Emergence of influenza A(H1N1)pdm09 6B.1A.5a.2a and 6B.1A.5a.2a.1 subclades leading to subtyping failure in a commercial molecular assay

Abstract

Background

During the 2023–2024 and early 2024–2025 influenza seasons, several influenza A-positive specimens in our laboratory failed subtyping for H1, H1pdm09, and H3 using the Allplex Respiratory Panel 1 (Allplex RP1) (Seegene Inc.). This study aimed to identify the cause of these subtyping failures.

Materials and methods

Between August 2023 and December 2024, 23 nasopharyngeal specimens tested positive for influenza A but were unsubtypeable for H1, H1pdm09, and H3. Confirmatory testing by the manufacturer included target-specific PCR for the M and HA genes, followed by sequencing to determine subclades.

Results

Among the 23 unsubtypeable specimens, 22 yielded PCR products for sequencing. Of these, 21 belonged to subclade 6B.1A.5a.2a.1 and one to 6B.1A.5a.2a. Sequence analysis revealed mismatches in the H1pdm09 primer/probe-binding regions of Allplex RP1, explaining the subtyping failures. Despite testing negative for H1pdm09 in Allplex RP1, sequencing confirmed their classification as H1N1pdm09 subclades with HA gene mutations.

Conclusions

Subclades 6B.1A.5a.2a.1 and 6B.1A.5a.2a harbour mutations that contributed to subtyping failures in some specimens tested with a commercial assay. While unsubtypeable influenza A results often raise concerns about emerging strains, sequencing confirmed that all unsubtypeable specimens tested with Allplex RP1 belonged to H1N1pdm09 within recognised subclades. Thus, such subtyping failures in this assay do not necessarily indicate a novel or zoonotic virus, though genomic surveillance remains essential.