The World Health Organization has recently declared Thailand a leishmaniasis hotspot in Southeast Asia due to the continuous increase in new symptomatic and asymptomatic cases over the years. This emerging parasitic disease is known to be caused by two autochthonous species of Leishmania belonging to the newly described subgenus Mundinia, namely L. martiniquensis and L. orientalis. In Thailand, clinical cases due to L. martiniquensis typically present with visceral leishmaniasis, whereas L. orientalis mainly causes localized cutaneous leishmaniasis. Although Leishmania species confirmation is essential for clinical diagnosis and treatment planning, the availability of highly accurate and rapid diagnostic methods remains limited. In this study, we developed a duplex TaqMan quantitative PCR assay using newly designed species-specific primers and probes based on sequences from the nucleotide and genome databases of Leishmania spp. retrieved from GenBank. The duplex qPCR assay was optimized to specifically amplify the internal transcribed spacer 1 (ITS1) of L. martiniquensis and the heat shock protein 70 (type I) intergenic region (HSP70-I IR) of L. orientalis with high amplification efficiencies. The performance of the optimized duplex qPCR was evaluated by analyzing 46 DNA samples obtained from cultures, and clinical and insect specimens, consistent with the results of the previously validated 18S rRNA-qPCR and ITS1-PCR. The duplex qPCR could detect both species of Leishmania at a limit of detection of one copy per reaction and did not cross-amplify with other pathogen DNA samples. Standard curves of the singleplex and duplex assays showed good linearity with excellent amplification efficiency. Using conventional ITS1-PCR and plasmid sequencing as a reference standard assay, the duplex qPCR showed diagnostic sensitivity and specificity of 100% and positive and negative predictive values of 100% for both Leishmania species with a perfect level of agreement (kappa = 1.0). The novel duplex TaqMan-based qPCR has shown to be a rapid, cost-effective, and highly accurate diagnostic tool for the simultaneous detection and identification of two autochthonous Leishmania spp. in a variety of clinical and entomological samples. This will greatly facilitate early diagnosis, treatment monitoring, and surveillance, especially in leishmaniasis-endemic areas where sequencing-based diagnosis is not routinely available.

Biomedical Technology Research Group for Vulnerable Populations Mae Fah Luang University Chiang Rai Thailand

Center of Excellence in Vector Biology and Vector Borne Disease Chulalongkorn University Thailand

Department of Parasitology Faculty of Medicine Chulalongkorn University Bangkok Thailand

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

Division of Infectious Diseases Department of Internal Medicine Hatyai Hospital Songkhla Thailand

Interdisciplinary Program of Biomedical Sciences Graduate School Chulalongkorn University Bangkok Thailand

School of Medicine Mae Fah Luang University Chiang Rai Thailand

Zobrazit více v PubMed

Akhoundi M., Kuhls K., Cannet A., Votýpka J., Marty P., Delaunay P., et al. A Historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl. Trop. Dis. 2016;10 PubMed PMC

Ampol R., Somwang P., Khositharattanakool P., Promrangsee C., Pataradool T., Tepboonreung P., et al. Nanopore-based surveillance of Leishmania parasites in Culicoides Latrielle (Diptera: Ceratopogonidae) caught from the affected community and Tham Phra Cave in Chiang Rai Province, the endemic area of leishmaniasis in Northern Thailand. Insects. 2024;15:327. PubMed PMC

Anugulruengkitt S., Songtaweesin W.N., Thepnarong N., Tangthanapalakul A., Sitthisan M., Chatproedprai S., et al. Case report: Simple nodular cutaneous leishmaniasis caused by autochthonous Leishmania (Mundinia) orientalis in an 18-month-old girl: the first pediatric case in Thailand and literature review. Am. J. Trop. Med. Hyg. 2022;108:44–50. PubMed PMC

Aronson N., Herwaldt B.L., Libman M., Pearson R., Lopez-Velez R., Weina P., et al. Diagnosis and treatment of leishmaniasis: Clinical practice guidelines by the infectious diseases society of America (IDSA) and the American society of tropical medicine and hygiene (ASTMH) Clin. Infect. Dis. 2016;63:1539–1557. PubMed

Arya M., Shergill I.S., Williamson M., Gommersall L., Arya N., Patel H.R. Basic principles of real-time quantitative PCR. Expert Rev. Mol. Diagn. 2005;5:209–219. PubMed

Bossolasco S., Gaiera G., Olchini D., Gulletta M., Martello L., Bestetti A., et al. Real-time PCR assay for clinical management of human immunodeficiency virus-infected patients with visceral leishmaniasis. J. Clin. Microbiol. 2003;41:5080–5084. PubMed PMC

Burza S., Croft S.L., Boelaert M. Leishmaniasis. Lancet. 2018;392:951–970. PubMed

Bustin S.A., Benes V., Garson J.A., Hellemans J., Huggett J., Kubista M., et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009;55:611–622. PubMed

Charyyeva A., Çetinkaya Ü., Özkan B., Şahin S., Yaprak N., Şahin I., et al. Genetic diversity of Leishmania tropica: Unexpectedly complex distribution pattern. Acta Trop. 2021;218 PubMed

Chen Y.F., Liao L.F., Wu N., Gao J.M., Zhang P., Wen Y.Z., et al. Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, the Peopleʼs Republic of China. PLoS Negl. Trop. Dis. 2021;15 PubMed PMC

Cohen J. A coefficient of agreement for nominal scales. Educ. Psychol. Meas. 1960;20:37–46.

da Silva L.A., de Sousa C.dosS., da Graça G.C., Porrozzi R., Cupolillo E. Sequence analysis and PCR-RFLP profiling of the hsp70 gene as a valuable tool for identifying Leishmania species associated with human leishmaniasis in Brazil. Infect. Genet. Evol. 2010;10:77–83. PubMed

Dougall A.M., Alexander B., Holt D.C., Harris T., Sultan A.H., Bates P.A., et al. Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia. Int. J. Parasitol. 2011;41:571–579. PubMed

Espinosa O.A., Serrano M.G., Camargo E.P., Teixeira M.M.G., Shaw J.J. An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum. Parasitology. 2018;145:430–442. PubMed

Folgueira C., Cañavate C., Chicharro C., Requena J.M. Genomic organization and expression of the HSP70 locus in New and Old World Leishmania species. Parasitology. 2007;134:369–377. PubMed

Folgueira C., Requena J.M. A postgenomic view of the heat shock proteins in kinetoplastids. FEMS Microbiol. Rev. 2007;31:359–377. PubMed

Fraga J., Montalvo A.M., De Doncker S., Dujardin J.C., Van der Auwera G. Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infect. Genet. Evol. 2010;10:238–245. PubMed

Galluzzi L., Ceccarelli M., Diotallevi A., Menotta M., Magnani M. Real-time PCR applications for diagnosis of leishmaniasis. Parasites Vectors. 2018;11:273. PubMed PMC

Garcia L., Kindt A., Bermudez H., Llanos-Cuentas A., De Doncker S., Arevalo J., et al. Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J. Clin. Microbiol. 2004;42:2294–2297. PubMed PMC

Hall T.A. BioEdit: A user-friendly biological sequence alignment editor analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999;41:95–98.

Hernández C., Alvarez C., González C., Ayala M.S., León C.M., Ramírez J.D. Identification of six New World Leishmania species through the implementation of a high-resolution melting (HRM) genotyping assay. Parasites Vectors. 2014;7:501. PubMed PMC

Hitakarun A., Tan-ariya P., Siripattanapipong S., Mungthin M., Piyaraj P., Naaglor T., et al. Comparison of PCR methods for detection of Leishmania siamensis infection. Parasites Vectors. 2014;7:458. PubMed PMC

Hoyos J., Rosales-Chilama M., León C., González C., Gómez M.A. Sequencing of hsp70 for discernment of species from the Leishmania (Viannia) guyanensis complex from endemic areas in Colombia. Parasites Vectors. 2022;15:406. PubMed PMC

Jariyapan N., Daroontum T., Jaiwong K., Chanmol W., Intakan N., Sor-suwan S., et al. Leishmania (Mundinia) orientalis n. sp. (Trypanosomatidae), a parasite from Thailand responsible for localized cutaneous leishmaniasis. Parasites Vectors. 2018;11:351. PubMed PMC

Kim H.J., Bathige S.D.N.K., Jeon H.B., Kim S.H., Yu C., Kang W., et al. Development of a duplex qPCR assay for the detection of coinfection in eels by Japanese eel endothelial cell-infecting virus and Anguillid herpesvirus-1. Aquaculture. 2024;578

Kralik P., Ricchi M. A basic guide to real time PCR in microbial diagnostics: definitions, parameters, and everything. Front. Microbiol. 2017;8:108. PubMed PMC

Kwakye-Nuako G., Mosore M.T., Boakye D., Bates P.A. Description, biology, and medical significance of Leishmania (Mundinia) chancei n. sp. (Kinetoplastea: Trypanosomatidae) from Ghana and Leishmania (Mundinia) procaviensis n. sp. (Kinetoplastea: Trypanosomatidae) from Namibia. J. Parasitol. 2023;109:43–50. PubMed

Kwakye-Nuako G., Mosore M.T., Duplessis C., Bates M.D., Puplampu N., Mensah-Attipoe I., et al. First isolation of a new species of Leishmania responsible for human cutaneous leishmaniasis in Ghana and classification in the Leishmania enriettii complex. Int. J. Parasitol. 2015;45:679–684. PubMed

Landis J.R., Koch G.G. An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers. Biometrics. 1977;33:363–374. PubMed

Leal C.A., Carvalho A.F., Leite R.C., Figueiredo H.C. Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp. BMC Vet. Res. 2014;10:150. PubMed PMC

Leelayoova S., Siripattanapipong S., Manomat J., Piyaraj P., Tan-ariya P., Bualert L., et al. Leishmaniasis in Thailand: A review of causative agents and situations. Am. J. Trop. Med. Hyg. 2017;96:534–542. PubMed PMC

Lyvannak S., Sreynich K., Heng S., Thyl M., Chandna A., Chanpheaktra N., et al. Case report: The first case report of visceral leishmaniasis in Cambodia. Am. J. Trop. Med. Hyg. 2022;107:336–338. PubMed PMC

Mann S., Frasca K., Scherrer S., Henao-Martínez A.F., Newman S., Ramanan P., et al. A review of leishmaniasis: Current knowledge and future directions. Curr. Trop. Med. Rep. 2021;8:121–132. PubMed PMC

Mathison B.A., Bradley B.T. Review of the clinical presentation, pathology, diagnosis, and treatment of leishmaniasis. Lab. Med. 2023;54:363–371. PubMed

Mungthin M., Leelayoova S., Siripattanapipong S. Taxonomy revision of Leishmania spp. in Thailand. J. Clin. Microbiol. 2021;59 21. PubMed PMC

Muniz J., Medina H. [Cutaneous leishmaniasis of the Guinea pig, Leishmania enriettii n. sp.] Hospital. 1948;33:7–25. In Portuguese. PubMed

Noppakun N., Kraivichian K., Siriyasatien P. Disseminated dermal leishmaniasis caused by Leishmania siamensis in a systemic steroid therapy patient. Am. J. Trop. Med. Hyg. 2014;91:869–870. PubMed PMC

Oh I.Y., Kim K.T., Jun J.H., Shin J.H., Sung H.J. Development of real-time PCR assays for detection of Dirofilaria immitis from infected dog blood. Korean J. Clin. Lab. Sci. 2016;48:88–93.

Ohta A., Nishi K., Hirota K., Matsuo Y. Using nanopore sequencing to identify fungi from clinical samples with high phylogenetic resolution. Sci. Rep. 2023;13:9785. PubMed PMC

Pothirat T., Tantiworawit A., Chaiwarith R., Jariyapan N., Wannasan A., Siriyasatien P., et al. First isolation of Leishmania from Northern Thailand: Case report, identification as Leishmania martiniquensis and phylogenetic position within the Leishmania enriettii complex. PLoS Negl. Trop. Dis. 2014;8 PubMed PMC

Preativatanyou K., Chinwirunsirisup K., Phumee A., Khositharattanakool P., Sunantaraporn S., Depaquit J., et al. Species diversity of phlebotomine sand flies and sympatric occurrence of Leishmania (Mundinia) martiniquensis, Leishmania (Leishmania) donovani complex, and Trypanosoma spp. in the visceral leishmaniasis focus of southern Thailand. Acta Trop. 2023;244 PubMed

Quijada L., Soto M., Alonso C., Requena J.M. Analysis of post-transcriptional regulation operating on transcription products of the tandemly linked Leishmania infantum hsp70 genes. J. Biol. Chem. 1997;272:4493–4499. PubMed

Requena J.M., Chicharro C., García L., Parrado R., Puerta C.J., Cañavate C. Sequence analysis of the 3’-untranslated region of HSP70 (type I) genes in the genus Leishmania: Its usefulness as a molecular marker for species identification. Parasites Vectors. 2012;5:87. PubMed PMC

Rezaei Z., Azarang E., Shahabi S., Omidian M., Pourabbas B., Sarkari B. Leishmania ITS1 is genetically divergent in asymptomatic and symptomatic visceral leishmaniasis: Results of a study in southern Iran. J. Trop. Med. 2020;2020 PubMed PMC

Rose K., Curtis J., Baldwin T., Mathis A., Kumar B., Sakthianandeswaren A., et al. Cutaneous leishmaniasis in red kangaroos: Isolation and characterisation of the causative organisms. Int. J. Parasitol. 2004;34:655–664. PubMed

Ruang-Areerate T., Ruang-Areerate P., Manomat J., Naaglor T., Piyaraj P., Mungthin M., et al. Genetic variation and geographic distribution of Leishmania orientalis and Leishmania martiniquensis among Leishmania/HIV co-infection in Thailand. Sci. Rep. 2023;13 PubMed PMC

Ruijter J.M., Barnewall R.J., Marsh I.B., Szentirmay A.N., Quinn J.C., van Houdt R., et al. Efficiency correction is required for accurate quantitative PCR analysis and reporting. Clin. Chem. 2021;67:829–842. PubMed

Saah A.J., Hoover D.R. “Sensitivity” and “specificity” reconsidered: The meaning of these terms in analytical and diagnostic settings. Ann. Intern. Med. 1997;126:91–94. PubMed

Sarasombath P.T. Leishmaniasis: An evolving public health concern in Thailand. Siriraj. Med. J. 2018;70:363–376.

Schönian G., Kuhls K., Mauricio I.L. Molecular approaches for a better understanding of the epidemiology and population genetics of Leishmania. Parasitology. 2011;138:405–425. PubMed

Šimundić A.M. Measures of diagnostic accuracy: Basic definitions. EJIFCC. 2009;19:203–211. PubMed PMC

Songumpai N., Promrangsee C., Noopetch P., Siriyasatien P., Preativatanyou K. 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. 2022;7:379. PubMed PMC

Spanakos G., Piperaki E.T., Menounos P.G., Tegos N., Flemetakis A., Vakalis N.C. Detection and species identification of Old World Leishmania in clinical samples using a PCR-based method. Trans. R. Soc. Trop. Med. Hyg. 2008;102:46–53. PubMed

Spotin A., Rouhani S., Haghighi A., Parvizi P. Low genetic heterogeneity of Leishmania major in different geographical regions of Iran. PLoS One. 2023;18 PubMed PMC

Srivarasat S., Brownell N., Siriyasatien P., Noppakun N., Asawanonda P., Rattanakorn K., et al. Case report: Autochthonous disseminated cutaneous, mucocutaneous, and visceral leishmaniasis caused by Leishmania martiniquensis in a patient with HIV/AIDS from Northern Thailand and literature review. Am. J. Trop. Med. Hyg. 2022;107:1196–1202. PubMed PMC

Talmi-Frank D., Nasereddin A., Schnur L.F., Schönian G., Töz S.O., Jaffe C.L., et al. Detection and identification of Old World Leishmania by high resolution melt analysis. PLoS Negl. Trop. Dis. 2010;4:e581. PubMed PMC

Tatti K.M., Sparks K.N., Boney K.O., Tondella M.L. Novel multitarget real-time PCR assay for rapid detection of Bordetella species in clinical specimens. J. Clin. Microbiol. 2011;49:4059–4066. PubMed PMC

Tien P. The 8th ASEAN Conference of Tropical Medicine and Parasitology (ACTMP) 2018. A case report of leishmaniasis and HIV coinfection in Hue Central Hospital.

Van der Auwera G., Dujardin J.C. Species typing in dermal leishmaniasis. Clin. Microbiol. Rev. 2015;28:265–294. PubMed PMC

van der Meide W., Guerra J., Schoone G., Farenhorst M., Coelho L., Faber W., et al. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites. J. Clin. Microbiol. 2008;46:73–78. PubMed PMC

Vu S.N., Tran H.S., Tran V.P., Tran C.T., Tran N.D., Dang D.A., et al. Taxonomical insights and ecology of sandfly (Diptera, Psychodidae) species in six provinces of Northern Vietnam. Parasite. 2021;28:85. PubMed PMC

WHO . World Health Organization; Geneva, Switzerland: 2023. Leishmaniasis.https://www.who.int/news-room/fact-sheets/detail/leishmaniasis