Rapid detection, quantification and speciation of Leishmania using real-time PCR and DNA sequencing at the rRNA Internal Transcribed Spacer 2

The ability to discriminate infection between closely related Leishmania species within the Viannia species complex, specifically L. braziliensis, L. guyanensis and L. panamensis is critical to inform the clinical diagnosis and determine the most efficacious treatment modality. We designed a nested primer set targeting the rRNA Internal Transcribed Spacer 2 (ITS2), located on Chromosome 27, to distinguish among all human infective Leishmania species. Separate nested and single primer pairs were developed for conventional and quantitative PCR approaches respectively. Species-specific single nucleotide polymorphisms and indels located within the PCR products were identified by Sanger sequencing. This single locus approach provides a sensitive and specific platform to identify the species of Leishmania causing infection.