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.

A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey.

Human visceral leishmaniasis (VL) caused by L. infantum and cutaneous leishmaniasis (CL) caused by L. tropica and L. infantum have been reported in Turkey. L. infantum is also responsible for canine leishmaniasis (CanL) and it is widely common in the country. The main aim of the present study was to design a real-time PCR method based on the internal transcribed spacer 1 (ITS1) region in the diagnosis of all clinical forms of leishmaniasis in Mediterranean, and to identify the species directly from clinical samples. Totally, 315 clinical specimens, human/canine visceral (blood, bone marrow, lymph node) and cutaneous (lesion aspiration) samples, and 51 Turkish Leishmania isolates typed by isoenzymatic method were included in the study. For optimization, DNA samples of the 34 strains were amplified by conventional ITS1-PCR and then sequenced for designing the primers and probes, allowing the species identification. Following the validation with the isolates, the test was applied on clinical samples and melting temperatures were used for genotyping. A group of PCR products were further sequenced for confirmation and assigning the inter- and intraspecies heterogeneity. The diagnosis of leishmaniasis is successfully achieved by the new real-time PCR method, and the test identified 80.43% of human and canine VL samples as L.infantum and 6.52% as L.tropica; 52.46% of CL samples as L. infantum and 26.90% as L. tropica. In 13.04% of visceral and 20.62% of cutaneous samples, two peaks were observed. However, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification. The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples. Genotypic variations of Leishmania parasites in Turkey within species and intraspecies were observed, and L. tropica is also found as causative agent of human and canine VL in Turkey.

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.

[The molecular diagnosis of cryptosporidiosis in fresh and formalin preserved fecal samples]. / Taze ve Formaldehitle Saklanmis Diski Orneklerinde Cryptosporidiosisin Moleküler Tanisi.

The acid-fast staining method is widely used in the diagnosis of cryptosporidiosis, a disease causing diarrhea in humans. However in this technique, some of Cryptosporidium spp oocysts can not be stained and seen as formed "ghost-like bodies" and which can only be evaluated by experienced microscopists. In the recent years, PCR technique which is proven to be also highly sensitive in diagnosis and genotyping, is used as an alternative method. In our study we aimed to evaluate the efficacy of PCR in diagnosis of cryptosporodiosis. Thirty-three stool samples, belonging to 22 patients who applied to Ege University Hospital, Parasitology Clinic between August 2001 - August 2003 and microscopically diagnosed as cryptosporodiosis has been included in the study. Twenty-three of these 33 samples were processed immediately, while ten samples were stored in 10% formalin. As the control group, 11 stool samples including 8 specimens with different parasites and 3 negative samples were selected. Nested-PCR is applied to all samples. The sensitivity and specifity of the method for fresh and formalin preserved samples were found to be 100% and 50%, respectively. As a conclusion, PCR technique is found to be useful for diagnosis in cryptosporodiosis patients with especially those including few oocysts in their fresh feces.

The infection risk of visceral leishmaniasis among household members of active patients.

Human visceral leishmaniasis (HVL), caused by Leishmania infantum is mainly observed as sporadic cases in Turkey and dogs are considered as the main reservoir of the disease. The incidence of visceral leishmaniasis among members of households where a HVL infection has already been diagnosed was studied in clusters around the diagnosed cases in different regions in Turkey. A total of 47 serum samples collected from the households of 11 proven visceral leishmaniasis patients were screened for anti-Leishmania antibodies by indirect immunofluorescent antibody test (IFAT). Three and one such household members belonging to the different families were found to be seropositive and borderline, respectively. Diagnosis was confirmed with the presence of amastigotes in bone marrow aspiration samples in all seropositives while the borderline case with slight and indefinitive symptoms of VL was followed only serologically at 3-month intervals and improved spontaneously in 1 year. Household members of individuals with previously confirmed visceral leishmaniasis were found to have higher frequency of the disease suggesting the household members should be included in the risk group for visceral leishmaniasis and serological screening should be performed for the detection of possible infection.

Diagnostic value of rK39 dipstick in zoonotic visceral leishmaniasis in Turkey.

K39 is a repetitive immunodominant epitope in a kinesin-related protein expressed predominantly in the amastigotes of visceral Leishmania spp. Enzyme immunoassays of patient's sera with recombinant K39 (rK39) proved to be highly specific and sensitive for diagnosis of active visceral leishmaniasis (VL, kala-azar). The same assays in dipstick format were also found effective for diagnosis of both human VL (HVL) and canine VL (CanVL) in most endemic areas of these diseases. Fifty-eight human patients and 22 dogs, clinically suspected of kala-azar, were screened with rK39 dipstick in comparison with the conventional methods of diagnosis, i.e., microscopic examinations of bone marrow and lymph node aspirates and immunofluorescent antibody tests (IFAT), respectively. Sixteen patients and 12 dogs were found to be rK39 dipstick positive. The results were corroborated with those of parasitological examinations, except 1, rK39-positive but smear-negative, case in each group. IFAT of the 2 discordant cases gave positive results. The rK39 dipstick is thus reliable for diagnosis of both HVL and canVL cases in Turkey.