8-Rahbarian

JRHS 2009; 9(2): 48-51

Copyright © Journal of Research in Health Sciences

Identification of Leishmania Species Isolated from Human Cutaneous Leishmaniasis Using PCR Method

Rahbarian N( PhD)a,  Mesgarian A( PhD)b,  Mahmoudi Rad M( PhD)a,  Hajaran H( PhD)c  Shahbazi F( PhD)b,  Mesgarian Z( BSc)d ,  Taghipour N(MsC)b

Cellular and Molecular Biology Research Center, Shahid Behesthi University, M.C., Tehran, Iran

b Department of Medical Parasitology and Mycology, Medicine Faculty, Shahid Behesthi University, M.C., Tehran, Iran

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

d Gonbad-e Qabus Health Center, Golestan, Iran

*Corresponding author:  Nourina Rahbarian, E-mail: nrahbarian@yahoo.com

Received: 2 July 2009; Accepted: 3 September 2009

Abstract

Background:  To determine the epidemiological status of cutaneous leishmaniasis outbreak, isolation and identification of the agent parasite, Leishmania, using PCR method in Gonbad-e Qabus County, north Iran, during 2006-2007.

Methods: Data were collected on the prevalence of scars and ulcers over a period of 3 months among 6990 inhabitants of five villages around Gonbad-e Qabus County, north Iran, during 2006-2007. Cul­tured promastigotes were identified using PCR technique. Its1 and its2 of Non Coding Transcribed re­gion at ribosomal DNA of 46 Leishmania isolates were amplified and the PCR products were sepa­rated by electrophoresis in 1.5% agarose gel (200 mA, 140 V), visualized by staining with ethidium bro­mide, and photographed.

Results: Among 6990 inhabitants of 5 villages, 62.9% were identified as scars and 1.5% as active le­sions. Individuals 11 to 20 years were the most highly infected age group. All the parasite isolates were Leishmania major.

Conclusion: Cutaneous leishmaniasis due to L. major is endemic in Gonbad-e Qabus County, north Iran.

Keywords: Cutaneous leishmaniasis, ITS-PCR, Epidemiology, Iran

Introduction

Leishmaniasis, a vector born disease, caused by obligate intramacrophage protozoa, is characterized by diversity and complexity. This parasite can cause a species dependent spectrum of diseases, ranging from self-healing cutaneous leishmaniasis to visceral infec­tions that are fatal. Human cutaneous and visceral leishmaniasis both occur in some parts of Iran. In Iran, L. major is causative agent of zoonotic cutaneous leishmaniasis (ZCL). L. tropica causes an­throponetic cutaneous leish-ma­niasis (ACL) considered as old endemic disease in many parts of Iran (1, 2).

This study was done to inspect cutaneous leishmaniasis (CL) in Gonbad-e Qabus bor­der villages using ITS-PCR method during 2006-2007.

Methods

To determine the CL species we did a de­scriptive research study in studied area. A total of 6990 inhabitants of five villages around Gonbad-e Qabus County, north Iran, were examined for CL and 124 patients, who had ulcers, were tested in public health cen­ter of this county, during 2006-2007. Culti­vated specimens were transported to Cellular and Molecular Biology Research Center in Shahid Beheshti University of Medical Sci­ences, Iran. Diagnosis was done by direct detection of parasites by microscopic exami­nation of clinical specimens and by cultiva­tion. The patients consented to their partici­pation in the study and the study was ap­proved by the Ethics Committee of Shahid Beheshti University of Medical Sciences. For each case having ulcers or scars, a form was completed to record the necessary in­forma­tion such as name, address, age, sex, number of ulcers or scars, site of ulcer(s) or scar(s), date and place of acquiring the dis­ease, etc.

Samples were taken from 46 patients (who did not have secondary infection) and cul­tured in NNN media, sloppy Evans medium and Schneider's medium with 10% (v/v) FBS, incubated at 22-25 °C and monitored every 3 d, from day 4 for growth. Cul­tures were exam­ined for 6 wk before be­ing con­sidered having no growth. All posi­tive cultures were subcul­tured in RPMI1640 media every 15 d (3).

For DNA extraction, the parasites were added in 500µL of lysis buffer (10mM Tris-HCl [pH 8.0], 0.32M sucrose, 5mM MgCl2 and 1% SDS), digested at 37 °C for 24 h, and then 500 µL phenol was added and sedi­mented at 8000 rpm for 5 min. The super­natants were diluted in 500µL chloroform and were sedi­mented at 8000rpm. DNA pre­cipitation was done in ethanol with 3 M so­dium acetate at -20 °C for 1h. The remaining pellet was dried at 37 °C and dissolved in 30 µL of TE buffer. The DNA concentration was determined by measuring optical ab­sorbance at 260 nm (4).

Specific oligonucleotide primer was de­signed based on GenBank accession no. AJ300485, LeishF (5-CAA CAC GCC GCC TCC TCT CT-3), LeishR (5-AAA CAA AGG TTG TCN GGG -3). PCR reaction was carried out based on the following con­ditions: initial denatura­tion at 94 °C for 5 min, followed by 30 cycles including dena­turation at 94°C for 35 S, an­nealing at 60°C for 35 s, extension at 72°C for 45 S, and final extension at 72°C for 5 min. At the end, 5 µl of the reaction mix was analyzed by 2% aga­rose gel electrophoresis. The primer was evaluated with Leishmania standard species including L. infantum (MCAN/ IR/97/LON490), L. major (MHOM/IR/75/ER), and L. tropica (MHOM/IR/o4/Mash10 (5).

Results

Electrophoresis patterns from each isolates were compared with reference strains of L. tropica, L. major and L. infantum. PCR prod­ucts from L. major, L. tropica and L. infan­tum were 626, 485, and 565 bp, respec­tively. The results showed that from 46 iso­lates, all (100%) were L. major in compari­son to ref­erence strains (Fig. 1).

Forty six isolates determined by ITS-PCR method as L. major, could produce ulcer at the base tail of BALB/c mice, 4-12 wk after in­oculation.

Figure. 1: Amplification of the ITS-1 and ITS-2 frag­ments in ribosomal DNA, using primers LeishF and LeishR. Six clinical isolates that were determined as L. major by ITS-PCR (lanes 1-6), negative control (lane 7), molecular weight marker (lane 8), reference strains of L. major, L. tropica, and L. infantum (lanes 9-11, respectively)

Discussion

Two species of Leishmania are involved in CL infections in Iran. L. major is causative agent of ZCL and L. tropica causes ACL. ZCL is found in many rural foci in the north, east and south of Iran (6).

The ability to distinguish among Leishmania is crucial when proscribing treatment, as well as in epidemiologic studies to determine possible control measures. The classic diag­nostic techniques for cutaneous leishmani­asis include microscopic diagnosis, the Mon­tenegro skin test, serological diagnosis, and parasite culture, all of which have im­portant limitations (7).

In this study, we used ITS-PCR method to determine species of Leishmania isolates from human patients clinically diagnosed as having tegumentary leishmaniasis. The re­sults indi­cated that L. major species was dominant in the studied areas of Gonbad-e Qabus County, north Iran, and ITS-PCR technique was a suitable tool for Leishmania characterization in epidemiological studies.

Bensossuan et al. (8) used restriction enzyme analysis of the ITS-PCR product and identi­fied 74.6% positive samples, which included strains of L. major (50.9%), L. tropica (47.2%), and L. braziliensis complex (1.9%). Cu­polillo (9) classified Leishmania species of new world using a ITS-PCR method. Jawabreh et al. (10) isolated 49 strains of Leishmania from the cutaneous lesions of Palestinians living in and around Jericho and applied ITS-PCR to their cultured promas­tigotes. Of 49 cultures, 28(57%) were L. major and 21(43%) L.  tropica.

Although CL has been present in Gonbad-e Qabus border villages in recent years, this is the first time that an endemic focus of zoono­tic CL has been identified. There are many active colonies of rodents around some of the in­fected villages. The occurrence of ZCL in Gonbad-e Qabus border villages seems to be the result of construction of buildings in farms near the colonies of ro­dents, storage of waste materials around the villages, which are suitable for building nests by rodents.

In conclusion, cutaneous leishmaniasis due to L. major is endemic in Gonbad-e Qabus County, north Iran.

Acknowledgements

The present study was conducted with the support of Cellular and Molecular Biology Research Center in Shahid Beheshti Univer­sity of Medical Sciences, Tehran, Iran.

References

  1. Talari SA, Rezvan Talaei R, Shajari GR. A clinical and epidemiology survey of cutaneous leishmaniasis in childhood. J Med Sci. 2006; 6(4):693-97.
  2. Nadim A. Leishmaniasis in: Epidemiol¬ogy and control of common disorders in Iran. Ed, Azizi F, Janghorbani M, Hatam H, 2nd ed. Endocrine & Metabolism Re¬search Center. Shaheed Beheshti Uni¬versity of Medical Sciences, 2000: pp. 524-34.
  3. Evans DA, Godfrey D, Lanham S, Lanotte G, Modabber F, Schnur L. Hand¬book on isolation, characterization and cryopreservation of Leishmania. World Health Organization, Geneva, Switzerland, 1989.
  4. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Man¬ual. Cold Spring Harbor Laboratory Press, NY, 1989.
  5. Shahbazi F, Shahabi S, Kazemi B, Mohebali M, Abadi AR, Zare Z. Evalua¬tion of PCR assay in diagnosis and identification of cutaneous leishmani¬asis: a comparison with the parasitologi¬cal methods. Parasitol Res. 2008; 103(5): 1159-62.
  6. Talari SA, Rezvan Talaei R, Shajari GR. Childhood cutaneous leishmani¬asis: report of 117 cases from Iran. Ko¬rean J Parasitol. 2006; 44(4):355-360.
  7. Schonian G, Nasereddin A, Dinse N. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diag Microbiol Infect. 2003; 4(1): 349-58.
  8. Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. Complete Record. J Clin Microbiol. 2006; 44(4):1435-39.
  9. Cupolillo E. Genetic relationships and evolution of new world Lieshmania: sug¬gestion of evolution link with En¬dot¬ry¬panum. Mem Inst Oswaldo cruz. 1998; 93:186.
  10. Jawabreh AO. The recent emergence of Leishmania tropica in Jericho (A'riha) and its environs, a classical focus of L. major. Trop Med Int Health. 2004; 9(7): 812-1


JRHS Office:

School of Public Health, Hamadan University of Medical Sciences, Shaheed Fahmideh Ave. Hamadan, Islamic Republic of Iran

Postal code: 6517838695, PO box: 65175-4171

Tel: +98 81 38380292, Fax: +98 81 38380509

E-mail: jrhs@umsha.ac.ir