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Background: 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. Methodology/Principal findings: 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. Conclusion and significance: 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. Author summary: Leishmaniasis is a disease caused by Leishmania parasites, transmitted by sand flies, and poses a major health risk in the Mediterranean region. Monitoring the spread of infected sand flies is crucial for controlling the disease. This study focused on improving the methods used to detect Leishmania in sand flies by comparing different DNA extraction techniques and assessing the accuracy of these methods across nine reference laboratories. All centers, except one, efficiently detected all Leishmania concentrations, demonstrating proficiency in diagnostic protocols. Moreover, we found that two specific DNA extraction kits, the EZ1 DSP Virus Kit and QIAamp DNA mini-kit, were the most effective for Leishmania detection. We also tested how sample preparation and shipping conditions affected the results, ensuring that our methods would work in real-world settings. Even under these conditions, the detection methods proved reliable. This work helps to standardize the detection of Leishmania, making surveillance more accurate and consistent. Continuous training and calibration are essential to ensure uniform diagnostic performance across laboratories, enhancing epidemiological surveillance and disease control strategies and enabling appropriate treatment. [ABSTRACT FROM AUTHOR]

• Imported cases of anthroponotic cutaneous leishmaniasis due to Leishmania tropica are increasingly documented in Europe. • Vector competence for an Asian L. tropica was investigated in Phlebotomus perniciosus , a southwestern European sand fly. • Leishmania tropica developed into abundant metacyclic promastigotes in 20% of laboratory-infected P. perniciosus. • Parasites have been successfully transmitted from infected to naive hamsters by the bite of P. perniciosus. • In particular settings P. perniciosus , a Leishmania infantum vector, may play the role of an occasional L. tropica vector. Imported cases of anthroponotic cutaneous leishmaniasis due to Leishmania tropica are increasingly documented in Europe. We investigated the ability of Phlebotomus perniciosus , a competent vector of Leishmania infantum widespread in southwestern Europe, to support the growth and transmissibility of an Asian strain of L. tropica recently isolated from a refugee. Parasite growth behavior was investigated in laboratory-reared sand flies fed artificially with promastigotes as well as in sand flies infected after biting on footpad lesions induced in hamsters by promastigote inoculation. The evolution of infection was checked by gut microscopy and quantitative real-time PCR, and it was found to be similar between promastigote- and amastigote-initiated infections. In 80% of infected sand flies, despite survival and flourishing growth of promastigotes after blood digestion and defecation, either the parasites died, or failed to migrate to the foregut and/or to mature into infective forms. However, in the remaining 20% L. tropica developed into abundant metacyclic promastigotes. The quantitative real-time PCR assay detected variable loads of gut promastigotes irrespective of morphological evidence of viability or progressive/final death. Parasite transmissibility was investigated by exposing naive hamsters to P. perniciosus previously infected on chronic lesions induced in hamsters which survived to take a second blood meal. Two months post exposure, lesions developed in skin sites bitten by sand flies confirmed to harbor metacyclic promastigotes; in the following months, the presence of viable and transmissible L. tropica parasites in lesions was demonstrated by xenodiagnosis assays. Our findings support the hypothesis that, in particular epidemiological situations, P. perniciosus may play the role of an occasional L. tropica vector. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis in Serbia was an endemic disease, and is considered to be eradicated for more than 40 years. In the past decade sporadic cases of canine leishmaniasis started to emerge for the first time in Vojvodina Province (previously non-endemic region of Serbia). Reports of introduced, and later on autochthonous cases of leishmaniasis alerted the possibility of disease emergence. The aim of this study was to bridge more than a half a century wide gap in entomological surveillance of sand fly vectors in Vojvodina, as well as to verify the presence of the vector species that could support Leishmania spp. circulation. Results: During the period 2013-2015, a total of 136 sand flies were collected from 48 of 80 surveyed locations. Four sand fly species of the genus Phlebotomus were detected: P. papatasi, P. perfiliewi, P. mascittii and P. neglectus. Detection of P. mascittii represents the first record of this species for the sand fly fauna in Vojvodina and in Serbia. All female specimens (n = 80) were tested for Leishmania spp. DNA, and three blood-fed P. papatasi specimens were positive (4%). One positive DNA sample was successfully amplified by ITS1 nPCR. The RFLP analysis of the resulting 350 bp fragment showed a typical pattern of L. infantum, and the ITS1 partial sequence blasted in GenBank confirmed 100% identity with L. infantum and L. donovani complex sequences. This result represents the first record of both Leishmania spp. and L. infantum DNA from sand flies in Vojvodina, and in Serbia. Conclusions: Presence of autochthonous canine leishmaniasis cases, records of Phlebotomus (Larroussius) species proven vectors of L. infantum (P. perfiliewi and P. neglectus) and detection of L. infantum DNA from wild caught (non-competent) vectors, prove that L. infantum is present in Vojvodina and indicates a probable circulation in the region. [ABSTRACT FROM AUTHOR]

The incidence of visceral leishmaniasis (VL) in Albania is higher than in other countries of southern Europe, however the role of local sand fly species in the transmission of Leishmania infantum was not addressed conclusively. In 2006, a country-wide collection of sand flies performed in 14 sites selected based on recent occurrence of VL cases showed that Phlebotomus neglectus was by far the most prevalent species (95.6%). Furthermore, 15% of pools made from 422 P. neglectus females tested positive for Leishmania sp. genomic DNA. In the same year, Culicoides trapping was performed for bluetongue disease surveillance in 91 sites of southern Albania, targeting livestock farms regardless recent occurrence of VL in the surveyed areas. In 35 sites where sand flies were collected along with midges, Phlebotomus perfiliewi was the most prevalent among the Phlebotomus species identified, however search for leishmanial DNA in females of this species was unsuccessful. In 2011, sand flies were trapped in 4 sites of north Albania characterized by high VL incidence, and females were dissected to search for Leishmania infections. Both P. neglectus and P. tobbi were collected at high densities. Two positive specimens were detected from a sample of 64 P. neglectus trapped in one site (3.1%). Parasites were successfully cultured from one specimen and characterized as belonging to Leishmania infantum zymodeme MON-1, the only zymodeme so far identified as the agent of human and canine leishmaniasis in the country. Altogether our studies indicate that P. neglectus is the main leishmaniasis vector in Albania. [ABSTRACT FROM AUTHOR]

Background Pioneering research on "Mediterranean Kala-Azar" carried out by Adler and Theodor early in the past century (~1930s) had identified Catania city (Sicily) as a major focus of the disease nowadays known as zoonotic visceral leishmaniasis (VL). Despite the fact that disease in both humans and dogs has continued to be highly prevalent in the Catania province up to the present times, research on Leishmania vectors in this urban focus dates back to that distant period. This study aimed to evaluate the persistence and current composition of the sand fly fauna in urban environments of Catania in recent years, 2006 and 2013. Methods In 2006 fifty-one suitable collecting sites were identified within 44 sub-units of a grid drawn to include the urban Catania area. In 2013 the survey was restricted to four of the most productive and representative sites resulting from the 2006 survey. In both periods 3 collections per month were performed using standard sticky traps set for 3 days in wall holes/cavities along public roads, from the end of April through December. Results 43/51 sites (84.3%) were found positive for sand flies. The 2006 collections accounted for a total of 4341 specimens including six species. Among competent Leishmania vector species, P. perniciosus was the most prevalent (36.5%) being identified in all sand fly-positive sites, with significant abundance in those of the old city centre. Other species of interest were P. sergenti (2.5%) and P. neglectus (1.5%). The 2013 survey produced 1130 sand flies, of which 39.5% were P. perniciosus, 1.6% P. sergenti and 0.7% P. neglectus. A search for Leishmania DNA in a small sample of 72 P. perniciosus females revealed 11% infection prevalence. Conclusions Our findings from an old urban focus of leishmaniasis demonstrate that phlebotomine sand flies have adapted fairly well to the drastic environmental changes that have occurred in cities of the Western world in the past century and still represent a potential risk for Leishmania transmission. [ABSTRACT FROM AUTHOR]

Abstract: Despite the recent reports of feline leishmaniosis from Southern Europe, cats are still regarded as unusual Leishmania hosts. A cat found chronically infected with Leishmania was submitted to xenodiagnosis. After being sedated, the animal was exposed to the bite of 100 laboratory-reared Phlebotomus perniciosus in a fine net cage for 90min. Four out of 19 blood-fed sandflies (21%) showed motile promastigotes at the dissection. Parasites cultured from cat''s lymph node and an infected fly were identical at PCR-RFLP genotyping and identified as Leishmania infantum MON-1, the main zymodeme responsible for human and canine leishmaniosis in Southern Europe. This is the first evidence of transmissibility of feline parasites to a proven vector, suggesting that cats may represent an additional domestic reservoir for L. infantum. [Copyright &y& Elsevier]