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.

Abbreviations: RT-PCR(s), reverse transcriptase polymerase chain reaction(s), WHO, World Health Organisation, RNA, ribonucleic acid, HA, hemagglutinin, NA, neuraminidase, H1/3, hemagglutinin type 1 or 3, N1/2, neuraminidase type 1 or 2, NAI(s), neuraminidase inhibitor(s), Ct, cycle threshold, SNP, single nucleotide polymorphisms, LNA, locked-nucleic acid, TCID₅₀, tissue culture infection dose 50, Ml, millilitre, Vp, viral particles, cDNA, complementary DNA, BHQ, black hole quencher, H275Y, histidine to tyrosine substitution at position 275, PDV, phocine distemper virus

Keywords: Pandemic, Influenza, Oseltamivir, Resistance, RT-PCR, H274Y