{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T15:03:07Z","timestamp":1791298987942,"version":"4.1.0"},"reference-count":78,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2021,7,9]],"date-time":"2021-07-09T00:00:00Z","timestamp":1625788800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Ministero della Salute","doi-asserted-by":"publisher","award":["IZS SI RC 02\/18"],"award-info":[{"award-number":["IZS SI RC 02\/18"]}],"id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to achieve a sensitivity of 1 parasite\/mL. For this purpose, we cloned the conserved kDNA fragment of 120 bp into competent cells and correlated them with serial dilutions of DNA extracted from reference parasite cultures calculating that a parasite cell contains approximately 36 molecules of kDNA. This assay was applied to estimate parasite load in clinical samples from visceral, cutaneous leishmaniasis patients and infected dogs and cats comparing with conventional diagnosis. The study aimed to propose a real-time PCR for the detection of Leishmania DNA from clinical samples trying to solve the diagnostic problems due to the low sensitivity of microscopic examination or the low predictive values of serology and resolve problems related to in vitro culture. The quantitative PCR assay in this study allowed detection of Leishmania DNA and quantification of considerably low parasite loads in samples that had been diagnosed negative by conventional techniques. In conclusion, this quantitative PCR can be used for the diagnosis of both human, canine and feline Leishmaniasis with high sensitivity and specificity, but also for evaluating treatment and the endpoint determination of leishmaniasis.<\/jats:p>","DOI":"10.3390\/pathogens10070865","type":"journal-article","created":{"date-parts":[[2021,7,9]],"date-time":"2021-07-09T10:50:38Z","timestamp":1625827838000},"page":"865","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":59,"title":["Molecular Diagnosis of Leishmaniasis: Quantification of Parasite Load by a Real-Time PCR Assay with High Sensitivity"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0464-4474","authenticated-orcid":false,"given":"Germano","family":"Castelli","sequence":"first","affiliation":[{"name":"Centro di Referenza Nazionale per le Leishmaniosi (C.Re.Na.L.), OIE Leishmania Reference Laboratory, Istituto Zooprofilattico Sperimentale della Sicilia, Via Gino Marinuzzi 3, 90129 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6181-6984","authenticated-orcid":false,"given":"Federica","family":"Bruno","sequence":"additional","affiliation":[{"name":"Centro di Referenza Nazionale per le Leishmaniosi (C.Re.Na.L.), OIE Leishmania Reference Laboratory, Istituto Zooprofilattico Sperimentale della Sicilia, Via Gino Marinuzzi 3, 90129 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefano","family":"Reale","sequence":"additional","affiliation":[{"name":"Laboratorio di Tecnologie Diagnostiche Innovative (TDI), Istituto Zooprofilattico Sperimentale della Sicilia, Via Gino Marinuzzi 3, 90129 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simone","family":"Catanzaro","sequence":"additional","affiliation":[{"name":"Centro di Referenza Nazionale per le Leishmaniosi (C.Re.Na.L.), OIE Leishmania Reference Laboratory, Istituto Zooprofilattico Sperimentale della Sicilia, Via Gino Marinuzzi 3, 90129 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viviana","family":"Valenza","sequence":"additional","affiliation":[{"name":"Centro di Referenza Nazionale per le Leishmaniosi (C.Re.Na.L.), OIE Leishmania Reference Laboratory, Istituto Zooprofilattico Sperimentale della Sicilia, Via Gino Marinuzzi 3, 90129 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabrizio","family":"Vitale","sequence":"additional","affiliation":[{"name":"Centro di Referenza Nazionale per le Leishmaniosi (C.Re.Na.L.), OIE Leishmania Reference Laboratory, Istituto Zooprofilattico Sperimentale della Sicilia, Via Gino Marinuzzi 3, 90129 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1111\/j.1550-7408.2000.tb00008.x","article-title":"Leishmania, Trypanosoma and monoxenous trypanosomatids as emerging opportunistic agents","volume":"47","author":"Dedet","year":"2000","journal-title":"J. Eukaryot. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.tvjl.2006.11.011","article-title":"Canine leishmaniasis: A diagnostic and clinical challenge","volume":"175","author":"Baneth","year":"2008","journal-title":"Vet. J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1186\/s13071-019-3386-y","article-title":"Leishmania infantum-specific IFN-\u03b3 production in stimulated blood from cats living in areas where canine leishmaniosis is endemic","volume":"12","author":"Priolo","year":"2019","journal-title":"Parasit Vectors"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Spada, E., Perego, R., Vitale, F., Bruno, F., Castelli, G., Tarantola, G., Baggiani, L., Magistrelli, S., and Proverbio, D. (2020). Feline Leishmania spp. Infection in a Non-Endemic Area of Northern Italy. Animals, 10.","DOI":"10.3390\/ani10050817"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"S54","DOI":"10.1016\/j.jcpa.2015.10.179","article-title":"Major Parasitic Zoonoses Associated with Dogs and Cats in Europe","volume":"155","author":"Baneth","year":"2016","journal-title":"J. Comp. Pathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.cimid.2016.03.001","article-title":"Prevalence of Leishmania infantum and co-infections in stray cats in northern Italy","volume":"45","author":"Spada","year":"2016","journal-title":"Comp. Immunol. Microbiol. Infect. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1053\/j.tcam.2009.06.006","article-title":"Leishmaniasis, an Emerging Disease Found in Companion Animals in the United States","volume":"24","author":"Petersen","year":"2009","journal-title":"Top. Companion Anim. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1111\/j.1529-8019.2009.01272.x","article-title":"Cutaneous and mucocutaneous leishmaniasis","volume":"22","author":"David","year":"2009","journal-title":"Dermatol. Ther."},{"key":"ref_9","first-page":"123","article-title":"Suspected cutaneous leishmaniasis in a sample of Westerns Sicily residents: What correlation with occupation?","volume":"108","author":"Verso","year":"2017","journal-title":"Med. Lav. Work Environ. Health"},{"key":"ref_10","first-page":"951","article-title":"Laboratory Diagnosis of Visceral Leishmaniasis","volume":"9","author":"Sundar","year":"2002","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"S179","DOI":"10.1016\/S0035-9203(02)90073-8","article-title":"Visceral leishmaniasis in India: Promises and pitfalls of a PCR-based blood test","volume":"96","author":"Adhya","year":"2002","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"622","DOI":"10.1016\/0035-9203(95)90416-6","article-title":"Detection of Leishmania in the blood of early kala-azar patients with the aid of the polymerase chain reaction","volume":"89","author":"Adhya","year":"1995","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"296","DOI":"10.4269\/ajtmh.1983.32.296","article-title":"Comparison of microscopy and culture in the detection of Leishmania donovani from splenic aspirates","volume":"32","author":"Lightner","year":"1983","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0001-706X(92)90002-F","article-title":"Recombinant kinetoplast DNA (kDNA) probe for identifying Leishmania tropica","volume":"52","author":"Massamba","year":"1992","journal-title":"Acta Trop."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.diagmicrobio.2014.01.031","article-title":"Parasite load estimation by qPCR differentiates between asymptomatic and symptomatic infection in Indian Visceral Leishmaniasis","volume":"80","author":"Sudarshan","year":"2014","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1739","DOI":"10.1056\/NEJMoa021556","article-title":"Oral Miltefosine for Indian Visceral Leishmaniasis","volume":"347","author":"Sundar","year":"2002","journal-title":"N. Engl. J. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1007\/s40291-018-0343-y","article-title":"Molecular Diagnosis of Visceral Leishmaniasis","volume":"22","author":"Sundar","year":"2018","journal-title":"Mol. Diagn. Ther."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.trstmh.2010.09.006","article-title":"Diagnosis of visceral leishmaniasis","volume":"105","author":"Srivastava","year":"2011","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_19","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":"Thakur","year":"2020","journal-title":"J. Parasit. Dis. Off. Organ Indian Soc. Parasitol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Reim\u00e3o, J.Q., Coser, E.M., Lee, M.R., and Coelho, A.C. (2020). Laboratory Diagnosis of Cutaneous and Visceral Leishmaniasis: Current and Future Methods. Microorganisms, 8.","DOI":"10.3390\/microorganisms8111632"},{"key":"ref_21","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":"Galluzzi","year":"2018","journal-title":"Parasites Vectors"},{"key":"ref_22","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":"Pereira","year":"2015","journal-title":"Cell Biosci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"12595","DOI":"10.1038\/srep12595","article-title":"Doubling Throughput of a Real-Time PCR","volume":"5","author":"Ahrberg","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.cca.2014.10.017","article-title":"Real-time PCR detection chemistry","volume":"439","author":"Navarro","year":"2015","journal-title":"Clin. Chim. Acta Int. J. Clin. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1017\/S0031182001007363","article-title":"Detection of Leishmania infantum by PCR, serology and cellular immune response in a cohort study of Brazilian dogs","volume":"122","author":"Quinnell","year":"2001","journal-title":"Parasitology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1053\/rvsc.2000.0419","article-title":"Canine leishmaniasis transmission: Higher infectivity amongst naturally infected dogs to sand flies is associated with lower proportions of T helper cells","volume":"69","author":"Guarga","year":"2000","journal-title":"Res. Vet. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1314","DOI":"10.1086\/344312","article-title":"Infectiousness in a cohort of brazilian dogs: Why culling fails to control visceral leishmaniasis in areas of high transmission","volume":"186","author":"Courtenay","year":"2002","journal-title":"J. Infect. Dis."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1136\/vr.156.11.346","article-title":"Efficacy of the treatment of dogs with leishmaniosis with a combination of metronidazole and spiramycin","volume":"156","author":"Pennisi","year":"2005","journal-title":"Vet. Rec."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1007\/s11259-005-0067-4","article-title":"Real-time PCR in dogs treated for leishmaniasis with allopurinol","volume":"29","author":"Pennisi","year":"2005","journal-title":"Vet. Res. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1101\/gr.6.10.986","article-title":"Real time quantitative PCR","volume":"6","author":"Heid","year":"1996","journal-title":"Genome Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1666","DOI":"10.1128\/JCM.40.5.1666-1669.2002","article-title":"Real-Time PCR for Detection and Quantitation of Leishmania in Mouse Tissues","volume":"40","author":"Nicolas","year":"2002","journal-title":"J. Clin. Microbiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"176","DOI":"10.2144\/97221pf02","article-title":"The LightCycler: A microvolume multisample fluorimeter with rapid temperature control","volume":"22","author":"Wittwer","year":"1997","journal-title":"BioTechniques"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"828","DOI":"10.1128\/CDLI.8.4.828-831.2001","article-title":"Real-time PCR as a new tool for quantifying Leishmania infantum in liver in infected mice","volume":"8","author":"Bretagne","year":"2001","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1529","DOI":"10.1128\/JCM.41.4.1529-1535.2003","article-title":"Detection, differentiation, and quantitation of pathogenic leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay","volume":"41","author":"Schulz","year":"2003","journal-title":"J. Clin. Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1038\/ng2053","article-title":"Comparative genomic analysis of three Leishmania species that cause diverse human disease","volume":"39","author":"Peacock","year":"2007","journal-title":"Nat. Genet."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1016\/j.ijpara.2007.05.015","article-title":"Comparative genomics: From genotype to disease phenotype in the leishmaniases","volume":"37","author":"Smith","year":"2007","journal-title":"Int. J. Parasitol."},{"key":"ref_37","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":"Weirather","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e7235","DOI":"10.7717\/peerj.7235","article-title":"Mitochondrial genomics of human pathogenic parasite Leishmania (Viannia) panamensis","volume":"7","author":"Urrea","year":"2019","journal-title":"PeerJ"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Ceccarelli, M., Buffi, G., Diotallevi, A., Andreoni, F., Bencardino, D., Vitale, F., Castelli, G., Bruno, F., Magnani, M., and Galluzzi, L. (2020). Evaluation of a KDNA-Based QPCR Assay for the Detection and Quantification of Old World Leishmania Species. Microorganisms, 8.","DOI":"10.3390\/microorganisms8122006"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1196\/annals.1307.018","article-title":"TaqMan-Based Detection of Leishmania infantum DNA Using Canine Samples","volume":"1026","author":"Vitale","year":"2004","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/j.tvjl.2007.04.013","article-title":"Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol","volume":"177","author":"Manna","year":"2008","journal-title":"Vet. J."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/1756-3305-5-15","article-title":"SYBR Green-based Real-Time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases","volume":"5","author":"Lins","year":"2012","journal-title":"Parasit Vectors"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1111\/j.1472-765X.2007.02294.x","article-title":"Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR","volume":"46","author":"Galluzzi","year":"2008","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1292","DOI":"10.1093\/nar\/30.6.1292","article-title":"Real-time PCR in virology","volume":"30","author":"Mackay","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1267","DOI":"10.1002\/iub.1894","article-title":"Massive mitochondrial DNA content in diplonemid and kinetoplastid protists","volume":"70","author":"Wheeler","year":"2018","journal-title":"IUBMB Life"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.mam.2016.11.012","article-title":"Leishmania infections: Molecular targets and diagnosis","volume":"57","author":"Akhoundi","year":"2017","journal-title":"Mol. Aspects Med."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1186\/s13071-017-2181-x","article-title":"The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis","volume":"10","author":"Ceccarelli","year":"2017","journal-title":"Parasit Vectors"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Ceccarelli, M., Galluzzi, L., Migliazzo, A., and Magnani, M. (2014). Detection and Characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR Green-Based Real-Time PCR and High Resolution Melt Analysis Targeting Kinetoplast Minicircle DNA. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0088845"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1186\/s13071-019-3409-8","article-title":"Emerging feline vector-borne pathogens in Italy","volume":"12","author":"Morganti","year":"2019","journal-title":"Parasit Vectors"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"15","DOI":"10.4103\/0972-9062.213320","article-title":"Molecular diagnosis of Old World leishmaniasis: Real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp.","volume":"49","author":"Khosravi","year":"2012","journal-title":"J. Vector Borne Dis."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1016\/j.ijid.2012.11.015","article-title":"Molecular characterization of cutaneous leishmaniasis in Al-Madinah Al-Munawarah Province, western Saudi Arabia","volume":"17","year":"2013","journal-title":"Int. J. Infect. Dis. IJID"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1016\/j.det.2015.03.018","article-title":"New World and Old World Leishmania Infections: A Practical Review","volume":"33","author":"Kevric","year":"2015","journal-title":"Dermatol. Clin."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/j.diagmicrobio.2004.10.005","article-title":"Leishmania (Viannia) subgenus kDNA amplification for the diagnosis of mucosal leishmaniasis","volume":"51","author":"Disch","year":"2005","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.vetpar.2012.11.007","article-title":"Detection of Leishmania infantum kinetoplast minicircle DNA by Real Time PCR in hair of dogs with leishmaniosis","volume":"192","author":"Iniesta","year":"2013","journal-title":"Vet. Parasitol."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/S0074-7696(08)61426-6","article-title":"Kinetoplast DNA in Trypanosomid Flagellates","volume":"99","author":"Simpson","year":"1986","journal-title":"Int. Rev. Cytol."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Castelli, G., Bruno, F., Caputo, V., Fiorella, S., Sammarco, I., Lupo, T., Migliazzo, A., Vitale, F., and Reale, S. (2020). Genetic tools discriminate strains of Leishmania infantum isolated from humans and dogs in Sicily, Italy. PLoS Negl. Trop. Dis., 14.","DOI":"10.1371\/journal.pntd.0008465"},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Mesa, L.E., Manrique, R., Muskus, C., and Robledo, S.M. (2020). Test accuracy of polymerase chain reaction methods against conventional diagnostic techniques for Cutaneous Leishmaniasis (CL) in patients with clinical or epidemiological suspicion of CL: Systematic review and meta-analysis. PLoS Negl. Trop. Dis., 14.","DOI":"10.1371\/journal.pntd.0007981"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"1435","DOI":"10.1128\/JCM.44.4.1435-1439.2006","article-title":"Comparison of PCR Assays for Diagnosis of Cutaneous Leishmaniasis","volume":"44","author":"Bensoussan","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"181","DOI":"10.2307\/3285616","article-title":"PCR detection and identification of Leishmania parasites in clinical specimens in Ecuador: A comparison with classical diagnostic methods","volume":"85","author":"Aviles","year":"1999","journal-title":"J. Parasitol."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1590\/S0100-879X2002000400002","article-title":"Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis","volume":"35","author":"Medeiros","year":"2002","journal-title":"Braz. J. Med. Biol. Res. Rev. Bras. Pesqui. Med. Biol."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"2246","DOI":"10.1128\/jcm.32.9.2246-2252.1994","article-title":"Diagnosis of cutaneous leishmaniasis and species discrimination of parasites by PCR and hybridization","volume":"32","author":"Guzman","year":"1994","journal-title":"J. Clin. Microbiol."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1159\/000063150","article-title":"Polymerase chain reaction for diagnosis of cutaneous leishmaniasis in histologically positive, suspicious and negative skin biopsies","volume":"205","author":"Safaei","year":"2002","journal-title":"Dermatology"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/S0035-9203(96)90112-1","article-title":"Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major: A comparison with direct microscopy of smears and sections from lesions","volume":"90","author":"Andresen","year":"1996","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/0035-9203(95)90537-5","article-title":"Detection of cutaneous Leishmania infection in paraffin-embedded skin biopsies using the polymerase chain reaction","volume":"89","author":"Laskay","year":"1995","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"251","DOI":"10.4269\/ajtmh.1995.53.251","article-title":"Comparison of the polymerase chain reaction and serology for the detection of canine visceral leishmaniasis","volume":"53","author":"Ashford","year":"1995","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_66","doi-asserted-by":"crossref","unstructured":"Adel, A., Berkvens, D., Abatih, E., Soukehal, A., Bianchini, J., and Saegerman, C. (2016). Evaluation of Immunofluorescence Antibody Test Used for the Diagnosis of Canine Leishmaniasis in the Mediterranean Basin: A Systematic Review and Meta-Analysis. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0161051"},{"key":"ref_67","first-page":"1409","article-title":"Specific serodiagnosis of canine leishmaniasis by indirect immunofluorescence, indirect hemagglutination, and counterimmunoelectrophoresis","volume":"49","author":"Mancianti","year":"1988","journal-title":"Am. J. Vet. Res."},{"key":"ref_68","unstructured":"(2020, March 26). Leishmaniosis. Available online: https:\/\/www.oie.int\/fileadmin\/Home\/eng\/Health_standards\/tahm\/3.01.11_LEISHMANIOSIS.pdf."},{"key":"ref_69","first-page":"286","article-title":"Leishmaniasis in immunosuppressed individuals","volume":"20","author":"Carrillo","year":"2014","journal-title":"Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1177\/1098612X19895067","article-title":"Serological, molecular and clinicopathological findings associated with Leishmania infantum infection in cats in Northern Italy","volume":"22","author":"Urbani","year":"2020","journal-title":"J. Feline Med. Surg."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1186\/s13071-015-0909-z","article-title":"LeishVet update and recommendations on feline leishmaniosis","volume":"8","author":"Pennisi","year":"2015","journal-title":"Parasit Vectors"},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1080\/15321810802116006","article-title":"Validation of a Recombinant Based Antibody ELISA for Diagnosis of Human and Canine Leishmaniasis","volume":"29","author":"Scalone","year":"2008","journal-title":"J. Immunoassay Immunochem."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1016\/j.cimid.2007.09.002","article-title":"Application of a recombinant protein for the serological diagnosis of canine leishmaniasis","volume":"31","author":"Boarino","year":"2008","journal-title":"Comp. Immunol. Microbiol. Infect. Dis."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"2931","DOI":"10.1128\/JCM.37.9.2931-2935.1999","article-title":"Detection of Leishmania infantum in Dogs by PCR with Lymph Node Aspirates and Blood","volume":"37","author":"Reale","year":"1999","journal-title":"J. Clin. Microbiol."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/S0022-1759(03)00223-0","article-title":"A method for the absolute quantification of cDNA using real-time PCR","volume":"278","author":"Whelan","year":"2003","journal-title":"J. Immunol. Methods"},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1186\/1756-3305-4-57","article-title":"Canine leishmaniasis: The key points for qPCR result interpretation","volume":"4","author":"Quilez","year":"2011","journal-title":"Parasites Vectors"},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1645\/13-253.1","article-title":"Evaluation of two modified culture media for Leishmania infantum cultivation versus different culture media","volume":"100","author":"Castelli","year":"2014","journal-title":"J. Parasitol."},{"key":"ref_78","doi-asserted-by":"crossref","unstructured":"Hossain, F., Ghosh, P., Khan, M.A.A., Duthie, M.S., Vallur, A.C., Picone, A., Howard, R.F., Reed, S.G., and Mondal, D. (2017). Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0185606"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/10\/7\/865\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:28:17Z","timestamp":1760164097000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/10\/7\/865"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,9]]},"references-count":78,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,7]]}},"alternative-id":["pathogens10070865"],"URL":"https:\/\/doi.org\/10.3390\/pathogens10070865","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,9]]}}}