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Molecular Diagnosis & Therapy

, Volume 22, Issue 4, pp 443–457 | Cite as

Molecular Diagnosis of Visceral Leishmaniasis

  • Shyam Sundar
  • Om Prakash Singh
Review Article

Abstract

Visceral leishmaniasis (VL), a deadly parasitic disease, is a major public health concern globally. Countries affected by VL have signed the London Declaration on Neglected Tropical Diseases and committed to eliminate VL as a public health problem by 2020. To achieve and sustain VL elimination, it will become progressively important not to miss any remaining cases in the community who can maintain transmission. This requires accurate identification of symptomatic and asymptomatic carriers using highly sensitive diagnostic tools at the primary health service setting. The rK39 rapid diagnostic test (RDT) is the most widely used tool and with its good sensitivity and specificity is the first choice for decentralized diagnosis of VL in endemic areas. However, this test cannot discriminate between current, subclinical, or past infections and is useless for diagnosis of relapses and as a prognostic (cure) test. Importantly, as the goal of elimination of VL as a public health problem is approaching, the number of people susceptible to infection will increase. Therefore, correct diagnosis using a highly sensitive diagnostic test is crucial for applying appropriate treatment and management of cases. Recent advances in molecular techniques have improved Leishmania detection and quantification, and therefore this technology has  become increasingly relevant due to its possible application in a variety of clinical sample types. Most importantly, given current problems in identifying asymptomatic individuals because of poor correlation between the main methods of detection, molecular tests are valuable for VL elimination programs, especially to monitor changes in burden of infection in specific communities. This review provides a comprehensive overview of the available VL diagnostics and discusses the usefulness of molecular methods in the diagnosis, quantification, and species differentiation as well as their clinical applications.

Notes

Compliance with Ethical Standards

Conflict of interest

SS and OPS declare no conflicts of interest.

Funding

This work was supported by the Bill & Melinda Gates Foundation (BMGF), USA (Grant no. OPP 1117011), Extramural Program of the National Institute of Allergy and Infectious Disease (NIAID), and National Institute of Health (TMRC [Tropical Medicine Research Centers] Grant no. U19AI074321). The funders had no role in the design, decision to publish, or preparation of this report.

References

  1. 1.
    Research priorities for Chagas disease, human African trypanosomiasis and leishmaniasis. World Health Organ Tech Rep Ser. 2012(975):v–xii, 1–100. http://apps.who.int/iris/bitstream/10665/77472/1/WHO_TRS_975_eng.pdf. Accessed 30 May 2018.
  2. 2.
    Tiwary P, Singh S, Kushwaha AK, Rowton E, Sacks D, Singh OP, et al. Establishing, expanding, and certifying a closed colony of Phlebotomus argentipes (Diptera: Psychodidae) for xenodiagnostic studies at the Kala Azar Medical Research Center, Muzaffarpur, Bihar, India. J Med Entomol. 2017;54(5):1129–39.PubMedGoogle Scholar
  3. 3.
    Guerin PJ, Olliaro P, Sundar S, Boelaert M, Croft SL, Desjeux P, et al. Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. Lancet Infect Dis. 2002;2(8):494–501.PubMedGoogle Scholar
  4. 4.
    Singh OP, Hasker E, Boelaert M, Sundar S. Elimination of visceral leishmaniasis on the Indian subcontinent. Lancet Infect Dis. 2016;16(12):e304–e309.Google Scholar
  5. 5.
    Le Rutte EA, Coffeng LE, Bontje DM, Hasker EC, Postigo JA, Argaw D, et al. Feasibility of eliminating visceral leishmaniasis from the Indian subcontinent: explorations with a set of deterministic age-structured transmission models. Parasit Vect. 2016;9:24.  https://doi.org/10.1186/s13071-016-1292-0.
  6. 6.
    Hollingsworth TD, Adams ER, Anderson RM, Atkins K, Bartsch S, Basáñez M-G, et al. Quantitative analyses and modelling to support achievement of the 2020 goals for nine neglected tropical diseases. Parasite Vect. 2015;8(1):630. http://www.parasitesandvectors.com/content/8/1/630. Accessed 30 May 2018.
  7. 7.
    Naghavi M, Wang H, Lozano R, Davis A, Liang X, Zhou M, et al. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;385(9963):117–71.  https://doi.org/10.1016/S0140-6736(14)61682-2.Google Scholar
  8. 8.
    Hirve S, Kroeger A, Matlashewski G, Mondal D, Banjara MR, Das P, et al. Towards elimination of visceral leishmaniasis in the Indian subcontinent: translating research to practice to public health. PLoS Negl Trop Dis. 2017;11(10):e0005889.Google Scholar
  9. 9.
    WHO/Regional Office for Europe. Manual on case management and surveillance of the leishmaniases in the WHO European Region. 2017. http://www.who.int/leishmaniasis/resources/978-92-89052-51-1/en/. Accessed 30 May 2018.
  10. 10.
    Reis LLD, Balieiro A, Fonseca FR, Goncalves MJF. Changes in the epidemiology of visceral leishmaniasis in Brazil from 2001 to 2014. Rev Soc Bras Med Trop. 2017;50(5):638–645.Google Scholar
  11. 11.
    Adam GK, Ali KM, Abdella YH, Omar SM, Ahmed MA, Abdalla TM, et al. Trend in cumulative cases and mortality rate among visceral leishmaniasis patients in Eastern Sudan: a 14-year registry, 2002–2015. Int J Infect Dis. 2016;51:81–4.PubMedGoogle Scholar
  12. 12.
    Stanley A, Engwerda C. Balancing immunity and pathology in visceral leishmaniasis. Immunol Cell Biol. 2007;85(2):138–147.Google Scholar
  13. 13.
    Pearson R, Sousa A. Clinical spectrum of leishmaniasis. Clin Infect Dis. 1996;22(1):1–11.PubMedGoogle Scholar
  14. 14.
    Dedet J, Pratlong F. Leishmaniasis. In: Cook GC, Zumla AI, editors. Manson’s tropical diseases. 22nd ed. London: Saunders; 2008. p. 1341–1365.  https://doi.org/10.1016/j.trstmh.2009.09.003.
  15. 15.
    Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22-26 March 2010. World Health Organisation Technical Report Series. 2011. http://apps.who.int/iris/handle/10665/44412. Accessed 30 May 2018.
  16. 16.
    Boelaert M, Meheus F, Sanchez A, Singh SP, Vanlerberghe V, Picado A, et al. The poorest of the poor: a poverty appraisal of households affected by visceral leishmaniasis in Bihar, India. Trop Med Int Health. 2009;14(6):639–44.PubMedGoogle Scholar
  17. 17.
    Boelaert M, Le Ray D, Der Van, Stuyft P. How better drugs could change kala-azar control. Lessons from a cost-effectiveness analysis. Trop Med Int Health. 2002;7(11):955–9.PubMedGoogle Scholar
  18. 18.
    Singh OP, Singh B, Chakravarty J, Sundar S. Current challenges in treatment options for visceral leishmaniasis in India: a public health perspective. Infect Dis Poverty. 2016;5:19.  https://doi.org/10.1186/s40249-016-0112-2.
  19. 19.
    London Declaration on Neglected Tropical Diseases 2012. http://unitingtocombatntds.org/london-declaration-neglected-tropical-diseases/. Accessed 30 May 2018.
  20. 20.
    Sundar S, Mondal D, Rijal S, Bhattacharya S, Ghalib H, Kroeger A, et al. Implementation research to support the initiative on the elimination of kala azar from Bangladesh, India and Nepal—the challenges for diagnosis and treatment. Trop Med Int Health. 2008;13(1):2–5.PubMedGoogle Scholar
  21. 21.
    Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol. 2002;9(5):951–8.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Burki T. East African countries struggle with visceral leishmaniasis. Lancet. 2009;374(9687):371–2.PubMedGoogle Scholar
  23. 23.
    Singh OP, Sundar S. Developments in diagnosis of visceral leishmaniasis in the elimination era. J Parasitol Res. 2015;239469.  https://doi.org/10.1155/2015/239469.
  24. 24.
    Chappuis F, Rijal S, Soto A, Menten J, Boelaert M. A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis. BMJ. 2006;333(7571):723.PubMedPubMedCentralGoogle Scholar
  25. 25.
    Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, et al. Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol. 2007;5(11):873–82.PubMedGoogle Scholar
  26. 26.
    Maurya R, Mehrotra S, Prajapati VK, Nylen S, Sacks D, Sundar S. Evaluation of blood agar microtiter plates for culturing Leishmania parasites to titrate parasite burden in spleen and peripheral blood of patients with visceral leishmaniasis. J Clin Microbiol. 2010;48(5):1932–4.PubMedPubMedCentralGoogle Scholar
  27. 27.
    Buxbaum L, Scott P. Interleukin 10- and Fcγ receptor-deficient mice resolve Leishmania mexicana Lesions. Infect Immun. 2005;73(4):2101–8.PubMedPubMedCentralGoogle Scholar
  28. 28.
    Gidwani K, Picado A, Ostyn B, Singh SP, Kumar R, Khanal B, et al. Persistence of Leishmania donovani antibodies in past visceral leishmaniasis cases in India. Clin Vaccine Immunol. 2011;18(2):346–8.PubMedGoogle Scholar
  29. 29.
    Jacquet D, Boelaert M, Seaman J, Rijal S, Sundar S, Menten J, et al. Comparative evaluation of freeze-dried and liquid antigens in the direct agglutination test for serodiagnosis of visceral leishmaniasis (ITMA-DAT/VL). Trop Med Int Health. 2006;11(12):1777–84.PubMedGoogle Scholar
  30. 30.
    Sundar S, Reed SG, Singh VP, Kumar PC, Murray HW. Rapid accurate field diagnosis of Indian visceral leishmaniasis. Lancet. 1998;351(9102):563–5.PubMedGoogle Scholar
  31. 31.
    Cunningham J, Hasker E, Das P, El Safi S, Goto H, Mondal D, et al. A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis. Clin Infect Dis. 2012;55(10):1312–9.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Boelaert M, El-Safi S, Hailu A, Mukhtar M, Rijal S, Sundar S, et al. Diagnostic tests for kala-azar: a multi-centre study of the freeze-dried DAT, rK39 strip test and KAtex in East Africa and the Indian subcontinent. Trans R Soc Trop Med Hyg. 2008;102(1):32–40.PubMedGoogle Scholar
  33. 33.
    Srividya G, Kulshrestha A, Singh R, Salotra P. Diagnosis of visceral leishmaniasis: developments over the last decade. Parasitol Res. 2011;110(3):1065–78.PubMedGoogle Scholar
  34. 34.
    Das VN, Siddiqui NA, Verma RB, Topno RK, Singh D, Das S, et al. Asymptomatic infection of visceral leishmaniasis in hyperendemic areas of Vaishali district, Bihar, India: a challenge to kala-azar elimination programmes. Trans R Soc Trop Med Hyg. 2011;105(11):661–6.PubMedGoogle Scholar
  35. 35.
    Gidwani K, Kumar R, Rai M, Sundar S. Longitudinal seroepidemiologic study of visceral leishmaniasis in hyperendemic regions of Bihar. India. Am J Trop Med Hyg. 2009;80(3):345–6.PubMedGoogle Scholar
  36. 36.
    Rijal S, Boelaert M, Regmi S, Karki BM, Jacquet D, Singh R, et al. Evaluation of a urinary antigen-based latex agglutination test in the diagnosis of kala-azar in eastern Nepal. Trop Med Int Health. 2004;9(6):724–9.PubMedGoogle Scholar
  37. 37.
    Sundar S, Agrawal S, Pai K, Chance M, Hommel M. Detection of leishmanial antigen in the urine of patients with visceral leishmaniasis by a latex agglutination test. Am J Trop Med Hyg. 2005;73(2):269–71.PubMedGoogle Scholar
  38. 38.
    Diro E, Techane Y, Tefera T, Assefa Y, Kebede T, Genetu A, et al. Field evaluation of FD-DAT, rK39 dipstick and KATEX (urine latex agglutination) for diagnosis of visceral leishmaniasis in northwest Ethiopia. Trans R Soc Trop Med Hyg. 2007;101(9):908–14.PubMedGoogle Scholar
  39. 39.
    Akhoundi M, Downing T, Votýpka J, Kuhls K, Lukeš J, Cannet A, et al. Leishmania infections: molecular targets and diagnosis. Mol Aspects Med. 2017;57:1–29.Google Scholar
  40. 40.
    de Ruiter CM, van der Veer C, Leeflang MM, Deborggraeve S, Lucas C, Adams ER. Molecular tools for diagnosis of visceral leishmaniasis: systematic review and meta-analysis of diagnostic test accuracy. J Clin Microbiol. 2014;52(9):3147–55.PubMedPubMedCentralGoogle Scholar
  41. 41.
    Saad AA, Ahmed NG, Osman OS, Al-Basheer AA, Hamad A, Deborggraeve S, et al. Diagnostic accuracy of the Leishmania OligoC-TesT and NASBA-Oligochromatography for diagnosis of leishmaniasis in Sudan. PLoS Negl Trop Dis. 2010;4(8):e776.PubMedPubMedCentralGoogle Scholar
  42. 42.
    Mugasa CM, Deborggraeve S, Schoone GJ, Laurent T, Leeflang MM, Ekangu RA, et al. Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania. Trop Med Int Health. 2010;15(7):800–5.PubMedGoogle Scholar
  43. 43.
    Mugasa CM, Laurent T, Schoone GJ, Basiye FL, Saad AA, El Safi S, et al. Simplified molecular detection of Leishmania parasites in various clinical samples from patients with leishmaniasis. Parasit Vectors. 2010;3(1):13.PubMedPubMedCentralGoogle Scholar
  44. 44.
    Srivastava P, Mehrotra S, Tiwary P, Chakravarty J, Sundar S. Diagnosis of Indian visceral leishmaniasis by nucleic acid detection using PCR. PLoS ONE. 2011;6(4):e19304.PubMedPubMedCentralGoogle Scholar
  45. 45.
    Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, et al. Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol. 2001;39(3):849–54.PubMedPubMedCentralGoogle Scholar
  46. 46.
    Maurya R, Singh RK, Kumar B, Salotra P, Rai M, Sundar S. Evaluation of PCR for diagnosis of Indian kala-azar and assessment of cure. J Clin Microbiol. 2005;43(7):3038–41.PubMedPubMedCentralGoogle Scholar
  47. 47.
    Molina R, Lopez-Velez R, Gutierrez-Solar B, Jimenez I, Alvar J. Isolation of Leishmania infantum from the blood of a patient with AIDS using sandflies. Trans R Soc Trop Med Hyg. 1992;86(5):516.Google Scholar
  48. 48.
    Dweik A, Schonian G, Mosleh IM, Karanis P. Evaluation of PCR-RFLP (based on ITS-1 and HaeIII) for the detection of Leishmania species, using Greek canine isolates and Jordanian clinical material. Ann Trop Med Parasitol. 2007;101(5):399–407.PubMedGoogle Scholar
  49. 49.
    da Silva MA, Pedrosa Soares CR, Medeiros RA, Medeiros Z, de Melo FL. Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis. Exp Parasitol. 2013;134(2):206–10.PubMedGoogle Scholar
  50. 50.
    Noyes HA, Reyburn H, Bailey JW, Smith D. A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. J Clin Microbiol. 1998;36(10):2877–81.PubMedPubMedCentralGoogle Scholar
  51. 51.
    Harris E, Kropp G, Belli A, Rodriguez B, Agabian N. Single-step multiplex PCR assay for characterization of new world Leishmania complexes. J Clin Microbiol. 1998;36(7):1989–95.PubMedPubMedCentralGoogle Scholar
  52. 52.
    Schonian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HD, Presber W, et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis. 2003;47(1):349–58.PubMedGoogle Scholar
  53. 53.
    Ceccarelli M, Galluzzi L, Diotallevi A, Andreoni F, Fowler H, Petersen C, et al. The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis. Parasit Vectors. 2017;10(1):239.  https://doi.org/10.1186/s13071-017-2181-x.
  54. 54.
    Flegontov PN, Strelkova MV, Kolesnikov AA. The Leishmania major maxicircle divergent region is variable in different isolates and cell types. Mol Biochem Parasitol. 2006;146(2):173–9.PubMedGoogle Scholar
  55. 55.
    Mauricio IL, Gaunt MW, Stothard JR, Miles MA. Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania. Int J Parasitol. 2007;37(5):565–76.PubMedGoogle Scholar
  56. 56.
    Lixia L, Jiping L, Hongtao J, Limin S, Bo L, Feng W, et al. Detection of Leishmania donovani infection using magnetic beads-based serum peptide profiling by MALDI-TOF MS in mice model. Parasitol Res. 2012;110(3):1287–90.Google Scholar
  57. 57.
    Schonian G, Kuhls K, Mauricio IL. Molecular approaches for a better understanding of the epidemiology and population genetics of Leishmania. Parasitology. 2010;138(4):405–25.PubMedGoogle Scholar
  58. 58.
    Montalvo AM, Fraga J, Monzote L, Montano I, De Doncker S, Dujardin JC, et al. Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the new and old world. Parasitology. 2010;137(8):1159–68.PubMedGoogle Scholar
  59. 59.
    da Silva LA, de Sousa Cdos S, da Graca GC, 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(1):77–83.PubMedGoogle Scholar
  60. 60.
    Cupolillo E, Grimaldi G Jr, Momen H. Discrimination of Leishmania isolates using a limited set of enzymatic loci. Ann Trop Med Parasitol. 1995;89(1):17–23.PubMedGoogle Scholar
  61. 61.
    Sudarshan M, Singh T, Singh AK, Chourasia A, Singh B, Wilson ME, et al. Quantitative PCR in epidemiology for early detection of visceral leishmaniasis cases in India. PLoS Negl Trop Dis. 2014;8(12):e3366.  https://doi.org/10.1371/journal.pntd.0003366.
  62. 62.
    Sudarshan M, Sundar S. Parasite load estimation by qPCR differentiates between asymptomatic and symptomatic infection in Indian visceral leishmaniasis. Diagn Microbiol Infect Dis. 2014;80(1):40–2.  https://doi.org/10.1016/j.diagmicrobio.2014.01.031.PubMedPubMedCentralGoogle Scholar
  63. 63.
    Weirather JL, Jeronimo SM, Gautam S, Sundar S, Kang M, Kurtz MA, et al. Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples. J Clin Microbiol. 2011;49(11):3892–904.PubMedPubMedCentralGoogle Scholar
  64. 64.
    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(1):73–8.PubMedGoogle Scholar
  65. 65.
    Kobets T, Badalova J, Grekov I, Havelkova H, Svobodova M, Lipoldova M. Leishmania parasite detection and quantification using PCR-ELISA. Nat Protoc. 2010;5(6):1074–80.PubMedGoogle Scholar
  66. 66.
    Mary C, Faraut F, Lascombe L, Dumon H. Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol. 2004;42(11):5249–55.PubMedPubMedCentralGoogle Scholar
  67. 67.
    Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M. Real-time PCR applications for diagnosis of leishmaniasis. Parasit Vect. 2018;11(1):273.  https://doi.org/10.1186/s13071-018-2859-8.
  68. 68.
    Boite MC, Mauricio IL, Miles MA, Cupolillo E. New insights on taxonomy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis. PLoS Negl Trop Dis. 2012;6(11):e1888.PubMedPubMedCentralGoogle Scholar
  69. 69.
    Mauricio IL, Yeo M, Baghaei M, Doto D, Pratlong F, Zemanova E, et al. Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD). Int J Parasitol. 2006;36(7):757–69.PubMedGoogle Scholar
  70. 70.
    Srivastava P, Singh T, Sundar S. Genetic heterogeneity in clinical isolates of Leishmania donovani from India. J Clin Microbiol. 2011;49(10):3687–90.PubMedPubMedCentralGoogle Scholar
  71. 71.
    Downing T, Imamura H, Decuypere S, Clark TG, Coombs GH, Cotton JA, et al. Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance. Genome Res. 2011;21(12):2143–56.PubMedPubMedCentralGoogle Scholar
  72. 72.
    Downing T, Stark O, Vanaerschot M, Imamura H, Sanders M, Decuypere S, et al. Genome-wide SNP and microsatellite variation illuminate population-level epidemiology in the Leishmania donovani species complex. Infect Genet Evol. 2012;12(1):149–59.PubMedPubMedCentralGoogle Scholar
  73. 73.
    Zhang CY, Lu XJ, Du XQ, Jian J, Shu L, Ma Y. Phylogenetic and evolutionary analysis of Chinese Leishmania isolates based on multilocus sequence typing. PLoS One. 2013;8(4):e63124.PubMedPubMedCentralGoogle Scholar
  74. 74.
    Kuhls K, Keilonat L, Ochsenreither S, Schaar M, Schweynoch C, Presber W, et al. Multilocus microsatellite typing (MLMT) reveals genetically isolated populations between and within the main endemic regions of visceral leishmaniasis. Microbes Infect. 2007;9(3):334–43.PubMedGoogle Scholar
  75. 75.
    Montalvo AM, Monzote L, Fraga J, Montano I, Muskus C, Marin M, et al. PCR-RFLP and RAPD for typing neotropical Leishmania. Biomedica. 2008;28(4):597–606.PubMedGoogle Scholar
  76. 76.
    Toledo A, Martin-Sanchez J, Pesson B, Sanchiz-Marin C, Morillas-Marquez F. Genetic variability within the species Leishmania infantum by RAPD. A lack of correlation with zymodeme structure. Mol Biochem Parasitol. 2002;119(2):257–64.PubMedGoogle Scholar
  77. 77.
    Fraga J, Montalvo AM, De Doncker S, Dujardin JC, Van der Auwera G. Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infect Genet Evol. 2010;10(2):238–45.PubMedGoogle Scholar
  78. 78.
    Alam MZ, Yasin G, Kato H, Sakurai T, Katakura K. PCR-based detection of Leishmania donovani DNA in a Stray dog from a visceral Leishmaniasis endemic focus in Bangladesh. J Vet Med Sci. 2012;75(1):75–8.PubMedGoogle Scholar
  79. 79.
    Blackwell JM. Leishmaniasis epidemiology: all down to the DNA. Parasitology. 1992;104(Suppl):S19–34.PubMedGoogle Scholar
  80. 80.
    Brustoloni YM, Lima RB, da Cunha RV, Dorval ME, Oshiro ET, de Oliveira AL, et al. Sensitivity and specificity of polymerase chain reaction in Giemsa-stained slides for diagnosis of visceral leishmaniasis in children. Mem Inst Oswaldo Cruz. 2007;102(4):497–500.PubMedGoogle Scholar
  81. 81.
    Schallig HD, Oskam L. Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification. Trop Med Int Health. 2002;7(8):641–51.PubMedGoogle Scholar
  82. 82.
    Reithinger R, Dujardin JC. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol. 2007;45(1):21–5.PubMedGoogle Scholar
  83. 83.
    Srivastava P, Dayama A, Mehrotra S, Sundar S. Diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg. 2010;105(1):1–6.PubMedPubMedCentralGoogle Scholar
  84. 84.
    Mathis A, Deplazes P. PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs. J Clin Microbiol. 1995;33(5):1145–9.PubMedPubMedCentralGoogle Scholar
  85. 85.
    Cortes S, Rolao N, Ramada J, Campino L. PCR as a rapid and sensitive tool in the diagnosis of human and canine leishmaniasis using Leishmania donovani s.l.-specific kinetoplastid primers. Trans R Soc Trop Med Hyg. 2004;98(1):12–7.PubMedGoogle Scholar
  86. 86.
    Katakura K, Kawazu S, Naya T, Nagakura K, Ito M, Aikawa M, et al. Diagnosis of kala-azar by nested PCR based on amplification of the Leishmania mini-exon gene. J Clin Microbiol. 1998;36(8):2173–7.PubMedPubMedCentralGoogle Scholar
  87. 87.
    Prina E, Roux E, Mattei D, Milon G. Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR. Microbes Infect. 2007;9(11):1307–15.PubMedGoogle Scholar
  88. 88.
    Costa CH, Stewart JM, Gomes RB, Garcez LM, Ramos PK, Bozza M, et al. Asymptomatic human carriers of Leishmania chagasi. Am J Trop Med Hyg. 2002;66(4):334–7.PubMedGoogle Scholar
  89. 89.
    Topno RK, Das VN, Ranjan A, Pandey K, Singh D, Kumar N, et al. Asymptomatic infection with visceral leishmaniasis in a disease-endemic area in Bihar, India. Am J Trop Med Hyg. 2010;83(3):502–6.PubMedPubMedCentralGoogle Scholar
  90. 90.
    Bhattarai NR, Van der Auwera G, Khanal B, De Doncker S, Rijal S, Das ML, et al. PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar. Trop Med Int Health. 2009;14(4):404–11.PubMedGoogle Scholar
  91. 91.
    Srivastava P, Gidwani K, Picado A, Van der Auwera G, Tiwary P, Ostyn B, et al. Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India. Trop Med Int Health. 2013;18(5):548–54.PubMedGoogle Scholar
  92. 92.
    Vallur AC, Duthie MS, Reinhart C, Tutterrow Y, Hamano S, Bhaskar KR, et al. Biomarkers for intracellular pathogens: establishing tools as vaccine and therapeutic endpoints for visceral leishmaniasis. Clin Microbiol Infect. 2013;20(6):O374–83.  https://doi.org/10.1111/1469-0691.12421.PubMedPubMedCentralGoogle Scholar
  93. 93.
    Biglino A, Bolla C, Concialdi E, Trisciuoglio A, Romano A, Ferroglio E. Asymptomatic Leishmania infantum infection in an area of northwestern Italy (Piedmont region) where such infections are traditionally nonendemic. J Clin Microbiol. 2009;48(1):131–6.PubMedPubMedCentralGoogle Scholar
  94. 94.
    Abbasi I, Aramin S, Hailu A, Shiferaw W, Kassahun A, Belay S, et al. Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia. BMC Infect Dis. 2013;27(13):153.Google Scholar
  95. 95.
    Fisa R, Riera C, Lopez-Chejade P, Molina I, Gallego M, Falco V, et al. Leishmania infantum DNA detection in urine from patients with visceral leishmaniasis and after treatment control. Am J Trop Med Hyg. 2008;78(5):741–4.PubMedGoogle Scholar
  96. 96.
    Vaish M, Singh OP, Chakravarty J, Sundar S. rK39 antigen for the diagnosis of visceral leishmaniasis by using human saliva. Am J Trop Med Hyg. 2012;86(4):598–600.PubMedPubMedCentralGoogle Scholar
  97. 97.
    Motazedian M, Fakhar M, Motazedian MH, Hatam G, Mikaeili F. A urine-based polymerase chain reaction method for the diagnosis of visceral leishmaniasis in immunocompetent patients. Diagn Microbiol Infect Dis. 2008;60(2):151–4.PubMedGoogle Scholar
  98. 98.
    Salam MA, Mondal D, Kabir M, Ekram AR, Haque R. PCR for diagnosis and assessment of cure in kala-azar patients in Bangladesh. Acta Trop. 2010;113(1):52–5.PubMedGoogle Scholar
  99. 99.
    Jorquera A, Gonzalez R, Marchan-Marcano E, Oviedo M, Matos M. Multiplex-PCR for detection of natural Leishmania infection in Lutzomyia spp. captured in an endemic region for cutaneous leishmaniasis in state of Sucre, Venezuela. Mem Inst Oswaldo Cruz. 2005;100(1):45–8.PubMedGoogle Scholar
  100. 100.
    Deborggraeve S, Laurent T, Espinosa D, Van der Auwera G, Mbuchi M, Wasunna M, et al. A simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis. J Infect Dis. 2008;198(10):1565–72.PubMedGoogle Scholar
  101. 101.
    De Doncker S, Hutse V, Abdellati S, Rijal S, Singh Karki BM, Decuypere S, et al. A new PCR-ELISA for diagnosis of visceral leishmaniasis in blood of HIV-negative subjects. Trans R Soc Trop Med Hyg. 2005;99(1):25–31.PubMedGoogle Scholar
  102. 102.
    Verma S, Avishek K, Sharma V, Negi NS, Ramesh V, Salotra P. Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis. Diagn Microbiol Infect Dis. 2013;75(4):390–5.PubMedGoogle Scholar
  103. 103.
    Khan MG, Bhaskar KR, Salam MA, Akther T, Pluschke G, Mondal D. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors. 2012;5:280.  https://doi.org/10.1186/1756-3305-5-280.PubMedPubMedCentralGoogle Scholar
  104. 104.
    Castellanos-Gonzalez A, Saldarriaga OA, Tartaglino L, Gacek R, Temple E, Sparks H, et al. A novel molecular test to diagnose canine visceral leishmaniasis at the point of care. Am J Trop Med Hyg. 2015;93(5):970–5.PubMedPubMedCentralGoogle Scholar
  105. 105.
    Mondal D, Ghosh P, Khan MA, Hossain F, Bohlken-Fascher S, Matlashewski G, et al. Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay. Parasit Vectors. 2016;9(1):281.PubMedPubMedCentralGoogle Scholar
  106. 106.
    Srivastava P, Gidwani K, Picado A, Van der Auwera G, Tiwary P, Ostyn B, et al. Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar. India. Trop Med Int Health. 2013;18(5):548–54.PubMedGoogle Scholar
  107. 107.
    Vaish M, Mehrotra S, Chakravarty J, Sundar S. Noninvasive molecular diagnosis of human visceral leishmaniasis. J Clin Microbiol. 2011;49(5):2003–5.PubMedPubMedCentralGoogle Scholar
  108. 108.
    Ferreira Sde A, Almeida GG, Silva Sde O, Vogas GP, Fujiwara RT, de Andrade AS, et al. Nasal, oral and ear swabs for canine visceral leishmaniasis diagnosis: new practical approaches for detection of Leishmania infantum DNA. PLoS Negl Trop Dis. 2013;7(4):e2150.PubMedGoogle Scholar
  109. 109.
    de Ruiter CM, van der Veer C, Leeflang MMG, Deborggraeve S, Lucas C, Adams ER. Molecular tools for diagnosis of visceral leishmaniasis: systematic review and meta-analysis of diagnostic test accuracy. J Clin Microbiol. 2014; 52(9):3147–55.Google Scholar
  110. 110.
    Adhya S, Chatterjee M, Hassan MQ, Mukherjee S, Sen S. Detection of Leishmania in the blood of early kala-azar patients with the aid of the polymerase chain reaction. Trans R Soc Trop Med Hyg. 1995;89(6):622–4.Google Scholar
  111. 111.
    Disch J, Maciel FC, de Oliveira MC, Orsini M, Rabello A. Detection of circulating Leishmania chagasi DNA for the non-invasive diagnosis of human infection. Trans R Soc Trop Med Hyg. 2003;97(4):391–5.Google Scholar
  112. 112.
    Wu Z, Bao Y, Ding Y, Yu M, Lu L, Zhang Y. An experimental study on application of PCR in detection of kala-azar. Southeast Asian J Trop Med Public Health. 1997;28(1):169–72.PubMedGoogle Scholar
  113. 113.
    Osman OF, Oskam L, Zijlstra EE, Kroon NC, Schoone GJ, Khalil ET, et al. Evaluation of PCR for diagnosis of visceral leishmaniasis. J Clin Microbiol. 1997;35(10):2454–7.PubMedPubMedCentralGoogle Scholar
  114. 114.
    Alam MZ, Shamsuzzaman AK, Kuhls K, Schonian G. PCR diagnosis of visceral leishmaniasis in an endemic region, Mymensingh district. Bangladesh. Trop Med Int Health. 2009;14(5):499–503.PubMedGoogle Scholar
  115. 115.
    Sudarshan M, Singh T, Chakravarty J, Sundar S. A correlative study of splenic parasite score and peripheral blood parasite load estimation by quantitative PCR in visceral leishmaniasis. J Clin Microbiol. 2015;53(12):3905–7.PubMedPubMedCentralGoogle Scholar
  116. 116.
    de Paiva-Cavalcanti M, de Morais RC, Pessoa-E-Silva R, Trajano-Silva LA, Gonçalves-de-Albuquerque SC, Tavares Dde H, et al. Leishmaniases diagnosis: an update on the use of immunological and molecular tools. Cell Biosci. 2015;5:31.  https://doi.org/10.1186/s13578-015-0021-2.
  117. 117.
    Galai Y, Chabchoub N, Ben-Abid M, Ben-Abda I, Ben-Alaya-Bouafif N, Amri F, et al. Diagnosis of mediterranean visceral leishmaniasis by detection of leishmania antibodies and leishmania DNA in oral fluid samples collected using an Oracol device. J Clin Microbiol. 2011;49(9):3150–3.PubMedPubMedCentralGoogle Scholar
  118. 118.
    Hu XS, Yang WT, Lu HG, Yan HP, Cheng JP, Ma Y, et al. Sequencing a specific kinetoplast DNA fragment of Leishmania donovani for polymerase chain reaction amplification in diagnosis of leishmaniasis in bone marrow and blood samples. J Parasitol. 2000;86(4):822–6.PubMedGoogle Scholar
  119. 119.
    Adams ER, Schoone GJ, Ageed AF, Safi SE, Schallig HD. Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples. Am J Trop Med Hyg. 2010;82(4):591–6.PubMedPubMedCentralGoogle Scholar
  120. 120.
    Takagi H, Itoh M, Islam MZ, Razzaque A, Ekram AR, Hashighuchi Y, et al. Sensitive, specific, and rapid detection of Leishmania donovani DNA by loop-mediated isothermal amplification. Am J Trop Med Hyg. 2009;81(4):578–82.PubMedGoogle Scholar
  121. 121.
    Adams ER, Schoone G, Versteeg I, Gomez MA, Diro E, Mori Y, et al. Development and evaluation of a novel LAMP assay for the diagnosis of cutaneous and visceral leishmaniasis. J Clin Microbiol. Epub. 2018.  https://doi.org/10.1128/JCM.00386-18.Google Scholar
  122. 122.
    Hendrickx S, Guerin PJ, Caljon G, Croft SL, Maes L. Evaluating drug resistance in visceral leishmaniasis: the challenges. Parasitology. 2018;145(4):453–63.PubMedGoogle Scholar
  123. 123.
    Singh OP, Singh B, Chakravarty J, Sundar S. Current challenges in treatment options for visceral leishmaniasis in India: a public health perspective. Infect Dis Poverty. 2016;8(5):19.  https://doi.org/10.1186/s40249-016-0112-2.Google Scholar
  124. 124.
    Freitas-Junior LH, Chatelain E, Kim HA, Siqueira-Neto JL. Visceral leishmaniasis treatment: what do we have, what do we need and how to deliver it? Int J Parasitol Drugs Drug Resist. 2012;2:11–9.PubMedPubMedCentralGoogle Scholar
  125. 125.
    Srivastava P, Prajapati VK, Rai M, Sundar S. Unusual case of resistance to amphotericin B in visceral leishmaniasis in a region in India where leishmaniasis is not endemic. J Clin Microbiol. 2011;49(8):3088–91.PubMedPubMedCentralGoogle Scholar
  126. 126.
    Gomes CM, Cesetti MV, de Paula NA, Vernal S, Gupta G, Sampaio RN, et al. Field validation of SYBR green- and TaqMan-based real-time PCR using biopsy and swab samples to diagnose american tegumentary leishmaniasis in an area where Leishmania (Viannia) braziliensis is endemic. J Clin Microbiol. 2017;55(2):526–34.  https://doi.org/10.1128/JCM.01954-16.PubMedPubMedCentralGoogle Scholar
  127. 127.
    Manna L, Reale S, Viola E, Vitale F, Foglia Manzillo V, Pavone LM, et al. Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Vet Parasitol. 2006;142(3–4):271–80.PubMedGoogle Scholar
  128. 128.
    Sudarshan M, Weirather JL, Wilson ME, Sundar S. Study of parasite kinetics with antileishmanial drugs using real-time quantitative PCR in Indian visceral leishmaniasis. J Antimicrob Chemother. 2011;66(8):1751–5.PubMedPubMedCentralGoogle Scholar
  129. 129.
    Hossain F, Ghosh P, Khan MAA, Duthie MS, Vallur AC, Picone A, et al. 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. 2017;12(9):e0185606.  https://doi.org/10.1371/journal.pone.0185606.PubMedPubMedCentralGoogle Scholar
  130. 130.
    de Paiva Cavalcanti M, Felinto de Brito ME, de Souza WV, de Miranda Gomes Y, Abath FG. The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood. Vet J. 2009;182(2):356–8.Google Scholar
  131. 131.
    Dantas-Torres F, da Silva Sales KG, Gomes da Silva L, Otranto D, Figueredo LA. 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. Mol Cell Probes. 2017;31:65–9.  https://doi.org/10.1016/j.mcp.2016.08.006.PubMedGoogle Scholar
  132. 132.
    Molina I, Fisa R, Riera C, Falco V, Elizalde A, Salvador F, et al. Ultrasensitive real-time PCR for the clinical management of visceral leishmaniasis in HIV-infected patients. Am J Trop Med Hyg. 2013;89(1):105–10.  https://doi.org/10.4269/ajtmh.12-0527.
  133. 133.
    Silva RC, Richini-Pereira VB, Kikuti M, Marson PM, Langoni H. Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers. Vet Q. 2017;37(1):23–30.Google Scholar
  134. 134.
    Ribeiro-Romão RP, Saavedra AF, Da-Cruz AM, Pinto EF, Moreira OC. Development of real-time PCR assays for evaluation of immune response and parasite load in golden hamster (Mesocricetus auratus) infected by Leishmania (Viannia) braziliensis. Parasit Vectors. 2016;9(1):361.  https://doi.org/10.1186/s13071-016-1647-6).
  135. 135.
    Suárez M, Valencia BM, Jara M, Alba M, Boggild AK, Dujardin JC, et al. Quantification of Leishmania (Viannia) kinetoplast DNA in ulcers of cutaneous leishmaniasis reveals inter-site and inter-sampling variability in parasite load. PLoS Negl Trop Dis. 2015;9(7):e0003936.  https://doi.org/10.1371/journal.pntd.0003936.
  136. 136.
    Jara M, Adaui V, Valencia BM, Martinez D, Alba M, Castrillon C, et al. 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. J Clin Microbiol. 2013;51(6):1826–33.PubMedPubMedCentralGoogle Scholar
  137. 137.
    Talmi-Frank D, Nasereddin A, Schnur LF, Schonian G, Toz SO, Jaffe CL, et al. Detection and identification of old world Leishmania by high resolution melt analysis. PLoS Negl Trop Dis. 2010;4(1):e581.PubMedPubMedCentralGoogle Scholar
  138. 138.
    de Almeida ME, Koru O, Steurer F, Herwaldt BL, da Silva AJ. Detection and differentiation of Leishmania spp. in clinical specimens by use of a SYBR green-based real-time PCR assay. J Clin Microbiol. 2016;55(1):281-290.  https://doi.org/10.1128/JCM.01764-16.
  139. 139.
    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(11):5080–4.PubMedPubMedCentralGoogle Scholar
  140. 140.
    Schulz A, Mellenthin K, Schonian G, Fleischer B, Drosten C. Detection, differentiation, and quantitation of pathogenic leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay. J Clin Microbiol. 2003;41(4):1529–35.PubMedPubMedCentralGoogle Scholar
  141. 141.
    Miro G, Oliva G, Cruz I, Canavate C, Mortarino M, Vischer C, et al. Multicentric, controlled clinical study to evaluate effectiveness and safety of miltefosine and allopurinol for canine leishmaniosis. Vet Dermatol. 2009;20(5–6):397–404.PubMedGoogle Scholar
  142. 142.
    Wortmann G, Hochberg L, Houng HH, Sweeney C, Zapor M, Aronson N, et al. Rapid identification of Leishmania complexes by a real-time PCR assay. Am J Trop Med Hyg. 2005;73(6):999–1004.PubMedGoogle Scholar
  143. 143.
    León CM, Muñoz M, Hernández C, Ayala MS, Flórez C, Teherán A, et al. Analytical performance of four polymerase chain reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia. Front Microbiol. 2017;8:1907.  https://doi.org/10.3389/fmicb.2017.01907.
  144. 144.
    Zampieri RA, Laranjeira-Silva MF, Muxel SM, Stocco de Lima AC, Shaw JJ, Floeter-Winter LM. High resolution melting analysis targeting hsp70 as a fast and efficient method for the discrimination of Leishmania species. PLoS Negl Trop Dis. 2016;10(2):e0004485.  https://doi.org/10.1371/journal.pntd.0004485.
  145. 145.
    Verma S, Kumar R, Katara GK, Singh LC, Negi NS, Ramesh V, et al. Quantification of parasite load in clinical samples of leishmaniasis patients: IL-10 level correlates with parasite load in visceral leishmaniasis. PLoS One. 2010;5(4):e10107.PubMedPubMedCentralGoogle Scholar
  146. 146.
    Leon CM, Munoz M, Hernandez C, Ayala MS, Florez C, Teheran A, et al. Analytical performance of four polymerase chain reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia. Front Microbiol. 2017;8:1907.PubMedPubMedCentralGoogle Scholar
  147. 147.
    Medeiros FA, Gomes LI, Oliveira E, de Souza CS, Mourao MV, Cota GF, et al. Development and validation of a PCR-ELISA for the diagnosis of symptomatic and asymptomatic infection by Leishmania (Leishmania) infantum. J Trop Med. 2017;2017:7364854.PubMedPubMedCentralGoogle Scholar
  148. 148.
    Sue MJ, Yeap SK, Omar AR, Tan SW. Application of PCR-ELISA in molecular diagnosis. Biomed Res Int. 2014;2014:653014.  https://doi.org/10.1155/2014/653014.PubMedPubMedCentralGoogle Scholar
  149. 149.
    Aara N, Khandelwal K, Bumb RA, Mehta RD, Ghiya BC, Jakhar R, et al. Clinco-epidemiologic study of cutaneous leishmaniasis in Bikaner, Rajasthan, India. Am J Trop Med Hyg. 2013;89(1):111–5.  https://doi.org/10.4269/ajtmh.12-0558.
  150. 150.
    Khandelwal K, Bumb RA, Mehta RD, Kaushal H, Lezama-Davila C, Salotra P, et al. A patient presenting with diffuse cutaneous leishmaniasis (DCL) as a first indicator of HIV infection in India. Am J Trop Med Hyg. 2011;85(1):64–5.PubMedPubMedCentralGoogle Scholar
  151. 151.
    Sharma NL, Mahajan VK, Kanga A, Sood A, Katoch VM, Mauricio I, et al. Localized cutaneous leishmaniasis due to Leishmania donovani and Leishmania tropica: preliminary findings of the study of 161 new cases from a new endemic focus in himachal pradesh. India. Am J Trop Med Hyg. 2005;72(6):819–24.PubMedGoogle Scholar
  152. 152.
    Zijlstra EE, Musa AM, Khalil EA. el-Hassan IM, el-Hassan AM. Post-kala-azar dermal leishmaniasis. Lancet Infect Dis. 2003;3(2):87–98.PubMedGoogle Scholar
  153. 153.
    Rahman KM, Islam S, Rahman MW, Kenah E, Ghalib CM, Zahid MM, et al. Increasing incidence of post-kala-azar dermal leishmaniasis in a population-based study in Bangladesh. Clin Infect Dis. 2009;50(1):73–6.Google Scholar
  154. 154.
    Cupolillo E, Grimaldi Junior G, Momen H, Beverley SM. Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol. 1995;73(1–2):145–55.PubMedGoogle Scholar
  155. 155.
    Mauricio IL, Stothard JR, Miles MA. Leishmania donovani complex: genotyping with the ribosomal internal transcribed spacer and the mini-exon. Parasitology. 2004;128(Pt 3):263–7.PubMedGoogle Scholar
  156. 156.
    Chargui N, Haouas N, Jaouadi K, Gorcii M, Pratlong F, Dedet JP, et al. Usefulness of a PCR-based method in the detection and species identification of Leishmania from clinical samples. Pathol Biol (Paris). 2012;60(6):e75–9.Google Scholar
  157. 157.
    Piarroux R, Azaiez R, Lossi AM, Reynier P, Muscatelli F, Gambarelli F, et al. Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction. Am J Trop Med Hyg. 1993;49(3):364–9.PubMedGoogle Scholar
  158. 158.
    Luyo-Acero GE, Uezato H, Oshiro M, Takei K, Kariya K, Katakura K, et al. Sequence variation of the cytochrome b gene of various human infecting members of the genus Leishmania and their phylogeny. Parasitology. 2004;128(Pt 5):483–91.PubMedGoogle Scholar
  159. 159.
    Yang BB, Chen DL, Chen JP, Liao L, Hu XS, Xu JN. Analysis of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference. Parasit Vectors. 2013;6:32.PubMedPubMedCentralGoogle Scholar
  160. 160.
    Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I. Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis. J Clin Microbiol. 2003;41(7):3147–53.PubMedPubMedCentralGoogle Scholar
  161. 161.
    Castilho TM, Shaw JJ, Floeter-Winter LM. New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species. J Clin Microbiol. 2003;41(2):540–6.PubMedPubMedCentralGoogle Scholar
  162. 162.
    Hide M, Banuls AL. Polymorphisms of cpb multicopy genes in the Leishmania (Leishmania) donovani complex. Trans R Soc Trop Med Hyg. 2008;102(2):105–6.PubMedGoogle Scholar
  163. 163.
    Hide M, Banuls AL. Species-specific PCR assay for L. infantum/L. donovani discrimination. Acta Trop. 2006;100(3):241–5.PubMedGoogle Scholar
  164. 164.
    Victoir K, Banuls AL, Arevalo J, Llanos-Cuentas A, Hamers R, Noel S, et al. The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia. Parasitology. 1998;117(Pt 1):1–13.PubMedGoogle Scholar
  165. 165.
    Fraga J, Montalvo AM, Van der Auwera G, Maes I, Dujardin JC, Requena JM. Evolution and species discrimination according to the Leishmania heat-shock protein 20 gene. Infect Genet Evol. 2013;28(18C):229–37.Google Scholar
  166. 166.
    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(5):2294–7.PubMedPubMedCentralGoogle Scholar
  167. 167.
    Hamad SH, Khalil EA, Musa AM, Ibrahim ME, Younis BM, Elfaki ME, et al. Leishmania donovani: genetic diversity of isolates from Sudan characterized by PCR-based RAPD. Exp Parasitol. 2010;125(4):389–93.PubMedGoogle Scholar
  168. 168.
    Mauricio IL, Howard MK, Stothard JR, Miles MA. Genomic diversity in the Leishmania donovani complex. Parasitology. 1999;119(Pt 3):237–46.PubMedGoogle Scholar
  169. 169.
    Martinez E, Alonso V, Quispe A, Thomas MC, Alonso R, Pinero JE, et al. RAPD method useful for distinguishing Leishmania species: design of specific primers for L. braziliensis. Parasitology. 2003;127(Pt 6):513–7.PubMedGoogle Scholar
  170. 170.
    Khanra S, Bandopadhyay SK, Chakraborty P, Datta S, Mondal D, Chatterjee M, et al. Characterization of the recent clinical isolates of Indian Kala-azar patients by RAPD-PCR method. J Parasit Dis. 2012;35(2):116–22.Google Scholar
  171. 171.
    Eisenberger CL, Jaffe CL. Leishmania: identification of Old World species using a permissively primed intergenic polymorphic-polymerase chain reaction. Exp Parasitol. 1999;91(1):70–7.PubMedGoogle Scholar
  172. 172.
    Kumar A, Boggula VR, Misra P, Sundar S, Shasany AK, Dube A. Amplified fragment length polymorphism (AFLP) analysis is useful for distinguishing Leishmania species of visceral and cutaneous forms. Acta Trop. 2010;113(2):202–6.PubMedGoogle Scholar
  173. 173.
    Rioux JA, Lanotte G, Serres E, Pratlong F, Bastien P, Perieres J. Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification. Ann Parasitol Hum Comp. 1990;65(3):111–25.PubMedGoogle Scholar
  174. 174.
    Banuls AL, Hide M, Prugnolle F. Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans. Adv Parasitol. 2007;64:1–109.PubMedGoogle Scholar
  175. 175.
    Hernandez C, Alvarez C, Gonzalez C, Ayala MS, Leon CM, Ramirez JD. Identification of six New World Leishmania species through the implementation of a high-resolution melting (HRM) genotyping assay. Parasit Vectors. 2014;14(7):501.Google Scholar
  176. 176.
    Berzunza-Cruz M, Cabrera N, Crippa-Rossi M, Sosa Cabrera T, Perez-Montfort R, Becker I. Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana. Parasitol Res. 2002;88(10):918–25.PubMedGoogle Scholar
  177. 177.
    Croan DG, Morrison DA, Ellis JT. Evolution of the genus Leishmania revealed by comparison of DNA and RNA polymerase gene sequences. Mol Biochem Parasitol. 1997;89(2):149–59.PubMedGoogle Scholar
  178. 178.
    Ibrahim ME, Barker DC. The origin and evolution of the Leishmania donovani complex as inferred from a mitochondrial cytochrome oxidase II gene sequence. Infect Genet Evol. 2001;1(1):61–8.PubMedGoogle Scholar
  179. 179.
    Asato Y, Oshiro M, Myint CK, Yamamoto Y, Kato H, Marco JD, et al. Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing. Exp Parasitol. 2009;121(4):352–61.PubMedGoogle Scholar
  180. 180.
    Hide M, Bras-Goncalves R, Banuls AL. Specific cpb copies within the Leishmania donovani complex: evolutionary interpretations and potential clinical implications in humans. Parasitology. 2007;134(Pt 3):379–89.PubMedGoogle Scholar
  181. 181.
    Zelazny AM, Fedorko DP, Li L, Neva FA, Fischer SH. Evaluation of 7SL RNA gene sequences for the identification of Leishmania spp. Am J Trop Med Hyg. 2005;72(4):415–20.PubMedGoogle Scholar
  182. 182.
    Pita-Pereira D, Lins R, Oliveira MP, Lima RB, Pereira BA, Moreira OC, Brazil RP, Britto C. 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. Parasit Vect. 2012;5:15.  https://doi.org/10.1186/1756-3305-5-15.Google Scholar

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© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Infectious Disease Research Laboratory, Department of Medicine, Institute of Medical SciencesBanaras Hindu UniversityVaranasiIndia

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