SYBR Green-based Real-Time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases

Abstract

BackgroundLeishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk.ResultsA SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95°C ± 0.01 and 77.36°C ± 0.02 for Leishmania and Viannia, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together L. (V.) braziliensis and L. (V.) shawii with a bootstrap value of 100%; while for L. (L.) infantum and L. (L.) amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus Leishmania (average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities.ConclusionsFor all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

“…As in most parasitic diseases, the challenge remains in having the ability to perform a rapid diagnosis of the disease and the characterization and genotyping of the different isolates obtained from infected individuals [11]. So far, the techniques typically employed to characterize and discriminate Leishmania species are Multi Locus Enzyme Electrophoresis (MLEE) [12] and multiple methods based on Polymerase Chain Reaction (PCR) such as Multilocus Sequence Typing (MLST) [13], PCRRestriction Fragment Length Polymorphism (PCR-RFLP) [14], Multiplex PCR [15] and PCR followed by sequencing [16].…”

Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

“…Primer pair choices were based on previously described sequences that amplify fragments of the Leishmania (V.) braziliensis minicircle kDNA (5,(32)(33)(34)(35)(36). We evaluated primer and probe specificity for Leishmania (V.) braziliensis using NCBI BLAST.…”

Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters

“…In contrast, multicopy kinetoplast DNA (kDNA) could boost this sensitivity. Indeed, different studies describing qPCR assays targeting kDNA proved highly sensitive for detection, species discrimination, and quantification of Leishmania in clinical specimens from patients with different forms of leishmaniasis (17,20,21).…”