DEVELOPMENT OF AN IDENTIFICATION METHOD FOR Leishmania spp BASED ON REAL-TIME PCR WITH HIGH-RESOLUTION MELTING
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Introduction
Leishmaniasis is a disease caused by parasites of the genus Leishmania . In Colombia, 6 pathogenic species have been reported. Traditional parasitological diagnosis based on the observation of parasites does not allow the species to be identified, which is why biochemical or molecular methods must be used, including conventional PCR, but this has some limitations and requires long periods of time to obtain results., which are sometimes inconclusive.
General Objective
Implement a methodology based on PCR-HRM, which allows the simultaneous detection and identification of parasites of the Leishmania genus in clinical samples from patients or parasites in cultures.
Materials and methods
A PCR-HRM was standardized, through which 237 clinical samples were evaluated, 98 classified as positive and 139 as parasitologically negative by direct and/or culture. The typings were compared with a previously standardized PCR-RFLP.
Results
It was possible to implement a PCR-HRM for the diagnosis and identification of Leishmania species, obtaining 100% agreement with the typings obtained by PCR-RFLP. It was even possible to detect and identify the parasite in samples diagnosed as negative by conventional methods. It was found that, with a reliability percentage greater than 95%, 91 samples out of 98 were typed, of which 81.63% of the cases were L. panamensis, 11.22% were L. braziliensis and 7.14 were indeterminate.% of cases.
Conclusions
PCR-HRM is a good method that allows the identification of the most prevalent species in Colombia, comparing specific mean denaturation temperatures according to the Leishmania species involved.
