Discrimination of Leishmania braziliensis Variants by kDNA Signatures Produced by LSSP-PCR

Abstract

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.

KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

“…In this sense, LSSP-PCR can be comparatively considered a simpler and more sensitive molecular tool for ascertaining genetic variability in Leishmania sp. since the kDNA signatures obtained in this and in previous studies, from analysis of L. infantum and L. braziliensis [35] , [37] strains, respectively, were very informative and composed of multiple fragments obtained in a single LSSP-PCR step, without the employment of DNA probes or restriction enzymes [10] , [24] , [45] – [46] .…”

“…However, despite of the effectiveness of LSSP-PCR to detect genetic variability in kinetoplastid parasites, only few studies have used LSSP-PCR for analyzing intraspecies polymorphisms in Leishmania [35]–[37].…”

“…This technique has been efficiently employed to ascertain genetic variability in protozoa parasites, such as Entamoeba histolytica [24] , Trypanosoma cruz i [27] – [33] and Trypanosoma rangeli [34] . However, despite of the effectiveness of LSSP-PCR to detect genetic variability in kinetoplastid parasites, only few studies have used LSSP-PCR for analyzing intraspecies polymorphisms in Leishmania [35] – [37] .…”

Evaluation of Genetic Polymorphism ofLeishmania (V.) braziliensisIsolates Obtained from the Same Patient before and after Therapeutic Failure or Reactivation of Cutaneous Lesions

“…DNA was extracted from the samples according to protocols previously described [11, 12]. DNA was amplified using a pair of primers (BI: 5′-GGGGTTGGTGTAATATAGTGG-3′ and B2: 5′-CTAATTGTGCACGGGGAGG-3′), directed against the variable region of kDNA minicircles (mitochondrial DNA) of Leishmania braziliensis complex species ( Viannia subgenus).…”

“…Amplification was performed like previously described [12]. The amplification products were analyzed on 2% agarose gel or 1.8% agarose (Sigma) gel visualized with ethidium bromide under UV light.…”