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.

Fig. 1

Linearity of the H1 and N1 subtype specific RT-PCRs for detection of pandemic influenza A/H1N1 (2009). 10-Fold serial dilutions of target RNA obtained from A/NL/602/2009(v) were analyzed by both sub type RT-PCRs in fourplex and plotted as Ct value versus the log of the calculated viral particles per milliliter in each dilution.

Fig. 2

H275Y endpoint fluorescence scatter plot. Mixtures of in vitro transcribed wild type and mutant RNA (total input 1.0 × 10⁵ vp/ml) were analyzed using the H275Y discrimination assay (black dots). Relative H275 wild type (465–533 nm) and 275Y mutant (533–580 nm) fluorescence emissions were plotted on the x-axis and y-axis, respectively. In addition, 61 clinical isolates from naïve pandemic influenza A/H1N1 (2009) infected patients were analyzed (white squares) to determine a threshold for detecting mutant genotypes in mixed virus populations (5%).