Diagnostic performance of ELISA kits and expanded antigen panels for hemagglutination inhibition assays in pig herds enzootically infected with porcine Influenza A viruses

A combined approach using both indirect and direct detection methods can improve the diagnostic efficiency of swine Influenza A virus (swIAV) infections. Despite its importance, limited research has been conducted on the diagnostic performance of serological tests commonly used for routine swIAV surveillance. Therefore, the objective of this study was to compare different serological assays, including hemagglutination inhibition (HI) tests using antigen panels of different breadth and three commercially available enzyme-linked immunosorbent assays (ELISAs) to evaluate their suitability as screening tools for identifying herds enzootically infected with swIAV. The diagnostic sensitivity and specificity of the ELISAs were assessed under field conditions, using the HI test as the reference standard. The HI test panel expanded with various swIAV pandemic strains of regional provenience (HI 3) demonstrated the highest sensitivity (97.77%), while the HI panel including only the four major swIAV subtypes (HI 1) showed the lowest sensitivity (93.74%) and negative predictive value (40.00% compared to HI 4 (incorporating all tested strains)). The level of agreement between the HIs and ELISA tests varied considerably, with the indirect ELISA exhibiting the highest concordance with HI assays. Among the ELISA assays, the indirect ELISA (ELISA 1) achieved the highest sensitivity (95.69%) and overall accuracy (94.26%), albeit with lower specificity (60.00%) when compared with the HI including all strains (HI 4). In contrast, the competitive ELISA (ELISA 2) and the blocking ELISA (ELISA 3) showed lower sensitivity (81.36% and 82.89%) but higher specificity (83.33% and 76.67%, respectively). This study demonstrates the value of combining different diagnostic tools to improve swIAV surveillance in enzootically infected herds. The indirect ELISA offered high sensitivity and was well suited for broad herd-level screening, while ELISAs based on competitive or blocking formats and HI assays offered higher specificity for confirmatory testing. Among all evaluated methods, the HI assay including locally circulating strains demonstrated the best overall performance and proved useful in detecting additional subtype-level information not identified by PCR. The integration of these approaches enhanced diagnostic precision and supported more effective surveillance strategies, albeit at the cost of increased resource demands.