اهلاً وسهلاً بالحضور الكرام

1. اهلاً وسهلاً بالحضور الكرام اهلاوسهلا بالحضور الكرام Welcome

2. Dr.SundusNsaifAL-Hucheimi College of Medicine University of Kufa

3. بسم الله الرحمن الرحيم سُبحَانَكَ لا عِلمَ لَنا إِلا ماَ عَلَمَّتناَ إِّنكَ أنت اْلعِلَيِم ألحكيِمَ صدق الله العلي العظيم))

4. Tracking of Cutaneous Leishmaniasis by Parasitological, Molecular and Biochemical Analysis

5. Introduction Leishmaniasis is one of the infectious parasitic diseases transmitted by biting sand flies with highest incidence in the world.WHOhas ranked leishmaniasis as one of the six important infectious diseases .

6. There are an estimated number of 12 million cases worldwide, with 2 million new cases added each year .90% of all cases occur in Afghanistan, Brazil, Iran ,Iraq, Saudi Arabia, and Syria . • *CL was mostly present in central Iraq and the Greater Baghdad area, but since the Gulf War the disease has extended to new areas rarely affected before, such as Missan, Thi-Qar, and Basrah . • *The number of CL cases doubled in study area after 2004,and caused several outbreak such as epidemic outbreaks have been reported in Qadessia province in 2008 . • *Leishmaniasis still constitute a major public health problem and underreported to a large degree.

7. Aims of study The present study aims at : 1.The specification of the different species and strain of genus Leishmania by nested –PCR and cellulose acetate electrophoresis. 2.The evaluation of the incidence and epidemiological aspects of cutaneous leishmaniasis in the study area . 3.The detection and containing epidemics in the early stages and providing early diagnosis .

8. Study design

9. The study included 126 patients with lesions clinically suggestive to be cutaneous leishmaniasis whom attended to hospitals from five Iraqi provinces as following: Al-Najaf , Babylon , Al-Qadisya, Karbala and Kut province . parasitology Culture NNN,Schneider Drosphila,RBMI1640 Direct smear examination positive positive negative negative biochemical molecular

10. Culture NNN,SchneiderDrosphila ,RBMI1640 Molecular test biochemical test DNA EXTRACTION by using the Bioneer Genomic DNA extraction Kit The comparisons were made by examination of the CAE of 4 soluble enzymes Nested-pcr LP 6PGDH GPI First step to detect leishmania genus Second step to identify leishmania spp. MPI

11. The result

12. Distribution of the type of infection according to type of lesions and age groups ofpatients. * S = Single , M = Multiple

13. Photograph of patient with cutaneous leishmaniasis (wet type)

14. Direct smear examination according to sex and age groups ofpatients.

15. Direct skin smear (Giemsa stain , 100 x) • showing amastigotesin lesion aspiration

16. Direct skin smear (Giemsa stain , 100 x) showing amastigotes in skin scrapings

17. Direct skin smear (Giemsa stain , 100 x) showing amastigotesin discharged of blood

18. Culture according to sex and age groups ofpatients.

19. Direct smear (Giemsa stain , 100 x) showing Promastigotes in the culture

20. Direct smear (Without stain) showing Promastigotes in the clture

21. Nested-PCR (first step) according to sex and age groups ofpatients.

22. Nested-PCR (second step) according to sex and age groups ofpatients.

23. Agarose gel electrophoresis ofLeishmaniaisolates in nested-PCR(first step) .Lane 1-15Leishmaniaspp , Lane 16,DNA size marker 100Pb

24. Agarose gel electrophoresis of Leishmania isolates in nested-PCR(second step) .Lane 1,13 ,DNA size marker 100 Pb;Lane 2,negative control ;Lane 3,6,7,11 (560Pb) L.majorisolates;Lane 4,5,8,9,10,12(750Pb)L. tropicaisolates

25. Cellulose acetate electrophoresis according to sex and age groups ofpatients.

26. Conclusion Nested-PCR is a reliable method for the diagnosis and identification of Leishmania species and can be applied in epidemiologic investigations. The cellulose acetate electrophoresis (isoenzymeanalysis) was an ideal method for discrimination of Leishmania. spp. variants.

27. Conclusion 3. Both L. major and L. tropicawere the causative agents of cutaneous leishmaniasis in study area . 4.L.major was the main species causing CL in comparing with L.tropica in the present study.

28. Recommendations 1. Opening number of research centres situated in the country offer DNA applications for so-called “parasite tracking.” After collection in the field, samples can be simply transferred to such a centre for further analysis.

29. Recommendations 2. More experimental studies for effective vaccine are developed for leishmaniasis.

30. Recommendations 3. Control of sand flies through residual insecticides spraying, Since L. major(the dominant species.) is an zoonotic parasite, therefore, it is emphasize on rodents eradication program and treatment of patients.

31. Recommendations 4. General physicians must be have a clear idea about diagnosis and treatment besides a fair knowledge about its pathophysiology specially in rural areas where this disease is most prevalent.

32. Recommendations 5. To understand the atypical behavior of Leishmania spp. in Iraq, studies need to be directed toward understanding the vector bionomics and reservoir hosts for this parasite.

34. THANK YOU