Yusuf Özbel

Spread of Vector-borne Diseases and Neglect of Leishmaniasis, Europe

Exotic vector-borne diseases are gaining attention at the expense of leishmaniasis.

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.

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.

Molecular homogeneity in diverse geographical populations of Phlebotomus papatasi (Diptera, Psychodidae) inferred from ND4 mtDNA and ITS2 rDNA Epidemiological consequences.

An intraspecific study on Phlebotomus papatasi, the main proven vector of Leishmania major among the members of the subgenus Phlebotomus, was performed. The internal transcribed spacer 2 (ITS 2) of rDNA and the ND4 gene of mt DNA were sequenced from 26 populations from 18 countries (Albania, Algeria, Cyprus, Egypt, Greece, India, Iran, Israel, Italy, Lebanon, Morocco, Saudi Arabia, Spain, Syria, Tunisia, Turkey, Yugoslavia and Yemen), and compared. Samples also included three other species belonging to the subgenus Phlebotomus: P. duboscqi, a proven vector of L. major in the south of Sahara (three populations from Burkina Faso, Kenya and Senegal), P. bergeroti, a suspected vector of L. major (three populations from Oman Sultanate, Iran and Egypt), and one population of P. salehi from Iran. A phylogenetic study was carried out on the subgenus Phlebotomus. Our results confirm the validity of the morphologically characterized taxa. The position of P. salehi is doubtful. Variability in P. papatasi contrasts with that observed within other species having a wide distribution like P. (Paraphlebotomus) sergenti in the Old World or Lutzomyia (Lutzomyia) longipalpis in the New World. Consequently, it could be hypothesized that all populations of P. papatasi over its distribution area have similar vectorial capacities. The limits of the distribution area of L. major are correlated with the distribution of common rodents acting as hosts of the parasites.

Sand Flies (Diptera: Phlebotominae) in Sanliurfa, Turkey Relationship of Phlebotomus sergenti with the Epidemic of Anthroponotic Cutaneous Leishmaniasis

Abstract 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. halepensis Theodor, 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.

Epidemiology, diagnosis and control of leishmaniasis in the Mediterranean region.

The leishmaniases are a widespread and medically important group of parasitic diseases, some of which pose a serious health threat in communities throughout the Mediterranean basin. In 1993, a joint, collaborative study of the Mediterranean leishmaniases was initiated by scientists from Israel, Turkey, Portugal and the Netherlands. The aim of this project was the development of a multi-component approach to the successful control of all forms of leishmaniasis, with special emphasis on the more severe, visceral leishmaniasis (VL). The need for highly sensitive and accurate new tools to facilitate diagnosis and epidemiological surveys of endemic areas and for studies on the immunology of VL in laboratory models (dogs and mice) was soon recognized. It is anticipated that the development of these tools and the associated technology will provide a better understanding of the disease and improve its control.

Multi-site DNA polymorphism analyses of Leishmania isolates define their genotypes predicting clinical epidemiology of leishmaniasis in a specific region.

Abstract Leishmania isolates from 57 cases of human cutaneous (CL), human visceral (VL), and canine visceral (CVL) leishmaniasis in Turkey were grouped by multi-site DNA polymorphism analyses into five genotypes. The initial grouping was based on DNA heterogeneity of the faster-evolving mitochondrion (kinetoplast) minicircles and the intergenic regions of two nuclear repetitive genes. Taxonomic affiliation and phylogenetic relationships of the five genotypes were inferred by comparing them with reference species for sequence heterogeneity in a ∼1.4 kb conserved single-copy gene, encoding N-acetylglucosamine-1-phosphate transferase (NAGT). Alignment of the available sequences revealed no gap, but up to 7% scattered base substitutions, suggesting that this functionally important gene is a suitable marker. Three genotypes are completely identical to the NAGTs of the reference species, identifying them as L. infantum, L. tropica, and L. major, respectively. The remaining two are recognized as L. major NAGT variants with one and four base substitutions, respectively. As expected, Maximum Likelihood analysis of the NAGT sequences separates them into three clades, corresponding to the three species. The majority of the isolates obtained are L. infantum and L. tropica, which have been known to cause infantile VL and anthroponotic CL in western and southeastern Turkey, respectively. Unexpected is the finding of Leishmania major variants and their dispersal, possibly as previously unrecognized clinico-epidemiologic entities of CL and VL.

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. Hovewer, 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.

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.

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.