The initial detection of Toscana virus in phlebotomine sandflies from Turkey.

Toscana virus (TOSV) is a prominent arthropod-borne viral agent of human central nervous system infections occurring in the Mediterranean region. The main transmission route to susceptible individuals involves sandflies as vectors. Despite several reports revealing widespread TOSV activity in Turkey, vectors remained unidentified. A sandfly field survey was carried out in five provinces in Central, Southeast and Mediterranean Anatolia in 2017 to identify TOSV and related sandfly-borne phleboviruses and Leishmania parasites, with evidence for circulation in the region. A total of 7136 sandfly specimens, collected via standard methods, were evaluated in 163 pools. TOSV was detected in 11 pools (6.7%), comprising Phlebotomus major sensu lato, Sergentomyia dentata and Phlebotomus papatasi species. TOSV partial L and S segment sequences were characterized, that phylogenetically clustered with local and global genotype A strains. An amino acid substitution outside the conserved motifs of the viral polymerase, also present in previous TOSV sequences in endemic regions, was observed. Leishmania tropica was detected in a single pool of Ph. sergentii (0.6%). This is the first report of TOSV in sandflies from Turkey, and this study further provides evidence for additional sandfly species with the potential to transmit TOSV.

Sandfly surveillance and investigation of Leishmania spp. DNA in sandflies in Kosovo.

In the past decade, leishmaniasis seems to be re-emerging in Balkan countries. There are serious implications that Kosovo is a visceral leishmaniasis endemic region with autochthonous transmission; nevertheless, surveillance of vectors, reservoirs or the disease is not yet established. Gaining knowledge about sandfly vector species is a prerequisite for the development of a monitoring and control plan in the future. After a long gap in research of over 70 years, sandfly studies in Kosovo were resumed in 2014. During this presence/absence study, nine sandfly species were detected: Phlebotomus papatasi, Ph. perfiliewi, Ph. tobbi, Ph. neglectus, Ph. simici, Ph. balcanicus, Ph. alexandri, Ph. mascittii and Sergentomyia minuta. Three species are new with regard to the fauna of Kosovo - Ph. alexandri, Ph. balcanicus and Ph. mascittii. Besides increased diversity, changes in the number of collected specimens and distribution range of species were recorded, with Ph. neglectus being the most dominant species with the widest distribution. Testing of randomly chosen females for Leishmania spp. DNA resulted the in detection of L. tropica in a specimen of Ph. neglectus. The presence of numerous vector species in the sandfly fauna of Kosovo pose a threat for the re-emergence of vector-borne diseases. Therefore, continuous surveillance is recommended with regular updates on vector distribution and abundance.

Molecular screening of Leishmania spp. infection and bloodmeals in sandflies from a leishmaniasis focus in southwestern Turkey.

Leishmaniasis is an arthropod-borne disease that affects approximately 2 million people worldwide annually. The aims of this study were to detect the presence of Leishmania (Kinetoplastida: Trypanosomatidae) DNA and the feeding preferences of probable vector species in an endemic focus of Leishmania infantum in Turkey. Entomological sampling was performed in August and October 2015 in Aydin province, where cases of human and canine leishmaniasis have been reported previously. A total of 1059 sandfly specimens comprising nine species belonging to two genera, Phlebotomus and Sergentomyia (both: Diptera: Psychodidae), and five subgenera of the Phlebotomus genus (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius and Transphlebotomus) were collected in five villages. Among all Phlebotomus specimens, Phlebotomus neglectus (39%) was noted as the most abundant species, followed by Phlebotomus tobbi (18%). Leishmania DNA was detected in pools from P. neglectus, P. tobbi and Sergentomyia dentata by kDNA polymerase chain reaction (PCR). Leishmania DNA from Phlebotomus specimens was identified as L. infantum, but Leishmania DNA from Sergentomyia spp. could not be identified to species level by ITS-1 real-time PCR. The detection of Leishmania DNA in wild-caught P. neglectus and the high percentage (24.2%) of human DNA in engorged specimens suggests that P. neglectus is probably an important vector species for L. infantum in Aydin province.

sandflyDST: a dynamic web-based decision support tool for the morphological identification of sandflies present in Anatolia and mainland Europe, and user study.

Species identification of sandflies is mainly performed according to morphological characters using classical written identification keys. This study introduces a new web-based decision support tool (sandflyDST) for guiding the morphological identification of sandfly species present in Anatolia and mainland Europe and classified in the Phlebotomus and Sergentomyia genera (both: Diptera: Psychodidae). The current version of the tool consists of 111 questions and 36 drawings obtained from classical written keys, and 107 photographs for the quick and easy identification of 26 species of the genus Phlebotomus and four species of the genus Sergentomyia. The tool guides users through a decision tree using yes/no questions about the morphological characters of the specimen. The tool was applied by 30 individuals, who then completed study questionnaires. The results of subsequent analyses indicated that the usability (x‾SUSScore=75.4) and users' level of appreciation (86.6%) of the tool were quite high; almost all of the participants considered recommending the tool to others. The tool may also be useful in training new entomologists and maintaining their level of expertise. This is a dynamic tool and can be improved or upgraded according to feedback. The tool is now available online at http://parasitology.ege.edu.tr/sandflyDST/index.php.

Sampling strategies for phlebotomine sand flies (Diptera: Psychodidae) in Europe.

The distribution of phlebotomine sand flies is widely reported to be changing in Europe. This can be attributed to either the discovery of sand flies in areas where they were previously overlooked (generally following an outbreak of leishmaniasis or other sand fly-related disease) or to true expansion of their range as a result of climatic or environmental changes. Routine surveillance for phlebotomines in Europe is localized, and often one of the challenges for entomologists working in non-leishmaniasis endemic countries is the lack of knowledge on how to conduct, plan and execute sampling for phlebotomines, or how to adapt on-going sampling strategies for other haematophagous diptera. This review brings together published and unpublished expert knowledge on sampling strategies for European phlebotomines of public health concern in order to provide practical advice on: how to conduct surveys; the collection and interpretation of field data; suitable techniques for the preservation of specimens obtained by different sampling methods; molecular techniques used for species identification; and the pathogens associated with sand flies and their detection methods.

Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey.

Infantile Mediterranean visceral leishmaniasis (IVL) and anthroponotic cutaneous leishmaniasis (ACL) have long been known to exist in the western and southeastern Turkey, respectively. To further study these and other related diseases, a recombinant antigen (rK39) specific to VL was used in an ELISA for serodiagnosis of selected patients and for screening dog reservoir populations in several endemic sites. Among 24 confirmed VL cases from western Turkey, the rK39 ELISA proved to be more sensitive than a combination of cultivation and microscopy of bone marrow aspirates. The specificity of rK39 for leishmaniasis was demonstrated by its lack of cross-reactivity with sera from other human diseases in the same sites. Interestingly, six of the 83 parasitologically proven ACL cases from southeast Turkey were also rK39 positive. The end point titers of the positive VL and CL cases vary from 10(-2) to 10(-5) and from 10(-2) to 10(-3), respectively. The rK39 ELISA was also used to screen 494 apparently healthy dogs from Urfa in southeast Turkey, Manisa/Alasehir near the Aegean Sea, and Karabuk near the Black Sea. Eighteen rK39-positive cases (3.6%), all from the latter two areas, were found to have varying endpoint titers (10(-2)-10(-4)). The high titers predicted increased severity and frequency of the clinical symptoms (i.e., lymphadenopathy, depilation, skin lesion, weight loss and/or death), which were manifested subsequently in 16 of these 18 cases. In addition, more positive canine cases were diagnosed by the rK39 ELISA preclinically than the procedures to detect parasites postsymptomatically in the lymph node aspirates. The use of the rK39 ELISA as a sensitive tool makes it possible to demonstrate coendemicity of canine and human VL, as expected in the case of IVL. The results also point to the possible presence of additional VL types in western Turkey and cutanovisceral type in the southeast part of this country.

Dog-DAT: a direct agglutination test using stabilized, freeze-dried antigen for the serodiagnosis of canine visceral leishmaniasis.

We have evaluated the use of an improved direct agglutination test (DAT) based on stable, freeze-dried antigen for the detection of anti-Leishmania antibodies in canine serum samples. With a cut-off value of 1:640, the sensitivity of the DAT was shown to be 100% and the specificity of the test was 98.8%.

Leishmaniasis in Turkey.

Leishmaniases are widespread in most countries in the Mediterranean basin, including Turkey. Two forms are observed in Turkey; Leishmania infantum is responsible from visceral leishmaniasis (VL), and L. tropica causes cutaneous leishmaniasis (CL). Phlebotomus sergenti, P. papatasi, P. major and P. syriacus are considered to be the probable vectors, and dogs are the main reservoir of L. infantum, while P. sergenti is the main suspected vector of L. tropica.VL is sporadically seen mainly in the Aegean, Mediterranean, and Central Anatolia Regions, but CL is endemic, especially in the Southeastern and Mediterranean Regions. Major touristic sites are free of both infections, and no infection is reported in any tourist. Mean number of annual VL and CL cases reported to Ministry of Health are 40 and 1,204, respectively, in the last four years. These data suggest that both VL and CL represent a public health problem in Turkey, but a decline is observed in the number of cases with both infections in recent years.

A survey on canine leishmaniasis in western Turkey by parasite, DNA and antibody detection assays.

Human visceral leishmaniasis (VL) caused by Leishmania infantum is found throughout the Mediterranean Region, including Turkey, where dogs are considered to be the main reservoir host for this parasite. In the district of Manisa, western Turkey, 37 human VL cases were reported from June 1993-August 1997. Twenty-four villages in this district were chosen for a survey of disease prevalence in dogs. The dogs, 490 in total, were examined using either the indirect immunofluoresence assay (IFAT) or direct agglutination test (DAT). Anti-Leishmania antibodies were found by at least one test in 5.3% (26/490) of the dogs. Infections were confirmed by parasitological examination of or polymerase chain reaction (PCR) on lymph node aspirates in 65% (13/20) and 76.4% (13/17) of the seropositive dogs tested, respectively. The confirmation rate was 85% by combining the results of PCR and microscopy. Our results demonstrate that canine VL is wide spread in western Turkey where human VL is endemic, and that serodiagnosis is a valuable tool for monitoring the infection.

Sand flies (Diptera: Phlebotominae) in Sanliurfa, Turkey: relationship of Phlebotomus sergenti with the epidemic of anthroponotic cutaneous leishmaniasis.

Sand fly (Diptera: Phlebotominae) fauna were surveyed in various districts of Sanliurfa in southeast Turkey for 3 yr immediately after an epidemic of cutaneous leishmaniasis (Leishmania tropica). Sticky papers and CDC light traps collected a total of 10,937 sand flies, of which 10,919 (4,158 females and 6,761 males) were identified as Phlebotomus and 18 (11 females and seven males) as Sergentomyia (S. theodori Parrot; S. adleri Theodor). Eight Phlebotomus spp. were identified: P. sergenti Parrot (72.3%), P. papatasi (Scopoli) (27.2%), P. brevis Theodor & Mesghali (0.20%), P. neglectus Leger & Pesson (0.13%), P. perfiliewi Parrot (0.05%), P. mascitti Grassi, P. halepensisTheodor, and P. alexandri Sinton (0.01%). Phlebotomus mascitti and P. neglectus, along with both Sergentomyia sp., have not been previously described from the study area. Similar results were obtained when both trapping methods were applied in the same houses, indicating that local P. sergenti and P. papatasi populations were equally attracted to the light. P. sergenti was consistently abundant, agreeing with the general view that this species is the vector of leishmaniasis in the region. There was no apparent decrease in the relative abundance of this vector versus the other species, suggesting that factor (s) other than a change in the dynamics of sand fly populations precipitated the decline of the human leishmaniasis epidemic in Sanliurfa.

Development of a fast agglutination screening test (FAST) for the detection of anti-Leishmania antibodies in dogs.

A fast agglutination screening test (FAST) for the detection of anti-Leishmania antibodies in serum samples from dogs with visceral leishmaniosis was developed. The test is based on the direct agglutination test (DAT), but combines a higher parasite concentration with a smaller test volume. In contrast to the DAT, the FAST makes use of only one serum dilution and the results can be read within 3 h as opposed to 18-20 h for the DAT. The FAST was evaluated using serum samples of confirmed cases of the disease and healthy controls collected in the most important endemic regions of canine visceral leishmaniosis, import cases of canine leishmaniosis in a non-endemic country, from non-endemic healthy controls and from dogs with other diseases. The performance of the FAST was compared with standard DAT. In the present study, the FAST had a sensitivity of 93.6% and a specificity of 89.0%. The DAT had a sensitivity of 88.6% and a specificity of 96.7%. Furthermore, using a large panel of serum samples of previously examined DAT positive or negative dogs it was shown that degree of agreement between the two tests was high (95.7%; kappa value = 0.91). The FAST offers the advantages of the DAT based on freeze-dried antigen with respect to stability of the antigen, sensitivity and specificity. Moreover, the FAST allows the rapid screening of a large number of samples, which makes the test very useful for epidemiological screening of large populations of dogs.

Cutaneous leishmaniasis infection in Balb/c mice using a Leishmania tropica strain isolated from Turkey.

Leishmania tropica, which is endemic in Turkey, is the causative agent of cutaneous leishmaniasis. Leishmania tropica promastigotes (2 x 10(7)) isolated from a patient with dermal leishmaniasis and reproduced in NNN medium were inoculated subcutaneously into the footpads of 10 Balb/c mice. Cutaneous leishmaniasis developed on the footpads of 4 mice approximately 45 days later. Leishmania tropica amastigotes were observed in smear slides and then cultivated in NNN medium. Balb/c mice are a suitable laboratory model for this isolate of L. tropica and thus a source of amastigotes for studies on the immunology, chemotherapy, and pathogenicity of cutaneous leishmaniasis.

A comparison of promastigote and amastigote antigens of Leishmania infantum in the diagnosis of visceral Leishmaniosis by ELISA and IFA tests in Turkey.

This study was designed to evaluate the ELISA and IFA tests for diagnosis of visceral leishmaniosis (VL) using soluble and crude antigens from promastigote and amastigote forms of Leishmania infantum. A total of 70 sera were tested, including 30 patients who were suspected for Leishmaniosis (10 healthy persons, 10 patients with Toxoplasmosis, 10 patients with Giardiosis). In the 30 patients suspected to have Leishmaniosis, smears were prepared from bone marrow aspirates and all material aspirated was cultured in NNN medium. The results were compared with those of parasitological and serological tests. While 2 patients were serologically positive and parasitologically negative from the 30 patients, 8 patients were serologically and parasitologically positive. The amastigote and promastigote antigens were 100% specific. Using these antigens with ELISA and IFA tests did not result and diagnosis different than VL.

Plastic detection comb better than visual screening for diagnosis of head louse infestation.

Finding lice can be difficult in head louse infestation. We compared a new louse detection comb with visual inspection. All children in two rural Turkish schools were screened by the two methods. Those with lice were offered treatment and the results monitored by detection combing. Children with nits only were re-screened to identify latent infestations. Using visual inspection we found 214/461 children (46%) with nits but only 30 (6.5%) with live lice. In contrast detection combing found 96 (21%) with live lice, of whom 20 had no nits. Detection combing was 3.84 times more effective than visual inspection for finding live lice. Only 10/138 (7.2%) children with nits and no lice were found to have active infestation by day 16. We found that the detection comb is significantly (P<0.001) more effective than visual screening for diagnosis; that nits are not a good indicator of active infestation; and that treatment with 1% permethrin was 89.6% effective.

Detection of species-specific antibody response of humans and mice bitten by sand flies.

Sand fly saliva plays an important role in Leishmania transmission. We characterized the host antibody response to saliva from 3 sand fly species. Specific IgG was observed in sera from experimentally bitten mice as well as in sera from individuals living in the endemic area of Leishmania tropica in Sanliurfa, Turkey. Sera of Sanliurfa inhabitants showed high IgG levels against saliva of Phlebotomus sergenti and P. papatasi, the 2 most abundant sand fly species in this area, but did not react with saliva of the New World sand fly, Lutzomyia longipalpis. Patients with active Le. tropica lesions possessed significantly higher anti-P. sergenti IgG levels than the healthy individuals from the same place while anti-P. papatasi IgG levels were equal in both groups. Major protein bands in P. papatasi and P. sergenti saliva reacted with both, human and mice sera; in P. papatasi, however, mouse IgG recognized preferentially the 42 kDa protein band while the human IgG reacted strongly with the 30 kDa band. Our data suggest that the antibody response to sand fly saliva could be used for monitoring the exposure of humans and other hosts to sand flies and might be used as a marker of risks for Leishmania transmission in endemic areas.

The sandflies (Diptera: Psychodidae) in the Turkish province of Hatay: some possible vectors of the parasites causing human cutaneous leishmaniasis.

The province of Hatay, which lies on the Mediterranean coast in south-eastern Turkey, forms an important endemic area for human cutaneous leishmaniasis (HCL). Between the June and November of 2001, the sandflies in eight areas of the province, Erzin, Dortyol, Iskenderun, Kirikhan, Antakya, Samandag, Altinozu and Yayladagi, were surveyed, using sticky-paper traps placed in goat, sheep, cow or chicken sheds close to houses. Although 217 of the 998 sandflies caught belonged to the genus Sergentomyia, the rest were of Phlebotomus species, including some species that, elsewhere, are known to act as vectors of the parasites causing HCL. The Sergentomyia caught were either S. theodori (59.9%) or S. dentata (40.1%). Most of the Phlebotomus were P. sergenti (60.8%) but P. papatasi (18.8%), P. syriacus (9.3%), P. neglectus (5.1%), P. tobbi (2.7%), P. simici (2.2%), P. alexandri (0.9%) and P. kyreniae (0.1%) were also identified (the latter three species for the first time in Hatay province). The identification of the main vector species of sandfly in the province is complicated because it is still uncertain which Leishmania species cause(s) the local HCL. It seems likely, however, that P. sergenti is the main vector in Antakya, Iskenderun, Dortyol, Erzin and Kirikhan. In areas such as Altinozu and Yayladagi, where, although P. sergenti is very rare or absent, HCL cases still occur each year, other species, such as the relatively abundant P. papatasi and P. syriacus, may be the main vectors.

Cultivation of Leishmania sp. in nutrient broth.

Bone-marrow aspiration and biopsy material samples obtained from two patients, one diagnosed as visceral and other as cutaneous leishmaniasis, were inoculated in Novy, McNeal, Nicolle (NNN) medium and nutrient broth (NB), containing fetal calf serum (FCS), penicillin and streptomycine. Both media were incubated at 27 degrees C for 10 days and observed daily for L. infantum and L. major promastigotes. Promastigotes were observed in nutrient broth after the first day, while in NNN media after the second or third day of incubation, indicating the effectiveness of nutrient broth in early diagnosis of both forms of leishmaniasis.

A new experimental in vitro culture medium for cultivation of Leishmania species.

A new liquid culture medium prepared with chemicals that can be obtained economically and commercially was tested in in vitro cultivation of Leishmania promastigotes to obtain a large number of organisms to use in serological studies. The number of Leishmania infantum and Leishmania tropica promastigotes taken from Novy-MacNeal-Nicolle (NNN) medium reached 1 x 10(7)/ml at the end of the 8th day in our new medium, though in NNN medium the number of organisms reached only 5 x 10(6)/ml. After 10 subsequent passages, the culture medium prepared was evaluated as being quite inexpensive, simple, and successful compared with other commercially available liquid culture media.

A sero-epidemiological study of visceral leishmaniosis in Izmir District, Turkey.

Visceral leishmaniosis caused by Leishmania infantum have been reported in Turkey. A total of 2055 blood samples collected in Izmir and six surrounding villages were tested for anti-Leishmania antibodies. Three established immunodiagnostic techniques, indirect fluorescence antibody (IFA), enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT), were employed using promastigotes and rK39 as antigens for the detection of anti-Leishmania antibodies. Five sero-positive cases (0.24%) were detected by the three tests used.

Evaluation of three new culture media for the cultivation and isolation of Leishmania parasites.

The aim of this study was to establish novel culture media for Leishmania parasites with a potential of obtaining high amounts of promastigotes with long-term viability, and consisting of ingredients that were available in microbiology or parasitology laboratories. Other features of these media included no requirement for blood, FCS (Fetal calf serum) or erythrocyte lysate, inexpensiveness and easiness in preparation. In addition, aspiration samples obtained from cutaneous leishmaniasis (CL) suspected patients were cultivated in these media. Three culture media were prepared; trypticase beef extract hemoglobine (TBH) medium, including trypticase, beef extract and yeast extract as the protein source, glucose as the carbohydrate source, FeNH4 and bovine hemoglobine; Peptone-Yeast extract medium (PY), found to be effective in our previous studies for cultuvation of on Leishmania parasites, with bovine hemoglobine (PYH) and Brain Heart medium, containing bovine hemoglobine (BKH). The number of promastigotes were the highest on day 8 and 13 in RPMI 1640 and BKH medium, respectively. In TBH and PYH, the peak level of reproduction was between day 16 and 19, and it was found to be higher in TBH medium after the day 20. The number of promastigotes were found to be close in BKH, TBH and RPMI-1640 media and lower in PYH medium. Examination of the cultivation of the aqueous lesion specimens of the 10 CL-suspected cases in media revealed reproduction in 9 flasks of RPMI-1640 containing 10% FCS, 7 TBH, 6 BKH and 4 PYH. The differences between the culture media were not found to be statistically significant. These results suggested that, three liquid culture media, assessed in this study, with no requirement for FCS or erythrocyte lysate, were effective in the reproduction of promastigotes, and could be used effectively in the patient isolation and field studies, as well.