Comparison of PCR Assays for Diagnosis of Cutaneous Leishmaniasis

Abstract

Three PCR assays for diagnosing leishmaniasis were compared and validated against parasite cultures and microscopic evaluation of stained tissue smears using 92 specimens from suspected cases of cutaneous leishmaniasis (CL) in Israel and the West Bank. Samples from imported and locally acquired disease were examined. The kinetoplast DNA (kDNA) PCR showed the highest sensitivity (98.7%) of any assay, correctly diagnosing 77/78 of the confirmed positive samples, followed by the rRNA gene internal transcribed spacer 1 (ITS1) PCR (71/78 positive, 91.0% sensitivity) and then the spliced leader mini-exon PCR (42/78 positive, 53.8% sensitivity). Either parasite culture or microscopy alone detected 62.8% (49/78) or 74.4% (58/78) of the positive specimens, respectively, while culture and microscopy together improved overall sensitivity to 83.3% (65/78). Except for the kDNA PCR that had six false positives, all other assays were 100% specific. Further, restriction enzyme analysis of the ITS1 PCR product enabled identification of 74.6% of the positive samples, which included strains of Leishmania major (50.9%), Leishmania tropica (47.2%), and the Leishmania braziliensis complex (1.9%). This suggests that a PCR using kDNA should be used for the diagnosis of CL and that an ITS1 PCR can be reliably used for the diagnosis of CL when rapid species identification is needed.

Retrospective Analysis of Canine Vector-borne Diseases (CVBD) in Germany with Emphasis on the Endemicity and Risk Factors of Leishmaniosis

“…This could be due to the different PCR protocols (nested / conventional / real-time PCR; gene target) of respective studies, or because swabs do not contain a uniform cell number, depending on the technique of sampling. The most sensitive PCR assay is the method using kinetoplast DNA (kDNA), detecting the copy number of the target regions between 50-and 250-fold higher than splice leader mini-exon (SLME) and the SSU rRNA (ITS1) (Bensoussan et al 2006). In the present study, Leishmania DNA was found in blood samples, skin scrapings, joint punctures and in bone marrow.…”

“…For this purpose, results of blood or serum samples sent by local veterinarians to a veterinary diagnostic laboratory were retrospectively analysed. Samples were examined for Leishmania spp., Babesia spp., Ehrlichia canis, Dirofilaria immitis and Anaplasma phagocytophilum during the years 2004-2006. Erythrocyte stages of large Babesia spp.…”

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

“…Our result for PCR sensitivity when conserved region kDNA was used, showed a higher value 69.6% (95% CI 59.2-72.6%) than when ITS rDNA was used as molecular target. This difference could be due to differences in the number of copies present in Leishmania: around 10,000 copies for kDNA and near 200 copies for ITS rDNA (Rodgers et al 1990, Pirmez et al 1999, Marfurt et al 2003, Bensoussan et al 2006. Also, differences in the detection limits for each molecular target have been reported.…”

Diagnosis of Leishmania (Leishmania) chagasi infection in dogs and the relationship with environmental and sanitary aspects in the municipality of Palmas, state of Tocantins, Brazil

“…As L. (L.) chagasi are intracellular parasites that infect cells of the mononuclear phagocytic system, like monocytes and macrophages, the use of the leukocyte layer instead of whole blood may increase sensitivity, reducing the interference of potential reaction inhibitors. PCR has gradually become the technique indicated to diagnose leishmaniasis, as conventional parasitology methods are not sufficiently sensitive 23,24 .…”