Two-Tiered Serological Testing for Borrelia Infection: Immunological Background and Analytical Limitations

Two-tiered serological testing is the recommended laboratory diagnostic approach for Borrelia infection, as outlined in international guidelines. This approach utilizes a screening test, typically an indirect immunofluorescence assay (IFA) or enzyme-linked immunosorbent assay (ELISA), followed by a confirmatory Western blot (immunoblot) technique. While effective in diagnosing complex manifestations of Lyme disease, such as Lyme arthritis and neuroborreliosis, this method exhibits lower sensitivity and specificity in early infection. Consequently, published data correlating serological results with clinical presentations are often variable. This study analyzed ELISA results from 878 patients, along with 181 IgM and 157 IgG immunoblot results, evaluating ELISA performance in relation to immunoblot outcomes. We also explored potential and practical factors contributing to discrepancies between ELISA and immunoblot results. The sensitivity and specificity of our anti-Borrelia IgM ELISA tests were 78.9% and 82.9%. For the IgG ELISA tests, sensitivity was 83.3% and specificity was 90%. The positive predictive values were 68.2% for IgM and 71.4% for IgG. The ELISA false positivity could have arised from cross-reactivity with antigens such as the p41 protein. Antibodies targeting immunodominant, pathogen-specific proteins often appear concurrently. Notably, antibodies against OspC proteins from multiple Borrelia species (B. burgdorferi sensu stricto, B. afzelii, B. garinii, and B. spielmannii) were detected in some immunoblot samples. Our study demonstrated adequate performance of IgM and IgG ELISA tests, consistent with published data. Optimizing test selection based on immunodominant antigens, minimizing false-positive and false-negative results, and ensuring appropriate application and timing of twotiered serological diagnostics require enhanced clinical expertise.