1. Molecular diagnosis of cutaneous leishmaniasis in Jordan
Nawal Hijjawi Department of Medical Laboratory Sciences Faculty of Allied Health Sciences Hashemite University
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2. Cutaneous leishmaniasis
Cutanoeus leishmaniasis is
an endemic disease in Jordan
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3. Leishmania species in Jordan
Two species
Leishamania major
Leishmania tropica

4. Leishmania major
Zoonotic cutaneous leishmaniasis by L. major is the main form of the disease in Jordan
Reservoir host: Primary, Psammomys obesus (Saliba et al., 1994)
Secondary, Meriones sp.
Vector: Phlebotomus papatasi (Janini et al., 1994)
Meriones
Psammomys obesus
Phlebotomus papatasi
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5. Leishmania tropica Has been restricted to few known foci in Jordan
Reservoir hosts: Hyraxes and Canines (suspected)
Vector: Ph. sergenti (Saliba et al., 1993; Kamhawi et al., 1995)
Hyraxes
Canine
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6. Local prevalence and distribution of cutaneous leishmaniasis

7. Cutaneous leishmaniasis is severely under-reported in Jordan Cutaneous leishmaniasis in the Jordanian side of the Jordan Valley: severe under-reporting and consequences on public health management Mosleh IM, Geith E, Natsheh L, Abdul-Dayem M, Abotteen N. Source Department of Biological Sciences, Faculty of Science, University of Jordan, Amman, Jordan. Trop Med Int Health Jun;13(6): Epub 2008 Mar Most reported cases mainly reflect patients seeking care in MOH facilities 2. No cases reported from the private sector or the RMS (Royal Medical services)
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8. Prevalence of Cutaneous leishmaniasis is severely underestimated
Other factors:
Nature of the disease (mild disease) and considered in some places as a normal event.
It occurs in remote and rural areas with limited resources for diagnosis and treatment.
Inadequate diagnosis
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9. Regional prevalence and distribution of cutaneous leishmaniasis
Du, R., Hotez, P. J., Al-Salem, W. S., and Acosta-Serrano, “Old World Cutaneous Leishmaniasis and
Refugee Crises in the Middle East and North Africa” PLOS Neglected Tropical Diseases, 10, 5, e (2016)

10. Cutaneous leishmaniasis and Refugee Crises in the Middle East
This map shows how the disease has spread out from Syria into countries such as Jordan, Iraq and Turkey

11. Distribution of sand fly and Leishmania species across Middle East countries
This graphic shows the spread of the sand flies across the Middle East that allowed the disease to thrive

12. There is an urgent need to strengthen the capabilities of diagnosis and also to differentiate the Leishmania species especially during outbreaks
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13. Diagnosis and identification of Leishmania species
in Jordan
1. Clinical picture of the lesion 2. Geographical distribution 3. Microscopy of Giemsa-stained smears 4. Molecular methods (PCR-based techniques) have never been used in Jordan
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14. Parasite detection and identification in Giemsa-stained smears
Amastigotes in macrophages
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15. Differences between cutaneous Leismania sp.
FEATURES
WET TYPE
DRY TYPE
Causative species
L. major
L. tropica
Incubation period
2-6 weeks
Heals in 3–5
months with
scarring
2-8 months
Heals over 1–2
years and scars, rarely spread
Geog. distribution
Rural
Urban
Reservoir
Mainly rodents
Dogs/ human
Type of lesions
Early ulcerative
Late ulcerative
Number of lesions
Often multiple
Often single sore, slowly
enlarges, crusty appearance
Sites of lesions
Usually legs & feet
Usually face
Natural course
Shorter (6 months)
Longer (≥ 1 year)
L. tropica
L. major
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16. Studies in Jordan for species discrimination using molecular-based assays
Two Studies

17. The identification of L
The identification of L. major as the causative species in considerable number of CL cases, shows that the JMidJV is a classic focus of L. major

19. No of Clinical samples examined Microscopy +ve (Amastigotes)
Molecular diagnosis
DNA-based assay using internal transcribed spacer1- polymerase chain reaction (ITS1-PCR) was applied for the diagnosis of leishmaniasis in Jordan
1: Specific diagnosis of the disease in clinical samples
No of Clinical samples examined
Microscopy +ve (Amastigotes)
ITS1-PCR +ve 41 28 30
Agarose gel electrophoresis (2%) of random PCR products ( bp) which were extracted from the 30 positive Leishmania samples. Lane M: 100 bp DNA ladder. Lanes 1-12: PCR products randomly selected from 30 clinical samples

20. 2: Identification of different Leishmania species Digestion of the ITS1 amplicons with the restriction enzyme Hae III
ITS1-PCR +ve
Leishmania major
Leishmania tropica 30 28 2

21. The Impact of Syrian Immigration on the Prevalence of Cutaneous leishmaniasis in Jordan???

22. Demographic data

23. Sampling sites
Jordan: (North Ghor, Irbid, AL- mashare`a, Jarash, Ajloun, Zarqa, Amman, Sahab, Arabah, Iraq Al- Amir, Dair alla, Dead Sea, Sowimeh, Ghor Alsaffe, South Shouna, Karak). Syrian refegees from Al-Azraq Camp: (Deir ez-zor, Aleppo, Palmyra, Hims, Idlib).

24. DNA based Leishmania typing methods
Three PCR-based typing techniques
Internal transcribed spacer 1 (ITS1)
Nested PCR of the ITS1-5.8S rDNA
Kinetoplast DNA (kDNA).

25. Criteria for species typing methods
Restriction fragment length polymorphism (RFLP)
A) Hae III restriction enzyme: discriminate L. major and L. tropica
B) Rsa 1 restriction enzyme: discriminate L. tropica subspecies
Sequencing and phylogenetic analysis

27. 20 L. major (14 Jordanian + 6 Syrian)

28. 9 L. major (6 Jordanian + 3 Syrian)

29. Sequencing and phylogenetic tree analysis of ITS1-5.8S rDNA gene

30. Sequencing and Phylogenetic tree analysis of ITS1 gene

31. Conclusion
L. tropica and L. major are the causative agent of CL among Jordanian and Syrian refugees and there is an increase in the risk of Leishmania transmission from Syrian refugees especially in the areas around the camps due to the presence of the sand-fly vector
Anthroponotic cutaneous leishmaniasis (ACL) due to L. tropica is dominant in Syria and there is a chance that his might be transmitted to Jordan with the presence of the sand fly vector P. sergenti.

32. The implementation of PCR based assays for the specific diagnosis of leishmaniasis is becoming an urgent need and species discrimination is important especially during outbreaks
a. ITS1-5.8S rDNA PCR proved to be a sensitive method for L. tropica discrimination.
b. ITS1-PCR RFLP showed high sensitivity to identify Leishmania species and highly sensitive for L. major sequencing.
c. kDNA minicircle gene showed lowest sensitivity compared to ITS1-5.8S rDNA PCR and ITS1-PCR RFLP diagnose and discriminate Leishmania species