Detection and species identification of Old World Leishmania in clinical samples using a PCR-based method

Abstract

The aim of this study was to develop a simple, low-cost method for the detection and species differentiation of Leishmania directly from clinical samples, for routine use in a parasitology laboratory. A total of 87 samples was used, including 60 peripheral blood, seven bone marrow and 17 skin lesion material samples, derived from Greek patients with visceral or cutaneous leishmaniasis, and three reference strains. PCR was performed using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene. Identification of the Leishmania species studied was achieved by digestion with a single restriction endonuclease (RFLP), single-strand conformational polymorphism (SSCP) and DNA sequencing of the PCR-generated fragments. Typing identified all visceral and one cutaneous leishmaniasis strains as L. infantum, twelve of the cutaneous leishmaniasis strains as L. tropica and four as L. major. The described PCR method proved efficient for the detection of pathogenic Leishmania species in various clinical samples, most importantly in peripheral blood samples. Furthermore, PCR followed by a simple RFLP using a single restriction endonuclease was capable of identifying all Leishmania species commonly encountered in Greece.

Disseminated Autochthonous Dermal Leishmaniasis Caused by Leishmania siamensis (PCM2 Trang) in a Patient from Central Thailand Infected with Human Immunodeficiency Virus

“…In terms of molecular diagnosis, we used primers designed from loci previously used for Leishmania identification. [10][11][12][13] ITS-1 region is the most variable marker that can be used to analyze the genetic difference among Leishmania species. 9 The clinical manifestations of novel leishmaniasis in Thailand can be categorized into three clinical forms: visceral, diffused cutaneous, and diffused cutaneous combined with visceral.…”

Species Typing in Dermal Leishmaniasis

“…5). Spanakos et al (114) used an alternative fragment also covering the ITS1 region (Fig. 4), but with ApoI as the restriction endonuclease.…”

Polymerase Chain Reaction Detection of Leishmania kDNA from the Urine of Peruvian Patients with Cutaneous and Mucocutaneous Leishmaniasis

“…1 Indeed, several lines of evidence support that lymphatogenous and hematogenous dissemination of the parasite may occur, 1,[6][7][8][9][10] and that nonfocal reservoirs of parasite persistence may actually protect against future reinfection. 9,11,12 Theoretically, then, parasites in both CL and ML would have access to the circulatory compartment of the host, as supported by numerous studies of CL showing Leishmania DNA in the blood, [7][8][9][10] and may therefore be detectable in urine. Cell-free DNA and small DNA fragments of 150-250 bp derived from human circulation can be detected in urine.…”