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.
Highlights
Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures
Considering an estimated length for the Leishmania minicircles conserved region of around 120 base pairs, we further investigated these minor differences on the melting temperature for each subgenus by sequencing the amplification products obtained from the analyzed Leishmania species
In order to verify if other Viannia species causing Cutaneous leishmaniasis (CL) in Brazil generated the characteristic melting profiles for the kinetoplastic DNA (kDNA) amplicons previously observed for this subgenus, the panel of Viannia reference strains was expanded by including L. (V.) guyanensis, L. (V.) naiffi and L. (V.) lainsoni promastigotes [see Additional file 2]
Summary
Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. The leishmaniases represent a group of diseases with worldwide distribution and a wide spectrum of clinical presentations, which constitute an important public health problem. Their control has been hampered by the unavailability of rapid means of detection and the lack of suitable therapeutic or prophylactic measures. Cutaneous leishmaniasis (CL) in Brazil is caused by a variety of dermotropic Leishmania species and a great diversity of these parasites is found in the Amazon Region. Except in primary forest areas in North Brazil and the Amazon Region, L. (V.) braziliensis is the main widespread etiologic agent of CL in Brazil [3]
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