title: Real-time PCR using FRET technology for Old World cutaneous leishmaniasis species differentiation.
abstract: 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 °C for L. aethiopica strains was distinguished from a single Tm at 57.4 ± 0.2 °C for L. major strains. A double curve with melting peaks at 66.6 ± 0.1 °C and 48.1 ± 0.5 °C or 55.8 ± 0.6 °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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal, QC, Canada.
authors.forename: Milli
authors.initials: M
authors.lastname: Nath-Chowdhury
authors.affiliation: National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal, QC, Canada.
authors.forename: Mugundhine
authors.initials: M
authors.lastname: Sangaralingam
authors.affiliation: Department of Parasitology-Mycology, Centre National de Référence des Leishmanioses, Centre Hospitalier Régional Universitaire of Montpellier and University Montpellier I (Faculty of Medicine), UMR CNRS 5290-IRD 224- UM1 et 2 "MIVEGEC", Montpellier, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bastien
authors.affiliation: Department of Parasitology-Mycology, Centre National de Référence des Leishmanioses, Centre Hospitalier Régional Universitaire of Montpellier and University Montpellier I (Faculty of Medicine), UMR CNRS 5290-IRD 224- UM1 et 2 "MIVEGEC", Montpellier, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Ravel
authors.affiliation: Department of Parasitology-Mycology, Centre National de Référence des Leishmanioses, Centre Hospitalier Régional Universitaire of Montpellier and University Montpellier I (Faculty of Medicine), UMR CNRS 5290-IRD 224- UM1 et 2 "MIVEGEC", Montpellier, France.
authors.forename: Francine
authors.initials: F
authors.lastname: Pratlong
authors.affiliation: Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Mendez
authors.affiliation: National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal, QC, Canada.|J.D. MacLean Centre for Tropical Diseases at McGill University, Montreal, QC, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Libman
authors.affiliation: National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal, QC, Canada. momar.ndao@mcgill.ca.|J.D. MacLean Centre for Tropical Diseases at McGill University, Montreal, QC, Canada. momar.ndao@mcgill.ca.
authors.forename: Momar
authors.initials: M
authors.lastname: Ndao
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000145:classification / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007891:Leishmania / Q000302:isolation & purification*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis / Q000469:parasitology*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D016054:DNA, Protozoan
keywords: Cutaneous; Diagnosis; FRET; Leishmania; Melting curve; Real-time PCR; qPCR