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Leishmaniasis, caused by Leishmania protozoan parasites transmitted by Phlebotomine sand flies, is a significant public health concern in the Mediterranean basin. Effective monitoring of Leishmania-infected sand flies requires standardized tools for comparing their distribution and infection prevalence. Consistent quantitative real-time PCR (qPCR) parameters and efficient DNA extraction protocols are crucial for reliable results over time and across regions. However, the absence of standardized technical recommendations for Leishmania DNA detection hinders effective surveillance. This study aimed to compare different DNA extraction protocols and conduct a qPCR-based External Quality Assessment (EQA) through a multicenter study involving nine reference laboratories, with a focus on optimizing Leishmania DNA detection in sand fly. EQA samples consisted of Leishmania infantum and L. major species, at concentrations ranging from 101 to 104 parasites/mL. All but one center detected all concentrations, demonstrating strong diagnostic proficiency. The ability to detect low concentrations highlighted the robustness of the qPCR assay used, though variations in Cq values indicated differences in sensitivity related to technical capabilities or DNA extraction kit performance. A comparative analysis of seven DNA extraction methods identified the EZ1 DSP Virus Kit and QIAamp DNA mini-kit as the most efficient, supporting their use in standardized protocols. The study also assessed the effects of lyophilization and shipment conditions, showing no significant compromise in Leishmania detection despite slight variations in Cq values. Experimentally infected sand flies were included to simulate field conditions, and all centers successfully detected positive samples with varying Cq values, probably reflecting differences in infection load. This study emphasizes the importance of standardized DNA extraction protocols and continuous quality assurance for accurate Leishmania DNA detection. The results highlight the superior performance of certain extraction kits and the need for ongoing technical training, essential for reliable leishmaniasis surveillance, particularly in field settings with low infection densities.

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA–positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.

Leishmaniasis is a significant public health concern in large parts of the world including Israel, with limited diagnostic tools available for effective surveillance and control. Traditional methods for sand fly species identification, Leishmania detection, and blood meal analysis are time-consuming and prone to errors. To address these challenges, this study aimed to develop PCR-high resolution melt (HRM) assays to accurately determine sand fly species, Leishmania infection and blood meal sources in sand flies. Field-collected sand flies from all regions of Israel were used for the validation of three PCR-HRM assays. These included 254 sand fly males and females identified morphologically for species verification; 1,120 unfed females for Leishmania detection, and 538 engorged females for blood meal identification. PCR products were subjected to HRM curve analysis, and results were compared to nucleotide sequencing and sand fly morphology. Eleven sand fly species, 25 different host species blood meals and four Leishmania species were discerned and each presented a specific HRM pattern. Of the 1,658 analyzed females, 16 (1%) were positive for Leishmania , and the species identified were: Leishmania major, L. tropica, L. infantum and L. donovani . Blood meal source was identified in 520 (96.7%) engorged females. Blood from four animal species (domestic cat, rock hyrax, European hare, cow) accounted for 53% of the sand fly blood meals and the remaining 47% came from 21 other animal species. The sand fly species distribution showed that L. major and L. donovani vectors were mostly prevalent in arid southern Israel while L. tropica and L. infantum vectors were abundant in central and northern Israel. These results present the current knowledge of the different Leishmania species life cycles, vectors, and host species present in Israel and substantiate the utility of the assays developed herein which combine the advantages of PCR and the discriminatory power of HRM.

Background Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of Leishmania spp., the causative agents of leishmaniasis. Since 2008, the Ministry of Health and the Ministry of Environmental Protection of Israel have conducted nationwide, periodic sand fly surveys. Initially, these surveys focused on localities with known endemic transmission of cutaneous leishmaniasis, but in recent years, entomological trapping has expanded to areas where prior information on sand flies was unavailable. Here we report the first confirmed occurrence of Phlebotomus (Larroussius) orientalis (Parrot, 1936) in Israel and place the Israeli material in a comparative phylogeographic context. Methods Entomological surveys by CO 2 trapping were conducted in the Negev Desert, southern Israel, between 2020 and 2024. Morphological sand fly identification was confirmed by sequencing fragments of the mitochondrial COI and Cytb / NADH1 genes. For a newly reported species, we inferred intraspecific phylogenetic relationships and divergence times between major clades. A subset of females was additionally screened for Leishmania DNA and vertebrate blood-meal sources by real-time polymerase chain reaction (PCR) coupled with high-resolution melt analysis. Results Targeted surveys and routine surveillance in the Negev region between 2020 and 2024 yielded 269 Phlebotomus orientalis (96 males, 173 females) among other species of local sand fly fauna from multiple wadi systems in the central Negev. These detections constitute the first confirmed records of Ph. orientalis in Israel. Species identification was confirmed through both morphological examination and molecular analyses of partial COI and Cytb / NADH1 genes. Phylogenetic analysis indicated that the Israeli Ph. orientalis specimens constitute a distinct lineage that diverged from East African conspecifics during the Early to Middle Pleistocene. Blood-meal analysis of engorged Ph. orientalis females identified the European hare as a vertebrate host, and none of the tested Ph. orientalis specimens were positive for Leishmania DNA. Conclusions Phlebotomus orientalis is a confirmed vector of L. donovani , the main agent of visceral leishmaniasis in East Africa. Its detection as a distinct and apparently long-established lineage in the Negev, in a region where parasites of the L. donovani complex are already involved in cutaneous leishmaniasis transmission, highlights the need to clarify the distribution, ecology, and host preferences of Ph. orientalis in Israel. Further studies are required to characterize its spatial and seasonal occurrence, evaluate its vector competence for L. donovani and L. infantum , and assess its potential contribution to current and future leishmaniasis transmission risks.

Zoonotic cutaneous leishmaniasis has long been endemic in Israel. In recent years reported incidence of cutaneous leishmaniasis increased and endemic transmission is being observed in a growing number of communities in regions previously considered free of the disease. Here we report the results of an intensive sand fly study carried out in a new endemic focus of Leishmania major. The main objective was to establish a method and to generate a data set to determine the exposure risk, sand fly populations' dynamics and evaluate the efficacy of an attempt to create \"cordon sanitaire\" devoid of active jird burrows around the residential area. Sand flies were trapped in three fixed reference sites and an additional 52 varying sites. To mark sand flies in the field, sugar solutions containing different food dyes were sprayed on vegetation in five sites. The catch was counted, identified, Leishmania DNA was detected in pooled female samples and the presence of marked specimens was noted. Phlebotomus papatasi, the vector of L. major in the region was the sole Phlebotomus species in the catch. Leishmania major DNA was detected in ~10% of the pooled samples and the highest risk of transmission was in September. Only a few specimens were collected in the residential area while sand fly numbers often exceeded 1,000 per catch in the agricultural fields. The maximal travel distance recorded was 1.91km for females and 1.51km for males. The calculated mean distance traveled (MDT) was 0.75km. The overall results indicate the presence of dense and mobile sand fly populations in the study area. There seem to be numerous scattered sand fly microsites suitable for development and resting in the agricultural fields. Sand flies apparently moved in all directions, and reached the residential area from the surrounding agricultural fields. The travel distance noted in the current work, supported previous findings that P. papatasi like P. ariasi, can have a relatively long flight range and does not always stay near breeding sites. Following the results, the width of the \"cordon sanitaire\" in which actions against the reservoir rodents were planned, was extended into the depth of the agricultural fields.

Background Artemisinin-based combination therapies (ACTs) are recommended as first-line treatment against uncomplicated Plasmodium falciparum infection. Mutations in the PfKelch13 (PF3D7_1343700) gene led to resistance to artemisinin in Southeast Asia. Mutations in the Pfcoronin (PF3D7_1251200) gene confer reduced artemisinin susceptibility in vitro to an African Plasmodium strain, but their role in clinical resistance has not been established. Methods We conducted a retrospective observational study of Israeli travellers returning from sub-Saharan Africa with P. falciparum malaria, including patients with artemether–lumefantrine (AL) failure. Blood samples from all malaria-positive patients are delivered to the national Parasitology Reference Laboratory along with personal information. Confirmation of malaria, species identification and comparative parasite load analysis were performed using real-time PCR. DNA extractions from stored leftover samples were analysed for the presence of mutations in Pfkelch13 and Pfcoronin. Age, weight, initial parasitaemia level and Pfcoronin status were compared in patients who failed treatment vs responders. Results During 2009–2020, 338 patients had P. falciparum malaria acquired in Africa. Of those, 15 (24–69 years old, 14 males) failed treatment with AL. Four were still parasitemic at the end of treatment, and 11 had malaria recrudescence. Treatment failure rates were 0% during 2009–2012, 9.1% during 2013–2016 and 17.4% during 2017–2020. In all patients, the Pfkelch13 propeller domain had a wild-type sequence. We did find the P76S mutation in the propeller domain of Pfcoronin in 4/15 (28.6%) of the treatment-failure cases compared to only 3/56 (5.5%) in the successfully treated patients (P = 0.027). Conclusion AL treatment failure emergence was not associated with mutations in Pfkelch13. However, P76S mutation in the Pfcoronin gene was more frequently present in the treatment-failure group and merits further investigation. The increase of malaria incidence in sub-Saharan-Africa partly attributed to the COVID-19 pandemic might also reflect a wider spread of ACT resistance.

Phlebotomine sand flies are the sole vector of leishmaniasis and other sand fly-borne diseases, and their populations are shaped by climate and human-driven environmental change. While warming is expected to expand sand fly ranges, the joint effects of long-term climatic variability and land-use change on population dynamics remain poorly understood, largely owing to limited long-term monitoring. We analyzed an 18-year dataset (2005-2021) from a site in semi-arid Ma'ale Adumim, Israel, using CO -baited traps. Land-cover change was quantified using remote sensing, focusing on the establishment of a managed urban park near the site. Climatic variables from the Israel Meteorological Service and ERA5 were related to interannual variation in abundance and seasonal phenology characters (median seasonal timing and peak timing). A marked land-cover transformation associated with an irrigated park was identified, increasing dry-season vegetation. Sand fly species showed contrasting long-term trends and phase-dependent responses: Phlebotomus sergenti and P. papatasi were most abundant immediately after landscape modification and declined later, whereas P. tobbi and P. syriacus increased after park establishment. Seasonal activity was strongly species-specific with limited interspecific synchrony. Climatic variability explained little interannual variation overall; only P. tobbi maximum abundance and timing of within-season population increase were associated with the timing of thermal onset. Anthropogenic land-use change dominated long-term sand fly population dynamics, often exceeding climatic effects, and altered habitat suitability in species-specific, phase-dependent ways. Integrating land management and spatial planning into prevention strategies is critical for reducing leishmaniasis risk in human-modified landscapes.

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA-positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA-positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.

Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of Leishmania spp., the causative agents of leishmaniasis. Since 2008, the Ministry of Health and the Ministry of Environmental Protection of Israel have conducted nationwide, periodic sand fly surveys. Initially, these surveys focused on localities with known endemic transmission of cutaneous leishmaniasis, but in recent years, entomological trapping has expanded to areas where prior information on sand flies was unavailable. Here we report the first confirmed occurrence of Phlebotomus(Larroussius)orientalis (Parrot, 1936) in Israel and place the Israeli material in a comparative phylogeographic context. Entomological surveys by CO.sub.2 trapping were conducted in the Negev Desert, southern Israel, between 2020 and 2024. Morphological sand fly identification was confirmed by sequencing fragments of the mitochondrial COI and Cytb/NADH1 genes. For a newly reported species, we inferred intraspecific phylogenetic relationships and divergence times between major clades. A subset of females was additionally screened for Leishmania DNA and vertebrate blood-meal sources by real-time polymerase chain reaction (PCR) coupled with high-resolution melt analysis. Targeted surveys and routine surveillance in the Negev region between 2020 and 2024 yielded 269 Phlebotomusorientalis (96 males, 173 females) among other species of local sand fly fauna from multiple wadi systems in the central Negev. These detections constitute the first confirmed records of Ph.orientalis in Israel. Species identification was confirmed through both morphological examination and molecular analyses of partial COI and Cytb/NADH1 genes. Phylogenetic analysis indicated that the Israeli Ph.orientalis specimens constitute a distinct lineage that diverged from East African conspecifics during the Early to Middle Pleistocene. Blood-meal analysis of engorged Ph.orientalis females identified the European hare as a vertebrate host, and none of the tested Ph.orientalis specimens were positive for Leishmania DNA. Phlebotomusorientalis is a confirmed vector of L.donovani, the main agent of visceral leishmaniasis in East Africa. Its detection as a distinct and apparently long-established lineage in the Negev, in a region where parasites of the L.donovani complex are already involved in cutaneous leishmaniasis transmission, highlights the need to clarify the distribution, ecology, and host preferences of Ph.orientalis in Israel. Further studies are required to characterize its spatial and seasonal occurrence, evaluate its vector competence for L.donovani and L.infantum, and assess its potential contribution to current and future leishmaniasis transmission risks.

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA-positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.