Extravascular Dermal Trypanosomes in Suspected and Confirmed Cases of gambiense Human African Trypanosomiasis.

BACKGROUND: The diagnosis of gambiense human African trypanosomiasis (gHAT) typically involves 2 steps: a serological screen, followed by the detection of living trypanosome parasites in the blood or lymph node aspirate. Live parasites can, however, remain undetected in some seropositive individuals, who, we hypothesize, are infected with Trypanosoma brucei gambiense parasites in their extravascular dermis. METHODS: To test this hypothesis, we conducted a prospective observational cohort study in the gHAT focus of Forecariah, Republic of Guinea. Of the 5417 subjects serologically screened for gHAT, 66 were enrolled into our study and underwent a dermatological examination. At enrollment, 11 seronegative, 8 unconfirmed seropositive, and 18 confirmed seropositive individuals had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immunohistological methods. RESULTS: In seropositive individuals, dermatological symptoms were significantly more frequent, relative to seronegative controls. T.b. gambiense parasites were present in the blood of all confirmed cases (n = 18) but not in unconfirmed seropositive individuals (n = 8). However, T. brucei parasites were detected in the extravascular dermis of all unconfirmed seropositive individuals and all confirmed cases. Skin biopsies of all treated cases and most seropositive untreated individuals progressively became negative for trypanosomes 6 and 20 months later. CONCLUSIONS: Our results highlight the skin as a potential reservoir for African trypanosomes, with implications for our understanding of this disease's epidemiology in the context of its planned elimination and underlining the skin as a novel target for gHAT diagnostics.

Analytical sensitivity of loopamp and quantitative real-time PCR on dried blood spots and their potential role in monitoring human African trypanosomiasis elimination.

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.

Human Leukocyte Antigen-G: A Promising Prognostic Marker of Disease Progression to Improve the Control of Human African Trypanosomiasis.

BACKGROUND: Human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense can be diagnosed in the early hemolymphatic stage (stage 1 [S1]) or meningoencephalitic stage (stage 2 [S2]). Importantly, individuals harbouring high and specific antibody responses to Tbg antigens but negative parasitology are also diagnosed in the field (seropositive [SERO]). Whereas some develop the disease in the months following their initial diagnosis (SERO/HAT), others remain parasitologically negative for long periods (SERO) and are apparently able to control infection. Human leucocyte antigen (HLA)-G, an immunosuppressive molecule, could play a critical role in this variability of progression between infection and disease. METHODS: Soluble HLA-G (sHLA-G) was measured in plasma for patients in the SERO (n = 65), SERO/HAT (n = 14), or HAT (n = 268) group and in cerebrospinal fluid for patients in S1 (n = 55), early S2 (n = 93), or late S2 (n = 110). Associations between these different statuses and the soluble level or genetic polymorphisms of HLA-G were explored. RESULTS: Plasma sHLA-G levels were significantly higher in HAT (P = 6 × 10-7) and SERO/HAT (P = .007) than SERO patients. No difference was observed between the SERO/HAT and HAT groups. Within the HAT group, specific haplotypes (HG010102 and HG0103) displayed increased frequencies in S1 (P = .013) and late S2 (P = .036), respectively. CONCLUSIONS: These results strongly suggest the involvement of HLA-G in HAT disease progression. Importantly, high plasma sHLA-G levels in SERO patients could be predictive of subsequent disease development and could represent a serological marker to help guide therapeutic decision making. Further studies are necessary to assess the predictive nature of HLA-G and to estimate both sensitivity and specificity.

Unravelling human trypanotolerance: IL8 is associated with infection control whereas IL10 and TNFα are associated with subsequent disease development.

In West Africa, Trypanosoma brucei gambiense, causing human African trypanosomiasis (HAT), is associated with a great diversity of infection outcomes. In addition to patients who can be diagnosed in the early hemolymphatic phase (stage 1) or meningoencephalitic phase (stage 2), a number of individuals can mount long-lasting specific serological responses while the results of microscopic investigations are negative (SERO TL+). Evidence is now increasing to indicate that these are asymptomatic subjects with low-grade parasitemia. The goal of our study was to investigate the type of immune response occurring in these "trypanotolerant" subjects. Cytokines levels were measured in healthy endemic controls (n = 40), stage 1 (n = 10), early stage 2 (n = 19), and late stage 2 patients (n = 23) and in a cohort of SERO TL+ individuals (n = 60) who were followed up for two years to assess the evolution of their parasitological and serological status. In contrast to HAT patients which T-cell responses appeared to be activated with increased levels of IL2, IL4, and IL10, SERO TL+ exhibited high levels of proinflammatory cytokines (IL6, IL8 and TNFα) and an almost absence of IL12p70. In SERO TL+, high levels of IL10 and low levels of TNFα were associated with an increased risk of developing HAT whereas high levels of IL8 predicted that serology would become negative. Further studies using high throughput technologies, hopefully will provide a more detailed view of the critical molecules or pathways underlying the trypanotolerant phenotype.

Trypanosoma brucei gambiense Spliced Leader RNA Is a More Specific Marker for Cure of Human African Trypanosomiasis Than T. b. gambiense DNA.

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.

Immune trypanolysis test with blood spotted on filter paper for epidemiological surveillance of sleeping sickness.

OBJECTIVES: The immune trypanolysis test (TL) is an accurate sero-diagnostic tool increasingly implemented for sleeping sickness medical surveillance, but it is restricted to the reference laboratories. To facilitate storage and transport of the test specimen, we developed a protocol for the examination of blood spotted on filter paper (TL-fp) that can be stored and shipped at ambient temperature. We compared its performance with the classical TL on plasma (TL-pl) that needs to be kept frozen until use. METHODS: The study was conducted in active foci of the Republic of Guinea. In total, 438 specimens from treated and untreated sleeping sickness patients and serological suspects were tested with both methods. RESULT: TL-fp gave significantly less positive results than TL-pl, but all the confirmed sleeping sickness cases were positive with the TL-fp protocol. CONCLUSION: TL-fp appears to offer a good compromise between feasibility and sensitivity to detect currently infected subjects who play a role in the transmission of Trypanosoma brucei gambiense and is useful for contributing to the elimination of gambiense sleeping sickness.

Conducting active screening for human African trypanosomiasis with rapid diagnostic tests: The Guinean experience (2016-2021).

Strategies to detect Human African Trypanosomiasis (HAT) cases rely on serological screening of populations exposed to trypanosomes. In Guinea, mass medical screening surveys performed with the Card Agglutination Test for Trypanosomiasis have been progressively replaced by door-to-door approaches using Rapid Diagnostic Tests (RDTs) since 2016. However, RDTs availability represents a major concern and medical teams must often adapt, even in the absence of prior RDT performance evaluation. For the last 5 years, the Guinean HAT National Control Program had to combine three different RDTs according to their availability and price: the SD Bioline HAT (not available anymore), the HAT Sero-K-SeT (most expensive), and recently the Abbott Bioline HAT 2.0 (limited field evaluation). Here, we assess the performance of these RDTs, alone or in different combinations, through the analysis of both prospective and retrospective data. A parallel assessment showed a higher positivity rate of Abbott Bioline HAT 2.0 (6.0%, n = 2,250) as compared to HAT Sero-K-SeT (1.9%), with a combined positive predictive value (PPV) of 20.0%. However, an evaluation of Abbott Bioline HAT 2.0 alone revealed a low PPV of 3.9% (n = 6,930) which was surpassed when using Abbott Bioline HAT 2.0 in first line and HAT Sero-K-SeT as a secondary test before confirmation, with a combined PPV reaching 44.4%. A retrospective evaluation of all 3 RDTs was then conducted on 189 plasma samples from the HAT-NCP biobank, confirming the higher sensitivity (94.0% [85.6-97.7%]) and lower specificity (83.6% [76.0-89.1%]) of Abbott Bioline HAT 2.0 as compared to SD Bioline HAT (Se 64.2% [52.2-74.6%]-Sp 98.4% [94.2-99.5%]) and HAT Sero-K-SeT (Se 88.1% [78.2-93.8%]-Sp 98.4% [94.2-99.5%]). A comparison of Abbott Bioline HAT 2.0 and malaria-RDT positivity rates on 479 subjects living in HAT-free malaria-endemic areas further revealed that a significantly higher proportion of subjects positive in Abbott Bioline HAT 2.0 were also positive in malaria-RDT, suggesting a possible cross-reaction of Abbott Bioline HAT 2.0 with malaria-related biological factors in about 10% of malaria cases. This would explain, at least in part, the limited specificity of Abbott Bioline HAT 2.0. Overall, Abbott Bioline HAT 2.0 seems suitable as first line RDT in combination with a second HAT RDT to prevent confirmatory lab overload and loss of suspects during referral for confirmation. A state-of-the-art prospective comparative study is further required for comparing all current and future HAT RDTs to propose an optimal combination of RDTs for door-to-door active screening.

Performance of clinical signs and symptoms, rapid and reference laboratory diagnostic tests for diagnosis of human African trypanosomiasis by passive screening in Guinea: a prospective diagnostic accuracy study.

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 reference laboratory tests on dried blood spots (DBS) for diagnosing HAT in Guinea. METHOD: The study took place in 14 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 (reference test) to confirm HAT and their DBS were tested in indirect enzyme-linked immunoassay (ELISA)/Trypanosoma brucei gambiense, trypanolysis, Loopamp Trypanosoma brucei Detection kit (LAMP) and m18S quantitative PCR (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% (48/2345, 95% CI: 1.5-2.7%). Odds ratios (OR) for HAT were increased for participants with swollen lymph nodes (OR = 96.7, 95% CI: 20.7-452.0), important weight loss (OR = 20.4, 95% CI: 7.05-58.9), severe itching (OR = 45.9, 95% CI: 7.3-288.7) or motor disorders (OR = 4.5, 95% CI: 0.89-22.5). Presence of at least one of these clinical presentations was 75.6% (95% CI: 73.8-77.4%) specific and 97.9% (95% CI: 88.9-99.9%) sensitive for HAT. HAT Sero-K-Set, rHAT Sero-Strip, and SD Bioline HAT were respectively 97.5% (95% CI: 96.8-98.1%), 99.4% (95% CI: 99.0-99.7%) and 97.9% (95% CI: 97.2-98.4%) specific, and 100% (95% CI: 92.5-100.0%), 59.6% (95% CI: 44.3-73.3%) and 93.8% (95% CI: 82.8-98.7%) sensitive for HAT. The RDT's positive and negative predictive values ranged from 45.2-66.7% and 99.2-100% respectively. 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% (95% CI: 68.9-95.0%) and 67.6% (95% CI: 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 HAT endemic areas 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 ).

Accelerating elimination of sleeping sickness from the Guinean littoral through enhanced screening in the post-Ebola context: A retrospective analysis.

BACKGROUND: Activities to control human African trypanosomiasis (HAT) in Guinea were severely hampered by the Ebola epidemic that hit this country between 2014 and 2016. Active screening was completely interrupted and passive screening could only be maintained in a few health facilities. At the end of the epidemic, medical interventions were progressively intensified to mitigate the risk of HAT resurgence and progress towards disease elimination. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective analysis was performed to evaluate the medical activities that were implemented in the three most endemic prefectures of Guinea (Boffa, Dubreka and Forecariah) between January 2016 and December 2018. Passive screening using rapid diagnostic tests (RDTs) was progressively resumed in one hundred and one health facilities, and active screening was intensified by visiting individual households and performing RDTs, and by conducting mass screening in villages by mobile teams using the Card Agglutination Test for Trypanosomiasis. A total of 1885, 4897 and 8023 clinical suspects were tested in passive, while 5743, 14442 and 21093 people were actively screened in 2016, 2017 and 2018, respectively. The number of HAT cases that were diagnosed first went up from 107 in 2016 to 140 in 2017, then subsequently decreased to only 73 in 2018. A progressive decrease in disease prevalence was observed in the populations that were tested in active and in passive between 2016 and 2018. CONCLUSIONS/SIGNIFICANCE: Intensified medical interventions in the post-Ebola context first resulted in an increase in the number of HAT cases, confirming the fear that the disease could resurge as a result of impaired control activities during the Ebola epidemic. On the other hand, the decrease in disease prevalence that was observed between 2016 and 2018 is encouraging, as it suggests that the current strategy combining enhanced diagnosis, treatment and vector control is appropriate to progress towards elimination of HAT in Guinea.

Sleeping sickness diagnosis: use of buffy coats improves the sensitivity of the mini anion exchange centrifugation test.

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 microl 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%, chi(2) = 6.93, P = 0.008) and lymph juice examination (77.2%, chi(2) = 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.

Sleeping sickness in the historical focus of forested Guinea: update using a geographically based method.

In 2017, 1447 new cases of Human African Trypanosomiasis (HAT) were reported, which reflects considerable progress towards the World Health Organisation's target of eliminating HAT as a public health problem by 2020. However, current epidemiological data are still lacking for a number of areas, including historical HAT foci. In order to update the HAT situation in the historical focus of forested Guinea, we implemented a geographically based methodology: Identification of Villages at Risk (IVR). The methodology is based on three sequential steps: Desk-based IVR (IVR-D), which selects villages at risk of HAT on the basis of HAT archives and geographical items; Field-based IVR (IVR-F), which consists in collecting additional epidemiological and geographical information in the field in villages at risk; and to be Medically surveyed IVR (IVR-M), a field data analysis through a Geographic Information System (GIS), to compile a list of the villages most at risk of HAT, suitable to guide active screening and passive surveillance. In an area of 2385 km2 with 1420,530 inhabitants distributed in 1884 settlements, 14 villages with a population of 11,236 inhabitants were identified as most at risk of HAT and selected for active screening. Although no HAT cases could be confirmed, subjects that had come into contact with Trypanosoma brucei gambiense were identified and two sentinel sites were chosen to implement passive surveillance. IVR, which could be applied to any gambiense areas where the situation needs to be clarified, could help to reach the objective of HAT elimination.

TITLE: Maladie du sommeil dans le foyer historique de Guinée forestière : actualisation grâce à une méthode géographique. ABSTRACT: En 2017, 1447 nouveaux cas de Trypanosomiase Humaine Africaine (THA) ont été rapportés, ce qui constitue une avancée importante pour atteindre l'objectif affiché par l'OMS d'éliminer la THA comme problème de santé publique d'ici 2020. Cependant, il existe toujours un manque d'informations épidémiologiques dans certaines zones, incluant des foyers historiques de THA. Afin d'actualiser la situation de la THA dans le foyer historique de Guinée forestière, nous avons appliqué une méthode géographique : l'Identification des Villages à Risque (IVR). La méthode s'effectue en 3 étapes successives : l'identification des villages à risque au bureau (IVR-D), qui sélectionne des villages à risque de THA sur la base d'archives de la THA et d'éléments géographiques ; l'identification des villages à risque sur le terrain (IVR-F), qui consiste à collecter des données épidémiologiques et géographiques des villages à risque sur le terrain ; l'identification des villages à risque à prospecter (IVR-M), une analyse des données de terrain, à travers un système d'information géographique, visant à dresser une liste de villages les plus à risque de THA, qui permettront d'orienter le dépistage actif et la surveillance passive. Dans une aire de 2385 km2, avec 1 420 530 habitants distribués dans 1884 peuplements, 14 villages d'une population de 11 236 habitants ont été identifiés comme les plus à risque de THA et sélectionnés pour un dépistage actif. Bien qu'aucun cas de THA n'ait été confirmé, des individus qui sont entrés en contact avec Trypanosoma brucei gambiense ont été identifiés et 2 sites sentinelles ont été retenus pour la surveillance passive. IVR, qui pourrait être appliquée dans n'importe quelle zone à gambiense où la situation nécessite d'être clarifiée, pourrait aider à atteindre l'objectif d'élimination de la THA.

Immune trypanolysis test as a promising bioassay to monitor the elimination of gambiense human African trypanosomiasis.

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.

TITLE: Le test immunitaire de tryanolyse comme biomarqueur prometteur pour le suivi de l'élimination de la trypanosomose humaine africaine à gambiense. ABSTRACT: 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.

Macrophage migrating inhibitory factor expression is associated with Trypanosoma brucei gambiense infection and is controlled by trans-acting expression quantitative trait loci in the Guinean population.

Infection by Trypanosoma brucei gambiense is characterized by a wide array of clinical outcomes, ranging from asymptomatic to acute disease and even spontaneous cure. In this study, we investigated the association between macrophage migrating inhibitory factor (MIF), an important pro-inflammatory cytokine that plays a central role in both innate and acquired immunity, and disease outcome during T. b. gambiense infection. A comparative expression analysis of patients, individuals with latent infection and controls found that MIF had significantly higher expression in patients (n = 141; 1.25 ±â€¯0.07; p < .0001) and latent infections (n = 25; 1.23 ±â€¯0.13; p = .0005) relative to controls (n = 46; 0.94 ±â€¯0.11). Furthermore, expression decreased significantly after treatment (patients before treatment n = 33; 1.40 ±â€¯0.18 versus patients after treatment n = 33; 0.99 ±â€¯0.10, p = .0001). We conducted a genome wide eQTL analysis on 29 controls, 128 cases and 15 latently infected individuals for whom expression and genotype data were both available. Four loci, including one containing the chemokine CXCL13, were found to associate with MIF expression. Genes at these loci are candidate regulators of increased expression of MIF after infection. Our study is the first data demonstrating that MIF expression is elevated in T. b. gambiense-infected human hosts but does not appear to contribute to pathology.

Differences in pathogenicity and virulence of Trypanosoma brucei gambiense field isolates in experimentally infected Balb/C mice.

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.

Impact of the Ebola outbreak on Trypanosoma brucei gambiense infection medical activities in coastal Guinea, 2014-2015: A retrospective analysis from the Guinean national Human African Trypanosomiasis control program.

BACKGROUND: The 2014-2015 Ebola outbreak massively hit Guinea. The coastal districts of Boffa, Dubreka and Forecariah, three major foci of Human African Trypanosomiasis (HAT), were particularly affected. We aimed to assess the impact of this epidemic on sleeping sickness screening and caring activities. METHODOLOGY/PRINCIPAL FINDINGS: We used preexisting data from the Guinean sleeping sickness control program, collected between 2012 and 2015. We described monthly: the number of persons (i) screened actively; (ii) or passively; (iii) treated for HAT; (iv) attending post-treatment follow-up visits. We compared clinical data, treatment characteristics and Disability Adjusted Life-Years (DALYs) before (February 2012 to December 2013) and during (January 2014 to October 2015) the Ebola outbreak period according to available data. Whereas 32,221 persons were actively screened from February 2012 to December 2013, before the official declaration of the first Ebola case in Guinea, no active screening campaigns could be performed during the Ebola outbreak. Following the reinforcement and extension of HAT passive surveillance system early in 2014, the number of persons tested passively by month increased from 7 to 286 between April and September 2014 and then abruptly decreased to 180 until January 2015 and to none after March 2015. 213 patients initiated HAT treatment, 154 (72%) before Ebola and 59 (28%) during the Ebola outbreak. Those initiating HAT therapy during Ebola outbreak were recruited through passive screening and diagnosed at a later stage 2 of the disease (96% vs. 55% before Ebola, p<0.0001). The proportion of patients attending the 3 months and 6 months post-treatment follow-up visits decreased from 44% to 10% (p <0.0001) and from 16% to 3% (p = 0.017) respectively. The DALYs generated before the Ebola outbreak were estimated to 48.7 (46.7-51.5) and increased up to 168.7 (162.7-174.7), 284.9 (277.1-292.8) and 466.3 (455.7-477.0) during Ebola assuming case fatality rates of 2%, 5% and 10% respectively among under-reported HAT cases. CONCLUSIONS/SIGNIFICANCE: The 2014-2015 Ebola outbreak deeply impacted HAT screening activities in Guinea. Active screening campaigns were stopped. Passive screening dramatically decreased during the Ebola period, but trends could not be compared with pre-Ebola period (data not available). Few patients were diagnosed with more advanced HAT during the Ebola period and retention rates in follow-up were lowered. The drop in newly diagnosed HAT cases during Ebola epidemic is unlikely due to a fall in HAT incidence. Even if we were unable to demonstrate it directly, it is much more probably the consequence of hampered screening activities and of the fear of the population on subsequent confirmation and linkage to care. Reinforced program monitoring, alternative control strategies and sustainable financial and human resources allocation are mandatory during post Ebola period to reduce HAT burden in Guinea.

Candidate gene polymorphisms study between human African trypanosomiasis clinical phenotypes in Guinea.

BACKGROUND: Human African trypanosomiasis (HAT), a lethal disease induced by Trypanosoma brucei gambiense, has a range of clinical outcomes in its human host in West Africa: an acute form progressing rapidly to second stage, spontaneous self-cure and individuals able to regulate parasitaemia at very low levels, have all been reported from endemic foci. In order to test if this clinical diversity is influenced by host genetic determinants, the association between candidate gene polymorphisms and HAT outcome was investigated in populations from HAT active foci in Guinea. METHODOLOGY AND RESULTS: Samples were collected from 425 individuals; comprising of 232 HAT cases, 79 subjects with long lasting positive and specific serology but negative parasitology and 114 endemic controls. Genotypes of 28 SNPs in eight genes passed quality control and were used for an association analysis. IL6 rs1818879 allele A (p = 0.0001, OR = 0.39, CI95 = [0.24-0.63], BONF = 0.0034) was associated with a lower risk of progressing from latent infection to active disease. MIF rs36086171 allele G seemed to be associated with an increased risk (p = 0.0239, OR = 1.65, CI95 = [1.07-2.53], BONF = 0.6697) but did not remain significant after Bonferroni correction. Similarly MIF rs12483859 C allele seems be associated with latent infections (p = 0.0077, OR = 1.86, CI95 = [1.18-2.95], BONF = 0.2157). We confirmed earlier observations that APOL1 G2 allele (DEL) (p = 0.0011, OR = 2.70, CI95 = [1.49-4.91], BONF = 0.0301) is associated with a higher risk and APOL1 G1 polymorphism (p = 0.0005, OR = 0.45, CI95 = [0.29-0.70], BONF = 0.0129) with a lower risk of developing HAT. No associations were found with other candidate genes. CONCLUSION: Our data show that host genes are involved in modulating Trypanosoma brucei gambiense infection outcome in infected individuals from Guinea with IL6 rs1818879 being associated with a lower risk of progressing to active HAT. These results enhance our understanding of host-parasite interactions and, ultimately, may lead to the development of new control tools.

APOL1 renal risk variants have contrasting resistance and susceptibility associations with African trypanosomiasis.

Reduced susceptibility to infectious disease can increase the frequency of otherwise deleterious alleles. In populations of African ancestry, two apolipoprotein-L1 (APOL1) variants with a recessive kidney disease risk, named G1 and G2, occur at high frequency. APOL1 is a trypanolytic protein that confers innate resistance to most African trypanosomes, but not Trypanosoma brucei rhodesiense or T.b. gambiense, which cause human African trypanosomiasis. In this case-control study, we test the prevailing hypothesis that these APOL1 variants reduce trypanosomiasis susceptibility, resulting in their positive selection in sub-Saharan Africa. We demonstrate a five-fold dominant protective association for G2 against T.b. rhodesiense infection. Furthermore, we report unpredicted strong opposing associations with T.b. gambiense disease outcome. G2 associates with faster progression of T.b. gambiense trypanosomiasis, while G1 associates with asymptomatic carriage and undetectable parasitemia. These results implicate both forms of human African trypanosomiasis in the selection and persistence of otherwise detrimental APOL1 kidney disease variants.

Accuracy of individual rapid tests for serodiagnosis of gambiense sleeping sickness in West Africa.

BACKGROUND: Individual rapid tests for serodiagnosis (RDT) of human African trypanosomiasis (HAT) are particularly suited for passive screening and surveillance. However, so far, no large scale evaluation of RDTs has been performed for diagnosis of Trypanosoma brucei gambiense HAT in West Africa. The objective of this study was to assess the diagnostic accuracy of 2 commercial HAT-RDTs on stored plasma samples from West Africa. METHODOLOGY/PRINCIPAL FINDINGS: SD Bioline HAT and HAT Sero-K-Set were performed on 722 plasma samples originating from Guinea and Côte d'Ivoire, including 231 parasitologically confirmed HAT patients, 257 healthy controls, and 234 unconfirmed individuals whose blood tested antibody positive in the card agglutination test but negative by parasitological tests. Immune trypanolysis was performed as a reference test for trypanosome specific antibody presence. Sensitivities in HAT patients were respectively 99.6% for SD Bioline HAT, and 99.1% for HAT Sero-K-Set, specificities in healthy controls were respectively 87.9% and 88.3%. Considering combined positivity in both RDTs, increased the specificity significantly (p ≤ 0.0003) to 93.4%, while 98.7% sensitivity was maintained. Specificities in controls were 98.7-99.6% for the combination of one or two RDTs with trypanolysis, maintaining a sensitivity of at least 98.1%. CONCLUSIONS/SIGNIFICANCE: The observed specificity of the single RDTs was relatively low. Serial application of SD Bioline HAT and HAT Sero-K-Set might offer superior specificity compared to a single RDT, maintaining high sensitivity. The combination of one or two RDTs with trypanolysis seems promising for HAT surveillance.

Reducing Human-Tsetse Contact Significantly Enhances the Efficacy of Sleeping Sickness Active Screening Campaigns: A Promising Result in the Context of Elimination.

BACKGROUND: Control of gambiense sleeping sickness, a neglected tropical disease targeted for elimination by 2020, relies mainly on mass screening of populations at risk and treatment of cases. This strategy is however challenged by the existence of undetected reservoirs of parasites that contribute to the maintenance of transmission. In this study, performed in the Boffa disease focus of Guinea, we evaluated the value of adding vector control to medical surveys and measured its impact on disease burden. METHODS: The focus was divided into two parts (screen and treat in the western part; screen and treat plus vector control in the eastern part) separated by the Rio Pongo river. Population census and baseline entomological data were collected from the entire focus at the beginning of the study and insecticide impregnated targets were deployed on the eastern bank only. Medical surveys were performed in both areas in 2012 and 2013. FINDINGS: In the vector control area, there was an 80% decrease in tsetse density, resulting in a significant decrease of human tsetse contacts, and a decrease of disease prevalence (from 0.3% to 0.1%; p=0.01), and an almost nil incidence of new infections (<0.1%). In contrast, incidence was 10 times higher in the area without vector control (>1%, p<0.0001) with a disease prevalence increasing slightly (from 0.5 to 0.7%, p=0.34). INTERPRETATION: Combining medical and vector control was decisive in reducing T. b. gambiense transmission and in speeding up progress towards elimination. Similar strategies could be applied in other foci.