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


Autoria(s): Graça,Grazielle Cardoso da; Volpini,Angela Cristina; Romero,Gustavo Adolfo Sierra; Oliveira Neto,Manoel Paes de; Hueb,Marcia; Porrozzi,Renato; Boité,Mariana Côrtes; Cupolillo,Elisa
Data(s)

01/08/2012

Resumo

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.

Formato

text/html

Identificador

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762012000500014

Idioma(s)

en

Publicador

Instituto Oswaldo Cruz, Ministério da Saúde

Fonte

Memórias do Instituto Oswaldo Cruz v.107 n.5 2012

Palavras-Chave #leishmaniasis #molecular diagnosis #species identification #polymerase chain reaction #RFLPs #hsp70
Tipo

journal article