Protocols
The detection of oseltamivir-resistant pandemic influenza A/H1N1 2009 viruses using a real-time RT-PCR assay

https://doi.org/10.1016/j.jviromet.2010.06.014Get rights and content

Abstract

A real-time reverse transcription PCR (rRT-PCR) assay was designed and evaluated for the detection of the point mutation in the influenza A N1 neuraminidase gene that results in a tyrosine to histidine substitution at amino acid position 275 (H275Y) causing resistance to oseltamivir, an antiviral neuraminidase inhibitor. The rRT-PCR assays detected the presence or absence of the H275Y mutation in 387/388 (99.7%) of clinical samples containing the pandemic influenza A/H1N1 2009 virus. The H275Y mutation was not detected in any of the community patient samples (0/132) but was detected in four hospitalized patients who had been treated with oseltamivir for several days. The sensitive rRT-PCR assays may be performed directly on patient specimens, can detect resistant virus at low levels, and therefore may provide early warning of developing resistance within individual patients or the wider population.

Introduction

Neuraminidase (NA) inhibitor resistance in influenza viruses had been uncommon until the emergence of oseltamivir-resistant seasonal influenza A (H1N1) viruses during the 2007/8 northern hemisphere influenza season. These resistant viruses spread and subsequently became the dominant seasonal H1N1 strain in many countries including Australia during 2008/09 (Hurt et al., 2009, Moscona, 2009). Oseltamivir resistance in these viruses was due to a point mutation in the NA gene that results in a tyrosine to histidine substitution at amino acid position 275 (H275Y). The pandemic influenza A/H1N1 2009 subtype that emerged in (Dawood et al., 2009) has now become the dominant influenza A strain worldwide, while the oseltamivir-resistant seasonal H1N1 strains are now rare in most of the world (World Health Organization, 2010a). However, there are concerns that oseltamivir resistance will develop in pandemic influenza A/H1N1 2009 viruses due to either mutations in the NA gene, or to reassortment with the resistant seasonal H1N1 virus, thereby reducing the effectiveness of oseltamivir for the treatment and prevention of this infection. So far, only a relatively small number of oseltamivir-resistant strains of pandemic influenza A/H1N1 2009, 268 from >20,000 samples investigated and all with the H275Y mutation, have been reported thus far from 20 countries including Australia (World Health Organization, 2010a, World Health Organization, 2010b, Speers et al., 2010). In order to monitor and detect such mutations, molecular assays are needed that can be performed directly on patient specimens and that are suitable for rapid, high throughput testing, especially those that can be incorporated as part of routine test protocols in diagnostic laboratories. This report describes a real-time reverse transcription PCR (rRT-PCR) assay for the rapid and reliable detection of the H275Y mutation associated with oseltamivir resistance that is suitable for incorporation into routine diagnostic testing protocols.

Section snippets

Laboratory strains

Virus stocks were prepared from clarified cell culture fluids of two local strains of pandemic influenza A/H1N1 2009 virus propagated in MDCK cells, A/Perth/29/2009 (oseltamivir-sensitive H275 strain) and A/Perth/261/2009 (oseltamivir-resistant H275Y strain). Identities were confirmed by rRT-PCR, DNA sequencing, and haemagglutination inhibition typing. Preliminary development of the assay used inactivated cell culture material from influenza A/Osaka/180/2009 virus (oseltamivir-resistant H275Y

Assay performance

The reaction efficiencies for the H275 and H275Y rRT-PCR assays were 91% and 93% and the LOD at a 95% confidence interval (CI) were 2.3 and 2.8 copies/reaction or 0.3 and 0.05 TCID50/reaction, respectively. The LOD at a 95% CI for the nested PCR assay was 2.7 and 3.8 copies/reaction or 0.7 and 0.3 TCID50/reaction for the H275 and H275Y virus strains, respectively.

The results for a collection of influenza A viruses tested in the rRT-PCR assays are shown in Table 2. The pandemic influenza A/H1N1

Discussion

The rRT-PCR assays were designed and implemented for the rapid detection of oseltamivir-resistant virus strains in patients with pandemic influenza A/H1N1 2009 infection. During the 2009 southern hemisphere influenza season the PathWest Laboratory Medicine Western Australia facility tested 22,354 samples for influenza viruses, with 5460 influenza positive samples, of which 4554 (83.4%) were pandemic influenza A/H1N1 2009 viruses. Samples examined in this study included all of those available

Conclusions

This report details the development and validation of a reliable and sensitive assay for the detection of the oseltamivir resistance mutation in pandemic influenza A/H1N1 2009 virus that can be performed directly on patient specimens. It has the potential to be used either as part of a targeted testing regimen directed at patients at higher risk of emergence of resistance, or as part of a more generalized primary testing protocol. It is able to detect resistant virus at low levels and therefore

Acknowledgement

The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health and Ageing.

References (11)

There are more references available in the full text version of this article.

Cited by (20)

  • Development and clinical testing of a simple, low-density gel element array for influenza identification, subtyping, and H275Y detection

    2014, Journal of Virological Methods
    Citation Excerpt :

    Nevertheless, influenza viruses continue to pose a significant health concern because of the genetic mutability of their RNA genomes, as well as their rapid and cross-species transmissibility. Numerous polymerase chain reaction (PCR)-based tests are available for detecting and sub-typing influenza virus, with many recently developed tests developed for detecting pandemic 2009 influenza A/H1N1 (A/H1N1pdm09) and/or antiviral drug-resistance mutations (Bolotin et al., 2009; Chidlow et al., 2010; Operario et al., 2010; Suzuki et al., 2010; Wang et al., 2010; Bennett et al., 2011; Lee et al., 2011; Tong et al., 2011; Wong et al., 2011; Arvia et al., 2012; Kawai et al., 2012; Redlberger-Fritz et al., 2012). Real-time PCR methods, however, are limited in their multiplexing capabilities in part because of limits on the number of unique fluorophores that can be detected simultaneously without significant optical cross-talk.

  • Detection of oseltamivir sensitive/resistant strains of pandemic influenza A virus (H1N1) from patients admitted to hospitals in Thailand

    2011, Journal of Virological Methods
    Citation Excerpt :

    None of the four patients had received oseltamivir as a prophylactic medication prior to the isolation of oseltamivir resistant H1N1 strains. Previously, several real-time RT-PCR assays for oseltamivir resistance detection were developed based on TaqMan MGB (minor groove binder) chemistry (Wong et al., 2011; Suzuki et al., 2010; Hindiyeh et al., 2010; Chidlow et al., 2010). In this study, the in-house assay based on real-time RT-PCR using specific LNA (locked nucleic acids) TaqMan probes was developed for the detection and determination of relative quantities of the oseltamivir resistant and oseltamivir sensitive strains of the pandemic influenza virus (H1N1).

  • Emerging oseltamivir resistance in seasonal and pandemic influenza A/H1N1

    2011, Journal of Clinical Virology
    Citation Excerpt :

    Each assay is designed to detect a single, known SNP, often within 1–2 h. PCR genotyping assays include primer specific assays (e.g., allele specific PCR), probe specific assays (e.g., allelic discrimination) and enzyme mediated assays (e.g., restriction fragment length polymorphism).17,18 Assays targeting the H275Y mutation for seasonal or pandemic H1N1 are summarized in Table 2.19–34 Comparative data evaluating various PCR genotyping protocols are not readily available, but most assays are highly sensitive.

  • Oseltamivir-resistant pandemic influenza a (H1N1) 2009 viruses in Spain

    2011, Journal of Clinical Virology
    Citation Excerpt :

    However, in comparison with these studies, other authors showed series with lower percentages of positivity near 3% between 148 severe patients17 or <1% between 1608 severe cases in Scotland.18 In Spain, a total of 413 hospitalised patients with clinical severe disease rendered frequency of viruses with H275Y substitution (1.93%) similar than prevalence reported by Harvala et al.18 and Chidlow et al.17 in severe patients. In the present study, six patients with resistant viruses detected were under immunosuppressive conditions and all of them had received oseltamivir treatment previously.

  • A comparison of pyrosequencing and neuraminidase inhibition assays for the detection of oseltamivir-resistant pandemic influenza A(H1N1) 2009 viruses

    2011, Antiviral Research
    Citation Excerpt :

    We also demonstrated that the ability of the NA inhibition assay to predict the percentage of oseltamivir resistance viruses in a mixed viral population was significantly improved when combined with a novel curve-fitting analysis. A total of 181 clinical specimens from 129 patients confirmed to be H1N1pdm positive by real-time RT-PCR were collected (Chidlow et al., 2010), including 50 specimens from 50 community out-patients, 50 from hospitalized patients, 45 from 13 intensive care unit (ICU) patients, and 36 from 16 deceased patients. Viruses were grown in Madin-Darby canine kidney (MDCK) cells (Barr et al., 2003), A/Auckland/1/2009 and A/Osaka/180/2009 were used as reference wild-type and H275Y mutant H1N1pdm viruses, respectively.

View all citing articles on Scopus
View full text