The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronic Trypanosoma brucei infection.

The meningeal space is a critical brain structure providing immunosurveillance for the central nervous system (CNS), but the impact of infections on the meningeal immune landscape is far from being fully understood. The extracellular protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT) or sleeping sickness, accumulates in the meningeal spaces, ultimately inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, by combining single-cell transcriptomics and mass cytometry by time-of-flight (CyTOF) with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges. These infection-induced ELAs were defined by the presence of ER-TR7+ fibroblastic reticular cells, CD21/35+ follicular dendritic cells (FDCs), CXCR5+ PD1+ T follicular helper-like phenotype, GL7+ CD95+ GC-like B cells, and plasmablasts/plasma cells. Furthermore, the B cells found in the infected meninges produced high-affinity autoantibodies able to recognise mouse brain antigens, in a process dependent on LTß signalling. A mid-throughput screening identified several host factors recognised by these autoantibodies, including myelin basic protein (MBP), coinciding with cortical demyelination and brain pathology. In humans, we identified the presence of autoreactive IgG antibodies in the cerebrospinal fluid (CSF) of second stage HAT patients that recognised human brain lysates and MBP, consistent with our findings in experimental infections. Lastly, we found that the pathological B cell responses we observed in the meninges required the presence of T. brucei in the CNS, as suramin treatment before the onset of the CNS stage prevented the accumulation of GL7+ CD95+ GC-like B cells and brain-specific autoantibody deposition. Taken together, our data provide evidence that the meningeal immune response during chronic T. brucei infection results in the acquisition of lymphoid tissue-like properties, broadening our understanding of meningeal immunity in the context of chronic infections. These findings have wider implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity in mice and humans, as observed in other autoimmune neurodegenerative disorders, including neuropsychiatric lupus and multiple sclerosis.

γδ T cells control murine skin inflammation and subcutaneous adipose wasting during chronic Trypanosoma brucei infection.

African trypanosomes colonise the skin to ensure parasite transmission. However, how the skin responds to trypanosome infection remains unresolved. Here, we investigate the local immune response of the skin in a murine model of infection using spatial and single cell transcriptomics. We detect expansion of dermal IL-17A-producing Vγ6+ cells during infection, which occurs in the subcutaneous adipose tissue. In silico cell-cell communication analysis suggests that subcutaneous interstitial preadipocytes trigger T cell activation via Cd40 and Tnfsf18 signalling, amongst others. In vivo, we observe that female mice deficient for IL-17A-producing Vγ6+ cells show extensive inflammation and limit subcutaneous adipose tissue wasting, independently of parasite burden. Based on these observations, we propose that subcutaneous adipocytes and Vγ6+ cells act in concert to limit skin inflammation and adipose tissue wasting. These studies provide new insights into the role of γδ T cell and subcutaneous adipocytes as homeostatic regulators of skin immunity during chronic infection.

Antibody Avidity Maturation Following Recovery From Infection or the Booster Vaccination Grants Breadth of SARS-CoV-2 Neutralizing Capacity.

BACKGROUND: Cross-neutralizing capacity of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is important in mitigating (re-)exposures. Role of antibody maturation, the process whereby selection of higher affinity antibodies augments host immunity, to determine SARS-CoV-2 neutralizing capacity was investigated. METHODS: Sera from SARS-CoV-2 convalescents at 2, 6, or 10 months postrecovery, and BNT162b2 vaccine recipients at 3 or 25 weeks postvaccination, were analyzed. Anti-spike IgG avidity was measured in urea-treated ELISAs. Neutralizing capacity was assessed by surrogate neutralization assays. Fold change between variant and wild-type neutralization inferred the breadth of neutralizing capacity. RESULTS: Compared with early-convalescent, avidity indices of late-convalescent sera were significantly higher (median, 37.7 [interquartile range 28.4-45.1] vs 64.9 [57.5-71.5], P < .0001). Urea-resistant, high-avidity IgG best predicted neutralizing capacity (Spearman r = 0.49 vs 0.67 [wild-type]; 0.18-0.52 vs 0.48-0.83 [variants]). Higher-avidity convalescent sera better cross-neutralized SARS-CoV-2 variants (P < .001 [Alpha]; P < .01 [Delta and Omicron]). Vaccinees only experienced meaningful avidity maturation following the booster dose, exhibiting rather limited cross-neutralizing capacity at week 25. CONCLUSIONS: Avidity maturation was progressive beyond acute recovery from infection, or became apparent after the booster vaccine dose, granting broader anti-SARS-CoV-2 neutralizing capacity. Understanding the maturation kinetics of the 2 building blocks of anti-SARS-CoV-2 humoral immunity is crucial.

DNA methylation patterns associated with konzo in Sub-Saharan Africa.

Konzo, a disease characterized by sudden, irreversible spastic paraparesis, affecting up to 10% of the population in some regions of Sub-Saharan Africa during outbreaks, is strongly associated with dietary exposure to cyanogenic bitter cassava. The molecular mechanisms underlying the development of konzo remain largely unknown. Here, through an analysis of 16 individuals with konzo and matched healthy controls from the same outbreak zones, we identified 117 differentially methylated loci involved in numerous biological processes that may identify cyanogenic-sensitive regions of the genome, providing the first study of epigenomic alterations associated with a clinical phenotype of konzo.

VSGs Expressed during Natural T. b. gambiense Infection Exhibit Extensive Sequence Divergence and a Subspecies-Specific Bias towards Type B N-Terminal Domains.

Trypanosoma brucei gambiense is the primary causative agent of human African trypanosomiasis (HAT), a vector-borne disease endemic to West and Central Africa. The extracellular parasite evades antibody recognition within the host bloodstream by altering its variant surface glycoprotein (VSG) coat through a process of antigenic variation. The serological tests that are widely used to screen for HAT use VSG as one of the target antigens. However, the VSGs expressed during human infection have not been characterized. Here, we use VSG sequencing (VSG-seq) to analyze the VSGs expressed in the blood of patients infected with T. b. gambiense and compared them to VSG expression in Trypanosoma brucei rhodesiense infections in humans as well as Trypanosoma brucei brucei infections in mice. The 44 VSGs expressed during T. b. gambiense infection revealed a striking bias toward expression of type B N termini (82% of detected VSGs). This bias is specific to T. b. gambiense, which is unique among T. brucei subspecies in its chronic clinical presentation and anthroponotic nature. The expressed T. b. gambiense VSGs also share very little similarity to sequences from 36 T. b. gambiense whole-genome sequencing data sets, particularly in areas of the VSG protein exposed to host antibodies, suggesting the antigen repertoire is under strong selective pressure to diversify. Overall, this work demonstrates new features of antigenic variation in T. brucei gambiense and highlights the importance of understanding VSG repertoires in nature. IMPORTANCE Human African trypanosomiasis is a neglected tropical disease primarily caused by the extracellular parasite Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their variant surface glycoprotein (VSG) coat. Despite the important role of VSGs in prolonging infection, VSG expression during human infections is poorly understood. A better understanding of natural VSG gene expression dynamics can clarify the mechanisms that T. brucei uses to alter its VSG coat. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that there are features of antigenic variation unique to human-infective T. brucei subspecies and that natural VSG repertoires may vary more than previously expected.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018.

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Therapeutic Efficacy of Artemisinin-Based Combination Therapies in Democratic Republic of the Congo and Investigation of Molecular Markers of Antimalarial Resistance.

Routine assessment of the efficacy of artemisinin-based combination therapies (ACTs) is critical for the early detection of antimalarial resistance. We evaluated the efficacy of ACTs recommended for treatment of uncomplicated malaria in five sites in Democratic Republic of the Congo (DRC): artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), and dihydroartemisinin-piperaquine (DP). Children aged 6-59 months with confirmed Plasmodium falciparum malaria were treated with one of the three ACTs and monitored. The primary endpoints were uncorrected and polymerase chain reaction (PCR)-corrected 28-day (AL and ASAQ) or 42-day (DP) cumulative efficacy. Molecular markers of resistance were investigated. Across the sites, uncorrected efficacy estimates ranged from 63% to 88% for AL, 73% to 100% for ASAQ, and 56% to 91% for DP. PCR-corrected efficacy estimates ranged from 86% to 98% for AL, 91% to 100% for ASAQ, and 84% to 100% for DP. No pfk13 mutations previously found to be associated with ACT resistance were observed. Statistically significant associations were found between certain pfmdr1 and pfcrt genotypes and treatment outcome. There is evidence of efficacy below the 90% cutoff recommended by WHO to consider a change in first-line treatment recommendations of two ACTs in one site not far from a monitoring site in Angola that has shown similar reduced efficacy for AL. Confirmation of these findings in future therapeutic efficacy monitoring in DRC is warranted.

Facial analysis technology for the detection of Down syndrome in the Democratic Republic of the Congo.

Down syndrome is one of the most common chromosomal anomalies affecting the world's population, with an estimated frequency of 1 in 700 live births. Despite its relatively high prevalence, diagnostic rates based on clinical features have remained under 70% for most of the developed world and even lower in countries with limited resources. While genetic and cytogenetic confirmation greatly increases the diagnostic rate, such resources are often non-existent in many low- and middle-income countries, particularly in Sub-Saharan Africa. To address the needs of countries with limited resources, the implementation of mobile, user-friendly and affordable technologies that aid in diagnosis would greatly increase the odds of success for a child born with a genetic condition. Given that the Democratic Republic of the Congo is estimated to have one of the highest rates of birth defects in the world, our team sought to determine if smartphone-based facial analysis technology could accurately detect Down syndrome in individuals of Congolese descent. Prior to technology training, we confirmed the presence of trisomy 21 using low-cost genomic applications that do not need advanced expertise to utilize and are available in many low-resourced countries. Our software technology trained on 132 Congolese subjects had a significantly improved performance (91.67% accuracy, 95.45% sensitivity, 87.88% specificity) when compared to previous technology trained on individuals who are not of Congolese origin (p < 5%). In addition, we provide the list of most discriminative facial features of Down syndrome and their ranges in the Congolese population. Collectively, our technology provides low-cost and accurate diagnosis of Down syndrome in the local population.

Molecular detection and species identification of Plasmodium spp. infection in adults in the Democratic Republic of Congo: A population-based study.

BACKGROUND: In efforts to control malaria infection, the Democratic Republic of Congo has implemented several strategies. Studies assessing their efficiency mainly involved at-risk groups, especially children under five years of age. This study aimed to determine the prevalence and identify the risk factors associated with Plasmodium spp. infection. METHODS: From October 2014 to March 2015, individuals aged at least 15 years were selected randomly and enrolled in a cross-sectional study conducted throughout the country. Microscopy and polymerase chain reaction (PCR) analysis were used for the detection of Plasmodium ssp. RESULTS: From 2286 individuals recruited, 1870 with valid laboratory results were included in the study for further analysis. The prevalence of Plasmodium spp. infection assessed by microscopy (355/ 1870 (19%) was lower than that estimated by PCR (580/1870 (31%). In addition, the difference between the two results was statistically significant (P < 0.0001). The most prevalent Plasmodium species was P. falciparum, either as mono-infection (96.3%; 95% C.I. 93.9-98.1) or combined with P. malariae (3.7%; 95% C.I. 2.8-5.9). The mean parasite density was 3272739 trophozoites/µL of blood. Women had higher risks of being infected than men (OR 2.03, 95% C.I.: 1.96. 2.62, P = 0.041)]. CONCLUSION: In this study, the molecular detection and species identification of Plasmodium spp. showed that, despite all efforts for malaria control, malaria remains a public health problem in the Democratic Republic of Congo. The high prevalence and parasite density of Plasmodium spp. in adults make this age group a potential parasitic infectious reservoir for the at-risk groups and supports the need to include this age group in further programs for malaria control.

High Levels of Genetic Diversity within Nilo-Saharan Populations: Implications for Human Adaptation.

Africa contains more human genetic variation than any other continent, but the majority of the population-scale analyses of the African peoples have focused on just two of the four major linguistic groups, the Niger-Congo and Afro-Asiatic, leaving the Nilo-Saharan and Khoisan populations under-represented. In order to assess genetic variation and signatures of selection within a Nilo-Saharan population and between the Nilo-Saharan and Niger-Congo and Afro-Asiatic, we sequenced 50 genomes from the Nilo-Saharan Lugbara population of North-West Uganda and 250 genomes from 6 previously unsequenced Niger-Congo populations. We compared these data to data from a further 16 Eurasian and African populations including the Gumuz, another putative Nilo-Saharan population from Ethiopia. Of the 21 million variants identified in the Nilo-Saharan population, 3.57 million (17%) were not represented in dbSNP and included predicted non-synonymous mutations with possible phenotypic effects. We found greater genetic differentiation between the Nilo-Saharan Lugbara and Gumuz populations than between any two Afro-Asiatic or Niger-Congo populations. F3 tests showed that Gumuz contributed a genetic component to most Niger-Congo B populations whereas Lugabara did not. We scanned the genomes of the Lugbara for evidence of selective sweeps. We found selective sweeps at four loci (SLC24A5, SNX13, TYRP1, and UVRAG) associated with skin pigmentation, three of which already have been reported to be under selection. These selective sweeps point toward adaptations to the intense UV radiation of the Sahel.

Cerebral malaria.

Malaria remains of significant public health concern under the tropics, causing millions of deaths annually. The disease is caused by protozoans of the Plasmodium genus, of which harbors several distinct species. Human infection occurs during the blood meal of an infected female mosquito belonging to the Anopheles genus. It is estimated that around 1% of children infected with Plasmodium falciparum develops a more severe form of malaria, which may eventually lead to cerebral complications including cerebral malaria (CM). CM can be positively diagnosed in patients unable to localize a painful stimulus, with peripheral asexual P. falciparum parasitemia and no other identifiable causes of an encephalopathy. Unarousable comas along with the presence of asexual forms of the parasite on a peripheral blood smear are hallmarks of the disease. While the molecular mechanisms underlying the pathogenesis of CM have yet be fully elucidated, the pathology in itself indicates a clear disease of the vascular endothelium. It is characterized by parasite sequestration, inflammatory cytokine production and vascular leakage, eventually resulting in brain hypoxia. The condition requires systemic health management consisting of focused nursing practices, supportive care, and anti-malarial drugs. The continued understanding of pathogenic mechanisms leading to the onset of CM is fundamental and key for the expansion and development of appropriate neuroprotective interventions. Future research perspectives may also include the development of field-based and rapid diagnostic tests for CM, understanding of host-pathogen interactions to advance development of prevention tools and therapies, and antimalarial drug trials.

Molecular identification of Plasmodium species in symptomatic children of Democratic Republic of Congo.

BACKGROUND: Worldwide, the highest malaria mortality is due to Plasmodium falciparum infection. However, other species of Plasmodium (Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi) can also cause malaria. Therefore, accurate identification of malaria species is crucial for patient management and epidemiological surveillance. This study aimed to determine the different Plasmodium species causing malaria in children under 5 years old in two provinces (Kinshasa and North Kivu) of the Democratic Republic of Congo (DRC). METHODS: From October to December 2015, a health-facility based cross-sectional study was conducted in General Reference Hospitals in Kinshasa and North Kivu. Four hundred and seven blood samples were collected from febrile children aged ≤ 5 years. Nested polymerase chain reaction assays were performed for Plasmodium species identification. RESULTS: Out of 407 children, 142 (34.9%) were infected with Plasmodium spp. and P. falciparum was the most prevalent species (99.2%). Among those infected children, 124 had a mono infection with P. falciparum and one with P. malariae. Mixed infections with P. falciparum/P. malariae and P. falciparum/P. vivax were observed in 6 (1.5%) and 8 (2.0%) children, respectively. The prevalence of infection was higher in females (64.8%) than in males (35.2%), p < 0.001. The age-specific distribution of infection showed that children of less than 2 years old were less infected (18.4%) compared to those aged above 2 years (81.6%), p < 0.001. CONCLUSION: Although this study showed clearly that the most prevalent species identified was P. falciparum, the findings demonstrate the existence of non-falciparum malaria, especially P. malariae and P. vivax among children aged ≤ 5 years living both Kinshasa and North Kivu Provinces in DRC.

Mortality trends and risk factors in advanced stage-2 Human African Trypanosomiasis: A critical appraisal of 23 years of experience in the Democratic Republic of Congo.

We conducted a retrospective study on mortality trends and risk factors in 781 naïve cases of advanced stage-2 sleeping sickness admitted between 1989 and 2012 at the National Reference Center for Human African Trypanosomiasis (HAT), Department of Neurology, Kinshasa University, Democratic Republic of Congo (DRC). Death was the outcome variable whereas age, gender, duration of disease, location of trypanosomes in body fluids, cytorachy, protidorachy, clinical status (assessed on a syndromic and functional basis) on admission, and treatment regimen were predictors in logistic regression models run at the 0.05 significance level. Death proportions were 17.2% in the standard melarsoprol schedule (3-series of intravenous melarsoprol on 3 successive days at 3.6 mg/kg/d, with a one-week interval between the series, ARS 9); 12.1% in the short schedule melarsoprol (10 consecutive days of intravenous melarsoprol at 2.2 mg/kg/d, ARS 10), 5.4% in the first-line eflornithine (14 days of eflornithine at 400 mg/kg/d in 4 infusions a day DFMO B), 9.1% in the NECT treatment regimen (eflornithine for 7 days at 400, mg/kg/d in 2 infusions a day combined with oral nifurtimox for 10 days at 15 mg/kg/d in 3 doses a day); and high (36%) in the group with select severely affected patients given eflornithine because of their clinical status on admission, at the time when this expensive drug was kept for treatment of relapses (14 days at 400 mg/kg/d in 4 infusions a day, DFMO A). After adjusting for treatment, death odds ratios were as follows: 10.40 [(95% CI: 6.55-16.51); p = .000] for clinical dysfunction (severely impaired clinical status) on admission, 2.14 [(95% CI: 1.35-3.39); p = .001] for high protidorachy, 1.99 [(95% CI: 1.18-3.37); p = .010] for the presence of parasites in the CSF and 1.70 [(95% CI: 1.03-2.81); p = .038] for high cytorachy. A multivariable analysis within treatment groups retained clinical status on admission (in ARS 9, ARS 10 and DFMO B groups) and high protidorachy (in ARS 10 and DFMO B groups) as significant predictors of death. The algorithm for initial clinical status assessment used in the present study may serve as the basis for further development of standardized assessment tools relevant to the clinical management of HAT and information exchange in epidemiological reports.

[Socioemotional disorders in children living in Konzo-affected areas, an epidemic paralytic disease associated with cyanide poisoning from food in sub-Saharan Africa]. / Troubles socio-émotionnels de l'enfant en milieu Konzo, un syndrome paralytique de nature épidémique associé à une intoxication cyanhydrique d'origine alimentaire en Afrique sub-saharienne.

INTRODUCTION: the aim of this study was to describe the socioemotional profile of children living in Konzo-affected areas, an epidemic toxico-nutritional palsy in sub-Saharan Africa. METHODS: we evaluated the socioemotional profile of 210 children, 123 with Konzo and 87 presumed to be healthy (4-17 years) based on a structured interview conducted with their parents during an epidemioclinic survey of Konzo in Congo-Kinshasa in 2011. Neurocognitive profile was identified by the KABC-II, the BOT-2 and the global neurological symptom index of Konzo. Associative tests were carried out by using chi-square test, logistic regression and, where applicable, generalized linear model, at the significance threshold of 0.05. RESULTS: in general, irritability, physical violence or inhibition with or without sadness were found in 46.0%, 30.2%, 18.7% of children respectively, with an increased risk of Konzo (OR = 2.6; CI95%: 1.4-4.8; p = 0.001). Socioemotional disorder was associated with underweight (OR: 0.49; CI95%: 0.31-0.78; p = 0.002) and with an elevated global neurological symptom index of Konzo (OR: 1.33; CI 95%: 1.1-1.63; p = 0.019); furthermore it exacerbated cognitive impairment in children with Konzo (interaction neurological status-socioemotional disorders D = 6.297; p = 0.013). High cognitive performances were observed in children without Konzo but with socioemotional disorders. The average concentration (standard deviation ± SD) of urinary thiocyanate was higher (554.8 ± 371.6 µmol/l) among children with Konzo associated with socioemotional disorders. CONCLUSION: children living in Konzo-affected areas have socioemotional disorders. Their psychopathological status and the effect of Konzo on cognition require in-depth studies.

The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes.

The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology.

Diagnostic accuracy of loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples.

BACKGROUND: Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT), but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME) DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP) in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC). METHODOLOGY/PRINCIPAL FINDINGS: 142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9-91.8%) in the first run and 93.0% (95% CI 87.5-96.1%) in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4-96.3%) in the first run and 96.4% (95% CI 91.1-98.6%) in the second run. Reproducibility was excellent with a kappa value of 0.81. CONCLUSIONS/SIGNIFICANCE: In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions.

New biomarkers for stage determination in Trypanosoma brucei rhodesiense sleeping sickness patients.

Accurate stage determination is crucial in the choice of treatment for patients suffering from sleeping sickness, also known as human African trypanosomiasis (HAT). Current staging methods, based on the counting of white blood cells (WBC) and the detection of parasites in the cerebrospinal fluid (CSF) have limited accuracy. We hypothesized that immune mediators reliable for staging T. b. gambiense HAT could also be used to stratify T. b. rhodesiense patients, the less common form of HAT.A population comprising 85 T. b. rhodesiense patients, 14 stage 1 (S1) and 71 stage 2 (S2) enrolled in Malawi and Uganda, was investigated. The CSF levels of IgM, MMP-9, CXCL13, CXCL10, ICAM-1, VCAM-1, neopterin and B2MG were measured and their staging performances evaluated using receiver operating characteristic (ROC) analyses.IgM, MMP-9 and CXCL13 were the most accurate markers for stage determination (partial AUC 88%, 86% and 85%, respectively). The combination in panels of three molecules comprising CXCL13-CXCL10-MMP-9 or CXCL13-CXCL10-IgM significantly increased their staging ability to partial AUC 94% (p value < 0.01).The present study highlighted new potential markers for stage determination of T. b. rhodesiense patients. Further investigations are needed to better evaluate these molecules, alone or in panels, as alternatives to WBC to make reliable choice of treatment.

Cerebrospinal fluid neopterin as marker of the meningo-encephalitic stage of Trypanosoma brucei gambiense sleeping sickness.

BACKGROUND: Sleeping sickness, or human African trypanosomiasis (HAT), is a protozoan disease that affects rural communities in sub-Saharan Africa. Determination of the disease stage, essential for correct treatment, represents a key issue in the management of patients. In the present study we evaluated the potential of CXCL10, CXCL13, ICAM-1, VCAM-1, MMP-9, B2MG, neopterin and IgM to complement current methods for staging Trypanosoma brucei gambiense patients. METHODS AND FINDINGS: Five hundred and twelve T. b. gambiense HAT patients originated from Angola, Chad and the Democratic Republic of the Congo (D.R.C.). Their classification as stage 2 (S2) was based on the number of white blood cells (WBC) (>5/µL) or presence of parasites in the cerebrospinal fluid (CSF). The CSF concentration of the eight markers was first measured on a training cohort encompassing 100 patients (44 S1 and 56 S2). IgM and neopterin were the best in discriminating between the two stages of disease with 86.4% and 84.1% specificity respectively, at 100% sensitivity. When a validation cohort (412 patients) was tested, neopterin (14.3 nmol/L) correctly classified 88% of S1 and S2 patients, confirming its high staging power. On this second cohort, neopterin also predicted both the presence of parasites, and of neurological signs, with the same ability as IgM and WBC, the current reference for staging. CONCLUSIONS: This study has demonstrated that neopterin is an excellent biomarker for staging T. b. gambiense HAT patients. A rapid diagnostic test for detecting this metabolite in CSF could help in more accurate stage determination.