Abstract The re-emergence in maternal and congenital syphilis (CS) infection in recent years has brought this sexually transmitted infection (STI) to the forefront of policy discussions among federal and state health and human services departments. The recommended tests at birth demonstrate poor sensitivity for the diagnosis of CS, necessitating longitudinal follow up for up to 18 months until infection can be definitively excluded. This strategy inevitably results in cases of delayed or even missed treatment, leading to serious adverse outcomes that could have been prevented. With data suggesting a missed diagnosis rate approaching 15%, there is an urgent need for improved diagnostic modalities to detect CS through direct identification ofT. pallidum using highly sensitive contemporary nucleic acid amplification tests (NAA Ts). Two potential options include: real-time quantitative polymerase chain reaction (qPCR) and the Aptima Treponema pallidum transcription-mediated amplification (TMA) assay. By directly detecting the pathogen in neonatal biospecimens, these assays may provide more timely detection of CS in order to initiate timely evaluations and treatment. The aim of this study is to conduct a large, multicenter, prospective observational cohort trial of 924 maternal and neonatal dyads at risk for CS to effectively evaluate the test performance of these NAATs compared to the 2021 STD CS infection categories assigned to that patient at birth ( confirmed proven/highly probable, possible CS, CS less likely, CS unlikely) to determine if a superior diagnostic algorithm exists. Longitudinal follow up confirming infection status will be performed up to 18 months of age. Based on existing scientific premise, our central hypothesis is that the contemporary NAA Ts will result in a more sensitive, specific and timely diagnosis of congenital infection with T. pallidum compared to the standard recommended testing algorithms. In addition, we plan to leverage the resulting clinical cohort to more accurately define adverse neurodevelopmental outcomes associated with CS. This study will be the first to evaluate neurodevelopmental outcomes of affected children by performing Bayley Scales of Infant and Toddler Development™ at 18 months on the offspring of infected women. Beyond the importance of this research in terms of pure science, the results could have far-reaching public health implications that may contribute to improved quality and guideline efforts.