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.     

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.     

Identification of peptide mimotopes of Trypanosoma brucei gambiense variant surface glycoproteins

Background

The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests.

Methodology/Principal Findings

PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05).

Conclusions/Significance

We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.     

Isolation of Trypanosoma brucei gambiense from cured and relapsed sleeping sickness patients and adaptation to laboratory mice

Background

Sleeping sickness due to Trypanosoma brucei (T.b.) gambiense is still a major public health problem in some central African countries. Historically, relapse rates around 5% have been observed for treatment with melarsoprol, widely used to treat second stage patients. Later, relapse rates of up to 50% have been recorded in some isolated foci in Angola, Sudan, Uganda and Democratic Republic of the Congo (DRC). Previous investigations are not conclusive on whether decreased sensitivity to melarsoprol is responsible for these high relapse rates. Therefore we aimed to establish a parasite collection isolated from cured as well as from relapsed patients for downstream comparative drug sensitivity profiling. A major constraint for this type of investigation is that T.b. gambiense is particularly difficult to isolate and adapt to classical laboratory rodents.

Methodology/Principal Findings

From 360 patients treated in Dipumba hospital, Mbuji-Mayi, D.R. Congo, blood and cerebrospinal fluid (CSF) was collected before treatment. From patients relapsing during the 24 months follow-up, the same specimens were collected. Specimens with confirmed parasite presence were frozen in liquid nitrogen in a mixture of Triladyl, egg yolk and phosphate buffered glucose solution. Isolation was achieved by inoculation of the cryopreserved specimens in Grammomys surdaster, Mastomys natalensis and SCID mice. Thus, 85 strains were isolated from blood and CSF of 55 patients. Isolation success was highest in Grammomys surdaster. Forty strains were adapted to mice. From 12 patients, matched strains were isolated before treatment and after relapse. All strains belong to T.b. gambiense type I.

Conclusions and Significance

We established a unique collection of T.b. gambiense from cured and relapsed patients, isolated in the same disease focus and within a limited period. This collection is available for genotypic and phenotypic characterisation to investigate the mechanism behind abnormally high treatment failure rates in Mbuji-Mayi, D.R. Congo.     

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions

Background

At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests.

Methodology/Principal Findings

A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79.

Conclusions/Significance

We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.     

Proteomic Selection of Immunodiagnostic Antigens for Human African Trypanosomiasis and Generation of a Prototype Lateral Flow Immunodiagnostic Device

Background

The diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens.

Methodology/Principal Findings

We took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device.

Conclusions/Significance

Using a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use.     

A CATT Negative Result after Treatment for Human African Trypanosomiasis Is No Indication for Cure

Background

Cure after treatment for human African trypanosomiasis (HAT) is assessed by examination of the cerebrospinal fluid every 6 months, for a total period of 2 years. So far, no markers for cure or treatment failure have been identified in blood. Trypanosome-specific antibodies are detectable in blood by the Card Agglutination Test for Trypanosomiasis (CATT). We studied the value of a normalising, negative post-treatment CATT result in treated Trypanosoma brucei (T.b.) gambiense sleeping sickness patients as a marker of cure.

Methodology/Principal Findings

The CATT/T.b. gambiense was performed on serum of a cohort of 360 T.b. gambiense patients, consisting of 242 primary and 118 retreatment cases. The CATT results during 2 years of post-treatment follow-up were studied in function of cure or treatment failure. At inclusion, sensitivity of CATT was 98% (234/238) in primary cases and only 78% (91/117) in retreatment cases. After treatment, the CATT titre decreased both in cured patients and in patients experiencing treatment failure.

Conclusions/Significance

Though CATT is a good test to detect HAT in primary cases, a normalising or negative CATT result after treatment for HAT does not indicate cure, therefore CATT cannot be used to monitor treatment outcome.     

Elevated Levels of IFN-γ in CSF and Serum of Patients with Amyotrophic Lateral Sclerosis

Objectives

To explore whether the levels of IFN-γ in cerebral spinal fluid (CSF) and serum are elevated in ALS patients and to analyze the correlations between the IFN-γ levels and disease progression.

Methods

CSF and serum samples were obtained from 52 ALS patients and 31 non-ALS patients. The levels of IFN-γ in CSF and serum were assessed, and disease progression parameters, including the disease interval (months from onset, MFO), the revised ALS Functional Rating Scale (ALSFRS-r) score and the disease progression rate (DPR) were analyzed by registered neurologists. All samples were measured using a commercial enzyme-linked immunosorbent assay. Statistical analyses were performed using Prism software.

Results

Compared to the non-ALS patients, the ALS patients displayed significantly increased levels of IFN-γ in both CSF and serum, and these values consistently correlated with disease progression.

Conclusions

These results demonstrated that IFN-γ in CSF may serve as a biomarker of ALS differentiation and progression. CSF IFN-γ was a more reliable biomarker of disease diagnosis and progression than serum IFN-γ.     

Stage Progression and Neurological Symptoms in Trypanosoma brucei rhodesiense Sleeping Sickness: Role of the CNS Inflammatory Response

Background

Human 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 Findings

This 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-α synthesis were associated with stage progression and were mirrored by a reduction in TGF-β 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.

Conclusions

Our 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.     

Identification of sVSG117 as an Immunodiagnostic Antigen and Evaluation of a Dual-Antigen Lateral Flow Test for the Diagnosis of Human African Trypanosomiasis

Background

The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.

Methodology/Principle Findings

Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.

Conclusion/Significance

In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use.     

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

Background

Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.

Methodology/Principal Findings

We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.

Conclusions/Significance

The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.     

Human and Animal Trypanosomes in C?te d'Ivoire Form a Single Breeding Population

Background

Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and contributes to the related veterinary disease, Nagana. T. brucei is segregated into three subspecies based on host specificity, geography and pathology. T. b. brucei is limited to animals (excluding some primates) throughout sub-Saharan Africa and is non-infective to humans due to trypanolytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species. T. b. gambiense is the more prevalent human, causing over 97% of human cases. Study of T. b. gambiense is complicated in that there are two distinct groups delineated by genetics and phenotype. The relationships between the two groups and local T. b. brucei are unclear and may have a bearing on the evolution of the human infectivity traits.

Methodology/Principal Findings

A collection of sympatric T. brucei isolates from Côte d’Ivoire, consisting of T. b. brucei and both groups of T. b. gambiense have previously been categorized by isoenzymes, RFLPs and Blood Incubation Infectivity Tests. These samples were further characterized using the group 1 specific marker, TgSGP, and seven microsatellites. The relationships between the T. b. brucei and T. b. gambiense isolates were determined using principal components analysis, neighbor-joining phylogenetics, STRUCTURE, F_ST, Hardy-Weinberg equilibrium and linkage disequilibrium.

Conclusions/Significance

Group 1 T. b. gambiense form a clonal genetic group, distinct from group 2 and T. b. brucei, whereas group 2 T. b. gambiense are genetically indistinguishable from local T. b. brucei. There is strong evidence for mating within and between group 2 T. b. gambiense and T. b. brucei. We found no evidence to support the hypothesis that group 2 T. b. gambiense are hybrids of group 1 and T. b. brucei, suggesting that human infectivity has evolved independently in groups 1 and 2 T. b. gambiense.     

The Genome Sequence of Trypanosoma brucei gambiense,Causative Agent of Chronic Human African Trypanosomiasis

Background

Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.

Methods and Findings

An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.

Conclusions

This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.     

Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults

Background

An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.

Methods

Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.

Results

Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.

Conclusions

CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.     

Melarsoprol Sensitivity Profile of Trypanosoma brucei gambiense Isolates from Cured and Relapsed Sleeping Sickness Patients from the Democratic Republic of the Congo

Background

Sleeping sickness caused by Trypanosoma brucei (T.b.) gambiense constitutes a serious health problem in sub-Sahara Africa. In some foci, alarmingly high relapse rates were observed in patients treated with melarsoprol, which used to be the first line treatment for patients in the neurological disease stage. Particularly problematic was the situation in Mbuji-Mayi, East Kasai Province in the Democratic Republic of the Congo with a 57% relapse rate compared to a 5% relapse rate in Masi-Manimba, Bandundu Province. The present study aimed at investigating the mechanisms underlying the high relapse rate in Mbuji-Mayi using an extended collection of recently isolated T.b. gambiense strains from Mbuji-Mayi and from Masi-Manimba.

Methodology/Principal Findings

Forty five T.b. gambiense strains were used. Forty one were isolated from patients that were cured or relapsed after melarsoprol treatment in Mbuji-Mayi. In vivo drug sensitivity tests provide evidence of reduced melarsoprol sensitivity in these strains. This reduced melarsoprol sensitivity was not attributable to mutations in TbAT1. However, in all these strains, irrespective of the patient treatment outcome, the two aquaglyceroporin (AQP) 2 and 3 genes are replaced by chimeric AQP2/3 genes that may be associated with resistance to pentamidine and melarsoprol. The 4 T.b. gambiense strains isolated in Masi-Manimba contain both wild-type AQP2 and a different chimeric AQP2/3. These findings suggest that the reduced in vivo melarsoprol sensitivity of the Mbuji-Mayi strains and the high relapse rates in that sleeping sickness focus are caused by mutations in the AQP2/AQP3 locus and not by mutations in TbAT1.

Conclusions/Significance

We conclude that mutations in the TbAQP2/3 locus of the local T.b. gambiense strains may explain the high melarsoprol relapse rates in the Mbuji-Mayi focus but other factors must also be involved in the treatment outcome of individual patients.     

Combination of Intratumoral Invariant Natural Killer T Cells and Interferon-Gamma Is Associated with Prognosis of Hepatocellular Carcinoma after Curative Resection

Purpose

To investigate the prognostic value of intratumoral invariant natural killer T (iNKT) cells and interferon-gamma (IFN-γ) in hepatocellular carcinoma (HCC) after curative resection.

Experimental Design

Expression of TRAV10, encoding the Vα24 domain of iNKT cells, and IFN-γ mRNA were assessed by quantitative real-time polymerase chain reaction in tumor from 224 HCC patients undergoing curative resection. The prognostic value of these two and other clinicopathologic factors was evaluated.

Results

Either intratumoral iNKT cells and IFN-γ alone or their combination was an independent prognostic factor for OS (P = 0.001) and RFS (P = 0.001) by multivariate Cox proportional hazards analysis. Patients with concurrent low levels of iNKT cells and IFN-γ had a hazard ratio (HR) of 2.784 for OS and 2.673 for RFS. The areas under the curve of iNKT cells, IFN-γand their combination were 0.618 vs 0.608 vs 0.654 for death and 0.591 vs 0.604 vs 0.633 for recurrence respectively by receiver operating characteristic curve analysis. The prognosis was the worst for HCC patients with concurrent low levels of iNKT cells and IFN-γ, which might be related with more advanced pTNM stage and more vascular invasion.

Conclusions

Combination of intratumoral iNKT cells and IFN-γ is a promising independent predictor for recurrence and survival in HCC, which has a better power to predict HCC patients’ outcome compared with intratumoral iNKT cells or IFN-γ alone.     

Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Background

The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) have been recently modified by coupling to oligochromatography (OC) for easy and fast visualisation of products. In this study we evaluate the sensitivity and specificity of the PCR-OC and NASBA-OC for diagnosis of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense human African trypanosomiasis (HAT).

Methodology and Results

Both tests were evaluated in a case-control design on 143 HAT patients and 187 endemic controls from the Democratic Republic of Congo (DRC) and Uganda. The overall sensitivity of PCR-OC was 81.8% and the specificity was 96.8%. The PCR-OC showed a sensitivity and specificity of 82.4% and 99.2% on the specimens from DRC and 81.3% and 92.3% on those from Uganda. NASBA-OC yielded an overall sensitivity of 90.2%, and a specificity of 98.9%. The sensitivity and specificity of NASBA-OC on the specimens from DRC was 97.1% and 99.2%, respectively. On the specimens from Uganda we observed a sensitivity of 84.0% and a specificity of 98.5%.

Conclusions/Significance

The tests showed good sensitivity and specificity for the T. b. gambiense HAT in DRC but rather a low sensitivity for T. b. rhodesiense HAT in Uganda.     

Importance of Reciprocal Balance of T Cell Immunity in Mycobacterium abscessus Complex Lung Disease

Background

Little is known about the nature of the host immune response to Mycobacterium abscessus complex (MABC) infection. The aim of the present study was to investigate whether alterations in serum immunomolecule levels after treating MABC lung disease patients with antibiotics can reflect the disease-associated characteristics.

Methods

A total of 22 immunomolecules in 24 MABC lung disease patients before and after antibiotic therapy were quantitatively analyzed using a multiplex bead-based system.

Results

In general, the pre-treatment levels of T helper type 1 (Th1)-related cytokines, i.e., interferon (IFN)-γ and interleukin (IL)-12, and Th2-related cytokines, i.e., IL-4 and IL-13, were significantly decreased in patients compared with control subjects. In contrast, the pre-treatment levels of Th17-related cytokines, i.e., IL-17 and IL-23, were significantly increased in MABC patients. Interestingly, significantly higher levels of IFN-γ-induced protein (IP)-10 and monokine induced by IFN-γprotein (MIG) were detected in patients with failure of sputum conversion at post-treatment compared to patients with successful sputum conversion.

Conclusion

Reduced Th1 and Th2 responses and enhanced Th17 responses in patients may perpetuate MABC lung disease, and the immunomolecules IP-10 and MIG, induced through IFN-γ, may serve as key markers for indicating the treatment outcome.     

Diagnostic accuracy of PCR in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study

Background

The polymerase chain reaction (PCR) has been proposed for diagnosis, staging and post-treatment follow-up of sleeping sickness but no large-scale clinical evaluations of its diagnostic accuracy have taken place yet.

Methodology/Principal Findings

An 18S ribosomal RNA gene targeting PCR was performed on blood and cerebrospinal fluid (CSF) of 360 T. brucei gambiense sleeping sickness patients and on blood of 129 endemic controls from the Democratic Republic of Congo. Sensitivity and specificity (with 95% confidence intervals) of PCR for diagnosis, disease staging and treatment failure over 2 years follow-up post-treatment were determined. Reference standard tests were trypanosome detection for diagnosis and trypanosome detection and/or increased white blood cell concentration in CSF for staging and detection of treatment failure. PCR on blood showed a sensitivity of 88.4% (84.4–92.5%) and a specificity of 99.2% (97.7–100%) for diagnosis, while for disease staging the sensitivity and specificity of PCR on cerebrospinal fluid were 88.4% (84.8–91.9%) and 82.9% (71.2–94.6%), respectively. During follow-up after treatment, PCR on blood had low sensitivity to detect treatment failure. In cerebrospinal fluid, PCR positivity vanished slowly and was observed until the end of the 2 year follow-up in around 20% of successfully treated patients.

Conclusions/Significance

For T.b. gambiense sleeping sickness diagnosis and staging, PCR performed better than, or similar to, the current parasite detection techniques but it cannot be used for post-treatment follow-up. Continued PCR positivity in one out of five cured patients points to persistence of living or dead parasites or their DNA after successful treatment and may necessitate the revision of some paradigms about the pathophysiology of sleeping sickness.