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

• Published in: Memórias do Instituto Oswaldo Cruz Vol. 107; no. 5; pp. 664 - 674
• Main Authors: da Graça, Grazielle Cardoso, Volpini, Angela Cristina, Romero, Gustavo Adolfo Sierra, Neto, Manoel Paes de Oliveira, Hueb, Marcia, Porrozzi, Renato, Boité, Mariana Côrtes, Cupolillo, Elisa
• Format: Journal Article
• Language: English
• Published: Brazil Fundação Oswaldo Cruz, Fiocruz 01.08.2012
• Subjects: DNA, Protozoan - genetics; hsp70; HSP70 Heat-Shock Proteins - genetics; Humans; Leishmania - genetics; Leishmania - isolation & purification; leishmaniasis; Leishmaniasis, Cutaneous - diagnosis; Leishmaniasis, Cutaneous - parasitology; molecular diagnosis; Polymerase Chain Reaction - methods; Polymorphism, Restriction Fragment Length; RFLPs; Sensitivity and Specificity; species identification - polymerase chain reaction
• ISSN: 1678-8060; 1678-8060
• DOI: 10.1590/S0074-02762012000500014

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.