Desenvolvimento de uma Proteína Quimérica Multiepítopo de Linfócitos B para uso como antígeno no diagnóstico sorológico da Leishmaniose Tegumentar Humana

Abstract

Leishmania (Viannia) braziliensis is a parasite of great importance responsible for most cases of human tegumentary leishmaniasis, especially in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including cutaneous or mucosal leishmaniasis. The association of clinical and epidemiological inquiries with laboratory tests (both direct and indirect methods) helps diagnosing human tegumentary leishmaniasis. Early diagnosis is essential to avoid permanent damage and severe functional sequelae, to identify cases of therapeutic failure and to prevent adverse effects caused by therapeutic approaches based on misdiagnosis. The high toxicity profile of the available drugs for treatment must be considered. None of the laboratory tests currently available can be considered the gold standard, as they do not have satisfactory accuracy for disease detection. Furthermore, the Montenegro Skin Test, which identifies a delayed-type hypersensitivity response to parasite antigens in infected patients, is no longer available in Brazil. This test Skin was an essential test to support infectologists in the differential diagnosis and clinical management of infected individuals. Therefore, there is an urgent need to discover new antigens for the development of high-quality diagnostic tests to be employed in endemic areas for Human tegumentar leishmaniasis. In this sense, the purpose of this study was to obtain a more refined antigen of L. braziliensis to be applied to the serological assays. To achieve this, we developed a gene containing the fusion of sequences encoding thirteen linear B-cell epitopes from proteins of L. braziliensis expressed in the amastigote stage, which are recognized by serum from patients with cutaneous and mucosal leishmaniasis, to produce a recombinant chimeric protein (rChiP). We also performed a comparative analysis with results obtained from ELISA employing soluble L. braziliensis antigen (SLb-Antigen) and a reference laboratory by the immunofluorescent assay (Bio- Manguinhos, Brazil). The rational strategy used in this work was successful in developing a potential antigen to be used in immunological assays to Human tegumentary leishmaniasis, confirmed by excellent sensitivity results (100.0%) in the detection of cutaneous and mucosal cases, simultaneously with excellent value of specificity (100.0%), which excluded the occurrence of false positive results in healthy individuals and in patients with Chagas' disease, commonly associated with cross-reactivity with Leishmania species in endemic areas. The present technology also opens a door for future investment for its use in field exams via an immunocromatographic test, which will be even more helpful in regions where laboratory structure is not available or scarce.