Update to the CDC influenza A(H3) subtyping assay for improved detection of influenza A(H3N2) viruses, including subclade K viruses

Influenza A(H3N2) viruses circulate globally and cause respiratory illness. The FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Influenza A (H3/H1pdm09) Subtyping assay is used to subtype seasonal influenza A viruses. However, emerging mutations in the H3 reverse primer region raised concerns about reduced assay sensitivity for contemporary circulating viruses. We updated the H3 assay by redesigning the reverse primer to target a more conserved region of the hemagglutinin gene, and incorporating minor adjustments into the forward primer and probe. Analytical sensitivity, inclusivity, and exclusivity evaluations were performed using cultured influenza viruses, synthetic RNA, and panels of non-influenza respiratory pathogens. Clinical performance was evaluated using seasonal A(H3N2) residual respiratory specimens. The updated assay (H3_v2) demonstrated analytical sensitivity comparable to or improved over the initial H3 assay, with a limit of detection of approximately 10 1.7 –10 2.1 EID 50 /mL and five copies of RNA per reaction. Improved performance was observed for viruses containing A21G with T23G, T23C, or T23A double mismatches in the reverse primer, with a measurable Ct value shift between the initial H3 and H3_v2 assay. The assay showed 100% agreement with expected results in 139 clinical specimens, and no cross-reactivity with other influenza subtypes or other respiratory pathogens. These findings indicate the updated H3_v2 assay improves sensitivity for contemporary and emerging seasonal A(H3N2) viruses while maintaining high specificity. This update ensures continued reliable detection of seasonal A(H3N2), including genetically drifted A(H3N2) viruses, such as the recently emerged A(H3N2) subclade K viruses, and supports global influenza surveillance and diagnostic preparedness. IMPORTANCE Accurate detection and subtyping of influenza A(H3N2) viruses are essential for clinical diagnostics, outbreak response, and global influenza surveillance. The emergence and fixation of mutations leading to primer mismatches in the FDA-cleared CDC A(H3) real-time RT-PCR assay raised concerns about reduced sensitivity for contemporary circulating A(H3N2) viruses. To address this, we redesigned the reverse primer and optimized additional assay components to improve detection of currently circulating seasonal A(H3N2) viruses. The updated H3_v2 assay restores and enhances sensitivity, including for viruses harboring key reverse primer mismatches, while maintaining high specificity and complete agreement with clinical specimens. This update ensures reliable detection of seasonal influenza A(H3N2) and A(H3N2) variant viruses, including recently emerged A(H3N2) subclade K viruses, and strengthens diagnostic preparedness and surveillance capacity nationally and internationally.