PROJECT SUMMARY: Development of improved serological diagnostic and parasite genotyping tools for congenital Chagas disease Chagas disease is a neglected disease caused by the protozoan parasite Trypanosoma cruzi. It is a serious public health issue in Latin America and the southern United States. Diagnosis of chronically infected patients, including congenital and maternal cases, is based on antibody detection. PA-18-031 priorities for Trypanosoma cruzi include the development of new diagnostic tests with high validity and reliability for timely detection of congenital and maternal T. cruzi infection. Indeed, the WHO recommends a minimum of two positive tests to establish diagnosis, due to limited sensitivity and specificity and frequent discordant results. Part of the discordances may be attributed to the very large genetic and antigenic diversity of T. cruzi, which has been divided into seven discrete typing units (DTUs) TcI-TcVI and Tcbat. Indeed, current serological tests are based on a very limited set of parasite antigens/strains which do not reflect the entire range of T. cruzi diversity and multiclonal infections. There is thus a critical need for more reliable tests based on panels of conserved antigens allowing detection of all T. cruzi genotypes. On the other hand, while parasite genotyping is straightforward in parasite culture and in vector samples, current PCR-based techniques have a low sensitivity with blood samples from chagasic patients. Another critical need is therefore to identify new molecular markers of T. cruzi DTUs, allowing a more sensitive genotyping in clinical samples. In aim 1, we will improve serological diagnostic of maternal and congenital T. cruzi infection identifying new conserved T. cruzi antigens. In Aim 2, we will improve the sensitivity of current molecular tools for genotyping by sequencing of T. cruzi in clinical samples from pregnant women and newborns. We will perform a bioinformatics screening of T. cruzi genome sequences from multiple DTUs for new antigens and molecular markers coupled with high-throughput immuno-screening with peptide arrays and multiplex PCR and sequencing, respectively. Antigens identified for diagnostic in an ELISA platform will be used in a rapid test platform for the accurate diagnostic of T. cruzi infection. We will also perform a retrospective analysis of parasite genotype and multi-strain infection on maternal and congenital Chagas disease. At the completion of these studies, we expect to have identified a set of conserved antigens for a novel rapid test for a reliable diagnostic and one or more molecular markers for genotyping by sequencing of all T. cruzi DTUs in pregnant women and newborns. These advances will also spur on molecular research on T. cruzi much beyond our own research focus, by further exploring parasite evolution and genetic diversity allowing us the to understand the potential relations between parasite characteristics, congenital transmission and clinical symptoms.