BackgroundTo improve tuberculosis (TB) diagnosis, the World Health Organisation (WHO) has called for a non-sputum based triage test to focus TB testing on people with a high likelihood of having... Show moreBackgroundTo improve tuberculosis (TB) diagnosis, the World Health Organisation (WHO) has called for a non-sputum based triage test to focus TB testing on people with a high likelihood of having active pulmonary tuberculosis (TB). Various host or pathogen biomarker-based testing devices are in design stage and require validity assessment. Host biomarkers have shown promise to accurately rule out active TB, but further research is required to determine generalisability. The TriageTB diagnostic test study aims to assess the accuracy of diagnostic test candidates, as well as field-test, finalise the design and biomarker signature, and validate a point-of-care multi-biomarker test (MBT).MethodsThis observational diagnostic study will evaluate sensitivity and specificity of biomarker-based diagnostic candidates including the MBT and Xpert® TB Fingerstick cartridge compared with a gold-standard composite TB outcome classification defined by symptoms, sputum GeneXpert® Ultra, smear and culture, radiological features, response to TB therapy and presence of an alternative diagnosis. The study will be conducted in research sites in South Africa, Uganda, The Gambia and Vietnam which all have high TB prevalence. The two-phase design allows for finalisation of the MBT in Phase 1 in which candidate host proteins will be evaluated on stored serum from Asia, South Africa and South America and on fingerstick blood from 50 newly recruited participants per site. The MBT test will then be locked down and validated in Phase 2 on 250 participants per site.DiscussionBy targeting confirmatory TB testing to those with a positive triage test, 75% of negative GXPU may be avoided, thereby reducing diagnostic costs and patient losses during the care cascade. This study builds on previous biomarker research and aims to identify a point-of-care test meeting or exceeding the minimum World Health Organisation target product profile of a 90% sensitivity and 70% specificity. Streamlining TB testing by identifying individuals with a high likelihood of TB should improve TB resources use and, in so doing, improve TB care. Show less
Introduction: The leptin signaling pathway plays an important role as a key regulator of glucose homeostasis, metabolism control and systemic inflammatory responses. However, the metabolic effects... Show moreIntroduction: The leptin signaling pathway plays an important role as a key regulator of glucose homeostasis, metabolism control and systemic inflammatory responses. However, the metabolic effects of leptin on infectious diseases, for example tuberculosis (TB), are still little known. Objectives: In this study, we aim to investigate the role of leptin on metabolism in the absence and presence of mycobacterial infection in zebrafish larvae and mice. Methods: Metabolites in entire zebrafish larvae and the blood of mice were studied using high-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and mass spectrometry, respectively. For transcriptome studies of zebrafish larvae, deep RNA sequencing was used. Results: The results show that leptin mutation leads to a similar metabolic syndrome as caused by mycobacterial infection in the two species, characterized by the decrease of 11 amine metabolites. In both species, this metabolic syndrome was not aggravated further when the leptin mutant was infected by mycobacteria. Therefore, we conclude that leptin and mycobacterial infection are both impacting metabolism non-synergistically. In addition, we studied the transcriptomes of lepb(ibl54) mutant zebrafish larvae and wild type (WT) siblings after mycobacterial infection. These studies showed that mycobacteria induced a very distinct transcriptome signature in the lepb(ibl54) mutant zebrafish compared to WT sibling control larvae. Furthermore, lepb(ibl55) Tg (pck1:luc1) zebrafish line was constructed and confirmed this difference in transcriptional responses. Conclusions: Leptin mutation and TB lead non-synergistically to a similar metabolic syndrome. Moreover, different transcriptomic responses in the lepb(ibl54) mutant and TB can lead to the similar metabolic end states. Show less
Doorn, C.L.R. van; Eckold, C.; Ronacher, K.; Ruslami, R.; Veen, S. van; Lee, J.S.; ... ; TANDEM Consortium 2022
Background Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients.... Show moreBackground Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients. Treatment success is lower among people with diabetes mellitus (DM). Predicting treatment outcome early after diagnosis, especially in TB-DM patients, would allow early treatment adaptation for individuals and may improve global TB control.Methods Samples were collected in a longitudinal cohort study of adult TB patients from South Africa (n = 94) and Indonesia (n = 81), who had concomitant DM (n = 59), intermediate hyperglycaemia (n = 79) or normal glycaemia/no DM (n = 37). Treatment outcome was monitored, and patients were categorized as having a good (cured) or poor (failed, recurrence, died) outcome during treatment and 12 months follow-up. Whole blood transcriptional profiles before, during and at the end of TB treatment were characterized using unbiased RNA-Seq and targeted gene dcRT-MLPA.Findings We report differences in whole blood transcriptome profiles, which were observed before initiation of treatment and throughout treatment, between patients with a good versus poor TB treatment outcome. An eight-gene and a 22-gene blood transcriptional signature distinguished patients with a good TB treatment outcome from patients with a poor TB treatment outcome at diagnosis (AUC = 0.815) or two weeks (AUC = 0.834) after initiation of TB treatment, respectively. High accuracy was obtained by cross-validating this signature in an external cohort (AUC = 0.749).Interpretation These findings suggest that transcriptional profiles can be used as a prognostic biomarker for treatment failure and success, even in patients with concomitant DM. Copyright (C) 2022 The Authors. Published by Elsevier B.V. Show less
Background: Antibodies against mycobacterial proteins are highly specific, but lack sensitivity, whereas cytokines have been shown to be sensitive but not very specific in the diagnosis of... Show moreBackground: Antibodies against mycobacterial proteins are highly specific, but lack sensitivity, whereas cytokines have been shown to be sensitive but not very specific in the diagnosis of tuberculosis (TB). We assessed combinations between antibodies and cytokines for diagnosing TB. Methods: Immuoglubulin (Ig) A and IgM antibody titres against selected mycobacterial antigens including Apa, NarL, Rv3019c, PstS1, LAM, "Kit 1" (MTP64 and Tpx)", and "Kit 2" (MPT64, Tpx and 19 kDa) were evaluated by ELISA in plasma samples obtained from individuals under clinical suspicion for TB. Combinations between the antibody titres and previously published cytokine responses in the same participants were assessed for diagnosing active TB. Results: Antibody responses were more promising when used in combination (AUC of 0.80), when all seven antibodies were combined. When anti-"Kit 1"-IgA levels were combined with five host cytokine biomarkers, the AUC increased to 97% (92-100%) with a sensitivity of 95% (95% CI, 73-100%), and specificity of 88.5% (95% CI, 68.7-97%) achieved after leave-one-out cross validation. Conclusion: When used in combination, IgA titres measured with ELISA against multiple Mycobacterium tuberculosis antigens may be useful in the diagnosis of TB. However, diagnostic accuracy may be improved if the antibodies are used in combination with cytokines. Show less
Gela, A.; Murphy, M.; Rodo, M.; Hadley, K.; Hanekom, W.A.; Boom, W.H.; ... ; Delayed BCG Study Team 2022
Background: Non-protein antigen classes can be presented to T cells by near-monomorphic antigen-presenting molecules such as CDI, MRI, and butyrophilin 3AI. Such T cells, referred to as donor... Show moreBackground: Non-protein antigen classes can be presented to T cells by near-monomorphic antigen-presenting molecules such as CDI, MRI, and butyrophilin 3AI. Such T cells, referred to as donor unrestricted T (DURT) cells, typically express stereotypic T cell receptors. The near-unrestricted nature of DURT cell antigen recognition is of particular interest for vaccine development, and we sought to define the roles of DURT cells, including MRI-restricted MAIT cells, CDth-restricted glucose monomycolate (GMM)-specific T cells, CDth-restricted NKT cells, and gamma delta T cells, in vaccination against Mycobacterium tuberculosis. Methods: We compared and characterized DURT cells following primary bacille Calmette-Guerin (BCG) vaccination in a cohort of vaccinated and unvaccinated infants, as well as before and after BCG-revaccination in adults. Findings: BCG (re)vaccination did not modulate peripheral blood frequencies, T cell activation or memory profiles of MAIT cells, CDth-restricted GMM-specific and germline-encoded mycolyl-reactive (GEM) cells or CDId-restricted NKT cells. By contrast, primary BCG vaccination was associated with increased frequencies of gamma delta T cells as well as a novel subset of CD26(+)CDI6I(+)TRAVI-2(-) IFN-gamma-expressing CD4(+) T cells in infants. Interpretation: Our findings, that most DURT cell populations were not modulated by BCG, do not preclude a role of BCG in modulating other qualitative aspects of DURT cells. More studies are required to understand the full potential of DURT cells in new TB vaccine strategies. Copyright (C) 2022 The Authors. Published by Elsevier B.V. Show less
The zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of... Show moreThe zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of granulomas, the inflammatory lesions characteristic of mycobacterial disease. The optically transparent zebrafish larvae provide a window on the initial stages of granuloma development in the context of innate immunity. Application of fluorescent dyes and transgenic markers enabled real-time visualization of how innate immune mechanisms, such as autophagy and inflammasomes, are activated in infected macrophages and how propagating calcium signals drive communication between macrophages during granuloma formation. A combination of imaging, genetic, and chemical approaches has revealed that the interplay between macrophages and mycobacteria is the main driver of tissue dissemination and granuloma development, while neutrophils have a protective function in early granulomas. Different chemokine signaling axes, conserved between humans and zebrafish, have been shown to recruit macrophages permissive to mycobacterial growth, control their microbicidal capacity, drive their spreading and aggregation, and mediate granuloma vascularization. Finally, zebrafish larvae are now exploited to explore cell death processes, emerging as crucial factors in granuloma expansion. In this review, we discuss recent advances in the understanding of mycobacterial pathogenesis contributed by zebrafish models. Show less
Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm)... Show moreTuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells. Show less
Objectives: IFN gamma-release assays (IGRAs) used for diagnosis of Mycobacterium (M.) tuberculosis infection have limited sensitivity. Alternative cytokines and M. tuberculosis latency-associated... Show moreObjectives: IFN gamma-release assays (IGRAs) used for diagnosis of Mycobacterium (M.) tuberculosis infection have limited sensitivity. Alternative cytokines and M. tuberculosis latency-associated antigens may improve immune-based tests.Methods: Multiplex cytokine analyses was done in culture supernatants after 6-day in vitro restimulation with M. tuberculosis IGRA and latency-associated antigens (i.e. Rv2628, Rv1733) in tuberculosis patients (n = 22) and asymptomatic contacts (AC)s (n = 20) from Ghana.Results: Four cytokines (i.e. IFN gamma, IP-10, IL-22 and IL-6) were significantly increased after IGRA-antigen specific restimulation. IFN gamma, IP-10, and IL-22 correlated positively and showed no differences between the study groups whereas IGRA-antigen induced IL-6 was significantly higher in tuberculosis patients. Using adjusted IGRA criteria, IL-6 showed the highest sensitivity for detection of tuberculosis patients (91%) and ACs (85%) as compared to IFN gamma, IP-10, and IL-22. Rv2628 and Rv1733 restimulation induced significantly higher IFN gamma, IP-10, and IL-22 concentrations in ACs. Combined antigen/cytokine analyses identified study group specific patterns and a combination of Rv2628/Rv1733 induced IFN gamma with IGRA-antigen induced IL-6 was optimal for classification of tuberculosis patients and ACs (AUC: 0.92, p<0.0001).Conclusions: We demonstrate the potency of alternative cytokines, especially IL-6, and latency-associated antigens Rv1733/Rv2628 to improve detection of M. tuberculosis infection and to classify tuberculosis patients and healthy contacts. (C) 2020 The British Infection Association. Published by Elsevier Ltd. All rights reserved. Show less
Background: Medical schools offer students the opportunity to perform international electives. This study aimed to assess health risks among medical students, to tailor institutional guidelines... Show moreBackground: Medical schools offer students the opportunity to perform international electives. This study aimed to assess health risks among medical students, to tailor institutional guidelines.Methods: Multicenter study at Dutch and Belgian universities, among medical students who visited low- or middle-income countries. Students completed four questionnaires: once before the elective and two weeks, three- and six months after return.Results: Data was complete for 479 students (follow-up rate 84%). Most traveled to Surinam (29%) and South-Africa (14%). Half of the students encountered difficulties in adapting to local culture. Almost 40% visited malaria endemic countries. Nearly all (87%) used chemoprophylaxis as prescribed. Definite needle-stick or splash injuries were reported by 7%. All were dealt with adequately in accordance with national guidelines. However, less than half of 24 possible incidents were handled adequately. Two-and-a-half percent had unprotected sex with a new partner. The incidence of travelers' diarrhea (TD) was 46%. In those with TD, the incidence of post-travel new-onset abdominal complaints was 3%. Three percent were involved in a minor traffic accident, 18% were injured during leisure activities, 5% were threatened or experienced physical violence. Only half of the students visiting a highly endemic country were screened for tuberculosis post-travel. For schistosomiasis this was 6%.Conclusions: Students abroad are exposed to medical and non-medical challenges, which should be addressed during pre-travel counseling. Contact details of a professional back home should be provided, so students can confer in case of problems while abroad. Lastly, we recommend a centrally organized post-travel health check. Show less
Background: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify... Show moreBackground: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify novel candidate host biomarkers. SERPING1, which encodes C1-inhibitor (C1-INH), the natural inhibitor of the C1-complex has emerged as candidate biomarker. Here we collated and analysed SERPING1 expression data and subsequently determined C1-INH protein levels in four cohorts of patients with TB.Methods: SERPING1 expression data were extracted from online deposited datasets. C1-INH protein levels were determined by ELISA in sera from individuals with active TB, LTBI as well as other disease controls in geographically diverse cohorts.Findings: SERPING1 expression was increased in patients with active TB compared to healthy controls (8/11 cohorts), LTBI (13/14 cohorts) and patients with other (non-TB) lung-diseases (7/7 cohorts). Serum levels of C1-INH were significantly increased in The Gambia and Italy in patients with active TB relative to the endemic controls but not in South Africa or Korea. In the largest cohort (n = 50), with samples collected longitudinally, normalization of C1-INH levels following successful TB treatment was observed. This cohort, also showed the most abundant increase in C1-INH, and a positive correlation between C1q and C1-INH levels. Combined presence of increased levels of both C1q and C1-INH had high specificity for active TB (96 %) but only very modest sensitivity 38 % compared to the endemic controls.Interpretation: SERPING1 transcript expression is increased in TB patients, while serum protein levels of C1-INH were increased in half of the cohorts analysed. Show less
Tanner, R.; Smith, S.G.; Meijgaarden, K.E. van; Giannoni, F.; Wilkie, M.; Gabriele, L.; ... ; McShane, H. 2019
A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of... Show moreA major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2 ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-gamma) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be < 5% coefficient of variation (CV), intermediate precision to be < 20% CV, and inter-site reproducibility to be < 30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development. Show less
Conclusions: We showed that five genes (CD8A, TIMP2, CCL22, FCGR1A and TNFRSF1A), specifically FCGR1A and CCL22 have the potential to discriminate active TB from non-active TB in HIV patients in... Show moreConclusions: We showed that five genes (CD8A, TIMP2, CCL22, FCGR1A and TNFRSF1A), specifically FCGR1A and CCL22 have the potential to discriminate active TB from non-active TB in HIV patients in Ethiopia and could be used to improve diagnostic tools for active TB in HIV patients, and to understand the pathogenesis of TB/HIV coinfection. (C) 2016 Published by Elsevier Ltd. Show less
In cancer and chronic infectious diseases, immune checkpoint-blockade of inhibitory receptors can enhance T-cell immunity. In tuberculosis (TB), a chronic infectious disease, prolonged antigen... Show moreIn cancer and chronic infectious diseases, immune checkpoint-blockade of inhibitory receptors can enhance T-cell immunity. In tuberculosis (TB), a chronic infectious disease, prolonged antigen exposure can potentially drive terminal T-cell differentiation towards functional 'exhaustion': in human TB T-cells express PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein-4). However, in murine TB not PD-1 but rather killer cell lectin-like receptor subfamily-G1 (KLRG1) was a superior indicator of terminal T-cell differentiation. We therefore compared expression of KLRG1, PD-1 and CTLA-4 on T-cells in different stages of human TB, and also analysed their induction following BCG-vaccination. KLRG1, PD-1 and CTLA-4-expression were highest on in vitro BCG-stimulated CD4(+) T-cells following recent TB-treatment; KLRG1 and PD-1-expression on CD4(+) T-cells in active - but not latent TB were only slightly increased compared to healthy donors. BCG-vaccination induced KLRG1-expression on BCG-stimulated CD8(+) but not CD4(+) T-cells, while neither PD-1 nor CTLA-4-expression increased. KLRG1-expressing CD8(+) T-cells exhibited markedly decreased proliferation, whereas PD-1(+) T-cells proliferated after in vitro BCG-stimulation. Thus, we demonstrate the presence of increased KLRG1-expressing T-cells in TB-treated individuals, and present KLRG1 as a marker of decreased human T-cell proliferation following BCG-vaccination. These results expand our understanding of cell-mediated immune control of mycobacterial infections. (c) 2015 Elsevier Ltd. All rights reserved. Show less
