Overcoming the bottleneck in Treponema pallidum quantification: a novel flow cytometric assay for rapid, precise, and...

Precise quantification of the causative agent of syphilis, Treponema pallidum (T. pallidum) is critical for advancing research in pathogenesis, treatment response, and vaccine development. However, current methods have certain limitations. Dark-field microscopy (DFM) suffers from low sensitivity, poor reproducibility, and strong operator dependence, while quantitative PCR (qPCR) offers high precision but is time-consuming, technically demanding, and reliant on high-quality, consistent commercial reagents. This methodological bottleneck highlights the urgent need for a technique that integrates the speed and simplicity of direct detection with the precision, objectivity, and throughput of an automated assay. Herein, to bridge this gap, we propose a strategy for rapid, high-throughput quantification of T. pallidum using a novel, fluorescence-based flow cytometric assay implemented on an automated urine analyzer (the Sysmex UF-5000 analyzer). The assay demonstrated a limit of detection of 7.02 × 10^³T. pallidum/mL and excellent precision (all coefficients of variation < 20%). It showed strong quantitative agreement with qPCR across a wide dynamic range (4.98 × 10³-2.10 × 10⁷T. pallidum/mL), with an excellent correlation (r = 0.9967), without significant proportional or constant bias (Passing-Bablok slope = 1.003). Bland-Altman analysis confirmed a close agreement (mean difference: -1.14 × 10⁵T.pallidum/mL). In contrast, DFM exhibited substantially higher variability (CVs 15.19-83.52%) and failed to detect low-concentration samples. Operationally, the flow cytometric assay provides results within 30 s per sample at a low consumable cost (approximately $0.35 per test), outperforming DFM in objectivity and throughput and qPCR in both speed and cost-effectiveness. In summary, this novel flow cytometric assay effectively overcomes the historical challenges associated with T.pallidum quantification. This automated, precise, and rapid assay integrates the simplicity of direct detection with the accuracy of molecular quantification, offering a standardized and practical tool to enhance research in syphilis microbiology, pharmacology, and immunology, paving the way for more reproducible and translatable scientific discoveries.