tropica as Dr. Jekyll and Mr. Hyde: Visceralization of a well-known cutaneous agent Ahmet Özbilgin1, Seray Töz2, Özgür Kurt3, Cumhur Gündüz4, İbrahim Çavuş1, Mehmet Karakuş2, Yusuf Özbel2 1 Celal Bayar University Faculty of Medicine Dept. of Parasitology, Manisa; 2 Ege University Faculty of Medicine Dept. of Parasitology, Izmir; 3 Acibadem University Faculty of Medicine Dept. of Medical Microbiology, Istanbul; 4 Ege University Faculty of Medicine Dept. of Medical Biology, Izmir, TURKEY

Abstract Introduction Materials and Methods Discussion

OBJECTIVES: Leishmaniasis may cause not only cutaneous lesions, but • Leishmaniasis may cause not only cutaneous or mucocutaneous • All patients examined are immunocompetent patients. • Pathogenicity of leishmaniasis may differ according to the also systemic, life-threatening infections in visceral organs. lesions, but also systemic, life-threatening infections that involve • Clinicians initially conducted bone marrow sampling to patients. virulence of the Leishmania strain infecting the patient as • In Turkey, (VL) caused by , visceral organs. • Bone marrow samples were stained with Giemsa, inoculated in NNN well as the immune competence of the individual. and (CL) caused by L. tropica and L. infantum and Enriched medium and used for Real Time PCR targeting ITS-1 • In Turkey, the visceral leishmaniasis (VL) caused by Leishmania (L.) have been recorded. region of Leishmania spp. • Genetic exchange has already been reported between infantum, and cutaneous leishmaniasis (CL) caused by L. tropica, L. • Here, we report three autochthonous VL cases caused by L. tropica • Bone marrow sample of one patient was partly inoculated in the Leishmania strains in nature, which yielded the infantum and recently L. major and L. donovani have been recorded. from an uncommon location, Manisa province in western Anatolia, peritoneal cavities of three Balb/C mice for diagnosis. development of hybrid strains. diagnosed with Real-Time PCR. • Development of infection in mice was observed regularly, and the • METHODS: Suspected of VL, clinicians extracted bone marrow samples • Cutaneous leishmaniasis (CL) is endemic in lesions and symptoms are recorded . Genomic and proteomic variations are and referred them to Parasitology Laboratory. certain spots in southeastern Anatolia, Turkey. identified in hybrid strains, which help • These samples were stained with Giemsa, inoculated in NNN and • Despite located in western Anatolia, Manisa Enriched medium and used for Real Time PCR targeting ITS-1 region of them adapt to their environment. province is also a site of CL in Turkey. Results Leishmania spp. • These hybrids may cause more severe • Bone marrow sample of one patient was partly inoculated in the • • peritoneal cavities of three Balb/C mice for diagnosis. Visceral leishmaniasis (VL), caused by L. infantum is relatively less Giemsa-stained smears => (+) Leishmania amastigotes. clinical manifestations, with worse common in Turkey, with 102 cases reported between 2007-2009. • Culture => Leishmania promastigotes. • Here, we report three autochthonous immunocompetent VL cases prognosis in patients. caused by L. tropica from Manisa province in western Anatolia, which • Real Time PCR targeting ITS-1 region => “Leishmania tropica” is an uncommon location, diagnosed with microscopy, culture and • Examination of L. tropica-infected Balb/C mice from Patient 1 • Another factor that may be effective in the pathogenicity Real-Time PCR. of Leishmania isolates is the RNA virus (LRV) present in some Leishmania isolates. RESULTS: Amastigotes and promastigotes of Leishmania spp. were visceral symptoms & cutaneous symptoms identified with microscopyBackgroundand culture, respectively. Materials and Methods Enlargement of liver and spleen & • First identified in 1988, LRV is associated with more • Application of Real Time PCR that targeted the ITS-1 region of desquamation, hair loss and cutaneous lesions severe clinical infections in laboratory mice with Leishmania spp. showed melting curves, which were concordant with L. PATIENT 1 (VL): Male, 50 years old. Admitted to leishmaniasis. tropica. hospital with fever, enlargement of both liver and • The parasites which were initially inoculated in the peritoneal cavities of spleen, nausea, diarrhea, vertigo, extreme sweating • In Leishmania guyanensis, nucleic acid of Leishmania RNA the mice showed not only visceral (enlargement of both liver and spleen) and fatigue. Examination of his blood smear showed virus (LRV1) is a strong immunogen. It may cause a hyper- but also dermotropic (desquamation, hair loss and cutaneous lesions) pancytopenia. VL was diagnosed with positive Giemsa- inflammatory immune response via toll-like receptor 3 manifestations. stained smears, culture and IFA test. CL was absent. L. tropica (TLR3). • Our next plan is to search for LRV in the Leishmania PATIENT 2 (VL): Male, 53 years old. Admitted to isolates we identified in Turkish patients. Up to date, there hospital with fever, enlargement of both liver and spleen, quick weight loss, extreme sweating and is no data about this issue. fatigue. Examination of his blood smear showed • Unveiling the genomic and proteomic content of CONCLUSION: CL-causing L. tropica is well-recognized in Turkey, with pancytopenia. VL was diagnosed with positive Giemsa- Leishmania strains by state-of-the-art methods will no cases identified previously in many provinces, including Manisa. stained smears, culture and IFA test. CL was absent. doubt help diagnosis and effective treatment of patients. • Interestingly, VL cases presented here were from Manisa but caused by L. tropica. PATIENT 3 (VL): Male, 10 years old. Admitted to • The differences in the genomic and proteomic components or hospital with fever, fatigue, enlargement of both liver References structures of these two groups of diversely-acting L. tropica isolates are and spleen, and failure to thrive. Examination of his Ok UZ, Balcıoglu IC, Taylan Ozkan A, et al. Leishmaniasis in Turkey. Acta Tropica 2002; 84: mysteries at the moment. However, based on these initial data, we plan blood smear showed pancytopenia. Leishmaniasis was 43–48. to investigate immediately the genomic and proteomic compositions of L. diagnosed with positive Giemsa-stained smears, tropica isolates of these three VL patients and compare them with three Hotez PJ et al. Neglected Tropical Diseases of the Middle East and North Africa: Review of culture and IFA test. CL was absent. Their Prevalence, Distribution, and Opportunities for Control. PLOS 2012; 6(2): e1475. different L. tropica isolates from previously-diagnosed CL patients from Manisa, with molecular methods and two-dimensional electrophoresis. Hartley MA, Ronet C, Zangger H, Beverley SM, Fasel N. Leishmania RNA virus: when the host • Identification of any genomic and/or proteomic variations in CL patient from Manisa: Female, 47 years old. First pays the toll. Front. Cell. Infect. Microbiol., 2012 (2): 99. 1-15. lesion started as an itching acne 10 months before these two different L. tropica isolate sets may point out not only Akopyants NS, Kimblin N, Secundino N, et al. Demonstration of genetic exchange during the shift either for visceralization of the parasites or their limited admission on the top of her nose, followed by three cyclical development of Leishmania in the sand fly vector. Science, 2009, 324(5924): 265– localizations inside the dermal regions of infected individuals, but lesions around it. Giemsa-stained smear (+), Culture (+) 268. also some suitable target molecules for vaccine development trials. Real Time PCR. => Leishmanania tropica. Volf P, Benkova I, Myskova J, et al. Increased transmission potential of /Leishmania infantum hybrids. Int J Parasitol, 2007, 37(6): 589-93. This project is financially supported by TUBITAK (Project no:111S179). Samples are cryopreserved in the Parasite Bank located in Celal Bayar University in Manisa, Turkey. Correspondence: Prof. Ahmet ÖZBİLGİN E-mail: [email protected]