Análise da rede de diagnóstico para leishmaniose tegumentar em laboratórios de referência do estado de Minas Gerais e desenvolvimento de dois protótipos: um teste rápido imunocromatográfico e uma formulação para intradermorreação

Abstract

Until 2016, one of the main diagnostic methods for tegumentary leishmaniasis (TL) in Brazil was the Montenegro Intradermal Reaction (IDRM). With its discontinuation, the state of Minas Gerais (MG) established a diagnostic network for TL, where the following tests would be carried out: direct parasitological examination (EPD) and polymerase chain reaction (PCR). This project evaluated the performance of this diagnostic network through analysis of the established diagnostic flow and proposed developing two prototype immunological tests: a rapid indirect immunochromatographic test (TRI) and a formulation for an intradermal reaction. In phase I of the project, the results of LT exams in the MG network were analyzed, registered in the Laboratory Environment Management System of the Ezequiel Dias Foundation (GAL/FUNED) from 2017 to 2020. 1,369 individuals and 704 (51, 4%) were confirmed cases: 610 (86.7%) by EPD and 94 (13.4%) by PCR. Additionally, 53 (25.3%) EPD with negative results had positive PCR results. In phase II, a chimeric protein was developed, created from the fusion of regions encoding multiepitopes from L. braziliensis B lymphocytes, to be used as an antigen in a new TRI. The antigen was evaluated in an indirect enzyme-linked immunosorbent assay (ELISA), using 79 LT cases and 59 control individuals, and presented 94.81% sensitivity and 91.67% specificity. In the evaluation using the TRI, with 40 cases of TL and 40 healthy individuals, it presented 55% sensitivity and 90% specificity. In Phase III, another chimeric protein was developed, containing the fusion of multiepitopes from CD4+ and CD8+ T lymphocytes from L. braziliensis and L. amazonensis, to be used in an intradermal reaction. The formulation was evaluated in a preclinical trial in a murine model, using control Balb/c mice control group and group infected with L. braziliensis and L. amazonensis. The intradermal reaction with the protein (concentration of 50µg) showed a papule diameter greater than the Montenegro standard antigen, with 48/72 hours. In evaluating the local inflammatory profile, the production index of CD4+ and CD8+ T lymphocytes of central memory (CD4+CD44HighCD62LHigh and CD8+CD44HighCD62LHigh) and effector memory (CD4+CD44HighCD62LLow and CD8+CD44HighCD62LLow) of the mice was verified and an increase in cells was observed of significant central and effector memory between the infected groups in both splenocytes and axillary satellite lymph nodes in relation to the control group. It can be concluded that the TL diagnosis flow in MG proved to be effective in guaranteeing the first examination carried out on the patient on site, allowing greater access and agility in treatment. Additionally, PCR proved to be a good confirmatory test. However, the importance of developing new alternatives for the diagnosis of TL is clearly demonstrated, which will favor the control of the disease and the implementation of adequate treatment in areas endemic to the disease. In relation to other phases, the results demonstrated a great potential of the antigens developed and used in the diagnostic tests, proposed in the present project (TRI and Intradermorreaction), to be used as promising alternatives in the diagnosis of TL, as they will contribute to the identification of cases and consequently in disease surveillance in the country.