title: Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identification of the parasite species.
abstract: 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 polymorphism (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.
authors.affiliation: Laboratório de Pesquisa em Leishmaniose, Coleção de Leishmania, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil.
authors.forename: Grazielle Cardoso da
authors.initials: GC
authors.lastname: Graça
authors.forename: Angela Cristina
authors.initials: AC
authors.lastname: Volpini
authors.forename: Gustavo Adolfo Sierra
authors.initials: GA
authors.lastname: Romero
authors.forename: Manoel Paes de
authors.initials: MP
authors.lastname: Oliveira Neto
authors.forename: Marcia
authors.initials: M
authors.lastname: Hueb
authors.forename: Renato
authors.initials: R
authors.lastname: Porrozzi
authors.forename: Mariana Côrtes
authors.initials: MC
authors.lastname: Boité
authors.forename: Elisa
authors.initials: E
authors.lastname: Cupolillo
mesh_terms: D016054:DNA, Protozoan / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D007891:Leishmania / Q000235:genetics* / Q000302:isolation & purification; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D018840:HSP70 Heat-Shock Proteins