Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

BACKGROUND: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. METHODS/PRINCIPAL FINDINGS: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. CONCLUSIONS: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

Evaluation of the efficacy of Olyset® Plus in a village-based cohort study in the Cukurova Plain, Turkey, in an area of hyperendemic cutaneous leishmaniasis.

The aim of this study was to measure the protective efficacy of Olyset(®) Plus, a new long-lasting factory-treated insecticidal net (LLIN) incorporated with 2% permethrin and 1% of the synergist piperonyl butoxide (PBO), against cutaneous leishmaniasis (CL) transmission under field conditions. A village-scale trial, promoting the use of LLIN by the local inhabitants of the study area was conducted as a pilot study in a new hyperendemic focus of CL caused by a Leishmania infantum/L. donovani hybrid parasite transmitted by proven vector species Phlebotomus tobbi in Cukurova Plain, Adana, Turkey, between May, 2013 and May, 2014. The study area comprised eight villages; two of them were selected as an intervention village with Olyset(®) Plus net (Kizillar) and a control village without net application (Malihidirli). Six villages with surrounding allopatric barriers were utilized as a buffer zone cluster between intervention and control villages. Monthly entomological surveys were performed in the intervention and control villages and Damyeri, representing the other six villages, to collect adults of Phlebotomus tobbi. Results showed a significant reduction in cutaneous leishmaniasis incidence in the intervention village from 4.78% to 0.37%. The protective efficacy rate of LLIN was 92.2%. In contrast, incidence rates increased in the control village from 3.67% to 4.69%. We also evaluated residual insecticide levels of used nets after six and 12 months of usage. It was determined that the nets had retained full insecticidal strength. These results highlight the value of real-world data on bed net effectiveness and longevity to guide decisions regarding sand fly control strategies. To the best of our knowledge, this is the first field study to evaluate Olyset(®) Plus efficacy in a hyperendemic cutaneous leishmaniasis area.

Seroprevalence of West Nile virus and tick-borne encephalitis virus in southeastern Turkey: first evidence for tick-borne encephalitis virus infections.

West Nile virus (WNV) and tick-borne encephalitis virus (TBEV) are among the medically important Flaviviruses that cause significant morbidity and mortality in humans. In this study, seroprevalence of WNV and TBEV in sera from two state medical hospitals from the southeastern part of Turkey was investigated. One hundred eighty-one serum samples were evaluated for WNV immunoglobulin G (IgG) by an indirect immunofluorescence test (IIFT) and for IgG antibodies against TBEV by a commercial enzyme-linked immunosorbent assay (ELISA) kit with enhanced sensitivity and specificity. Sera positive for WNV IgG were further analyzed by plaque reduction neutralization assay (PRNA). TBEV IgM was also investigated by ELISA in all seroreactive samples. Of 181 sera, 29 (16%) were positive for WNV IgG by IIFT and 17 of 179 (9.5%) were confirmed by PRNA. Nineteen of 181 (10.5%) sera were detected to have TBEV IgG. Mean titer of TBEV IgG was 43.0 RU/mL (median, 33.9 RU/mL; cutoff: 20 RU/mL). Four samples with WNV IgG antibodies were also positive for TBEV IgG antibodies. TBEV IgM was detected in 9 of 39 (23%) of all seroreactive sera, where IgM positivity were accompanied by IgG for 6 samples. These results suggest the presence of possible human WNV and TBEV infections in southeastern Turkey where vector activity have previously been detected.

West Nile virus studies in the Sanliurfa Province of Turkey.

We attempted to isolate West Nile virus from mosquitoes collected in the field for the first time in Turkey. A total of 6,457 mosquito specimens from Culex pipiens Linnaeus, Ochlerotatus caspius (Pallas) and Aedes spp. species were included in this study. Culex pipiens samples made up 56% of the total species, O. caspius 24% and Aedes spp 20%. There were no positive results after studying mosquito samples using Real-time PCR, VecTest, and Vero cell culture. In serological tests of 181 human serum samples, 29 (16%) were found to be West Nile positive. On the basis of these results, we intend to collect more mosquito samples especially from those areas from which positive serum samples were obtained.

Phenotypic variation among local populations of phlebotomine sand flies (Diptera: Psychodidae) in southern Turkey.

The wing-shape morphology of local populations of the medically important phlebotomine sand flies, Phlebotomus sergenti, P. papatasi, P. tobbi, and P. similis, were examined in both sexes by using geometric morphometrics. There are three major mountain ranges that may serve as geographical barriers for species distribution in the study area and four main gaps were recognized among these barriers. We found no statistically important differences in wing morphology in all examined species in both sexes for all local populations. These results show that the barriers are not sufficient to stop gene flow among local populations of sand flies. The graphical depiction of PCA, CVA, and F-test confirmed our morphometric study suggesting that the difference in wing morphology between P. similis and P. sergenti indicates that these are clearly different species. These two show sympatric distribution in the Konya Plain of Anatolia.

Distribution and altitudinal structuring of phlebotomine sand flies (Diptera: Psychodidae) in southern Anatolia, Turkey: their relation to human cutaneous leishmaniasis.

The two Old World genera, Phlebotomus and Sergentomyia, were both recorded in southern Anatolia in Turkey. Phlebotomus species predominated and comprised about 93% of the entire collection (3,172 specimens). Out of the sixteen species identified, two belonged to the genus Sergentomyia: S. dentata and S. theodori. The remaining fourteen species in the genus Phlebotomus were grouped under four subgenera including some species that are elsewhere known to act as vectors of human cutaneous leishmaniasis. Most of the Phlebotomus were P. tobbi (32.5%), but P. papatasi, P. transcaucasicus, P. halepensis, P. galilaeus, P. sergenti, P. syriacus, P. neglectus, P. simici, P. alexandri, P. similis, P jacusieli, P. perfiliewi, and P. brevis were also identified. There were two associations of sand fly fauna with altitudinal gradient; the first one at relatively higher altitudes and the second one at lower altitudes. The transition between these two assemblages was within the range of 800-1,000 m. It is likely that Adana and Hatay provinces are transitional areas between western and eastern Anatolia. Mountains do not appear to be important geographical barriers for sand fly distribution. We also found that the proven vector P. sergenti is a widely distributed species throughout southern Anatolia and this species, together with its closely related species P. similis, shows sympatry in Konya Province.

Field evaluation of an area repellent system (Thermacell) against Phlebotomus papatasi (Diptera: Psychodidae) and Ochlerotatus caspius (Diptera: Culicidae) in Sanliurfa Province, Turkey.

A field evaluation of a new area repellent system, Thermacell Mosquito Repellent (TMR, cis-trans allethrin), was conducted against phlebotomine sand flies and mosquitoes in Cunpolat village, Sanliurfa Province, in southeastern Anatolia, Turkey, an area historically endemic for cutaneous leishmaniasis and high sand fly populations. The objective of this study was to determine the efficacy, duration of protection, and spatial characteristics of protection (downwind distance from point of release), of the TMR. Twelve adult volunteers (nine in the treatment and three controls) made collections from ankle to knee for 25 min every hour from 2100 to 0300 on six consecutive nights in August 2002. Treatment consisted of placing a TMR unit at the middle of the village and then placing human bait collectors at 2.3, 4.6, and 7.6 m away from the repellent unit. Results from the field tests showed highly significant protection provided by the TMR from attack by Phlebotomus papatasi (Scopoli) (P < 0.001) and Ochlerotatus caspius (Pallas) (P < 0.001) for up to 4 h postapplication. In the six nights that tests were done, a total of 949 sand flies and 1095 mosquitoes were collected from the untreated control sites. Only 86 sand flies and 83 mosquitoes were collected at all distances from the places treated with TMR. These results indicated that reduction in sand fly and mosquito biting rates in treated groups ranged from 87.5 to 97.7% (mean protection 92%) and 90.2-97.4% (mean protection 93%), respectively. The percentage reduction values were maintained above 90.0% for 6 h of the assessment period. Overall, the number of bites by the cutaneous leishmaniasis vector Ph. papatasi and also Oc. caspius was reduced > 11-fold and 13-fold, respectively, by the TMR.