Project Summary Congenital cytomegalovirus (cCMV) and neonatal herpes simplex virus (nHSV) impose major health threats on neonates. Although CMV and HSV are lifelong infections with periods of latency and reactivation, most maternal infections remain undiagnosed due to nonspecific or absent clinical symptoms. In the United States, a child is permanently disabled by cCMV infection every hour, even though 9 of 10 infants are asymptomatic after birth and remain undiagnosed. Invasive nHSV, on the other hand, is a rare neonatal infection that presents with a broad range of clinical symptoms, including those that may be life-threatening. Viral culture and DNA detection by polymerase chain reaction (PCR) have become the “Gold Standard” for the diagnosis of cCMV and nHSV infection, despite poor sensitivity of PCR assays in neonates and time consuming culture techniques (up to 5-7 days). Universal genotyping of pathogen genomic sequences using High Resolution Melt (U-HRM) provides a simple, low cost, rapid, and modern alternative to viral cultures and PCR techniques. By measuring the fluorescence of an intercalating dye as PCR-amplified pathogen DNA or RNA fragments are heated and disassociate, sequence defined melt curves, or “fingerprints”, are generated with single-nucleotide resolution in a closed-tube reaction. These unique microbial “fingerprints” are then automatically identified and quantified using machine learning technology, with an accuracy of 99-100% on minimal blood volume (1 mL), in a platform called NeoChip. Presently, we have established unique signature melt curves for 40 bacterial species and antimicrobial resistance genes that commonly infect neonates. Additionally, NeoChip has been expanded to distinguish individually amplified melt curve signatures for multiple pathogen identification and quantification, as required for polymicrobial infection. In this proposal, we will build out NeoChip’s comprehensive database by incorporating clinical strains of CMV and HSV with actionable antiviral resistance genes. Because NeoChip identifies variances in nucleic acid sequences, individual differentiation and quantification of CMV and HSV strains are possible. We will also translate the NeoChip for specific and rapid diagnosis of cCMV and nHSV infection in a large prospective clinical study of pregnant women and their offspring(s), as well as directly compare the platform to standard quantitative nucleic acid test (QNAT) assays, IgG/IgM antibody testing, and clinical outcome measures for statistical concordance (predictive value). Finally, we will validate and translate NeoChip for cCMV detection and clinical correlation using dried blood spot (DBS) samples for incorporation into standard universal newborn screening programs. NeoChip’s goal is to provide an accurate and valid test for the timely diagnosis of pathogen etiology (viral, bacterial, and fungal) in a single test with efficacy on broad tissue matrices and capacity to inform microbial...