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Abstract Background First detected in 1996 in geese in China, Highly Pathogenic Avian Influenza (HPAI) A(H5N1) virus cause severe illness with high mortality rates among infected poultry. Detection in human first occurred in 1997 during poultry outbreaks in Hong Kong and have since been detected in humans sporadically in more than 20 countries causing serious illness and death. In 2021, the HPAI A (H5N1) strain was detected in North America, primarily infecting wild birds and commercial poultry, though it has been spreading to wild terrestrial, marine, and even domestic animals. Human infections have also occurred both from exposure to infected birds and/or other animals, but also through unknown exposures. While no human-to-human transmission has occurred to date, the Center for Disease Control (CDC) considers HPAI A (H5N1) virus to be possible of causing severe disease in infected humans. Rapid testing of persons presenting with respiratory illness provides high value to health care providers, however the novel strain of HPAI A(H5N1) has been largely untested against diagnostic assays. This study evaluates laboratory detection of HPAI A(H5N1) using commercially available Nucleic Acid Amplification Tests (NAAT) with liquid-stable, qualitative proficiency samples containing whole genome H5N1 cDNA. Methods American Proficiency Institute, an independent proficiency testing provider, studied the recovery of HPAI A(H5N1) in simulated patient samples in a pilot event in 2025. Two blinded liquid samples (PIA-01 and PIA-02) supplied by Microbix Biosystems were mailed to 74 unique laboratories utilizing 15 different methodologies. Laboratories were instructed to process the sample as instructed by their Original Equipment Manufacturer’s (OEMs) Instructions for Use. Sample PIA-01 contained HPAI A (H5N1) and Sample PIA-02 contained H1N1. Sample PIA-01 was comprised of whole genome H5N1 cDNA template created using synthetic biology and Gibson assembly. The consensus sequence included was based on published data from the first human patients infected with H5N1 (clade 2.3.4.4b). Sequence was confirmed by NGS, quantified by ddPCR, and qualified for released by the Cepheid Gene Xpert SARS/Flu/RSV assay. Results Participants demonstrated they were able to accurately detect Influenza A, with only a limited number of laboratories being able to subtype to the level of H1N1 and H5N1. Conclusion Based on the results reported by participants for American Proficiency Institute’s pilot event, laboratories are able to detect HPAI A(H5N1) in NAAT assays as a positive result confirming Influenza A infection. While many methods do include limited subtyping A(H1) and A(H3), novel influenza A virus such as A(H5) are not commonly included in commercially available assays. Including subtypes is imperative to provide the necessary tracking in outbreaks.