Objectives: Continuous genetic drift in the hemagglutinin gene of influenza A(H3N2) viruses compromises the reliability of standard molecular diagnostics, resulting in target dropouts and epidemiological surveillance gaps. This study evaluated the clinical performance of the World Health Organization (WHO)-recommended (2024) real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) sets against emerging Peruvian A(H3N2) subclades J.2 and K, and developed an optimized dual-target multiplex assay to restore diagnostic sensitivity.

Methods: Genomic sequencing was used to identify mutational profiles in circulating Peruvian A(H3N2) subclades J.2 and K. Standard WHO (2024) oligonucleotides were tested against 20 A(H3N2)-confirmed clinical samples. The dual-target multiplex RT-qPCR assay was then redesigned with targeted bioinformatic modifications and validated.

Results: Genomic analysis identified critical oligonucleotide mismatches in both subclades. Consequently, the standard WHO assay failed to detect the virus in any tested isolate. The optimized multiplex assay successfully restored amplification in all 20 sequencing-confirmed A(H3N2) viruses. It demonstrated high analytical concordance (Pearson´s r = 0.70, P < 0.001) between the two targets without competitive inhibition. Furthermore, both the J.2 and the highly mutated K subclades were amplified with equivalent diagnostic performance.

Conclusion: The optimized dual-target RT-qPCR assay successfully overcomes the diagnostic failure of standard probes, providing a reliable, fail-safe mechanism for detecting currently circulating H3N2 variants. These findings emphasize the necessity of routine genomic audits to maintain current molecular protocols designed for detection.