Volume 73, Issue 6
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645


A real-time PCR assay for the detection of four complexes (, , , and ) was developed and evaluated. The assay was developed to detect the glucosephosphate isomerase gene and capitalizes on DNA sequence variability within that gene for complex identification. Primer/probe sets were created and tested against a panel of 21 known negative controls and on DNA extracted from cultured promastigotes or from tissue biopsies from patients with cutaneous leishmaniasis. The assay was highly specific, as no amplification products were detected in the negative control samples while simultaneously retaining a high degree of complex-specific diagnostic accuracy for cultured organisms and patient clinical samples. Real-time PCR offers rapid (within hours) identification of to the complex level and provides a useful molecular tool to assist both epidemiologists and clinicians.


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  1. Herwaldt BL, 1999. Leishmaniasis. Lancet 354 : 1191–1199.
  2. World Health Organization, 2000. Fact sheet no. 116. Available at http://www.who.int/inffs/en/fact116.html.
  3. Cupolillo E, Medina-Costa E, Noyes H, Momen H, Grimaldi G Jr, 2000. A revised classification for Leishmania and Endotrypanum. Parasitol Today 16 : 142–144.
  4. Berman JD, 1997. Human leishmaniasis: clinical, diagnostic and therapeutic developments in the last 10 years. Clin Infect Dis 24 : 684–703.
  5. Chance JL, 1985. The biochemical and immunological taxonomy of Leishmania. Chang K-P, Bray RS, eds. Leishmaniasis. Amsterdam: Elsevier, 93–110.
  6. McMahon-Pratt D, David JR, 1981. Monoclonal antibodies that distinguish between New World species of Leishmania. Nature 291 : 581–583.
  7. Harris E, Kropp G, Belli A, Rodriguez B, Agabian N, 1998. Single-step multiplex PCR assay for characterization of New World Leishmania complexes. J Clin Microbiol 36 : 1989–1995.
  8. van Eys G, Meredith S, 1996. Detection and characterization of Leishmania parasites by DNA-based methods. Southern blotting and PCR. Methods Mol Biol 50 : 227–242.
  9. Castilho T, Shaw J, Floeter-Winter L, 2003. New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species. J Clin Microbiol 41 : 540–546.
  10. Mauricio I, Gaunt M, Stothard J, Miles M, 2001. Genetic typing and phylogeny of the Leishmania donovani complex by restriction analysis of PCR amplified gp63 intergenic regions. Parasitology 122 : 393–403.
  11. Marfurt J, Nasereddin A, Niederwieser I, Jaffe C, Beck H, Felger I, 2003. 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 41 : 3147–3153.
  12. Schulz A, Mellenthin K, Gabriele S, Fleischer B, Drosten C, 2003. Detection, differentiation, and quantitation of pathogenic Leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay. J Clin Microbiol 41 : 1529–1535.
  13. Nicolas L, Milon G, Prina E, 2002. Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 51 : 295–299.
  14. Wortmann G, Sweeney C, Houng H, Aronson N, Stiteler J, Jackson J, Ockenhouse C, 2001. Rapid identification of leishmaniasis by fluorogenic polymerase chain reaction. Am J Trop Med Hyg 65 : 583–587.
  15. Aronson N, Coleman R, Coyne P, Rowton E, Hack D, Polhemus M, Wortmann G, Cox K, Weina P, Herwaldt B, 2003. Cutaneous leishmaniasis in U.S. military personnel-Southwest/Central Asia, 2002–2003. MMWR 52 (42): 1009–1012.
  16. Hendricks L, Wright N, 1979. Diagnosis of cutaneous leishmaniasis by in vitro cultivation of saline aspirates in Schneider’s Drosophila medium. Am J Trop Med Hyg 28 : 962–964.
  17. Kreutzer RD, Christensen HA, 1980. Characterization of Leishmania spp. by isozyme electrophoresis. Am J Trop Med Hyg 29 : 199–208.
  18. Kreutzer RD, Souraty N, Semko ME, 1987. Biochemical identities and differences among Leishmania species and subspecies. Am J Trop Med Hyg 36 : 22–32.
  19. Nyame K, Do-Thi C, Opperdoes F, Michels P, 1994. Subcellular distribution and characterization of glucosephosphate isomerase in Leishmania mexicana mexicana. Mol Biochem Parasitol 67 : 269–279.
  20. Aviles H, Belli A, Armijos R, Monroy F, Harris E, 1999. PCR detection and identification of Leishmania parasites in clinical specimens in Ecuador: a comparison with classical diagnostic methods. J Parasitol 85 : 181–187.
  21. de Oliveira C, Bafica A, Oliveira F, Favali C, Correa T, Freitas L, Nascimento E, Costa J, Barral A, 2003. Clinical utility of polymerase chain reaction-based detection of Leishmania in the diagnosis of American cutaneous leishmaniasis. Clin Infect Dis 37 : e149–e153.
  22. Perandin F, Manca N, Calderaro A, Piccolo G, Galati L, Ricci L, Medici M, Arcangeletti CM, Snounou G, Dettori G, Chezzi C, 2004. Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale for routine clinical diagnosis J Clin Microbiol 42 : 1214–1219.
  23. Faix D, Houng H, Gaydos J, Liu S, Connors J, Brown X, Asher L, Vaughn D, Binn L, 2004. Evaluation of a rapid quantitative diagnostic test for adenovirus type 4. Clin Infect Dis 38 : 391–397.
  24. Hochberg L, Aronson N, Neafie R, McEvoy P, Gilmore L, Fishbain J, Hawkes C, Weina P, Wortmann G, 2004. A comparison of diagnostic methods for Old World cutaneous leishmaniasis [Abstract P-429]. Program and Abstracts of the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy (Washington, DC). Washington, DC: American Society for Microbiology, 453.
  25. Navin TR, Arana BA, Arana FE, Berman JD, Chajon JF, 1992. Placebo-controlled clinical trial of sodium stibogluconate (Pentostam) versus ketoconazole for treating cutaneous leishmaniasis in Guatemala. J Infect Dis 165 : 528–534.
  26. Alrajhi A, Ibrahim E, De Vol E, Khairat M, Faris R, Maguire J, 2002. Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major. N Engl J Med 346 : 891–895.
  27. Bossolasco S, Gaiera G, Olchini D, Gulletta M, Martello L, Bestetti A, Bossi L, Germagnoli L, Lazzarin A, Uberti-Foppa C, Cinque P, 2003. Real-time PCR assay for clinical management of human immunodeficiency virus-infected patients with visceral leishmaniasis. J Clin Microbiol 41 : 5080–5084.

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  • Received : 09 Oct 2004
  • Accepted : 17 Aug 2005

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