Real-time PCR using FRET technology for Old World cutaneous leishmaniasis species differentiation

Background: Recently, there has been a re-emergence of cutaneous leishmaniasis in endemic countries and an increase in imported cases in non-endemic countries by travelers, workers, expatriates, immigrants, and military force personnel. Old World cutaneous leishmaniasis is caused primarily by Leishmania major, L. tropica and L. aethiopica. Despite their low sensitivity, diagnosis traditionally includes microscopic and histopathological examinations, and in vitro cultivation. Several conventional PCR techniques have been developed for species identification, which are time-consuming and labour-intensive. Real-time PCR using SYBR green dye, although provides rapid detection, may generate false positive signals. Therefore, a rapid and easy method such as a FRET-based real-time PCR would improve not only the turn-around time of diagnosing Old World cutaneous Leishmania species but will also increase its specificity and sensitivity. Methods: A FRET-based real-time PCR assay which amplifies the cathepsin L-like cysteine protease B gene encoding a major Leishmania antigen was developed to differentiate L. major, L. tropica, and L. aethiopica in one single step using one set of primers and probes. Assay performance was tested on cutaneous and visceral strains of Leishmania parasite cultures and isolates of other protozoan parasites as well as human biopsy specimen. Results: The assay readily differentiates between the three Old World cutaneous leishmaniasis species based on their melting curve characteristics. A single Tm at 55.2 +/- 0.5 degrees C for L. aethiopica strains was distinguished from a single Tm at 57.4 +/- 0.2 degrees C for L. major strains. A double curve with melting peaks at 66.6 +/- 0.1 degrees C and 48.1 +/- 0.5 degrees C or 55.8 +/- 0.6 degrees C was observed for all L. tropica strains. The assay was further tested on biopsy specimens, which showed 100 % agreement with results obtained from isoenzyme electrophoresis and Sanger sequencing. Conclusion: Currently, there are no published data on real-time PCR using FRET technology to differentiate between Old World cutaneous Leishmania species. In summary, our assay based on specific hybridization addresses the limitations of previous PCR technology and provides a single step, reliable method of species identification and rapid diagnostic applications.