Toward diagnostic preparedness: detection of highly pathogenic avian influenza A(H5N1) in contrived nasal swab specimens using rapid antigen and point-of-care molecular tests

Abstract

Highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b was first detected in birds in 2021 in the United States (U.S.). An ongoing outbreak in dairy cattle began in early 2024 involving genotype B3.13. At least 70 U.S. cases have been identified in humans with exposure to infected cattle, poultry, and wild birds. No human-to-human transmission has yet been documented. However, for diagnostic preparedness, we evaluated the ability of currently available influenza tests to detect 2024 U.S. H5N1 genotypes, B3.13 and D1.1, predominant in wild birds and poultry. Contrived nasal swab samples were prepared using live or inactivated virus and used to test 12 rapid antigen tests (lateral flow assays, [LFA]), including 10 commercially available influenza A and two H5-specific LFAs. Five point-of-care (POC) molecular assays were also tested. An inclusivity testing protocol utilizing a predetermined dilution series was used to evaluate each assay, enabling a head-to-head comparison of performances. All LFAs and POC molecular tests were able to detect bovine 2024 H5N1 genotype B3.13. Sensitivity for the POC molecular tests (heat-inactivated B3.13) ranged from 1.55 to 7.75 TCID₅₀/swab. For 11/12 LFAs, 10 commercial influenza A, and an RUO H5 assay, the sensitivity (live B3.13) ranged from 78 to 1550 TCID₅₀/swab. The 12th LFA detected 77,500 TCID₅₀/mL transport media. Testing of four LFAs confirmed inclusivity for genotype D1.1. Available influenza tests can detect 2024 U.S. H5N1 strains in contrived samples with a wide range of analytical sensitivity. In the event of human-to-human transmission, clinical performance and optimal sample types would need to be established.IMPORTANCETo date, at least 70 human cases of highly pathogenic avian influenza A (HPAI) H5N1 have been reported. Human-to-human transmission has not yet been reported, but there is ongoing HPAI H5N1 spread within animal and bird populations. As part of pandemic preparedness, it is critical to know whether available tests capable of detecting seasonal influenza A can also detect HPAI H5N1. Our paper describes results from the first study to assess and compare the ability of 10 commercially available influenza A lateral flow assays (LFAs), two LFAs specific for H5 influenza, and five POC molecular tests from three manufacturers to detect the H5N1 virus circulating in U.S. cattle (genotype B3.13). We found that all tests detect HPAI H5N1 in contrived nasal swab specimens, with varying degrees of analytical sensitivity. We also tested a subset of LFAs (three 510k-cleared COVID/flu multiplex tests and an RUO H5-specific LFA) to confirm that they were able to detect a D1.1 genotype (the predominant genotype in wild birds and poultry) strain isolated from a human case of H5N1 in Washington State. Given the persistent circulation of HPAI H5N1 cases, it is critical to know that available tests can analytically detect H5N1. Clinical performance and optimal sample types should be evaluated if human-to-human transmission is observed.

Keywords: OTC; POC; bird flu; influenza A; lateral flow assays.