{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T20:33:27Z","timestamp":1779309207075,"version":"3.51.4"},"reference-count":45,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2024,8,26]],"date-time":"2024-08-26T00:00:00Z","timestamp":1724630400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004156","name":"Mahidol University","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004156","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Leishmaniasis, a neglected tropical disease caused by parasitic protozoa of the <jats:italic>Leishmania<\/jats:italic> genus, remains a global health concern with significant morbidity and mortality. In Thailand, the rising incidence of autochthonous leishmaniasis cases involving <jats:italic>Leishmania<\/jats:italic> (<jats:italic>Mundinia<\/jats:italic>) <jats:italic>martiniquensis<\/jats:italic> and novel <jats:italic>Leishmania<\/jats:italic> (<jats:italic>Mundinia<\/jats:italic>) <jats:italic>orientalis<\/jats:italic> underscores the critical need for accurate diagnosis and effective control strategies. This study presents a sensitive and specific nucleic acid lateral flow immunoassay (NALFIA) that integrates a duplex PCR assay with a lateral flow device (LFD) strip format. Targeting the internal transcribed spacer 1 (ITS1) region, known for its unique combination of conserved and variable sequences, this assay employs primers labeled with biotin, digoxigenin, and fluorescein isothiocyanate (FITC) markers, enabling precise species identification and differentiation of these two <jats:italic>Leishmania<\/jats:italic> species. Remarkably, the assay achieves a sensitivity that surpasses agarose gel electrophoresis, detecting as few as 10<jats:sup>\u22122<\/jats:sup> parasite\/\u03bcL for <jats:italic>L<\/jats:italic>. <jats:italic>martiniquensis<\/jats:italic> and 10<jats:sup>\u22124<\/jats:sup> parasite\/\u03bcL for <jats:italic>L<\/jats:italic>. <jats:italic>orientalis<\/jats:italic>. Notably, the assay exhibited reliable specificity, revealing no cross-amplification with other major viscerotropic <jats:italic>Leishmania<\/jats:italic> species or reference organisms. Evaluation using 62 clinical samples further confirms the effectiveness of the PCR-LFD assay, with a sensitivity of 100% for <jats:italic>L<\/jats:italic>. <jats:italic>martiniquensis<\/jats:italic> and 83.3% for <jats:italic>L<\/jats:italic>. <jats:italic>orientalis<\/jats:italic>, and an excellent agreement (<jats:italic>\u03ba<\/jats:italic> value = 0.948) with nested PCR. This integrated assay represents a promising advancement in diagnostic tools, offering rapid and accurate results that can significantly contribute to effective disease management and control. Given the increasing relevance of these <jats:italic>Leishmania<\/jats:italic> species in current public health scenarios, this assay serves as a valuable tool for both diagnostic and research applications.<\/jats:p>","DOI":"10.1371\/journal.pone.0307601","type":"journal-article","created":{"date-parts":[[2024,8,26]],"date-time":"2024-08-26T20:22:51Z","timestamp":1724703771000},"page":"e0307601","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of nucleic acid lateral flow immunoassay for duplex detection of Leishmania martiniquensis and Leishmania orientalis in asymptomatic patients with HIV"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4318-2510","authenticated-orcid":true,"given":"Namfon","family":"Nawattanapaibool","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Toon","family":"Ruang-areerate","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Phunlerd","family":"Piyaraj","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Saovanee","family":"Leelayoova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mathirut","family":"Mungthin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2807-1412","authenticated-orcid":true,"given":"Suradej","family":"Siripattanapipong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2024,8,26]]},"reference":[{"key":"pone.0307601.ref001","doi-asserted-by":"crossref","first-page":"750","DOI":"10.12688\/f1000research.11120.1","article-title":"Leishmaniasis: a review.","volume":"6","author":"E Torres-Guerrero","year":"2017","journal-title":"F1000Research"},{"key":"pone.0307601.ref002","doi-asserted-by":"crossref","first-page":"622","DOI":"10.1016\/B978-0-12-818731-9.00178-6","volume-title":"Encyclopedia of Infection and Immunity","author":"S Antinori","year":"2022"},{"key":"pone.0307601.ref003","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1533\/9781908818737.227","volume-title":"Imported Infectious Diseases","author":"F. Cobo","year":"2014"},{"key":"pone.0307601.ref004","doi-asserted-by":"crossref","first-page":"12-","DOI":"10.1051\/parasite\/2014011","article-title":"Leishmania (Leishmania) martiniquensis n. sp. (Kinetoplastida: Trypanosomatidae), description of the parasite responsible for cutaneous leishmaniasis in Martinique Island (French West Indies).","volume":"21","author":"N Desbois","year":"2014","journal-title":"Parasite (Paris, France)."},{"issue":"5","key":"pone.0307601.ref005","doi-asserted-by":"crossref","first-page":"821","DOI":"10.4269\/ajtmh.2012.11-0707","article-title":"Autochthonous disseminated dermal and visceral leishmaniasis in an AIDS patient, southern Thailand, caused by Leishmania siamensis","volume":"86","author":"L Bualert","year":"2012","journal-title":"Am J Trop Med Hyg"},{"issue":"1","key":"pone.0307601.ref006","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1186\/s13071-018-2908-3","article-title":"Leishmania (Mundinia) orientalis n. sp. (Trypanosomatidae), a parasite from Thailand responsible for localised cutaneous leishmaniasis.","volume":"11","author":"N Jariyapan","year":"2018","journal-title":"Parasit Vectors."},{"issue":"5","key":"pone.0307601.ref007","doi-asserted-by":"crossref","first-page":"10.1128\/jcm.00217-21","DOI":"10.1128\/JCM.00217-21","article-title":"Taxonomy Revision of Leishmania spp. in Thailand","volume":"59","author":"M Mungthin","year":"2021","journal-title":"Journal of Clinical Microbiology"},{"issue":"3","key":"pone.0307601.ref008","doi-asserted-by":"crossref","first-page":"534","DOI":"10.4269\/ajtmh.16-0604","article-title":"Leishmaniasis in Thailand: A Review of Causative Agents and Situations","volume":"96","author":"S Leelayoova","year":"2017","journal-title":"Am J Trop Med Hyg"},{"issue":"6","key":"pone.0307601.ref009","doi-asserted-by":"crossref","first-page":"e1009654","DOI":"10.1371\/journal.ppat.1009654","article-title":"Experimental transmission of Leishmania (Mundinia) parasites by biting midges (Diptera: Ceratopogonidae).","volume":"17","author":"T Becvar","year":"2021","journal-title":"PLoS Pathog."},{"issue":"11","key":"pone.0307601.ref010","doi-asserted-by":"crossref","first-page":"379","DOI":"10.3390\/tropicalmed7110379","article-title":"First Evidence of Co-Circulation of Emerging Leishmania martiniquensis, Leishmania orientalis, and Crithidia sp. in Culicoides Biting Midges (Diptera: Ceratopogonidae), the Putative Vectors for Autochthonous Transmission in Southern Thailand","volume":"7","author":"N Songumpai","year":"2022","journal-title":"Tropical Medicine and Infectious Disease"},{"key":"pone.0307601.ref011","first-page":"14","article-title":"Natural infection with Leishmania (Mundinia) martiniquensis supports Culicoides peregrinus (Diptera: Ceratopogonidae) as a potential vector of leishmaniasis and characterization of a Crithidia sp. isolated from the midges","author":"S Kaewmee","year":"2023","journal-title":"Frontiers in Microbiology"},{"issue":"7","key":"pone.0307601.ref012","doi-asserted-by":"crossref","first-page":"e0009545","DOI":"10.1371\/journal.pntd.0009545","article-title":"Prevalence and associated risk factors of Leishmania infection among immunocompetent hosts, a community-based study in Chiang Rai, Thailand.","volume":"15","author":"P Sriwongpan","year":"2021","journal-title":"PLoS Negl Trop Dis"},{"key":"pone.0307601.ref013","author":"T Ruang-areerate","year":"2021","journal-title":"Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients."},{"issue":"11","key":"pone.0307601.ref014","doi-asserted-by":"crossref","first-page":"e0006095","DOI":"10.1371\/journal.pntd.0006095","article-title":"Prevalence and risk factors associated with Leishmania infection in Trang Province, southern Thailand.","volume":"11","author":"J Manomat","year":"2017","journal-title":"PLoS Negl Trop Dis"},{"issue":"4","key":"pone.0307601.ref015","doi-asserted-by":"crossref","first-page":"1502","DOI":"10.4269\/ajtmh.20-0332","article-title":"Risk Factors of Leishmania Infection among HIV-Infected Patients in Trang Province, Southern Thailand: A Study on Three Prevalent Species","volume":"103","author":"S Charoensakulchai","year":"2020","journal-title":"The American Journal of Tropical Medicine and Hygiene"},{"key":"pone.0307601.ref016","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1186\/s13578-015-0021-2","article-title":"Leishmaniases diagnosis: an update on the use of immunological and molecular tools","volume":"5","author":"M de Paiva-Cavalcanti","year":"2015","journal-title":"Cell Biosci"},{"issue":"46","key":"pone.0307601.ref017","first-page":"e89","article-title":"Evidence map of diagnosis, treatment, prognosis, prevention, and control in visceral leishmaniasis.","volume":"2022","author":"BF Ibiapina AB","year":"2022","journal-title":"Rev Panam Salud Publica"},{"issue":"6","key":"pone.0307601.ref018","doi-asserted-by":"crossref","first-page":"820","DOI":"10.1111\/jeu.12415","article-title":"Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection","volume":"64","author":"S Siripattanapipong","year":"2017","journal-title":"Journal of Eukaryotic Microbiology"},{"issue":"1","key":"pone.0307601.ref019","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.actatropica.2009.09.005","article-title":"Ekram ARMS, Haque R. PCR for diagnosis and assessment of cure in kala-azar patients in Bangladesh","volume":"113","author":"MA Salam","year":"2010","journal-title":"Acta Tropica"},{"issue":"1","key":"pone.0307601.ref020","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/0166-6851(96)02629-1","article-title":"Phylogenetic relationships between Leishmania, Viannia and Sauroleishmania inferred from comparison of a variable domain within the RNA polymerase II largest subunit gene","volume":"79","author":"D Croan","year":"1996","journal-title":"Molecular and Biochemical Parasitology"},{"issue":"8","key":"pone.0307601.ref021","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1046\/j.1365-3156.2002.00911.x","article-title":"Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification","volume":"7","author":"HD Schallig","year":"2002","journal-title":"Trop Med Int Health"},{"issue":"4","key":"pone.0307601.ref022","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1007\/s40291-018-0343-y","article-title":"Molecular Diagnosis of Visceral Leishmaniasis","volume":"22","author":"S Sundar","year":"2018","journal-title":"Molecular Diagnosis & Therapy."},{"issue":"1","key":"pone.0307601.ref023","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1016\/S0732-8893(03)00093-2","article-title":"PCR diagnosis and characterization of Leishmania in local and imported clinical samples","volume":"47","author":"G Sch\u00f6nian","year":"2003","journal-title":"Diagn Microbiol Infect Dis"},{"key":"pone.0307601.ref024","doi-asserted-by":"crossref","first-page":"607287","DOI":"10.1155\/2014\/607287","article-title":"ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and Strains from Cases of Human Cutaneous Leishmaniasis in States of the Mexican Southeast.","volume":"2014","author":"A Monroy-Ostria","year":"2014","journal-title":"Interdisciplinary perspectives on infectious diseases."},{"issue":"1","key":"pone.0307601.ref025","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1006\/expr.2001.4592","article-title":"Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing","volume":"97","author":"NO El Tai","year":"2001","journal-title":"Exp Parasitol"},{"issue":"1","key":"pone.0307601.ref026","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1186\/s13071-014-0458-x","article-title":"Comparison of PCR methods for detection of Leishmania siamensis infection.","volume":"7","author":"A Hitakarun","year":"2014","journal-title":"Parasites & Vectors."},{"issue":"2","key":"pone.0307601.ref027","first-page":"495","article-title":"Reliable interpretation and long-term stability using SYBRTM safe fluorescent assay for loop-mediated isothermal amplification (LAMP) detection of Leishmania spp.","volume":"36","author":"T Thita","year":"2019","journal-title":"Trop Biomed"},{"key":"pone.0307601.ref028","doi-asserted-by":"crossref","first-page":"114178","DOI":"10.1016\/j.jpba.2021.114178","article-title":"Heat-enhancing aggregation of gold nanoparticles combined with loop-mediated isothermal amplification (HAG-LAMP) for Plasmodium falciparum detection.","volume":"203","author":"P Suwannin","year":"2021","journal-title":"Journal of Pharmaceutical and Biomedical Analysis"},{"key":"pone.0307601.ref029","author":"T Ruang-areerate","year":"2022","journal-title":"Distance-based paper device using combined SYBR safe and gold nanoparticle probe LAMP assay to detect Leishmania among patients with HIV"},{"issue":"24","key":"pone.0307601.ref030","doi-asserted-by":"crossref","first-page":"8077","DOI":"10.1039\/D0AN01306D","article-title":"Development of a fluorescent distance-based paper device using loop-mediated isothermal amplification to detect Escherichia coli in urine","volume":"145","author":"N Saengsawang","year":"2020","journal-title":"Analyst"},{"issue":"1","key":"pone.0307601.ref031","doi-asserted-by":"crossref","first-page":"18781","DOI":"10.1038\/s41598-023-46001-6","article-title":"Validation of quantitative loop-mediated isothermal amplification assay using a fluorescent distance-based paper device for detection of Escherichia coli in urine.","volume":"13","author":"N Saengsawang","year":"2023","journal-title":"Sci Rep."},{"issue":"11","key":"pone.0307601.ref032","doi-asserted-by":"crossref","first-page":"e0009982","DOI":"10.1371\/journal.pntd.0009982","article-title":"Molecular identification of two newly identified human pathogens causing leishmaniasis using PCR-based methods on the 3\u2019 untranslated region of the heat shock protein 70 (type I) gene.","volume":"15","author":"N Jariyapan","year":"2021","journal-title":"PLoS Negl Trop Dis."},{"key":"pone.0307601.ref033","first-page":"2022.10.06.511237","volume-title":"Novel triplex nucleic acid lateral flow immuno-assay (NALFIA) for rapid detection of Nipah virus, Middle East respiratory syndrome coronavirus and Reston ebolavirus.","author":"S Chingtham","year":"2022"},{"issue":"1","key":"pone.0307601.ref034","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":"Web Server issue","key":"pone.0307601.ref035","doi-asserted-by":"crossref","first-page":"W43","DOI":"10.1093\/nar\/gkm234","article-title":"OligoCalc: an online oligonucleotide properties calculator","volume":"35","author":"WA Kibbe","year":"2007","journal-title":"Nucleic Acids Res"},{"key":"pone.0307601.ref036","doi-asserted-by":"crossref","first-page":"105651","DOI":"10.1016\/j.actatropica.2020.105651","article-title":"Simplified closed tube loop mediated isothermal amplification (LAMP) assay for visual diagnosis of Leishmania infection.","volume":"212","author":"C Sukphattanaudomchoke","year":"2020","journal-title":"Acta Trop"},{"issue":"1","key":"pone.0307601.ref037","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1186\/1471-2180-13-60","article-title":"Multilocus characterization and phylogenetic analysis of Leishmania siamensis isolated from autochthonous visceral leishmaniasis cases, southern Thailand","volume":"13","author":"S Leelayoova","year":"2013","journal-title":"BMC Microbiology"},{"issue":"4","key":"pone.0307601.ref038","first-page":"736","article-title":"Detection of Leishmania martiniquensis DNA in various clinical samples by quantitative PCR","volume":"32","author":"S Jungudomjaroen","year":"2015","journal-title":"Trop Biomed"},{"issue":"2","key":"pone.0307601.ref039","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1007\/s10096-010-1071-3","article-title":"Universal PCR assays for the differential detection of all Old World Leishmania species","volume":"30","author":"SO Odiwuor","year":"2011","journal-title":"Eur J Clin Microbiol Infect Dis"},{"issue":"Spec. Iss.","key":"pone.0307601.ref040","doi-asserted-by":"crossref","first-page":"S350","DOI":"10.17221\/959-CJFS","article-title":"Nucleic Acid Lateral Flow Immunoassay for the Detection of Pathogenic Bacteria from Food","volume":"27","author":"M Blazkova","year":"2009","journal-title":"Czech Journal of Food Science"},{"issue":"11","key":"pone.0307601.ref041","doi-asserted-by":"crossref","first-page":"3520","DOI":"10.1128\/JCM.01426-12","article-title":"Direct blood PCR in combination with nucleic acid lateral flow immunoassay for detection of Plasmodium species in settings where malaria is endemic","volume":"50","author":"PF Mens","year":"2012","journal-title":"J Clin Microbiol"},{"issue":"1","key":"pone.0307601.ref042","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":"The Journal of Molecular Diagnostics"},{"issue":"1","key":"pone.0307601.ref043","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1128\/CMR.19.1.111-126.2006","article-title":"Drug resistance in leishmaniasis","volume":"19","author":"SL Croft","year":"2006","journal-title":"Clin Microbiol Rev"},{"issue":"10","key":"pone.0307601.ref044","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1016\/j.pt.2018.07.012","article-title":"Advances toward Diagnostic Tools for Managing Zoonotic Visceral Leishmaniasis","volume":"34","author":"MS Duthie","year":"2018","journal-title":"Trends Parasitol"},{"key":"pone.0307601.ref045","doi-asserted-by":"crossref","first-page":"104073","DOI":"10.1016\/j.meegid.2019.104073","article-title":"ISC1, a new Leishmania donovani population emerging in the Indian sub-continent: Vector competence of Phlebotomus argentipes","volume":"76","author":"V Seblova","year":"2019","journal-title":"Infection, Genetics and Evolution"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0307601","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,8,26]],"date-time":"2024-08-26T20:23:18Z","timestamp":1724703798000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0307601"}},"subtitle":[],"editor":[{"given":"Alireza","family":"Badirzadeh","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2024,8,26]]},"references-count":45,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2024,8,26]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0307601","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,26]]}}}