B-216 Development and Performance Evaluation of a Molecular Reference Material for Influenza H5N1 Strain Screening

Abstract Background Influenza A H5N1 is a highly pathogenic avian influenza virus strain that poses a significant threat to global public health due to its potential to cause severe illness and widespread outbreaks in both humans and animals. While transmission of H5N1 from animals to humans remains rare, the virus has shown the capacity to mutate, potentially increasing its transmissibility among humans and leading to a global pandemic. This highlights the urgent need for effective control measures, including early detection. Preventing the spread of H5N1 requires robust surveillance and continuous monitoring to mitigate the risk of devastating outbreak. This study describes work to develop a positive reference material for influenza A H5N1 for nucleic acids tests. Methods A human plasma-based matrix was tested in accordance with FDA guidelines and found non-reactive for HBsAg, anti-HIV 1/2, anti-HCV, HIV-1 RNA, and HCV RNA. TaqMan® assays targeting the PB1, H5b, and N1 genes were designed and validated for digital PCR quantification using the Bio-Rad QX200 Droplet Digital™ PCR system. Sequences covering the full genome of A/cattle/Texas/56283/2024(H5N1) were designed and verified by Sanger sequencing and then in vitro transcribed (IVT). The IVT RNAs were used to produce AccuPlex™ recombinant replicant-deficient H5N1 virus stocks, which were then combined to create the final product. Digital PCR was used to monitor formulation, and the final reference material was tested using the Cepheid Xpert® Xpress CoV-2/Flu/RSV plus kit, Abbott ID Now Inluenza A & B 2 kit using a 200 µL input volume to replicate sample volumes from nasopharynx swabs, as well as other test platforms at external laboratories. Results One lot of reference material was produced, and the concentration of each target gene was measured at approximately 6,000 copies per milliliter (cp/mL) using the Bio-Rad QX200 Droplet Digital™ PCR system. Positive detection was observed when using the Cepheid and Abbott ID Now kits indicating compatibility with point-of-care (POC) instruments. The material was also evaluated by external laboratories where positive detection was observed on various platforms. Both real-time and accelerated stability studies conducted on our proprietary AccuPlex™ technology have demonstrated maintained stability at 2 – 8°C for up to two years, ensuring consistent performance and reliability over time. Conclusion LGC Clinical Diagnostics has developed an influenza A H5N1 molecular reference material that will assist assay developers in bringing new screening methods to market quicker. This reference material may also support optimization and validation of nucleic acid tests that detect influenza A H5N1 in a clinical setting. Supporting H5N1 testing is critical as, ultimately, monitoring the virus’s spread is crucial for mitigating the risk of H5N1 outbreaks.