In the mangroves of Guinea, where the most active foci of human African trypanosomiasis in West Africa are located, vector control against tsetse flies using insecticide-impregnated Tiny Targets was first introduced in 2012. While annual deployments of Tiny Targets have resulted in an overall 90

Human African trypanosomiasis (HAT), or sleeping sickness, is currently targeted for elimination. The etiologic agent of HAT is a trypanosome belonging to the species Trypanosoma brucei ( Tb ) s.l., a unicellular parasite transmitted by tsetse flies. Tb s.l. consists of three subspecies: T. b. brucei ( Tbb ), T. b. gambiense ( Tbg ) and T. b. rhodesiense ( Tbr ). These subspecies are morphologically indistinguishable and classified according to host in which they are found, type of disease and geographical distribution. During the last few decades, there has been considerable effort to genetically characterize Tb s.l. isolated from domestic and wild animals in order to better evaluate the impact of animal reservoirs on the epidemiology of HAT. To assess genetic diversity of Tb s.l. strains circulating in three endemic or historical HAT foci in Côte d’Ivoire, we conducted a population genetics study of these parasites. Biological and isolated stock samples collected from pigs and reference stocks were tested with the primers of the Trypanosoma gambiense- specific-glycoprotein gene (TgsGP) and were genetically characterized with eighteen microsatellite primers. TgsGP positive samples did not fit into Tbg as regard to their microsatellite profile. We also found that in Ivoirian foci, Tbb populations (animal trypanosomes) were structured as several strongly isolated units that propagate clonally. This is in variance with other published data on that subspecies. This study confirms the need to develop better tools to explore the relationships between Tbb and Tbg and to study the epidemiological role of potential animal reservoir for Tbg . ### Competing Interest Statement The authors have declared no competing interest.

A simple method for accurately identifying Glossina spp in the field is a challenge to sustain the future elimination of Human African Trypanosomiasis (HAT) as a public health scourge, as well as for the sustainable management of African Animal Trypanosomiasis (AAT). Current methods for Glossina species identification heavily rely on a few well-trained experts. Methodologies that rely on molecular methodologies like DNA barcoding or mass spectrometry protein profiling (MALDI TOFF) haven’t been thoroughly investigated for Glossina sp. Nevertheless, because they are destructive, costly, time-consuming, and expensive in infrastructure and materials, they might not be well adapted for the survey of arthropod vectors involved in the transmission of pathogens responsible for Neglected Tropical Diseases, like HAT. This study demonstrates a new type of methodology to classify Glossina species. In conjunction with a deep learning architecture, a database of Wing Interference Patterns (WIPs) representative of the Glossina species involved in the transmission of HAT and AAT was used. This database has 1766 pictures representing 23 Glossina species. This cost-effective methodology, which requires mounting wings on slides and using a commercially available microscope, demonstrates that WIPs are an excellent medium to automatically recognize Glossina species with very high accuracy.

Even if the number of Human African Trypanosomiasis (HAT) cases from Kinshasa province in DRC is going towards elimination for the last decade, cases still occur in the periphery of the city. The diagnosis of 21 cases in the south periphery of Kinshasa, between 2015 and 2017 gives evidence of the existence of an active focus in this area. Here, we present the results of a punctual entomological survey that was realized in july 2014 in the outskirts of the southeast of Kinshasa. Using pyramidal traps, we caught tsetse flies during 2 days, dissecting the fresh ones for further molecular analysis. The average Apparent Density of flies per Trap and per Day was three with a maximum of 5.6 flies in Nganda PIO. Polymerase chain reaction analysis of the midguts provided evidence of a high prevalence (57.2%) of infected flies. Ninety three percent of the trypanosomes that were identified belonged to the Nanomonas species, but Trypanozoon trypanosomes were also present in 24% of the infected flies, including mixed infections with Nanomonas, including 3 flies carrying Trypanosoma brucei gambiense, the human pathogen of trypanosomiasis. These results show that at the time of the field's study there was an active reservoir of trypanosomes, closed to pigsties, knowing that pig is a potential animal reservoir. It also demonstrates that xenomonitoring using the entomological approach can be an efficient tool for monitoring sleeping sickness. Finally, results are discussed in the frame of WHO's HAT elimination project. Regarding Kinshasa, it points out the need of regular epidemiologic surveys.

Trypanosoma brucei causes human African trypanosomiasis (HAT). Three subspecies were described: T. b. gambiense (Tbg) and T. b. rhodesiense (Tbr) in humans, and T. b. brucei (Tbb) in animals. Molecular markers subdivided Tbg into two groups: Tbg1 and Tbg2, of which the latter is different from Tbg1 and Tbr (absence of the SRA gene), but indistinguishable from Tbb. Tbg2 is considered to be a zoonotic form of HAT in West Africa. Tbg2 was found mainly in Cote d'Ivoire between 1978 and 1992, but the latest description was made in Ghana in 2013. New molecular tools would be welcome to characterize such infections and determine their origins (resistance to human serum or patient immunodeficiency) in the current context of HAT elimination.

Population genetics is a convenient tool to study the population biology of non-model and hard to sample species. This is particularly true for parasites and vectors. Heterozygote deficits and/or linkage disequilibrium often occur in such studies and detecting the origin of those (Wahlund effect, reproductive system or amplification problems) is uneasy. We used new tools (correlation between the number of times a locus is found in significant linkage disequilibrium and its genetic diversity, correlations between Wright's F-IS and F-ST, F-IS and number of missing data, F-IT and allele size and standard errors comparisons) for the first time on a real data set of tsetse flies, a vector of dangerous diseases to humans and domestic animals in sub-Saharan Africa. With these new tools, and cleaning data from null allele, temporal heterogeneity and short allele dominance effects, we unveiled the coexistence of two highly divergent cryptic clades in the same sites. These results are in line with other studies suggesting that the biodiversity of many taxa still largely remain undescribed, in particular pathogenic agents and their vectors. Our results also advocate that including individuals from different cohorts tends to bias subdivision measures and that keeping loci with short allele dominance and/or too frequent missing data seriously jeopardize parameter's estimations. Finally, separated analyses of the two clades suggest very small tsetse densities and relatively large dispersal.

Trypanosoma brucei gambiense causes human African trypanosomiasis (HAT). Between 1990 and 2015, almost 440000 cases were reported. Large-scale screening of populations at risk, drug donations, and efforts by national and international stakeholders have brought the epidemic under control with <2200 cases in 2016. The World Health Organization (WHO) has set the goals of gambiense-HAT elimination as a public health problem for 2020, and of interruption of transmission to humans for 2030. Latent human infections and possible animal reservoirs may challenge these goals. It remains largely unknown whether, and to what extend, they have an impact on gambiense-HAT transmission. We argue that a better understanding of the contribution of human and putative animal reservoirs to gambiense-HAT epidemiology is mandatory to inform elimination strategies.

Background The sleeping sickness focus of Campo in South Cameroon is still active, at a low endemic level, for more than a century, despite a regular medical surveillance. The present study focuses on the spatial distribution of xenomonitoring information obtained from an entomological survey performed in the dry season 2012. It appears that humans constitute a third of the blood meals and that the flies’ densities were coherent with those classically observed in the different biotopes. Paradoxically, the epicenter of the focus is the place where the risk indicators are the lowest ones. Methods Particular attention was paid to the entomological device so that it covered the main part of human activities in the study area. One hundred and sixty-two pyramidal traps were used to catch tsetse flies twice a day that were identified, counted, dissected. Molecular analysis using classical and specific molecular markers was conducted to determine the importance of trypanosome infections and the nature of the feeding hosts. This information was used to calculate a Transmission Risk Index and to define a gradient of risk that was projected into a Geographical Information System. Results Conventional entomological indicators such as species identification of tsetse flies or the Apparent Density per Trap per day, show that Glossina palpalis palpalis is the main species in the campo area which is classically distributed into the different biotopes of the study area. Molecular analysis reveals that humans constitute a third of the blood feeding hosts and that 20 % of the dissected flies were infected with trypanosomes, principally with Nannomonas . Nevertheless, one fly was carrying Trypanosoma brucei gambiense , the pathogen agent of sleeping sickness, showing that the reservoir is still active in the epicenter of the focus. Paradoxically, the Transmission Risk Index is not important in the epicenter, demonstrating that endemic events are not only depending on the man/vector contact. Conclusion Xenomonitoring provides a valuable guide/tool to determine places at higher risk for vector/human contact and to identify trypanosomes species circulating in the focus. This information from xenomonitoring demonstrates that decision makers should include a veterinary device in a control strategy.

Although Human African Trypanosomiasis is largely considered to be in the process of extinction today, the persistence of human and animal reservoirs, as well as the vector, necessitates a laborious elimination process. In this context, modeling could be an effective tool to evaluate the ability of different public health interventions to control the disease. Using the Cormas® system, we developed HATSim, an agent-based model capable of simulating the possible endemic evolutions of sleeping sickness and the ability of National Control Programs to eliminate the disease. This model takes into account the analysis of epidemiological, entomological, and ecological data from field studies conducted during the last decade, making it possible to predict the evolution of the disease within this area over a 5-year span. In this article, we first present HATSim according to the Overview, Design concepts, and Details (ODD) protocol that is classically used to describe agent-based models, then, in a second part, we present predictive results concerning the evolution of Human African Trypanosomiasis in the village of Lambi (Cameroon), in order to illustrate the interest of such a tool. Our results are consistent with what was observed in the field by the Cameroonian National Control Program (CNCP). Our simulations also revealed that regular screening can be sufficient, although vector control applied to all areas with human activities could be significantly more efficient. Our results indicate that the current model can already help decision-makers in planning the elimination of the disease in foci.

Trypanosomes are bloodstream protozoan parasites, which are pathogens of veterinary and medical importance. Several mammalian species, including humans, can be infected by different species of the genus Trypanosoma (T. congolense, T. evansi, T. brucei, T. vivax) exhibiting more or less virulent and pathogenic phenotypes. A previous screening of the excreted-secreted proteins of T. congolense demonstrated an overexpression of several proteins correlated with the virulence and pathogenicity of the strain. Of these proteins, calreticulin (CRT) has shown differential expression between two T. congolense strains with opposite infectious behavior and has been selected as a target molecule based on its immune potential functions in parasitic diseases. In this study, we set out to determine the role of T. congolense calreticulin as an immune target. Immunization of mice with recombinant T. congolense calreticulin induced antibody production, which was associated with delayed parasitemia and increased survival of the challenged animal. These results strongly suggest that some excreted-secreted proteins of T. congolense are a worthwhile target candidate to interfere with the infectious process.

To identify miRNAs whose expression are differentially regulated during trypanosome infections a microarray targeting more than 600 rat miRNA was used to analyze the miRNA expression profiles between uninfected rats and animals infected by Trypanosoma congolense and Trypanosoma brucei s.l. The potential targets of dysregulated miRNAs as well as their biological pathways and functions were predicted using several bioinformatics software tools.Irrespective of the infecting trypanosome species, eight miRNAs (seven up- and one down-regulated) were dysregulated during infections. Moreover, other miRNAs were differentially regulated in rats infected by specific trypanosome species. Functional analyses of differentially regulated miRNAs indicated their involvement in diverse biological processes. Among these, transcription repressor activity, gene expression control as well as protein transporter activity were predominant. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of dysregulated miRNAs revealed their involvement in several biological pathways and disease conditions. This suggests possible modulation of such pathways following trypanosome infection; for example, the MAPK signaling pathway which is known to play vital roles in apoptosis, innate immune response and response to viral infections was highly affected. Axon guidance was equally highly impacted and may indicate a cross reactivity between pathogen proteins and guidance molecules representing one pathological mechanism as it has been observed with influenza HA. Furthermore, Ingenuity pathway analyses of dysregulated miRNAs and potential targets indicated strong association with inflammatory responses, cell death and survival as well as infectious diseases.The data generated here provide valuable information to understand the regulatory function of miRNAs during trypanosome infections. They improved our knowledge on host-parasite cross-talks and provide a framework for investigations to understand the development of trypanosomes in their hosts as well as the differences in the clinical and pathological evolutions of the disease. (C) 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

The sleeping sickness focus of Campo lies along the Atlantic coast and extends along the Ntem River, which constitutes the Cameroonian and Equatorial Guinean border. It is a hypo-endemic focus with the disease prevalence varying from 0.3 to 0.86% during the last few decades. Investigations on animal reservoirs revealed a prevalence of Trypanosoma brucei gambiense of 0.6% in wild animals and 4.83% in domestic animals of this focus. From 2001 to 2012, about 19 931 tsetse were collected in this focus and five tsetse species including Glossina palpalis palpalis, G. pallicera, G. nigrofusca, G. tabaniformis and G. caliginea were identified. The analysis of blood meals of these flies showed that they feed on human, pig, goat, sheep, and wild animals such as antelope, duiker, wild pig, turtle and snake. The percentage of blood meals taken on these hosts varies according to sampling periods. For instance, 6.8% of blood meals from pig were reported in 2004 and 22% in 2008. This variation is subjected to considerable evolutions because the Campo HAT focus is submitted to socio-economic mutations including the reopening of a new wood company, the construction of autonomous port at "Kribi" as well as the dam at "Memve ele". These activities will bring more that 3000 inhabitants around Campo and induce the deforestation for the implementation of farmlands as well as breeding of domestic animals. Such mutations have impacts on the transmission and the epidemiology of sleeping sickness due to the modification of the fauna composition, the nutritional behavior of tsetse, the zoophilic/anthropophilic index. To achieve the elimination goal in the sleeping sickness focus of Campo, we report in this paper the current epidemiological situation of the disease, the research findings of the last decades notably on the population genetics of trypanosomes, the modifications of nutritional behavior of tsetse, the prevalence of T. b. gambiense in humans, domestic and wild animals. An overview on the types of mutations occurring in the region has been raised and a discussion on the strategies that can be implemented to achieve the elimination of the disease has been made.

African trypanosomiases, including the human disease referred to as 'sleeping sickness' and the animal diseases such as nagana, surra and dourine, are neglected vector-borne diseases that after years of research still need improved diagnosis and chemotherapy. Advances in proteomics offer new tools to define biomarkers, whose expression may reflect host-parasite interactions occurring during the infection. In this review, the authors first describe the current diagnostic tools used to detect a trypanosome infection during field surveys, and then discuss their interests, limits and further evolutions. The authors also report on the contribution of molecular diagnostics, and the recent advances and developments that make it suitable for fieldwork. The authors then explore the recent uses of proteomics technology to define host and parasite biomarkers that allow detection of the infection, the power and constraints of the technology. The authors conclude by discussing the urgent need to use the biomarkers discovered in order to develop tools to improve trypanosomiasis control in the near future.

Human African trypanosomiasis, or sleeping sickness, is a neglected vector-borne parasitic disease caused by protozoa of the species Trypanosoma brucei sensu lato. Within this complex species, T. b. gambiense is responsible for the chronic form of sleeping sickness in Western and Central Africa, whereas T. b. rhodesiense causes the acute form of the disease in East Africa. Presently, 1.5 million disability-adjusted life years (DALYs) per year are lost due to sleeping sickness. In addition, on the basis of the mortality, the disease is ranked ninth out of 25 human infectious and parasitic diseases in Africa. Diagnosis is complex and needs the intervention of a specialized skilled staff; treatment is difficult and expensive and has potentially life-threatening side effects. The use of transcriptomic and proteomic technologies, currently in rapid development and increasing in sensitivity and discriminating power, is already generating a large panel of promising results. The objective of these technologies is to significantly increase our knowledge of the molecular mechanisms governing the parasite establishment in its vector, the development cycle of the parasite during the parasite's intra-vector life, its interactions with the fly and the other microbial inhabitants of the gut, and finally human host-trypanosome interactions. Such fundamental investigations are expected to provide opportunities to identify key molecular events that would constitute accurate targets for further development of tools dedicated to field work for early, sensitive, and stage-discriminant diagnosis, epidemiology, new chemotherapy, and potentially vaccine development, all of which will contribute to fighting the disease. The present review highlights the contributions of the transcriptomic and proteomic analyses developed thus far in order to identify potential targets (genes or proteins) and biological pathways that may constitute a critical step in the identification of new targets for the development of new tools for diagnostic and therapeutic purposes.

Human and animal African trypanosomoses, or sleeping sickness and Nagana, are neglected vector-borne parasitic diseases caused by protozoa belonging to the Trypanosoma genus. Advances in proteomics offer new tools to better understand host-vector-parasite crosstalks occurring during the complex parasitic developmental cycle, and to determine the outcome of both transmission and infection. In this review, we summarize proteomics studies performed on African trypanosomes and on the interactions with their vector and mammalian hosts. We discuss the contributions and pitfalls of using diverse proteomics tools, and argue about the interest of pathogenoproteomics, both to generate advances in basic research on the best knowledge and understanding of host-vector-pathogen interactions, and to lead to the concrete development of new tools to improve diagnosis and treatment management of trypanosomoses in the near future.

To characterize the potential transmission sites of sleeping sickness in Kinshasa, two entomologic surveys were carried out during the dry and the rainy seasons in rural and periurban areas of Kinshasa in 2005. About 610 pyramidal traps were set up, and 897 Glossina fuscipes quanzensis were captured. Environmental and biologic factors were reported, and relationships between these factors were evaluated using logistic regression and multiple correspondence analysis. The biologic factors (the presence of tsetse flies, human blood meals, and teneral flies) were progressively accumulated at each capture site to permit the characterization of the sleeping sickness transmission risk. The dry season was found to be a more favorable period for the disease transmission than the rainy season. Moreover, the landscapes characterized by the presence of argillaceous soils, raised ground cover with forest residues and rivers, were identified as types of environments with greater risk of sleeping sickness transmission. Pig breeding appeared as an important factor increasing the disease transmission. If vector control is continuously performed along rivers segments at high risk, the transmission of sleeping sickness in rural and periurban areas of Kinshasa will considerably decrease.

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south–east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted–secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host–parasite interactions. We used a combined proteomic approach (one-dimensional SDS–PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.