Background <p>Reverse sequence algorithms are increasingly used for the serological diagnosis of syphilis in clinical practice; however, differences between the approaches recommended by the U.S. Centers for Disease Control and Prevention (CDC) and the European Centre for Disease Prevention and Control (ECDC) may influence test interpretation.</p> Methods <p>We performed a retrospective laboratory-based study including 82 serum samples with reactive <i>Treponema pallidum </i>electrochemiluminescence immunoassay (ECLIA) results collected at a tertiary-care university hospital. Samples were evaluated according to CDC and ECDC reverse sequence algorithms using rapid plasma reagin (RPR) and <i>Treponema pallidum</i> hemagglutination assay (TPHA) in the respective testing sequence. Agreement between algorithms was assessed using percentage concordance and Cohen’s κ statistic, and discordant results were further evaluated using available clinical and follow-up data.</p> Results <p>Discordant serological patterns were identified between the two algorithms. Although overall agreement between the algorithms was high (96.3%; κ = 0.709), the wide confidence interval of Cohen’s κ suggested statistical uncertainty, likely related to the limited number of discordant cases. Using the ECDC algorithm, three of 82 (3.7%) ECLIA-reactive samples with reactive RPR but nonreactive TPHA results did not fulfill the serological criteria of the testing sequence at the time of evaluation; follow-up serological findings in two of these cases were considered compatible with recent or early active syphilis.</p> Conclusions <p>Although overall agreement between the algorithms was high, interpretation of discordant serological patterns may vary depending on testing sequence and assay selection. These findings suggest that early incorporation of nontreponemal testing may aid interpretation of discordant serological patterns in routine clinical practice.</p> Clinical trial number <p>Not applicable.</p>