Emergence of circulating influenza A H3N2 viruses with genetic drift in the matrix gene: be alert of false-negative test results

Abstract

In March 2022, we observed samples with a negative fluorescent signal (60.5%, n = 43) for the influenza A matrix gene and a stronger positive signal for subtype A(H3N2). Forty-three samples were positive in InfA (H3N2) (mean Cq 30.9, range 23.9-35.1), and 26 of the 43 samples were negative in InfA matrix (mean Cq 28.0, range 23.2-30.6). Our multiplex test is a laboratory-developed four-target, four-color influenza A reverse-transcription PCR assay targeting the matrix gene, subtypes A(H3N2) and A(H1N1)pdm09. Several samples were negative when retested on commercial influenza Point-of-Care assays. As the matrix gene is a stand-alone target in most commercial diagnostic assays, we caution against false-negative subtype A test results.

Keywords: Assay; H3N2; M gene; RT-PCR; diagnostic; genetic drift; mutations; sequencing; surveillance.

Fig. 1

Section (nucleotide position 1–200) of an alignment showing LDT primer and probe binding sites in the influenza A M gene. Alignment of the M gene from seven different circulating influenza A variants belonging to clade 3C.2a1b.2a.2 and one circulating influenza A variant belonging to clade 3C.2a1b.1a (“A_Denmark_120_2021”) was performed by use of the CLC Main WorkBench 21.0.5 software (QIAGEN). NC_007367.1 was used as reference sequence for A(H3N2) and NC_026431.1 as reference sequence for A(H1N1) (https://www.ncbi.nlm.nih.gov/genbank/). Primer binding sites are marked with a red arrow, and the probe binding site is marked with a green arrow. Mutations in the primer binding sites are highlighted with a blue square.