Influenza diagnosis, surveillance and monitoring, is currently often based on detection and characterisation of influenza virus by molecular tests. However, in some cases, serological analysis become the preferred option due to lack of access to samples from acutely affected animals, due to convenient access to blood samples collected and used for the screening of other diseases or, for seroprevalence studies. Serological diagnostic approach usually consists of antibody detection by Enzyme-Linked Immunosorbent Assay (ELISA) usually targeting the nucleoprotein of influenza A virus (IAV) and/or Hemagglutination Inhibition (HI) test targeting hemagglutinin (HA)-antigen. ELISA is a highly sensitive test and several kits are commercially available. HI allows for subtyping based on HA but it is highly dependent on the antigen used in the test. ELISA is therefore mainly applied for testing freedom of infection and prevalence determination purposes, while HI is recommended for the determination of the immune status in individual animals or populations post-vaccination (WOAH, 2023. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 12th edition). Due to the known high diversity and constant evolution of IAV strains, reference antigens used in the HI test aiming for antigens with broad cross reactivity and reflecting contemporary circulating field strains should be ideally updated. A logic consequence is the limited harmonisation of diagnostic protocols across labs and countries, all adjusted to the characteristics of regional circulating strains, challenging the cross[1]country sero-surveillance of swine influenza.
Proficiency testing schemes (PTS) are inter-laboratory testing schemes to determine the performance of individual laboratories for specific tests and to monitor laboratories’ continuing performance. These programs are of special interest for viruses such as Influenza or Porcine Respiratory and Reproductive Syndrome that evolve so rapidly. In these cases, it is essential to assess the availability of the tests to properly detect new emerged strains. Royal GD (GD) is accredited according to NEN-EN-ISO/IEC 17043:2010 to perform PTS. Every year, GD organises a PTS for swine influenza antibody detection for both ELISA and HI. The aim of this study was to analyse the results of the participating laboratories in the PTS organised by GD for the ELISA and HI of the last six years in order to get insights into the performance of the serological tests throughout the years, as well as to detect potential challenges on the detection of swine influenza infections.
Methods:
Data Set & Analysis
Anonymised data from GD PTS Swine Influenza Virus antibody detection in serum results from 2018 to 2023 were used. Annually, eight lyophilized samples (selected from a set of 12 available samples) were sent to participating laboratories to be tested in duplicate. All samples originated from animal experiments with known infection and or vaccination moment. The average participation rate, overall agreement of results between laboratories and laboratory performance were assessed. In addition, the qualitative agreement between ELISA and HI were determined. Sample-agreement was determined separately for screening ELISAs and HI subtype-specific (H1N1, H1N2 and H3N2) individually. The sample-agreement (%) was assessed by determining the qualitative sample status (e.g. positive or negative), number of participants with true qualitative results and total of reported qualitative results for that sample. The sample-agreement (%) was then calculated by dividing the number of participants with true qualitative results by the total of reported qualitative results by test system for that sample. Best-case sample-agreement (%) where samples reported as ‘suspected’ were evaluated according to the true qualitative value of the sample and worst-case agreement where samples reported as ‘suspected’ were defined as false.
