Interleukin (IL)-6 and IL-10 Are Up Regulated in Late Stage Trypanosoma brucei rhodesiense Sleeping Sickness

BackgroundSleeping sickness due to Trypanosoma brucei rhodesiense has a wide spectrum of clinical presentations coupled with differences in disease progression and severity across East and Southern Africa. The disease progresses from an early (hemo-lymphatic) stage to the late (meningoencephalitic) stage characterized by presence of parasites in the central nervous system. We hypothesized that disease progression and severity of the neurological response is modulated by cytokines.MethodsA total of 55 sleeping sickness cases and 41 healthy controls were recruited passively at Lwala hospital, in Northern Uganda. A panel of six cytokines (IFN-γ, IL1-β, TNF-α, IL-6, TGF-β and IL-10) were assayed from paired plasma and cerebrospinal fluid (CSF) samples. Cytokine concentrations were analyzed in relation to disease progression, clinical presentation and severity of neurological responses.ResultsMedian plasma levels (pg/ml) of IFN-γ (46.3), IL-6 (61.7), TGF-β (8755) and IL-10 (256.6) were significantly higher in cases compared to controls (p< 0.0001). When early stage and late stage CSF cytokines were compared, IL-10 and IL-6 were up regulated in late stage patients and were associated with a reduction in tremors and cranioneuropathy. IL-10 had a higher staging accuracy with a sensitivity of 85.7% (95% CI, 63.7%-97%) and a specificity of 100% (95% CI, 39.8%-100%) while for IL-6, a specificity of 100% (95% CI, 47.8%-100%) gave a sensitivity of 83.3% (95% CI, 62.2%-95.3%).ConclusionOur study demonstrates the role of host inflammatory cytokines in modulating the progression and severity of neurological responses in sleeping sickness. We demonstrate here an up-regulation of IL-6 and IL-10 during the late stage with a potential as adjunct stage biomarkers. Given that both cytokines could potentially be elevated by other CNS infections, our findings should be further validated in a large cohort of patients including those with other inflammatory diseases such as cerebral malaria.     

Clinical Profiles,Disease Outcome and Co-Morbidities among T. b. rhodesiense Sleeping Sickness Patients in Uganda

BackgroundThe acute form of Human African Trypanosomiasis (HAT, also known as Sleeping sickness) caused by Trypanosoma brucei rhodesiense has been shown to have a wide spectrum of focus specific clinical presentation and severity in East and Southern Africa. Indeed HAT occurs in regions endemic for other tropical diseases, however data on how these co-morbidities might complicate the clinical picture and affect disease outcome remains largely scanty. We here describe the clinical presentation, presence of co-infections, and how the latter impact on HAT prognosis.ConclusionsWe show a wide spectrum of sleeping sickness clinical presentation and disease outcome that was apparently not significantly influenced by concurrent infections. It would thus be interesting to determine the host and/or parasite factors that might be responsible for the observed diverse clinical presentation.     

Predicting which tropical tree species are vulnerable to forest disturbances

Tropical forest management often focuses on a few high‐value timber species because they are thought to be the most vulnerable in logged forests. However, other tree species may be vulnerable to secondary effects of logging, like loss of vertebrate dispersers. We examined vulnerability of tree species to loss of vertebrate dispersers in Mabira, a heavily disturbed tropical rainforest in Uganda. Fruit characteristics and shade tolerance regimes of 269 tree species were compiled. Stem densities of tree species producing fruits of various sizes and having different shade tolerance regimes were computed for Mabira and compared with densities of conspecifics in Budongo, a less disturbed forest with similar floral composition. Seventy per cent of tree species in Mabira are animal‐dispersed, of which 10% are large‐fruited light demanders. These species are the most vulnerable because they rarely recruit beneath adult conspecifics and are exclusively dispersed by large vertebrates, also vulnerable in heavily disturbed forests. Comparison of densities between Mabira and Budongo showed that large‐fruited light demanders had a lower density in Mabira. Other categories of tree species had similar densities in both forests. It is plausible that the low density of large‐fruited light demanders is due to limited recruitment caused by dispersal limitations.     

Discovery and Verification of Osteopontin and Beta-2-microglobulin as Promising Markers for Staging Human African Trypanosomiasis

Human African trypanosomiasis, or sleeping sickness, is a parasitic disease endemic in sub-Saharan Africa, transmitted to humans through the bite of a tsetse fly. The first or hemolymphatic stage of the disease is associated with presence of parasites in the bloodstream, lymphatic system, and body tissues. If patients are left untreated, parasites cross the blood-brain barrier and invade the cerebrospinal fluid and the brain parenchyma, giving rise to the second or meningoencephalitic stage. Stage determination is a crucial step in guiding the choice of treatment, as drugs used for S2 are potentially dangerous. Current staging methods, based on counting white blood cells and demonstrating trypanosomes in cerebrospinal fluid, lack specificity and/or sensitivity. In the present study, we used several proteomic strategies to discover new markers with potential for staging human African trypanosomiasis. Cerebrospinal fluid (CSF) samples were collected from patients infected with Trypanosoma brucei gambiense in the Democratic Republic of Congo. The stage was determined following the guidelines of the national control program. The proteome of the samples was analyzed by two-dimensional gel electrophoresis (n = 9), and by sixplex tandem mass tag (TMT) isobaric labeling (n = 6) quantitative mass spectrometry. Overall, 73 proteins were overexpressed in patients presenting the second stage of the disease. Two of these, osteopontin and β-2-microglobulin, were confirmed to be potential markers for staging human African trypanosomiasis (HAT) by Western blot and ELISA. The two proteins significantly discriminated between S1 and S2 patients with high sensitivity (68% and 78%, respectively) for 100% specificity, and a combination of both improved the sensitivity to 91%. The levels of osteopontin and β-2-microglobulin in CSF of S2 patients (μg/ml range), as well as the fold increased concentration in S2 compared with S1 (3.8 and 5.5 respectively) make the two markers good candidates for the development of a test for staging HAT patients.Human African trypanosomiasis (HAT), or sleeping sickness, is caused by an extracellular protozoan parasite of the genus Trypanosoma, which is transmitted through the bite of a tsetse fly (genus Glossina). Two morphologically identical subspecies of the parasite, are responsible for the two geographically and clinically different forms of HAT: a chronic form, widespread in West and Central Africa, caused by T. b. gambiense, and an acute form, endemic in eastern Africa, caused by T. b. rhodesiense (1). In both forms of the disease, parasites are initially localized in the blood stream, lymph, and peripheral tissues; this is the first or hemolymphatic stage (S1). During this stage, patients present generic clinical features that are common to other infectious diseases such as human immunodeficiency virus (HIV), malaria, and tuberculosis (TB), which can coexist with HAT, thus making its early diagnosis difficult (2). If treatment is not carried out, the disease progresses to the second or meningoencephalitic stage (S2) after trypanosomes cross the blood-brain barrier (BBB) and invade the central nervous system (CNS). This phase is characterized by a broad range of neurological signs that are indicative of CNS involvement (1). Diagnosis of HAT is based on parasitological demonstration of parasites in blood or lymph-node aspirate (3). All positive or suspect patients have to undergo a lumbar puncture and cerebrospinal fluid (CSF)1 examination, to determine whether they have second stage disease (4). According to the World Health Organization (WHO) guidelines, the meningoencephalitic stage is defined by the presence of parasites in CSF and/or a white blood cell (WBC) count of more than 5 cells per μl (5). Other parameters, such as intrathecal IgM production could also provide additional information to determine whether the CNS is involved (6, 7).Treatment of HAT patients varies depending on the infecting parasite and the stage of disease (5, 8). S2 drugs in current use, including melarsoprol, eflornithine, and a combination of nifurtimox and eflornithine have several limitations, such as a high rate of toxicity (melarsoprol causes death to 5% of treated patients) (9), complex logistics, and mode of administration (6, 10). Consequently, staging is a vital step in the diagnosis and treatment of HAT. However, the poor specificity or sensitivity of WBC counting and of parasitological techniques for demonstration of parasites in CSF, highlight the need for discovery of better tools for staging the disease.Several attempts have been made during the last decade to identify potential biomarkers able to discriminate between the two stages of sleeping sickness. Most of the efforts focused on cytokines and chemokines, because the patient''s immune system plays a crucial role in the brain pathology (11–14).Proteomic approaches are increasingly being applied in biomedical research and clinical medicine to investigate body fluids as a source of biomarkers (15), including the diagnosis of neurological disorders such as Alzheimer''s disease (16), Parkinson''s disease (17), and multiple sclerosis (18, 19). The protein composition of CSF is strictly regulated and can reflect the physiological or pathological state of the CNS (15). Thus in the present study, we addressed the challenge of staging HAT by analyzing CSF from T. b. gambiense patients using two complementary proteomic strategies: a classical approach based on two-dimensional gel electrophoresis (2-DE), and quantitative mass spectrometry (MS) using isobaric tandem mass tag (TMT) technology (sixplex TMT® MS/MS) (20).     

Trypanosoma brucei rhodesiense transmitted by a single tsetse fly bite in vervet monkeys as a model of human African trypanosomiasis

We have investigated the pathogenicity of tsetse (Glossina pallidipes)-transmitted cloned strains of Trypanosoma brucei rhodesiense in vervet monkeys. Tsetse flies were confirmed to have mature trypanosome infections by xenodiagnosis, after which nine monkeys were infected via the bite of a single infected fly. Chancres developed in five of the nine (55.6%) monkeys within 4 to 8 days post infection (dpi). All nine individuals were successfully infected, with a median pre-patent period of 4 (range = 4-10) days, indicating that trypanosomes migrated from the site of fly bite to the systemic circulation rapidly and independently of the development of the chancre. The time lag to detection of parasites in cerebrospinal fluid (CSF) was a median 16 (range = 8-40) days, marking the onset of central nervous system (CNS, late) stage disease. Subsequently, CSF white cell numbers increased above the pre-infection median count of 2 (range = 0-9) cells/microl, with a positive linear association between their numbers and that of CSF trypanosomes. Haematological changes showed that the monkeys experienced an early microcytic-hypochromic anaemia and severe progressive thrombocytopaenia. Despite a 3-fold increase in granulocyte numbers by 4 dpi, leucopaenia occurred early (8 dpi) in the monkey infection, determined mainly by reductions in lymphocyte numbers. Terminally, leucocytosis was observed in three of nine (33%) individuals. The duration of infection was a median of 68 (range = 22-120) days. Strain and individual differences were observed in the severity of the clinical and clinical pathology findings, with two strains (KETRI 3741 and 3801) producing a more acute disease than the other two (KETRI 3804 and 3928). The study shows that the fly-transmitted model accurately mimics the human disease and is therefore a suitable gateway to understanding human African trypanosomiasis (HAT; sleeping sickness).     

Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal

Background

The development of arsenical and diamidine resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 purine transporter as a result of alterations in the corresponding T. brucei adenosine transporter 1 gene (TbAT1). Previously, specific TbAT1 mutant type alleles linked to melarsoprol treatment failure were significantly more prevalent in T. b. gambiense from relapse patients at Omugo health centre in Arua district. Relapse rates of up to 30% prompted a shift from melarsoprol to eflornithine (α-difluoromethylornithine, DFMO) as first-line treatment at this centre. The aim of this study was to determine the status of TbAT1 in recent isolates collected from T. b. gambiense sleeping sickness patients from Arua and Moyo districts in Northwestern Uganda after this shift in first-line drug choice.

Methodology and results

Blood and cerebrospinal fluids of consenting patients were collected for DNA preparation and subsequent amplification. All of the 105 isolates from Omugo that we successfully analysed by PCR-RFLP possessed the TbAT1 wild type allele. In addition, PCR/RFLP analysis was performed for 74 samples from Moyo, where melarsoprol is still the first line drug; 61 samples displayed the wild genotype while six were mutant and seven had a mixed pattern of both mutant and wild-type TbAT1. The melarsoprol treatment failure rate at Moyo over the same period was nine out of 101 stage II cases that were followed up at least once. Five of the relapse cases harboured mutant TbAT1, one had the wild type, while no amplification was achieved from the remaining three samples.

Conclusions/significance

The apparent disappearance of mutant alleles at Omugo may correlate with melarsoprol withdrawal as first-line treatment. Our results suggest that melarsoprol could successfully be reintroduced following a time lag subsequent to its replacement. A field-applicable test to predict melarsoprol treatment outcome and identify patients for whom the drug can still be beneficial is clearly required. This will facilitate cost-effective management of HAT in rural resource-poor settings, given that eflornithine has a much higher logistical requirement for its application.     

A Combined CXCL10, CXCL8 and H-FABP Panel for the Staging of Human African Trypanosomiasis Patients

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic tropical disease. It progresses from the first, haemolymphatic stage to a neurological second stage due to invasion of parasites into the central nervous system (CNS). As treatment depends on the stage of disease, there is a critical need for tools that efficiently discriminate the two stages of HAT. We hypothesized that markers of brain damage discovered by proteomic strategies and inflammation-related proteins could individually or in combination indicate the CNS invasion by the parasite.

Methods

Cerebrospinal fluid (CSF) originated from parasitologically confirmed Trypanosoma brucei gambiense patients. Patients were staged on the basis of CSF white blood cell (WBC) count and presence of parasites in CSF. One hundred samples were analysed: 21 from stage 1 (no trypanosomes in CSF and ≤5 WBC/µL) and 79 from stage 2 (trypanosomes in CSF and/or >5 WBC/µL) patients. The concentration of H-FABP, GSTP-1 and S100β in CSF was measured by ELISA. The levels of thirteen inflammation-related proteins (IL-1ra, IL-1β, IL-6, IL-9, IL-10, G-CSF, VEGF, IFN-γ, TNF-α, CCL2, CCL4, CXCL8 and CXCL10) were determined by bead suspension arrays.

Results

CXCL10 most accurately distinguished stage 1 and stage 2 patients, with a sensitivity of 84% and specificity of 100%. Rule Induction Like (RIL) analysis defined a panel characterized by CXCL10, CXCL8 and H-FABP that improved the detection of stage 2 patients to 97% sensitivity and 100% specificity.

Conclusion

This study highlights the value of CXCL10 as a single biomarker for staging T. b. gambiense-infected HAT patients. Further combination of CXCL10 with H-FABP and CXCL8 results in a panel that efficiently rules in stage 2 HAT patients. As these molecules could potentially be markers of other CNS infections and disorders, these results should be validated in a larger multi-centric cohort including other inflammatory diseases such as cerebral malaria and active tuberculosis.     

Identification of coding sequences from a freshly prepared Trypanosoma brucei brucei expression library by polymerase chain reaction

Animal African trypanosomiasis (AAT) also known as Nagana is a devastating disease among domestic animals in large parts of Sub-Saharan Africa causing loses in milk and meat production as well as traction power. However, there is currently no commercial vaccine against AAT. The parasites have also developed resistance to some of the drugs in use. Moreover, the use of affordable computer-aided wet bench methods in the search for vaccine and/or new drug targets against this disease have not yet been fully explored in developing countries. This study, therefore, explored the use of PCR to screen a freshly prepared bloodstream form Trypanosoma brucei brucei (T. b. brucei) expression library for coding sequences followed by bioinformatics analyses specifying the functions and importance of these proteins to parasite survival. Eleven protein coding sequences were identified from twenty nine purified clones. The putative retro transposon hot spot protein 4 (RHSP 4) was the only protein with a fully annotated DNA sequence. All the others were hypothetical or had partial or unqualified annotations. RHSP 4 and pyruvate dehydrogenase E1 component, alpha sub-unit (PDE1α) are involved in aerobic respiration whereas succinyl-Co A-3-ketoacid-coenzyme A transferase mitochondrial precursor (SKTMP) is predicted to be involved in ketone body catabolism. Cystathionine beta-synthase (CBS) and alpha-1,3-mannosyltransferase (αMT) have been predicted in cysteine biosynthesis and vesicular transport respectively. The functions of the hypothetical proteins encountered have neither been experimentally determined nor predicted. We hypothesize that both CBS and PDE1α are good drug targets. Overall, about 300 plates are required to PCR screen the entire Trypanosoma brucei genome in approximately eight months. This method is therefore, applicable and affordable in the search for new drug targets under conditions of limited resources among developing countries.