rLci5 and rKDDR: recombinant antigens with high diagnostic potential for human visceral leishmaniasis

Human visceral leishmaniasis (HVL) is a major public health threat given its broad geographic distribution and potentially fatal course, underscoring the need for early and accurate diagnosis. This study evaluated the performance of the recombinant proteins rLci5 and rKDDR-plus (a kinesin-derived degenerate repeat recombinant protein) for the serological diagnosis of HVL. Indirect ELISAs using rLci5 and rKDDR-plus were standardized with different antigen concentrations, primary and secondary antibody dilutions, and peroxidase-conjugated Protein. The standardized indirect ELISAs were evaluated alongside the LSH Ab ECO Teste, a commercial rK39-based rapid immunochromatographic test, using a panel of human serum samples with confirmed HVL-positive (n=59) and HVL-negative (n=81) status, as well as sera from individuals with other infections (n=85). The reference standard for positivity in HVL diagnosis of serum panel was a positive parasitological examination of bone marrow aspirate, either alone or in combination with a positive rK39 rapid test; HVL-negative status was defined by negative results using the same criteria. Results were normalized using the reactivity index (RI). Diagnostic performance was assessed by sensitivity, specificity, ROC curves analysis, McNemar's test, and Cohen's Kappa index. ELISA-rLci5 showed 91.5% sensitivity, 97.5% specificity, whereas ELISA-rLci5 using Protein A showed 89.9% sensitivity, and 95.0% specificity. ELISA-rKDDR-plus presented 98.3% sensitivity, 98.8% specificity; ELISA-rKDDR-plus using the Protein A showed 98.3% sensitivity, and 98.8% specificity. Although protein A increased mean optical density values, it did not translate into improved diagnostic accuracy. The LSH Ab ECO Teste showed 91.5% sensitivity and 100% specificity. Both recombinant antigens showed similar diagnostic performance of the LSH Ab ECO Teste, a rK39-based rapid test used in Brazil. Protein A proved to be a viable alternative to conventional secondary antibodies, increasing signal intensity, but not influencing the diagnostic performance. These findings support rLci5 and rKDDR-plus as promising candidates for improved HVL serodiagnosis.