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In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of <jats:italic>Leishmania aethiopica<\/jats:italic>. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the \u2018Mary kDNA PCR\u2019 and a newly designed \u2018LC kDNA PCR\u2019 for improved <jats:italic>L<\/jats:italic>. <jats:italic>aethiopica<\/jats:italic> detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of <jats:italic>L<\/jats:italic>. <jats:italic>aethiopica<\/jats:italic> on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.<\/jats:p>","DOI":"10.1371\/journal.pntd.0008903","type":"journal-article","created":{"date-parts":[[2021,1,12]],"date-time":"2021-01-12T22:51:49Z","timestamp":1610491909000},"page":"e0008903","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":31,"title":["Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia"],"prefix":"10.1371","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8378-241X","authenticated-orcid":true,"given":"Behailu","family":"Merdekios","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8918-3901","authenticated-orcid":true,"given":"Myrthe","family":"Pareyn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3770-1957","authenticated-orcid":true,"given":"Dagimawie","family":"Tadesse","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nigatu","family":"Eligo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3612-5716","authenticated-orcid":true,"given":"Mekibib","family":"Kassa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4677-0911","authenticated-orcid":true,"given":"Bart K. 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