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Identification of Trypanosome proteins in plasma from African sleeping sickness patients infected with T. b. rhodesiense.

ABSTRACT: Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a 'deep-mining" proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification.

SUBMITTER: Eyford BA

PROVIDER: S-EPMC3738533 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of Trypanosome proteins in plasma from African sleeping sickness patients infected with T. b. rhodesiense.

Eyford Brett A BA Ahmad Rushdy R Enyaru John C JC Carr Steven A SA Pearson Terry W TW

PloS one 20130808 8

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a 'deep-mining" proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma ...[more]

PMID: 23951171

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Project description:BackgroundEquine trypanosomiases are complex infectious diseases with overlapping clinical signs defined by their mode of transmission. Despite their economic impacts, these diseases have been neglected by the scientific community, the veterinary authorities and regulatory organizations. To fill the observed knowledge gap, we undertook the identification of different trypanosome species and subspecies naturally infecting horses and donkeys within the Chadian sleeping sickness focus. The objective of the study was to investigate the potential role of these domestic animals as reservoirs of the human-infective Trypanosoma brucei gambiense.MethodBlood samples were collected from 155 donkeys and 131 horses in three human African trypanosomiasis (HAT) foci in Chad. Rapid diagnostic test (RDT) and capillary tube centrifugation (CTC) test were used to search for trypanosome infections. DNA was extracted from each blood sample and different trypanosome species and subspecies were identified with molecular tools.ResultsFrom 286 blood samples collected, 54 (18.9%) and 36 (12.6%) were positive for RDT and CTC, respectively. PCR revealed 101 (35.3%) animals with trypanosome infections. The Cohen's kappa coefficient used to evaluate the concordance between the diagnostic methods were low; ranging from 0.09?±?0.05 to 0.48?±?0.07. Trypanosomes of the subgenus Trypanozoon were the most prevalent (29.4%), followed by T. congolense forest (11.5%), Trypanosoma congolense savannah (4.9%) and Trypanosoma vivax (4.5%). Two donkeys and one horse from the Maro HAT focus were found with T. b. gambiense infections. No significant differences were observed in the infection rates of different trypanosomes between animal species and HAT foci.ConclusionsThis study revealed several trypanosome species and subspecies in donkeys and horses, highlighting the existence of AAT in HAT foci in Chad. The identification of T. b. gambiense in donkeys and horses suggests considering these animals as potential reservoir for HAT in Chad. The presence of both human-infective and human non-infective trypanosomes species highlights the need for developing joint control strategies for HAT and AAT.

Project description:BackgroundHuman African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late (meningoencephalitic) stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance.Methodology/principal findingsThis was a retrospective study on a cohort of 115 T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-? synthesis were associated with stage progression and were mirrored by a reduction in TGF-? levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF.ConclusionsOur results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value.

Project description: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-?, IL1-?, TNF-?, IL-6, TGF-? 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-? (46.3), IL-6 (61.7), TGF-? (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.

Dataset Information

Identification of Trypanosome proteins in plasma from African sleeping sickness patients infected with T. b. rhodesiense.

Publications

Identification of Trypanosome proteins in plasma from African sleeping sickness patients infected with T. b. rhodesiense.

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