{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T22:13:47Z","timestamp":1790806427535,"version":"4.1.0"},"reference-count":35,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2024,5,7]],"date-time":"2024-05-07T00:00:00Z","timestamp":1715040000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001713","name":"European and Developing Countries Clinical Trials Partnership","doi-asserted-by":"publisher","award":["TMA2016SF- 1437"],"award-info":[{"award-number":["TMA2016SF- 1437"]}],"id":[{"id":"10.13039\/501100001713","id-type":"DOI","asserted-by":"publisher"}]}],"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>Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methodology\/Principle findings<\/jats:title>\n<jats:p>The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for <jats:italic>Leishmania donovani<\/jats:italic> and <jats:italic>Leishmania infantum<\/jats:italic>, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Conclusions\/Significance<\/jats:title>\n<jats:p>Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0011637","type":"journal-article","created":{"date-parts":[[2024,5,7]],"date-time":"2024-05-07T17:21:41Z","timestamp":1715102501000},"page":"e0011637","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":15,"title":["Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4747-090X","authenticated-orcid":true,"given":"Norbert J.","family":"van Dijk","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dawit Gebreegziabiher","family":"Hagos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela M.","family":"Huggins","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eugenia","family":"Carrillo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophia","family":"Ajala","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen","family":"Chicharro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Kiptanui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose Carlos","family":"Solana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edwin","family":"Abner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dawit","family":"Wolday","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henk D. F. H.","family":"Schallig","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,5,7]]},"reference":[{"issue":"4","key":"pntd.0011637.ref001","doi-asserted-by":"crossref","first-page":"759","DOI":"10.3390\/microorganisms9040759","article-title":"Effects of Visceralising Leishmania on the Spleen, Liver, and Bone Marrow: A Pathophysiological Perspective","volume":"9","author":"A Poulaki","year":"2021","journal-title":"Microorganisms"},{"key":"pntd.0011637.ref002","unstructured":"Leishmaniasis: World Health Organization; 2023 [updated 12-01-2023; cited 2023 12-04-2023]. https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/leishmaniasis#:~:text=Visceral%20leishmaniasis%20(VL)%2C%20also,East%20Africa%20and%20in%20India."},{"issue":"6","key":"pntd.0011637.ref003","article-title":"Rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease","volume":"2014","author":"M Boelaert","year":"2014","journal-title":"Cochrane Database Syst Rev"},{"issue":"1","key":"pntd.0011637.ref004","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1186\/s12879-020-05558-7","article-title":"The diagnostic accuracy of direct agglutination test for serodiagnosis of human visceral leishmaniasis: a systematic review with meta-analysis","volume":"20","author":"M Mohebali","year":"2020","journal-title":"BMC Infectious Diseases"},{"issue":"1","key":"pntd.0011637.ref005","doi-asserted-by":"crossref","first-page":"1166","DOI":"10.1186\/s12879-021-06826-w","article-title":"Performance of rapid rk39 tests for the diagnosis of visceral leishmaniasis in Ethiopia: a systematic review and meta-analysis","volume":"21","author":"DG Hagos","year":"2021","journal-title":"BMC Infectious Diseases"},{"issue":"5","key":"pntd.0011637.ref006","doi-asserted-by":"crossref","first-page":"e1665","DOI":"10.1371\/journal.pntd.0001665","article-title":"The diagnostic accuracy of serologic and molecular methods for detecting visceral leishmaniasis in HIV infected patients: meta-analysis","volume":"6","author":"GF Cota","year":"2012","journal-title":"PLoS Negl Trop Dis"},{"issue":"3","key":"pntd.0011637.ref007","doi-asserted-by":"crossref","first-page":"e0008139","DOI":"10.1371\/journal.pntd.0008139","article-title":"Evaluation of six commercial kits for the serological diagnosis of Mediterranean visceral leishmaniasis","volume":"14","author":"MF Leveque","year":"2020","journal-title":"PLoS Negl Trop Dis"},{"issue":"6","key":"pntd.0011637.ref008","doi-asserted-by":"crossref","first-page":"929","DOI":"10.4269\/ajtmh.2009.80.929","article-title":"Field evaluation of rK39 test and direct agglutination test for diagnosis of visceral leishmaniasis in a population with high prevalence of human immunodeficiency virus in Ethiopia","volume":"80","author":"R ter Horst","year":"2009","journal-title":"Am J Trop Med Hyg"},{"issue":"6","key":"pntd.0011637.ref009","doi-asserted-by":"crossref","first-page":"e2869","DOI":"10.1371\/journal.pntd.0002869","article-title":"Visceral Leishmaniasis and HIV coinfection in East Africa","volume":"8","author":"E Diro","year":"2014","journal-title":"PLoS Negl Trop Dis"},{"issue":"1","key":"pntd.0011637.ref010","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/AAC.04298-14","article-title":"Systematic review of biomarkers to monitor therapeutic response in leishmaniasis","volume":"59","author":"AE Kip","year":"2015","journal-title":"Antimicrob Agents Chemother"},{"issue":"2","key":"pntd.0011637.ref011","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1046\/j.1365-3156.2001.00680.x","article-title":"Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan","volume":"6","author":"EE Zijlstra","year":"2001","journal-title":"Trop Med Int Health"},{"issue":"2","key":"pntd.0011637.ref012","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1007\/s12639-020-01212-w","article-title":"Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods","volume":"44","author":"S Thakur","year":"2020","journal-title":"Journal of Parasitic Diseases"},{"issue":"5","key":"pntd.0011637.ref013","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1016\/0035-9203(92)90086-R","article-title":"Kala-azar: a comparative study of parasitological methods and the direct agglutination test in diagnosis","volume":"86","author":"EE Zijlstra","year":"1992","journal-title":"Trans R Soc Trop Med Hyg"},{"issue":"9","key":"pntd.0011637.ref014","doi-asserted-by":"crossref","first-page":"3147","DOI":"10.1128\/JCM.00372-14","article-title":"Molecular tools for diagnosis of visceral leishmaniasis: Systematic review and meta-analysis of diagnostic test accuracy","volume":"52","author":"CM De Ruiter","year":"2014","journal-title":"Journal of Clinical Microbiology"},{"issue":"1","key":"pntd.0011637.ref015","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1186\/s13071-018-2859-8","article-title":"Real-time PCR applications for diagnosis of leishmaniasis","volume":"11","author":"L Galluzzi","year":"2018","journal-title":"Parasites & Vectors"},{"issue":"5","key":"pntd.0011637.ref016","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1093\/cid\/ciab124","article-title":"Blood Parasite Load as an Early Marker to Predict Treatment Response in Visceral Leishmaniasis in Eastern Africa","volume":"73","author":"L Verrest","year":"2021","journal-title":"Clin Infect Dis"},{"key":"pntd.0011637.ref017","first-page":"8240784","article-title":"Development and Clinical Evaluation of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Brazil","volume":"2019","author":"DM de Avelar","year":"2019","journal-title":"Biomed Res Int"},{"issue":"7","key":"pntd.0011637.ref018","doi-asserted-by":"crossref","DOI":"10.1128\/JCM.00386-18","article-title":"Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis","volume":"56","author":"ER Adams","year":"2018","journal-title":"J Clin Microbiol"},{"issue":"1","key":"pntd.0011637.ref019","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1186\/s13071-021-05133-2","article-title":"Diagnosis of visceral and cutaneous leishmaniasis using loop-mediated isothermal amplification (LAMP) protocols: a systematic review and meta-analysis","volume":"15","author":"AC Erber","year":"2022","journal-title":"Parasites & Vectors"},{"key":"pntd.0011637.ref020","doi-asserted-by":"crossref","DOI":"10.3389\/fcimb.2021.670759","article-title":"Evaluation of Loopamp\u2122 Leishmania Detection Kit and Leishmania Antigen ELISA for Post-Elimination Detection and Management of Visceral Leishmaniasis in Bangladesh","volume":"11","author":"F Hossain","year":"2021","journal-title":"Frontiers in Cellular and Infection Microbiology"},{"issue":"1","key":"pntd.0011637.ref021","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.jmoldx.2017.09.004","article-title":"Plasmodium Detection and Differentiation by Direct-on-Blood PCR Nucleic Acid Lateral Flow Immunoassay: Development, Validation, and Evaluation","volume":"20","author":"JM Roth","year":"2018","journal-title":"J Mol Diagn"},{"issue":"1","key":"pntd.0011637.ref022","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1186\/s12936-023-04496-4","article-title":"Laboratory evaluation of the miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA), a simplified molecular diagnostic test for Plasmodium","volume":"22","author":"NJ van Dijk","year":"2023","journal-title":"Malaria Journal"},{"issue":"2","key":"pntd.0011637.ref023","doi-asserted-by":"crossref","first-page":"356","DOI":"10.1016\/j.tvjl.2008.05.018","article-title":"The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood","volume":"182","author":"M de Paiva Cavalcanti","year":"2009","journal-title":"Vet J"},{"issue":"9","key":"pntd.0011637.ref024","doi-asserted-by":"crossref","first-page":"1100","DOI":"10.1046\/j.1537-2995.2001.41091100.x","article-title":"A novel RT-PCR for reliable and rapid HCV RNA screening of blood donations","volume":"41","author":"H Hennig","year":"2001","journal-title":"Transfusion"},{"key":"pntd.0011637.ref025","doi-asserted-by":"crossref","first-page":"e0335422","DOI":"10.1128\/spectrum.03354-22","article-title":"Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp","author":"C Chicharro","year":"2023","journal-title":"Microbiol Spectr"},{"issue":"1","key":"pntd.0011637.ref026","doi-asserted-by":"crossref","first-page":"33","DOI":"10.4269\/ajtmh.13-0150","article-title":"Clinical epidemiology, diagnosis and treatment of visceral leishmaniasis in the Pokot endemic area of Uganda and Kenya","volume":"90","author":"YK Mueller","year":"2014","journal-title":"Am J Trop Med Hyg"},{"issue":"11","key":"pntd.0011637.ref027","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":"11","key":"pntd.0011637.ref028","doi-asserted-by":"crossref","first-page":"3892","DOI":"10.1128\/JCM.r00764-11","article-title":"Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples","volume":"49","author":"JL Weirather","year":"2011","journal-title":"J Clin Microbiol"},{"issue":"3","key":"pntd.0011637.ref029","doi-asserted-by":"crossref","first-page":"e0006277","DOI":"10.1371\/journal.pntd.0006277","article-title":"Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain","volume":"12","author":"M Bangert","year":"2018","journal-title":"PLOS Neglected Tropical Diseases"},{"issue":"12","key":"pntd.0011637.ref030","doi-asserted-by":"crossref","first-page":"5973","DOI":"10.1128\/JCM.43.12.5973-5977.2005","article-title":"Diagnostic accuracy of two rK39 antigen-based dipsticks and the formol gel test for rapid diagnosis of visceral leishmaniasis in northeastern Uganda","volume":"43","author":"F Chappuis","year":"2005","journal-title":"J Clin Microbiol"},{"issue":"10","key":"pntd.0011637.ref031","doi-asserted-by":"crossref","first-page":"1312","DOI":"10.1093\/cid\/cis716","article-title":"A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis","volume":"55","author":"J Cunningham","year":"2012","journal-title":"Clin Infect Dis"},{"key":"pntd.0011637.ref032","doi-asserted-by":"crossref","first-page":"3569704","DOI":"10.1155\/2022\/3569704","article-title":"Diagnostic Performance of Three rK39 Rapid Diagnostic Tests and Two Direct Agglutination Tests for the Diagnosis of Visceral Leishmaniasis in Southern Iran","volume":"2022","author":"Z Rezaei","year":"2022","journal-title":"J Trop Med"},{"issue":"9","key":"pntd.0011637.ref033","doi-asserted-by":"crossref","first-page":"e2441","DOI":"10.1371\/journal.pntd.0002441","article-title":"Validation of two rapid diagnostic tests for visceral leishmaniasis in Kenya","volume":"7","author":"J Mbui","year":"2013","journal-title":"PLoS Negl Trop Dis"},{"issue":"8","key":"pntd.0011637.ref034","doi-asserted-by":"crossref","first-page":"387","DOI":"10.3390\/vetsci9080387","article-title":"Canine Leishmaniasis: Update on Epidemiology, Diagnosis, Treatment, and Prevention","volume":"9","author":"M Morales-Yuste","year":"2022","journal-title":"Veterinary Sciences"},{"issue":"10","key":"pntd.0011637.ref035","doi-asserted-by":"crossref","first-page":"e0008741","DOI":"10.1371\/journal.pntd.0008741","article-title":"Cost-effectiveness of serological tests for human visceral leishmaniasis in the Brazilian scenario","volume":"14","author":"ML Freire","year":"2020","journal-title":"PLOS Neglected Tropical Diseases"}],"container-title":["PLOS Neglected Tropical Diseases"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0011637","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,5,7]],"date-time":"2024-05-07T17:22:11Z","timestamp":1715102531000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0011637"}},"subtitle":[],"editor":[{"given":"Abhay R","family":"Satoskar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2024,5,7]]},"references-count":35,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2024,5,7]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pntd.0011637","relation":{},"ISSN":["1935-2735"],"issn-type":[{"value":"1935-2735","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,5,7]]}}}