Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP

Abstract

Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist.

Identificación molecular de aislamientos clínicos de Leishmania spp. procedentes de Colombia con base en el gen hsp70

“…La discriminación entre L. panamensis y L. guyanensis usando la restricción del producto amplificado de la hsp70 con BccI, resulta muy evidente si se sigue el protocolo propuesto, el cual se evaluó previamente en cepas de referencia, aislamientos y muestras clínicas procedentes también de Colombia (16).…”

“…En este contexto, la variante PCR-hsp70/RFLP basada en el gen hsp70, propuesta por García, et al (11), y modificada posteriormente, detecta 14 especies de importancia médica usando un solo par de cebadores y, a partir de un único producto de amplificación, logra identificar individualmente la mayoría de dichas especies mediante una o dos restricciones enzimáticas (15). Esta variante se ha aplicado tanto al análisis de cepas como al de muestras clínicas humanas (16).…”

Imported new world cutaneous leishmaniasis in a traveller from Slovakia

“…The complex Leishmania (Viannia) panamensis/guyanensis is the predominant aetiologic agent of CL and MCL in Ecuador, and was the agent identifi ed in our patient. In addition to this complex, a further fi ve species of Leishmania have been recorded in Ecuador (1,11,19,20).…”

“…Diagnosis is confi rmed by a microscopic and cultivation evidence of the parasite from biopsies of skin lesions. Because Leishmania species are indistinguishable microscopically, differentiation is based on molecular, biological (polymerase chain reaction (PCR)) and biochemical techniques (8,9,10,11).…”

Application of the Eco-Epidemiological Method in the Study of Leishmaniasis Transmission Foci

“…For detection of parasites in phlebotomines, samples should include a pool of at least ive insects or the anterior portion of an insect, including the irst abdominal tergite area that includes parasites atached to the stomodeal valve [12,13]. PCR can also distinguish between parasite species using a technique known as PCR-RFLP, which consists of determining the length of the restriction fragments of the ampliied product when it is subjected to digestion by diferent restriction enzymes [14,15].…”