenza A (H1N1) viruses in the Netherlands during the winter 2007/2008.

Background: Continuous monitoring of influenza antiviral susceptibility has become more important since the introduction of the neuraminidase inhibitors (NAI) in 1999, in addition to the existing adamantane M2 channel inhibitors (M2I). Since the 2005/2006 winter season the Dutch National Influenza Centre (ErasmusMC and RIVM) systematically monitors the antiviral susceptibility of influenza viruses derived from patients with influenza like illness or acute respiratory infection who consult a general practitioner of the influenza sentinel network (coordinated by NIVEL). Only sporadically viruses with a lower susceptibility for the NAI were found in the Netherlands in the winter seasons 2005/2006 and 2006/2007, whilst an increasing proportion of A(H3N2) viruses were found resistant to M2I. Methods: During the 2007/2008 season, influenza viruses detected in clinical specimens from sentinel and non-sentinel (through hospital and peripheral laboratories) patients were tested for antiviral susceptibility. Fifty percent inhibitory concentrations (IC50s) for the NAIs oseltamivir and zanamivir were determined using a fluorescent inhibition assay. Sequencing of the neuraminidase (NA) gene and the M2 ion channel gene was used to identify mutations previously associated with antiviral resistance to NAI and M2I respectively. To determine the possible impact of oseltamivir resistant A(H1N1) viruses on the severity of disease, basic descriptive variables and information on symptoms, complications and exposure to antivirals were collected. Results: A(H1N1) influenza viruses (91% of all subtyped influenza A viruses) dominated the first part of the 2007/2008 winter season followed by type B viruses from week 9/2008 onward (Figure 1). By the end of March 2008, 132 isolates [119 A( H1N1), 1 A(H3N2), 12 B] were analyzed for antiviral resistance. Resistant strains were only detected among the A(H1N1) viruses from sentinel and non-sentinel sources; 33 (28%) were found resistant and contained the H274Y substitution in the neuraminidase that has been associated with oseltamivir resistance previously. None of the oseltamivir resistant viruses tested for zanamivir and amantadine susceptibility were resistant to these antivirals. Further analysis of A(H1N1) viruses for which the NA nucleotide sequence was available showed that the resistant viruses (n=28) had glycine (G) at position 354, whereas the sensitive viruses (n=75) had aspartic acid (D) at that position. This substitution does not have known association with oseltamivir resistance. By four-week period, the proportion resistant viruses increased from 0% in weeks 40-51/2007 to 14% in weeks 52/2007-3/2008, 34% in weeks 4-7/2008 and 35% in weeks 8-11/2008. For a break down by week see Figure 1. Basic descriptive variables were available for all 119 patients with A(H1N1) virus and clinical data for 30 (all sentinel patients). The proportion resistant strains among viruses from sentinel and non-sentinel patients was not significantly different, 20% and 30% respectively (X2=1.196, p=0.274). Furthermore, no differences (p>0.05) were found between patients infected with oseltamivir resistant or sensitive virus with respect to age, gender, symptoms, complications or death. None of the 30 patients for which clinical data were available used oseltamivir prior to virus isolation. Conclusions Similar to many countries in Europe and worldwide, oseltamivir resistant A(H1N1) influenza viruses started to spread in the Netherlands during the 2007/2008 winter season with an overall prevalence of 28% among 119 A(H1N1) viruses analyzed by March 2008. Based on the current data, no differences in clinical impact between resistant and sensitive viruses were found. The analysis will be repeated when more data become available. Acknowledgements: We thank all patients and their general practitioners or hospital physicians for providing information and specimens for investigation, the Dutch virologists for sending non-sentinel virus isolates to the NIC, and the Dutch Society for Clinical Virology for their help in designing the clinical study among non-sentinel patients