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Although quantitative serology is the primary indirect diagnostic approach, polymerase chain reaction (PCR) is the most sensitive direct diagnostic method for confirming the infection by detecting\n                      <jats:italic>Leishmania<\/jats:italic>\n                      DNA in biological samples, but its performance varies according to the analysed tissue and molecular assay. This study aimed to validate a duplex real-time PCR assay and compare its diagnostic performance with a nested PCR in six different canine tissues, as well as to define the diagnostic value of non-invasive samples in clinical staging and monitoring in naturally infected dogs before and after specific treatment.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      <jats:italic>Leishmania<\/jats:italic>\n                      nested PCR and quantitative PCR targeting the small subunit (SSU) ribosomal RNA (rRNA) gene were performed on lymph node, blood, oral and conjunctival swabs, urine and hair samples from 66 dogs (20 non-infected, 15 clinically healthy infected and 31 naturally infected sick dogs classified according to LeishVet clinical staging). Sick dogs classified as stage II\u2013III were re-evaluated 30\u00a0days after therapy to assess clinical improvement and changes in parasite load during follow-up.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Lymph node samples showed the highest sensitivity and parasite loads, correlating well with clinical severity, IFAT titres and albumin\/globulin ratio. Among non-invasive samples, oral swabs showed the best performance, particularly in dogs with clinically relevant disease, while conjunctival swabs showed moderate sensitivity. By contrast, blood showed limited and stage-dependent diagnostic value, while urine and hair demonstrated very low sensitivity. Quantitative PCR showed slightly higher sensitivity than nPCR in most tissues, except for lymph node and blood samples. A\n                      <jats:italic>C<\/jats:italic>\n                      <jats:sub>T<\/jats:sub>\n                      cut-off of 39.00 was applied to minimise non-specific amplification, which was particularly pronounced in urine qPCR, achieving 100% specificity across all other tissues. The qPCR showed a reaction efficiency of 85.4% (\n                      <jats:italic>R<\/jats:italic>\n                      <jats:sup>2<\/jats:sup>\n                      \u2009=\u20090.997), with a limit of detection of 0.5 parasite equivalents\/reaction. Parasite load increased significantly from stage I to stage II dogs, but did not differ between stages II and III. After treatment, clinical scores and parasite loads decreased significantly in lymph node, oral and conjunctival swab samples.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>PCR performance in canine leishmaniosis is strongly influenced by tissue, technique and clinical stage. nPCR should be the test of choice for diagnostic confirmation, with lymph node as the gold standard sample, while oral swabs represent the most reliable non-invasive alternative, particularly in dogs with clinically relevant disease. For clinical follow-up, qPCR is preferable due to its ability to monitor parasite load changes in appropriate tissues, particularly lymph node and oral swab samples. The limited reliability of PCR from non-invasive samples in early stages of infection reinforces the need for a multimodal diagnostic approach in the clinical management of canine leishmaniosis.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13071-026-07490-2","type":"journal-article","created":{"date-parts":[[2026,6,21]],"date-time":"2026-06-21T05:53:22Z","timestamp":1782021202000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Validation of a duplex real-time PCR (qPCR) in clinically classified dogs naturally infected with Leishmania infantum before and after specific treatment: preliminary results"],"prefix":"10.1186","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-2472-3549","authenticated-orcid":false,"given":"Clara","family":"G\u00f3mez-Velasco","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1911-8642","authenticated-orcid":false,"given":"Ana","family":"Montoya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2419-1254","authenticated-orcid":false,"given":"Roc\u00edo","family":"Checa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaime","family":"Heredia-Caldeiro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7682-9083","authenticated-orcid":false,"given":"Juan Pedro","family":"Barrera","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7505-9495","authenticated-orcid":false,"given":"Efr\u00e9n","family":"Est\u00e9vez-S\u00e1nchez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6967-6733","authenticated-orcid":false,"given":"Juliana","family":"Sarquis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela","family":"Garc\u00eda-Rup\u00e9rez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-8421-566X","authenticated-orcid":false,"given":"Pablo","family":"Moraleda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0981-2470","authenticated-orcid":false,"given":"Guadalupe","family":"Mir\u00f3","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,21]]},"reference":[{"key":"7490_CR1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.vetpar.2009.05.022","volume":"165","author":"L Solano-Gallego","year":"2009","unstructured":"Solano-Gallego L, Koutinas A, Mir\u00f3 G, Cardoso L, Pennisi MG, Ferrer L, et al. 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