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.

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.