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PURPOSE: The boom in Burkina Faso's artisanal gold mining since 2007 has attracted populations from Côte d'Ivoire and Guinea, which are the West African countries most affected by human African trypanosomiasis (HAT) and therefore increases its risk of re-emergence. Our aim was to update the HAT data in Burkina Faso in the risk of the re-emergence context with the advent of artisanal gold mining.METHODS: The study was carried out in the southwestern Burkina Faso where entomological surveys were conducted using biconical traps in March 2017. Follow by an active medical survey in April 2017, which was targeted the gold panners in 7 villages closer to artisanal gold sites, using CATT, mini-anion exchange centrifugation technique, trypanolysis test (TL) and ELISA test to measure human/tsetse contacts. The buffy coat technique and the TL were also applied in pigs to check their reservoir role of human trypanosomes.RESULTS: Our results have shown no case of HAT among 958 individuals tested and all the 50 pigs were also negative, but the level of antibodies against tsetse saliva evidenced by ELISA revealed low human/tsetse contact. Moreover, gold panners practise agriculture and breeding in an infected tsetse area, which are increased the risk.CONCLUSION: Our results illustrate that the risk of re-emergence is low. The passive surveillance system implemented in 2015 in southwestern Burkina Faso is needed to increase the sentinel sites to better cover this area by taking into account the gold mining. Finally, awareness-raising activities are needed among populations about HAT.

Background: Passive diagnosis of human African trypanosomiasis (HAT) at the health facility level is a major component of HAT control in Guinea. We examined which clinical signs and symptoms are associated with HAT, and assessed the performance of selected clinical presentations, of rapid diagnostic tests (RDT), and of laboratory tests on dried blood spots (DBS) for diagnosing HAT. Method: The study took place in 11 health facilities in Guinea, where 2345 clinical suspects were tested with RDTs HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT. Seropositives underwent parasitological examination to confirm HAT and their DBS were tested in indirect ELISA/T.b. gambiense, trypanolysis, LAMP and m18S qPCR. Multivariable regression analysis assessed association of clinical presentation with HAT. Sensitivity, specificity, positive and negative predictive values of key clinical presentations, of the RDTs and of the DBS tests for HAT diagnosis were determined. Results: The HAT prevalence, as confirmed parasitologically, was 2.0% (1.5-2.7%). Odds ratios (OR) for HAT were increased for participants with swollen lymph nodes (OR 96.7), important weight loss (OR 20.4), severe itching (OR 45.9) or motor disorders (OR 4.5). Presence of at least one of these clinical presentations was 75.6% (73.8-77.4%) specific and 97.9% (88.9-99.9%) sensitive for HAT. HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT were respectively 97.5% (96.8-98.1%), 99.4% (99.0-99.7%) and 97.9% (97.2-98.4%) specific, and 100% (92.5-100.0%), 59.6% (44.3-73.3%) and 93.8% (82.8-98.7%) sensitive for HAT. All DBS tests had specificities ≥ 92.9%. While LAMP and m18S qPCR sensitivities were below 50%, trypanolysis and ELISA/T.b. gambiense had sensitivities of 85.3% (68.9-95.0%) and 67.6% (49.5-82.6%). Conclusions: Presence of swollen lymph nodes, important weight loss, severe itching or motor disorders are simple but accurate clinical criteria for HAT referral in Guinea. Diagnostic performances of HAT Sero-K-Set and SD Bioline HAT are sufficient for referring positives to microscopy. Trypanolysis on DBS may discriminate HAT patients from false RDT positives. Trial registration: The trial was registered under NCT03356665 in clinicaltrials.gov (November 29, 2017, retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03356665).

The World Health Organisation has targeted the elimination of human African trypanosomiasis (HAT) as zero transmission by 2030. Continued surveillance needs to be in place for early detection of re-emergent cases. In this context, the performance of diagnostic tests and testing algorithms for detection of the re-emergence of Trypanosoma brucei gambiense HAT remains to be assessed. We carried out a door-to-door active medical survey for HAT in the historical focus of Batié, South-West Burkina Faso. Screening was done using three rapid diagnostic tests (RDTs). Two laboratory tests (ELISA/T. b. gambiense and immune trypanolysis) and parasitological examination were performed on RDT positives only. In total, 5883 participants were screened, among which 842 (14%) tested positive in at least one RDT. Blood from 519 RDT positives was examined microscopically but no trypanosomes were observed. The HAT Sero-K-Set test showed the lowest specificity of 89%, while the specificities of SD Bioline HAT and rHAT Sero-Strip were 92% and 99%, respectively. The specificity of ELISA/T. b. gambiense and trypanolysis was 99% (98-99%) and 100% (99-100%), respectively. Our results suggest that T. b. gambiense is no longer circulating in the study area and that zero transmission has probably been attained. While a least cost analysis is still required, our study showed that RDT preselection followed by trypanolysis may be a useful strategy for post-elimination surveillance in Burkina Faso.Suivi de l’élimination de la Trypanosomiase Humaine Africaine dans le foyer historique de Batié au sud-ouest du Burkina Faso.L’Organisation mondiale de la santé a ciblé l’élimination de la trypanosomiase humaine africaine (THA) comme transmission zéro d’ici 2030. Une surveillance continue doit être mise en place pour la détection précoce des cas réémergents. Dans ce contexte, la performance des tests de diagnostic et des algorithmes de test pour la détection de la réémergence de la THA de Trypanosoma brucei gambiense reste à évaluer. Nous avons réalisé une enquête médicale en porte-à-porte pour la THA dans le foyer historique de Batié, au sud-ouest du Burkina Faso. Le dépistage a été effectué à l’aide de trois tests de diagnostic rapide (TDR). Deux tests de laboratoire (ELISA/T. b. gambiense et trypanolyse immunitaire) et un examen parasitologique ont été effectués uniquement sur les TDR positifs. Au total, 5883 participants ont été dépistés, parmi lesquels 842 (14 %) ont été testés positifs dans au moins un TDR. Le sang de 519 TDR positifs a été examiné au microscope mais aucun trypanosome n’a été observé. Le test HAT Sero-K-Set a montré la spécificité la plus faible de 89 %, tandis que les spécificités de SD Bioline HAT et rHAT Sero-Strip étaient de 92 % et 99 %, respectivement. La spécificité d’ELISA/T. b. gambiense et de la trypanolyse étaient respectivement de 99 % (98–99 %) et 100 % (99–100 %). Nos résultats suggèrent que T. b. gambiense ne circule plus dans la zone d’étude et que la transmission zéro a probablement été atteinte. Bien qu’une analyse de moindre coût soit toujours nécessaire, notre étude a montré qu’une présélection par TDR suivie d’une trypanolyse peut être une stratégie utile pour la surveillance post-élimination au Burkina Faso.

After intensive control efforts, human African trypanosomiasis (HAT) was declared eliminated in Côte d'Ivoire as a public health problem in December 2020 and the current objective is to achieve the interruption of the transmission (zero cases). Reaching this objective could be hindered by the existence of an animal reservoir of Trypanosoma (T.) brucei (b.) gambiense. In the framework of a study led in 2013 to assess the role of domestic animals in the epidemiology of HAT in the two last active foci from Côte d'Ivoire (Bonon and Sinfra), plasmas were sampled from four species of domestic animals for parasitological (microscopic examination by the buffy coat technique (BCT)), serological (immune trypanolysis (TL)) and molecular (specific PCR: TBR for T. brucei s.l., TCF for T. congolense forest type, TVW for T. vivax and PCR for T. b. gambiense) testing. In order to improve the understanding of the involvement/role of these animals in the transmission of T. b. gambiense, we have quantified in this study the IgG response to whole saliva extracts of Glossina palpalis gambiensis in order to perform an association analysis between anti-saliva responses and the positivity of diagnostic tests. Cattle and pigs had significantly higher rates of anti-tsetse saliva responses compared to goats and sheep (p 0.01). In addition, the anti-tsetse saliva responses were strongly associated with the parasitology (BCT+), serology (TL+) and PCR (TBR+ and TCF+) results (p 0.001). These associations indicate a high level of contacts between the positive/infected animals and tsetse flies. Our findings suggest that protecting cattle and pigs against tsetse bites could have a significant impact in reducing transmission of both animal and human trypanosome species, and advocates for a "One health" approach to better control African trypanosomosis in Côte d'Ivoire.

Background Little is known about the diagnostic performance of rapid diagnostic tests (RDTs) for passive screening of human African trypanosomiasis (HAT) in Côte d’Ivoire. We determined HAT prevalence among clinical suspects, identified clinical symptoms and signs associated with HAT RDT positivity, and assessed the diagnostic tests’ specificity, positive predictive value and agreement. Methods Clinical suspects were screened with SD Bioline HAT, HAT Sero-K-Set and rHAT Sero-Strip. Seropositives were parasitologically examined, and their dried blood spots tested in trypanolysis, ELISA/Tbg, m18S-qPCR and LAMP. The HAT prevalence in the study population was calculated based on RDT positivity followed by parasitological confirmation. The association between clinical symptoms and signs and RDT positivity was determined using multivariable logistic regression. The tests’ Positive Predictive Value (PPV), specificity and agreement were determined. Results Over 29 months, 3433 clinical suspects were tested. The RDT positivity rate was 2.83%, HAT prevalence 0.06%. Individuals with sleep disturbances (p, Author summary As human African trypanosomiasis (HAT) or sleeping sickness is approaching elimination, case management is progressively transferred from specialized teams to front line health care centres. This approach raises practical questions. What clinical symptoms and signs should trigger HAT testing? What rapid diagnostic tests (RDT) are suitable for screening? Which unconfirmed serological suspects should be examined further? During this study conducted in Côte d’Ivoire, individuals with sleep disturbances, motor disorders, convulsions, severe weight loss, or psychiatric problems were more often positive in RDTs. These symptoms and signs should trigger referral for HAT screening. Our results confirm appropriateness of the existing HAT screening strategy with SD Bioline HAT or HAT Sero-K-Set having specificities of 97.8% and 98.9%. Subsequent tests on dried blood spots from RDT positives were 93.3% to 98.9% specific, and increased the positive predictive value from below 15% up to 67%. For selection of RDT seropositives for additional parasitological examinations, trypanolysis on dried blood spots is suitable, but could be replaced by ELISA, which can be performed locally. The optimal diagnostic test algorithm for Côte d’Ivoire, in terms of cost-effectiveness, remains to be determined.

The objective set by WHO to reach elimination of human African trypanosomiasis (HAT) as a public health problem by 2020 is being achieved. The next target is the interruption of gambiense-HAT transmission in humans by 2030. To monitor progress towards this target, in areas where specialized local HAT control capacities will disappear, is a major challenge. Test specimens should be easily collectable and safely transportable such as dried blood spots (DBS). Monitoring tests performed in regional reference centres should be reliable, cheap and allow analysis of large numbers of specimens in a high-throughput format. The aim of this study was to assess the analytical sensitivity of Loopamp, M18S quantitative real-time PCR (M18S qPCR) and TgsGP qPCR as molecular diagnostic tests for the presence of Trypanosoma brucei gambiense in DBS. The sensitivity of the Loopamp test, with a detection limit of 100 trypanosomes/mL, was in the range of parasitaemias commonly observed in HAT patients, while detection limits for M18S and TgsGP qPCR were respectively 1000 and 10,000 trypanosomes/mL. None of the tests was entirely suitable for high-throughput use and further development and implementation of sensitive high-throughput molecular tools for monitoring HAT elimination are needed.

Gambiense human African trypanosomiasis (g-HAT) is the chronic form of sleeping sickness caused by Trypanosoma brucei gambiense in West and Central Africa, while Trypanosoma brucei rhodesiense causes an acute form in eastern Africa. g-HAT is targeted for elimination as a public health problem by 2020 and 0 transmission by 2030 [1,2]. Control of g-HAT is largely based on identification and treatment of infected individuals, supplemented by control of the tsetse fly vectors [3]. There has been growing evidence that when both tsetse control and case identification activities are carried out simultaneously in the same geographies, elimination of the disease is accelerated [4–6]. Here, we describe how the Trypa-NO! Partnership is using novel and classical tools to drive g-HAT elimination in an integrated approach, progress made, lessons learnt, and future directions.

International audience; Control of human African trypanosomiasis (HAT) is highly dependent on the ability to detect and treat infected individuals. However, a number of individuals exposed to Trypanosoma brucei gambiense are able to control infection to undetectable levels in blood. They are long-term potential reservoirs and thus a threat for control strategies. Cytokine responses in whole blood stimulation assays were quantified in individuals with contrasting HAT status. Trypanosome-induced IFN-gamma production was only observed in "trypanotolerant" subjects suspected of harboring latent infections. This result contributes new insights into the immune responses associated with infection control and opens novel diagnosis perspectives regarding HAT elimination.

Burkina Faso belongs to a group of countries in which human African trypanosomiasis (HAT), caused by Trypanosoma brucei gambiense, is no longer considered to be a public health problem. Although no native cases have been detected since 1993, there is still the risk of HAT re-emergence due to significant population movements between Burkina Faso and active HAT foci in Côte d’Ivoire. Since 2014, Burkina Faso receives support from the WHO to implement a passive surveillance program. This resulted in the detection in 2015 of the first putative native HAT case since two decades. However, epidemiological entomological and molecular biology investigations have not been able to identify with certainty the origin of this infection or to confirm that it was due to T. b. gambiense. This case emphasises the need to strengthen passive surveillance of the disease for sustained elimination of HAT as a public health problem in Burkina Faso., Author summary In 2012, the roadmap for the Control of Neglected Tropical Diseases (NTD) of the World Health Organization (WHO) included human African trypanosomiasis (HAT) to be eliminated as a public health problem by 2020. To reach this ambitious objective in Burkina Faso, where the vector (and consequently a risk of HAT re-emergence) is still present, a passive surveillance system based on sentinel sites was established in the southwestern part of the country, considered to be the most at-risk area. The implementation of this system recently resulted in the diagnosis of the first putative native sleeping sickness case since two decades. Although the origin of this infection and how the patient was infected could not be identified, the detection of this native case confirms that HAT re-emergence in Burkina Faso is still a risk. This demonstrates the importance of implementing, maintaining and reinforcing passive surveillance programs in at-risk areas.

Trypanosoma brucei gambiense (T. b. gambiense) is the major causative agent of human African trypanosomiasis (HAT). A great variety of clinical outcomes have been observed in West African foci, probably due to complex host-parasite interactions. In order to separate the roles of parasite genetic diversity and host variability, we have chosen to precisely characterize the pathogenicity and virulence of T. b. gambiense field isolates in a mouse model. Thirteen T. b. gambiense strains were studied in experimental infections, with 20 Balb/C infected mice per isolate. Mice were monitored for 30 days, in which mortality, parasitemia, anemia, and weight were recorded. Mortality rate, prepatent period, and maximum parasitemia were estimated, and a survival analysis was performed to compare strain pathogenicity. Mixed models were used to assess parasitemia dynamics, weight, and changes in Packed Cell Volume (PCV). Finally, a multivariate analysis was performed to infer relationships between all variables. A large phenotypic diversity was observed. Pathogenicity was highly variable, ranging from strains that kill their host within 9 days to a non-pathogenic strain (no deaths during the experiment). Virulence was also variable, with maximum parasitemia values ranging from 42 million to 1 billion trypanosomes/ml. Reduced PCV and weight occurred in the first two weeks of the infection, with the exception of two strains. Finally, the global analysis highlighted three groups of strains: a first group with highly pathogenic strains showing an early mortality associated with a short prepatent period; a second group of highly virulent strains with intermediate pathogenicity; and a third group of isolates characterized by low pathogenicity and virulence patterns. Such biological differences could be related to the observed clinical diversity in HAT. A better understanding of the biological pathways underlying the observed phenotypic diversity could thus help to clarify the complex nature of the host-parasite interactions that determine the resistance/susceptibility status to T. brucei gambiense.

BackgroundHuman African trypanosomiasis, or sleeping sickness, remains a serious problem in tropical Africa. Timely diagnosis of this disease requires systematic population screening, particularly for Trypanosoma brucei gambiense, which has a long asymptomatic period.The lack of sensitivity and specificity of conventional diagnostic tests has led in recent years to the use of molecular tools. Amplification of parasite-specific DNA sequences significantly improved diagnosis of infection. However, these molecular tools still have some limitations especially in the case of low parasitaemia. Furthermore, research is still needed to make molecular detection a real control tool for the fight against sleeping sickness. The purpose of this study is to determine the threshold of sensitivity of real-time PCR using the 18S and TgsGp primers and of the LAMP technique, applied in the DiTECT-HAT project as molecular reference tests.MethodsWe used serial dilutions containing 0, 1, 10, 100, 103, 104, 105, 106 parasites per ml of blood. Samples were extracted, and DNA was amplified.ResultsThe analytical sensitivity of the 18S real-time PCR with the Taqman probe of the filter paper samples is 100 parasites/ml and that of the TgsGp real-time PCR with the Taqman probe of filter paper samples is 104 parasites/ml. For Lamp technique, the analytical sensitivity is 103 parasites/ml.ConclusionThis study shows that a ‘negative PCR’ would not mean ‘no parasite’. It suggests that DNA detection techniques should still be improved.

The World Health Organization (WHO) has set the goal of gambiense-Human African trypanosomiasis (HAT) elimination as a public health problem for 2020 and interruption of transmission in humans for 2030. In this context, it is crucial to monitor progress towards these targets using accurate tools to assess the level of transmission in a given area. The aim of this study was to investigate the relevance of the immune trypanolysis test (TL) as a population-based bioassay to evaluate Trypanosoma brucei gambiense transmission in various epidemiological contexts. Significant correlations were observed between HAT endemicity levels and the percentage of TL-positive individuals in the population. TL therefore appears to be a suitable population-based biomarker of the intensity of transmission. In addition to being used as a tool to assess the HAT status at an individual level, assessing the proportion of TL positive individuals in the population appears as a promising and easy alternative to monitor the elimination of gambiense HAT in a given area.Le test immunitaire de tryanolyse comme biomarqueur prometteur pour le suivi de l’élimination de la trypanosomose humaine africaine à gambiense.L’Organisation mondiale de la santé a fixé comme objectif l’élimination de la trypanosomose humaine africaine (THA) à gambiense en tant que problème de santé publique à l’horizon 2020 et l’interruption de la transmission humaine pour 2030. Dans ce contexte, il est crucial de suivre les progrès accomplis vers ces objectifs à l’aide d’outils précis pour évaluer le niveau de transmission dans une zone donnée. Le but de ce travail était d’étudier la pertinence du test immunitaire de trypanolyse (TL) en tant que marqueur biologique populationnel pour évaluer la transmission de Trypanosoma brucei gambiense dans divers contextes épidémiologiques. Des corrélations significatives ont été observées entre les niveaux d’endémicité de la THA et le pourcentage d’individus positifs à la TL dans la population. La TL apparaît donc comme un biomarqueur populationnel de l’intensité de la transmission. En plus d’être utilisé comme un outil pour évaluer le statut de la THA au niveau individuel, l’évaluation de la proportion d’individus positifs à la TL dans la population apparaît comme une alternative simple et prometteuse pour surveiller l’élimination de la THA à gambiense dans une zone donnée.

To assess the efficacy of treatment for human African trypanosomiasis, accurate tests that can discriminate relapse from cure are needed. We report the first data that the spliced leader (SL) RNA is a more specific marker for cure of human African trypanosomiasis than parasite DNA. In blood samples obtained from 61 patients in whom human African trypanosomiasis was cured, SL RNA detection had specificities of 98.4%-100%, while DNA detection had a specificity of only 77%. Data from our proof-of-concept study show that SL RNA detection has high potential as a test of cure.

At a time when human African trypanosomiasis (HAT) elimination again seems a reachable goal in many parts of sub-Saharan Africa, it is becoming increasingly important to characterise the factors involved in disease resurgence or maintenance to develop sustainable control strategies. In this study conducted in the Forecariah mangrove focus in Guinea, HAT patients and serological suspects (SERO) were identified through mass screening of the population with the Card Agglutination Test for Trypanosomiasis (CATT) and were followed up for up to 2 years. Analysis of the samples collected during the follow-up of HAT patients and SERO was performed with PCR (TBR1/TBR2) and the trypanolysis serological test (TL) in order to clarify the role played by these individuals in the epidemiology of HAT. PCR positivity was higher in TL + than in SERO TL − (50% vs. 18%, respectively). Whereas CATT plasma titres decreased both in treated HAT patients and SERO TL − , SERO TL + maintained high CATT titres. Four out of 17 SERO TL + developed HAT during the study. These results strongly suggest that SERO TL + individuals are asymptomatic carriers. In the context where disease prevalence is sufficiently low, treating SERO TL + individual may thus be of crucial importance in order to cut transmission.

Summary Objectives To evaluate a modification of the mini anion exchange centrifugation test (mAECT) for the diagnosis of Trypanosoma brucei (T.b.) gambiense human African trypanosomiasis (HAT). To increase its sensitivity, this test uses 350 μl of buffy coat withdrawn from 5 ml of blood instead of blood. Methods The new protocol was first tested experimentally on serial dilution of trypanosomes and was then further evaluated under field conditions on 57 patients with HAT diagnosed during a medical survey in Guinea. Results Experimentally, the use of buffy coats improved mAECT sensitivity at least five fold and enabled to consistently detect parasites in blood at a concentration of 10 trypanosomes/ml. During the field evaluation, more patients tested positive by mAECT-bc (96.5%) than by mAECT-blood (78.9%, χ2 = 6.93, P = 0.008) and lymph juice examination (77.2%, χ2 = 7.67, P = 0.005). Furthermore, the number of parasites per collectors was significantly higher (7.2 vs. 2.6, P = 0.001) when buffy coats were used instead of blood. Conclusion The use of the mAECT-bc protocol enabled a significant improvement of HAT parasitological diagnosis in Guinea, without any additional costs. It would deserve to be tested in other T.b. gambiense endemic areas. Diagnostic de maladie du sommeil: l’utilisation de ‘Buffy Coats’ ameliore la sensibilite du test de la mini centrifugation aechangeuse d’anion Objectifs: Evaluer une modification du test de la mini centrifugation aechangeuse d’anion (mAECT) pour le diagnostic de la trypanosomiase humaine africaine (THA) aTrypanosoma brucei gambiense. Afin d’augmenter sa sensibilite, ce test utilise 350 μl de couenne leuco-plaquettaire (buffy-coat), prelevee sur 5 ml de sang au lieu de sang total. Methodes: Le nouveau protocole a ete teste sur des dilutions en serie de trypanosomes et a ensuite eteevalue dans des conditions de terrain sur 57 patients THA diagnostiques lors d’une enquete medicale en Guinee. Resultats: Experimentalement, l’utilisation de ‘buffy coats’ a ameliore la sensibilite du test mAECT d’au moins cinq fois et a permis de detecter de facon consistante des parasites dans le sang a une concentration de 10 trypanosomes/ml. Au cours de l’evaluation sur le terrain, plus de patients ont ete testes positifs par mAECT-bc (96,5%) que par mAECT-sang (78,9%; Chi2 = 6,93; p = 0,008) et l’examen du liquide lymphatique (77,2%; Chi2 = 7,67; p = 0,005). Le nombre de parasites par collecteur etait significativement plus eleve (7,2 vs 2,6; p = 0,001) lorsque le ‘buffy coats’ plutot que du sang etaient utilise. Conclusion: L’utilisation du protocole mAECT-bc a permis une amelioration significative du diagnostic parasitologique de la THA en Guinee sans frais supplementaires. Il merite d’etre teste sur Tb gambiense dans d’autres regions endemiques. Diagnostico de la enfermedad del sueno: el uso de la Capa Leucocitaria mejora la sensibilidad de la tecnica de filtracion en mini-columna de intercambio anionico (mAECT) Objetivos: Evaluar una modificacion de la tecnica de filtracion en mini-columna de intercambio anionico (mAECT) para el diagnostico de la tripanosomiasis humana Africana (THA) por Trypanosoma brucei gambiense. Con el fin de aumentar su sensibilidad, se utilizaron 350 μl de la capa leucocitaria (mAECT-cl) tomada a partir de 5 ml de sangre, en vez de sangre (mAECT-s). Metodos: El nuevo protocolo se probo primero experimentalmente en una dilucion seriada de tripanosomas y despues fue evaluado bajo condiciones de campo en 57 pacientes con THA, diagnosticados durante un estudio medico en Guinea. Resultados: Experimentalmente, el uso de la capa leucocitaria mejoro la sensibilidad del mAECT en al menos cinco veces, y permitio detectar consistentemente parasitos en sangre a una concentracion 10 tripanosomas/ml. Durante la evaluacion de campo, mas pacientes dieron positivo por mAECT-cl (96.5%) que por mAECT-s (78.9%, Chi2=6.93, p=0.008) y examen de linfa (77.2%, Chi2=7.67, p=0.005). El numero de parasitos por colector fue significativamente mayor (7.2 vs 2.6, p=0.001) cuando se recolecto la capa leucocitaria en vez de utilizar la sangre. Conclusion: El uso del protocolo mAECT-cl permitio mejoras significativas en el diagnostico parasitologico de THA en Guinea, sin costes adicionales. Vale la pena probarlo en otras areas endemicas para T.b. gambiense.

Trypanosomatidae are a dangerous family of Euglenobionta parasites that threaten the health and economy of millions of people around the world. More precisely describing the population biology and reproductive mode of such pests is not only a matter of pure science, but can also be useful for understanding parasite adaptation, as well as how parasitism, specialization (parasite specificity), and complex life cycles evolve over time. Studying this parasite’s reproductive strategies and population structure can also contribute key information to the understanding of the epidemiology of associated diseases; it can also provide clues for elaborating control programs and predicting the probability of success for control campaigns (such as vaccines and drug therapies), along with emergence or re-emergence risks. Population genetics tools, if appropriately used, can provide precise and useful information in these investigations. In this paper, we revisit recent data collected during population genetics surveys of different Trypanosoma species in sub-Saharan Africa. Reproductive modes and population structure depend not only on the taxon but also on the geographical location and data quality (absence or presence of DNA amplification failures). We conclude on issues regarding future directions of research, in particular vis-à-vis genotyping and sampling strategies, which are still relevant yet, too often, neglected issues.

Evolutionary theory predicts that the lack of recombination and chromosomal re-assortment in strictly asexual organisms results in homologous chromosomes irreversibly accumulating mutations and thus evolving independently of each other, a phenomenon termed the Meselson effect. We apply a population genomics approach to examine this effect in an important human pathogen, Trypanosoma brucei gambiense. We determine that T.b. gambiense is evolving strictly asexually and is derived from a single progenitor, which emerged within the last 10,000 years. We demonstrate the Meselson effect for the first time at the genome-wide level in any organism and show large regions of loss of heterozygosity, which we hypothesise to be a short-term compensatory mechanism for counteracting deleterious mutations. Our study sheds new light on the genomic and evolutionary consequences of strict asexuality, which this pathogen uses as it exploits a new biological niche, the human population. DOI: http://dx.doi.org/10.7554/eLife.11473.001, eLife digest An organism’s genetic ‘blueprint’ is encoded in DNA packaged within structures called chromosomes. Most organisms have two copies of each chromosome and, through sexual reproduction, the DNA within a pair of chromosomes can recombine randomly in a process that could be likened to shuffling a deck of cards. This process generates genetic diversity and means that any undesirable combinations of genes or mutations can be eliminated from the population by natural selection. While these activities are thought to promote the long-term survival of a species, some organisms appear not to have sex at all. Evolutionary theory predicts that ‘asexual’ organisms should face extinction in the long-term and that a lack of sexual recombination should leave a characteristic genetic ‘signature’ in their DNA. The theory also predicts that pairs of chromosomes will evolve independently, a phenomenon that is termed the ‘Meselson effect’. However, while it was first predicted almost twenty years ago, evidence for this signature has been elusive. Now, Weir et al. have used the asexual parasite (Trypanosoma brucei gambiense), which causes African sleeping sickness in humans, to search for signs of the Meselson effect. Sequencing the whole genome of a large number of parasites revealed that the population of this parasite arose from a single individual within the last 10,000 years. Over this time, mutations have built up and the lack of sexual recombination means that the two chromosomes in each pair have evolved independently of the other. These results provide the first demonstration of the Meselson effect at a genome-wide level in any organism. Weir et al. also uncovered evidence that this parasite uses a mechanism called “gene conversion” to compensate for its lack of sex. This mechanism essentially repairs the inferior, or mutated, copy of a gene on a chromosome by ‘copying and pasting’ the superior copy from the chromosome’s partner. The findings also suggest that gene conversion can only go some way to compensating for a lack of sex. A future challenge will be to investigate how effective this mechanism can be in the long term and to predict whether the parasite will ultimately become extinct. DOI: http://dx.doi.org/10.7554/eLife.11473.002

To assess the efficacy of treatment for human African trypanosomiasis, accurate tests that can discriminate relapse from cure are needed. We report the first data that the spliced leader (SL) RNA is a more specific marker for cure of human African trypanosomiasis than parasite DNA. In blood samples obtained from 61 patients in whom human African trypanosomiasis was cured, SL RNA detection had specificities of 98.4%-100%, while DNA detection had a specificity of only 77%. Data from our proof-of-concept study show that SL RNA detection has high potential as a test of cure.

Human African trypanosomiasis (HAT) in the Democratic Republic of the Congo (DRC) is caused by the protozoan Trypanosoma brucei gambiense. Until recently, all patients in the second or neurological stage of the disease were treated with melarsoprol. At the end of the past and the beginning of the present century, alarmingly high relapse rates in patients treated with melarsoprol were reported in isolated HAT foci. In the Mbuji-Mayi focus of DRC, a particular mutation that confers cross resistance for pentamidine and melarsoprol was recently found for all strains studied. Nevertheless, treatment successfully cured a significant proportion of patients. To check for the existence of other possible genetic factors of the parasites, we genotyped trypanosomes isolated from patients before and after treatment (relapsing patients) with eight microsatellite markers. We found no evidence of any genetic correlation between parasite genotype and treatment outcome and we concluded that relapse or cure probably depend more on patients' factors such as disease progression, nutritional or immunological status or co-infections with other pathogens. The existence of a melarsoprol and pentamidine resistance associated mutation at such high rates highlights an increasing problem, even for other drugs, especially those using the same transporters as melarsoprol and pentamidine.

Microsatellite genotyping of Trypanosoma brucei gambiense, the causative agent of human African trypanosomiasis or sleeping sickness, and population genetics tools, are useful for inferring population parameters such as population size and dispersal. Amplifying parasite DNA directly from body fluids (i.e., blood, lymph or cerebrospinal fluid) allows avoiding costly and tedious isolation phases. It is however associated to increased frequencies of amplification failures (allelic dropouts and/or null alleles) at some loci. In this paper, we present a study focused on three T. brucei gambiense microsatellite loci suspected to present amplification problems when amplified from body fluids sampled in Guinean sleeping sickness foci. We checked for the real nature of blank and apparent homozygous genotypes of parasite DNA directly amplified from body fluids and tested the effect of three different DNA quantities of trypanosomes. Our results show that some initially blank and homozygous genotypes happen to be actual heterozygous genotypes. In Guinea, lymph from the cervical nymph nodes, known to contain the highest concentrations of parasites, appeared to provide the best amplification results. Simply repeating the PCR may be enough to retrieve the correct genotype, but we also show that increasing initial DNA content provides better results while undertaking first amplification. We finally propose an optimal protocol for amplifying trypanosome’s DNA directly from body fluids that should be adapted to local characteristics and/or constraints.

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.

The final outcome of infection by Trypanosoma brucei gambiense, the main agent of sleeping sickness, has always been considered as invariably fatal. While scarce and old reports have mentioned cases of self-cure in untreated patients, these studies suffered from the lack of accurate diagnostic tools available at that time. Here, using the most specific and sensitive tools available to date, we report on a long-term follow-up (15 years) of a cohort of 50 human African trypanosomiasis (HAT) patients from the Ivory Coast among whom 11 refused treatment after their initial diagnosis. In 10 out of 11 subjects who continued to refuse treatment despite repeated visits, parasite clearance was observed using both microscopy and polymerase chain reaction (PCR). Most of these subjects (7/10) also displayed decreasing serological responses, becoming progressively negative to trypanosome variable antigens (LiTat 1.3, 1.5 and 1.6). Hence, in addition to the “classic” lethal outcome of HAT, we show that alternative natural progressions of HAT may occur: progression to an apparently aparasitaemic and asymptomatic infection associated with strong long-lasting serological responses and progression to an apparently spontaneous resolution of infection (with negative results in parasitological tests and PCR) associated with a progressive drop in antibody titres as observed in treated cases. While this study does not precisely estimate the frequency of the alternative courses for this infection, it is noteworthy that in the field national control programs encounter a significant proportion of subjects displaying positive serologic test results but negative results in parasitological testing. These findings demonstrate that a number of these subjects display such infection courses. From our point of view, recognising that trypanotolerance exists in humans, as is now widely accepted for animals, is a major step forward for future research in the field of HAT., Author Summary The existence of a diversity of infection outcomes – ranging from self-cure to asymptomatic, severe or fatal cases – is now widely recognised for most parasitic and infectious diseases. The dogma concerning sleeping sickness, however, is still that infection is 100% fatal. Here we describe a 15-year follow-up of patients diagnosed with human African trypanosomiasis (HAT) in the Ivory Coast but who refused treatment. Our results, based on clinical, serological, molecular, and parasitological investigations, combining diagnostic tools for the field and highly specific and sensitive laboratory tests, constitute the most comprehensive study on the natural evolution of Trypanosoma brucei gambiense infection in its human host. At least two alternative natural progressions of HAT to the “classic” fatal disease were identified: a progression to an apparently aparasitaemic and asymptomatic infection and a progression to an apparently spontaneous resolution of infection. We believe that recognising that trypanotolerance exists in humans is a major step forward for future research aimed at identifying human-specific defence and immune mechanisms involved in the control of T.b. gambiense infection and thus new candidate therapeutic or prophylactic targets.

Thanks to its sensitivity and its ease of use in the field, the card agglutination test for trypanosomiasis (CATT) is widely used for serological screening of Trypanosoma brucei gambiense human African trypanosomiasis (HAT). Positive subjects are then examined by microscopy to confirm the disease. However, the CATT exhibits false-positive results raising the question of whether CATT-positive subjects who are not confirmed by microscopic detection of trypanosomes (SERO) are truly exposed to T.b. gambiense infection. For this purpose, we applied microsatellite genotyping on DNA extracted from blood of both HAT confirmed patients and SERO subjects in Guinea and Côte d'Ivoire since microsatellite genotyping has proved useful for the study of T.b. gambiense genetic diversity. Problems of amplification failures raise the question of the sensitivity of microsatellite markers when applied on biological samples especially from SERO subjects for who low blood parasitaemia are suspected. Nevertheless, we have shown that the trypanosomes from SERO individuals that have been genotyped belong to T.b. gambiense group 1 and were identical to those found in HAT patients. These results constitute the first evidences that at least some SERO are indeed infected by T.b. gambiense group 1 and that they may constitute a human reservoir of parasite in HAT foci. Whether these individuals should undergo treatment remains an open question as long as their role in HAT transmission is unknown. Our results strongly recommend the follow-up of such subjects to improve control strategies.

Human African trypanosomiasis (HAT) or sleeping sickness is a major public health problem in sub-Saharan Africa and is due to the kinetoplastid parasite Trypanosoma brucei gambiense in West and Central Africa. The exact role of multiple infections, the basis of clinical diversity observed in patients and the determinism that leads trypanosomes into different body fluids of the host remain opened questions to date. In this paper we investigate, in three Guinean foci, whether strains found in blood, lymph or cerebrospinal fluid (CSF) or in patients at different phase of HAT (phase 1, early phase 2 and late phase 2) are representative of the focus they belong to. Amplifications of parasites directly from body fluids led to substantial amounts of allelic drop outs, especially so for blood and CSF samples, which required data recoding of all homozygous sites into missing data. While controlling for geography, date of sampling and patient's phase of the disease, we found no effect of body fluids in the genetic structure of T. b. gambiense despite the presence of mixed infections. On the contrary, we found that the strains found in patients in different phase of the disease differed genetically, with early phase patients being more likely to be infected with more recent strains than patients at a more advanced phase of the disease. Thus, the combination of date of sampling and patient's status represents a parameter to be controlled for in population genetic structure analyses. Additional studies will also be required to explore further the phenomenon of mixed infections and its consequences.

Background Because of its high sensitivity and its ease of use in the field, the card agglutination test for trypanosomiasis (CATT) is widely used for mass screening of sleeping sickness. However, the CATT exhibits false-positive results (i) raising the question of whether CATT-positive subjects who are negative in parasitology are truly exposed to infection and (ii) making it difficult to evaluate whether Trypanosoma brucei (T.b.) gambiense is still circulating in areas of low endemicity. The objective of this study was to assess the value of the immune trypanolysis test (TL) in characterising the HAT status of CATT-positive subjects and to monitor HAT elimination in West Africa. Methodology/Principal Findings TL was performed on plasma collected from CATT-positive persons identified within medical surveys in several West African HAT foci in Guinea, Côte d'Ivoire and Burkina Faso with diverse epidemiological statuses (active, latent, or historical). All HAT cases were TL+. All subjects living in a nonendemic area were TL−. CATT prevalence was not correlated with HAT prevalence in the study areas, whereas a significant correlation was found using TL. Conclusion and Significance TL appears to be a marker for contact with T.b. gambiense. TL can be a tool (i) at an individual level to identify nonparasitologically confirmed CATT-positive subjects as well as those who had contact with T.b. gambiense and should be followed up, (ii) at a population level to identify priority areas for intervention, and (iii) in the context of HAT elimination to identify areas free of HAT., Author Summary Human African trypanosomiasis (HAT) due to Trypanosoma brucei (T.b.) gambiense is usually diagnosed using two sequential steps: first the card agglutination test for trypanosomiasis (CATT) used for serological screening, followed by parasitological methods to confirm the disease. Currently, CATT will continue to be used as a test for mass screening because of its simplicity and high sensitivity; however, its performance as a tool of surveillance in areas where prevalence is low is poor because of its limited specificity. Hence in the context of HAT elimination, there is a crucial need for a better marker of contact with T.b. gambiense in humans. We evaluated here an existing highly specific serological tool, the trypanolysis test (TL). We evaluated TL in active, latent and historical HAT foci in Guinea, Côte d'Ivoire and Burkina Faso. We found that TL was a marker for exposure to T.b. gambiense. We propose that TL should be used as a surveillance tool to monitor HAT elimination.

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by two subspecies of the protozoan parasite Trypanosoma brucei (T.b.) and transmitted through tsetse flies (Glossina sp.). T.b. gambiense occurs from West to Central sub-Sahara Africa, while T.b. rhodesiense is endemic in East sub-Sahara Africa. Other closely related taxa (T.b. brucei, T. evansi, T. equiperdum, T. congolense, T. vivax) cause animal African trypanosomiases in domestic animals like cattle, horse, camel, goat, sheep, and buffalo in Africa, Latin America, Europe, and Asia [1]. Approximately 10,000 sleeping sickness patients are diagnosed and treated each year [2]. This is only a fraction of those carrying this lethal infection since (i) the population at risk lives mainly in remote areas outside the action radius of health centres or mobile teams, and (ii) the diagnostic tests suffer from limited sensitivity [3],[4]. There is a consensus to (i) only treat patients with confirmed diagnosis, i.e., in whom the parasite has been demonstrated, except in particular situations, and (ii) to examine the cerebrospinal fluid (CSF) for white blood cell counts and the presence of trypanosomes in order to decide which drugs to administer. To determine whether a patient is cured, examination of CSF is repeated each semester for up to 2 years [4]. In T.b. gambiense sleeping sickness, general low parasite loads necessitate the use of concentration techniques to reveal infection. Capillary tube centrifugation (CTC, WOO) [5], quantitative buffy coat (QBC) [6], and mini anion exchange centrifugation technique (mAECT) [7] are applied on blood. mAECT is the most sensitive method for trypanosome detection in blood and is based on a purification technique first described by Lanham et al. and later adapted for diagnosis of sleeping sickness and of animal infections with T. brucei and T. evansi [7]–[10]. In mAECT, trypanosomes are separated from 350 µl of blood by anion exchange chromatography on diethylaminoethyl cellulose (DEAE). Eluted trypanosomes are then concentrated by low speed centrifugation followed by direct microscopic examination of the sediment in a transparent collector tube. The large volume of blood examined allows detection of fewer than 50 trypanosomes/ml. For sensitive detection of trypanosomes in CSF, single and double centrifugation (DC) and modified single centrifugation (MSC) are applied, with MSC being easier to perform and at least as sensitive as DC [11]. For several years, mAECT was produced at the Projet de Recherches Cliniques sur la Trypanosomiase (PRCT) in Daloa, Cote d'Ivoire, with financial support from the World Health Organization (WHO), and then for some time at Institut Pierre Richet (IPR) in Bouake, Cote d'Ivoire. The PRCT model is still produced on demand in this country (B. Miezan, personal commmunication). Several attempts to establish a production unit elsewhere were discontinued. At the request of the Belgian Technical Cooperation (BTC) for the Programme National de Lutte contre la Trypanosomiase Humaine Africaine (PNLTHA) in the Democratic Republic of the Congo (DRC), the Institute of Tropical Medicine (ITM, Belgium) and the Institut National de Recherche Biomedicale (INRB, DRC) developed a modified version of the mAECT. With financial support from the Belgian Directorate General for Development Cooperation and WHO, production at INRB started in 2003. At the end of 2005, production stopped since raw material for the 9-mm diameter glass collector tube was not commercially available anymore. From 2006 on, with support from the Foundation for Innovative New Diagnostics (FIND), the column rack, microscope viewing chamber, and collector tube were redesigned. Important advantages of the new collector tube and the viewing chamber are their robustness and the fact that microscopic examination of the sediment is possible without mounting the tip of the collector tube under water (Figures 1–​3).3). Improvements were made to the packing trays (Figure S1), filter material (Figure S2), sterilisation procedure, and instruction leaflets. In addition, the production infrastructure at INRB and the quality control system were upgraded. Actually, quality control is performed internally at INRB and externally at ITM before a batch is released. Detailed production standard operating procedures were written and are collated in an mAECT handbook that can be found on the FIND Web site [12]. Figure 1 Column rack with mounted columns and trypanosomes being eluted from the blood in the collector tubes. Figure 2 Collector tube mounted in the mAECT viewing chamber under the microscope. Figure 3 Detail of collector tube tip. In 2007, a comparison between the first model of mAECT produced at INRB and the PRCT model was carried out at the Centre International de Recherche-Developpement sur l'Elevage en zone Sub-humide (CIRDES) in Bobo-Dioulasso, Burkina Faso, on 198 columns of each model. Columns received 350 µl of human blood containing on average 35 T.b. gambiense trypanosomes (100 trypanosomes per ml of blood). The time to run a column was recorded and trypanosomes in each collector tube were counted by two independent observers. Averages were compared using paired t-test and showed a significantly shorter run time for the INRB than the PRCT model (29 and 42 min, respectively, p

Background: Pathogens and their vectors are organisms whose ecology is often only accessible through population genetics tools based on spatio-temporal variability of molecular markers. However, molecular tools may present technical difficulties due to the masking of some alleles (allelic dropouts and/or null alleles), which tends to bias the estimation of heterozygosity and thus the inferences concerning the breeding system of the organism under study. This is especially critical in clonal organisms in which deviation from panmixia, as measured by Wright's FIS, can, in principle, be used to infer both the extent of clonality and structure in a given population. In particular, null alleles and allelic dropouts are locus specific and likely produce high variance of Wright's FISacross loci, as rare sex is expected to do. In this paper we propose a tool enabling to discriminate between consequences of these technical problems and those of rare sex. Methods. We have performed various simulations of clonal and partially clonal populations. We introduce allelic dropouts and null alleles in clonal data sets and compare the results with those that exhibit increasing rates of sexual recombination. We use the narrow relationship that links Wright's FISto genetic diversity in purely clonal populations as assessment criterion, since this relationship disappears faster with sexual recombination than with amplification problems of certain alleles. Results: We show that the relevance of our criterion for detecting poorly amplified alleles depends partly on the population structure, the level of homoplasy and/or mutation rate. However, the interpretation of data becomes difficult when the number of poorly amplified alleles is above 50%. The application of this method to reinterpret published data sets of pathogenic clonal microbes (yeast and trypanosomes) confirms its usefulness and allows refining previous estimates concerning important pathogenic agents. Conclusion: Our criterion of superimposing between the FISexpected under clonality and the observed FIS, is effective when amplification difficulties occur in low to moderate frequencies (20-30%). © 2014 Séré et al.; licensee BioMed Central Ltd.