Molecular diagnosis of Old World leishmaniasis: Real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp.

ABSTRACT

Background & objectives: Rapid and accurate diagnosis and identification of Leishmania sp causing cutaneous leishmaniasis is crucial in control and therapeutic programs. The problem of diagnosis with traditional methods is that they have a low sensitivity or time consuming but molecular techniques would be an alternative method for rapid and accurate diagnosis. In this work, tryparedoxine peroxidase gene-based real-time PCR was used for accurate identification of Leishmania spp causing Old-World cutaneous leishmaniasis. Methods: In this study, biopsies of specimens were taken from the ulcerative sites in 100 patients and used for direct microscopy, culture in NNN or fixed in alcohol for identification of Leishmania spp using tryparedoxin peroxidase gene-based realtime PCR (qPCR). Results: Using direct microscopy and culture method, Leishmania parasites were isolated from 68 out of 100 patient samples. However, 13 patients with negative finding on traditional tests, had positive results on RT-PCR test. After melting curve analysis of PCR product, Leishmania major in 75 and L. tropica in 4 cases were identified. The sensitivity and specificity of RT-PCR for diagnosis of cutaneous leishmaniasis was 98.7 and 59.8%, respectively. Conclusion: Results of this study showed that RT-PCR was the most sensitive diagnostic test for cutaneous leishmaniasis and represents a tool for rapid species identification.