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Human visceral leishmaniasis (VL) caused by  L. infantum  and cutaneous leishmaniasis (CL) caused by  L. tropica  and  L. infantum  have been reported in Turkey.  L. infantum  is also responsible for canine leishmaniasis (CanL) and it is widely common in the country. The main aim of the present study was to design a real-time PCR method based on the internal transcribed spacer 1 (ITS1) region in the diagnosis of all clinical forms of leishmaniasis in Mediterranean, and to identify the species directly from clinical samples. Totally, 315 clinical specimens, human/canine visceral (blood, bone marrow, lymph node) and cutaneous (lesion aspiration) samples, and 51 Turkish  Leishmania  isolates typed by isoenzymatic method were included in the study. For optimization, DNA samples of the 34 strains were amplified by conventional ITS1-PCR and then sequenced for designing the primers and probes, allowing the species identification. Following the validation with the isolates, the test was applied on clinical samples and melting temperatures were used for genotyping. A group of PCR products were further sequenced for confirmation and assigning the inter- and intraspecies heterogeneity. The diagnosis of leishmaniasis is successfully achieved by the new real-time PCR method, and the test identified 80.43% of human and canine VL samples as  L.infantum  and 6.52% as  L.tropica ; 52.46% of CL samples as  L. infantum  and 26.90% as  L. tropica . In 13.04% of visceral and 20.62% of cutaneous samples, two peaks were observed. Hovewer, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification. The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples. Genotypic variations of  Leishmania  parasites in Turkey within species and intraspecies were observed, and  L. tropica  is also found as causative agent of human and canine VL in Turkey.

A Real-Time ITS1-PCR Based Method in the Diagnosis and Species Identification of Leishmania Parasite from Human and Dog Clinical Samples in Turkey