Detection, genotyping, and phylogenetic analysis of Leishmania isolates collected from infected Jordanian residents and Syrian refugees who suffered from cutaneous leishmaniasis

  • Kamal J. F. HijawiEmail author
  • Nawal S. Hijjawi
  • Jwan H. Ibbini
Genetics, Evolution, and Phylogeny - Original Paper


Leishmania is a parasitic protozoan which is transmitted to humans through the bite of an infected female Phlebotomus and Lutzomyia sand flies. Cutaneous leishmaniasis (CL), caused by Leishmania major and L. tropica, is an endemic disease in many areas of Jordan and considered as a major public health problem. The political instability in the Syrian Arab Republic has resulted in the immigration of large number of refugees into Jordan where most of them resided in camps near the Syrian borders. Therefore, the main objective of the present study was to inspect Leishmania species/genotypes which are responsible for CL infections among Syrian refugees and compare them with the recovered species/genotypes isolated from Jordanian patients. Three molecular-based assays (ITS1-PCR-RFLP, Nested ITS1-5.8S rDNA PCR, and Kinetoplast DNA PCR) followed by sequencing and phylogenetic analysis were undertaken and compared for their efficiency to confirm CL diagnosis and genotype the infecting Leishmania species. Thereafter, the evolutionary relationships among various Leishmania isolates from Syrian and Jordanian CL patients were elucidated. Results from the present study indicated that 20 and 9 out of the inspected 66 patients (39 Jordanian and 27 Syrian) were infected with L. major and L. tropica respectively. ITS1-PCR RFLP typing proved to be more sensitive in the detection of Leishmania species (positive in 44% of the isolates) compared to both ITS1-5.8S rDNA gene and Kinetoplast DNA PCR which were successful in identifying Leishmania species only in 23% and 33% of the isolates respectively. Sequencing and phylogenetic analysis of ITS1 and ITS1-5.8S rDNA genes revealed high levels of heterogeneity among the sequenced isolates. One sample typed as L. tropica from Jordanian patient showed high similarity with L. tropica sample isolated from a Syrian patient in a Lebanon refugee camp; therefore, the need for comprehensive studies to confirm if any new L. tropica strains might be introduced to Jordan by Syrian refugees is urgently indicated. These observations highlighted the need for further studies to clarify the risk status of species and strains which might be introduced from Syria to Jordan.


Leishmania Cutaneous leishmaniasis (CL) ITS1 5.8S rDNA gene PCR RFLP Sequence analysis 



This project was funded from the Deanship of Research and Graduate Studies at Hashemite University and the IBDAA Humanitarian Foundation in Amman, Jordan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Ajaoud M, Es-Sette N, Hamdi S et al (2013) Detection and molecular typing of Leishmania tropica from Phlebotomus sergenti and lesions of cutaneous leishmaniasis in an emerging focus of Morocco. Parasit Vectors 6:1–9. CrossRefGoogle Scholar
  2. Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P, Delaunay P, Sereno D (2016) A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl Trop Dis 10:1–40. CrossRefGoogle Scholar
  3. Al-Abdallat M, Shadfan B, Kanani KA (2015) Recent collection of sandflies of the genus Phlebotomus (Diptera : Psychodidae) from Jordan , with a checklist of previous records. Jordan J Biol Sci 8:193–197. CrossRefGoogle Scholar
  4. Al-Dakhil AA, Al-Ajmi RA, Siddiqi NJ, Ayaad TH (2017) Molecular typing of phlebotomine sand flies in al-Madinah and Asir regions, Saudi Arabia using PCR–RFLP of 18S ribosomal RNA gene. Saudi J Biol Sci 24:1697–1703. CrossRefGoogle Scholar
  5. Al-Nahhas SA, Kaldas RM (2013) Characterization of Leishmania species isolated from cutaneous human samples from central region of Syria by RFLP analysis. ISRN Parasitol 2013:1–5. CrossRefGoogle Scholar
  6. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, Boer M, the WHO Leishmaniasis Control Team (2012) Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7:e35671. CrossRefGoogle Scholar
  7. Amro A, Gashout A, Al-Dwibe H et al (2012) First molecular epidemiological study of cutaneous Leishmaniasis in Libya. PLoS Negl Trop Dis 6:e1700. CrossRefGoogle Scholar
  8. Arbaji AK, Gradoni L, Gramiccia M (1993) Leishmanin skin test survey in a focus of high endemicity of Leishmania major in Jordan. Acta Trop 54:77–79. CrossRefGoogle Scholar
  9. Ashford RW (2000) The leishmaniases as emerging and reemerging zoonoses. Int J Parasitol 30:1269–1281. CrossRefGoogle Scholar
  10. Azmi K, Schnur L, Schonian G, Nasereddin A, Pratlong F, el Baidouri F, Ravel C, Dedet JP, Ereqat S, Abdeen Z (2012) Genetic, serological and biochemical characterization of Leishmania tropica from foci in northern Palestine and discovery of zymodeme MON-307. Parasit Vectors 5:1–14. CrossRefGoogle Scholar
  11. Bates PA (2007) Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int J Parasitol 37:1097–1106. CrossRefGoogle Scholar
  12. Bensoussan E, Nasereddin A, Schnur LF et al (2006) Comparison of PCR assays for diagnosis of cutaneous leishmaniasis comparison of PCR assays for diagnosis of cutaneous Leishmaniasis. J Clin Microbiol 44:1435–1439. CrossRefGoogle Scholar
  13. Boggild AK, Valencia BM, Espinosa D, Veland N, Ramos AP, Arevalo J, Llanos-Cuentas A, Low DE (2010) Detection and species identification of Leishmania DNA from filter paper lesion impressions for patients with American cutaneous leishmaniasis. Clin Infect Dis 50:e1–e6. CrossRefGoogle Scholar
  14. Bordbar A, Parvizi P (2014) High infection frequency, low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran. Acta Trop 133:69–72. CrossRefGoogle Scholar
  15. De Almeida ME, Steurer FJ, Koru O et al (2011) Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2. J Clin Microbiol 49:3143–3149. CrossRefGoogle Scholar
  16. de Vries HJC, Reedijk SH, Schallig HDFH (2015) Cutaneous leishmaniasis: recent developments in diagnosis and management. Am J Clin Dermatol 16:99–109. CrossRefGoogle Scholar
  17. Desjeux P (2001) The increase in risk factors for leishmaniasis worldwide. Trans R Soc Trop Med Hyg 95:239–243. CrossRefGoogle Scholar
  18. Desjeux P (2004) Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27:305–318. CrossRefGoogle Scholar
  19. Doroodgar A, Sadr F, Razavi MR, Doroodgar M, Asmar M, Doroodgar M (2015) A new focus of zoonotic cutaneous leishmaniasis in Isfahan Province, Central Iran. Asian Pac J Trop Dis 5:S54–S58. CrossRefGoogle Scholar
  20. Du R, Hotez PJ, Al-Salem WS, Acosta-Serrano A (2016) Old World cutaneous leishmaniasis and refugee crises in the Middle East and North Africa. PLoS Negl Trop Dis 10:1–11. CrossRefGoogle Scholar
  21. El-Beshbishy HA, Al-Ali KH, El-Badry AA (2013) Molecular characterization of cutaneous leishmaniasis in Al-Madinah Al-Munawarah province, western Saudi Arabia. Int J Infect Dis 17:e334–e338. CrossRefGoogle Scholar
  22. Elmahallawy EK, Sampedro Martínez A, Rodriguez-Granger J et al (2014) Diagnosis of leishmaniasis. J Infect Dev Ctries 8:961–972. CrossRefGoogle Scholar
  23. Es-Sette N, Ajaoud M, Laamrani-Idrissi A, Mellouki F, Lemrani M (2014) Molecular detection and identification of Leishmania infection in naturally infected sand flies in a focus of cutaneous leishmaniasis in northern Morocco. Parasit Vectors 7:1–8. CrossRefGoogle Scholar
  24. Guerbouj S, Mkada Driss I, Guizani I (2014) Molecular tools for understanding eco-epidemiology, diversity and pathogenesis of Leishmania parasites. In: Leishmaniasis - trends epidemiol diagnosis and treatment. InTech, Claborn, pp 62–88Google Scholar
  25. Haddad N, Saliba H, Altawil A, Villinsky J, al-Nahhas S (2015) Cutaneous leishmaniasis in the central provinces of Hama and Edlib in Syria: vector identification and parasite typing. Parasit Vectors 8(1).
  26. Hajjaran H, Mohebali M, Abaei MR, Oshaghi MA, Zarei Z, Charehdar S, Mirjalali H, Sharifdini M, Teimouri A (2013) Natural infection and phylogenetic classification of Leishmania spp. infecting Rhombomys opimus, a primary reservoir host of zoonotic cutaneous leishmaniasis in northeast Iran. Trans R Soc Trop Med Hyg 107:550–557. CrossRefGoogle Scholar
  27. Hayani K, Dandashli A, Weisshaar E (2015) Cutaneous leishmaniasis in Syria: clinical features, current status and the effects of war. Acta Derm Venereol 95:62–66. CrossRefGoogle Scholar
  28. Hijjawi N, Kanani KA, Rasheed M, Atoum M, Abdel-Dayem M, Irhimeh MR (2016) Molecular diagnosis and identification of leishmania species in Jordan from saved dry samples. Biomed Res Int 2016:1–7. CrossRefGoogle Scholar
  29. Jafari R, Najafzadeh N, Sedaghat MM, Parvizi P (2013) Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province, Iran. Asian Pac J Trop Med 6:792–797. CrossRefGoogle Scholar
  30. Jalouk L, Al Ahmed M, Gradoni L, Maroli M (2007) Insecticide-treated bednets to prevent anthroponotic cutaneous leishmaniasis in Aleppo governorate, Syria: results from two trials. Trans R Soc Trop Med Hyg 101:360–367. CrossRefGoogle Scholar
  31. Jumaian N, Kamhawi SA, Halalsheh M, Abdel-Hafez SK (1998) Short report: outbreak of cutaneous leishmaniasis in a nonimmune population of soldiers in Wadi Araba, Jordan. Am J Trop Med Hyg 58:160–162. CrossRefGoogle Scholar
  32. Kamhawi S, Abdel-Hafez SK, Arbagi A (1995a) A new focus of cutaneous leishmaniasis caused by Leishmania tropica in northern Jordan. Trans R Soc Trop Med Hyg 89(3):255–257CrossRefGoogle Scholar
  33. Kamhawi S, Abdelhafez SK, Molyneux DH (1995b) A comprehensive account of species composition, distribution and ecology of Phlebotomine sandflies in Jordan. Parasite 2:163–172. CrossRefGoogle Scholar
  34. Kamhawi S, Modi GB, Pimenta PFP et al (2000) The vectorial competence of Phlebotomus sergenti is specific for Leishmania tropica and is controlled by species-specific, lipophosphoglycan-mediated midgut attachment. Parasitology 121:25–33. CrossRefGoogle Scholar
  35. Maroli M, Feliciangeli MD, Bichaud L et al (2013) Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol 27:123–147. CrossRefGoogle Scholar
  36. Maslov DA, Yurchenko VY, Jirku M, Lukes J (2010) Two new species of trypanosomatid parasites isolated from heteroptera in Costa Rica. J Eukaryot Microbiol 57:177–188. CrossRefGoogle Scholar
  37. Monroy-ostria A, Nasereddin A, Monteon VM, et al (2014) ITS1 PCR-RFLP diagnosis and characterization of leishmania in clinical samples and strains from cases of human cutaneous leishmaniasis in States of the Mexican Southeast.
  38. Mosleh IM, Geith E, Natsheh L, Abdul-Dayem M, Abotteen N (2008) Cutaneous leishmaniasis in the Jordanian side of the Jordan Valley: severe under-reporting and consequences on public health management. Trop Med Int Health 13:855–860. CrossRefGoogle Scholar
  39. Mosleh IM, Shönian G, Geith E, al-Jawabreh A, Natsheh L (2015) The Jordanian Mid Jordan Valley is a classic focus of leishmania major as revealed by RFLP of 56 isolates and 173 ITS-1-PCR-positive clinical samples. Exp Parasitol 148:81–85. CrossRefGoogle Scholar
  40. Motazedian H, Karamian M, Noyes HA, Ardehali S (2002) DNA extraction and amplification of Leishmania from archived, Giemsa-stained slides, for the diagnosis of cutaneous leishmaniasis by PCR. Ann Trop Med Parasitol 96:31–34. CrossRefGoogle Scholar
  41. Mouttaki T, Morales-Yuste M, Merino-Espinosa G, Chiheb S, Fellah H, Martin-Sanchez J, Riyad M (2014) Molecular diagnosis of cutaneous leishmaniasis and identification of the causative Leishmania species in Morocco by using three PCR-based assays. Parasit Vectors 7:1–9. CrossRefGoogle Scholar
  42. Noyes HA, Reyburn H, Bailey JW, Smith D (1998) A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. J Clin Microbiol 36:2877–2881Google Scholar
  43. Ozaras R, Leblebicioglu H, Sunbul M, Tabak F, Balkan II, Yemisen M, Sencan I, Ozturk R (2016) The Syrian conflict and infectious diseases. Expert Rev Anti-Infect Ther 14:547–555. CrossRefGoogle Scholar
  44. Parvizi P, Moradi G, Akbari G, Farahmand M, Ready PD, Piazak N, Assmar M, Amirkhani A (2008) PCR detection and sequencing of parasite ITS-rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central Iran. Parasitol Res 103(6):1273–1278. CrossRefGoogle Scholar
  45. Postigo JAR (2010) Leishmaniasis in the World Health Organization Eastern Mediterranean Region. Int J Antimicrob Agents 36:S62–S65. CrossRefGoogle Scholar
  46. Reithinger R, Mohsen M, Aadil K, Sidiqi M, Erasmus P, Coleman PG (2003) Anthroponotic cutaneous leishmaniasis, Kabul, Afghanistan. Emerg Infect Dis 9:727–729. CrossRefGoogle Scholar
  47. Reithinger R, Dujardin J, Louzir H et al (2007) Cutaneous leishmaniasis. Clin Dermatol 25:203–211. CrossRefGoogle Scholar
  48. 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. CrossRefGoogle Scholar
  49. Salam N, Al-Shaqha WM, Azzi A (2014) Leishmaniasis in the Middle East: incidence and epidemiology. PLoS Negl Trop Dis 8:1–8. CrossRefGoogle Scholar
  50. Saliba EK, Hagishi GI, Yates JA, Oumeish OY (1988) Cutaneous leishmaniasis in Jordan: biochemical identification of human and Psammomys obesus isolates as Leishmania major. Ann Trop Med Parasitol 82:21–25. CrossRefGoogle Scholar
  51. Saliba E, Saleh N, Bisharat Z, Oumeish O, Khoury S, Gramiccia M, Gradoni L (1993) Cutaneous leishmaniasis due to Leishmania tropica in Jordan. Trans R Soc Trop Med Hyg 87(6):633. CrossRefGoogle Scholar
  52. Salloum T, Khalifeh I, Tokajian S (2016) Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon. Parasite Epidemiology and Control 1:159–168. CrossRefGoogle Scholar
  53. Schnur LF, Nasereddin A, Schwenkenbecher JM et al (2004) The recent emergence of Leishmania tropica in Jericho (A’ riha) and its environs, a classical focus of L. major. Tropical Med Int Health 9:812–816CrossRefGoogle Scholar
  54. Schönian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HDFH, Presber W, Jaffe CL (2003) PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 47:349–358. CrossRefGoogle Scholar
  55. Schwenkenbecher JM, Wirth T, Schnur LF, Jaffe CL, Schallig H, al-Jawabreh A, Hamarsheh O, Azmi K, Pratlong F, Schönian G (2006) Microsatellite analysis reveals genetic structure of Leishmania tropica. Int J Parasitol 36:237–246. CrossRefGoogle Scholar
  56. Sharifi I, Fekri AR, Aflatonian MR, Khamesipour A, Nadim A, Mousavi MRA, Momeni AZ, Dowlati Y, Godal T, Zicker F, Smith PG, Modabber F (1998) Randomised vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniasis in Bam, Iran. Lancet 351:1540–1543. CrossRefGoogle Scholar
  57. Swick BL (2012) Polymerase chain reaction-based molecular diagnosis of cutaneous infections in Dermatopathology. Semin Cutan Med Surg 31:241–246. CrossRefGoogle Scholar
  58. Talmi-Frank D, Jaffe CL, Nasereddin A et al (2010) Leishmania tropica in rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis. Am J Trop Med Hyg 82:814–818. CrossRefGoogle Scholar
  59. Teixeira DE, Benchimol M, Rodrigues JCF, Crepaldi PH, Pimenta PFP, de Souza W (2013) The cell biology of Leishmania: how to teach using animations. PLoS Pathog 9:8–11. CrossRefGoogle Scholar
  60. Tsokana, CN, Labrini VA, George V, Vassiliki S, Katerina M, Charalambos B (2014) Molecular diagnosis of leishmaniasis, species identification and phylogenetic analysis. In leishmaniasis-trends in epidemiology, diagnosis and treatment. InTech 161–176Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Medical Laboratory Sciences, Faculty of Applied Health SciencesThe Hashemite UniversityZarqaJordan
  2. 2.Department of Land Management and Environment, Faculty of Natural Resources and EnvironmentHashemite UniversityZarqaJordan

Personalised recommendations