Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmani
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arasites & Vectors Open Access
RESEARCH
Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis Mahmoud Nateghi Rostami1*, Fatemeh Darzi1, Mahin Farahmand1, Mohsen Aghaei2 and Parviz Parvizi3
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. Keywords: Leishmania, PCR, ITS, Diagnosis, Cutaneous leishmaniasis Background Cutaneous leishmaniasis (CL) is usually manifested as a nodule which gradually develops to a self-healing lesion leaving a scar, but a polymorphism is seen in lesion characteristics, and diverse atypical forms are reported [1]. *Correspondence: [email protected] 1 Laboratory of Host‑Parasite Interactions, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran Full list of author information is available at the end of the article
The host’s immune response, Leishmania species, and inter- and intra-species genetic diversity of Leishmania might be involved in this clinical polymorphism [2, 3]. CL is a geographically extensive disease and in the Old World can be caused by any of the four different species: Leishman
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