Cytochrome b and Molecular Typing of Leishmania spp. in a Passive Sampling of Suspected Patients with Cutaneous Leishmaniasis in Sistan and Baluchestan Province, Eastern Iran

  • Gholamreza MOTALLEB Dept. of Biology, Faculty of Sciences, University of Zabol, Zabol, Iran
  • Hadi MIRAHMADI Infectious Diseases and Tropical Medicine Research Center, Resistant Tuberculosis Institute, Zahedan University of Medical Sciences, Zahedan, Iran Dept. of Parasitology and Mycology, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran
  • Ahmad ZARE-ZADEH Dept. of Biology, Faculty of Sciences, University Campus, Graduate School, University of Zabol, Zabol, Iran
  • Ahmad MEHRAVARAN Infectious Diseases and Tropical Medicine Research Center, Resistant Tuberculosis Institute, Zahedan University of Medical Sciences, Zahedan, Iran Dept. of Parasitology and Mycology, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran
Keywords: Leishmania major, Leishmania tropica, Cytochrome b, PCR, DNA sequencing, Iran

Abstract

Background: Despite the high prevalence and drug resistance of disease in Sistan and Baluchestan Province of Iran, the species of cutaneous leishmaniasis (CL) has not been identified. In the present study, cytochrome b (Cyt b) was used in Sistan and Baluchestan to find species of Leishmania in suspected patients of CL using PCR-RFLP and DNA sequencing. Methods: This study was conducted from Oct 2015 to Oct 2016. The samples were collected from the individuals clinically suspected to CL and referred to Iran Shahr, Chabahar, Khash, Zabol, Zahedan, Mirjaveh, and Nikshahr health centers. Overall, 700 Giemsa-Stained slides from the wound of patients suspected of CL were passive collected and examined under a light microscope at×1000. After DNA extraction, positive samples were used for Cyt b detection by PCR-RFLP to determine the parasite species. One hundred positive slides were selected for molecular studies. Among positive samples, 20% were sequenced. To compare the results of sequences, molecular evolutionary genetic analysis (MEGA6) was used.Results: Overall, 53 samples were identified as L.‎ major and 47 samples (47%) L. tropica. Cyt b in L. major and L. tropica is converted to 400 and 480 bp and 130, 215 and 535 bp pieces respectively. In the isolated L. tropica and L. major, nucleotide changes were 3-5 (mainly in wobble site). Conclusion: Infection was more related to L. major. PCR-RFLP method has a high sensitivity for diagnosis of Leishmania species. 

References

World Health Organization. 2016. Leishmaniasis: Epidemiological situation. http://www.who.int/leishmaniasis/burden/en

Gholamrezaei M, Mohebali M, Hanafi-Bojd AA, Sedaghat MM, Shirzadi MR. Ecological Niche Modeling of main reservoir hosts of zoonoticcutaneous leishmaniasis in Iran. Acta Trop. 2016; 160:44-52.

Pourmohammadi B, Motazedian MH, Hatam GR, Kalantari M, Habibi P, Sarkari B. Comparison of Three Methods for diagnosis of cutaneous leishmaniasis. Iran J Parasitol.2010;5(4):1-8.

Shirzadi MR, Esfahani SB, Mohebali M, Ershadi MRY, Gharachorlo F, Razavia MR, Postigo JA. Epidemiological status of leishmaniasis in the Islamic Republic of Iran, 1983–2012. East Mediterr Health J.2015;21(10):736-742.

Asato Y, Oshiro M, Myint CK et al. Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing. Exp Parasitol.2009;121(4),352-361.

Luyo-Acero GE, Uezato H, Oshiro M et al. Sequence variation of the cytochrome b gene of various human infecting members of the genus Leishmania and their phylogeny. Parasitol. 2004;128:483-491.

Schalling HD, Oskam L. Molecular biological application in the diagnosis and control of leishmaniasis and parasite identification. Trop Med Int Health.2002;7(8):641-51.

Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I. Identification and differentiation of Leishmania species in clinical sample by PCR amplification of the miniexone sequence and subsequence re-striction fragment length polimorfism analysis. J Clin Microbiol.2003;41(7):3147-53.

Schonian G, Nasereddin A, Dinse N et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis.2003;3;47(1):349-58.

Baily MS, Lockwood DN. Cutaneous of leishmaniasis. Clin Dermatol.2007; 25(2):203-11.

Tashakori M, Kuhls K, Al-Jawabreh A, Mauricio I, Schönian G, Farajnia S, limohamadian MH. Leishmania major: Genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer. Acta Trop.2006; 98:52-58.

Al-Jawebreh A, Schoenian G, Hamarsheh O, Presber W. Clinical diagnosis of leishmaniasis: A comparison study between standardized graded direct microscopy and ITS-1 PCR of gimsa stained smears. Acta Trop. 2006; 99(1):55-61.

Fakhr M, Mikkaeili F, Hatam GR, Habibi P, Karamian M, Motazedian MH. Molecular epidemiology survey of cutaneous leishmaniasis in referral patient parasitology lab at Shiraz School of Medicine and importance application of PCR for diagnosis of disease. J Jahrom Univ Med Sci.2010;8(1):1-5.

Mansueto P, Vitale G, Di Lorenzo G, Rini GB, Mansueto S, Cillari E. Immunopathology of leishmaniasis: an update. Int J Immunopathol Pharmacol.2007;20(3):435-45.

Joshi MB, Dwyer DM. Molecular and functional analyses of a novel class I secretory nuclease from the human pathogen, Leishmania donovani. J Biol Chem.2007; 282(13):10079-95.

Published
2017-12-26
How to Cite
1.
MOTALLEB G, MIRAHMADI H, ZARE-ZADEH A, MEHRAVARAN A. Cytochrome b and Molecular Typing of Leishmania spp. in a Passive Sampling of Suspected Patients with Cutaneous Leishmaniasis in Sistan and Baluchestan Province, Eastern Iran. IJPA. 12(4):534-43.
Section
Original Article(s)