celal bayar university vocational school of health

Introduction

Turkey as a Crossroads for Leishmania species:

Genotypic Identification Reveals the Presence of Four Leishmania species in Autochthonous Cases of Cutaneous LeishmaniasisAhmet Özbilgin1, Fulya Okudan2, Suhan Günasti Topal3, Soner Uzun4, Mehmet Harman5, Hatice Ertabaklar6, Cumhur Gündüz7, İpek Östan8, Özgür Kurt9

Sema Ertuğ6, Ayşegül Erat4, Dilek Güngör3, Çağla Kayabaşı8,İbrahim Çavuş1, Yusuf Özbel10

1 Celal Bayar University Medical School Department of Parasitology, Manisa; 2 Alanya State Hospital Department of Dermatology, Antalya, 3 Çukurova University Medical School Department of Dermatology, Adana, 4 Akdeniz University Medical School Department of Dermatology, Antalya, 5 Dicle University Medical School Department of Dermatology, Diyarbakir, 6 Adnan Menderes University Medical School Department of Parasitology, Aydin

7 Ege University Medical School Department of Medical Biology, İzmir, 8 Celal Bayar University Vocational School of Health Sciences, Manisa, 9 Acibadem University Medical School Dept. of Medical Microbiology, Istanbul, 10 Ege University Medical School Department of Parasitology, İzmir, TURKEY

Methods

AbstractTurkey is situated on the crossroads betweenAsia, Africa and Europe. Its subtropical climatemakes it a suitable environment for leishmaniasis,where almost 15,000 cutaneous leishmaniasis (CL)cases were reported between 2005 and 2012.

Results

Conclusions

References1. Özbel Y, Özensoy Töz S., 2007, “Leishmanosis. Tıbbi Parazit Hastalıkları” (Eds. Özcel MA, Özbel Y, Ak M.), Türkiye Parazitoloji Derneği Yayını No: 22, S.197-

2412. Miles MA, Yeo M, Mauricio IL. Genetics. Leishmania exploit sex. Science, 2009, 324(5924):187-9.3. Akopyants NS, Kimblin N, Secundino N, et al. Demonstration of genetic exchange during cyclical development of Leishmania in the sand fly vector. Science,

2009, 324(5924): 265–268.4. Ok UZ, Balcioğlu IC, Taylan Ozkan A, Ozensoy S, Ozbel Y. Leishmaniasis in Turkey. Acta Tropica. 2002; 84: 43–48.5. Serin MS, Waki K, Chang KP, Aslan G, Direkel S, Otag F, Kayar B, Koksal F, Emekdas G. Consistence of mini exon polymerase chain reaction restriction

fragment length polymorphism and single-copy gene sequence analyses in discriminating Leishmania genotypes. Diagn Microbiol Infect Dis. 2007; 57(3):295-299.

6. Uzun S, Uslular C, Yücel A. Cutaneous leishmaniasis: evaluation of 3074 cases in the Cukurova region of Turkey. Br J Dermatol. 1999; 140: 347–350.7. Dea-Ayuela MA, Rama-Iñiguez S, Bolás-Fernández F. Proteomic analysis of antigens from Leishmania infantum promastigotes. Proteomics. 2006; 6(14):

4187-4194.

The number of Leishmania species identified in the study and the provinces of Turkeywhere they were diagnosed were shown in Figure 1.

Results

Presence of all four Leishmania species may causeelevation of autochthous VL cases in coming years.

L. donovani/infantum hybrids (+) => genetic exchangehappen between Leishmania species in Turkey.

The study was supported by TUBITAK (Project No: 111S179). Samples are cryopreserved in the Parasite Bank located in Celal Bayar University in Manisa, Turkey.

Turkey’s unique location makes it a “meeting point” for people fromvarious countries where some serious infectious diseases are endemic.

Cutaneous leishmaniasis (CL) is a vector-borne disease, transmitted to2 million new cases annually, most of whom live in developing countries.

CL has been endemic in southeastern Turkey; however, it is reportedfrom many foci in western Anatolia, together with higher numbers ofcases and vectors.

For Correspondence: Prof. Ahmet ÖZBİLGİN E-mail: a.ozbilgin@yahoo.com

Patients were admitted and diagnosed with CL in various health care centers in Turkey. (n=250)

Diagnosis => Giemsa-stained smears (MICROSCOPY) Cultivation in NNN and Enriched Medium (CULTURE)

Materials and Methods

Promastigotes

Amastigotes

Reference strains of L. tropica, L. major, L. donovani and L. infantumwere used for comparisons of results.

All four causative agents of leishmaniasis (L. tropica, L. donovani, L. infantum andL. major) were found to be present in Turkey. Indeed, hybrid species (L. donovani/L. infantum hybrid) were also identified.Proteinic differences were found between hybrid and non-hybrid species, whichprobably a cause for different clinical manifestations in mice.

Different clinical manifestations of the same Leishmania species maybe due to their genomic or proteomic characteristics. These are the partsof further research we plan to conduct in the coming years.

Understanding the mysteries of leishmaniosis by such studies will nodoubt contribute to the efforts for its diagnosis, treatment and breakingof vector-host transmission processess.

The only causative agents of CL were Leishmania tropica and L. infantum inTurkey.L. major and L. donovani were recently identified in CL cases, but withoutany information whether they were local or imported cases. Determination ofLeishmania species in clinical cases is crucial for the management oftreatment.Here, 250 autochthonous CL patients diagnosed in Turkey recently, arepresented together with their basic features and genotypic assessmentresults.METHODS: Patients admitted to hospitals across Turkey, with skin lesions.CL diagnosis => clinical evaluation, microscopic examination of Giemsa-stained samples and cultivation in NNN and Enriched Medium and ITS-1 Realtime PCR.Atypical lesion samples were inoculated in Balb/C mice for diagnosis.

RESULTS: Totally, 250 CL cases were identified in different provinces inTurkey (Figure 1). Male patients were slightly more than females (133 vs.117), and the leading lesion site was the face (n=159; 63.6%). Number oflesions on patients were mostly one or two (n=199 in total; 79.6%).

RT-PCT results indicated L. tropica (n=184; 72.4%), L. major (n=30;12.8%), L. infantum (n=23; 8.8%), L. donovani (n=11; 4.4%) and L.infantum/ L. donovani hybrids (n=2; 1.2). All atypical cases inoculated intomice were found to be hybrid Leishmania isolates.

CONCLUSION: All four causative agents of CL as well as hybrid isolateswere found in autochthonous cases in Turkey.

The second leading species was L. major, which is well-documented indebilitating CL in mice, and should be considered during treatments. CL cases caused by L. major in our study group showed seriousulcerating skin lesions, in contrast to L. major cases reported from MiddleEastern countries, where self-limiting, non-ulcerating lesions are observed.The underlying factors of this different clinical outcomes worth furtherresearch.The presence of L. donovani and L. major may be seen as a sign ofpotential elevation of autochthonous visceral leishmaniasis cases incoming years, which are relatively low today.Public health measures should urgently be implemented to prevent thetransmission of leishmaniasis together with large-scale projects that willreveal the reservoirs and vectors of leishmaniasis in Turkey.

Despite the causative agents have been known as Leishmaniatropica and L. infantum, autochthonous cases due to L. majorand L. donovani have recently been reported. Indeed, hybridLeishmania isolates have been reported, which may neither bediagnosed nor eradicated with current methods. Here, we aimed to present 250 CL cases diagnosed indifferent provinces of Turkey and assess the current situation ofCL in Turkey, through both causative agents and individualfeatures of patients.

CL cases in Turkey

1990-2010: 46.003

2013 only: ~4.500* * almost half of themare Syrian refugees

Real Time PCR => ITS-1 Region of Leishmania spp. is amplified both from thelesion and culture material.

Sequence analyses: Typing was done after amplification, including hsp70gene and cpb genes of L. infantum and L. donovani, if necessary.

2D Electrophoresis => To analyze proteinic differences.

Inoculation in Laboratory Animals => To assess the clinical outcome ofinfections in vivo.

Presence of CL, VL and canine leishmaniasis caused also byL. donovani and L. major in western Anatolia indicates ahigh risk of the spread of these infections into Europe.

• In Manisa province, which is aneighbor of Izmir in westernAnatolia, these agents wereidentified as the causative agentsof CL, VL and canine leishmaniasis.

Causative Agent in Manisa* Infection

Leishmania tropica CL + VL+ CanL

Leishmania infantum CL + VL+ CanL

Leishmania major CL

Leishmania donovani VL + CanL

L. infantum/L. donovani hybrid CL

45 min by plane(Izmir-Athens)

15 min by ferry(Izmir-Chios)

L. donovani and L. major: Knocking on Europe’s Door?

Leishmania tropica

Leishmania major

Leishmania infantum

Leishmania. donovani

Hybrids (L.don/L. inf)

184

30 2311

2

Autochthnous CL cases in Turkey