Trypanosoma brucei gambiense excreted/secreted factors impair lipopolysaccharide-induced maturation and activation of human monocyte-derived dendritic cells.

Trypanosoma brucei gambiense, an extracellular eukaryotic flagellate parasite, is the main etiological agent of human African trypanosomiasis (HAT) or sleeping sickness. Dendritic cells (DCs) play a pivotal role at the interface between innate and adaptive immune response and are implicated during HAT. In this study, we investigated the effects of T gambiense and its excreted/secreted factors (ESF) on the phenotype of human monocyte-derived DCs (Mo-DCs). Mo-DCs were cultured with trypanosomes, lipopolysaccharide (LPS), ESF derived from T gambiense bloodstream strain Biyamina (MHOM/SD/82), or both ESF and LPS. Importantly, ESF reduced the expression of the maturation markers HLA-DR and CD83, as well as the secretion of IL-12, TNF-alpha and IL-10, in LPS-stimulated Mo-DCs. During mixed-leucocyte reactions, LPS- plus ESF-exposed DCs induced a non-significant decrease in the IFN-gamma/IL-10 ratio of CD4 + T-cell cytokines. Based on the results presented here, we raise the hypothesis that T gambiense has developed an immune escape strategy through the secretion of paracrine mediators in order to limit maturation and activation of human DCs. The identification of the factor(s) in the T gambiense ESF and of the DCs signalling pathway(s) involved may be important in the development of new therapeutic targets.

Gambiense human african trypanosomiasis and immunological memory: effect on phenotypic lymphocyte profiles and humoral immunity.

In mice, experimental infection with Trypanosoma brucei causes decreased bone marrow B-cell development, abolished splenic B-cell maturation and loss of antibody mediated protection including vaccine induced memory responses. Nothing is known about this phenomenon in human African trypanosomiasis (HAT), but if occurring, it would imply the need of revaccination of HAT patients after therapy and abolish hope for a HAT vaccine. The effect of gambiense HAT on peripheral blood memory T- and B-cells and on innate and vaccine induced antibody levels was examined. The percentage of memory B- and T-cells was quantified in peripheral blood, prospectively collected in DR Congo from 117 Trypanosoma brucei gambiense infected HAT patients before and six months after treatment and 117 controls at the same time points. Antibodies against carbohydrate antigens on red blood cells and against measles were quantified. Before treatment, significantly higher percentages of memory B-cells, mainly T-independent memory B-cells, were observed in HAT patients compared to controls (CD20+CD27+IgM+, 13.0% versus 2.0%, p<0.001). The percentage of memory T-cells, mainly early effector/memory T-cells, was higher in HAT (CD3+CD45RO+CD27+, 19.4% versus 16.7%, p = 0.003). After treatment, the percentage of memory T-cells normalized, the percentage of memory B-cells did not. The median anti-red blood cell carbohydrate IgM level was one titer lower in HAT patients than in controls (p<0.004), and partially normalized after treatment. Anti-measles antibody concentrations were lower in HAT patients than in controls (medians of 1500 versus 2250 mIU/ml, p = 0.02), and remained so after treatment, but were above the cut-off level assumed to provide protection in 94.8% of HAT patients, before and after treatment (versus 98.3% of controls, p = 0.3). Although functionality of the B-cells was not verified, the results suggest that immunity was conserved in T.b. gambiense infected HAT patients and that B-cell dysfunction might not be that severe as in mouse models.

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.

Maintenance of B cells during chronic murine Trypanosoma brucei gambiense infection.

African trypanosomosis is a debilitating parasitic disease occurring in large parts of sub-Saharan Africa. Trypanosoma brucei gambiense accounts for 98% of the reported HAT infections and causes a chronic, gradually progressing disease. Multiple experimental murine models for trypanosomosis have demonstrated inflammation-dependent apoptosis of splenic follicular B (FoB) cells and the destruction of B-cell memory against previously encountered pathogens. Here, we report that during murine infection with a chronic T. b. gambiense field isolate, FoB cells are retained. This coincided with reduced levels of IFN-γ and TNF-α during the acute phase of the infection. This result suggests that in chronic infections with low virulent parasites, less inflammation is elicited and consequently no FoB cell destruction occurs.

Structure of the trypanosome haptoglobin-hemoglobin receptor and implications for nutrient uptake and innate immunity.

African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin-hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three α-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake.

DNA break site at fragile subtelomeres determines probability and mechanism of antigenic variation in African trypanosomes.

Antigenic variation in African trypanosomes requires monoallelic transcription and switching of variant surface glycoprotein (VSG) genes. The transcribed VSG, always flanked by '70 bp'-repeats and telomeric-repeats, is either replaced through DNA double-strand break (DSB) repair or transcriptionally inactivated. However, little is known about the subtelomeric DSBs that naturally trigger antigenic variation in Trypanosoma brucei, the subsequent DNA damage responses, or how these responses determine the mechanism of VSG switching. We found that DSBs naturally accumulate close to both transcribed and non-transcribed telomeres. We then induced high-efficiency meganuclease-mediated DSBs and monitored DSB-responses and DSB-survivors. By inducing breaks at distinct sites within both transcribed and silent VSG transcription units and assessing local DNA resection, histone modification, G2/M-checkpoint activation, and both RAD51-dependent and independent repair, we reveal how breaks at different sites trigger distinct responses and, in 'active-site' survivors, different switching mechanisms. At the active site, we find that promoter-adjacent breaks typically failed to trigger switching, 70 bp-repeat-adjacent breaks almost always triggered switching through 70 bp-repeat recombination (∼60% RAD51-dependent), and telomere-repeat-adjacent breaks triggered switching through loss of the VSG expression site (25% of survivors). Expression site loss was associated with G2/M-checkpoint bypass, while 70 bp-repeat-recombination was associated with DNA-resection, γH2A-focus assembly and a G2/M-checkpoint. Thus, the probability and mechanism of antigenic switching are highly dependent upon the location of the break. We conclude that 70 bp-repeat-adjacent and telomere-repeat-adjacent breaks trigger distinct checkpoint responses and VSG switching pathways. Our results show how subtelomere fragility can generate the triggers for the major antigenic variation mechanisms in the African trypanosome.

A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding.

Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.

Antigenic variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression control.

African trypanosomes are lethal human and animal parasites that use antigenic variation for evasion of host adaptive immunity. To facilitate antigenic variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic variation requires transcription of a single VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organization all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context.

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.

Specificity of serological screening tests and reference laboratory tests to diagnose gambiense human African trypanosomiasis: a prospective clinical performance study.

BACKGROUND: Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future "screen and treat" strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. METHODS: During active screening, venous blood samples from 1095 individuals from Côte d'Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. RESULTS: One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1-99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5-88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7-84.2%); DCN HAT RDT 78.2% (95% CI: 75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100% (n = 399) and 93.0-100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. CONCLUSIONS: Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based "screen and treat" strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. TRIAL REGISTRATION: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.

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

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

A LiTat 1.5 variant surface glycoprotein-derived peptide with diagnostic potential for Trypanosoma brucei gambiense.

OBJECTIVE: To evaluate the accuracy of a peptide, corresponding to the variant surface glycoprotein (VSG) LiTat 1.5 amino acid (AA) sequence 268-281 and identified through alignment of monoclonal antibody selected mimotopes, for diagnosis of Trypanosoma brucei gambiense sleeping sickness. METHODS: A synthetic biotinylated peptide (peptide 1.5/268-281), native VSG LiTat 1.3 and VSG LiTat 1.5 were tested in an indirect ELISA with 102 sera from patients with HAT and 102 endemic HAT-negative controls. RESULTS: The area under the curve (AUC) of peptide 1.5/268-281 was 0.954 (95% confidence interval 0.918-0.980), indicating diagnostic potential. The areas under the curve of VSG LiTat 1.3 and LiTat 1.5 were 1.000 (0.982-1.000) and 0.997 (0.973-1.000), respectively, and significantly higher than the AUC of peptide 1.5/268-281. On a model of VSG LiTat 1.5, peptide 1.5/268-281 was mapped near the top of the VSG. CONCLUSIONS: A biotinylated peptide corresponding to AA 268-281 of VSG LiTat 1.5 may replace the native VSG in serodiagnostic tests, but the diagnostic accuracy is lower than for the full-length native VSG LiTat 1.3 and VSG LiTat 1.5.

Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor.

Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1-resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucei is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1-resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1-resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense.

Human African trypanosomiasis.

Human African trypanosomiasis (sleeping sickness) occurs in sub-Saharan Africa. It is caused by the protozoan parasite Trypanosoma brucei, transmitted by tsetse flies. Almost all cases are due to Trypanosoma brucei gambiense, which is indigenous to west and central Africa. Prevalence is strongly dependent on control measures, which are often neglected during periods of political instability, thus leading to resurgence. With fewer than 12 000 cases of this disabling and fatal disease reported per year, trypanosomiasis belongs to the most neglected tropical diseases. The clinical presentation is complex, and diagnosis and treatment difficult. The available drugs are old, complicated to administer, and can cause severe adverse reactions. New diagnostic methods and safe and effective drugs are urgently needed. Vector control, to reduce the number of flies in existing foci, needs to be organised on a pan-African basis. WHO has stated that if national control programmes, international organisations, research institutes, and philanthropic partners engage in concerted action, elimination of this disease might even be possible.

[Molecular dialogue between African trypanosomes and humans]. / Dialogue moléculaire entre trypanosomes Africains et l'homme.

The evolutionary origin of Man in the African continent has imposed the requirement to resist endemic parasites, in particular African trypanosomes (prototype: Trypanosoma brucei). Therefore, human serum is provided with an efficient system of innate immunity against these parasites, as discovered by A. Laveran in 1902. However, two T. brucei clones, termed T. b. rhodesiense and T. b. gambiense, managed to escape this immunity system, enabling them to grow in humans where they cause sleeping sickness. We have identified the gene allowing T. b. rhodesiense to resist trypanolysis by human serum, which led us to discover that the trypanolytic factor is apolipoprotein L1 (apoL1). ApoL1 is a human-specific serum protein bound to HDL particles that also contain another human-specific protein termed "haptoglobin-related protein " (Hpr). Following the binding of hemoglobin (Hb) to Hpr, the apoL1-bearing HDL particles are avidly taken up by the trypanosome through their binding to a parasite surface receptor for the Hp-Hb complex. After endocytosis apoL1 kills the parasite by generating anionic pores in the lysosomal membrane. In our laboratory, mutant versions of apoL1 have been constructed, which are no longer neutralized by the resistance protein of T. b. rhodesiense and are therefore able to kill this human pathogen. Unexpectedly, we have recently discovered that similar mutants do actually exist in nature : in Africans and Americans of recent African origin, even a single allele of these mutants allows protection against infection by T. b. rhodesiense, but the price to pay is a high frequency of end-stage renal disease when doubly allelic. The evidence of natural selection of these apoL1 mutations despite their deleterious potential for kidneys highlights the importance of the resistance to trypanosomes in the evolution of Man. The mechanism by which mutant apoL1 triggers end-stage renal disease is currently studied.

Multiplex Bead Assay for the Detection of Human IgG Antibody Responses to African Trypanosomes.

The recent introduction of large-scale, population-based serologic surveys in several nations where human African trypanosomiasis (HAT) remains endemic could provide an opportunity to better map the remaining disease foci and to identify asymptomatic, seropositive individuals who are infected with the more chronic form of the parasite, Trypanosoma brucei gambiense (gHAT). We have incorporated a soluble form of variant surface glycoprotein 117 and a recombinant invariant surface glycoprotein 65.1 into a multiplex bead assay (MBA) method that is commonly used for the detection of IgG antibody responses to other neglected tropical diseases. A positive result was defined as reactivity to both antigens. MBA sensitivity and specificity for gHAT infection were 92% and 96%, respectively. Assay specificity for the acute form of disease caused by T.b. rhodesiense (rHAT) was 94%, but the sensitivity was only 63.6%. In the future, additional antigens could be incorporated into the multiplex assay to improve rHAT sensitivity.

Genetic and immunological basis of human African trypanosomiasis.

Human African trypanosomiasis, or sleeping sickness, results from infection by two subspecies of the protozoan flagellate parasite Trypanosoma brucei, termed Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, prevalent in western and eastern Africa respectively. These subspecies escape the trypanolytic potential of human serum, which efficiently acts against the prototype species Trypanosoma brucei brucei, responsible for the Nagana disease in cattle. We review the various strategies and components used by trypanosomes to counteract the immune defences of their host, highlighting the adaptive genomic evolution that occurred in both parasite and host to take the lead in this battle. The main parasite surface antigen, named Variant Surface Glycoprotein or VSG, appears to play a key role in different processes involved in the dialogue with the host.

Passive surveillance of human African trypanosomiasis in Côte d'Ivoire: Understanding prevalence, clinical symptoms and signs, and diagnostic test characteristics.

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<0.001), motor disorders (p = 0.002), convulsions (p = 0.02), severe weight loss (p = 0.02) or psychiatric problems (p = 0.04) had an increased odds (odds ratios 1.7-4.6) of being HAT RDT seropositive. Specificities ranged between 97.8%-99.6% for individual RDTs, and 93.3-98.9% for subsequent tests on dried blood spots. The PPV of the individual RDTs was below 14.3% (CI 2-43), increased to 33.3% (CI 4-78) for serial RDT combinations, and reached 67% for LAMP and ELISA/Tbg on RDT positives. Agreement between diagnostic tests was poor to moderate (Kappa ≤ 0.60), except for LAMP and ELISA/Tbg (Kappa = 0.66). CONCLUSION: Identification of five key clinical symptoms and signs may simplify referral for HAT RDT screening. The results confirm the appropriateness of the diagnostic algorithm presently applied, with screening by SD Bioline HAT or HAT Sero-K-Set, supplemented with trypanolysis. ELISA/Tbg could replace trypanolysis and is simpler to perform. TRIAL REGISTRATION: ClinicalTrials.gov NCT03356665.

Type I IFNs play a role in early resistance, but subsequent susceptibility, to the African trypanosomes.

Macrophages express a spectrum of proinflammatory and regulatory mediators during African trypanosomiasis. Microarray analyses revealed similar profiles of induced genes in macrophages stimulated with the trypanosome soluble variant surface glycoprotein in vitro and in macrophages taken from infected mice. Genes associated with the acute phase response and with type I IFN responses were prominent components of the macrophage activation profiles expressed within 72 h in vitro and in vivo. Thus, induction of proinflammatory gene expression is a characteristic of early trypanosome infection that is driven primarily by soluble variant surface glycoprotein exposure, and it may be that IFN-alpha/beta plays a central role in regulation of early resistance to trypanosomes. To test this hypothesis, we assessed parameters of infection in mouse strains with genetic alterations in the IFN-alpha/beta response pathway. We found that Ifnar1(-/-) mice, which lack the receptor for type I IFNs, exhibited delayed control of parasite burden during the first week of infection and died earlier than did wild-type controls. However, infection of Ubp43(-/-) mice, which are hyperresponsive to type I IFNs, did not exhibit enhanced resistance to trypanosomes. Instead, these animals also failed to control parasite burden and were more susceptible than wild-type animals. Additionally, the Ubp43(-/-) mice exhibited a significant defect in IFN-gamma production, which is definitively linked to host resistance in trypanosomiasis. These results show that type I IFNs play a role in early control of parasites in infected mice but may contribute to down-regulation of IFN-gamma production and subsequent loss of host resistance later in infection.