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Several studies have discovered monoxenous trypanosomatids (Leptomonas and Crithidia) in patients with VL. In different situations of leishmaniasis, investigations have examined cases of co-infection between Leishmania spp. and Crithidia spp. These coinfections have been observed in a wide range of vertebrate hosts, indicating that they are not rare. Diagnostic techniques require improvements and more robust tools to accurately detect the causative agent of VL. This study aimed to develop a real-time quantitative dye-based PCR (qPCR) assay capable of distinguishing Leishmania infantum from Crithidia-related species and to estimate the parasite load in samples of VL from humans and animals. The primer LinJ31_2420 targets an exclusive phosphatase of L. infantum; the primer Catalase_LVH60-12060_1F targets the catalase gene of Crithidia. Therefore, primers were designed to detect L. infantum and Crithidia sp. LVH60A (a novel trypanosomatid isolated from VL patients in Brazil), in samples related to VL. These primers were considered species-specific, based on sequence analysis using genome data retrieved from the TriTryp database and the genome assembling of Crithidia sp. LVH60A strain, in addition to experimental and clinical data presented herein. This novel qPCR assay was highly accurate in identifying and quantifying L. infantum and Crithidia sp. LVH60A in samples obtained experimentally (in vitro and in vivo) or collected from hosts (humans, dogs, cats, and vectors). Importantly, the screening of 62 cultured isolates from VL patients using these primers surprisingly revealed that 51 parasite cultures were PCR+ for Crithidia sp. In addition, qPCR assays identified the co-infection of L. infantum with Crithidia sp. LVH60A in two new VL cases in Brazil, confirming the suspicion of co-infection in a previously reported case of fatal VL. We believe that the species-specific genes targeted in this study can be helpful for the molecular diagnosis of VL, as well as for elucidating suspected co-infections with monoxenous-like trypanosomatids, which is a neglected fact of a neglected disease.<\/jats:p>","DOI":"10.3390\/tropicalmed8080405","type":"journal-article","created":{"date-parts":[[2023,8,8]],"date-time":"2023-08-08T12:38:59Z","timestamp":1691498339000},"page":"405","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1831-1638","authenticated-orcid":false,"given":"Nayore Tamie","family":"Takamiya","sequence":"first","affiliation":[{"name":"Department of Genetics and Evolution, Center for Biological Sciences and Health, Federal University of S\u00e3o Carlos (UFSCar), S\u00e3o Carlos 13565-905, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luana Aparecida","family":"Rogerio","sequence":"additional","affiliation":[{"name":"Department of Genetics and Evolution, Center for Biological Sciences and Health, Federal University of S\u00e3o Carlos (UFSCar), S\u00e3o Carlos 13565-905, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Caroline","family":"Torres","sequence":"additional","affiliation":[{"name":"Department of Genetics and Evolution, Center for Biological Sciences and Health, Federal University of S\u00e3o Carlos (UFSCar), S\u00e3o Carlos 13565-905, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jo\u00e3o Augusto Franco","family":"Leonel","sequence":"additional","affiliation":[{"name":"Post-Graduate Program in Experimental Epidemiology Applied to Zoonoses at the Faculty of Veterinary Medicine and Animal Science, University of S\u00e3o Paulo, S\u00e3o Paulo 05508-270, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7762-5453","authenticated-orcid":false,"given":"Geovanna","family":"Vioti","sequence":"additional","affiliation":[{"name":"Post-Graduate Program in Experimental Epidemiology Applied to Zoonoses at the Faculty of Veterinary Medicine and Animal Science, University of S\u00e3o Paulo, S\u00e3o Paulo 05508-270, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3916-3600","authenticated-orcid":false,"given":"Tricia Maria Ferreira","family":"de Sousa Oliveira","sequence":"additional","affiliation":[{"name":"Post-Graduate Program in Experimental Epidemiology Applied to Zoonoses at the Faculty of Veterinary Medicine and Animal Science, University of S\u00e3o Paulo, S\u00e3o Paulo 05508-270, SP, Brazil"},{"name":"Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of S\u00e3o Paulo, Pirassununga 13635-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karoline Camila","family":"Valeriano","sequence":"additional","affiliation":[{"name":"Ribeir\u00e3o Preto Medical School, University of S\u00e3o Paulo, FMRP-USP, Ribeir\u00e3o Preto 14049-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3407-3123","authenticated-orcid":false,"given":"Gabriane Nascimento","family":"Porcino","sequence":"additional","affiliation":[{"name":"Ribeir\u00e3o Preto Medical School, University of S\u00e3o Paulo, FMRP-USP, Ribeir\u00e3o Preto 14049-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Isabel Kinney Ferreira","family":"de Miranda Santos","sequence":"additional","affiliation":[{"name":"Ribeir\u00e3o Preto Medical School, University of S\u00e3o Paulo, FMRP-USP, Ribeir\u00e3o Preto 14049-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7302-2006","authenticated-orcid":false,"given":"Carlos H. N.","family":"Costa","sequence":"additional","affiliation":[{"name":"Natan Portela Institute of Tropical Diseases, Teresina 64002-510, PI, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dorcas Lamounier","family":"Costa","sequence":"additional","affiliation":[{"name":"Natan Portela Institute of Tropical Diseases, Teresina 64002-510, PI, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tauana Sousa","family":"Ferreira","sequence":"additional","affiliation":[{"name":"Laboratory of Medical Parasitology and Vector Biology, Faculty of Medicine, University of Bras\u00edlia, Bras\u00edlia 70910-900, DF, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8252-8690","authenticated-orcid":false,"given":"Rodrigo","family":"Gurgel-Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"Laboratory of Medical Parasitology and Vector Biology, Faculty of Medicine, University of Bras\u00edlia, Bras\u00edlia 70910-900, DF, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3410-3927","authenticated-orcid":false,"given":"Jo\u00e3o Santana","family":"da Silva","sequence":"additional","affiliation":[{"name":"Fiocruz-Bi-Institutional Translational Medicine Project, Oswaldo Cruz Foundation, Ribeir\u00e3o Preto 14040-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Felipe Roberti","family":"Teixeira","sequence":"additional","affiliation":[{"name":"Department of Genetics and Evolution, Center for Biological Sciences and Health, Federal University of S\u00e3o Carlos (UFSCar), S\u00e3o Carlos 13565-905, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roque Pacheco","family":"De Almeida","sequence":"additional","affiliation":[{"name":"Department of Medicine, Center for Biology and Health Sciences, Federal University of Sergipe (UFS), Aracaju 49060-108, SE, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jos\u00e9 M. 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Microorganisms, 9.","DOI":"10.3390\/microorganisms9051101"},{"key":"ref_2","unstructured":"(2020, March 23). WHO|Leishmaniasis. Available online: http:\/\/www.who.int\/gho\/neglected_diseases\/leishmaniasis\/en\/."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Alvar, J., V\u00e9lez, I.D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J., den Boer, M., and The WHO Leishmaniasis Control Team (2012). Leishmaniasis Worldwide and Global Estimates of Its Incidence. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0035671"},{"key":"ref_4","unstructured":"Centers for Disease Control and Prevention (2020, March 26). Parasites\u2014Leishmaniasis, Available online: https:\/\/www.cdc.gov\/parasites\/leishmaniasis\/index.html."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.actatropica.2011.05.012","article-title":"Importance of Worldwide Asymptomatic Carriers of Leishmania infantum (L. chagasi) in Human","volume":"119","author":"Michel","year":"2011","journal-title":"Acta Trop."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Gardinassi, L.G., Garcia, G.R., Costa, C.H.N., Silva, V.C., and Santos, I.K.F. (2016). de M. Blood Transcriptional Profiling Reveals Immunological Signatures of Distinct States of Infection of Humans with Leishmania infantum. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0005123"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"784463","DOI":"10.3389\/fimmu.2022.784463","article-title":"Insight Into the Long Noncoding RNA and MRNA Coexpression Profile in the Human Blood Transcriptome Upon Leishmania infantum Infection","volume":"13","author":"Maruyama","year":"2022","journal-title":"Front. Immunol."},{"key":"ref_8","unstructured":"(2022, January 19). Visceral Leishmaniasis\u2014PAHO\/WHO|Pan American Health Organization. Available online: https:\/\/www.paho.org\/en\/topics\/leishmaniasis\/visceral-leishmaniasis."},{"key":"ref_9","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_10","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_11","doi-asserted-by":"crossref","unstructured":"Sudarshan, M., Singh, T., Singh, A.K., Chourasia, A., Singh, B., Wilson, M.E., Chakravarty, J., and Sundar, S. (2014). Quantitative PCR in Epidemiology for Early Detection of Visceral Leishmaniasis Cases in India. PLoS Negl. Trop. Dis., 8.","DOI":"10.1371\/journal.pntd.0003366"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"18069","DOI":"10.1038\/s41598-022-21497-6","article-title":"Gauging the Skin Resident Leishmania Parasites through a Loop Mediated Isothermal Amplification (LAMP) Assay in Post-Kala-Azar Dermal Leishmaniasis","volume":"12","author":"Ghosh","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e00386-18","DOI":"10.1128\/JCM.00386-18","article-title":"Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis","volume":"56","author":"Adams","year":"2018","journal-title":"J. Clin. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Nzelu, C.O., Kato, H., and Peters, N.C. (2019). Loop-Mediated Isothermal Amplification (LAMP): An Advanced Molecular Point-of-Care Technique for the Detection of Leishmania Infection. PLoS Negl. Trop. Dis., 13.","DOI":"10.1371\/journal.pntd.0007698"},{"key":"ref_15","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":"Tavares","year":"2015","journal-title":"Cell Biosci."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Porcino, G.N., Carvalho, K.S.S., Braz, D.C., Silva, V.C., Costa, C.H.N., and de Miranda Santos, I.K.F. (2019). Evaluation of Methods for Detection of Asymptomatic Individuals Infected with Leishmania infantum in the State of Piau\u00ed, Brazil. PLoS Negl. Trop. Dis., 13.","DOI":"10.1371\/journal.pntd.0007493"},{"key":"ref_17","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_18","doi-asserted-by":"crossref","first-page":"279","DOI":"10.4103\/2221-1691.233010","article-title":"Co-Detection and Isolation of Leishmania and Crithidia among Naturally Infected Tatera Indica (Rodentia: Muridae) in Fars Province, Southern Iran","volume":"8","author":"Kalantari","year":"2018","journal-title":"Asian Pac. J. Trop. Biomed."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"804707","DOI":"10.3389\/fcimb.2022.804707","article-title":"A Stroll Through the History of Monoxenous Trypanosomatids Infection in Vertebrate Hosts","volume":"12","author":"Boucinha","year":"2022","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1111\/tmi.13042","article-title":"Isolation of Crithidia Spp. from Lesions of Immunocompetent Patients with Suspected Cutaneous Leishmaniasis in Iran","volume":"24","author":"Ghobakhloo","year":"2019","journal-title":"Trop. Med. Int. Health"},{"key":"ref_21","first-page":"2285","article-title":"Super Infection of Cutaneous Leishmaniasis Caused by Leishmania Major and L. Tropica to Crithidia fasciculata in Shiraz, Iran","volume":"48","author":"Mirzapour","year":"2019","journal-title":"Iran. J. Public Health"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Doudi, M., Karami, M., Eslami, G., and Setorki, M. (2015). A Study of Genetic Polymorphism of Crithidia in Isfahan, Iran. Zahedan J. Res. Med. Sci., 17.","DOI":"10.17795\/zjrms971"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"52","DOI":"10.4103\/0972-9062.309518","article-title":"DNA-Based Detection of Leishmania and Crithidia Species Isolated from Humans in Cutaneous and Post-Kala-Azar Dermal Leishmaniasis from Shiraz and Kharameh, Southern Iran","volume":"57","author":"Kalantari","year":"2020","journal-title":"J. Vector Borne Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.ijid.2023.05.012","article-title":"Co-Infection of Leishmania infantum and a Crithidia-Related Species in a Case of Refractory Relapsed Visceral Leishmaniasis with Non-Ulcerated Cutaneous Manifestation in Brazil","volume":"133","author":"Rogerio","year":"2023","journal-title":"Int. J. Infect. Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2329","DOI":"10.1002\/vms3.929","article-title":"Domestic Dogs Carriers of Leishmania infantum, Leishmania tropica and Crithidia fasciculata as Potential Reservoirs for Human Visceral Leishmaniasis in Northeastern Iran","volume":"8","author":"Fakhar","year":"2022","journal-title":"Vet. Med. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/j.ijppaw.2021.04.003","article-title":"Crithidia Mellificae Infection in Different Mammalian Species in Brazil","volume":"15","author":"Dario","year":"2021","journal-title":"Int. J. Parasitol. Parasites Wildl."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Songumpai, N., Promrangsee, C., Noopetch, P., Siriyasatien, P., and Preativatanyou, K. (2022). 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. Trop. Med. Infect. Dis., 7.","DOI":"10.3390\/tropicalmed7110379"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2088","DOI":"10.3201\/eid2511.181548","article-title":"Non-Leishmania parasite in fatal visceral leishmaniasis\u2013like disease, Brazil","volume":"25","author":"Maruyama","year":"2019","journal-title":"Emerg. Infect. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1409","DOI":"10.1017\/S0031182016000949","article-title":"Leishmania Mexicana: Promastigotes and Amastigotes Secrete Protein Phosphatases and This Correlates with the Production of Inflammatory Cytokines in Macrophages","volume":"143","author":"Becker","year":"2016","journal-title":"Parasitology"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1016\/j.exppara.2006.04.011","article-title":"Leishmania Amazonensis: Characterization of an Ecto-Phosphatase Activity","volume":"114","year":"2006","journal-title":"Exp. Parasitol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"964","DOI":"10.1111\/febs.15083","article-title":"Catalase Compromises the Development of the Insect and Mammalian Stages of Trypanosoma Brucei","volume":"287","author":"Kraeva","year":"2020","journal-title":"FEBS J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.meegid.2016.06.054","article-title":"Catalase in Leishmaniinae: With Me or against Me?","volume":"50","author":"Kraeva","year":"2017","journal-title":"Infect. Genet. Evol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Thakur, L., Kushwaha, H.R., Negi, A., Jain, A., and Jain, M. (2020). Leptomonas Seymouri Co-Infection in Cutaneous Leishmaniasis Cases Caused by Leishmania Donovani From Himachal Pradesh, India. Front. Cell. Infect. Microbiol., 10.","DOI":"10.3389\/fcimb.2020.00345"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2774","DOI":"10.1128\/JCM.00966-12","article-title":"Coinfection of Leptomonas Seymouri and Leishmania Donovani in Indian Leishmaniasis","volume":"50","author":"Ghosh","year":"2012","journal-title":"J. Clin. Microbiol."},{"key":"ref_35","first-page":"1145","article-title":"Detection of Leptomonas Sp. Parasites in Clinical Isolates of Kala-Azar Patients from India","volume":"10","author":"Srivastava","year":"2010","journal-title":"Infect. Genet. Evol. J. Mol. Epidemiol. Evol. Genet. Infect. Dis."},{"key":"ref_36","first-page":"198","article-title":"Molecular Identification of Species Caused Cutaneous Leishmaniasis in Southern Zone of Iran","volume":"13","author":"Barazesh","year":"2019","journal-title":"J. Arthropod-Borne Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2129","DOI":"10.1101\/gr.122945.111","article-title":"Chromosome and Gene Copy Number Variation Allow Major Structural Change between Species and Strains of Leishmania","volume":"21","author":"Rogers","year":"2011","journal-title":"Genome Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"D457","DOI":"10.1093\/nar\/gkp851","article-title":"TriTrypDB: A Functional Genomic Resource for the Trypanosomatidae","volume":"38","author":"Aslett","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., and Madden, T.L. (2012). Primer-BLAST: A Tool to Design Target-Specific Primers for Polymerase Chain Reaction. BMC Bioinformatics, 13.","DOI":"10.1186\/1471-2105-13-134"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Vioti, G., da Silva, M.D., Galvis-Ovallos, F., Alves, M.L., da Silva, D.T., Leonel, J.A.F., Pereira, N.W.B., Benassi, J.C., Spada, J.C.P., and Maia, C. (2021). Xenodiagnosis in Four Domestic Cats Naturally Infected by Leishmania infantum. Transbound. Emerg. Dis.","DOI":"10.1111\/tbed.14216"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/infdis\/1.1.1","article-title":"On the Cultivation of Trypanosoma Brucei","volume":"1","author":"Novy","year":"1904","journal-title":"J. Infect. Dis."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1590\/S0037-86821994000300004","article-title":"Hemoculture: Sensitive technique in the detection of Trypanosoma cruzi in chagasic patients in the chronic phase of Chagas disease","volume":"27","author":"Luz","year":"1994","journal-title":"Rev. Soc. Bras. Med. Trop."},{"key":"ref_43","first-page":"O374","article-title":"Biomarkers for Intracellular Pathogens: Establishing Tools as Vaccine and Therapeutic Endpoints for Visceral Leishmaniasis","volume":"20","author":"Vallur","year":"2014","journal-title":"Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis."},{"key":"ref_44","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"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Ghosh, P., Hasnain, M.G., Hossain, F., Khan, M.A.A., Chowdhury, R., Faisal, K., Mural, M.A., Baker, J., Nath, R., and Ghosh, D. (2018). Evaluation of Real-Time PCR for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis in Endemic Foci of Bangladesh. Open Forum Infect. Dis., 5.","DOI":"10.1093\/ofid\/ofy234"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Chowdhury, R., Ghosh, P., Khan, M.A.A., Hossain, F., Faisal, K., Nath, R., Baker, J., Wahed, A.A.E., Maruf, S., and Nath, P. (2020). Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis. Trop. Med. Infect. Dis., 5.","DOI":"10.3390\/tropicalmed5020095"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/S0166-0934(00)00244-5","article-title":"A Quantification of Human Cells Using an ERV-3 Real Time PCR Assay","volume":"91","author":"Miley","year":"2001","journal-title":"J. Virol. Methods"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1080\/13550280601039634","article-title":"SYBR Green\u2013Based Quantitation of Human T-Lymphotropic Virus Type 1 Proviral Load in Peruvian Patients with Neurological Disease and Asymptomatic Carriers: Influence of Clinical Status, Sex, and Familial Relatedness","volume":"12","author":"Adaui","year":"2006","journal-title":"J. Neurovirol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1016\/j.vetmic.2009.07.006","article-title":"Development and Use of Real-Time PCR to Detect and Quantify Mycoplasma Haemocanis and \u201cCandidatus Mycoplasma Haematoparvum\u201d in Dogs","volume":"140","author":"Barker","year":"2010","journal-title":"Vet. Microbiol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1016\/j.vetpar.2013.07.006","article-title":"Molecular Diagnosis of Canine Visceral Leishmaniasis: A Comparative Study of Three Methods Using Skin and Spleen from Dogs with Natural Leishmania infantum Infection","volume":"197","author":"Reis","year":"2013","journal-title":"Vet. Parasitol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1046\/j.1365-2583.2002.00315.x","article-title":"Molecular Evolution of the Cacophony IVS6 Region in Sandflies","volume":"11","author":"Lins","year":"2002","journal-title":"Insect Mol. Biol."},{"key":"ref_52","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_53","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1146\/annurev.mi.49.100195.001001","article-title":"The Structure and Replication of Kinetoplast DNA","volume":"49","author":"Shapiro","year":"1995","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_54","unstructured":"Takahashi, T.Y. (2021, April 30). Triagem de Isolados Cl\u00ednicos de Leishmania sp. para Sequenciamento Gen\u00f4mico. Available online: https:\/\/repositorio.ufscar.br\/handle\/ufscar\/8195\/browse?type=author&value=Takahashi%2C+Talita+Yuri."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"315","DOI":"10.2144\/btn-2018-0089","article-title":"Spike-in Genomic DNA for Validating Performance of Metagenomics Workflows","volume":"65","author":"Venkataraman","year":"2018","journal-title":"BioTechniques"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1826","DOI":"10.1128\/JCM.00208-13","article-title":"Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters","volume":"51","author":"Jara","year":"2013","journal-title":"J. Clin. Microbiol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/S0020-7519(98)00167-2","article-title":"A Nested PCR for the SsrRNA Gene Detects Trypanosoma Binneyi in the Platypus and Trypanosoma Sp. in Wombats and Kangaroos in Australia1","volume":"29","author":"Noyes","year":"1999","journal-title":"Int. J. Parasitol."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"2725","DOI":"10.1093\/molbev\/mst197","article-title":"MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0","volume":"30","author":"Tamura","year":"2013","journal-title":"Mol. Biol. Evol."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1002\/ijc.2910260208","article-title":"Establishment and Characterization of a Human Acute Monocytic Leukemia Cell Line (THP-1)","volume":"26","author":"Tsuchiya","year":"1980","journal-title":"Int. J. Cancer"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/j.vetpar.2018.07.004","article-title":"High Molecular Prevalence of Leishmania in Phlebotomine Sand Flies Fed on Chicken Blood in Brazil","volume":"259","author":"Neiva","year":"2018","journal-title":"Vet. Parasitol."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.mcp.2016.08.006","article-title":"Leishmania-FAST15: A Rapid, Sensitive and Low-Cost Real-Time PCR Assay for the Detection of Leishmania infantum and Leishmania Braziliensis Kinetoplast DNA in Canine Blood Samples","volume":"31","author":"Otranto","year":"2017","journal-title":"Mol. Cell. Probes"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1177\/104063871102300102","article-title":"Selection of a Cutoff Value for Real-Time Polymerase Chain Reaction Results to Fit a Diagnostic Purpose: Analytical and Epidemiologic Approaches","volume":"23","author":"Caraguel","year":"2011","journal-title":"J. Vet. Diagn. Investig. Off. Publ. Am. Assoc. Vet. Lab. Diagn. Inc"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"388","DOI":"10.3201\/eid2602.191428","article-title":"Comment letter: Non-Leishmania Parasite in Fatal Visceral Leishmaniasis\u2013like Disease, Brazi","volume":"26","author":"Domagalska","year":"2020","journal-title":"Emerg. Infect. Dis."},{"key":"ref_64","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_65","doi-asserted-by":"crossref","first-page":"5351098","DOI":"10.1155\/2020\/5351098","article-title":"Leishmania ITS1 Is Genetically Divergent in Asymptomatic and Symptomatic Visceral Leishmaniasis: Results of a Study in Southern Iran","volume":"2020","author":"Rezaei","year":"2020","journal-title":"J. Trop. Med."},{"key":"ref_66","doi-asserted-by":"crossref","unstructured":"Yurchenko, V., Butenko, A., and Kostygov, A.Y. (2021). Genomics of Trypanosomatidae: Where We Stand and What Needs to Be Done?. Pathogens, 10.","DOI":"10.3390\/pathogens10091124"},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Kraeva, N., Butenko, A., Hlav\u00e1\u010dov\u00e1, J., Kostygov, A., My\u0161kova, J., Grybchuk, D., Le\u0161tinov\u00e1, T., Vot\u00fdpka, J., Volf, P., and Opperdoes, F. (2015). Leptomonas Seymouri: Adaptations to the Dixenous Life Cycle Analyzed by Genome Sequencing, Transcriptome Profiling and Co-Infection with Leishmania Donovani. PLoS Pathog., 11.","DOI":"10.1371\/journal.ppat.1005127"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1017\/S0031182020001936","article-title":"Identification of Leishmania infantum in Blood Donors from Endemic Regions for Visceral Leishmaniasis","volume":"148","author":"Pereira","year":"2021","journal-title":"Parasitology"},{"key":"ref_69","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_70","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_71","doi-asserted-by":"crossref","unstructured":"Zampieri, R.A., Laranjeira-Silva, M.F., Muxel, S.M., Stocco de Lima, A.C., Shaw, J.J., and Floeter-Winter, L.M. (2016). High Resolution Melting Analysis Targeting Hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004485"},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.mcp.2013.01.003","article-title":"Quantitative Real Time PCR Assays for the Detection of Leishmania (Viannia) Braziliensis in Animals and Humans","volume":"27","author":"Otranto","year":"2013","journal-title":"Mol. Cell. Probes"},{"key":"ref_73","doi-asserted-by":"crossref","unstructured":"Filgueira, C.P.B., Moreira, O.C., Cantanh\u00eade, L.M., de Farias, H.M.T., Porrozzi, R., Britto, C., Boit\u00e9, M.C., and Cupolillo, E. (2020). Comparison and Clinical Validation of QPCR Assays Targeting Leishmania 18S RDNA and HSP70 Genes in Patients with American Tegumentary Leishmaniasis. PLoS Negl. Trop. Dis., 14.","DOI":"10.1371\/journal.pntd.0008750"},{"key":"ref_74","doi-asserted-by":"crossref","unstructured":"Reina, A.M., Mewa, J.C., Calzada, J.E., and Salda\u00f1a, A. (2022). Characterization of Leishmania Spp. Causing Cutaneous Lesions with a Negative Parasitological Diagnosis in Panama. Trop. Med. Infect. Dis., 7.","DOI":"10.3390\/tropicalmed7100282"},{"key":"ref_75","doi-asserted-by":"crossref","unstructured":"Singh, N., Chikara, S., and Sundar, S. (2013). SOLiDTM Sequencing of Genomes of Clinical Isolates of Leishmania Donovani from India Confirm Leptomonas Co-Infection and Raise Some Key Questions. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0055738"},{"key":"ref_76","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 Agents1","volume":"47","author":"Dedet","year":"2000","journal-title":"J. Eukaryot. Microbiol."}],"container-title":["Tropical Medicine and Infectious Disease"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2414-6366\/8\/8\/405\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:28:10Z","timestamp":1760128090000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2414-6366\/8\/8\/405"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,8]]},"references-count":76,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2023,8]]}},"alternative-id":["tropicalmed8080405"],"URL":"https:\/\/doi.org\/10.3390\/tropicalmed8080405","relation":{},"ISSN":["2414-6366"],"issn-type":[{"type":"electronic","value":"2414-6366"}],"subject":[],"published":{"date-parts":[[2023,8,8]]}}}