Digital PCR quantification of Treponema pallidum burden in primary syphilis lesion swabs reveals variable performance across direct detection methods.

BACKGROUND: Syphilis incidence in the United States has reached its highest level since 1950, yet diagnosis of primary syphilis remains constrained by the limitations of serology, darkfield microscopy (DFM), and quantitative PCR (qPCR). Absolute organism burden in primary lesions is incompletely characterized, and existing estimates derived from qPCR standard curves may not capture its true range. We applied digital PCR (dPCR) to directly quantify Treponema pallidum burden and compare diagnostic performance across modalities. METHODS: Lesion swabs from 21 specimens (19 participants) with high clinical suspicion of primary syphilis were tested by RPR, DFM, a three-target qPCR panel (polA, tpp47, and multi-copy tpa3), and tpp47 single-target dPCR on the QuantStudio Absolute Q platform. Performance was assessed against a composite reference and swab organism burden was normalized to organisms per mL of swab media. RESULTS: Median organism burden was found to be 38,796 organisms/mL (range 5,745 to 582,388), spanning nearly two orders of magnitude and revealing greater lesional heterogeneity than prior estimates. tpp47 Ct correlated strongly with dPCR burden, whereas RPR titer showed no correlation. Digital PCR and multi-target qPCR each demonstrated 100% sensitivity and specificity against a composite reference standard of multiple syphilis detection methods, compared with 87.5% sensitivity for single-target tpp47 qPCR and 81.2% for DFM. CONCLUSIONS: Digital PCR provides precise quantification of T. pallidum in primary lesions swabs and reveals substantial swab burden heterogeneity not captured by prior methods. General validation in a larger cohort is needed for clinical adoption of dPCR, but these findings may help inform minimum detection thresholds for future syphilis screening assays.