Toward improved Lyme disease testing in Canada, beyond standard two-tiered testing and Borrelia burgdorferi : A pilot project

Background: Accurate diagnosis of Lyme disease remains challenging as early clinical manifestations are often nonspecific and erythema migrans lesions are not always present, necessitating reliable serological testing. In this study, we aimed to compare the agreements of various simulated serological standard two-tier testing (STTT) and modified two-tier testing (MTTT) algorithms, with particular focus on identifying trends and potential redundancies between assay configurations. Methods: A total of 342 patient serum samples were collected between 2021 and 2024 and subjected to testing using various kits. Evaluated enzyme immunoassays (EIAs) included ZEUS (VlsE1/pepC10 IgG/IgM, Whole-Cell IgG/IgM, IgG, and IgM) and DiaSorin LIAISON (Total Antibody Plus, IgG, and IgM). Immunoblots included Mikrogen IgG/IgM and Euroimmun IgG/IgM assays targeting Borrelia burgdorferi sensu lato . Twelve MTTT algorithms and six STTT algorithms were evaluated. Overall percentage agreements were calculated between algorithms to assess concordance in the final serological classification. Results: Overall seroreactivity varied by algorithm. Although one STTT algorithm yielded the greatest number of interpreted positives, MTTT algorithms on average yielded a greater number of samples interpreted as positive (165/342, 48.2%, 95% CI 43.0% to 53.5%) compared with the STTT algorithms (148/342, 43.3%, 95% CI 38.0% to 48.5%). The highest overall percentage agreements (>95%) among MTTT algorithms were only observed when both algorithms employed the same EIAs detecting combined IgG/IgM antibodies, but applied in reverse order (ie, first- and second-tier tests interchanged). Conclusion: MTTT algorithms demonstrated higher and more consistent positivity rates than STTT algorithms, although this should not be interpreted as evidence of superior accuracy in the absence of a reference standard. Concerningly, certain configurations of MTTTs showed high agreement with one another, likely attributable to shared antigen targets. These findings underscore the need for careful validation of assay combinations to avoid redundancy and optimize harmonized Lyme disease diagnostic strategies in Canada.