Discrimination of Leishmania braziliensis variants by kDNA signatures produced by LSSP-PCR.

The conventional methods for identification and typing of Leishmania species depend on previous culture isolation of the parasites. Not infrequently, culture is unsuccessful and may result in misrepresentation of the heterogeneity of the original isolate. Thus, more reliable and precise identification of genotypes of Leishmania spp. is important for a better clinical and epidemiological understanding of the disease. We evaluated the potential of LSSP-PCR targeting kDNA minicircles in discriminating different variants of the parasite with the use of clinical samples directly or cultivated parasites. The 1st step of this procedure consists of the amplification of the minicircles by conventional PCR; the 2nd step is low-stringency amplification of the minicircles previously amplified, with the use of 1 of the primers. Although LSSP-PCR produced complex and distinct kDNA signatures for isolates representing different species, further experiments demonstrated that the approach had the potential for discriminating intraspecific variants of L. braziliensis. Thus, the generated profiles were too variable to be useful as markers for species identification. Moreover, we demonstrated that the approach can be directly applied to clinical samples. In conclusion, LSSP-PCR targeting kDNA minicircles produces profiles that reflect polymorphisms of the predominant classes of minicircles, and can be useful for studies aimed at discriminating Leishmania braziliensis genotypes without the need for previous cultivation of the parasite.

The compositional landscape of minicircle sequences isolated from active lesions and scars of American cutaneous leishmaniasis.

BACKGROUND: American cutaneous leishmaniasis (ACL) is characterized by cutaneous lesions that heal spontaneously or after specific treatment. This paper reports on the analysis of kDNA minicircle sequences from clinical samples (typical lesions and scars) that were PCR-amplified with specific primers for Leishmania species of the subgenus Viannia. METHODS: From 56 clinical isolates we obtained a single amplified fragment (ca. 790 bp), which after cloning and sequencing resulted in 290 minicircle sequences from both active lesions and scars. We aimed to get a compositional profile of these sequences in clinical samples and evaluate the corresponding compositional changes. Sequences were analyzed with the compseq and wordcount (Emboss package) to get the composition of di-, tri-, tetra-, penta- and hexanucleotides. Additionally, we built a nucleotide dictionary with words of 7, 8, 9 and 10 nucleotides. RESULTS: This compositional analysis showed that minicircles amplified from active cutaneous lesions and scars have a distinct compositional profile as viewed by nucleotide composition of words up to 10mer. With regard to the most frequent nucleotide words above length 6, there is also a distinct pattern for 7, 8, 9 and 10mer. CONCLUSION: These results indicate that minicircle sequences can be monitored upon direct exposure to a selection/stressing environment (e.g. chemical action) by evaluating their nucleotide compositional profile. It might be useful as a molecular tool in research concerning the evolution of infecting Leishmania in both vector and vertebrate hosts.