EVALUATION OF MOLECULAR TECHNIQUES FOR DISCRIMINATION OF SPECIES OF Leishmania spp. FROM THE BRAZILIAN AMAZON AND EXTRA-AMAZON REGION, WITH POTENTIAL APPLICATION IN CLINICAL PRACTICE.

• Grant number: 23/16087-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: October 01, 2024
• End date: September 30, 2026
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: José Angelo Lauletta Lindoso
• Grantee: Rayane Miranda Dias
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Introduction: Leishmania is the protozoan that causes leishmaniasis, a non-contagious infectious disease that can be classified as visceral or integumentary and can be transmitted by the bite of sandflies. Several diagnostic methods for American cutaneous leishmaniasis have been developed, but none of the currently available tests can be considered the gold standard. Molecular tests are an alternative for the diagnosis of cutaneous leishmaniasis, as they enable the detection of the parasite's DNA, and depending on the test used, it is possible to identify different species and the Hsp-70 gene is an important target for discriminating species of Leishmania, as it presents conserved regions, with little variability between different species. Objective: To evaluate the use of different molecular techniques, in clinical practice, that can be applied in the species-specific diagnosis of American cutaneous leishmaniasis. Methodology: Samples from patients at the Emílio Institute of Infectious Diseases and the Zoonosis Control Center of Santarém-PA will be used. The clinical samples will be subjected to DNA extraction, and a polymerase chain reaction will be performed using Hsp-70 as the target gene and the products will be visualized on a 2% agarose gel. The products obtained from PCR-Hsp70 will be subjected to restriction enzyme digestion (HAE III) and subsequent visualization of the fragmented product on a 2% agarose gel. The PCR-Hsp 70 fragments will also be subjected to sequencing using the Sanger technique and aligned with sequences deposited in genBank, using the BioEdit program. Samples will also be subjected to a real-time PCR reaction, using the Hsp70 gene as a target, and subsequently, high-resolution dissociation profile analysis (HRM=High Resolution Melting) will be carried out.