Anne-Laure Bañuls

Clinical pleiomorphism in human leishmaniases, with special mention of asymptomatic infection

This review gives an update of current knowledge on the clinical pleiomorphism of Leishmania, with a special emphasis on the case of asymptomatic carriage. The first part describes the numerous unusual expressions of the disease that occur besides the classic (visceral, cutaneous, and mucocutaneous) forms of leishmaniases. The second part deals with progress in the understanding of disease outcome in humans, and the possible future approaches to improve our knowledge in the field. The third part highlights the role of the too often neglected asymptomatic carrier compartment. This group could be key to understanding infraspecific differences in virulence and pathogenicity of the parasite, as well as identifying the genetic determinants involved in the expression of the disease.

Reproductive strategies and population structure in Leishmania: substantial amount of sex in Leishmania Viannia guyanensis

Leishmania species of the subgenus Viannia and especially Leishmania Viannia guyanensis are responsible for a large proportion of New World leishmaniasis cases. Since a recent publication on Leishmania Viannia braziliensis, the debate on the mode of reproduction of Leishmania parasites has been reopened. A predominant endogamic reproductive mode (mating with relatives), together with strong Wahlund effects (sampling of strains from heterogeneous subpopulations), was indeed evidenced. To determine whether this hypothesis can be generalized to other Leishmania Viannia species, we performed a population genetic study on 153 human strains of L. (V.) guyanensis from French Guiana based on 12 microsatellite loci. The results revealed important homozygosity and very modest linkage disequilibrium, which is in agreement with a high level of sexual recombination and substantial endogamy. These results also revealed a significant isolation by distance with relatively small neighbourhoods and hence substantial viscosity of Leishmania populations in French Guiana. These results are of epidemiological relevance and suggest a major role for natural hosts and/or vectors in parasite strain diffusion across the country as compared to human hosts.

Ecology of Phlebotomine Sand Flies in the Rural Community of Mont Rolland (Thiès Region, Senegal): Area of Transmission of Canine Leishmaniasis

Background Different epidemiological studies previously indicated that canine leishmaniasis is present in the region of Thiès (Senegal). However, the risks to human health, the transmission cycle and particularly the implicated vectors are unknown. Methodology/Principal Findings To improve our knowledge on the population of phlebotomine sand flies and the potential vectors of canine leishmaniasis, sand flies were collected using sticky traps, light traps and indoor spraying method using pyrethroid insecticides in 16 villages of the rural community of Mont Rolland (Thiès region) between March and July 2005. The 3788 phlebotomine sand flies we collected (2044 males, 1744 females) were distributed among 9 species of which 2 belonged to the genus Phlebotomus: P. duboscqi (vector of cutaneous leishmaniasis in Senegal) and P. rodhaini. The other species belonged to the genus Sergentomyia: S. adleri, S. clydei, S. antennata, S. buxtoni, S. dubia, S. schwetzi and S. magna. The number of individuals and the species composition differed according to the type of trap, suggesting variable, species-related degrees of endophily or exophily. The two species of the genus Phlebotomus were markedly under-represented in comparison to the species of the genus Sergentomyia. This study also shows a heterogeneous spatial distribution within the rural community that could be explained by the different ecosystems and particularly the soil characteristics of this community. Finally, the presence of the S. dubia species appeared to be significantly associated with canine leishmaniasis seroprevalence in dogs. Conclusions/Significance Our data allow us to hypothesize that the species of the genus Sergentomyia and particularly the species S. dubia and S. schwetzi might be capable of transmitting canine leishmaniasis. These results challenge the dogma that leishmaniasis is exclusively transmitted by species of the genus Phlebotomus in the Old World. This hypothesis should be more thoroughly evaluated.

Identification of phlebotomine sand flies using one MALDI-TOF MS reference database and two mass spectrometer systems

BackgroundRapid, accurate and high-throughput identification of vector arthropods is of paramount importance in surveillance programmes that are becoming more common due to the changing geographic occurrence and extent of many arthropod-borne diseases. Protein profiling by MALDI-TOF mass spectrometry fulfils these requirements for identification, and reference databases have recently been established for several vector taxa, mostly with specimens from laboratory colonies.MethodsWe established and validated a reference database containing 20 phlebotomine sand fly (Diptera: Psychodidae, Phlebotominae) species by using specimens from colonies or field-collections that had been stored for various periods of time.ResultsIdentical biomarker mass patterns (‘superspectra’) were obtained with colony- or field-derived specimens of the same species. In the validation study, high quality spectra (i.e. more than 30 evaluable masses) were obtained with all fresh insects from colonies, and with 55/59 insects deep-frozen (liquid nitrogen/-80xa0°C) for up to 25xa0years. In contrast, only 36/52 specimens stored in ethanol could be identified. This resulted in an overall sensitivity of 87 % (140/161); specificity was 100 %. Duration of storage impaired data counts in the high mass range, and thus cluster analyses of closely related specimens might reflect their storage conditions rather than phenotypic distinctness. A major drawback of MALDI-TOF MS is the restricted availability of in-house databases and the fact that mass spectrometers from 2 companies (Bruker, Shimadzu) are widely being used. We have analysed fingerprints of phlebotomine sand flies obtained by automatic routine procedure on a Bruker instrument by using our database and the software established on a Shimadzu system. The sensitivity with 312 specimens from 8 sand fly species from laboratory colonies when evaluating only high quality spectra was 98.3 %; the specificity was 100 %. The corresponding diagnostic values with 55 field-collected specimens from 4 species were 94.7 % and 97.4 %, respectively.ConclusionsA centralized high-quality database (created by expert taxonomists and experienced users of mass spectrometers) that is easily amenable to customer-oriented identification services is a highly desirable resource. As shown in the present work, spectra obtained from different specimens with different instruments can be analysed using a centralized database, which should be available in the near future via an online platform in a cost-efficient manner.

First clinical case of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis in a domestic cat from French Guiana

We report the first case of natural infection of a domestic female cat (Felis catus) by Leishmania (Viannia) braziliensis in French Guiana. The infected animal had a cutaneous ulcer on the nose and nodules of different sizes in the ears. The diagnosis was confirmed by molecular analysis of cutaneous samples that detected the presence of Leishmania parasites and allowed identifying the Leishmania (Viannia) braziliensis species. The discovery of a cat infected by L. (V.) braziliensis suggests the possibility that cats could be potential secondary reservoirs of Leishmania parasites in French Guiana. Thus, it would be important to investigate the possible epidemiological role of domestic cats in domestic foci of Leishmania in this region.

Multifaceted Population Structure and Reproductive Strategy in Leishmania donovani Complex in One Sudanese Village

Leishmania species of the subgenus Leishmania and especially L. donovani are responsible for a large proportion of visceral leishmaniasis cases. The debate on the mode of reproduction and population structure of Leishmania parasites remains opened. It has been suggested that Leishmania parasites could alternate different modes of reproduction, more particularly clonality and frequent recombinations either between related individuals (endogamy) or between unrelated individuals (outcrossing) within strongly isolated subpopulations. To determine whether this assumption is generalized to other species, a population genetics analysis within Leishmania donovani complex strains was conducted within a single village. The results suggest that a mixed-mating reproduction system exists, an important heterogeneity of subsamples and the coexistence of several genetic entities in Sudanese L. donovani. Indeed, results showed significant genetic differentiation between the three taxa (L. donovani, L. infantum and L. archibaldi) and between the human or canine strains of such taxa, suggesting that there may be different imbricated transmission cycles involving either dogs or humans. Results also are in agreement with an almost strict specificity of L. donovani stricto sensu to human hosts. This empirical study demonstrates the complexity of population structure in the genus Leishmania and the need to pursue such kind of analyses at the smallest possible spatio-temporal and ecological scales.

Transmission of Leishmania infantum in the Canine Leishmaniasis Focus of Mont-Rolland, Senegal: Ecological, Parasitological and Molecular Evidence for a Possible Role of Sergentomyia Sand Flies

Leishmania (L.) infantum is the causative agent in an endemic focus of canine leishmaniasis in the Mont-Rolland district (Thiès, Senegal). In this area, the transmission cycle is well established and more than 30% of dogs and 20% of humans are seropositive for L. infantum. However, the sand fly species involved in L. infantum transmission cycle are still unknown. Between 2007 and 2010, 3654 sand flies were collected from different environments (indoor, peridomestic, farming and sylvatic areas) to identify the main L. infantum vector(s). Nine sand fly species were identified. The Phlebotomus genus (n = 54 specimens; Phlebotomus (Ph) duboscqi and Phlebotomus (Ph). rodhaini) was markedly under-represented in comparison to the Sergentomyia genus (n = 3600 specimens; Sergentomyia (Se) adleri, Se. clydei, Se. antennata, Se. buxtoni, Se. dubia, Se. schwetzi and Se. magna). Se. dubia and Se. schwetzi were the dominant species indoor and in peridomestic environments, near humans and dogs. Blood-meal analysis indicated their anthropophilic behavior. Some Se. schwetzi specimens fed also on dogs. The dissection of females in the field allowed isolating L. infantum from sand flies of the Sergentomyia genus (0.4% of Se. dubia and 0.79% of Se. schwetzi females). It is worth noting that one Se. dubia female not engorged and not gravid revealed highly motile metacyclic of L. infantum in the anterior part of the midgut. PCR-based diagnosis and sequencing targeting Leishmania kinetoplast DNA (kDNA) highlighted a high rate of L. infantum-positive females (5.38% of Se. dubia, 4.19% of Se. schwetzi and 3.64% of Se. magna). More than 2% of these positive females were unfed, suggesting the parasite survival after blood-meal digestion or egg laying. L. infantum prevalence in Se. schwetzi was associated with its seroprevalence in dogs and humans and L. infantum prevalence in Se. dubia was associated with its seroprevalence in humans. These evidences altogether strongly suggest that species of the Sergentomyia genus are probably the vectors of canine leishmaniasis in the Mont-Rolland area and challenge one more time the dogma that in the Old World, leishmaniasis is exclusively transmitted by species of the Phlebotomus genus.

Vector soup: high‐throughput identification of Neotropical phlebotomine sand flies using metabarcoding

Phlebotomine sand flies are haematophagous dipterans of primary medical importance. They represent the only proven vectors of leishmaniasis worldwide and are involved in the transmission of various other pathogens. Studying the ecology of sand flies is crucial to understand the epidemiology of leishmaniasis and further control this disease. A major limitation in this regard is that traditional morphological‐based methods for sand fly species identifications are time‐consuming and require taxonomic expertise. DNA metabarcoding holds great promise in overcoming this issue by allowing the identification of multiple species from a single bulk sample. Here, we assessed the reliability of a short insect metabarcode located in the mitochondrial 16S rRNA for the identification of Neotropical sand flies, and constructed a reference database for 40 species found in French Guiana. Then, we conducted a metabarcoding experiment on sand flies mixtures of known content and showed that the method allows an accurate identification of specimens in pools. Finally, we applied metabarcoding to field samples caught in a 1‐ha forest plot in French Guiana. Besides providing reliable molecular data for species‐level assignations of phlebotomine sand flies, our study proves the efficiency of metabarcoding based on the mitochondrial 16S rRNA for studying sand fly diversity from bulk samples. The application of this high‐throughput identification procedure to field samples can provide great opportunities for vector monitoring and eco‐epidemiological studies.

Ecology and spatiotemporal dynamics of sandflies in the Mediterranean Languedoc region (Roquedur area, Gard, France)

BackgroundPhlebotomine sandflies are hematophagous insects widely present in Western Mediterranean countries and known for their role as Leishmania vectors. During the last ten years, the risk of leishmaniasis re-emergence has increased in France. However, sandfly biology and ecology in the South of France remain poorly known because the last detailed study on their spatiotemporal dynamics was performed over 30xa0years ago. The aim of the present study was to update our knowledge on sandfly ecology by determining their spatiotemporal dynamics and by investigating the relationship between environmental/climatic factors and the presence and abundance of sandflies in the South of France.MethodsAn entomological survey was carried out during three years (2011–2013) along a 14 kilometer-long transect. The findings were compared with the data collected along the same transect in 1977. Data loggers were placed in each station and programmed to record temperature and relative humidity every six hours between April 2011 and November 2014. Several environmental factors (such as altitude, slope and wall orientation (North, East, West and South)) were characterized at each station.ResultsFour sandfly species were collected: Phlebotomus ariasi and Sergentomyia minuta, which were predominant, Ph. perniciosus and Ph. mascittii. Sandfly activity within the studied area started in May and ended in October with peaks in July-August at the optimum average temperature. We found a positive effect of altitude and temperature and a negative effect of relative humidity on Ph. ariasi and Se. minuta presence. We detected interspecific differences and non-linear effects of these climatic variables on sandfly abundance. Although the environment has considerably changed in 30xa0years, no significant difference in sandfly dynamics and species diversity was found by comparing the 1977 and 2011–2013 data.ConclusionOur study shows that this area maintains a rich sandfly fauna with high Ph. ariasi population density during the active season. This represents a risk for Leishmania transmission. The analysis revealed that the presence and abundance of Ph. ariasi and Se. minuta were differently correlated with the environmental and climatic factors. Comparison with the data collected in 1977 highlighted the sandfly population stability, suggesting that they can adapt, in the short and long term, to changing ecosystems.

Complete mitochondrial genome of Lutzomyia (Nyssomyia) umbratilis (Diptera : Psychodidae), the main vector of Leishmania guyanensis

Abstract The nearly complete mitochondrial genome of Lutzomyia umbratilis Ward & Fraiha, 1977 (Psychodidae: Phlebotominae), considered as the main vector of Leishmania guyanensis, is presented. The sequencing has been performed on an Illumina Hiseq 2500 platform, with a genome skimming strategy. The full nuclear ribosomal RNA segment was also assembled. The mitogenome of L. umbratilis was determined to be at least 15,717u2009bp-long and presents an architecture found in many mitogenomes of insect (13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one non-coding region also referred as the control region). The control region contains a large repeated element of c. 370u2009bp and a poly-AT region of unknown length. This is the first mitogenome of Psychodidae to be described.