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Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase.

ABSTRACT: BACKGROUND:Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed. OBJECTIVES:We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene. STUDY DESIGN:Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza. RESULTS:121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected. CONCLUSIONS:We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms.

SUBMITTER: van der Vries E

PROVIDER: S-EPMC7185517 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase.

van der Vries E E Jonges M M Herfst S S Maaskant J J Van der Linden A A Guldemeester J J Aron G I GI Bestebroer T M TM Koopmans M M Meijer A A Fouchier R A M RA Osterhaus A D M E AD Boucher C A CA Schutten M M

Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20091025 1

<h4>Background</h4>Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed.<h4>Objectives</h4>We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition ...[more]

PMID: 19857993

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Project description:Pandemic (H1N1) 2009 influenza is affecting countries in all five continents, with most cases so far having been reported in North and South America and Europe, and children and young adults being the most susceptible age groups. To date, the clinical course of disease is typically mild, with low hospitalization and mortality rates. Pandemic (H1N1) 2009 is susceptible to oseltamivir and, although few clinical data are yet available, current information suggests that treatment with oseltamivir appears to be beneficial. Only isolated cases of resistance to the drug have been reported to date, in keeping with the low frequency observed in clinical studies involving patients infected with seasonal influenza viruses. Current health authority guidelines recommend the use of oseltamivir in infected adults and children who have or are at elevated risk for severe disease, including pregnant women; use during the pandemic in infants < 1 year has also been authorized in Europe and a number of other countries, including the USA and Canada. Before the onset of the current pandemic, F. Hoffmann-La Roche Ltd expanded annual production capacity for oseltamivir to 400 million treatment courses per year to meet anticipated demand. However, during an influenza pandemic, and despite increased production capabilities, resources are nonetheless likely to be initially in short supply. For this reason, Roche, in line with WHO recommendations, has advocated advance stockpiling of antivirals by governments as a pandemic preparedness measure. Between 2004 and December 2009, 350 million treatment courses were supplied to governments worldwide. Support for developing countries has been a priority. Roche has established a cluster of initiatives aimed at increasing access to Tamiflu for the world's developing economies, including, making donations to the WHO, establishing the Tamiflu Reserves Program (TRP) and sub-licensing and manufacturing contracts with local companies in Asia and Africa. Furthermore, Roche has published a document outlining how it would allocate limited supplies of Tamiflu during a pandemic, which are in line with WHO recommendations stating that 'resources should be used to provide the maximum possible health benefit'. Roche is also offering support such as reprocessing of expiring capsule stocks (in development) and shelf-life extension to support governments in the management of their stockpiles. Clinical studies, either sponsored by or supported by Roche, are in progress. These trials are designed to investigate the effectiveness of oseltamivir in patients infected with the pandemic virus in greater depth, and include high-dose studies, assessment of natural and drug-induced resistance, and response to treatment in high-risk populations such as young infants, immunocompromised patients and the severely ill.

Project description:BackgroundResistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility.MethodsThe NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0-15) and zanamivir (days 15-25 and 70-80).ResultsCompared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6-69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin.ConclusionsReduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines the importance of close monitoring of treated patients especially those immunocompromised.

Project description:The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007-2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.

Dataset Information

Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase.

Publications

Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase.

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