Enhancing diagnostic preparedness for H5N1: a validation study of H5 single-plex assay and detection across multiple...

Area of Science:

• Clinical microbiology
• Infectious disease diagnostics
• Public health surveillance

Background:

• Increasing transmission of highly pathogenic avian influenza (HPAI) H5N1 from animals to humans necessitates improved diagnostic tools.
• Current commercial multiplex respiratory panels may detect H5N1 but lack specific H5N1 subtyping capabilities, with unverified real-world performance.

Purpose of the Study:

• To evaluate the limit of detection (LoD) of three commercial molecular diagnostic assays for H5N1.
• To develop and validate a real-time RT-PCR assay for H5 subtype detection on the Hologic Panther Fusion platform.

Main Methods:

• Evaluation of H5N1 LoD using bioMérieux BioFire Respiratory 2.1 Panel, Cepheid Xpert Xpress CoV-2/Flu/RSV Plus, and Hologic Panther Fusion SARS-CoV-2/Flu A/B/RSV assays.
• Development and validation of a real-time RT-PCR H5 single-plex assay for reflex testing on the Hologic Panther Fusion Open Access platform.

Main Results:

• All three evaluated commercial assays reliably detected H5N1 at low viral concentrations.
• The developed real-time RT-PCR H5 assay demonstrated capability for H5 subtype differentiation.

Conclusions:

• Current commercial assays, while detecting H5N1, do not differentiate the H5 subtype.
• Integration of validated commercial assays with a targeted H5 assay enhances clinical decision-making and public health surveillance for H5N1.
• Improved diagnostic strategies reduce the risk of missed H5N1 infection cases.