Identification of highly conserved <i>Trypanosoma cruzi</i> antigens for the development of a universal serological diagnostic assay

Chagas Disease is an important neglected tropical disease caused by <i>Trypanosoma cruzi</i>. There is no gold standard for diagnosis and commercial serological tests perform poorly in certain locations. By aligning <i>T. cruzi</i> genomes covering parasite genetic and geographic diversity, we identified highly conserved proteins that could serve as universal antigens for improved diagnosis. Their antigenicity was tested in high-density peptide microarrays using well-characterized plasma samples, including samples presenting true infections but discordant serology. Individual and combination of epitopes were also evaluated in peptide-ELISAs. We identified &gt;1400 highly conserved <i>T. cruzi</i> proteins evaluated in microarrays. Remarkably, <i>T. cruzi</i> positive controls had a different epitope recognition profile compared to serologically discordant samples. In particular, multiple <i>T. cruzi</i> antigens used in current tests and their strain-variants, and novel epitopes thought to be broadly antigenic failed to be recognized by discordant samples. Nonetheless, &gt;2000 epitopes specifically recognized by IgGs from both positive controls and discordant samples were identified. Evaluation of selected peptides in ELISA further illustrated the extensive variation in antibody profiles among subjects and a peptide combination could outperform a commercial ELISA, increasing assay sensitivity from 52.3% to 72.7%. Individual variation in antibody profiles rather than <i>T. cruzi</i> diversity appears to be the main factor driving differences in serological diagnostic performance according to geography, which will be important to further elucidate. ELISA with a combination of peptides recognized by a greater number of individuals could better capture infections, and further development may lead to an optimal antigen mixture for a universal diagnostic assay.