Evaluation of PCR-RFLP (based on ITS-1 and HaeIII) for the detection of Leishmania species, using Greek canine isolates and Jordanian clinical material

Abstract

It is possible to detect and distinguish Leishmania parasites using PCR-RLFP — a combination of PCR and analysis of the fragment-length polymorphism seen when the amplicons are digested with one or more restriction enzymes. In the present study, clinical samples from 24 Jordanians suspected to have cutaneous leishmaniasis and cultures set up using leishmanial parasites from five Greek dogs were investigated using PCR, in which the internal-transcribed-spacer-1 (ITS1) region of the parasites' ribosomal-RNA gene was amplified, followed by HaeIII digestion of the resulting amplicons. The cultures, which were all maintained in Leibowitz L-15 medium with 20% foetal calf serum, were each investigated as serial dilutions. Using the PCR-RLFP analysis, each culture was identified as L. donovani and each was found positive for this parasite with a mean sensitivity of 66%–100% (depending on the culture dilution tested), a specificity of 100%, a mean positive predictive value of 100%, and a negative predictive value of 74.6%–100%. When simulated clinical samples, created by mixing human blood with known numbers of L. donovani promastigotes, were investigated, the PCR-RFLP gave optimal results (with a value of 100% each for sensitivity, specificity and positive and negative predictive values). When the real clinical samples (25 lesion aspirates and 20 samples of peripheral blood from 24 Jordanian patients) were investigated using the molecular method, 20 (84%) of the patients were found to have lesion aspirates that were PCR-RFLP-positive for L. major (although, by microscopy, only six were found to have amastigote-positive lesion aspirates). None of the blood samples from the Jordanian patients, however, was found PCR-positive.