Wild rabbits are Leishmania infantum reservoirs in southeastern Spain.

OBJECTIVE: We contribute to the understanding of the transmission dynamics of Leishmania infantum suggesting the involvement of rabbits as wild reservoirs. RESULTS: The prevalence of infection was 86.0% (270/314 wild rabbits) ranging from 18.2% to 100% in natural geographical regions. The estimated average parasite load was 324.8 [CI 95% 95.3-554.3] parasites per mg of ear lobe ranging from 0 to 91,597 parasites/mg per tissue section. CONCLUSIONS: A positive correlation was found between skin parasite load in wild rabbits and human incidence with evidence of the presence of the same L. infantum genotypes in rabbits and humans, providing new epidemiological and biological basis for the consideration of wild rabbits as a relevant L. infantum wild reservoir. Molecular parasite surveillance reflects the great genotypic variability of the parasite population in wild rabbits. Most of these genotypes have also been found to infect humans, dogs and sandflies in the region. Our findings also highlight that direct genotyping of the parasite in host tissues should be used for molecular surveillance of the parasite instead of cultured isolates.

Morphology does not allow differentiating the species of the Phlebotomus perniciosus complex: Molecular characterization and investigation of their natural infection by Leishmania infantum in Morocco.

Morphological and DNA-based complemented approaches were applied for characterization of sympatric populations of Phlebotomus longicuspis and Phlebotomus perniciosus in Morocco. Both sand fly species are generally recorded in sympatry in North Africa but on few occasions have been molecularly characterized. The diagnostic confusion of these species has led to errors in their geographical distribution and probably, in the assignment of their role in the transmission of L. infantum. Sand flies were caught inside households in El Borouj, central Morocco, in 2014-2015. For female sand flies, detection of L. infantum natural infection and blood meal identification were carried out. According to morphological identification, Phlebotomus longicuspis s.l. (34.7%) was the second most abundant Phlebotomus species after P. sergenti, followed by atypical Phlebotomus perniciosus (7.1%); 11.6% of the male specimens of P. longicuspis s.l. were identified as P. longicuspis LCx according to the number of coxite setae. The density of Larroussius species was very high (31 Larroussius/light trap/night) in the peripheral neighbourhood of Oulad Bouchair (p = 0.001) where the first case of cutaneous leishmaniasis due to Leishmania infantum was detected in 2017. Phylogenetic trees based on three independent genes highlighted three well-supported clusters within P. perniciosus complex that could be interpreted as corresponding to P. perniciosus, P. longicuspis s.s. and an undescribed species, all coexisting in sympatry. Some females with typical morphology of P. longicuspis were genetically homologous to P. perniciosus. The taxa cannot be differentiated by morphological methods but characterized by a distinctive genetic lineage for which the synapomorphic characters are described. Leishmania infantum was detected in females of all clusters with a low parasite load. Population genetics will help to assess the threat of the geographical spread of L. infantum in Morocco by determining the density, abundance and vector role of the species of the P. perniciosus complex identified correctly.

Effectiveness of an O-Alkyl Hydroxamate in Dogs with Naturally Acquired Canine Leishmaniosis: An Exploratory Clinical Trial.

Canine leishmaniosis is a challenge in veterinary medicine and no drug to date has achieved parasite clearance in dogs. Histone deacetylase inhibitors are a drug class widely used in cancer chemotherapy. We have successfully used O-alkyl hydroxamates (vorinostat derivatives) in the treatment of a laboratory model of visceral leishmaniasis without showing toxicity. In order to test the effectiveness of a particular compound, MTC-305, a parallel-group, randomized, single-centre, exploratory study was designed in naturally infected dogs. In this clinical trial, 18 dogs were allocated into 3 groups and were treated with either meglumine antimoniate (104 mg SbV/kg), MTC-305 (3.75 mg/kg) or a combination of both using a lower MTC-305 dose (1.5 mg/kg) through a subcutaneous route for 2 treatment courses of 30 days, separated by a 30-day rest period. After treatment, a follow-up time of 4 months was established. Parasite burden in bone marrow, lymph node and peripheral blood were quantified through qPCR. Antibody titres were determined through an immunofluorescence antibody test, and cytokine expression values were calculated through RT-qPCR. Treatment safety was evaluated through the assessment of haematological and biochemical parameters in blood, weight, and gastrointestinal alterations. Assessment was carried out before, between and after treatment series. Treatment with MTC-305 was effective at reducing parasite burdens and improving the animals' clinical picture. Dogs treated with this compound did not present significant toxicity signs. These results were superior to those obtained using the reference drug, meglumine antimoniate, in monotherapy. These results would support a broader clinical trial, optimised dosage, and an expanded follow-up stage to confirm the efficacy of this drug.

Leishmaniasis vectors in the environment of treated leishmaniasis cases in Spain.

Transmission of leishmaniasis in endemic areas is characterized by microfocality related to the presence of the vector. Most entomological studies in southwestern Europe have focused on sylvatic areas and town outskirts, very few have sampled town or urban centres, and no survey has investigated inside households. The aim of this study was to determine the sand fly species diversity and vector density in the surroundings of human leishmaniasis cases compared with environments in which there was no association. Sand flies were captured in 26 households associated with recently treated leishmaniasis patients, 15 neighbouring houses without associated cases, and in others environments. Overall 7495 sand flies belonging to six species were captured. The highest sand fly density was found in farmhouses where there is a great availability of blood sources and breeding sites. In the environment of human leishmaniasis cases, Sergentomyia minuta was the most prevalent species followed by Phlebotomus perniciosus. Nevertheless, lower Leishmania infantum infection rates and lower intensity of infection were detected in S. minuta sand flies than in P. perniciosus. The density of P. perniciosus in households with recently treated leishmaniasis patients varies between 0 and 108 sand flies per light trap/night, with the maximum values corresponding to farmhouses. This species appears to be adapted to both indoors and outdoors domestic biotopes, including urban households.

Canine Leishmaniasis: Update on Epidemiology, Diagnosis, Treatment, and Prevention.

Dog are the main reservoir of Leishmania infantum, causing canine leishmaniasis, an incurable multisystemic disease that leads to death in symptomatic dogs, when not treated. This parasite causes visceral, cutaneous, and mucosal leishmaniasis in people in the Mediterranean Basin, North Africa, South America, and West Asia. This disease is mostly unknown by veterinarians outside the endemic areas, but the disease is expanding in the Northern Hemisphere due to travel and climate change. New methodologies to study the epidemiology of the disease have found new hosts of leishmaniasis and drawn a completely new picture of the parasite biological cycle. Canine leishmaniasis diagnosis has evolved over the years through the analysis of new samples using novel molecular techniques. Given the neglected nature of leishmaniasis, progress in drug discovery is slow, and the few drugs that reach clinical stages in humans are unlikely to be commercialised for dogs, but several approaches have been developed to support chemotherapy. New-generation vaccines developed during the last decade are now widely used, along with novel prevention strategies. The implications of the epidemiology, diagnosis, treatment, and prevention of canine leishmaniasis are fundamental to public health.

Understanding the factors that determine the emergence of anthroponotic cutaneous leishmaniasis due to Leishmania tropica in Morocco: Density and mitochondrial lineage of Phlebotomus sergenti in endemic and free areas of leishmaniasis.

Anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica is spreading to new areas in Morocco. Exposure to the vector, Phlebotomus sergenti, is the only proven risk factor. Our objective was to compare the densities and genetic characteristics of P. sergenti populations in two nearby localities in Morocco, one in an ACL endemic area (El Borouj) and another in a nonendemic area (Sidi Hajjaj). P. sergenti density was significantly higher in the endemic area than in the nonendemic town (p = 0.032). A different predominant P. sergenti mitochondrial lineage was evidenced in each one of the two localities, and for the first time, the P. sergenti lineage acting as a vector of L. tropica has been identified. Bioclimatic differences were detected between both localities. In conclusion we found differences in both the density and the mitochondrial lineage of P. sergenti populations that may explain the different epidemiological situation. Given that the density of P. sergenti in the locality without ACL cases seems sufficient to allow transmission, the main factor that would justify its nonendemic character could be the absence of P. sergenti Lineage IV, which seems to prefer warmer and drier climates.

Role of wild rabbits as reservoirs of leishmaniasis in a non-epidemic Mediterranean hot spot in Spain.

There is limited information regarding the role of wild mammals in the transmission dynamics of Leishmania infantum. A potential human leishmaniasis hot spot was detected in southern Spain that could not be explained solely by canine leishmaniasis prevalence. The aim of this work was to analyse the involvement of wild rabbits as the main factor affecting this Mediterranean hot spot. A survey of wild rabbits, dogs and sand flies was conducted in the human cases environment. A nearby region without clinical leishmaniasis cases was used as reference control. 51 wild rabbits shot by hunters were analysed by molecular techniques. 1100 sand flies were captured and morphologically identified. Blood collected from patients' relatives/ neighbours (n = 9) and dogs (n = 66) was used for molecular analysis and serology. In Mediterranean leishmaniasis hot spots such as Montefrío municipality (average incidence of 16.8 human cases per 100,000 inhabitants/year), wild rabbits (n = 40) support high L. infantum infection rates (100%) and heavy parasite burdens (average value: 503 parasites/mg) in apparently normal ear skin directly accessible to sand flies, enabling the existence of heavily parasitized Phlebotomus perniciosus females (12.5% prevalence). The prevalence of infection and median parasite load were very low among rabbits captured in Huéscar (n = 11), a human clinical leishmaniasis-free area for the last 18 years. P. perniciosus was the most abundant Phlebotomus species in all the domestic/peridomestic microhabitats sampled, both indoors and outdoors. Accordingly, leishmaniasis is clustering in space and time at this local scale represented by Montefrío due to the proximity of two competent host reservoirs (dogs and heavily parasitized wild rabbits) associated with overlapping sylvatic and domestic transmission cycles through the main vector, P. perniciosus. We highlight the usefulness of determining the prevalence of infection and parasite burden in wild rabbits as a control leishmaniasis measure with the advantage that the use of the ear offers.

Intra and peridomiciliary comparison of density, sex ratio and gonotrophic stage of Phlebotomus sergenti in an active anthroponotic cutaneous leishmaniasis focus in Morocco.

Cutaneous leishmaniasis due to Leishmania tropica represents a major public health problem due to its ability to spread into non-endemic areas by means of its vectors, and the associated dramatic psychosocial impact. The objective of this work was to compare the intra and extradomiciliary density, sex ratio and gonotrophic stage of sand flies from a recent active focus in Morocco. This field study is based on the need to optimize the effectiveness of control programs. Two different capture methods, CDC light traps and sticky traps, were used at two different times of the year, corresponding with the peaks of sand fly abundance. 7,815 sand flies were captured and classified into 13 species belonging to genera Sergentomyia (50.8%) and Phlebotomus (49.2%). Phlebotomus sergenti was the most abundant and frequent species of the genus Phlebotomus both inside (49.3%) and outside houses (52.1%) and it showed the highest density in extradomiciliary captures in June. The proportion of blood-fed females was similar indoors and outdoors (21.5% and 26.3%, respectively). Females in the three gonotrophic stages were found in 26% houses and this was significantly associated with some factors related to housing conditions. Therefore, P. sergenti seems well adapted to both indoors and outdoors biotopes where these females coexist with males. These findings suggest that the adoption of additional measures could benefit the strategy of the Moroccan health authorities, currently consisting of indoor insecticide spraying, given that transmission may also occur outdoors.

Seasonal dynamics of phlebotomine sand flies and autochthonous transmission of Leishmania infantum in high-altitude ecosystems in southern Spain.

Leishmaniasis is a vector-borne disease transmitted by sand flies. A dozen species have been involved in the transmission of Leishmania infantum in the Mediterranean region. Climate change may alter sand fly distribution at particular altitudes and latitudes. The objective of this study was to interrogate the existence of stable populations of sand flies in high-altitude ecosystems and evaluate if these populations are enough to support autochthonous transmission of leishmaniasis. These altitudinal conditions can be found in Sierra Nevada (southern Spain). Therefore, we have determined the sand fly population dynamics in different biotopes located at elevations above 1,300 m a.s.l. and searched for evidence of leishmaniasis transmission. Five collecting sites above 1,300 m a.s.l. containing large livestock concentrations were selected. Sand flies were caught using CDC light traps from May to November, annually from 2008 to 2013, and these were morphologically identified. Association between sand fly density or presence and temperature/humidity was estimated by linear and logistic regression, respectively. Leishmania infantum detection in female sand flies was performed by PCR. Diagnosis of canine leishmaniasis (CanL) was carried out by indirect immunofluorescence and PCR. A total of 2,973 specimens of 5 sand fly species were collected from June to October. Phlebotomus perniciosus was the most frequent (100%), abundant (80.1%) and densest species (9.8 sand flies/trap). The minimum temperature on the day of capture was the most important variable factor for sand fly presence and P. perniciosus density. An increase in altitude showed a negative effect over the sand fly diversity and activity period, driving changes in seasonal dynamics similar to those reported by latitudinal changes. CanL prevalence was 23%, a similar rate to previous surveys carried out on randomly selected dogs from towns in southern Spain. A successful host-vector-pathogen network was found at this altitude characterised by 9.9% L. infantum infection rate in non-blood fed P. perniciosus and Phlebotomus ariasi females and high CanL prevalence that entails an increase in the leishmaniasis risk area driven by sand fly colonization.

A multi-restriction fragment length polymorphism genotyping approach including the beta-tubulin gene as a new differential nuclear marker for the recognition of the cryptic species Anisakis simplex s.s. and Anisakis pegreffii and their hybridization events.

The detection of Anisakis simplex s.s./A. pegreffii putative hybrids has been a controversial issue in spite of the fact that natural hybridization is an extended process across free living and parasitic organisms. Differential traits of biomedical and ecological importance, such as invasive and allergenic potential have been demonstrated in both cryptic species. Therefore, in this work, we discuss about the potential for hybridization between these anisakid species in sympatric zones, implementing a multi-marker Restriction fragment length polymorphism (RFLP) genotyping approach based on the ribosomal DNA internal transcribed spacer 1 (ITS1), the mitochondrial cytochrome C oxidase 2 (Cox-2) and a new nuclear marker, the highly conserved ß-tubulin gene (ß-TUB). The two cryptic species differed at least in 7 bp in the ß-TUB gene and some larvae with heterozygous genotypes at the 7 diagnostic nucleotide positions were found. Taxonomic, population and genealogical analyses served to support the occurrence of hybridization between both species. Predicted restriction endonucleases enzymes were assayed for Cox-2 and ß-TUB markers. The implemented multi-marker PCR-RFLP allowed us to detect the two pure parental species, F1 hybrids, hybrid backcrossed progeny and individuals with nuclear-mitochondrial discordance, being a useful, simple and reproducible procedure in any laboratory for epidemiological studies.

O-Alkyl Hydroxamates Display Potent and Selective Antileishmanial Activity.

Leishmania (L.) infantum causes visceral, cutaneous, and mucosal leishmaniasis in humans and canine leishmaniasis in dogs. Herein, we describe that O-alkyl hydroxamate derivatives displayed potent and selective in vitro activity against the amastigote stage of L. infantum while no activity was observed against promastigotes. Compound 5 showed potent in vivo activity against L. infantum. Moreover, the combination of compound 5 supported on gold nanoparticles and meglumine antimoniate was also effective in vivo and improved the activity of these compounds compared to that of the individual treatment. Docking studies showed that compound 5 did not reach highly conserved pocket C and established interactions with the semiconserved residues V44, A45, R242, and E243 in pocket A of LiSIR2rp1. The surface space determined by these four amino acids is not conserved in human sirtuins. Compound 5 represents a new class of selective ligands with antileishmanial activity.

Leishmaniasis due to Leishmania infantum: Integration of human, animal and environmental data through a One Health approach.

The aim of this study was to explore Leishmania infantum epidemiology through a One Health approach that promotes a better estimation of leishmaniasis burden and a deeper understanding of the spatial distribution of the key actors of the parasite life cycle (vectors, reservoirs and humans). We conducted a 14-year mixed retrospective and prospective study of leishmaniasis cases in an endemic area in southern Spain (Granada province), to estimate the human incidence and its association with the vector presence, cryptic leishmaniasis rates and canine leishmaniasis prevalence. We found an annual linear increase in the incidence that cannot be fully explained by active case surveillance and the improvement of PCR diagnostic techniques. 49.4% of cases were not reported to the surveillance system. Approximately half of the human cases correspond to the visceral form that occurred more frequently in men; cutaneous, mucosal and cryptic forms were also detected. Leishmaniasis is no longer a disease of young children, accounting for a quarter of immunocompetent patients and most infected people remained asymptomatic. Human and canine leishmaniasis, cryptic or symptomatic, are present in the whole province, where there is a medium/high risk of the presence of Phlebotomus perniciosus, the main vector. We found association between the incidence of human leishmaniasis and the presence of the vector, but not with the prevalence of canine leishmaniasis and cryptic human leishmaniasis. A potential hot spot was also found, where high leishmaniasis incidence may be associated to the involvement of host species other than dogs.

Possible introduction of Leishmania tropica to urban areas determined by epidemiological and clinical profiles of patients with cutaneous leishmaniasis in Casablanca (Morocco).

Leishmaniases are a group of infectious diseases caused by protozoan Leishmania parasites and are transmitted by the bites of infected phlebotomine sandflies. The heterogeneity of these diseases is influenced by both parasitic properties and host factors. Cutaneous leishmaniasis (CL) is a major public health problem in Morocco, where the geographical expansion of CL (particularly CL caused by Leishmania tropica), the heterogeneous appearance of lesions and the difficulty in diagnosing CL contribute to late diagnosis of CL and delayed treatment of patients. Therefore, the main objective of this study was to describe the epidemiological and clinical profiles of patients with CL diagnosed in Casablanca (Morocco), which is a non-endemic area for CL. A cross-sectional study was conducted between 2010 and 2016, during which epidemiological and clinical data were collected from patients that met the inclusion criteria through an information sheet. Then, samples were obtained from each patient for parasitological and molecular diagnosis, and only patients with positive polymerase chain reaction and genotyping results were included in the study. Overall, 106 cases of CL were genotyped, of which 61 (57.5%) were caused by L. tropica, 38 (35.9%) by L. major and 7 (6.6%) by L. infantum. While all age groups were affected, CL cases wherein L. tropica was the causative agent were most frequently diagnosed in children aged 0-9 years (p = 0.005), whereas those caused by L. major were more frequently diagnosed in elderly patients (p = 0.004). Multivariate logistic regression analysis showed that two clinical variables were significantly associated with CL caused by L. tropica: lesion size (p = 0.002) and occurrence of lesion on the face (p = 0.005). Furthermore, the results of our survey highlighted the association of Leishmania infection when travelling to endemic areas. The high number of endemic foci where patients with CL were infected with L. tropica illustrated the tendency of this form to spread and generate epidemics, exposing young people to a greater degree to the disease. The epidemic status of CL caused by L. tropica in Morocco and the increased movement of the population from rural to urban areas indicate a possible introduction of this species to urban areas.

Vertical transmission may play a greater role in the spread of Leishmania infantum in synanthropic Mus musculus rodents than previously believed.

Vertical transmission of Leishmania infantum was demonstrated in domestic mice captured close to the home of a patient with leishmaniasis. Leishmania infantum DNA was detected in 88.9% of synanthropic Mus musculus adult rodents and 29.2% of their unborn foetuses. Mother-to-infant transmission was observed in all females whose gestational stage was sufficiently advanced to allow foetal analysis (foetal length 2-2.5 cm). The infection rate in foetal samples ranged from 11.1% to 50.0%, with parasite loads of up to 6,481 parasites/5 mg tissue. A low density of Phlebotomus perniciosus was also found (0.2 specimen/CDC trap). Six infected mice captured in March were only 1.5 months old and could thus not have had contact with the vector. Vertical transmission thus appears to play a greater role in the spread of leishmaniasis than previously thought, particularly since rodents are natural hosts for the parasite and are prolific in nature.

Asymptomatic Leishmania infection in blood donors from the Southern of Spain.

OBJECTIVES: To investigate the proportion of asymptomatic infection among blood donors in a region endemic for Leishmania; and to ascertain epidemiological and genetic factors associated with this condition. METHODS: We studied 1260 blood donors in the Province of Granada in the Southern Spain. After obtaining informed consent in each participant, a poll about habits, housing and contact with animals were carried out. Blood samples were obtained for determining antileishmanial antibodies and a PCR assay. HLA typing was performed in a randomly sample among the donors with positive serology. RESULTS: We have found that L. infantum antibodies were present in 7.9% of blood donors and DNA in blood was detected in 2.5% of donors. There was no concordance between both determinations, except in one patient. Taking into consideration both techniques, 129 participants were considered to have asymptomatic Leishmania infection. No participant in this study developed clinical leishmaniasis during a follow-up period of 2 years. HLA were typed in 51 donors. Asymptomatic Leishmania infection might be associated with certain HLA antigens. A multivariate analysis was done with the variables obtained through the participants' interview. The contact with livestock (goats, pigs, and sheep), but not dogs, either at home or in the environment, was significantly and independently associated with asymptomatic leishmania infection. CONCLUSIONS: Asymptomatic leishmanial infection among blood donors is frequent in the Granada Province, south of Spain. The presence of livestock in this region is related to this infection, perhaps influencing vector density of this disease. Some HLA genes might be associated with asymptomatic leishmanial state.

A nanodelivered Vorinostat derivative is a promising oral compound for the treatment of visceral leishmaniasis.

There is currently no satisfactory treatment for visceral leishmaniasis; the disease is thus in desperate need of novel drugs. The ideal candidate should be effective, safe, affordable, and administered via the oral route. Histone deacetylases (HDACs) are involved in silencing critical regulatory pathways, including pro-apoptotic programs, and represent potential therapeutic targets for pharmacological interventions. O-alkyl hydroxamates have traditionally been considered to exert no effect on mammal HDACs. The aim of this study was to evaluate the effect of MDG, a SAHA derivative of the O-alkyl hydroxamate family with no activity on human histone deacetylase enzymes, on the visceral leishmaniasis causative agents and in a murine model of the disease. The effects of vorinostat, tubacin and valproic acid (well-known mammal HDAC inhibitors) on the parasite were also evaluated. MDG was found to be highly active against Leishmania infantum and L. donovani intracellular amastigotes in vitro but not against the promastigote stage. In contrast, vorinostat, tubacin and valproic acid showed no activity against the parasite. Assays investigating hERG and Cav1.2 channels in vitro found no evidence of MDG-driven cardiotoxicity. MDG showed neither hepatotoxicity nor mutagenicity, nor did it exert activity on cytochrome P450 enzymes. MDG was adsorbed onto gold nanoparticles for the in vivo experiments, performed on infected Balb/c mice. MDG was effective at reducing the parasite load in major target tissues (bone marrow, spleen and liver) in more than 70% at 25 mg/kg through both the oral and intraperitoneal route, proving more active than the reference compounds (meglumine antimoniate, MA) without showing toxicity. In addition, the combination of MDG and MA was very effective.

Risk factors for the expansion of cutaneous leishmaniasis by Leishmania tropica: Possible implications for control programmes.

Cutaneous leishmaniasis (CL) caused by Leishmania tropica is emerging in new areas, initially as outbreaks and then establishing endemic foci. There is little evidence of the risk factors and effectiveness of existing control measures, what limits our ability to generalize in different epidemiological settings. The disease is described as anthroponotic; however, zoonotic outbreaks have been reported in some countries. Our aim was to identify risk factors in a recently reported endemic focus in Morocco in order to design more effective control programmes. A case-control study was conducted from September 2014 to October 2015 for epidemiological data collection from families with and without CL cases. Sandflies were captured and L. tropica infection determined. The presence of potential animal reservoirs was evaluated. 71 CL cases (44 diagnosed between 2013 and 2015) and 137 healthy people were surveyed. The average age of the new cases was 33.1 ± 22.3 years, and 69.0% were women. Phlebotomus sergenti was the most abundant species with a density of 4.27 sandflies/trap/night and differences between houses with and without CL cases were detected (p-value = 0.014). Overall, 2.7% female P. sergenti and 3.0% dogs were positive for L. tropica. Human, cat, rabbit and bird blood was detected in blood-fed P. sergenti females. 45% people used preventive measures that were not translated into a reduction in the individual risk of acquiring CL. Exposure to P. sergenti was the only risk factor found, and the reduction in its density could be achieved through the improvement of water wells management, organic fertilizers' disposal and dogs control. The lack of effectiveness of indoor residual spraying and treated nets are attributable to poor compliance and misuse of them. In addition, result optimization of the awareness campaigns on the public is possible by involving patients with CL to explain their own experience.

Concomitant visceral and localized cutaneous leishmaniasis in two Moroccan infants.

BACKGROUND: Leishmaniases are vector-borne diseases caused by the protozoa of the Leishmania genus. The clinical spectrum of these diseases extends from benign dermal lesions to visceral forms. In the Mediterranean region, zoonotic visceral leishmaniasis (ZVL) is caused by L. infantum. If untreated within two years, the disease usually leads to death. In Morocco, ZVL is endemic in the north, with a hundred cases notified each year, mostly in children aged below five years. Here, we report on two clinical observations in infants presenting unusual concomitant VL and cutaneous leishmaniasis (CL) in Morocco. CASE PRESENTATION: In this case study, we report on two infants aged nine and 12 months old. They both have a history of febrile splenomegaly, anemia, and pallor of mucous membranes. Visceral leishmaniasis was confirmed by parasitological diagnosis (positive bone marrow smear and screening of anti-L. infantum antibodies). However, the clinical examination also showed cutaneous lesions that suggested the presence of CL. This was reinforced by the patients having a history of living or traveling to endemic foci. Thus, direct examination, culture, and PCR-RFLP (ITS1-Hae 3) were carried out on the patients' dermal exudates. In one of the infants, CL was associated with L. infantum, while in the other it was associated with L. tropica. The infants were treated as according to the recommendations of the Ministry of Health. Both patients were cured in two months; defervescence, reduction of splenomegaly, and healing of cutaneous lesions were all observed. CONCLUSIONS: These singular patients illustrate the clinical polymorphism of CL and the necessity of updating the differential diagnosis of leukemia-like syndromes, including VL, in children living in or travelling to known endemic areas. These observations suggest a change in the Mediterranean VL phenotype that may be associated with CL.

Molecular diagnosis of Pseudoterranova decipiens s.s in human, France.

BACKGROUND: Anisakis and Pseudoterranova are the main genera involved in human infections caused by nematodes of the Anisakidae family. Species identification is complicated due to the lack of differential morphological characteristics at the larval stage, thus requiring molecular differentiation. Pseudoterranova larvae ingested through raw fish are spontaneously eliminated in most cases, but mechanical removal by means of endoscopy might be required. To date, only very few cases of Pseudoterranova infection have been reported in France. CASE PRESENTATION: A 19-year-old woman from Northeastern France detected, while brushing her teeth, a larva exiting through her mouth. The patient who presented with headache, diarrhea, and abdominal cramps reported having eaten baked cod. The worm was a fourth-stage larva with a size of 22 × 0.9 mm, and molecular biology identified it as Pseudoterranova decipiens sensu stricto (s. s.). In a second P. decipiens infection case, occurring a few months later, a worm exited through the patient's nose after she had eaten raw sea bream. CONCLUSION: These two cases demonstrate that Pseudoterranova infection is not uncommon among French patients. Therefore, molecular techniques should be more widely applied for a better characterization of anisakidosis epidemiology in France.

Phlebotomine sandflies (Diptera, Phlebotomidae) of Lanzarote Island (Canary Islands, Spain): Ecological survey and evaluation of the risk of Leishmania transmission.

Phlebotomine sandflies are natural vectors of Leishmania spp. and their expansion throughout has been evidenced in the last few years due to the global warming and changes in human behavior, worsening leishmaniasis problem. However, phlebotomine sandflies have been captured in small numbers on the Canary Islands, particularly on the island of Lanzarote, where only one limited survey was carried out almost thirty years ago. The proximity of this island to Morocco, in addition to the high number of tourists, sometimes accompanied by their dogs, from leishmaniasis endemic regions, highlights the importance of studying the sandfly fauna on this island in order to determine the transmission risk of leishmaniasis Thirty-eight sampling sites spread across the island were studied, and ecological features were gathered to identify the ecological traits associated to the presence of sandflies. Only 85 sandfly specimens were captured (1.18/m2) with the following species distribution: Sergentomyia minuta (0.15 specimens/m2), which was reported for the first time on this island, and S. fallax (1.03/m2). Sandfly captured were achieved in only 7 out of 38 stations. No specimen of the Phlebotomus genus was captured and given that none of the species captured has been demonstrated vectors of human pathogenic Leishmania and considering that they were captured in low frequency and density, it can be concluded that the current leishmaniasis transmission risk is null.