Preparing the outbreak assistance laboratory network in the Netherlands for the detection of the influenza virus A(H1N1) variant

Abstract 

Background

Late April 2009, human infection with variant influenza virus A(H1N1)v emerged in the Northern Americas posing a threat that this virus may become the next pandemic influenza virus.

Objectives

To prepare laboratories for surge capacity for molecular diagnosis of patients suspected for A(H1N1)v infection in the Netherlands.

Study design

A panel of 10 blinded specimens containing seasonal A(H1N1) or A(H3N2), or A/Netherlands/602/2009(H1N1)v influenza virus, or negative control was distributed to the outbreak assistance laboratories (OAL) together with influenza virus A (M-gene), swine influenza virus A (NP-gene) and influenza virus A(H1N1)v (H1v-gene) specific primers and probes and protocol (CDC Atlanta, USA). Laboratories were asked to implement and test this protocol.

Results

All OAL were able to detect A(H1N1)v using the CDC M-gene reagents, the majority with similar sensitivity as the in-house M-gene based assays. RT-PCRs used in routine diagnostic setting in the OAL specifically designed to detect H1, H3, or NS1 from seasonal influenza A viruses, did not or at very low level cross-react with A(H1N1)v. The CDC swine NP-gene and H1v-gene RT-PCRs showed somewhat reduced sensitivity compared to the CDC and in-house M-gene RT-PCRs. In contrast, in-house developed A(H1N1)v specific H1v-gene and N1v-gene RT-PCRs showed equal sensitivity to CDC and in-house M-gene RT-PCRs.

Conclusions

The Dutch OAL are prepared for detection and specific identification of A(H1N1)v, although some level of cross-reactivity was observed with seasonal influenza viruses. Additionally, M-gene based generic influenza A virus detection is recommended to be able to detect emerging influenza A viruses in routine settings.

Abbreviations: A(H1N1)v, variant influenza A(H1N1) virus of swine origin, naming according to latest WHO decision, M, gene encoding the influenza virus matrix protein, HA, gene encoding the influenza virus hemagglutinin, NA, gene encoding the influenza virus neuraminidase, Swine NP, gene encoding the swine influenza virus type A specific nucleoprotein, NS1, gene encoding the influenza virus type A non-structural protein 1, H1v, gene encoding the influenza virus A(H1N1)v specific HA, N1v, gene encoding the influenza virus A(H1N1)v specific NA, NIC, National Influenza Centre, PCR, polymerase chain reaction, RT, reverse transcriptase, RT-PCR, reverse transcriptase polymerase chain reaction, Ct value, cycle threshold value is the number of cycles required for the fluorescent signal to cross the threshold, i.e. exceeds the background level, OAL, outbreak assistance laboratories

Keywords: Influenza, Pandemic, A(H1N1)v, Molecular diagnostics, Laboratory network, Proficiency