Inter- and Intraspecific Variations of Leishmania Strains Isolated from Patients with Cutaneous and Visceral Leishmaniases in Fars Province, South of Iran

Document Type: Original Article(s)

Authors

1 Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

2 Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroentrology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Faculty of Pharmacy, University of Reims Champagne-Ardenne, France

4 Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Abstract

Background: Cutaneous and visceral leishmaniases are present in Fars Province in the south of Iran. The current study aimed to evaluate the inter- and intragenic diversities of Leishmania species isolated from patients with leishmaniasis in Fars Province, using PCR-based analyses and DNA sequencing of  the N-acetylglucosamine-1-phosphate transferase (nagt) gene.Methods: Clinical samples were taken from the skin lesions of 120 individuals with clinical suspicion of cutaneous leishmaniasis (CL) referred to the major health centers of Shiraz. Along with microscopic examination, a part of each sample was used for in vitro cultivation. DNA was extracted from the cultured parasites and the nagt gene was PCR-amplified. For RFLP analysis, the PCR product of the nagt gene was digested with the Acc1 restriction enzyme. Moreover, the PCR products of 23 isolates were sequenced and analyzed, using MEGA5.Results: From the 120 patients with clinical suspicion of CL, 110 (91.7%) cases were found to be positive by direct microscopy while 77 (64.1%) of the cultures were positive. Digestion of the PCR product with the Acc1 restriction enzyme detected L. major in 57 out of the 77 (74.1%) and L. tropica, in 20 out of the 77 (25.9%) cases with CL. Phylogenetic analysis grouped the Leishmania isolates into 3 main clades, representing L. major, L. infantum, and L. tropica, encompassing 2, 2, and 2 haplotypes, respectively. Within the clades, the L. tropica intraspecies divergence was more pronounced in L. major.Conclusion: The findings of this study demonstrated that the causative agent of CL in Fars Province was mainly L. major and that there was considerable heterogeneity between the Leishmania species and also within the L. major isolates.

Keywords


  1. - Antinori S, Schifanella L, Corbellino M. Leishmaniasis: new insights from an old and neglected disease. Eur J Clin Microbiol Infect Dis. 2012;31:109-18. doi: 10.1007/s10096-011-1276-0. PubMed PMID: 21533874.
  2. - Postigo JA. Leishmaniasis in the World Health Organization Eastern Mediterranean Region. Int J Antimicrob Agents. 2010;36:S62-5. doi: 10.1016/j.ijantimicag.2010.06.023. PubMed PMID: 20728317.
  3. - Motazedian H, Noamanpoor B, Ardehali S. Characterization of Leishmania parasites isolated from provinces of the Islamic Republic of Iran. East Mediterr Health J. 2002;8:338-44. PubMed PMID: 15339122.
  4. - Hajjaran H, Mohebali M, Mamishi S, Vasigheh F, Oshaghi MA, Naddaf SR, et al. Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran. Biomed Res Int. 2013;2013:789326. doi: 10.1155/2013/789326. PubMed PMID: 24286085; PubMed Central PMCID: PMC3826333.
  5. - Mohebali M. Visceral leishmaniasis in Iran: Review of the epidemiological and clinical features. Iran J Parasitol. 2013;8:348-58. PubMed PMID: 24454426; PubMed Central PMCID: PMC3887234.
  6. - Asgari Q, Motazedian MH, Mehrabani D, Oryan A, Hatam GR, Owji SM, et al. Zoonotic cutaneous leishmaniasis in Shiraz, Southern Iran: A molecular, isoenzyme and morphologic approach. J Res Med Sci. 2007;12:7-15.
  7. - Razmjou S, Hejazy H, Motazedian MH, Baghaei M, Emamy M, Kalantary M. A new focus of zoonotic cutaneous leishmaniasis in Shiraz, Iran. Trans R Soc Trop Med Hyg. 2009;103:727-30. doi: 10.1016/j.trstmh.2008.12.013. PubMed PMID: 19223055.
  8. - Davami MH, Motazedian MH, Sarkari B. The changing profile of cutaneous leishmaniasis in a focus of the disease in Jahrom district, southern Iran. Ann Trop Med Parasitol. 2010;104:377-82. doi: 10.1179/136485910X12786389891083. PubMed PMID: 20819305.
  9. - Oryan A, Shirian S, Tabandeh MR, Hatam GR, Randau G, Daneshbod Y. Genetic diversity of Leishmania major strains isolated from different clinical forms of cutaneous leishmaniasis in southern Iran based on minicircle kDNA. Infect Genet Evol. 2013;19:226-31. doi: 10.1016/j.meegid.2013.07.021. PubMed PMID: 23892374.
  10. - Mohebali M, Edrissian GH, Shirzadi MR, Akhoundi B, Hajjaran H, Zarei Z, et al. An observational study on the current distribution of visceral leishmaniasis in different geographical zones of Iran and implication to health policy. Travel Med Infect Dis. 2011;9:67-74. doi: 10.1016/j.tmaid.2011.02.003. PubMed PMID: 21419708.
  11. - Hatam GR, Adnani SJ, Asgari Q, Fallah E, Motazedian MH, Sadjjadi SM, et al. First report of natural infection in cats with Leishmania infantum in Iran. Vector Borne Zoonotic Dis. 2010;10:313-6. doi: 10.1089/vbz.2009.0023. PubMed PMID: 19492951.
  12. - Sarkari B, Hatam GR, Adnani SJ, Asgari Q. Seroprevalence of feline leishmaniasis in areas of Iran where Leishmania infantum is endemic. Ann Trop Med Parasitol. 2009;103:275-7. doi: 10.1179/136485909X398276. PubMed PMID: 19341541.
  13. - Sarkari B, Hatam G, Ghatee M. Epidemiological features of visceral leishmaniasis in fars province, southern iran. Iran J Public Health. 2012;41:94-9. PubMed PMID: 23113170; PubMed Central PMCID: PMC3481615.
  14. - Mirzaei A, Schweynoch C, Rouhani S, Parvizi P, Schonian G. Diversity of Leishmania species and of strains of Leishmania major isolated from desert rodents in different foci of cutaneous leishmaniasis in Iran. Trans R Soc Trop Med Hyg. 2014;108:502-12. doi: 10.1093/trstmh/tru085. PubMed PMID: 24980555.
  15. - Akhoundi M, Hajjaran H, Baghaei A, Mohebali M. Geographical distribution of leishmania species of human cutaneous leishmaniasis in fars province, southern iran. Iran J Parasitol. 2013;8:85-91. PubMed PMID: 23682265; PubMed Central PMCID: PMC3655245.
  16. - Azizi K, Badzohreh A, Sarkari B, Fakoorziba MR, Kalantari M, Moemenbellah-Fard MD, et al. Nested polymerase chain reaction and sequence- based detection of leishmania infection of sand flies in recently emerged endemic focus of zoonotic cutaneous leishmaniasis, southern iran. Iran J Med Sci. 2013;38:156-62. PubMed PMID: 24031105; PubMed Central PMCID: PMC3771217.
  17. - Davami MH, Motazedian MH, Kalantari M, Asgari Q, Badzohre A, Mohammadpour I. First microscopical and molecular-based characterization of Leishmania major within naturally infected Phlebotomus salehi (Diptera; Psychodidae) in Fars province, southern Iran. Ann Trop Med Parasitol. 2011;105:485-91. doi: 10.1179/1364859411Y.0000000037. PubMed PMID: 22185942; PubMed Central PMCID: PMC4100315.
  18. - Fakhar M, Mikaeili F, Hatam G, Habibi P, Karamian M, Motazedian M, et al. A molecular epidemiology survey of cutaneous leishmaniasis in patient referring to parasitology lab at shiraz school of medicine and the importance of pcr assay. jjums. 2010; 8:1-6.
  19. - Motazedian H, Karamian M, Noyes HA, Ardehali S. DNA extraction and amplification of leishmania from archived, Giemsa-stained slides, for the diagnosis of cutaneous Leishmaniasis by PCR. Ann Trop Med Parasitol. 2002;96:31-4. doi: 10.1179/000349802125000484. PubMed PMID: 11989531.
  20. - Schonian G, Kuhls K, Mauricio IL. Molecular approaches for a better understanding of the epidemiology and population genetics of Leishmania. Parasitology. 2011;138:405-25. doi: 10.1017/S0031182010001538. PubMed PMID: 21078222.
  21. - 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;47:349-58. doi: 10.1016/S0732-8893(03)00093-2. PubMed PMID: 12967749.
  22. - Reithinger R, Dujardin JC. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol. 2007;45:21-5. doi: 10.1128/JCM.02029-06. PubMed PMID: 17093038; PubMed Central PMCID: PMC1828971.
  23. - Waki K, Dutta S, Ray D, Kolli BK, Akman L, Kawazu S, et al. Transmembrane molecules for phylogenetic analyses of pathogenic protists: Leishmania-specific informative sites in hydrophilic loops of trans- endoplasmic reticulum N-acetylglucosamine-1-phosphate transferase. Eukaryot Cell. 2007;6:198-210. doi: 10.1128/EC.00282-06. PubMed PMID: 17142569; PubMed Central PMCID: PMC1797956.
  24. - Liu X, Chang KP. N-acetylglucosamine-1-phosphate transferase gene is conserved in five Leishmania spp. and overexpressed due to its amplification in their tunicamycin-resistant variants. Mol Biochem Parasitol. 1993;59:177-9. doi: 10.1016/0166-6851(93)90020-X. PubMed PMID: 8390612.
  25. - Hajjaran H, Mohebali M, Teimouri A, Oshaghi MA, Mirjalali H, Kazemi-Rad E, et al. Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene. Infect Genet Evol. 2014;26:203-12. doi: 10.1016/j.meegid.2014.05.026. PubMed PMID: 24911282.
  26. - Mahmoudzadeh-Niknam H, Ajdary S, Riazi-Rad F, Mirzadegan E, Rezaeian A, Khaze V, et al. Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran. Trop Med Int Health. 2012;17:1335-44. doi: 10.1111/j.1365-3156.2012.03078.x. PubMed PMID: 22947226.
  27. - Tashakori M, Kuhls K, Al-Jawabreh A, Mauricio IL, Schonian 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. doi: 10.1016/j.actatropica.2006.01.010. PubMed PMID: 16513079.
  28. - Ghatee MA, Sharifi I, Kuhls K, Kanannejad Z, Harandi MF, de Almeida ME, et al. Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran. Exp Parasitol. 2014;144:44-51. doi: 10.1016/j.exppara.2014.06.003. PubMed PMID: 24932536.