Desenvolvimento de marcador molecular para o diagnóstico de variedades de leishmania circulantes na Região Norte do Brasil.

Abstract

The determination of the prevailing species responsible for American Tegumentary Leishmaniasis (ATL) and the detection at species level of the varieties of the protozoan parasite agent of this endemic disease at the Amazon region are important for determination of the clinical evolution and to better indicate the therapeutic conduct due to the different responses to chemotherapeutic drugs by the Leishmania species. By now we are not aware of an available test to Leishmania (Viannia) guyanensis capable of detecting its intra-specific varieties obtained from human clinical presentations or from vertebrate and invertebrate hosts. We succeed to discriminate Leishmania (V.) guyanensis from Leishmania (V.) braziliensis and also from Leishmania (V.) panamensis through a polimerase chain reaction direct genomic DNA amplification aimed at the transcribed internal spacer (ITS) of the ribosomal RNA gene (rDNA). The positive reaction is visualized at the agarose gel electrophoresis by the presence of an aproximatedly 229 nucleotides size band for L. guyanensis that is absent for the two other species L. braziliensis and L. panamensis. The diagnosis can be done using culture material, skin biopsy or squashing the sand fly vector and having the DNA extracted. In a second step of the investigation, the ITS rDNA was sequenced in L. lainsoni; L. naiffi and four L. guyanensis strains, two of them presenting a muco-cutaneous form of Leishmaniasis. These sequences were compared to others available at the Genbank Database, confirming the previous data from multilocus enzyme electrophoresis and ITS restriction fragment lenght polymorphisms analysis, positioning L. lainsoni and L. naiffi as more divergent species as compared to L. braziliensis, L. panamensis and L. guyanensis species inside de Viannia sub-genus. A correlation was observed in grouping nearer the two L. guyanensis strains, respectively IM4243 and IM4235, responsible for the muco-cutaneous form. This protozoan belonging to sub-genus Viannia show clinical and epidemiological importance in South America north region, particularly the Amazon region and this study can be considered an advance in providing a tool to better understand the parasite species silvatic cycle and in providing tools to characterize the main species responsible for the clinical form presentation of tegumentary leishmaniasis in the human population at this region.