Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

• Autor(a) principal: Graça,Grazielle Cardoso da
• Data de Publicação: 2012
• Outros Autores: Volpini,Angela Cristina, Romero,Gustavo Adolfo Sierra, Oliveira Neto,Manoel Paes de, Hueb,Marcia, Porrozzi,Renato, Boité,Mariana Côrtes, Cupolillo,Elisa
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Memórias do Instituto Oswaldo Cruz
• Texto Completo: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762012000500014
• topic: leishmaniasis; molecular diagnosis; species identification; polymerase chain reaction; RFLPs; hsp70

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.