Discovery and fine epitope mapping of novel serology-based markers for diagnosis of CongenitalDiscovery and fine epitope mapping of novel serology-based markers for diagnosis of Congenital Chagas Disease Chagas Disease using high-density peptide chips.

MOSCATELLI, G; FRASCH, ACC; ALTCHEH, J; MUCCI, JS; CAMPETELLA, O; BRACCO, L; TEKIEL, V; BUSCAGLIA, CA; NIELSEN, M; AGUERO, F

New Orleans, Louisiana USA

Congreso; ASTMH 67th Ann Meeting,, October 28 - November 1; 2018

American Society of Tropical Medicine and Hygiene

Discovery and fine epitope mapping of novel serology-based markers for diagnosis of Congenital Chagas Disease using high-density peptide chipsChagas Disease, caused by Trypanosoma cruzi, affects 6-8 million people in the Americas and at least 2 million women of fertile age are estimated to be chronically infected with T. cruzi. Congenital infections with T. cruzi represent a global problem, occurring on average in 5% of children born from chronically infected mothers, both in endemic and non-endemic areas. Current diagnostic protocols and techniques for Congenital Chagas Disease (CCD) are cumbersome, have poor performance or require re-testing of newborns and infants during the first year of age. Therefore, new and improved diagnostics that can be deployed in primary health centers are urgently needed. Serology-based assays are preferred due to their low cost and simplicity, but currently suffer of poor performance in newborns due to the presence of confounding maternal antibodies. Furthermore, there is a very limited set of T. cruzi antigens characterized from congenital infections. Here we present a set of defined antigens and epitopes with and excellent profile for diagnosis of CCD. We have developed a highly parallelized screening platform based on high-density peptide-arrays displaying >175,000 short peptides derived from 457 T. cruzi proteins (Carmona SJ et al 2015). We have screened these peptide chips with 2 pools of sera from 10 infected newborns, and with 2 paired pools of sera from their mothers. Each microarray slide was assayed with anti-human IgG (Cy3 labeled), and anti-human IgM (Cy5 labeled) secondary antibodies, and imaged in a fluorescence scanner. After data normalization we reconstructed the antibody-binding profiles for each protein in the array and validated the assay by checking the profiles of known antigens. Comparative analysis of IgG reactivity between paired sera pools of newborns and their mothers allowed the identification of candidate antigens for diagnosis of CCD. These display high IgG reactivity in infected newborns, and low or null reactivity in the paired samples from their mothers. Analysis of IgM antibody-binding also led to the identification of candidate antigens with strong IgM reactivity in the samples from infected newborns. From these data we prioritized 125 highly reactive peptides from the top 5% of both IgG and IgM sets, and selected 48 short peptides for further validation and assessment of their seroprevalence in standard ELISA format in 96-well plates (currently underway). This study represents the largest screening so far for discovery of congenital markers of infection with T. cruzi.