Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis

Abstract

Background: The characterization of Leishmania species is important for clinical management of the diseases and the epidemiological control of the parasite distribution. Most of the published polymerase chain reaction (PCR) amplification methods lack the ability to identify all species complexes, have low performance and usually need post-PCR procedures. There is a need for improving the diagnosis of CL by development of an accurate affordable PCR method to identify all Leishmania species in clinical specimens. Methods: We designed an optimized a PCR amplifying the internal transcribed spacer 2 sequence of the ribosomal RNA gene (ITS2) aligned from different strains of CL-causing Leishmania species in the Old World. The performance of the method was evaluated on lesion samples from several CL suspected patients and the limit of detection (LOD) was determined on DNA of promastigotes from reference strains. Results: The universal PCR enabled simultaneous discrimination of L. major, L. tropica and L. infantum using one primer pair in one reaction. Stained smear microscopy and Novy-MacNeal-Nicolle (NNN) medium culture alone detected 77.27% (17/22) and 72.73% (16/22) of the positive CL samples, respectively. The PCR assay showed 100% sensitivity (22/22) (95% CI: 84.56-100%) and 100% specificity (3/3) (95% CI: 29.24-100%) for species identification on isolates from lesion scraping/exudate and 100% sensitivity (13/13) (95% CI: 75.29-100%) and 100% specificity (11/11) (95% CI: 71.51-100%) for species identification on biopsy samples of CL patients, while being capable to successfully amplify as little as 0.01-0.1 pg of Leishmania DNA from cultured promastigotes. Conclusions: We present a validated easy-to-use affordable universal PCR assay to identify the most common Old World Leishmania species causing CL. This PCR assay could be used as a sensitive/specific technique to diagnose CLcausing Leishmania species in clinical samples with high accuracy.

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

“…This method can be time consuming, requires an array of equipment and, as the PCR tube containing amplicons must be opened for electrophoresis, is associated with a risk of contamination [ 169 ]. Clinical sensitivity and specificity of up to 100% each have been achieved for lesion samples, with detection of as low as 0.01–0.1 pg of cultured Leishmania promastigote DNA [ 170 ].…”

Multilocus sequence typing analysis of Leishmania clinical isolates from cutaneous leishmaniasis patients of Iran

“…Based on PCR analysis, 28 of 41 (68.3%) lesional samples were characterized as L. major and the remaining 13 isolates (31.7%) were identified as L. tropica. The identity of the strains was confirmed with molecular methods (Nateghi Rostami et al, 2020).…”

“…The DNAs were kept at -20 °C until further use. Species identification of the isolates was performed by species specific primers with multiplex PCR method targeting kinetoplast DNA and a nested-PCR restriction fragment length polymorphism (RFLP) targeting internal transcribed spacer 2 (ITS2) region as previously described (Nateghi Rostami et al, 2020).…”

Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species

“…Moreover, a pan- Leishmania assay can also be exploited in eco-epidemiological studies, including identification of vectors and reservoirs [ 24 ]. In recent years, only a few attempts to develop pan- Leishmania assays based on nucleic acids amplification (i.e., SL RNA, rDNA or kDNA minicircles) have been made [ 24 , 25 , 26 , 27 ]. We previously showed that qPCR-ML was able to detect Old World L. ( L .)…”