A study on African animal trypanosomosis in four areas of Senegal.

In Senegal, several areas provide great potential for agriculture and animal production, but African animal trypanosomosis (AAT) is one of the major constraints to the development of more effective livestock production systems. A study was conducted to assess the current situation of AAT in this country. Surveys were carried out between June 2011 and September 2012 in four different areas: Dakar, Sine Saloum, Kedougou region and Basse Casamance in several animal species: dogs (152), donkeys (23), horses (63), sheep (43), goats (52) and cattle (104), distributed in the four sites. Molecular tools (PCR) indicated 3.4% positive animals including dogs, donkeys, a goat and cattle. The savannah type of Trypanosoma congolense Broden, 1904 (53% of positive cases) and the forest type of T. congolense (subgenus Nannomonas Hoare, 1964) were predominant. Trypanosoma vivax Ziemann, 1905 (subgenus Duttonella Chalmers, 1918) was only present in one animal and no trypanosome of the subgenus Trypanozoon Lühe, 1906 was found. Half of the positive cases were detected in Sine Saloum, where T. congolense savannah-type was predominant, and the other half in Basse Casamance, where T. congolense forest-type was predominant; no cases were found in Dakar or in the Kedougou region. A high risk of infection in dogs with T. congolense savannah-type was shown in Sine Saloum, requiring prevention and control of dogs in this area. The involvement of tsetse flies in the transmission of T. congolense in Sine Saloum and Basse Casamance is discussed.

Population genetics of Trypanosoma brucei circulating in Glossina palpalis palpalis and domestic animals of the Fontem sleeping sickness focus of Cameroon.

BACKGROUND: Human African Trypanosomiasis is still a public health threat in Cameroon. To assess Trypanosoma brucei strains circulating in the Fontem sleeping sickness focus, we conducted a genetic structure study using microsatellites to assess genotypes circulating in both tsetse flies and domestic animals. METHOD: For this study, pyramidal traps were set up and 2695 tsetse flies were collected and 1535 (57%) living flies were dissected and their mid-guts collected. Furthermore, blood samples were collected from 397 domestic animals (pigs, goats, sheep and dogs). DNA was extracted from midguts and blood samples, and specific primers were used to identify trypanosomes of the subgenus Trypanozoon. All positive samples were genetically characterized with seven microsatellite markers. RESULTS: Seventy five (4.7%) midguts of tsetse flies and 140 (35.2%) domestic animals were found infected by trypanosomes of the subgenus Trypanozoon. The genetic characterization of 215 Trypanozoon positive samples (75 from tsetse and 140 from animals) revealed a genetic diversity between Trypanosoma brucei circulating in tsetse and domestic animals. Of these positive samples, 87 (40.5%) single infections were used here to investigate the population genetics of Trypanosoma brucei circulating in tsetse and domestic animals. The dendrogram illustrating the genetic similarities between Trypanosoma brucei genotypes was subdivided into four clusters. The samples from tsetse belonged to the same cluster whereas the samples from domestic animals and espcially pigs were distributed in the four clusters. CONCLUSION: Pigs appeared as the animal species harboring the highest number of different Trypanosoma brucei strains. They may play an important role in the propagation of different genotypes. The FST values revealed a sub structuration of Trypanosoma brucei according to hosts and sometimes villages. The data obtained from this study may have considerable importance for the understanding of the transmission and the spread of specific genotypes of Trypanosoma brucei.

Evaluation of trypanocidal drugs used for human African trypanosomosis against Trypanosoma lewisi.

Trypanosomes from animals are potential pathogens for humans. Several human cases infected by Trypanosoma lewisi, a parasite of rats, have been reported. The number of these infections is possibly underestimated. Some infections were self-cured, others required treatment with drugs used in human African trypanosomosis. An in vitro evaluation of these drugs and fexinidazole, a new oral drug candidate, has been performed against T. lewisi in comparison with T. brucei gambiense. All have comparable activities against the two parasites. Suramin was not effective. In vivo, drugs were tested in rats immunosuppressed by cyclophosphamide. The best efficacy was obtained for fexinidazole, and pentamidine (15 mg/kg): rats were cured in 7 and 10 days respectively. Rats receiving nifurtimox-eflornithine combination therapy (NECT) or pentamidine (4 mg/kg) were cured after 28 days, while melarsoprol was weakly active. The identification of efficient drugs with reduced toxicity will help in the management of new cases of atypical trypanosomosis.

Atypical human infections by animal trypanosomes.

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

Spatial and temporal variations relevant to tsetse control in the Bipindi focus of southern Cameroon.

BACKGROUND: Human African Trypanosomiasis (HAT) remains a public health problem in many poor countries. Due to lack of financial resources in these countries, cost-effective strategies are needed for efficient control of this scourge, especially the tsetse vector. It was shown that perennial water sources maintain a favourable biotope for tsetse flies and thus the transmission dynamics of sleeping sickness. The present paper aimed at assessing the transmission dynamics of HAT in a forest environment where the hydrographic network is important. METHODS: Two entomological surveys were carried out in July 2009 and March 2010 in the Bipindi sleeping sickness focus of the South Region of Cameroon. Entomological and parasitological data were collected during both trapping periods (including the climate variations throughout a year) and compared to each other. The level of risk for transmission of the disease during each trapping period was also evaluated at the trap level and materialised on the map of the Bipindi focus. RESULTS: Glossina palpalis palpalis was the most prevalent tsetse fly species captured in this focus. The overall densities of tsetse flies as well as the risk for transmission of HAT in the Bipindi focus were significantly higher in July than in March. At the trap level, we observed that these parameters were almost constant, whatever the trapping period, when the biotope included perennial water sources. CONCLUSIONS: This study shows that the spatial distribution of traps, as well as the temporal climatic variations might influence entomological and parasitological parameters of HAT and that the presence of perennial water sources in biotopes would favour the development of tsetse flies and thus the transmission of sleeping sickness. These factors should, therefore, be taken into account in order to provide more efficient vector control.

The Trypanosoma brucei gambiense secretome impairs lipopolysaccharide-induced maturation, cytokine production, and allostimulatory capacity of dendritic cells.

Trypanosoma brucei gambiense, a parasitic protozoan belonging to kinetoplastids, is the main etiological agent of human African trypanosomiasis (HAT), or sleeping sickness. One major characteristic of this disease is the dysregulation of the host immune system. The present study demonstrates that the secretome (excreted-secreted proteins) of T. b. gambiense impairs the lipopolysaccharide (LPS)-induced maturation of murine dendritic cells (DCs). The upregulation of major histocompatibility complex class II, CD40, CD80, and CD86 molecules, as well as the secretion of cytokines such as tumor necrosis factor alpha, interleukin-10 (IL-10), and IL-6, which are normally released at high levels by LPS-stimulated DCs, is significantly reduced when these cells are cultured in the presence of the T. b. gambiense secretome. Moreover, the inhibition of DC maturation results in the loss of their allostimulatory capacity, leading to a dramatic decrease in Th1/Th2 cytokine production by cocultured lymphocytes. These results provide new insights into a novel efficient immunosuppressive mechanism directly involving the alteration of DC function which might be used by T. b. gambiense to interfere with the host immune responses in HAT and promote the infectious process.

Secreted proteases of Trypanosoma brucei gambiense: possible targets for sleeping sickness control?

Human African trypanosomiasis (HAT) is caused by trypanosomes of the species Trypanosoma brucei and belongs to the neglected tropical diseases. Presently, WHO has listed 36 countries as being endemic for sleeping sickness. No vaccine is available, and disease treatment is difficult and has life-threatening side effects. Therefore, there is a crucial need to search for new therapeutic targets against the parasite. Trypanosome excreted-secreted proteins could be promising targets, as the total secretome was shown to inhibit, in vitro, host dendritic cell maturation and their ability to induce lymphocytic allogenic responses. The secretome was found surprisingly rich in various proteins and unexpectedly rich in diverse peptidases, covering more than ten peptidase families or subfamilies. Given their abundance, one may speculate that they would play a genuine role not only in classical "housekeeping" tasks but also in pathogenesis. The paper reviews the deleterious role of proteases from trypanosomes, owing to their capacity to degrade host circulating or structural proteins, as well as proteic hormones, causing severe damage and preventing host immune response. In addition, proteases account for a number of drug targets, such drugs being used to treat severe diseases such AIDS. This review underlines the importance of secreted proteins and especially of secreted proteases as potential targets in HAT-fighting strategies. It points out the need to conduct further investigations on the specific role of each of these various proteases in order to identify those playing a central role in sleeping sickness and would be suitable for drug targeting.

Identification and genetic characterization of Trypanosoma congolense in domestic animals of Fontem in the South-West region of Cameroon.

To understand the circulation and the spread of Trypanosoma congolense genotypes in animals of Fontem in the southwest region of Cameroon, T. congolense forest and T. congolense savannah were investigated in 397 domestic animals in eight villages. Out of the 397 domestic animals, 86 (21.7%) were found infected by trypanosomes, using the capillary tube centrifugation test. The PCR with specific primers identified 163 (41.1%) and 81 (20.4%) animals infected by T. congolense forest and T. congolense savannah, respectively; showing for the first time the circulation of T. congolense savannah in the Fontem region. No infection with T. congolense savannah was found in pigs whereas goats and sheep were infected by T. congolense forest and/or T. congolense savannah. The prevalence of trypanosomes varied significantly amongst villages and animal species. The genotyping of T. congolense forest positive samples using microsatellites markers showed that multiple genotypes occurred in 27.2% (44/163) of animals sampled, whereas single genotypes were found in 73.8% (119/163) of samples. Some alleles were found in all animal species as well as in all villages and were responsible for major genotypes, whereas others (rare alleles) were identified only in some animals of few villages. These rare alleles were characteristic of specific genotypes, assimilated to minor genotypes which can be spread in the region through tsetse flies. The microsatellite markers show a low genetic variability and an absence of sub-structuration within T. congolense forest. The analysis of the microsatellite data revealed a predominant clonal reproduction within T. congolense forest. Pigs were the animal species with the highest number of different genotypes of T. congolense forest. They seem to play an important epidemiological role in the propagation and spread of different genotypes of T. congolense.

Chemoprophylaxis and treatment of African canine trypanosomosis in French military working dogs: a retrospective study.

African trypanosomosis is a major threat to livestock production in sub-Saharan Africa. Although the disease mainly concerns cattle, dogs can also be infected by Trypanosoma spp. transmitted by tsetse flies. Between 1997 and 2003, the parasite Trypanosoma congolense was identified in French military dogs sent to Africa. On infected dogs, the diagnosis was made during the mission or just after the return to France, depending on when the symptoms appeared. The high incidence and mortality rate among these dogs led veterinarians of the French Health Service to implement a systematic chemoprophylaxis beginning in 2004. Between 2004 and 2011, the chemoprophylaxis was carried out on more than 400 military dogs. The protocol of chemoprophylaxis relies on the use of isometamidium chloride (Trypamidium(®), Merial). The drug has been used successfully at the dosage of 1mg/kg body weight by deep intramuscular injection, every two or three months. In addition, dogs are given collars impregnated with deltamethrin (Scalibor(®), MSD Animal Health). Isometamidium chloride was also used successfully in the treatment of military dogs infected with T. congolense, with a full recovery and without any relapses.

Population dynamics of Glossina palpalis gambiensis symbionts, Sodalis glossinidius, and Wigglesworthia glossinidia, throughout host-fly development.

The tsetse fly (Diptera: Glossinidae), the vector of trypanosomes causing human and animal trypanosomiasis, harbors symbiotic microorganisms including the primary symbiont Wigglesworthia glossinidia, involved in the fly's nutrition and fertility, and the secondary symbiont Sodalis glossinidius, involved in the trypanosome establishment in the fly's midgut. Both symbionts are maternally transmitted to the intrauterine progeny through the fly's milk gland secretions. In this study, we investigated the population dynamics of these symbionts during fly development. Wigglesworthia and Sodalis densities were estimated using quantitative PCR performed on Glossina palpalis gambiensis at different developmental stages. The results showed that the density of the primary Wigglesworthia symbiont was higher than that of Sodalis for all host developmental stages. Sodalis densities remained constant in pupae, but increased significantly in adult flies. The opposite situation was observed for Wigglesworthia, whose density increased in pupae and remained constant during the female adult stage. Moreover, Wigglesworthia density increased significantly during the transition from the pupal to the teneral stage, while mating had a contradictory effect depending on the age of the fly. Finally, tsetse fly colonization by both symbionts appears as a continuous and adaptive process throughout the insect's development. Last, the study demonstrated both symbionts of G. p. gambiensis, the vector of the chronic form of human African trypanosomiasis, to be permanent inhabitants of the colony flies throughout their life span. This was expected for the primary symbiont, Wigglesworthia, but not necessarily for the secondary symbiont, S. glossinidius, whose permanent presence is not required for the fly's survival. This result is of importance as Sodalis could be involved in the tsetse fly vector competence and may constitute a target in the frame of sleeping sickness fighting strategies.

The bacterial flora of tsetse fly midgut and its effect on trypanosome transmission.

The tsetse fly, Glossina palpalis is a vector of the trypanosome that causes sleeping sickness in humans and nagana in cattle along with associated human health problems and massive economic losses. The insect is also known to carry a number of symbionts such as Sodalis, Wigglesworthia, Wolbachia whose effects on the physiology of the insect have been studied in depth. However, effects of other bacterial flora on the physiology of the host and vector competence have received little attention. Epidemiological studies on tsetse fly populations from different geographic sites revealed the presence of a variety of bacteria in the midgut. The most common of the flora belong to the genera Entrobacter (most common), Enterococcus, and Acinetobacter. It was a little surprising to find such diversity in the tsetse midgut since the insect is monophagous consuming vertebrate blood only. Diversity of bacteria is normally associated with polyphagous insects. In contrast to the symbionts, the role of resident midgut bacterial flora on the physiology of the fly and vector competence remains to be elucidated. With regard, Sodalis glossinidius, our data showed that flies harbouring this symbiont have three times greater probability of being infected by trypanosomes than flies without the symbiont. The data delineated in these studies under score the need to carry out detailed investigations on the role of resident bacteria on the physiology of the fly and vector competence.

APOL1 expression is induced by Trypanosoma brucei gambiense infection but is not associated with differential susceptibility to sleeping sickness.

Most African trypanosome species are sensitive to trypanolytic factors (TLFs) present in human serum. Trypanosome lysis was demonstrated to be associated with apolipoprotein L-I (APOL1). Trypanosoma brucei (T. b.) gambiense and Trypanosoma brucei rhodesiense, the two human infective trypanosome species, have both developed distinct resistance mechanisms to APOL1 mediated lysis. Whereas T. b. rhodesiense resistance is linked with the expression of the serum resistance associated (SRA) protein that interacts with APOL1 inside the parasite lysosome, inhibiting its lytic action; T. b. gambiense resistance is rather controlled by a reduced expression of the parasite HpHb receptor, limiting APOL1 absorption by trypanosomes. Based on this last observation we hypothesised that variation in the host APOL1 environment could significantly alter T. b. gambiense growth and thus resistance/susceptibility to sleeping sickness. To test this hypothesis, we have measured blood APOL1 relative expression in HAT patients, uninfected endemic controls and serologically positive subjects (SERO TL(+)) that are suspected to control infection to parasitological levels that are undetectable by the available test used in the field. All RNA samples were obtained from medical surveys led in the HAT mangrove foci of Coastal Guinea. Results indicate that APOL1 expression is a complex trait dependant on a variety of factors that need to be taken into account in the analysis. Nevertheless, multivariate analysis showed that APOL1 expression levels were significantly higher in both HAT and SERO TL(+) subject as compared to endemic controls (p=0.006). This result suggests that APOL1 expression is likely induced by T. b. gambiense, but is not related to resistance/susceptibility in its human host.

Genetic characterization of Trypanosoma brucei circulating in domestic animals of the Fontem sleeping sickness of Cameroon.

To improve our knowledge on the transmission dynamics of trypanosomes, Trypanosoma brucei was identified in domestic animals of the Fontem sleeping sickness focus of Cameroon and their genetic characterizations were performed using seven polymorphic microsatellite markers. About 397 domestic animals including 225 pigs, 87 goats, 65 sheep and 20 dogs were sampled. The card agglutination test for trypanosomiasis was positive for 254 (63.98%) animals while the parasitological examinations (thin blood film and capillary tube centrifugation) revealed 86 (21.66%) trypanosome infections. The PCR based method revealed 140 (35.26%) infections of trypanosomes of the subgenus Trypanozoon. The genetic characterization of these 140 positive samples revealed 89 different alleles: 82 in pigs, 72 in goat, 60 in sheep and 48 in dog. Whatever the microsatellite marker used, most of positive samples were amplified. However, the sensitivity (percentage of samples amplified for each marker) of these markers varies significantly between them (χ(2) = 120.32; P < 0.0001). This study showed a high level (80.00%) of mixed genotypes as well as a wide range of T. brucei genotypes circulating in domestic animals of the Fontem sleeping sickness focus of Cameroon. This indicates that several T. brucei genotypes can naturally be transmitted simultaneously to tsetse flies during a single blood meal.

Multiple infections of Trypanosoma brucei gambiense in blood and cerebrospinal fluid of human African trypanosomosis patients from Angola: consequences on clinical course and treatment outcome.

Human African trypanosomosis, caused by Trypanosoma brucei gambiense, is a chronic disease, although various clinical patterns have been observed, from asymptomatic to acute forms. Since 2001 in Angola, 80% of patients have been found to be in the meningoencephalitic stage of the disease. The existence of an acute form of the disease caused by virulent strains of trypanosomes was suspected. To test this hypothesis, four sensitive and polymorphic microsatellite markers were used to characterize the trypanosome DNA extracted from the blood and cerebrospinal fluid of 100 patients in the meningoencephalitic stage. Twenty-three patients were found with mixed T. b. gambiense genotypes in the blood and/or cerebrospinal fluid. The absence of association between the number of infecting genotypes, the presence of neurological signs and white blood cell counts in the cerebrospinal fluid, seems to indicate, at least in the context of the present study, the absence of virulent strains. However, out of five patients who died from encephalopathy syndrome during treatment with eflornithine, three harbored multiple infections.

Genetic diversity and population structure of the secondary symbiont of tsetse flies, Sodalis glossinidius, in sleeping sickness foci in Cameroon.

BACKGROUND: Previous studies have shown substantial differences in Sodalis glossinidius and trypanosome infection rates between Glossina palpalis palpalis populations from two Cameroonian foci of human African trypanosomiasis (HAT), Bipindi and Campo. We hypothesized that the geographical isolation of the two foci may have induced independent evolution in the two areas, resulting in the diversification of symbiont genotypes. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we investigated the symbiont genetic structure using the allelic size variation at four specific microsatellite loci. Classical analysis of molecular variance (AMOVA) and differentiation statistics revealed that most of the genetic diversity was observed among individuals within populations and frequent haplotypes were shared between populations. The structure of genetic diversity varied at different geographical scales, with almost no differentiation within the Campo HAT focus and a low but significant differentiation between the Campo and Bipindi HAT foci. CONCLUSIONS/SIGNIFICANCE: The data provided new information on the genetic diversity of the secondary symbiont population revealing mild structuring. Possible interactions between S. glossinidius subpopulations and Glossina species that could favor tsetse fly infections by a given trypanosome species should be further investigated.

Bacterial diversity associated with populations of Glossina spp. from Cameroon and distribution within the Campo sleeping sickness focus.

Tsetse flies were sampled in three villages of the Campo sleeping sickness focus in South Cameroon. The aim of this study was to investigate the flies' gut bacterial composition using culture-dependent techniques. Out of the 32 flies analyzed (27 Glossina palpalis palpalis, two Glossina pallicera, one Glossina nigrofusca, and two Glossina caliginea), 17 were shown to be inhabited by diverse bacteria belonging to the Proteobacteria, the Firmicutes, or the Bacteroidetes phyla. Phylogenetic analysis based on 16S rRNA gene sequences indicated the presence of 16 bacteria belonging to the genera Acinetobacter (4), Enterobacter (4), Enterococcus (2), Providencia (1), Sphingobacterium (1), Chryseobacterium (1), Lactococcus (1), Staphylococcus (1), and Pseudomonas (1). Using identical bacterial isolation and identification processes, the diversity of the inhabiting bacteria analyzed in tsetse flies sampled in Cameroon was much higher than the diversity found previously in flies collected in Angola. Furthermore, bacterial infection rates differed greatly between the flies from the three sampling areas (Akak, Campo Beach/Ipono, and Mabiogo). Last, the geographic distribution of the different bacteria was highly uneven; two of them identified as Sphingobacterium spp. and Chryseobacterium spp. were only found in Mabiogo. Among the bacteria identified, several are known for their capability to affect the survival of their insect hosts and/or insect vector competence. In some cases, bacteria belonging to a given genus were shown to cluster separately in phylogenetic trees; they could be novel species within their corresponding genus. Therefore, such investigations deserve to be pursued in expanded sampling areas within and outside Cameroon to provide greater insight into the diverse bacteria able to infect tsetse flies given the severe human and animal sickness they transmit.

Trypanosoma brucei s.l.: Microsatellite markers revealed high level of multiple genotypes in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus of Cameroon.

To identify Trypanosoma brucei genotypes which are potentially transmitted in a sleeping sickness focus, microsatellite markers were used to characterize T. brucei found in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus in Cameroon. For this study, two entomological surveys were performed during which 2685 tsetse flies were collected and 1596 (59.2%) were dissected. Microscopic examination revealed 1.19% (19/1596) mid-gut infections with trypanosomes; the PCR method identified 4.7% (75/1596) infections with T. brucei in the mid-guts. Of these 75 trypanosomes identified in the mid-guts, Trypanosoma brucei gambiense represented 0.81% (13/1596) of them, confirming the circulation of human infective parasite in the Fontem focus. Genetic characterization of the 75 T. brucei samples using five microsatellite markers revealed not only multiple T. brucei genotypes (47%), but also single genotypes (53%) in the mid-guts of the wild tsetse flies. These results show that there is a wide range of trypanosome genotypes circulating in the mid-guts of wild tsetse flies from the Fontem sleeping sickness focus. They open new avenues to undertake investigations on the maturation of multiple infections observed in the tsetse fly mid-guts. Such investigations may allow to understand how the multiple infections evolve from the tsetse flies mid-guts to the salivary glands and also to understand the consequence of these evolutions on the dynamic (which genotype is transmitted to mammals) of trypanosomes transmission.

Microsatellite genotyping reveals diversity within populations of Sodalis glossinidius, the secondary symbiont of tsetse flies.

The aim of this study was to develop a PCR-based microsatellite genotyping method for identifying genetic diversity in Sodalis glossinidius, a symbiont associated with tsetse fly infection by trypanosomes causing human and animal trypanosomiasis. Allelic polymorphism at three loci, investigated on 40 fly gut extracts, evidenced eight alleles and the existence of five genotypes. This novel approach was shown to be efficient and suitable for routine large-scale genotyping of S. glossinidius present in the biologically complex tsetse fly extracts; it could favor progress in the fields of diagnosis, epidemiology, population genetics, and fly/symbiont/trypanosome interactions.

Transcriptomics and proteomics in human African trypanosomiasis: current status and perspectives.

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.

Intertwining threshold settings, biological data and database knowledge to optimize the selection of differentially expressed genes from microarray.

BACKGROUND: Many tools used to analyze microarrays in different conditions have been described. However, the integration of deregulated genes within coherent metabolic pathways is lacking. Currently no objective selection criterion based on biological functions exists to determine a threshold demonstrating that a gene is indeed differentially expressed. METHODOLOGY/PRINCIPAL FINDINGS: To improve transcriptomic analysis of microarrays, we propose a new statistical approach that takes into account biological parameters. We present an iterative method to optimise the selection of differentially expressed genes in two experimental conditions. The stringency level of gene selection was associated simultaneously with the p-value of expression variation and the occurrence rate parameter associated with the percentage of donors whose transcriptomic profile is similar. Our method intertwines stringency level settings, biological data and a knowledge database to highlight molecular interactions using networks and pathways. Analysis performed during iterations helped us to select the optimal threshold required for the most pertinent selection of differentially expressed genes. CONCLUSIONS/SIGNIFICANCE: We have applied this approach to the well documented mechanism of human macrophage response to lipopolysaccharide stimulation. We thus verified that our method was able to determine with the highest degree of accuracy the best threshold for selecting genes that are truly differentially expressed.