Original Article

Intra-Species Diversity of Leishmania major and L. tropica from Clinical Isolates of Cutaneous Leishmaniasis in Southwest Iran Inferred by ITS1-rDNA


Background: Cutaneous leishmaniasis (CL) as a public health concern is increasingly circulating by causative agents of Leishmania tropica and L. major in Iran. As regard to recent treatment failure and controlling problems, the accurate elucidation of heterogeneity traits and taxonomic status of Leishmania spp. should be broadly addressed by policymakers. This study was designed to determine the genetic variability and molecular characterization of L. major and L. tropica from Iranian CL patients.

Methods: One hundred positive Giemsa-stained slides were taken from clinical isolates of CL at Pol-e-Dokhtar County, Southwest Iran, from May 2014 to Sep 2016. DNAs were directly extracted and amplified by targeting ribosomal internal transcribed spacer (ITS) gene following microscopic observation. To identify Leishmania spp. amplicons were digested by restriction enzyme HaeIII subsequent PCR-RFLP technique. To reconfirm, the isolates were directly sequenced to conduct diversity indices and phylogenetic analysis.

Results: Based upon the RFLP patterns, 84 and 16 isolates were explicitly identified to L. tropica and L. major respectively. No co-infection was found in clinical isolates. The high genetic diversity of L. tropica (Haplotype diversity 0.9) was characterized compared to L. major isolates (Hd 0.476). The intra-species diversity for L. tropica and L. major isolates corresponded to 3%-3.9% and 0%-0.4%, respectively.

Conclusion: Findings indicate the L. tropica isolates with remarkable heterogeneity than L. major are predominantly circulating at Pol-e-Dokhtar County. Occurrence of high genetic variability of L. tropica may be noticed in probable treatment failure and/or emerging of new haplotypes; however, more studies are warranted from various geographic regions of Southwest Iran, using large sample size.



1. Desjeux P (2001). The increase in risk factors for leishmaniasis worldwide. Trans R Soc Trop Med Hyg, 95:239-243.
2. Alvar J, Vélez ID, Bern C et al (2012). Leishmaniasis worldwide and global estimates of its incidence. PLoS One, 7:e35671.
3. Spotin A, Rouhani S, Parvizi P (2014). The associations of Leishmania major and Leishmania tropica aspects by focusing their morphological and molecular features on clinical appearances in Khuzestan province, Iran. BioMed Res Int, 2014: 913510.
4. Badirzadeh A, Mohebali M, Asadgol Z et al (2017). The burden of leishmaniasis in Iran: findings from the global burden of disease from 1990 to 2010. Asian Pac J Trop Dis, 7:513–518.
5. Bordbar A, Parvizi P (2014). High density of Leishmania major and rarity of other mammals' Leishmania in zoonotic cutaneous leishmaniasis foci, Iran. Trop Med Int Health, 19:355-363.
6. Spotin A, Rouhani S, Ghaemmaghami P et al (2015). Different morphologies of Leishmania major amastigotes with no molecular diversity in a neglected endemic area of zoonotic cutaneous leishmaniasis in Iran. Iran Biomed J, 19:149-59.
7. Sharbatkhori M, Spotin A, Taherkhani H et al (2014). Molecular variation in Leishmania parasites from sandflies species of a zoonotic cutaneous leishmaniasis in northeast of Iran. J Vector Borne Dis, 51:16-21.
8. Rouhani S, Mirzaei A, Spotin A, Parvizi P (2014). Novel identification of Leishmania major in Hemiechinus auritus and molecular detection of this parasite in Meriones libycus from an important foci of zoonotic cutaneous leishmaniasis in Iran. J Infect Public Health, 7:210-217.
9. Spotin A, Parvizi P (2016). Comparative study of viscerotropic pathogenicity of Leishmania major amastigotes and promastigotes based on identification of mitochondrial and nucleus sequences. Parasitol Res, 115:1221-1228.
10. Kheirandish F, Sharafi AC, Kazemi B et al (2013). First molecular identification of Leishmania species in a new endemic area of cutaneous leishmaniasis in Lorestan, Iran. Asian Pac J Trop Med, 6:713-717.
11. Cupolillo E, Brahim LR, Toaldo CB et al (2003). Genetic polymorphism and molecular epidemiology of Leishmania (Viannia) braziliensis from different hosts and geographic areas in Brazil. J Clin Microbiol, 41:3126-3132.
12. Yatawara L, Le TH, Wickramasinghe S, Agatsuma T (2008). Maxicircle (mitochondrial) genome sequence (partial) of Leishmania major: gene content, arrangement and composition compared with Leishmania tarentolae. Gene, 424:80-86.
13. Parvizi P, Ready P (2008). Nested PCRs and sequencing of nuclear ITS‐rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. Trop Med Int Health, 13:1159-1171.
14. Montalvo A, Fraga J, Monzote L et al (2010). Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World. Parasitology, 137:1159-1168.
15. Rastaghi Are, Spotin A, Khataminezhad Mr et al (2017). Evaluative Assay of Nuclear and Mitochondrial Genes to Diagnose Leishmania Species in Clinical Specimens. Iran J Public Health, 46:1422-1429.
16. Seyyedtabaei Sj, Rostami A, Haghighi A et al (2017). Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases. Iran J Parasitol, 12:206-214.
17. Moshrefi M, Spotin A, Kafil HS et al (2017). Tumor suppressor p53 induces apoptosis of host lymphocytes experimentally infected by Leishmania major, by activation of Bax and caspase-3: a possible survival mechanism for the parasite. Parasitol Res, 116:2159-2166.
18. Hillis DM, Moritz C, Porter CA, Baker RJ (1991). Evidence for biased gene conversion in concerted evolution of ribosomal DNA. Science, 251:308-10.
19. Schönian G, Nasereddin A, Dinse N et al (2003). PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis, 47:349-358.
20. Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R (2003). DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics, 19:2496-2497.
21. Ivens AC, Peacock CS, Worthey EA et al (2005). The genome of the kinetoplastid parasite, Leishmania major. Science, 309:436-442.
22. Schönian G, Schnur L, El Fari M et al (2001). Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods. Trans R Soc Trop Med Hyg, 95:217-224.
23. Van Herwerden L, Gasser RB, Blair D (2000). ITS-1 ribosomal DNA sequence variants are maintained in different species and strains of Echinococcus. Int J Parasitol, 30:157-169.
24. Oryan A, Shirian S, Tabandeh M-R et al (2013). Genetic diversity of Leishmania major strains isolated from different clinical forms of cutaneous leishmaniasis in southern Iran based on minicircle kDNA. Infect Genet Evol, 19:226-231.
25. Hajjaran H, Mohebali M, Mamishi S et al (2013). Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran. BioMed Res Int, 2013: 789326.
26. Shirian S, Oryan A, Hatam GR et al (2016). Correlation of genetic heterogeneity with cytopathological and epidemiological findings of Leishmania major isolated from cutaneous leishmaniasis in Southern Iran. Acta Cytol, 60:97-106.
27. Tashakori M, Kuhls K, Al-Jawabreh A et al (2006). Leishmania major: genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer. Acta Trop, 98:52-58.
28. Dabirzade M, Sadeghi HMM, Hejazi H, Baghaii M (2012). Genetic polymorphism within the Leishmania major in two hyper endemic areas in Iran. Arch Iran Med,15(3):151-6.
29. Eslami G, Salehi R, Khosravi S, Doudi M (2012). Genetic analysis of clinical isolates of Leishmania major from Isfahan, Iran. J Vector Borne Dis, 49:168-74.
30. Mahmoudzadeh Niknam H, Ajdary S, Riazi-Rad F et la (2012). Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran. Trop Med Int Health, 17:1335-1344.
31. Kheirandish F, Sharafi AC, Kazemi B et al (2013). Identification of Leishmania species using PCR assay on giemsa-stained slides prepared from cutaneous leishmaniasis patients. Iran J Parasitol, 8:382-8.
IssueVol 48 No 5 (2019) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijph.v48i5.1806
Cutaneous leishmaniasis Genetic diversity PCR-RFLP Leishmania tropica Leishmania major Iran

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
MIRZAPOUR A, SPOTIN A, BEHNIAFAR H, AZIZI H, MALEKI B, SHAKERAMINIA H, SEYYED TABAEI SJ. Intra-Species Diversity of Leishmania major and L. tropica from Clinical Isolates of Cutaneous Leishmaniasis in Southwest Iran Inferred by ITS1-rDNA. Iran J Public Health. 2019;48(5):893-901.