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.

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.

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.

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.

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.

Topical Treatment of Leishmania tropica Infection Using (-)-α-Bisabolol Ointment in a Hamster Model: Effectiveness and Safety Assessment.

There is currently no reliable treatment for the management of cutaneous leishmaniasis, and intralesional antimonial injections remain the main treatment. The present work aims at evaluating the antileishmanial effectiveness and safety of (-)-α-bisabolol (1) in a novel topical formulation on a cutaneous leishmaniasis model involving Leishmania tropica-infected Syrian hamsters. The topical treatment with 1 reduced lesion thickness to 56% at 2.5%, showing a higher efficacy than the reference control, meglumine antimoniate. Other regimens (ointment at 1% and 5% and oral treatment at 200 mg/kg) reduced the footpad thickness as well. The skin parasite load decreased after the experiment in all treatment groups, particularly in those animals treated with the 2.5% formulation (83.2%). Treatment with (-)-α-bisabolol at different concentrations or through an oral route did not lead to the appearance of toxicity or side effects in healthy hamsters or infected animals. Therefore, topical (-)-α-bisabolol was more effective than meglumine antimoniate in this cutaneous leishmaniasis model without showing toxicity effects on the hamsters. These results are of great interest and might be used for the development of alternatives for the treatment of cutaneous leishmaniasis, either in monotherapy or in combination with other drugs whose skin permeability could be enhanced by this sesquiterpene.