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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 <jats:italic>Leishmania<\/jats:italic> species in clinical specimens.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Methods<\/jats:title>\n<jats:p>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 <jats:italic>Leishmania<\/jats:italic> 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.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Results<\/jats:title>\n<jats:p>The universal PCR enabled simultaneous discrimination of <jats:italic>L. major<\/jats:italic>, <jats:italic>L. tropica<\/jats:italic> and <jats:italic>L. infantum<\/jats:italic> using one primer pair in one reaction. Stained smear microscopy and Novy-MacNeal-Nicolle (NNN) medium\u00a0culture 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\u2013100%) and 100% specificity (3\/3) (95% CI: 29.24\u2013100%) for species identification on isolates from lesion scraping\/exudate and 100% sensitivity (13\/13) (95% CI: 75.29\u2013100%) and 100% specificity (11\/11) (95% CI: 71.51\u2013100%) for species identification on biopsy samples of CL patients, while being capable to successfully amplify as little as 0.01\u20130.1 pg of <jats:italic>Leishmania<\/jats:italic> DNA from cultured promastigotes.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Conclusions<\/jats:title>\n<jats:p>We present a validated easy-to-use affordable universal PCR assay to identify the most common Old World <jats:italic>Leishmania<\/jats:italic> species causing CL. This PCR assay could be used as a sensitive\/specific technique to diagnose CL-causing <jats:italic>Leishmania<\/jats:italic> species in clinical samples with high accuracy.<\/jats:p>\n<\/jats:sec>","DOI":"10.1186\/s13071-020-04261-5","type":"journal-article","created":{"date-parts":[[2020,8,27]],"date-time":"2020-08-27T13:04:15Z","timestamp":1598533455000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis"],"prefix":"10.1186","volume":"13","author":[{"given":"Mahmoud","family":"Nateghi Rostami","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fatemeh","family":"Darzi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahin","family":"Farahmand","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohsen","family":"Aghaei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Parviz","family":"Parvizi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,8,27]]},"reference":[{"key":"4261_CR1","doi-asserted-by":"publisher","first-page":"240","DOI":"10.1016\/j.actatropica.2017.05.022","volume":"172","author":"CB Meireles","year":"2017","unstructured":"Meireles CB, Maia LC, Soares GC, Teodoro IPP, Gadelha M, da Silva CGL, et al. 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All procedures were in accordance with the ethical standards of human experimentation of 1964 Declaration of Helsinki and its later amendments (World Medical Association, 64th WMA General Assembly, Fortaleza, Brazil, October 2013). The same samples which were taken during routine diagnostics were used, however, informed consent was obtained from each volunteer participated in the study.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"431"}}