Identification, selection and testing of new antigens for tegumentary Leishmaniasis serological diagnosis

Tegumentary Leishmaniasis (TL), caused by several Leishmania species, presents a broad spectrum of clinical manifestations. This diversity complicates early and accurate diagnosis, which is crucial for effective treatment a preventing chronic or disfiguring outcomes. Conventional diagnostic methods often have significant limitations related to sample collection, technical execution, and interpretation of results. This study employed an immunoproteomic strategy to identify novel antigens, aiming to improve serological diagnostic methods for TL. Candidate peptides were selected using in silico linear B-cell epitope prediction tools (BCPreds and ABCpred), leading to the synthesis of 20 peptides from six proteins. ELISA assays identified six peptides (HS1, HS3, HS5, BT7, EL1, and EL2) with promising diagnostic performance. The highest ELISA performance was observed with the BT7 peptide, as well as with the combination of BT7 and EL1 peptides. The ELISA using BT7 alone achieved an area under the curve (AUC) of 0.967, with 88% sensitivity, 97% specificity, 0.97 positive predictive value (PPV), and 0.88 negative predictive value (NPV) (p < 0.0001). It showed minimal cross-reactivity with visceral leishmaniasis (3%) and no reactivity with sera from patients with paracoccidioidomycosis or sporotrichosis. Performance varied by geographic origin of the sera: BT7 performed best with samples from the Southeast region, whereas EL1 performed better with samples from the Central-west region. Consequently, we tested a cocktail of both peptides. Notably, the combination of BT7 and EL1 achieved the highest diagnostic performance, with an AUC of 0.971, 96% sensitivity, and 88% specificity, enabling accurate detection of positive cases across different geographic regions. These findings highlight the potential of synthetic peptides as valuable targets for developing precise serological diagnostics for TL.