Serological testing for Lyme disease: current assays, diagnostic performance, and challenges

Lyme disease, caused by Borrelia burgdorferi sensu lato, remains the most common vector-borne infection in temperate regions of the Northern Hemisphere. Laboratory diagnosis relies primarily on serologic testing, as current direct detection methods generally lack sensitivity in routine clinical specimens. Current serological diagnosis of Lyme disease relies on a two-tiered testing algorithm. In the article by E. J. Horn, B. Menefee, A. M. Schotthoefer, G. Dempsey, et al. (J Clin Microbiol 64:e01187-25, 2026, https://doi.org/10.1128/jcm.01187-25), the authors further demonstrated that the modified two-tiered testing (MTTT) algorithm has increased sensitivity compared with the standard two-tiered testing (STTT) algorithm for detecting antibodies from early Lyme disease samples. Despite the improved sensitivity using MTTT, important limitations persist, including limited sensitivity in early infection and the inability to distinguish active from past infection. This commentary briefly summarizes the current landscape of Lyme disease serologic assays, outlines established and emerging diagnostic algorithms, and discusses key challenges that continue to impact diagnostic accuracy and clinical decision-making.