Direct quantitative PCR detects genetic biomarkers of antileishmanial drug resistance in clinical samples from dogs with leishmaniosis

Direct quantitative PCR detects genetic biomarkers of antileishmanial drug resistance in clinical samples from dogs with leishmaniosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Direct quantitative PCR detects genetic biomarkers of antileishmanial drug resistance in clinical samples from dogs with leishmaniosis Marina Carrasco Martin, Clàudia Viñeta, Joan Martí Carreras, Xavier Roura, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8843866/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Treatment response in canine leishmaniosis is driven by the dog host, the Leishmania parasite, and pharmacological factors, with drug resistance increasingly undermining the effectiveness of therapy. A direct quantitative PCR test (LeishGenR™) was applied to 104 clinical samples from 95 dogs in the Mediterranean area diagnosed with leishmaniosis in veterinary clinical settings and testing positive for Leishmania infantum by PCR. The assay enabled rapid detection of genetic drug-resistance biomarkers for allopurinol ( metk ), meglumine antimoniate ( mrpa ), and miltefosine ( LdMT ), providing a clinically relevant, timely alternative to culture-based approaches by directly analyzing circulating Leishmania infantum amastigotes. Results The assay (LeishGenR™) showed high specificity (100%) and sensitivity (> 87.5%) for genetic drug-resistance profile assignment and a strong correlation with whole-genome sequencing for gene copy number assessment ( metk : r = 0.878; mrpa : r = 0.943 and LdMT = 0.691). Genetic drug-resistance biomarkers were detected in 24.3% of L. infantum DNA from clinical samples analyzed (20/82; 95% CI 16.3–34.6)), most commonly for allopurinol (13.4%; 95% CI 7.6–22.4), then meglumine antimoniate (9.4%; 95% CI 4.6–18.2), and for miltefosine (5.4%; 95% CI 1.8–14.8). Prevalence was higher in dogs previously treated for leishmaniosis. Conclusion This study demonstrates the ability to detect genetic biomarkers of drug resistance in L. infantum directly from clinical samples of dogs with leishmaniosis. This method enables rapid, precise detection of genomic biomarkers, circumventing delays associated with culture-based methods and supporting more effective clinical management and surveillance. Among dogs with high parasitemia referred to clinics in Mediterranean regions sampled in this study, the findings reveal a significant prevalence of circulating L. infantum strains carrying genomic drug resistance biomarkers to standard treatments for canine leishmaniosis. Leishmania infantum canine allopurinol meglumine antimoniate miltefosine Full Text Additional Declarations Competing interest reported. MCM and JMC are current employees of Nano1Health SL. Supplementary Files AdditionalFile1.xlsx AdditionalFile2.xlsx AdditionalFile3.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 11 May, 2026 Reviews received at journal 23 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviews received at journal 30 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers agreed at journal 25 Feb, 2026 Reviewers invited by journal 25 Feb, 2026 Editor assigned by journal 23 Feb, 2026 Editor invited by journal 23 Feb, 2026 Submission checks completed at journal 23 Feb, 2026 First submitted to journal 23 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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MCM and JMC are current employees of Nano1Health SL.","formattedTitle":"Direct quantitative PCR detects genetic biomarkers of antileishmanial drug resistance in clinical samples from dogs with leishmaniosis","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Leishmania infantum, canine, allopurinol, meglumine antimoniate, miltefosine","lastPublishedDoi":"10.21203/rs.3.rs-8843866/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8843866/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTreatment response in canine leishmaniosis is driven by the dog host, the \u003cem\u003eLeishmania\u003c/em\u003e parasite, and pharmacological factors, with drug resistance increasingly undermining the effectiveness of therapy. A direct quantitative PCR test (LeishGenR\u0026trade;) was applied to 104 clinical samples from 95 dogs in the Mediterranean area diagnosed with leishmaniosis in veterinary clinical settings and testing positive for \u003cem\u003eLeishmania infantum\u003c/em\u003e by PCR. The assay enabled rapid detection of genetic drug-resistance biomarkers for allopurinol (\u003cem\u003emetk\u003c/em\u003e), meglumine antimoniate (\u003cem\u003emrpa\u003c/em\u003e), and miltefosine (\u003cem\u003eLdMT\u003c/em\u003e), providing a clinically relevant, timely alternative to culture-based approaches by directly analyzing circulating \u003cem\u003eLeishmania infantum\u003c/em\u003e amastigotes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe assay (LeishGenR\u0026trade;) showed high specificity (100%) and sensitivity (\u0026gt;\u0026thinsp;87.5%) for genetic drug-resistance profile assignment and a strong correlation with whole-genome sequencing for gene copy number assessment (\u003cem\u003emetk\u003c/em\u003e: r\u0026thinsp;=\u0026thinsp;0.878; \u003cem\u003emrpa\u003c/em\u003e: r\u0026thinsp;=\u0026thinsp;0.943 and \u003cem\u003eLdMT\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.691). Genetic drug-resistance biomarkers were detected in 24.3% of \u003cem\u003eL. infantum\u003c/em\u003e DNA from clinical samples analyzed (20/82; 95% CI 16.3\u0026ndash;34.6)), most commonly for allopurinol (13.4%; 95% CI 7.6\u0026ndash;22.4), then meglumine antimoniate (9.4%; 95% CI 4.6\u0026ndash;18.2), and for miltefosine (5.4%; 95% CI 1.8\u0026ndash;14.8). Prevalence was higher in dogs previously treated for leishmaniosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study demonstrates the ability to detect genetic biomarkers of drug resistance in \u003cem\u003eL. infantum\u003c/em\u003e directly from clinical samples of dogs with leishmaniosis. This method enables rapid, precise detection of genomic biomarkers, circumventing delays associated with culture-based methods and supporting more effective clinical management and surveillance. Among dogs with high parasitemia referred to clinics in Mediterranean regions sampled in this study, the findings reveal a significant prevalence of circulating \u003cem\u003eL. infantum\u003c/em\u003e strains carrying genomic drug resistance biomarkers to standard treatments for canine leishmaniosis.\u003c/p\u003e","manuscriptTitle":"Direct quantitative PCR detects genetic biomarkers of antileishmanial drug resistance in clinical samples from dogs with leishmaniosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 12:47:59","doi":"10.21203/rs.3.rs-8843866/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"303798600870220721493404215465539572370","date":"2026-05-11T08:58:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T05:08:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144278884472977263103533428369083033201","date":"2026-04-03T00:34:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T15:28:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67545104589907586109393358197015891387","date":"2026-03-11T08:37:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58846138210003627503214850069371282913","date":"2026-02-25T13:22:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T10:41:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-24T03:35:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-24T02:42:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-23T17:19:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2026-02-23T17:14:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0a24c070-6209-471c-870d-e9fd7829547c","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"303798600870220721493404215465539572370","date":"2026-05-11T08:58:51+00:00","index":60,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-27T12:47:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 12:47:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8843866","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8843866","identity":"rs-8843866","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}