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Öğe The current clinical and geographical situation of cutaneous leishmaniasis based on species identification in Turkey(Elsevier, 2019) Ozbilgin, Ahmet; Toz, Seray; Harman, Mehmet; Topal, Suhan Gunasti; Uzun, Soner; Okudan, Fulya; Gungor, DilekLeishmaniases are a group of vector-borne diseases caused by the members of Leishrnania genus, and there are three main clinical forms of the infection as visceral, cutaneous, and mucocutaneous. Cutaneous leishmaniasis is a growing public health problem in Turkey due to increasing detection of autochthonous cases caused by L. major and L. donovani in some regions in addition to Syrian imported cases. For this reason, we aimed to evaluate the current epidemiological situation of CL in the view of causative agents and their geographical distribution throughout Turkey. The samples were collected from 356 CL patients admitted to different centers in 18 provinces between January 2013 and December 2016. Direct microscopy, culture (regular and enriched NNN) and molecular techniques (real-time ITS1 PCR and hsp70 PCR/sequencing) were performed. By molecular techniques, 299, 28, 19 and 10 isolates/clinical samples were identified as L. tropica, L. major, L. infant= and L. donovani, respectively. Most of the patients (65.73%) had one lesion usually on their face/head. Dry-nodular type lesions (n = 291) were mainly associated with L. tropica while L. major was mainly found related to wet-ulcerative ones. Leishmaniasis recidivans was also detected in 2.52% among 356 patients. L. tropica was detected as most widespread species causing CL in Turkey. L. infantum and L. major was also found in one third of the provinces. Enriched NNN culture was worked well for isolating the parasite and 346 isolates were successfully grown and stored in liquid nitrogen. The comparison of all diagnostic techniques showed that the parasitological positivity rate could increase if the combination of direct microscopy and real-time ITS1 PCR is used. Besides well-known anthroponotic L. tropica cases, the increasing detection of CL cases caused by zoonotic species, L. infantum and L. major, is one of the most important findings in the present study. In our opinion to ensure timely and accurate diagnosis, proper treatment and countrywide effective control of CL in Turkey a systematic approach is needed on the base of information about characteristics of lesions and patients and epidemiological features of the disease.Öğe The Diagnostic Value of Lesional Skin Smears Performed by Experienced Specialist in Cutaneous Leishmaniasis and Routine Microbiology Laboratory(Wolters Kluwer Medknow Publications, 2019) An, Isa; Harman, Mehmet; Cavus, Ibrahim; Ozbilgin, AhmetObjective: Leishmaniasis is a common vector-borne infection affecting 12 million people in 98 countries. The most frequently used method in diagnosis is the microscopic investigation of the leishmania smears. The diagnostic value of this method varies according to the experience of the evaluator. In this prospective study, it was aimed to emphasize the importance of experience in the evaluation of lesional smears used in the diagnosis of cutaneous leishmaniasis. Methods: In this study, patients who were admitted to Dicle University Medical Faculty Hospital Dermatological and Venereal Diseases Outpatient Clinic between January and December 2016 and who had lesions with suspicious cutaneous leishmaniasis were included. For all the cases, both in the routine microbiology laboratory and in the diagnosis and treatment of cutaneous leishmaniasis, separate smears were performed by an experienced specialist and evaluated independently from each other. Results: In 70 of 98 cases studied, the diagnosis of cutaneous leishmaniasis was confirmed by laboratory evaluations. The rate of positivity was significantly higher in the smears analyzed by experienced specialist in the clinical and diagnosis of cutaneous leishmaniasis (95.7%) than in the smears analyzed by the routine microbiology laboratory (42.9%) (p<0.001). Conclusion: The data in our study showed that smears should be performed and evaluated by experienced specialists in the clinical and diagnosis of cutaneous leishmaniasis.Öğe Leishmaniasis in Turkey: first clinical isolation of Leishmania major from 18 autochthonous cases of cutaneous leishmaniasis in four geographical regions(Wiley, 2016) Ozbilgin, Ahmet; Culha, Guelnaz; Uzun, Soner; Harman, Mehmet; Topal, Suhan Gunasti; Okudan, Fulya; Zeyrek, FadileObjectiveTo report isolation of Leishmaniamajor strains obtained from 18 Turkish autochthonous cutaneous leishmaniasis (CL) patients infected with L.major between 2011 and 2014. MethodsInitial diagnosis relied on microscopy and culture in enriched medium, prepared by adding specific amounts of liver extract, protein and lipid sources to NNN medium. Promastigotes were then transferred to RPMI medium including 10% of foetal calf serum for mass culture. Species-specific real-time PCR targeting ITS1 region of Leishmania spp. was performed using both lesion aspiration samples and cultured promastigotes. Two of 18 isolates were identified by isoenzyme analysis in the Leishmaniasis Reference Center in Montpellier, France. Each isolate was inoculated into the footpads of six mice to observe the pathogenicity of L.major. Developing lesions were observed, and the thickening of footpads was measured weekly. ResultsMelting curve analyses of 18 isolates showed a peak concordant with L.major, and two of them were confirmed by isoenzyme analyses as L.major zymodeme MON103. In the mouse model, acute lesions seen on day 21 were accepted as an indication of heavy infection. Severe impairments were observed on all mouse footpads over 3weeks, which even progressed to extremity amputation. ConclusionCutaneous leishmaniasis-causing L.major was recently identified in Adana province in southern Turkey, with PCR. Our study shows that such CL cases are not limited to Adana but currently present from western to Southeastern Anatolia, and along the Mediterranean coast. The role of small mammals, the main reservoirs of L.major in Anatolia, needs to be elucidated, as do the underlying factors that cause severe clinical manifestations in L.major infections in Turkey, contrary to the infections in neighbouring countries. ObjectifRapporter sur l'isolement de souches de Leishmania major obtenues a partir de 18 cas de leishmaniose cutanee (LC) de patients turcs autochtones infectes par L. major entre 2011 et 2014. MethodesLe diagnostic initial a porte sur la microscopie et la culture sur un milieu enrichi, prepare en ajoutant des quantites specifiques d'extrait de foie, de proteines et de sources de lipides au milieu NNN. Les promastigotes ont ensuite ete transferes dans le milieu RPMI contenant 10% de serum fOEtal de veau pour la culture de masse. La PCR en temps reel specifique de l'espece et ciblant la region ITS1 de Leishmania spp. a ete realisee en utilisant a la fois les echantillons d'aspiration de la lesion et de promastigotes cultives. Deux des 18 isolats ont ete identifies par analyse des isoenzymes dans le Centre de reference de la leishmaniose a Montpellier, en France. Chaque isolat a ete inocule dans les coussinets plantaires de six souris pour observer la pathogenicite de L. major. Les lesions en developpement ont ete observees et l'epaississement des coussinets plantaires ont ete mesures chaque semaine. ResultatsLes analyses de courbe de fusion des 18 isolats ont montre un pic concordant avec L. major et deux d'entre eux ont ete confirmes par des analyses d'isoenzyme comme L. major de zymodeme MON103. Dans le modele murin, des lesions aigues observees au jour 21 ont ete acceptees comme une indication de forte infection. Des deficiences severes ont ete observees sur tous les coussinets plantaires des souris pendant plus de trois semaines, qui ont meme progresse jusqu'a l'amputation de l'extremite. ConclusionL. major causant la LC a ete recemment identifie dans la province d'Adana dans le sud de la Turquie par la PCR. Notre etude montre que de tels cas de LC ne sont pas limites a Adana, mais sont actuellement presents dans l'ouest et dans le sud-est de l'Anatolie, et le long de la cote mediterraneenne. Le role des petits mammiferes, principaux reservoirs de L. major en Anatolie, devrait etre elucide, de meme que les facteurs sous-jacents qui causent les manifestations cliniques severes dans les infections a L. major en Turquie, contrairement aux infections dans les pays voisins. ObjetivoReportar el aislamiento de cepas de L. major obtenidas de 18 pacientes turcos con leishmaniosis cutanea (LC) autoctona, infectados con Leishmania major entre 2011 y 2014. MetodosEl diagnostico inicial se realizo mediante microscopia y cultivo en medio enriquecido, preparado mediante la adicion de cantidades especificas de extracto de higado, proteina y fuentes de lipido al medio NNN. Los promastigotes fueron transferidos al medio RPMI con un 10% de suero fetal para su cultivo masivo. Se realizo PCR en tiempo real, especie-especifica, que detecta la region ITS1 de Leishmania spp., utilizando tanto muestras aspiradas de las lesiones como promastigotes de cultivo. Dos de los 18 aislados se identificaron mediante analisis isoenzimatico en el Centro de Referencia de la Leishmaniosis en Montpellier, Francia. Cada aislado fue inoculado en las almohadillas de las patas de seis ratones para observar la patogenicidad de L. major. Se observo el desarrollo de las lesiones y se midio semanalmente el engrosamiento de las almohadillas. ResultadosEl analisis de la curva de fusion de los 18 aislados mostro un pico de concordancia con Leishmania major, y dos de ellos fueron confirmados mediante un analisis isoenzimatico como L. major zymodeme MON103. En el modelo de raton, las lesiones agudas observadas en el dia 21 se aceptaron como indicativas de una infeccion masiva. Se observaron danos graves en todas las almohadillas de los ratones a lo largo de tres semanas, que progresaron hasta la amputacion de las extremidades. ConclusionRecientemente se ha identificado, mediante PCR, LC causada por L. major en la provincia de Adana al sur de Turquia. Nuestro estudio muestra que estos casos de LC no estan limitados a Adana y que actualmente existen desde el oeste al sudeste de Anatolia y a lo largo de la costa Mediterranea. Es necesario aclarar el papel que en Anatolia juegan los pequenos mamiferos, principales reservorios de L. major, al igual que el de los factores que hay detras de las manifestaciones clinicas severas en infecciones por L. major en Turquia, al contrario del de las infecciones presentes en paises vecinos.Öğe Leishmaniasis in Turkey: Visceral and cutaneous leishmaniasis caused by Leishmania donovani in Turkey(Elsevier Science Bv, 2017) Ozbilgin, Ahmet; Harman, Mehmet; Karakus, Mehmet; Bart, Aldert; Toz, Seray; Kurt, Ozgur; Cavus, IbrahimIn Turkey, the main causative agents are Leishmania tropica (L. tropica) and Leishmania infantum (L. infant:tun) for cutaneous leishmaniasis (CL) and L. infantum for visceral leishmaniasis (VL). In this study, we investigated leishmaniasis cases caused by L. donovani and established animal models for understanding its tropism in in vivo conditions. Clinical samples (lesion aspirations and bone marrow) obtained from CL/VL patients were investigated using parasitological (smear/NNN) and DNA-based techniques. For species identification, a real time ITS1-PCR was performed using isolates and results were confirmed by hsp70 PCR-N/sequencing and cpb gene PCR/sequencing in order to reveal Leishmania donovani and Leishmania infantum discrimination. Clinical materials from CL and VL patients were also inoculated into two experimental groups (Group CL and Group VL) of Balb/C mice intraperitoneally for creating clinical picture of Turkish L. donovani strains. After 45 days, the samples from visible sores of the skin were taken, and spleens and livers were removed. Measurements of the internal organs were done and touch preparations were prepared for checking the presence of amastigotes. The strains were isolated from all patients and amastigotes were seen in all smears of the patients, and then isolates were immediately stored in liquid nitrogen. In real time ITS1-PCR, the melting temperatures of all samples were out of range of L. infcrnuttn, L. tropica and L. major. Sequencing of hsp70 PCR-N showed that all isolates highly identical to previously submitted L. donovani sequences in GenBank, and cpb gene sequencing showed five isolates had longer cpbF allele, whereas one isolate contained a mixed sequence of both cpbF and cpbE. All mice in both experimental groups became infected. Compared to controls, the length and width of both liver and spleen were significantly elevated (p < 0.001) in both groups of mice. However, the weight of the liver increased significantly in all mice whereas the weight of spleen increased only in VL group. Amastigotes were also seen in all touch preparations prepared from skin sores, spleen and liver. L. donovani strain was isolated from autocutaneous a VL patient first time in Turkey. Animal models using clinical samples were successfully established and important clinical differences of the isolated strains were observed.Öğe Overcoming the Challenge;In VivoEfficacy of Miltefosine for Chronic Cutaneous Leishmaniasis(Springer Int Publ Ag, 2021) Tunali, Varol; Harman, Mehmet; cavus, Ibrahim; Gunduz, Cumhur; Ozbilgin, Ahmet; Turgay, NevinBackground Cutaneous Leishmaniasis (CL) is the most common form of leishmaniasis. CL can be divided into two major groups: acute CL (ACL) and chronic CL (CCL). The aim of this study is to compare the efficacy of miltefosin and pentavalent antimony compoundsin vivowith the CCL patient samples. Materials Three study groups were formed, each consisting of five male Mus musculus (Balb/C) mice. In this model, promastigotes from the culture of a CCL patient were utilized. 100 mu LL. tropicapromastigote suspension with a density of 10(8)promastigotes/ml were injected into the hint-right footpad of each experimental animal intradermally. Footpads of the mice were measured every two weeks until 24th week. From the 13th week, miltefosin 50 mg/kg/day was administered orally using gavage for 21 days, Meglumin antimoniate (MA) was administered by intramuscular (IM) injection daily for 21 days at 50 mg/kg/day and saline was administered IM for 21 days for the miltefosine, MA and control group, respectively. Results The footpad measurements of the miltefosine group were lower than the control group statistically. Between the MA group and the miltefosine group and MA group and the control group, there was no statistically significant difference. Giemsa stained slides revealed amastigotes in one, two and all of the slides for the miltefosine, MA and control group, respectively. Molecular tests were performed with the Rotor-Gene device andL. tropicaconsistent peaks were obtained in one of the miltefosine group, four in the MA group and all mice in the control group. Conclusions Demonstration of both clinical and laboratory improvement in four of the five experimental animals provides strong evidence that miltefosine is an effective drug in the treatment of CCL. In the literature, no clinical or laboratory studies using miltefosine have been performed with CCL patients only.Öğe Refugees at the Crossroads of Continents: A Molecular Approach for Cutaneous Leishmaniasis Among Refugees in Turkey(Springer Int Publ Ag, 2020) Ozbilgin, Ahmet; Gencoglan, Gulsum; Tunali, Varol; Cavus, Ibrahim; Yildirim, Ahmet; Guenduez, Cumhur; Harman, MehmetPurpose Due to mass population movements driven by internal conflicts and wars, cutaneous leishmaniasis (CL) is becoming increasingly important in Turkey. This study is aimed at determining the clinical aspects, diagnosis and genotyping of CL patients coming to Turkey from abroad. Methods In our study, the clinical materials obtained from the patients or sent for diagnostic purposes from other centers to our laboratory between years 2012 and 2016 were assessed retrospectively. In total, there were 38 patients from Syria, Iraq, Afghanistan, Iran, and Turkmenistan. Results 29 (76%), 28 (73%) and 33 (87%) samples were positive by light microscopy, Novy-McNeal-Nicolle(NNN), and enriched medium, respectively. By ITS-1 gene region PCR, 31 (81%) of the cases were positive. 35 of the patients were tested positive by at least one of the diagnostic methods. By genotyping, 21 Leishmania tropica, 8 Leishmania major, 3 Leismania infantum, 2 Leishmania donovani, and 1 Leishmania aethopica were detected. Conclusion This study is aimed at informing the clinicians working in the field for the import CL cases and recording the changing epidemiological features of CL in the region as well as discussing the possible focus for L. aethiopica infection which has not been shown in the region before.
