Loop-mediated isothermal amplification assays for the rapid discrimination of Treponema pallidum lineages and...

Molecular surveillance aimed at identifying the subspecies and lineages of Treponema pallidum is important. Conventional molecular methods for genotyping are effective but are also time-consuming and costly. This study aimed to develop and evaluate a novel loop-mediated isothermal amplification (LAMP) method using quenching probes to quickly distinguish T. pallidum subsp. pallidum lineages (Nichols, SS14Ω-A, SS14Ω-B 1A, and SS14-East Asia) and T. pallidum subsp. endemicum circulating in East Asia. We developed novel LAMP assays for genotyping the tp0705 (amino acid variants of 506, 625, and 708) and tp0548 genes (a 9 bp insertion region). The LAMP assays were used to genotype 133 clinical samples (Nichols, n = 9; SS14Ω-A, n = 7; SS14Ω-B 1A, n = 2; SS14-East Asia, n = 110; and T. pallidum subsp. endemicum, n = 5) collected from adult patients diagnosed with syphilis in Japan. The genotyping results of the LAMP assays were compared with the results from Sanger or whole-genome sequencing. The lower detection limit of the LAMP assays for tp0705 and tp0548 genotyping was 20 DNA copies/reaction. Of the 133 samples analyzed, the genotypes, lineages, and subspecies of T. pallidum were identified in 129 (97.0%) samples by the LAMP assays. The genotyping results of the LAMP assays were completely consistent with those of Sanger and whole-genome sequencing. Our novel LAMP genotyping assays can rapidly and easily discriminate the lineages and subspecies of T. pallidum circulating in East Asia. This method will contribute to the epidemiological surveillance of T. pallidum in East Asia.

Syphilis remains a significant global public health concern. Molecular surveillance is essential for understanding the transmission dynamics and spread of various Treponema pallidum subsp. pallidum lineages, including emerging strains such as Treponema pallidum subsp. endemicum (TEN). However, conventional genotyping methods like multi-locus sequence typing and whole-genome sequencing are costly, time-consuming, and require specialized equipment and expertise. In this study, we developed a novel loop-mediated isothermal amplification assay with quenching probes that enables rapid, accurate, and low-cost identification of major T. pallidum lineages and subspecies. Our method demonstrated high sensitivity and specificity using clinical specimens and successfully distinguished among Nichols, SS14 variants, and TEN. This assay is well suited for large-scale epidemiological studies and may support more effective and timely public health responses to the rising incidence of syphilis in East Asia and beyond.