Avaliação do desempenho de proteínas recombinantes no diagnóstico de leishmaniose visceral humana (estudo de fase I)

Abstract

Introduction: Leishmaniasis is a group of diseases caused by protozoa of the genus Leishmania, considered one of the 20 neglected tropical diseases, and presents varied clinical manifestations: cutaneous, mucocutaneous, visceral and post-kala-azar dermal. Human visceral leishmaniasis (HVL) is a serious public health problem due to its wide distribution and potentially fatal clinical course, which makes early and accurate diagnosis essential. Objective: To evaluate the performance of recombinant proteins (rLci5 and KDDR-PLUS) in detecting seropositivity in individuals infected with Leishmania infantum. Methodology: The project was approved by the Ethics Committee of the Gonçalo Moniz Institute (CAAE: 43012821.6.0000.0040). The indirect ELISA was standardized with different concentrations of antigen, primary and secondary antibodies, and with protein A conjugated to peroxidase. The results were normalized by the reactivity index (RI). Accuracy was assessed using 2x2 contingency tables, sensitivity, specificity, and ROC curves with 95% Confidence Interval. McNemar's test and Cohen's Kappa index were applied for statistical analysis. Results: Standardization of ELISA with rLci5 (50ng/well) resulted in 100% sensitivity and specificity (95% CI 84.6-100). With protein A, the best combination was 50ng of antigen and 1:20,000 dilution. ELISA with rLci5 obtained 91.5% sensitivity, 97.5% specificity, and 97.4% AUC; with protein A, sensitivity of 89.9%, 95% specificity, and 97.4% AUC. ELISA with rKDDR-PLUS showed 98.3% sensitivity, 98.8% specificity, and 99.8% AUC; with protein A, sensitivity of 98.3%, specificity of 98.8% and AUC of 98.5%. The rapid test TR LSH AB ECO detected IgG in 54 of 59 positive samples (sensitivity of 91.5%), but specificity of only 40.96%. Protein A increased the mean OD for rLci5 (1.945 ± 1.139) and rKDDR-PLUS (2.605 ± 0.776), showing an increase in detection capacity. Conclusion: The results demonstrate the high diagnostic performance of the recombinant proteins rLci5 and rKDDR-PLUS. The replacement of the secondary antibody by protein A conjugated to peroxidase proved to be effective, especially with the rKDDR-PLUS protein. The study confirms the superiority of these proteins in relation to the rapid test with rK39 used in the current protocol of the Unified Health System.