{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T07:42:50Z","timestamp":1785310970582,"version":"3.55.0"},"update-to":[{"DOI":"10.1371\/journal.pntd.0012543","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T00:00:00Z","timestamp":1736208000000}}],"reference-count":28,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2024,12,23]],"date-time":"2024-12-23T00:00:00Z","timestamp":1734912000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000780","name":"European Commission","doi-asserted-by":"publisher","award":["101057690"],"award-info":[{"award-number":["101057690"]}],"id":[{"id":"10.13039\/501100000780","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014013","name":"UKRI","doi-asserted-by":"crossref","award":["grants 10038150 and 10039289"],"award-info":[{"award-number":["grants 10038150 and 10039289"]}],"id":[{"id":"10.13039\/100014013","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Climos project"},{"DOI":"10.13039\/501100001677","name":"Inserm","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100001677","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background<\/jats:title>\n<jats:p>Leishmaniasis, caused by <jats:italic>Leishmania<\/jats:italic> protozoan parasites transmitted by Phlebotomine sand flies, is a significant public health concern in the Mediterranean basin. Effective monitoring of <jats:italic>Leishmania<\/jats:italic>-infected sand flies requires standardized tools for comparing their distribution and infection prevalence. Consistent quantitative real-time PCR (qPCR) parameters and efficient DNA extraction protocols are crucial for reliable results over time and across regions. However, the absence of standardized technical recommendations for <jats:italic>Leishmania<\/jats:italic> DNA detection hinders effective surveillance. This study aimed to compare different DNA extraction protocols and conduct a qPCR-based External Quality Assessment (EQA) through a multicenter study involving nine reference laboratories, with a focus on optimizing <jats:italic>Leishmania<\/jats:italic> DNA detection in sand fly.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methodology\/Principal findings<\/jats:title>\n<jats:p>EQA samples consisted of <jats:italic>Leishmania infantum<\/jats:italic> and <jats:italic>L<\/jats:italic>. <jats:italic>major<\/jats:italic> species, at concentrations ranging from 10<jats:sup>1<\/jats:sup> to 10<jats:sup>4<\/jats:sup> parasites\/mL. All but one center detected all concentrations, demonstrating strong diagnostic proficiency. The ability to detect low concentrations highlighted the robustness of the qPCR assay used, though variations in Cq values indicated differences in sensitivity related to technical capabilities or DNA extraction kit performance. A comparative analysis of seven DNA extraction methods identified the EZ1 DSP Virus Kit and QIAamp DNA mini-kit as the most efficient, supporting their use in standardized protocols. The study also assessed the effects of lyophilization and shipment conditions, showing no significant compromise in <jats:italic>Leishmania<\/jats:italic> detection despite slight variations in Cq values. Experimentally infected sand flies were included to simulate field conditions, and all centers successfully detected positive samples with varying Cq values, probably reflecting differences in infection load.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Conclusion and significance<\/jats:title>\n<jats:p>This study emphasizes the importance of standardized DNA extraction protocols and continuous quality assurance for accurate <jats:italic>Leishmania<\/jats:italic> DNA detection. The results highlight the superior performance of certain extraction kits and the need for ongoing technical training, essential for reliable leishmaniasis surveillance, particularly in field settings with low infection densities.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0012543","type":"journal-article","created":{"date-parts":[[2024,12,23]],"date-time":"2024-12-23T18:42:01Z","timestamp":1734979321000},"page":"e0012543","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":7,"title":["Performance evaluation of nine reference centers and comparison of DNA extraction protocols for effective surveillance of Leishmania-infected Phlebotomine sand flies: Basis for technical recommendations"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3672-2692","authenticated-orcid":true,"given":"Jorian","family":"Prudhomme","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aymeric","family":"Delabarre","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bulent","family":"Alten","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Umut","family":"Berberoglu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eduardo","family":"Berriatua","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gioia","family":"Bongiorno","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9 Manuel","family":"Cristovao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maya","family":"Davidovich-Cohen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trentina","family":"Di Muccio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ozge","family":"Erisoz Kasap","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eleonora","family":"Fiorentino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Oscar","family":"D. Kirstein","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edwin","family":"Kniha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carla","family":"Maia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mesut","family":"Mungan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clara","family":"Mu\u00f1oz-Hern\u00e1ndez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammed","family":"Nal\u00e7aci","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gizem","family":"Oguz Kaskan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yusuf","family":"Ozbel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seray","family":"Ozensoy Toz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ricardo","family":"Parreira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharina","family":"Platzgummer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ceylan","family":"Polat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9","family":"Risue\u00f1o","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liora","family":"Studentsky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gamze","family":"Varol","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Walochnik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kardelen","family":"Yeti\u015fmi\u015f","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Florence","family":"Robert-Gangneux","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,12,23]]},"reference":[{"issue":"30","key":"pntd.0012543.ref001","doi-asserted-by":"crossref","first-page":"20540","DOI":"10.2807\/1560-7917.ES2013.18.30.20540","article-title":"The role of indigenous phlebotomine sandflies and mammals in the spreading of leishmaniasis agents in the Mediterranean region.","volume":"18","author":"M Antoniou","year":"2013","journal-title":"Euro Surveill."},{"issue":"3","key":"pntd.0012543.ref002","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1007\/s00281-020-00788-y","article-title":"Leishmaniasis immunopathology\u2014impact on design and use of vaccines, diagnostics and drugs.","volume":"42","author":"PM Kaye","year":"2020","journal-title":"Seminars in Immunopathology"},{"key":"pntd.0012543.ref003","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1007\/s40475-021-00232-7","article-title":"A review of leishmaniasis: current knowledge and future directions","volume":"8","author":"S Mann","year":"2021","journal-title":"Current Tropical Medicine Reports"},{"issue":"7","key":"pntd.0012543.ref004","doi-asserted-by":"crossref","first-page":"e0011497","DOI":"10.1371\/journal.pntd.0011497","article-title":"The estimated distribution of autochthonous leishmaniasis by Leishmania infantum in Europe in 2005\u20132020.","volume":"17","author":"C Maia","year":"2023","journal-title":"PLoS Negl Trop Dis"},{"issue":"1\u20132","key":"pntd.0012543.ref005","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.vetpar.2015.05.003","article-title":"Spread of Leishmania infantum in Europe with dog travelling","volume":"213","author":"C Maia","year":"2015","journal-title":"Vet Parasitol"},{"issue":"14","key":"pntd.0012543.ref006","first-page":"1","article-title":"Cutaneous leishmaniasis in North Africa: a review.","volume":"21","author":"K Aoun","year":"2014","journal-title":"Parasite"},{"key":"pntd.0012543.ref007","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.actatropica.2018.11.001","article-title":"The current clinical and geographical situation of cutaneous leishmaniasis based on species identification in Turkey","volume":"190","author":"A Ozbilgin","year":"2019","journal-title":"Acta Trop"},{"issue":"273","key":"pntd.0012543.ref008","first-page":"1","article-title":"Real-time PCR applications for diagnosis of leishmaniasis.","volume":"11","author":"L Galluzzi","year":"2018","journal-title":"Parasit Vectors"},{"issue":"9","key":"pntd.0012543.ref009","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1111\/myc.13603","article-title":"A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis.","volume":"66","author":"D Wilmes","year":"2023","journal-title":"Mycoses"},{"issue":"6","key":"pntd.0012543.ref010","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1242\/jeb.037564","article-title":"Challenges in predicting climate and environmental effects on vector-borne disease episystems in a changing world","volume":"213","author":"W. Tabachnick","year":"2010","journal-title":"J Exp Biol"},{"issue":"1\u20132","key":"pntd.0012543.ref011","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/j.cca.2006.06.016","article-title":"Multicenter proficiency study for detection of Toxoplasma gondii in amniotic fluid by nucleic acid amplification methods","volume":"375","author":"K Kaiser","year":"2007","journal-title":"Clin Chim Acta"},{"issue":"9","key":"pntd.0012543.ref012","doi-asserted-by":"crossref","first-page":"3216","DOI":"10.1128\/JCM.02500-09","article-title":"Multicentric comparative analytical performance study for molecular detection of low amounts of Toxoplasma gondii from simulated specimens","volume":"48","author":"Y Sterkers","year":"2010","journal-title":"J Clin Microbiol"},{"issue":"6","key":"pntd.0012543.ref013","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1093\/mmy\/myz115","article-title":"The fungal PCR initiative\u2019s evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: toward a standard for a diagnostics assay","volume":"58","author":"M Gits-Muselli","year":"2020","journal-title":"Med Mycol"},{"issue":"49","key":"pntd.0012543.ref014","doi-asserted-by":"crossref","first-page":"1","DOI":"10.2807\/1560-7917.ES.2016.21.49.30418","article-title":"Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014.","volume":"21","author":"G Van der Auwera","year":"2016","journal-title":"Euro Surveill."},{"issue":"12","key":"pntd.0012543.ref015","doi-asserted-by":"crossref","first-page":"3881","DOI":"10.1128\/JCM.01164-09","article-title":"Multicenter comparative evaluation of five commercial methods for Toxoplasma DNA extraction from amniotic fluid","volume":"47","author":"H Yera","year":"2009","journal-title":"J Clin Microbiol"},{"issue":"10","key":"pntd.0012543.ref016","doi-asserted-by":"crossref","first-page":"1100.e7","DOI":"10.1016\/j.cmi.2018.01.014","article-title":"Evaluation of five automated and one manual method for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid","volume":"24","author":"H Yera","year":"2018","journal-title":"Clin Microbiol Infect"},{"issue":"11","key":"pntd.0012543.ref017","doi-asserted-by":"crossref","first-page":"5249","DOI":"10.1128\/JCM.42.11.5249-5255.2004","article-title":"Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity","volume":"42","author":"C Mary","year":"2004","journal-title":"J Clin Microbiol"},{"issue":"7","key":"pntd.0012543.ref018","doi-asserted-by":"crossref","first-page":"826","DOI":"10.3390\/pathogens10070826","article-title":"Comparison of three in-house real PCR assays targeting kinetoplast DNA, the small subunit ribosomal RNA gene and the glucose-6-phosphate isomerase gene for the detection of Leishmania spp. in human serum.","volume":"10","author":"K Tanida","year":"2021","journal-title":"Pathogens"},{"issue":"8","key":"pntd.0012543.ref019","doi-asserted-by":"crossref","first-page":"e0010676","DOI":"10.1371\/journal.pntd.0010676","article-title":"Test combination to detect latent Leishmania infection: A prevalence study in a newly endemic area for L. infantum, northeastern Italy.","volume":"16","author":"AM De Pascali","year":"2022","journal-title":"PLoS Negl Trop Dis"},{"issue":"1","key":"pntd.0012543.ref020","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1186\/s13071-022-05524-z","article-title":"Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods.","volume":"15","author":"I Gow","year":"2022","journal-title":"Parasit Vectors"},{"issue":"2","key":"pntd.0012543.ref021","doi-asserted-by":"crossref","first-page":"e0011128","DOI":"10.1371\/journal.pntd.0011128","article-title":"PCR for detection of Leishmania donovani from microscopically negative tissue smears of suspected patients in Gondar, Ethiopia.","volume":"17","author":"R Melkamu","year":"2023","journal-title":"PLoS Negl Trop Dis"},{"issue":"2","key":"pntd.0012543.ref022","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1093\/jme\/tjz199","article-title":"Comparative study of promastigote- and amastigote-Initiated infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) conducted in different biosafety level laboratories.","volume":"57","author":"S Vaselek","year":"2020","journal-title":"J Med Entomol"},{"issue":"1","key":"pntd.0012543.ref023","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.tvjl.2006.10.019","article-title":"Diagnosis of canine visceral leishmaniasis: biotechnological advances","volume":"175","author":"Y Gomes","year":"2008","journal-title":"The Veterinary Journal"},{"issue":"6","key":"pntd.0012543.ref024","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1016\/j.jmoldx.2022.03.009","article-title":"Molecular diagnosis of toxoplasmosis: multicenter evaluation of the Toxoplasma RealCycler universal PCR assay on 168 characterized human samples","volume":"24","author":"M-P Brenier-Pinchart","year":"2022","journal-title":"The Journal of Molecular Diagnostics"},{"issue":"8","key":"pntd.0012543.ref025","doi-asserted-by":"crossref","first-page":"1000","DOI":"10.1016\/j.jmoldx.2021.05.010","article-title":"Multicenter comparative assessment of the TIB MolBiol Toxoplasma gondii detection kit and four laboratory-developed PCR assays for molecular diagnosis of toxoplasmosis","volume":"23","author":"M-P Brenier-Pinchart","year":"2021","journal-title":"The Journal of Molecular Diagnostics"},{"issue":"5","key":"pntd.0012543.ref026","doi-asserted-by":"crossref","first-page":"1369","DOI":"10.1128\/JCM.02379-16","article-title":"Evaluation of Toxoplasma ELITe MGB real-time PCR assay for diagnosis of toxoplasmosis","volume":"55","author":"F Robert-Gangneux","year":"2017","journal-title":"J Clin Microbiol"},{"issue":"12","key":"pntd.0012543.ref027","doi-asserted-by":"crossref","first-page":"2006","DOI":"10.3390\/microorganisms8122006","article-title":"Evaluation of a kDNA-based qPCR assay for the detection and quantification of old world Leishmania species.","volume":"8","author":"M Ceccarelli","year":"2020","journal-title":"Microorganisms"},{"issue":"1","key":"pntd.0012543.ref028","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13071-024-06418-y","article-title":"Sand fly blood meal volumes and their relation to female body weight under experimental conditions","volume":"17","author":"V Volfov\u00e1","year":"2024","journal-title":"Parasit Vectors"}],"updated-by":[{"DOI":"10.1371\/journal.pntd.0012543","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T00:00:00Z","timestamp":1736208000000}}],"container-title":["PLOS Neglected Tropical Diseases"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0012543","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T18:50:49Z","timestamp":1736275849000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0012543"}},"subtitle":[],"editor":[{"given":"Clarence","family":"Mang\u2019era","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2024,12,23]]},"references-count":28,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2024,12,23]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pntd.0012543","relation":{"new_version":[{"id-type":"doi","id":"10.1371\/journal.pntd.0012543","asserted-by":"object"}]},"ISSN":["1935-2735"],"issn-type":[{"value":"1935-2735","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,12,23]]}}}