Abstract
BackgroundThe clinical and epidemiological significance of Leishmania DNA in extralesional sites is obscured by uncertainty of whether the DNA derives from viable parasites. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients.MethodsThe feasibility of discriminating parasite viability by PCR of Leishmania 7SLRNA was evaluated in relation with luciferase activity of luc transfected intracellular amastigotes in dose-response assays of Glucantime cytotoxicity. Monocytes, tonsil swabs, aspirates of normal skin and lesions of 28 cutaneous and 2 mucocutaneous leishmaniasis patients were screened by kDNA amplification/Southern blot. Positive samples were analyzed by quantitative PCR of Leishmania 7SLRNA genes and transcripts.Results7SLRNA amplification coincided with luciferase activity, confirming discrimination of parasite viability. Of 22 patients presenting kDNA in extralesional samples, Leishmania 7SLRNA genes or transcripts were detected in one or more kDNA positive samples in 100% and 73% of patients, respectively. Gene and transcript copy number amplified from extralesional tissues were comparable to lesions. 7SLRNA transcripts were detected in 13/19 (68%) monocyte samples, 5/12 (42%) tonsil swabs, 4/11 (36%) normal skin aspirates, and 22/25 (88%) lesions; genes were quantifiable in 15/19 (79%) monocyte samples, 12/13 (92%) tonsil swabs, 8/11 (73%) normal skin aspirates.ConclusionViable parasites are present in extralesional sites, including blood monocytes, tonsils and normal skin of dermal leishmaniasis patients. Leishmania 7SLRNA is an informative target for clinical and epidemiologic investigations of human leishmaniasis.
Highlights
Prevention and control of dermal leishmaniasis in the New World continues to elude measures based on case identification and treatment
We show that 7SLRNA transcript copy number is proportional to the number of live parasites and demonstrate the presence of Leishmania 7SLRNA genes and transcripts in blood monocytes, normal skin and tonsilar mucosa of patients with dermal leishmaniasis
Participants in this study were patients consulting at Centro Internacional de Entrenamiento e Investigaciones Medicas (CIDEIM) in Cali, Colombia, and who were included in a prior investigation [5]
Summary
Prevention and control of dermal leishmaniasis in the New World continues to elude measures based on case identification and treatment. The persistence of Leishmania infection following resolution of disease has been supported by serologic, immunohistochemical and molecular evidence. Leishmania kDNA has been the principal molecular target, yielding evidence of parasites in extralesional tissues including scars, normal skin, and blood monocytes [5,6,7]. The high stability of DNA molecules and the possibility of its persistence following parasite death has raised questions concerning the interpretation of its amplification from blood and apparently healthy tissues as indicative of the presence of viable Leishmania [8]. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients
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