PROJECT SUMMARY Since 2014, Congenital Syphilis (CS) cases in the United States, caused by the transplacental transmission of the bacterium Treponema pallidum from mother to child, have increased at an alarming rate. Targeted surveillance studies indicate a rise >185% from 2014 to 2018 alone. Women who acquire syphilis within 4 years of pregnancy will transmit the infection to 80% of their fetuses, and 4 in 10 pregnancies will end in stillbirth or infant death. Infants contracting CS may be asymptomatic, but they can also suffer from serious early- (< 3 months) and late-stage (>2 years) manifestations of multisystemic syphilis infection, including skin lesions, bone deformities, liver abnormalities, and blindness. Treponema strains are extremely difficult to culture, thus sensitive and specific diagnosis requires multi-step testing paradigms that are labor intensive, subject to false-positives and false-negatives, and limited in scalability. No single test is currently commercially available that enables accurate, rapid, inexpensive, and simple widespread testing for syphilis infection. Yet, such a test could reduce syphilis-associated stillbirth and perinatal death by up to 75%. Here we propose to extend the capabilities of “MeltSeq”, our novel pathogen identification platform that is specifically geared towards neonatal infections and small-volume blood samples. Phase I builds upon our previous work in broad-based pathogen identification at single-genome levels to develop a fast and actionable Treponema detection and strain differentiating assay. In addition, we will reduce testing times from ~3 hours to <1 hour, the typical time of an office visit, by using our unique device to achieve rapid amplification and by optimizing our novel blood sample preparation technology. These advances will enable syphilis screening during pregnancy. Finally, we will advance microbial DNA extraction from dried blood spot (DBS), which are collected on virtually all newborns in the United States, to enable universal newborn syphilis screening. Phase II involves both retrospective and prospective clinical evaluation for 510(k) approval and commercialization of a point-of-care device. Our multidisciplinary team approach combines expertise in clinical neonatology, bioengineering, and commercial product development to create a simple and inexpensive syphilis test that can be utilized for pregnant women and newborn screening programs.