Arnaud Cannet

A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies

Background The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Methodology and Principal Findings Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? Conclusions and Significance We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

Leishmania infections: Molecular targets and diagnosis

Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs.

A review of data on laboratory colonies of bed bugs (Cimicidae), an insect of emerging medical relevance.

Cimicidae are hematophagous Heteroptera, feeding on human blood, that have been the subject of significant medical investigation. In particular, they have been colonized under laboratory conditions to study their medical relevance. Laboratory colonization of these bugs is a multifactorial phenomenon. Our goal was to conduct a comparative literature review to classify the published data, demonstrating preferred bed bug colony conditions. We show that physical factors including temperature, relative humidity and photoperiod, and physiological factors such as type and frequency of blood meals play important roles in laboratory colonies. Any change in these factors produces changes in life-cycle duration. Temperature and blood meal are the most important factors, with a marked impact on the life-cycle of laboratory populations, depending on the species. A wide range of temperatures (15–34 °C) and relative humidity (46–75%) with an average of 25 °C and 59% were found for these colonies. Two widely used blood sources for the colonies were rabbits and humans.

Spatial genetic structure and restricted gene flow in bed bugs (Cimex lectularius) populations in France.

Bed bugs (Cimex lectularius) are resurgent blood-sucking ectoparasites that are currently increasing at a rapid rate, particularly in industrialized countries, such as France. Despite the rapid spread of bed bugs, there is a lack of knowledge concerning the population structure and gene flow among C. lectularius populations in France. To fill this gap, a genetic study was conducted using 183 C. lectularius from 14 populations of bed bugs collected in a hotel and in individual apartments in the French Riviera and in the Saint Ouen suburb of Paris. The samples were genotyped using an isolated set of six polymorphic microsatellite loci, including five new loci which were newly isolated and chosen based on prior successful amplification, and one previously described loci (bb15b). The low genetic diversity observed in the samples (of one to five alleles) suggested that most of prospected populations were established by only a few individuals, possibly from a single mated female. The overall genetic differentiation was high and statistically significant (FST=0.556, p<0.0001). Pairwise analysis of the populations indicated significant genetic differentiation for 24 out of the 45 (53%) population pairs associated with FST, ranging from 0.0042 to 0.862. No obvious relationship between the level of genetic differentiation and the geographic distance was observed when considering all samples. Analysis with Structure software identified nine distinct genetic clusters within the dataset. These preliminary results help to elucidate the genetic structure and gene flow of C. lectularius populations in France; however, the available information should be expanded in further studies.

New microsatellite markers for multi-scale genetic studies on Phlebotomus ariasi Tonnoir, vector of Leishmania infantum in the Mediterranean area

The population structure of Phlebotomus ariasi, a proven vector of Leishmania infantum in the Mediterranean area, is still poorly understood. Previously, only two microsatellite loci had been developed to study the population genetics of this species. Herein we use these loci and determined fourteen novel microsatellite loci, useful for the characterization of P. ariasi populations. These loci were tested on three populations of P. ariasi, two from France and one from Portugal. In addition, the usefulness of these markers was also evaluated on seven other sandfly species. We show, that for P. ariasi, 15 of the 16 loci selected were polymorphic, with a mean of 4.25 alleles and an observed heterozygosity of 0.299. Within the P. ariasi population of France, 11 loci were polymorphic, with an average of 2.44 alleles and an observed heterozygosity of 0.2177. The fixation index was moderate among the French populations but high between French and Portuguese populations. In addition, eight loci were also found to be amplifiable in six other Phlebotomus species. These results demonstrate the usefulness of this new set of microsatellite loci for population structure and molecular ecology studies of P. ariasi at various spatial scales, but also of other sandfly species.

Experimental infection of Phlebotomus perniciosus by bioluminescent Leishmania infantum using murine model and artificial feeder

Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. In the present study, we carried out a screening on the experimental infection of Phlebotomus pernioucus by bioluminescent Leishmania infantum using murine model and artificial feeder. We developed a real-time polymerase chain reaction (RT-PCR)-based method to determine individually the number of Leishmania promastigotes fed by infected flies. Among 1840 new emerged female sand flies, 428 were fed on the infected mice. After their death, they were analysed individually by RT-PCR. Our results demonstrated just a single Leishmania positive female at sixth day post meal. A total of 1070 female sand flies were exposed in contact with artificial feeder containing the human blood with two different quantities of Leishmania parasites: 2.106/mL and 1.107/mL. A blood meal including 1.107/mL LUC-promastigotes was proposed to 270 females and 75 (28%) flies were engorged. Among them, 44 (59%) were positive by RT-PCR analysis, with a relative average of 50551 Leishmania parasites. In case of blood feeding of females with 2.106/mL promastigotes, 57 out of 800 (7%) females succeed to feed from artificial feeder which 22 (39%) were positive with a relative average of 6487 parasites.

Immunodetection and molecular determination of visceral and cutaneous Leishmania infection using patients' urine

The diagnosis of leishmaniasis relies mainly on the use of invasive processes, to collect the biological material for detecting Leishmania parasites. Body fluids, which can be collected by non-invasive process, would greatly facilitate the leishmaniasis diagnosis. In the present study, we investigated the potency of urine immunoblotting to diagnose cutaneous and visceral leishmaniasis and we compared with routine molecular methods. A total of 80 samples, including 40 sera and their 40 corresponding urine samples were collected from 37 suspected patients with cutaneous and visceral leishmaniasis, and 3 healthy individuals (as control), in Ilam and Ardabil provinces of Iran. All sera and urine samples were analyzed, using immunoblotting. The confirmation of leishmaniasis infection was performed, using conventional and quantitative PCRs as well as by sequencing the amplicons. Among 37 suspected patients, 23 patients presented cutaneous lesions (CL) and 14 exhibited clinical symptoms reminiscent of visceral leishmaniasis (L. infantum). Among cutaneous patients, 15 were positive for zoonotic cutaneous leishmaniasis (L. major), and eight for anthroponotic cutaneous leishmaniasis (L. tropica). Molecular quantification of Leishmania parasites was performed on sera, urines and cutaneous biopsies of CL and VL patients, demonstrating that parasite load is lower in urines, compared to sera or biopsy. DNA can be detected in 20 out of 23 (86.9%) CL urine samples and in 13 out of 14 (92.8%) VL urine samples. Immunodetection analysis demonstrates that 22 out of 23 (95.6%) sera from CL patients and all patients suspected with VL are positive. For urine samples, 18 out of 23 (78.2%) urine of CL patients and 13 out of 14 (92.8%) urine of VL patients were positive, using Western blot. Therefore, immunodetection and molecular analysis using urine samples can be used as a diagnostic tool for surveying cutaneous and visceral leishmaniasis.

An old lady with Pediculosis pubis on the head hair.

to isotretinoin or acitretin, it exerts strong anti-inflammatory and immune-modulatory effects without suppressing the activity of the sebaceous glands. It is used mainly for the treatment of chronic hand eczema, leading to durable remissions. Additional clinical efficacy has been reported in a variety of other cutaneous diseases such as ichthyosis or Darier’s disease. In the case presented here, alitretinoin showed rapid and robust activity in treating the papules without aggravating the xerosis. Taken together, we identify alitretinoin as a novel effective treatment option for cutaneous amyloidosis, specifically in patients with strong pruritus and sebostatic skin conditions.