Determination of sand fly fauna and molecular detection of Leishmania in sand flies in Antalya Province, Southern Turkey.

Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are diseases transmitted by infected female sand flies. Since the eradication of malaria in Turkey, CL is the main vector-borne disease in the country, with more than 2000 cases per year, making it a significant public health problem. The aims of this study were to carry out an entomological survey in Antalya Province, an endemic area for CL in the Mediterranean Region of Turkey, to identify sand fly fauna and to screen female specimens for the presence of Leishmania parasites (Leishmania infantum, L. tropica, L. major, and L. donovani) using molecular analysis. Sand flies were collected in 42 localities of seven districts in Antalya Province using CDC miniature light traps in two different periods, June 2012 and September 2013. The specimens were kept in 96% ethanol until the dissection was done. The head and genitalia of the specimens were cut for preparing individual slides for species identification. The rest of the body of female specimens was kept separately. The specimens were identified at the species level, and 27 pools were generated according to the locations and species for screening the presence of Leishmania. A commercial kit was used for DNA extractions. Real-time and conventional polymerase chain reaction (PCR) targeting the internal transcribed spacer region (ITS1) were then performed. In total, 1306 specimens comprising nine species belonging to the Phlebotomus genus were collected in the study region, with Phlebotomus neglectus/syriacus (38.82%) the most abundant, followed by P. alexandri (21.67%) and P. tobbi (20.44%). In the 27 pools, Leishmania infantum DNA was detected in four pools containing P. neglectus/syriacus and one pool containing P. tobbi. In conclusion, the sand fly fauna in the Antalya Province is diverse. The probable vector sand fly species are P. neglectus/syriacus and P. tobbi with high dominance (59.26%), which indicates a high risk of CL transmission. The data presented here may help to shed more light on the transmission cycles of the Leishmania parasite in this CL endemic area.

The current clinical and geographical situation of cutaneous leishmaniasis based on species identification in Turkey.

Leishmaniases are a group of vector-borne diseases caused by the members of Leishmania 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. infantum 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.

Evaluation of conjunctival swab sampling in the diagnosis of canine leishmaniasis: A two-year follow-up study in Çukurova Plain, Turkey.

The diagnosis of canine leishmaniasis (CanL) in symptomatic and asymptomatic dogs is a very important and problematic public health issue in Turkey. A longitudinal study was carried out on dogs in selected villages in the Çukurova Plain in Turkey, from July 2011 to June 2013, where cutaneous (CL) and visceral (VL) leishmaniasis is endemic. The study aimed to determine the prevalence of CanL and to evaluate the early diagnostic performance of the non-invasive conjunctival swab nested PCR (CS n-PCR) test in comparison with the Indirect Fluorescent Antibody Test (IFAT). The consecutive blood and CS samples from a representative number of dogs (80-100 dogs/each survey) were collected in a cohort of 6 villages located in the area. Clinical symptoms, demographic and physical features about each dog were noted and lymph node aspiration samples were obtained from selected dogs with lymphadenopathy. In four surveys during the period, a total of 338 sets (blood and CS) of samples from 206 dogs were obtained, such that 83 dogs were sampled more than once. In the cross-sectional analysis, the CanL prevalence was found to be 27.18% (between 7.14% and 39.13%) by IFAT and 41.74% (between 29.03% and 46.66%) by CS n-PCR. The isolated strains were identified as Leishmania infantum MON-1 (n=9) and MON-98 (n=2) by MLEE analysis. Genetic studies targeting the Hsp70 and ITS1 regions performed on 11 dog isolates also showed two clear separate groups. According to IFAT results, 24 of the 83 dogs sampled more than once showed seroconversion (n=19) or a four-fold increase in Ab titers (n=5), while 17 were positive in the initial screening. Forty-two dogs stayed negative during the whole period. The natural Leishmania exposure rate was detected as 31.14% in the study area. CS n-PCR only detected Leishmania infection earlier than IFAT in 8 dogs. No statistical difference was found after the analysis of demographical and physical data. The results indicated that (i) circulation of the dog population is very common in settlements in the Çukurova Plain, but the disease prevalence is high and stable, (ii) the performance of CS n-PCR for detecting Leishmania-dog contact is higher than IFAT, (iii) and some of the parasites isolated from dogs have different zymodemes and/or genotypes from previous human and sand fly isolates; suggesting the probability of two different cycles of leishmaniasis in this particular area. This hypothesis should be supported by future studies targeting vectors and reservoirs.

Seroepidemiological and entomological survey in a new focus of zoonotic visceral leishmaniasis in Kars province, Northeastern Turkey.

Visceral leishmaniasis (VL) has now been recorded from 38 provinces of Turkey. Twenty-one VL cases were reported within six years from settlements located in most northeastern Turkey and we therefore aimed to carry out an entomological and seroepidemiological survey in this new focus for clarifying risk factors. Blood samples from 290 children and 165 dogs were collected. Sera samples were investigated for anti-Leishmania antibodies using indirect fluorescent antibody test. Sand fly collection for determining the fauna and seasonal activity was performed in all settlements by CDC light traps between June and September 2006. Although no seropositive child was detected during the survey the overall seroprevalence rate of canine leishmaniasis was found as 7.2%. A total of 4154 sand flies were collected and 10 species of genus Phlebotomus were identified belonging to Adlerius, Larroussius, Paraphlebotomus and Phlebotomus subgenera. Among them Phlebotomus kandelakii s.l. (55.44%), Phlebotomus balcanicus (12.62%) and Phlebotomus neglectus (4.40%) was detected as probable vector species for this new focus. The poor sanitation, very high population size of sand flies, probably because of very short season, no control measures for sand flies as well as dogs, and presence of microclimate suitable for sand flies were considered as main risk factors in the area.

[Cutaneous leishmaniasis in Turkey]. / Türkiye'de Kutanöz Leishmaniasisin Durumu.

Cutaneous leishmaniasis (CL) caused by Leishmania protozoon parasites is a disease which is characterized by long-term nodulo-ulcerative lesions healing spontaneously with scarring. The disease has been well-known in Anatolia for centuries and has different names such as; Urfa boil, Antep boil, year boil, Halep boil, oriental sore and beauty scar. The causative agents are Leishmania tropica and Leishmania tropica/Leishmania infantum in Southeastern Anatolia and East Mediterranean, respectively. CL is a notifiable disease in Turkey and, according to the Ministry of Health official records, 46.003 new cases were reported between 1990 and 2010. Among those cases, 96% of them were reported from the Sanliurfa, Adana, Osmaniye, Hatay, Diyarbakir, Içel and Kahramanmaras provinces. Although 45% of cases were notified from Sanliurfa in the past 20 years, its ratio is currently decreasing while other regions' ratios have been showing an increasing trend. Easier transportation between cities, increased travel migration of the population from rural areas to the peripheral suburbs with inadequate infrastructure and unhealthy housing are thought to be the main factors for spreading the disease from Southeastern Anatolia to other regions of Turkey. Lack of treatment of patients as reservoir hosts because of different reasons and ineffective and inadequate use of insecticides against vector sand flies have also played an important role in spreading the disease. Neglect of this disease by patients and health institutions can also be considered as other factors for the spreading. We believe that, after the strategic plan for leishmaniasis prepared by the Turkish Ministry of Health with the contribution of scientists in 2011 is put into practice, the control of the disease will be more effective.

Spatial distribution of phlebotomine sand flies in the Aydin Mountains and surroundings: the main focus of cutaneous leishmaniasis in western Turkey.

An entomological survey was conducted to determine the spatial distribution of phlebotomine fauna and understand the effect of environmental factors. The entomological survey was carried out during 2006-2007 in a study area in the rural area of Aydin province, near the Kusadasi town where VL, CL, and canine leishmaniasis (CanL) are endemic. In 2006 and 2007, 132 locations were sampled using sticky traps mainly on embankments. Detailed environmental and meteorological information was also collected for each location. The results of entomological studies indicated that the probable vectors are Phlebotomus tobbi and P. neglectus for VL and CanL, and P. similis for CL in this western leishmaniasis focus. The data revealed a correlation between their presence and spatial variables such as altitude, sampling site location, and humidity. The distribution areas of probable vector species in this study area allowed the identification of risk levels, which may provide useful information to guide the leishmaniasis research in endemic regions.