Leishmaniasis in Turkey: Emerging Leishmania major infections in Anatolia

Ahmet Özbilgin1, Gülnaz Çulha2, Soner Uzun 3, Mehmet Harman4, Suhan Günastı Topal5, Fulya Okudan6, Fadile Zeyrek7, Cumhur Gündüz8, İpek Östan9, Seray Töz10, Özgür Kurt11 Işın Akyar11, Ayşegül Erat 3, Dilek Güngör5, Çağla Kayabaşı 8, İbrahim Çavuş1, Patrick Bastien12, Francine Pratlong12, Tanıl Kocagöz11, Yusuf Özbel10

1 Celal Bayar University Medical School Dept. of Parasitology, Manisa; 2 Mustafa Kemal University Medical School Dept. of Microbiology and Clinical Microbiology, Hatay; 3 Akdeniz University Medical School Deparment of Dermatology,Antalya; 4 Dicle University Medical School Deparment of Dermatology,Diyarbakir; 5 Çukurova University Medical School Deparment of Dermatology,Adana; 6 Alanya State Hospital Deparment of Dermatology,Alanya, 7 Harran University Medical School Dept. of Microbiology and Clinical Microbiology, Sanliurfa; 8 Ege University Medical School Dept. of Medical Biology, İzmir; 9 Celal Bayar University Vocational School of Health Sciences, Manisa ; 10 Ege University Medical School Dept. of Parasitology, İzmir; 11 Acıbadem University Medical School Dept. of Medical Microbiology, İstanbul; - TURKEY; 12 Departement de Parasitologie-Mycologie Centre National de Reference des Leishmanioses Faculte de Médecine, Université Montpellier 1 39 Av. Charles Flahault 34295 Montpellier Cedex 5, FRANCE Abstract Materials & Methods Discussion

Objective: Here, we report 16 new cases of Cutaneous Leishmaniasis (CL) by Leishmania major, which may cause a severe clinical disease in patients, • Cutaneous leishmaniasis due to L. major, the and discuss the current threat posed by this aggresive protozoon in Turkey. zoonotic disease, have been documented in North Africa and Middle East, especially in rural Cutaneous lesions caused by L. tropica (on the left) and L. arid regions. major (on the right). Note that the skin lesion is intact with • In Iran, Saudi Arabia, Morocco, Tunisia, Syria, L. tropica while a serious lesion is observed with L. major. Israel, and Iraq, many CL cases due to L. major Materials and Methods: have been reported, where Phlebotomus • Lesion samples taken from 13 autochthonous cases living in provinces from • The melting curve analyses of southern and southeastern Anatolia such as, Antalya (n=4), Adana (n=3), Mardin ITS-1 PCR showed a peak papatasi was the vector, and the great gerbil (n=2), Diyarbakir (n=2), Bitlis (n=1) and Hatay (n=1), and three imported cases concordant with Leishmania major. (Rhombomys opimus) or the rock hyrax from Iran, Iraq and Syria, were inoculated into NNN medium and consequently to The map shows the number of CL cases identified in Turkish provinces. Note RPMI medium including 20% of Fetal Calf Serum (FCS) for mass culture. • DNA Sequence analyses also (Procavia capensis) were the reservoirs. that the blues indicate the provinces of imported cases, while the reds indicate • One Leishmania isolate was injected into the footpads of six Golden Syrian the provinces where autochthonous L. major infections were identified. indicated L. major as the causative hamsters to establish an animal model. agent. • Hamsters were physically examined once a week for 3 months. • To identify the species of the parasites, real-time ITS-1 PCR was performed both with the clinical material and the promastigotes obtained from culture. • Melting curve analyses of real-time PCR using primers and probes designed for the ITS-1 region of Leishmania spp. were conducted. Results: • Active and acute lesions developed in 7-10 day old hamsters, which were evaluated as very short and heavy. Irreversible impairments were observed on hamster skins in time, leadingBackgroundto total amputation of the extremity. • Lesion samples were inoculated in NNN medium and then • The melting curve analyses of ITS-1 PCR and the sequence analyses of the showed a peak concordant with Leishmania major. transferred to RPMI medium containing 20% of fetal calf serum • Species of two autochthonous isolates were confirmed by isoenzyme analyses (FCS) for mass production. Developing L. major infection on the footpad of hamster in Leishmaniasis Reference Center of WHO in Montpellier (France) as L. major • A total of 107 promastigotes of each isolate were Two samples were sent to WHO Leishmaniasis Reference MON-103. Center in Montpellier (France) for isoenzyme analysis and Conclusion: There has been emerging CL cases caused by L. major in eastern inoculated into the footpads of six hamsters. both of them were found to be L. major MON-103. • CL cases due to L. major have been emerging in Turkey, which is estimated to prevail. L. major causes a more severe infection in • A recently-developed ITS-1 Real-Time PCR assay was applied patients and its treatment is harder than L. tropica. Public health measures should include L. major infections while researchers plan field studies to identify both with the lesion sample and culture material. Melting curve Table 1. Overall data of 16 CL patients infected with L. major. the last years. The cases are from eastern the vectors and reservoirs of L. major. analyses were also done (Töz SO, 2013). . Number Anatolia, but it is well-known that both the Province Code for Registration Initials Sex Site of Sore(s) Genotype of Sores vectors and the reservoirs of the agent are Background Results Antalya MHOM/TR/2013/CBUANT009 R.Y. M 3 Hand,Arm, Finger L. major present in rural regions of whole Anatolia. Antalya MHOM/TR/2013/CBUANT012 M.A.G. M 1 Leg L. major • A large-scale study that will focus on both Antalya MHOM/TR/2013/CBUANT014 Ş.Z. F 1 Face L. major Leishmania infantum is the the reservoirs and vectors of leishmaniasis in causative agent of visceral Antalya MHOM/TR/2013/CBUALA016 N.K. F 1 Face L. major leishmaniasis (VL), while both L. Adana MHOM/TR/2013/CBUALA005 S.Ö. F 2 Lip, Shoulder L. major whole Anatolia is urgently needed to infantum and L. tropica causes Adana MHOM/TR/2013/CBUADN019 D.C. M 1 Arm L. major overcome the growing public health problem cutaneous infection (CL) in Adana MHOM/TR/2013/CBUADN021 N.Ş. 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This project is financially supported by TUBITAK (Project no:111S179). For Correspondence: Prof. Ahmet ÖZBİLGİN E-mail: [email protected]