In individuals with tuberculosis-infection – until recently referred to as latent tuberculosis infection – the risk of progression to active tuberculosis (reactivation) varies strongly. Among those... Show moreIn individuals with tuberculosis-infection – until recently referred to as latent tuberculosis infection – the risk of progression to active tuberculosis (reactivation) varies strongly. Among those at increased risk of reactivation are patients with an impaired immune system, e.g. due to immunosuppressive therapy. Therefore, prior to planned immunosuppression, patients are screened for tuberculosis-infection and subsequently treated in case of infection. Current screening methods include the Mantoux test, Interferon-γ release assays (i.e., the QuantiFERON-TB Gold Plus and T-SPOT.TB) and chest X-ray. However, despite screening, cases of reactivation continue to occur – in part due to the lack of a gold standard test for tuberculosis-infection. Therefore, the aims of this thesis were to increase the diagnostic sensitivity for tuberculosis-infection prior to immunosuppression. Using various (novel) methods we showed that approximately two-thirds of all QuantiFERON-TB Gold Plus results just below the manufacturer’s cut-off (in the borderline range) are caused by Mycobacterium tuberculosis-infection, which now warrants preventive treatment in patients with such a result. Furthermore, we quantified the diagnostic accuracy of chest X-ray for tuberculosis-infection and showed that using a novel ultra-low dose CT scanning technique, sensitivity for tuberculosis-infection could be significantly increased by three-fold compared to chest X-ray. 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
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
Belay, M.; Legesse, M.; Mihret, A.; Ottenhoff, T.H.M.; Franken, K.L.; Bjune, G.; Abebe, F. 2016
