Molecular identification of Leishmania isolates obtained from patients suspected as having cutaneous leishmaniasis referred to reference laboratories from Yazd province in central Iran


1 Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan; Research Centre of Molecular Science and Biotechnology, Shahid Sadoughi University of Medical Sciences, Daneshjoo Blv, Iran

2 Research Centre of Molecular Science and Biotechnology, Shahid Sadoughi University of Medical Sciences, Daneshjoo Blv; Department of Parasitology and Mycology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

3 Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Cutaneous leishmaniasis (CL) continues to be an increasing public health problem in Iran. The dominant etiologic agents of CL in the Old World are Leishmania major and Leishmania tropica. One of the important endemic foci of CL in Iran is Yazd. Recently, previous studies showed the equal prevalence of L. major and Ltropica as the agents of cutaneous leishmaniasis in this area. This prompted us to identify the genotype of L. major isolates obtained from patients with cutaneous leishmaniasis.
Materials and Methods: After completing a clinical/epidemiologic data questionnaire for 218 patients with suspected skin lesions, scraping samples were collected, and each specimen was examined using both direct microscopy and molecular assay of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
Results : Results showed that of the 218 samples, Leishman body was observed in 77 by direct smear and 104 by PCR assay. Molecular assay indicated 50 cases as L. major, 52 cases as L. tropica, and two cases as unknown. Molecular characterization of L. major isolates showed four patterns, named LmA1, LmA2, LmA3, and LmA4.
Conclusion: Our study is the first report for molecular characterization of L. major from one of the important central province of Iran that could affect the control strategies in this field.


1. Grimaldi G Jr, Tesh RB. Leishmaniases of the New World: Current concepts and implications for future research. Clin Microbiol Rev 1993;6:230-50.  Back to cited text no. 1
2. Gomes AH, Armelin IM, Menon SZ, Pereira-Chioccola VL. Leishmania (V.) braziliensis: Detection by PCR in biopsies from patients with cutaneous leishmaniasis. Exp Parasitol 2008;119:319-24.  Back to cited text no. 2
3. Evans D. Handbook on isolation, characterization and cryopreservation of Leishmania. Geneva: World Health Organization; 1989.  Back to cited text no. 3
4. Nadim A, Rashti AS, Faghih A. Epidemiology of cutaneous leishmaniasis in Iran: B. Khorassan. 3. Human infection. Bull Soc Pathol Exot Filiales 1969;62:702-10.  Back to cited text no. 4
5. Lopes UG, Wirth DF. Identification of visceral Leishmania species with cloned sequences of kinetoplast DNA. Mol Biochem Parasitol 1986;20:77-84.  Back to cited text no. 5
6. Bailey MS, Lockwood DN. Cutaneous leishmaniasis. Clin Dermatol 2007;25:203-11.  Back to cited text no. 6
7. Tavares CA, Fernandes AP, MeloMN. Molecular diagnosis of leishmaniasis. Expert Rev Mol Diagn 2003;3:657-67.  Back to cited text no. 7
8. Singh S, Sivakumar R. Recent advances in the diagnosis of leishmaniasis. J Postg Med 2003;49:55-60.  Back to cited text no. 8
9. Vega-López F. Diagnosis of cutaneous leishmaniasis. Curr Opin Infect Dis 2003;16:97-101.  Back to cited text no. 9
10. Weigle KA, Labrada LA, Lozano C, Santrich C, Barker DC. PCR-based diagnosis of acute and chronic cutaneous leishmaniasis caused by Leishmania (Viannia). J Clin Microbiol 2002;40:601-6.  Back to cited text no. 10
11. Garcia AL, Kindt A, Quispe-Tintaya KW, Bermudez H, Llanos A, Arevalo J, et al. American tegumentary leishmaniasis: Antigen-gene polymorphism, taxonomy and clinical pleomorphism. Infect Genet Evol 2005;5:109-16.  Back to cited text no. 11
12. Berzunza-Cruz M, Cabrera N, Crippa-Rossi M, Sosa Cabrera T, Pérez-Montfort R, Becker I. Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana. Parasitol Res 2002;88:918-25.  Back to cited text no. 12
13. Cupolillo E, Brahim LR, Toaldo CB, de Oliveira-Neto MP, de Brito ME, Falqueto A, et al. Genetic polymorphism and molecular epidemiology of Leishmania (Viannia) braziliensis from different hosts and geographic areas in Brazil. J Clin Microbiol 2003;41:3126-32.  Back to cited text no. 13
14. Rodriguez-Bonfante C, Bonfante-Garrido R, Grimaldi G Jr, Momen H, Cupolillo E. Genotypically distinct Leishmania colombiensis isolates from Venezuela cause both cutaneous and visceral leishmaniasis in humans. Infect Genet Evol 2003;3:119-24.  Back to cited text no. 14
15. Kelly JM. Isolation of DNA and RNA from Leishmania. Methods Mol Biol 1993;21:123-31.  Back to cited text no. 15
16. Doudi M, Hejazi SH, Razavi MR, Narimani M, Khanjani S, Eslami G. Comparative molecular epidemiology of Leishmania major and Leishmania tropica by PCR-RFLP technique in hyper endemic cities of Isfahan and Bam, Iran. Med Sci Monit 2010;16:CR530-5.  Back to cited text no. 16
17. Veland N, Boggild AK, Valencia C, Valencia BM, Llanos-Cuentas A, Van der Auwera G, et al. Leishmania (Viannia) species identification on clinical samples from cutaneous leishmaniasis patients in Peru: Assessment of a molecular stepwise approach. J Clin Microbiol 2012;50:495-8.  Back to cited text no. 17
18. Parhizkari M, Motazedian MH, Asqari Q, Mehrabani D. The PCR-based detection of Leishmania major in Mus musculus and other rodents caught in southern Iran: A guide to sample selection. Ann Trop Med Parasitol 2011;105:319-23.  Back to cited text no. 18
19. 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;47:3892-904.  Back to cited text no. 19
20. Schönian 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;47:349-58.  Back to cited text no. 20
21. Jaffe CL, Baneth G, Abdeen ZA, Schlein Y, Warburg A. Leishmaniasis in Israel and the Palestinian Authority. Trends Parasitol 2004;20:328-32.  Back to cited text no. 21
22. Victoir K, Bañuls AL, Arevalo J, Llanos-Cuentas A, Hamers R, Noël 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.  Back to cited text no. 22
23. Ferroglio E, Romano A, Trisciuoglio A, Poggi M, Ghiggi E, Sacchi P, et al. Characterization of Leishmania infantum strains in blood samples from infected dogs and humans by PCR-RFLP. Trans R Soc Trop Med Hyg 2006;100:636-41.  Back to cited text no. 23
24. de Bruijn MH, Barker DC. Diagnosis of New World leishmaniasis: Specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA. Acta Trop 1992;52:45-58.  Back to cited text no. 24
25. Hajjaran H, Mohebali M, Razavi M, Rezaei S, Kazemi B, Edrissian GH, et al. Identification of Leishmania species isolated from human cutaneous leishmaniasis, using random amplified polymorphic DNA (RAPD-PCR). Iran J Public Health 2004;33:8-15.  Back to cited text no. 25
26. 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;41:3147-53.  Back to cited text no. 26
27. Gadisa E, Genetu A, Kuru T, Jirata D, Dagne K, Aseffa A, et al. Leishmania (Kinetoplastida): Species typing with isoenzyme and PCR-RFLP from cutaneous leishmaniasis patients in Ethiopia. Exp Parasitol 2007;115:339-43.  Back to cited text no. 27
28. Tashakori M, Kuhls K, Al-Jawabreh A, Mauricio IL, Schönian G, Farajnia S, et al. Leishmania major: Genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer. Acta Trop 2006;98:52-8.  Back to cited text no. 28
29. Cupolillo E, Momen H, Grimaldi G Jr. Genetic diversity in natural populations of New World Leishmania. Mem Inst Oswaldo Cruz 1998;93:663-8.  Back to cited text no. 29
30. Kumar R, Bumb RA, Ansari NA, Mehta RD, Salotra P. Cutaneous leishmaniasis caused by Leishmania tropica in Bikaner, India: Parasite identification and characterization using molecular and immunologic tools. Am J Trop Med Hyg 2007;76:896-901.  Back to cited text no. 30
31. Rotureau B, Ravel C, Couppié P, Pratlong F, Nacher M, Dedet JP, et al. Use of PCR-restriction fragment length polymorphism analysis to identify the main new world Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples. J Clin Microbiol 2006;44:459-67.  Back to cited text no. 31
32. Schönian G, Schnur L, el Fari M, Oskam L, Kolesnikov AA, Sokolowska-Köhler W, et al. Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods. Trans R Soc Trop Med Hyg 2001;95:217-24.  Back to cited text no. 32
33. Kazemi-Rad E, Mohebali M, Hajjaran H, Rezaei S, Mamishi S. Diagnosis and characterization of Leishmania species in Giemsa-stained slides by PCR-RFLP. Iran J Public Health 2008;37:54-60.  Back to cited text no. 33
34. Dabirzadeh M, Mirmohammad Sadeghi H, Baghaie M, Hejazi H. Genetic Polymorphism of Leishmania major in two hyper endemic regions of Iran revealed by PPIP-PCR and ITS-RFLP. Arch Iran Med 2012;15:151-6.  Back to cited text no. 34
35. Delgado O, Cupolillo E, Bonfante-Garrido R, Silva S, Belfort E, Grimaldi Júnior G, et al. Cutaneous leishmaniasis in Venezuela caused by infection with a new hybrid between Leishmania (Viannia) braziliensis and L. (V.) guyanensis. Mem Inst Oswaldo Cruz 1997;92:581-2.  Back to cited text no. 35
36. Calvopina M, Gomez EA, Uezato H, Kato H, Nonaka S, Hashiguchi Y. Atypical clinical variants in New World cutaneous leishmaniasis: Disseminated, erysipeloid, and recidiva cutis due to Leishmania (V.) panamensis. Am J Trop Med Hyg 2005;73:281-4.  Back to cited text no. 36
37. Baghaei M, Mehdizadeh M, Afarid M, Attarzadeh A, Farzin D, Hosseini S. Intraspecific variation in Leishmania major isolated from different forms of zoonotic cutaneous Leishmaniasis. Iran J Med Sci 2005;30:51-4.  Back to cited text no. 37