Virology
Severe sensitivity loss in an influenza A molecular assay due to antigenic drift variants during the 2014/15 influenza season

https://doi.org/10.1016/j.diagmicrobio.2016.02.004Get rights and content

Highlights

  • During the 2014–2015 influenza season, a decreased sensitivity for the detection of influenza A(H3N2)/3C.2a using a commercially available multiplex assay was encountered.

  • Gene sequencing of the influenza A matrix protein showed a point mutation (C163T) leading to a mismatch with the assay probes.

  • The known risk of false-negative results in molecular assays due to genomic variation is emphasized, especially in microorganisms subject to great variation such as influenza A.

  • Intensive monitoring of incidence, epidemiological data, and clinical context can give a first indication of missed influenza A strains.

Abstract

The 2014–2015 influenza season in Belgium was dominated by the circulation of 2 influenza A(H3N2) subgroups: 3C.2a and 3C.3b. Analysis of 166 nasopharyngeal aspirates, collected in patients with respiratory illness at the start of the epidemic season, showed a decreased sensitivity for the detection of influenza A(H3N2)/3C.2a using a commercially available multiplex assay. Gene sequencing of the matrix protein showed a point mutation (C163T) leading to a mismatch with the assay probes.

Introduction

Influenza virus is an important cause of respiratory illness, annually causing serious morbidity and mortality in both adults and children (Poehling et al., 2006, Reed et al., 2014). Rapid and accurate diagnosis is important for patient management, including discontinuation of antibiotics, initiation of antiviral therapy, and infection control measures. Diagnosis solely based on clinical symptoms can be challenging, due to their nonspecific nature such as fever, cough, headache, and malaise (Dugas et al., 2015). Therefore, diagnostic tests can be helpful. These tests should focus on virus detection rather than antibody response (Kumar and Henrickson, 2012). The use of viral cell culture, the traditional gold standard, is abandoned in most clinical laboratories because it can take up to 14 days to obtain the final result. It is replaced by antigen detection tests and/or nucleic acid amplification tests (NAATs). Antigen detection tests, based on direct fluorescence or the immunochromatographic principle, yield results within 10 minutes to 1 hour and are widely used. However, sensitivities vary between different manufacturers and patient populations (Chartrand et al., 2012). Despite the fact that the latest generation of these tests, some with a digital read-out, provide acceptable results (Chartrand et al., 2012, Peaper and Landry, 2014), a higher sensitivity and specificity are attained with NAAT. Consequently, the latter have become the new gold standard (Kumar and Henrickson, 2012). Recently, multiplex molecular assays have reached the market, allowing a syndrome based approach by the detection of multiple respiratory pathogens, both viruses and bacteria, in a single test (Peaper and Landry, 2014, Salez et al., 2015). The latest evolution in influenza testing is the emergence of “rapid NAAT”, combining the speed and flexibility of an antigen test and the superior sensitivity and specificity of a molecular assay. Some of these test are easy to perform and can even be used in a point-of-care setting (Dunn and Ginocchio, 2015).

Influenza A is classified in different subtypes based on its surface glycoproteins neuraminidase (NA) and hemagglutinin (HA). During influenza replication, minor genetic changes in the NA or HA appear and accumulate, leading to so-called antigenic drift. Antigenic shift variants can arise following reassortment of NA and HA genes, occasionally resulting in pandemics. Even previously infected or vaccinated individuals have very little or no immunity to these new strains (Treanor, 2010).

Genetic variation should be taken into account when primers and probes are designed for molecular assays, since a mismatch (e.g., due to a point mutations) could lead to false-negative results (Steensels et al., 2013). Therefore, most molecular assays target conserved gene sequences of the influenza A virus such as the matrix (M) or nucleoprotein genes (Peaper and Landry, 2014, Steensels et al., 2013, Yang et al., 2014). The World Health Organization advises to target the M gene region 144 to 251 (start codon of the M1 gene was designated 1) (Ward et al., 2004, World Health Organization (WHO), 2009).

At the start of the influenza season 2014–2015, we presumed a decreased sensitivity for the detection of influenza A using our routine molecular detection method. Samples of several clinically suspect cases of influenza did not yield positive PCR results or showed small but distinct peaks below the threshold of the assay.

Section snippets

Samples and setting

From December 11, 2014, until February 3, 2015, 184 consecutive nasopharyngeal aspirates from patients presenting with respiratory illness in our acute secondary care hospital were analyzed. Samples were stored straight at −80 °C immediately after testing. Eighteen samples were excluded for further analysis due to insufficient sample volume, resulting in 166 analyzed samples.

Methods

All 166 samples were tested using RespiFinder RG (Qiagen, manufactured by PathoFinder, Maastricht, The Netherlands) and

Results

The results of the Xpert Flu/RSV XC (n = 23) were concordant with results of the in-house PCR. The positive results obtained by Xpert Flu/RSV XC in 16 samples and designated as negative by RespiFinder RG were confirmed to be positive. Furthermore, 2 samples that were positive with both Xpert Flu/RSV XC and RespiFinder and 5 that were negative in both assays were analyzed. Hence, true positives were defined as positive with Xpert Flu/RSV XC. False negatives were samples that were negative by

Discussion

The influenza A virus is extremely subjective to genetic variation. In 2014, 3 influenza A(H3N2) subgroups have emerged, 1 in subdivision 3C.2 and 3C.2a and 2 in 3C.3, 3C.3a, and 3C.3b (European Centre for Disease Prevention and Control, 2015). In Belgium, the 2014/15 influenza season was dominated by the circulation of influenza A(H3N2) viruses belonging to subgroups 3C.2a and 3C.3b (NRC Influenza, unpublished data), while the A/Texas/50/2012 strain used in the 2014/15 northern hemisphere

Conclusions

The 2014/15 influenza season in Belgium was dominated by influenza A(H3N2) subgroup 3C.2a. Our routine diagnostic assay for respiratory pathogens, the RespiFinder RG, showed a decreased sensitivity for this subgroup. This emphasizes the known risk of false-negative results in molecular assays due to genomic variation, especially in microorganisms subject to great variation such as influenza A. Several measures can be implemented to address this problem. Molecular tests should be carefully

Acknowledgements

We acknowledge the skillful assistance of the technicians of the microbiology and molecular laboratory at AZ Sint-Lucas Ghent. We would also like to thank Anne Vankeerberghen (Laboratory of Clinical Microbiology, Onze-Lieve-Vrouwziekenhuis, Aalst, Belgium), who was responsible for in-house PCR analysis. We acknowledge the authors, originating and submitting laboratories of the sequences from GISAID's EpiFlu Database on which the phylogenetic analysis is based (Fig. 1; accession numbers shown).

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    Conflict of interest: Guus Simons and Antoinette Brink are employees of PathoFinder B.V.

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