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Trypanothione reductase (TR) is a suitable target for drug discovery approaches against leishmaniasis, although the identification of potent inhibitors is still challenging. Herein, we harnessed a fragment-based drug discovery (FBDD) strategy to develop new TR inhibitors. Previous crystallographic screening identified fragments 1 - 3 , which provided ideal starting points for a medicinal chemistry campaign. In silico investigations revealed critical hotspots in the TR binding site, guiding our structure- and ligand-based structure-actvity relationship (SAR) exploration that yielded fragment-derived compounds 4 - 14 . A trend of improvement in Leishmania infantum TR inhibition was detected along the optimization and confirmed by the crystal structures of 9 , 10 , and 14 in complex with Trypanosoma brucei TR. Compound 10 showed the best TR inhibitory profile ( K i = 0.2 μM), whereas 9 was the best one in terms of in vitro and ex vivo activity. Although further fine-tuning is needed to improve selectivity, we demonstrated the potentiality of FBDD on a classic but difficult target for leishmaniasis.

Leishmania spp. are responsible for up to 1 million new cases each year. The current therapeutic arsenal against Leishmania is largely inadequate, and there is an urgent need for better drugs. Trypanothione reductase (TR) represents a druggable target since it is essential for the parasite and not shared by the human host. Here, we report the optimization of a novel class of potent and selective Li TR inhibitors realized through a concerted effort involving X-ray crystallography, synthesis, structure-activity relationship (SAR) investigation, molecular modeling, and in vitro phenotypic assays. 5-Nitrothiophene-2-carboxamides 3 , 6e , and 8 were among the most potent and selective TR inhibitors identified in this study. 6e and 8 displayed leishmanicidal activity in the low micromolar range coupled to SI > 50. Our studies could pave the way for the use of TR inhibitors not only against leishmaniasis but also against other trypanosomatidae due to the structural similarity of TR enzymes.

BackgroundSurveillance of human leishmaniasis in Europe is mostly limited to country-specific information from autochthonous infections in the southern part. As at the end of 2021, no integrated analysis has been performed for cases seen across centres in different European countries.AimTo provide a broad perspective on autochthonous and imported leishmaniasis cases in endemic and non-endemic countries in Europe.MethodsWe retrospectively collected records from cutaneous, mucosal and visceral leishmaniasis cases diagnosed in 15 centres between 2014 and 2019. Centres were located in 11 countries: Belgium, France, Germany, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Data on country of infection, reason for travelling, infecting species, age and sex were analysed.ResultsWe obtained diagnostic files from 1,142 cases, of which 76%, 21% and 3% had cutaneous, visceral, and mucosal disease, respectively. Of these, 68% were men, and 32% women, with the median age of 37 years (range: 0-90) at diagnosis. Visceral leishmaniasis was mainly acquired in Europe (88%; 167/190), while cutaneous leishmaniasis was primarily imported from outside Europe (77%; 575/749). Sixty-two percent of cutaneous leishmaniasis cases from outside Europe were from the Old World, and 38% from the New World. Geographic species distribution largely confirmed known epidemiology, with notable exceptions.ConclusionsOur study confirms previous reports regarding geographic origin, species, and traveller subgroups importing leishmaniasis into Europe. We demonstrate the importance of pooling species typing data from many centres, even from areas where the aetiology is presumably known, to monitor changing epidemiology.

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777-TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.

The management of mucosal leishmaniasis in immunocompromised patients is not standardized and limited data are available on the use of miltefosine for treatment and secondary prophylaxis. We describe a case of mucosal leishmaniasis in an HIV-coinfected patient treated with miltefosine due to a severe allergic reaction to liposomal amphotericin B.

Eighty years after the last published record of human leishmaniasis from Dagestan, Russian Federation, we report two recent cases which were most probably acquired locally: one case of visceral leishmaniasis in a 2-year old child, and one cutaneous leishmaniasis case in a 39-year-old man co-infected with HIV, both resident in Dagestan., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The epidemiology of feline vector-borne pathogens (FeVBPs) has been less investigated in cats than in dogs. The present study assessed the prevalence of Rickettsia spp., Babesia spp., Cytauxzoon spp. and Leishmania infantum infections in cat populations living in central Italy, by molecular and serological tools., Results: A total of 286 healthy cats were randomly selected from catteries and colonies in central Italy. Peripheral blood and conjunctival swab (CS) samples were collected during surgical procedures for regional neutering projects. Sera were analysed by IFAT to detect anti-Rickettsia felis, R. conorii, Babesia microti and Leishmania IgG antibodies using commercial and home-made antigens. DNA extracted from buffy coats (BCs) was tested for Rickettsia spp., and Piroplasmida species, including Cytauxzoon spp. and Babesia spp. by PCR. Buffy coats and CS samples were assayed by a nested (n)-PCR for Leishmania spp. Sixty-two cats (21.67%) were seropositive to at least one of the tested pathogens. The serological assay revealed 23 (8.04%) and 18 (6.29%) positive cats for R. felis and R. conorii, respectively, with low titers (1/64-1/128). No antibodies against B. microti were detected. Neither Rickettsia nor Piroplasmida DNA were amplified using the specific PCR assays. Thirty-one cats (10.83%) tested positive to anti-Leishmania IgG, with titers ranging from 1:40 to 1:160 and 45 animals (15.73%) tested positive to Leishmania CS n-PCR, whereas none of the animals tested positive to BC n-PCR. Considering the results obtained by IFAT and CS n-PCR, a moderate agreement between the two tests was detected (κ = 0.27)., Conclusions: The results of the serological and molecular surveys showed a moderate exposure to Leishmania in the investigated cats and highlighted the limited molecular diagnostic value of BC versus CS samples for this pathogen. Conversely no evidence supported the circulation of Cytauxzoon spp. in domestic cats, in contrast with previous detections in European wild cats in the same areas monitored. The low positive titres for R. felis in association with no DNA BC amplification prevent speculation on the exposure of feline populations to this FeVBP due to the cross-reactivity existing within spotted fever group rickettsiosis (SFGR).

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., (Copyright © 2019. Published by Elsevier Ltd.)

Visceral leishmaniasis has been recognized as an opportunistic infection affecting people with cellular-immunity impairment, including hematopoietic cell transplantation (HCT) recipients. We describe the case of a young Italian man with Hodgkin lymphoma, who developed visceral leishmaniasis after multiple lines of chemotherapy and allogenic HCT. Literature review of visceral leishmaniasis in HCT recipients was also performed. Eleven patients (median age 50 years, 9 male) developed visceral leishmaniasis after allogenic (n = 9) and autologous (n = 2) HCT. Most of them presented with fever and pancytopenia. Bone marrow examination was the main diagnostic technique; liposomal amphotericin B was the treatment of choice. Four out of eight patients (for whom data are available) experienced visceral leishmaniasis relapse. Visceral leishmaniasis in HCT recipients is a rare event that should be suspected in patients with persistent fever, pancytopenia and possible exposure to Leishmania spp., remembering that - as well as South-East Asia, East Africa and South America - it is endemic in several European regions., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compounds against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compounds. We identified a novel chemical class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compound from this class (compound 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compound 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts., Competing Interests: The authors have declared that no competing interests exist.

The study presented here aimed at identifying a new class of compounds acting against Leishmania parasites, the causative agent of Leishmaniasis. For this purpose, the thioether derivatives of our in-house library have been evaluated in whole-cell screening assays in order to determine their in vitro activity against Leishmania protozoan. Among them, promising results have been achieved with compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine) (IC 50  =   29.43 µM), which is able to impair the mechanism of the parasite defence against the reactive oxygen species by inhibiting the trypanothione reductase (TR) with high efficiency (K i 0.25 ± 0.18 µM). The X-ray structure of L. infantum TR in complex with RDS 777 disclosed the mechanism of action of this compound that binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding and avoiding its reduction.

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.

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.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally., Competing Interests: Conflicts of Interest: None declared., (This article is copyright of The Authors, 2016.)

Southern Italy, particularly Campania region, is an area where canine leishmaniasis (CanL) and zoonotic human visceral leishmaniasis (VL) are endemic. The red fox (Vulpes vulpes) has been hypothesized to play a role in occurrence of CanL in Italy but specific studies are poor. The aim of the present survey was to investigate the prevalence of Leishmania infection in dogs and foxes living in the same rural area (Picentini hills). 123 sera from autochthonous fox-hunting dogs were examined by immunofluorescent-antibody test (IFAT) using a cut-off of 1:160. The seroprevalence of dogs examined was 17.9%. Moreover, 48 foxes were examined after having been shooted by hunters or road accidents. Spleen, liver and lymph node samples were analyzed by specific Leishmania nested PCR (n-PCR). 10 foxes were found infected by L. infantum (20.8%) of which 4 animals in spleen, 2 in lymph nodes and 4 both in spleen and lymph nodes. The overall n-PCR positivity was 17.4% for spleen samples and 13.3% for lymph nodes; all liver samples resulted negative. In positive PCR foxes no signs clearly referable to leishmaniasis were recorded at necropsy. The results confirmed the presence of L. infantum infection in red foxes from Southern Italy, with a moderate level of exposure. Because large proportions of dogs with ascertained progressive leishmaniasis show a prolonged "subpatent condition" during which they are only positive to n-PCR before seroconversion, our results allow to assume that exposure risk in foxes is lower than hunting dogs living in the studied area.

Background: Cutaneous leishmaniasis (CL) is widespread in Yemen but has not been fully documented., Objectives: This study aimed to investigate the clinicoepidemiologic and geographic aspects of CL in northwest Yemen and the taxonomic profile of the causative Leishmania species., Methods: All CL cases diagnosed at the Dermatology Clinic of the Saudi Hospital at Hajjah during 1997-2012 were reviewed. Diagnoses were based on clinical, microscopic and, occasionally, histopathologic examinations. Leishmania species identification was carried out in 712 microscopically positive samples by multi-locus enzyme electrophoresis and polymerase chain reaction-restriction fragment length polymorphism., Results: During the surveillance period, 1343 cases of CL were diagnosed. Lesions per patient ranged from one to 71, but most patients had a single facial lesion, classified as representing the "dry type" in 1315 (97.9%) and "wet type" in 28 (2.1%) patients. Leishmania typing in 576 cases identified Leishmania tropica as the main species responsible (n = 529), followed by Leishmania infantum (n = 20), Leishmania donovani (n = 11), and members of the L. donovani complex (n = 8). Atypical molecular patterns were observed in eight CL cases diagnosed in areas in which the three Leishmania species were found sympatrically., Conclusions: Cutaneous leishmaniasis appears to be endemic in northwest Yemen, where its incidence has recently increased abruptly. The disease presents clinically as the "dry type" and is caused mainly by L. tropica and occasionally by L. infantum, L. donovani, and L. donovani complex species. A sympatric diffusion of the three species is present in some governorates., (© 2016 The International Society of Dermatology.)

In the past decade, the number of imported leishmaniasis cases has increased in countries of Western Europe. The trend is associated with increasing travels, ecotourism activity, military operations and immigration. While in endemic countries leishmaniasis is usually well diagnosed, accurate patient history and parasite identification are necessary to distinguish between autochthonous and imported cases. This is particularly important, as new Leishmania species/genotypes may be introduced and transmitted by local phlebotomine vectors without appropriate surveillance, with unpredictable consequences. We report on the surveillance of imported leishmaniasis performed by the Leishmania Identification Reference Centre of Rome from 1986 through 2012, involving health care centres from 16/20 Italian regions. Suspected imported cases were analyzed and conclusions were based on clinical, epidemiological and diagnostic findings. Over the years, different parasite identification methods were employed, including MultiLocus Enzyme Electrophoresis and molecular techniques combining disease diagnosis (SSU rDNA nested-PCR) and Leishmania typing (nuclear repetitive sequence and ITS-1 PCR-RFLPs). A total of 105 imported cases were recorded (annual range: 0-20) of which 36 were visceral (VL) (16 HIV-coinfections) and 69 cutaneous (CL) cases; 85 cases (52 CL) were from the Old World and 20 (17 CL) from the New World. Eight Leishmania species were identified, of which 7 were exotic to Italy. VL importation until 1995 was associated with the spread of Mediterranean Leishmania-HIV co-infections in early 1990s. Following the introduction of HAART treatment, such cases became occasional in Italians but relatively frequent among immigrants. In contrast, a steady increase of CL cases was observed from different areas of the Old and New Worlds, that in recent years included mainly immigrants 'visiting friends and relatives' and Italian tourists. This positive trend likely depends on better diagnosis and reporting; however, we suspect that many CL cases remained unrecognized. Given the relatively low incidence of leishmaniasis importation, the risk of introduction of exotic parasites appears limited, although the detection of anthroponotic species requires attention.

Background: Visceral leishmaniasis (VL) is a severe disease caused by Leishmania infantum in the Mediterranean basin, and is associated with considerable morbidity and mortality. Infantile VL may begin suddenly, with high fever and vomiting, or insidiously, with irregular daily fever, anorexia, and marked splenomegaly. Delays in diagnosis of VL are common, highlighting the need for increased awareness of clinicians for VL in endemic European countries., Case Presentation: We report 4 cases of young children in northern Italy presenting with persistent fever of unknown origin and diagnosed with VL by serological and molecular methods. At the time of diagnosis, these patients showed an unusual echographic pattern characterized by multiple iso-hypoechoic nodules associated with splenomegaly., Conclusion: We suggest that detection of spleen nodules represents a signature of VL in infants, thus helping to diagnose systemic Leishmania infantum infection in children.

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.

Canine leishmaniasis is an important zoonosis caused by uncontrolled infection with Leishmania infantum, where an inappropriate immune response is not only responsible for permitting this intracellular parasite to multiply, but is also responsible for several of the pathological processes seen in this disease. Effective canine vaccines are therefore a highly desirable prevention tool. In this randomised, double-blinded, controlled trial, the efficacy of the LiESP/QA-21 vaccine (CaniLeish, Virbac, France) was assessed by exposing 90 naïve dogs to natural L. infantum infection during 2 consecutive transmission seasons, in two highly endemic areas of the Mediterranean basin. Regular PCR, culture, serological and clinical examinations were performed, and the infection/disease status of the dogs was classified at each examination. The vaccine was well-tolerated, and provided a significant reduction in the risk of progressing to uncontrolled active infection (p = 0.025) or symptomatic disease (p = 0.046), with an efficacy of 68.4% and a protection rate of 92.7%. The probability of becoming PCR positive was similar between groups, but the probability of returning to a PCR negative condition was higher in the vaccinated group (p = 0.04). In conclusion, we confirmed the interest of using this vaccine as part of a comprehensive control program for canine leishmaniasis, and validated the use of a protocol based on regular in-depth assessments over time to assess the efficacy of a canine leishmaniasis vaccine.

An increased number of autochthonous visceral leishmaniasis (VL) cases has recently been reported in Bologna Province in northern Italy. Over six months from November 2012 to May 2013, 14 cases occurred, whereas the average number of cases per year was 2.6 (range: 0-8) in 2008 to 2012. VL was diagnosed in a median of 40 days (range: 15-120) from disease onset. This delay in diagnosis shows the need for heightened awareness of clinicians for autochthonous VL in Europe.

Starting from 1989 Italy experienced an increase of visceral leishmaniasis (VL) cases over a baseline of 10 to 30 cases reported annually. The number of cases peaked in 2000 and 2004 with more than 200 cases/year, and subsequently declined to reach on average one third of the 2000 peak value in the period after 2010. A retrospective analysis from 1982 to 2012 showed that the multi-annual epidemic consisted of major components including (i) an outbreak involving infants and immunocompetent adults in parts of the Campania region (southern peninsular Italy) and that appears to have declined naturally, (ii) a second outbreak affecting human immunodeficiency virus (HIV)-infected individuals throughout the country, that declined owing to the use of highly active antiretroviral therapies (HAART), (iii) a generalised increase of VL cases in immunocompetent individuals and patients affected by associated conditions other than HIV from endemic regions of peninsular and insular Italy (other than Campania), which was due to a geographical spreading of VL foci, with no major case-clusters or outbreak features. A minor component consisted in the appearance of a few autochthonous cases in formerly non-endemic areas, starting from the early 1990s. Epidemic determinants and reasons for VL decline in the Campania region remain largely unexplained, despite the information available on canine reservoir and phlebotomine vectors in Italy.

The incidence of clinical and clinicopathological signs associated with the progression of infection was evaluated prospectively in 329 naïve young dogs exposed to Leishmania infantum transmission and examined periodically during 22 months (M). The dogs were part of Leishmania vaccine investigations performed under natural conditions. Vaccinated groups were considered in the evaluation when the vaccine resulted non-protective and the appearance and progression of signs did not differ statistically from controls at each time point, otherwise only control groups were included. 115 beagles were part of 3 studies (A to C) performed in the same kennel; 214 owned dogs (29 breeds, 2.3% beagles) were included in a study (D) performed in 45 endemic sites. At M22 the prevalence of any Leishmania infection stage classified as subpatent, active asymptomatic, or symptomatic was 59.8% in studies A-C and 29.2% in study D. Despite different breed composition and infection incidence, the relative proportion of active infections and the progression and type of clinical and clinicopathological signs have been similar in both study sets. All asymptomatic active infections recorded have invariably progressed to full-blown disease, resulting in 56 sick dogs at M22. In these dogs, lymph nodes enlargement and weight loss--recorded from M12--were the most common signs. Cutaneous signs were seen late (M18) and less frequently. Ocular signs appeared even later, being sporadically recorded at M22. Most clinicopathological alterations became evident from M12, although a few cases of thrombocytopenia or mild non-regenerative anemia were already observed at M6. Albumin/globulin inversions were recorded from M12 and urea/creatinine increase appeared mostly from M18. Altogether our findings indicate that any susceptible young dogs naturally infected by L. infantum present a common pattern of progression of signs during 2 years post infection, providing clues for medical and epidemiological applied aspects.

The objective of the present study was to evaluate the diagnostic performance of a noninvasive assay, conjunctival swab (CS) nested-PCR (n-PCR), for diagnosing canine leishmaniasis (CanL) in different stages of infection in comparison to the performance of the indirect immunofluorescence antibody test (IFAT), lymph node microscopy, and buffy coat n-PCR. To this end, we performed a cross-sectional survey among 253 nonselected dogs in areas of endemicity in central Italy. We also performed a longitudinal study of CS n-PCR among 20 sick dogs undergoing antileishmanial treatment. In the first study, among the 72 animals that were positive by at least one test (28.45%), CS n-PCR showed the best relative performance (76.38%), with a high concordance in comparison to standard IFAT serology (κ = 0.75). The highest positivity rates using CS n-PCR were found in asymptomatic infected dogs (84.2%) and sick dogs (77.8%); however, the sensitivity of the assay was not associated with the presence of clinical signs. In the follow-up study on treated sick dogs, CS n-PCR was the most sensitive assay, with promising prognostic value for relapses. The univariate analysis of risk factors for CanL based on CS n-PCR findings showed a significant correlation with age (P = 0.012), breed size (P = 0.026), habitat (P = 4.9 × 10(-4)), and previous therapy (P = 0.014). Overall, the results indicated that CS n-PCR was the most sensitive assay of the less invasive diagnostic methods and could represent a good option for the early and simple diagnosis of CanL infection in asymptomatic animals and for monitoring relapses in drug-treated dogs.

Epidemiological studies on the distribution of leishmaniasis caused by Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatidae) have been based principally on serological surveys of the canine reservoir. This methodology is useful due to the facility of sampling, the rapidity in obtaining results, its consistency and because it allows the detection of heterogeneous foci of canine leishmaniasis (CanL) even in small areas. Other investigations have analysed Leishmania parasitism in sandflies (Diptera: Psychodidae: Phlebotominae) by using classical dissection techniques. These techniques allow the vector species to be incriminated in different foci, although they suffer from being very time consuming. Lately, studies in this field are increasingly using molecular techniques, which are faster and easier to perform. In the present work, we applied a nested-PCR in a study of natural infection of sandflies by Leishmania in three isolated farms where serological data on canine leishmaniasis of local dogs were also obtained. The analysis allowed the detection of 38.7% of females with positive nested-PCR (78%, 18% and 0%, respectively, in the different isolated farms). The positive Leishmania DNA samples were genotyped and identified as L. infantum. The results of this work provide new data for the vectorial capacity of Phlebotomus ariasi in a Pyrenean area, which can be considered at risk of becoming a new focus of CanL. The females with positive nested-PCR displayed blood in the midgut at different degrees of digestion, and/or were gravid. According to the multivariate logistic regression analysis, the risk of nested-PCR-positivity increased significantly with the degree of blood digestion (OR = 1.3; P value = 0.025). The Phlebotomus species and the presence of eggs were not statistically associated with nested-PCR positivity (P value of >0.05). The correlation of positive nested-PCR results with the presence of seropositive dogs in the farm confirms the utility of this technique in the study of the distribution and intensity of leishmaniasis foci. Also, the importance of sandfly blood-meal digestion for epidemiological surveys of leishmaniasis foci has been demonstrated.

Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin-trypanothione reductase-NADPH complex was solved at 3.5 Å resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis.

In Leishmania the glutathione/glutathione reductase eukaryotic redox sys-tem is replaced by the unique trypanothione/trypanothione reductase (TR) system. In vitro, silver is a more effective TR inhibitor than antimony, the first line drug against leishmaniasis in most endemic countries, and its mechanism of inhibition is similar to that of Sb(III). In particular, silver binds with high affinity to the catalytic triad Cys52, Cys57, and His461', thereby inhibiting TR. Here, Ag(0) activity was tested on the promastigote and amastigote stages of Leishmania infantum using a drug-delivery system consisting in Ag(0) nanoparticles encapsulated by ferritin molecules (PfFt-AgNPs). These were able to induce an antiproliferative effect on the parasites at metal concentrations lower than those used with antimony.

A longitudinal study was carried out on kennelled stray dogs in a canine leishmaniasis (CanL) endemic area, to evaluate early and late diagnostic performance of a non-invasive conjunctival swab (CS) nested (n)-PCR analysis for Leishmania detection in 2 cohorts of dogs, respectively. (A) Sixty-five IFAT- and CS n-PCR-negative dogs exposed to, and followed up once or twice a month during a full sand fly season (July-November 2008). In parallel, a sand fly survey was performed on site using standard sticky traps set twice a month, for a cumulative surface of 63 m(2). (B) Seventeen IFAT- and CS n-PCR-negative dogs found positive in July 2008 at the peripheral blood buffy-coat (BC) n-PCR. These dogs were examined again by BC n-PCR in September and November 2008, and before the subsequent transmission season (May 2009) along with CS n-PCR and IFAT. None of the cohort (A) dogs converted to positive CS n-PCR during the transmission season. Although approximately 2500 phlebotomine specimens were collected with peaks of 100-147 specimens/m(2) sticky trap, the cumulative density of the only proven CanL vector in the area (Phlebotomus perniciosus) was found to be very low (0.5/m(2)). All cohort (B) dogs remained substantially seronegative; BC n-PCR showed an intermittent positive trend during the period surveyed, resulting in 82% conversions to negative by the end of the study, in contrast with 71% conversions to positive at the CS n-PCR analysis. In conclusion, while CS n-PCR was not found effective for the early detection of Leishmania contacts in dogs exposed to a low pressure of vectorial transmission, this assay showed to slowly convert to positive in a high rate of dogs, in the absence of seroconversion. CS n-PCR technique can be a suitable marker for assessing Leishmania exposure in dogs as a non-invasive alternative to current serological and molecular tools., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Cutaneous leishmaniasis (CL) is widespread in Yemen, but not fully documented., Objective: To study the clinicoepidemiologic profile of CL in the northwestern region of Yemen Republic and to identify the responsible Leishmania species by molecular methods., Methods: All 265 CL cases (176 males and 89 females) were subjected to detailed analysis. Diagnosis was based on clinical features, positive slit skin smear, and histopathologic findings in some cases. In 198 cases, positive smears were examined at the Leishmania Reference Centre of Istituto Superiore di Sanità, Rome (Italy), by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for Leishmania typing., Results: All patients were Yemeni nationals, originated from 10 governorates of northwestern Yemen. Most of the patients had a single noduloulcerative lesion on the face suggestive of "dry"-type CL. Slit skin smear was positive in 255 cases (96.23%). Leishmania sp PCR was positive in all 198 cases examined; the RFLP analysis was positive in 155 samples with the following identification results: L. tropica in 133 cases (85.80%), L. infantum in 17 (10.97%), and L. donovani in 5 (3.23%)., Limitations: This was a prospective study of CL cases at one center only; hence, the full extent of the disease in the entire region cannot be predicted., Conclusion: CL appears to be endemic in northwestern region of Yemen, clinically presenting as 'dry' type, caused mainly by L. tropica (85.8% of cases) and occasionally by L. infantum (10.97%) and L. donovani (3.23%). There is a need for a multicenter study to evaluate the extent of the disease and diffusion of each Leishmania responsible species.

We report a visceral leishmaniasis case in an immunocompetent immigrant with acute renal failure. Parasites were demonstrated in bone marrow, peripheral blood, and kidney samples. A collapsing focal segmental glomerulosclerosis was documented, which was successfully treated with a single infusion of 10 mg/kg liposomal amphotericin B.

A 2-year survey aimed to study seasonal phenology, host-blood feeding preferences and Leishmania infections of Phlebotomus perniciosus, was carried out in a high-endemic focus of canine leishmaniasis in Rome province, Italy. Sandfly densities were monitored by sticky traps, while CDC light traps and hand collections were used for individual sandfly analyses. Four species were identified, three belonging to Phlebotomus (P. perniciosus, Phlebotomus mascittii and Phlebotomus papatasi) and one to Sergentomyia (Sergentomyia minuta) genera. In sticky traps, P. perniciosus (53.5%) and S. minuta (46.0%) were the prevalent species, whereas specimens collected by CDC light traps and hand collections consisted mostly of P. perniciosus. The study showed a markedly different sandfly density between 2003 and 2002 collections (5024 specimens versus 644 specimens, respectively), which was attributed to different climatic conditions. In 2003 adults were active during 7 months (May-November) with a typical bimodal distribution, while in 2002 their activity was shorter (5 months, June-October) without clear density peaks. In this low-density year, the prevalence of P. perniciosus was significantly higher in domestic than in wild resting sites. Blood meal tests showed that P. perniciosus had no preferences for any of the two Leishmania susceptible hosts, being the forage ratio rate 0.7 and 1.0 for man and dog, respectively. A significant number of P. perniciosus was found with avian (60.3%) or ovine (24.2%) blood, that was in relation with the habitats where fed specimens were collected. The rate of specimens of P. perniciosus without blood and found positive for Leishmania DNA, detected by a sensitive nested-PCR technique, was much higher (27.6%) than the promastigote infection rate determined by microscopy in the dissected females (1.4%). The intense Leishmania transmission in the study area was confirmed by the high prevalence of anti-leishmanial antibodies in dogs (33.3%).

Eight patients with cutaneous ulcers were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected cutaneous leishmaniasis (CL). Diagnosis was confirmed parasitologically by both positive Giemsa-stained smears and successful culture of Leishmania promastigotes in NNN medium. The eight parasite isolates were shown to belong to the Leishmania donovani complex by kDNA PCR. Isoenzyme typing of three isolates revealed that they were identical to the L. donovani MON-82 reference strain, and the gp63 PCR-RFLP profile showed similar patterns to a reference strain of MON-82. CL is endemic in most regions of Sudan and has been reported previously as being caused by L. major MON-74. The results of this study suggest that L. donovani is also a cause of CL in Sudan and that further study of isolates from Sudanese patients with cutaneous ulcers is warranted to ascertain whether L. donovani or L. major is the causative agent.

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 90 min. 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.

Most experience in the comparison of diagnostic tools for canine leishmaniasis comes from cross-sectional surveys of dogs of different ages and breeds and in cases with unknown onset and duration of leishmaniasis. A longitudinal study was performed on 43 beagle dogs exposed to three transmission seasons (2002 to 2004) of Mediterranean leishmaniasis and examined periodically over 32 months through bone marrow microscopy and nested PCR (n-PCR), lymph node culture, serology (immunofluorescent-antibody test), and evaluation of clinical parameters. Starting from January 2003, the highest rate of positives was detected by n-PCR at all assessments (from 23.3% to 97.3%). Sensitivities of serologic and parasitological techniques were lower but increased with time, from 15.8% to 75.0 to 77.8%. Some dogs that tested positive by n-PCR but negative by other tests ("subpatent infection") remained so until the end of the study or converted to negative in subsequent assessments, whereas all dogs with positive serology and/or microscopy/culture ("asymptomatic patent infection") exhibited progressive leishmaniasis; 68% of them developed clinical disease ("symptomatic patent infection") during the study, at 7 (range, 3 to 14) months after being positive to all tests. Postexposure infection incidences were high and were significantly different between 2002 and 2003 exposures (39.5% and 91.7%, respectively). The time course of infection was highly variable in each dog, with three patterns being identified: (i) rapid establishment of a patent condition (0 to 2 months from detection of infection); (ii) a prolonged subpatent condition (4 to 22 months) before progression; and (iii) a transient subpatent condition followed by 10 to 21 months of apparent Leishmania-negative status before progression.

Cutaneous leishmaniasis (CL) in the Old World is caused mainly by three species of Leishmania: L. major, L. tropica and L. aethiopica, and sporadically by L. infantum and L. donovani. In Ethiopia, zoonotic cutaneous leishmaniasis, caused by L. aethiopica, is a major public health problem affecting thousands of people in the highlands. By contrast, little is known about the existence and epidemiology of CL due to L. tropica. In this report, we provide the first well-documented case of CL in Ethiopia caused by L. tropica. The patient acquired the infection in Awash valley of the Ethiopian Rift Valley (northeastern Ethiopia), where Phlebotomus sergenti and P. saevus have previously been found infected by L. tropica. Using the isoenzyme electrophoresis technique, the isolate was found to belong to a variant of L. tropica zymodeme MON-71, one of the new zymodemes found in Ethiopia from P. sergenti in the same region so far. The epidemiological implications of the finding are discussed.