The accurate diagnosis of Lyme disease, caused by Borrelia burgdorferi, is critical for effective treatment and prevention of disabling long-term effects. This study investigates discrepancies in serological testing methods for Borrelia infections, focusing on the performance of different assays in annual Lyme screening for high-risk populations. We compared the results of two-tier protocols, including first-tier assays from EUROIMMUN, ZEUS, SERION (all ELISA) and Diasorin (CLIA). Our findings reveal significant variability in test sensitivity: EUROIMMUN combined with ZEUS ELISA detected more positive cases compared to the Diasorin and SERION tests. Notably, 19% of recent Borrelia infections were missed due to test selection, highlighting the clinical implications of these discrepancies. Case studies illustrate the potential for misdiagnosis and delayed treatment, emphasizing the need for standardized and optimized testing protocols. We recommend the adoption of more sensitive and comprehensive methods, and the practice of comparing current results with previous samples from the same individual to improve diagnostic accuracy and patient outcomes.

AG is a senior officer and shareholder, and EV, YS, JL and MK are employees of Innatoss Laboratories B.V., which provides diagnostic screening for infectious diseases including Borrelia infections.

no external funding was received.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

All blood/serum samples and data reported in this article were submitted to Innatoss for diagnostic purposes. The Medisch Ethische Toetsingscommissie Brabant, Tilburg, the Netherlands has waived the need for IRB approval of reuse of surplus diagnostics materials (NW2020-77). The sample/patient IDs cannot be used to reveal identity of the study subjects, and they are not known to anyone outside the research group.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Samples All blood/serum samples and data reported in this article were submitted to Innatoss for diagnostic purposes. The Medisch Ethische Toetsingscommissie Brabant, Tilburg, the Netherlands has waived the need for IRB approval of reuse of surplus diagnostics materials (NW2020-77). The sample/patient IDs cannot be used to reveal identity of the study subjects, and they are not known to anyone outside the research group.

Conflict of interest AG is a senior officer and shareholder, and EV, YS, JL and MK are employees of Innatoss Laboratories B.V., which provides diagnostic screening for infectious diseases including Borrelia infections.

All data produced in the present study are available upon reasonable request to the authors