Characterization of loci with allelic variation in the Leishmania braziliensis genome that could be useful in diagnostics tests and epidemiological studies

by Sousa, Rosana Santos

American tegumentary leishmaniasis (ATL) in Bahia, Brazil is an infectiousdisease caused by L. braziliensis. This species has a high degree of geneticvariability, as well as a large spectrum of clinical manifestations of illness. A previousstudy of parasites isolated from patients from an endemic area demonstrated that thepopulation of L. braziliensis was multiclonal and contained different clades whichwere associated with specific clinical outcomes. In this study, we wanted to identifythe allelic variation in the DNA fragments generated by our three RAPD eletrophoreticprofiles (ABI-04, A8 and ABI-18). We used six L. braziliensis isolates from differentclades and typed two isolates with each RAPD profile. From the profiles, wesubmitted six distinct bands for sequencing (ABI-04: band B1 and B2, A8: band B1,B2, B3 and ABI-18: band B1). Of 24 samples submitted, 22 were successfullysequenced. The sequences generated from each RAPD profile separately andbetween the pairs of isolates were aligned and compared for: (a) nucleotide sequenceidentity: characterization as homologous loci; (b) the presence of polymorphisms; and(c) the nature of the detected polymorphisms. We found that the sequences for twocorresponding bands of the ABI-04 RAPD protocol and for the three bands of the A8RAPD protocol had around 90% sequence identity, that corresponded to homologousloci with some allelic variation. The detected polymorphisms were 97 SNPs, 227indels and 08 microsatellites. We also analyzed the sequences for potential restrictiondigest sites to obtain polymorphic sites that could be used to develop an RFLP basedtyping method, and found up to 120 different sites between the nine loci. In the future,the allelic variations identified between these loci will be useful for the development ofepidemiological and diagnostic tools with prognostic value on the human infections