{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T01:16:40Z","timestamp":1788225400235,"version":"build-2803163510"},"reference-count":34,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"NextGenerationEU-MUR PNRR","award":["PE00000007, INF-ACT"],"award-info":[{"award-number":["PE00000007, INF-ACT"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,7,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Among\n                      <jats:italic toggle=\"yes\">Leishmania<\/jats:italic>\n                      spp. transmitted by phlebotomine sand flies,\n                      <jats:italic toggle=\"yes\">Leishmania infantum<\/jats:italic>\n                      is regarded as the most widely distributed species worldwide and the primary causative agent of canine leishmaniasis. However, in regions where\n                      <jats:italic toggle=\"yes\">L. Infantum<\/jats:italic>\n                      is endemic the sympatric occurrence of the herpetophilic\n                      <jats:italic toggle=\"yes\">Leishmania tarentolae<\/jats:italic>\n                      in dogs, cats, and humans has prompted the development of sensitive and specific diagnostic assays. To evaluate the clinical and analytical sensitivities of a quantitative PCR (qPCR) assay targeting the minicircle kDNA fragment of both species, three devices with different technologies (lab-on-chip real-time PCR applied on the portable Q3-Plus V2 platform, CFX96 Real-Time System, and droplet digital PCR [ddPCR]) were tested. The sensitivity (limit of detection [LOD]) and specificity of the assay were assessed by processing DNA samples from cultures of both\n                      <jats:italic toggle=\"yes\">Leishmania<\/jats:italic>\n                      spp. and from biological samples of\n                      <jats:italic toggle=\"yes\">Sergentomyia minuta<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 120; midgut), of lizards (\n                      <jats:italic toggle=\"yes\">Podarcis siculus<\/jats:italic>\n                      ;\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 29; blood, eggs, heart, intestine, kidneys, liver, lungs, and spleen), and of dogs (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 101; blood, bone marrow, conjunctival swab, lymph node, and skin). The performance of the assay applied on all devices was evaluated by comparing the quantification cycle values obtained by qPCR with the DNA copy number determined by ddPCR. The LOD of serial dilutions of DNA from each\n                      <jats:italic toggle=\"yes\">Leishmania<\/jats:italic>\n                      spp., spiked DNA, and biological samples were overlapping across the devices, whereas receiver operating characteristic analysis showed a shift in sensitivity for ddPCR (AUC, 0.9858) compared to CFX96 and Q3 qPCRs (AUC, 0.9517). The assay developed is an efficient and sensitive tool regardless of the device tested, though ddPCR showed higher sensitivity in detecting low quantities of DNA in all samples, as supported by the highest AUC value.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        The development of sensitive and specific molecular tools for detecting\n                        <jats:italic toggle=\"yes\">Leishmania infantum<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">Leishmania tarentolae<\/jats:italic>\n                        has been evaluated in a duplex qPCR assay applied on three devices with different technologies (lab-on-chip real-time PCR applied on the portable Q3-Plus V2 platform, CFX96 Real-Time System, and droplet digital PCR). Both\n                        <jats:italic toggle=\"yes\">Leishmania<\/jats:italic>\n                        spp. were tested in different tissues of various animal hosts, and the clinical sensitivity and specificity were evaluated. The duplex qPCR assay developed herein proved to be an efficient tool, regardless of the device used, and will be useful in improving current knowledge on the distribution of\n                        <jats:italic toggle=\"yes\">L. infantum<\/jats:italic>\n                        and of\n                        <jats:italic toggle=\"yes\">L. tarentolae<\/jats:italic>\n                        and in better understanding the potential role of new hosts\/reservoirs or vectors for these\n                        <jats:italic toggle=\"yes\">Leishmania<\/jats:italic>\n                        spp.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03854-25","type":"journal-article","created":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T13:01:48Z","timestamp":1781528508000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Performance assessment of a duplex quantitative PCR assay for detecting\n                    <i>Leishmania infantum<\/i>\n                    and\n                    <i>Leishmania tarentolae<\/i>\n                    using three qPCR devices"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9061-437X","authenticated-orcid":true,"given":"Maria Stefania","family":"Latrofa","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027ynra39","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary Medicine, University of Bari","place":["Bari, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucas","family":"Cafferati-Beltrame","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027ynra39","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary Medicine, University of Bari","place":["Bari, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pietro","family":"D'Addabbo","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027ynra39","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biosciences, Biotechnologies and Environment, University of Bari","place":["Bari, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Cereda","sequence":"additional","affiliation":[{"name":"STMicroelectronics Srl","place":["Agrate Brianza, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viviane Noll","family":"Louzada-Flores","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027ynra39","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary Medicine, University of Bari","place":["Bari, Italy"]},{"id":[{"id":"https:\/\/ror.org\/05f82e368","id-type":"ROR","asserted-by":"publisher"}],"name":"Unit\u00e9 de Parasitologie Mol\u00e9culaire et Signalisation, Institut Pasteur, Universit\u00e9 Paris Cit\u00e9, INSERM 1201","place":["Paris, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ilaria Varotto","family":"Boccazzi","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00wjc7c48","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biosciences, University of Milan","place":["Milan, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8807-4802","authenticated-orcid":true,"given":"Sara","family":"Epis","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00wjc7c48","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biosciences, University of Milan","place":["Milan, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jairo Alfonso","family":"Mendoza-Roldan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027ynra39","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary Medicine, University of Bari","place":["Bari, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7518-476X","authenticated-orcid":true,"given":"Domenico","family":"Otranto","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027ynra39","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary Medicine, University of Bari","place":["Bari, Italy"]},{"id":[{"id":"https:\/\/ror.org\/03hj2gd47","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary Clinical Sciences, City University of Hong Kong","place":["Kowloon Tong, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0035671"},{"key":"e_1_3_3_3_2","unstructured":"World Health Organization (WHO). 2023. 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