Browsing by Author "Scriba, Thomas"
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- ItemOpen AccessA retrospective review of risk factors for recalcitrant peptic structures(2025) Ndlebe, Babalwa; Chinnery, Galya; Scriba, ThomasIntroduction: Peptic strictures (PS) are a common benign cause of dysphagia, but a scarcity of local data is available as regards identifying risk factors associated with recalcitrancy. Methods: Single centre retrospective audit of PS undergoing endoscopic management between 1st March 2018 and 1st March 2022, aiming to identify recalcitrancy risk factors. Results: Of 69 patients (37 male, 53.4%) with PS, 27 (39.1%) were diagnosed with recalcitrant strictures. Most strictures were positioned distally (53; 76.8%) with an associated hiatus hernia in 52 (75.4%). While comorbidities were not associated with recalcitrancy, younger age was a risk factor (recalcitrant stricture group median age 51 (IQR 38.5-61.0 years) versus non-recalcitrant group median age 62.5 (IQR 48.5-70.8 years); p=0.044). Although HIV status did not affect recalcitrancy risk, taking oral antiretrovirals (ARVs) was significantly associated with PS recalcitrancy (p=0.032; OR 4.55). Presenting degree of dysphagia (p<0.001; OR 16), requiring more than 3 dilatations (p<0.001), and smaller index residual oesophageal lumen (p<0.001) were all significantly associated with stricture recalcitrancy. Fourteen patients were temporarily stented (having a total of 24 stents placed). Thirteen patients had post endoscopic complications with most of these complications occurring amongst the recalcitrant group (n=11). Four complications occurred during endoscopy, two partial thickness tears managed endoscopically, a gastric perforation requiring an over-the-scope-clip closure and one sedation related hypoxia requiring a short period of bag-mask-valve ventilation and sedation-reversal. Two deaths occurred in the cohort; one from a suspected aspiration pneumonia five days after last dilatation and one from a suspected missed oesophageal perforation (2.3% immediate endoscopic intervention complication rate for 265 dilatations performed). Conclusion: Locally pill oesophagitis related to ARVs has been identified as a potential cause of recalcitrancy; identifying at-risk patients early may allow for management adjustments to improve outcomes.
- ItemOpen AccessBCG-specific T cell proliferation and cytotoxic capacity in infants as risk of tuberculosis disease, following newborn BCG vaccination(2014) Keyser, Alana; Hanekom, Willem A; Scriba, ThomasBCG is the only vaccine against tuberculosis and has been used for over 90 years. BCG efficacy is variable, especially in countries with high TB prevalence, where over a million deaths due to tuberculosis, are still reported annually. New TB vaccines are under development to either replace or boost the BCG vaccine. However, our understanding of the immune response required for protection against TB disease, remains inadequate. Identification of a protective immune response is only possible in a clinical trial of an efficacious vaccine, allowing comparison of vaccine-induced immune responses in protected and unprotected individuals. In the absence of such a vaccine, as is the case with TB, we can only explore biomarkers of risk of disease. The most commonly measured outcomes of anti-mycobacterial immunity in clinical trials, specific Th1 cells, are typically thought to be protective in TB. However, to date, human mycobacteria-specific Th1 responses have not correlated with risk of TB disease. New approaches are urgently required to identify other factors at play in conferring protection against TB. In this thesis, we explored BCG-specific cytotoxic T cells as candidate correlates of risk of TB disease in BCG-vaccinated infants. We hypothesized that reduced production of cytotoxic molecules by T cells in response to BCG are associated with risk of developing TB disease. We designed a case/control study nested within a large trial of newborn BCG-vaccination.Blood was collected at 10 weeks and infants, were followed up for two years.We compared outcomes in infants ultimately diagnosed with TB (at risk of TB disease) and two groups of healthy infants (not at risk of TB disease), the first group had household contact with TB cases, the second group were randomly selected from the community, which is endemic for TB. Amongst these groups, we designated a training and a test cohort to allow validation of candidate correlates of risk of TB.
- ItemOpen AccessCharacterisation of a public T cell clonotype enriched in m.tb infected individuals who do not progress to tuberculosis(2025) Matela, Motshidisi; Musvosvi, Munyaradzi; Scriba, ThomasIntroduction: T cells are known to play an important role in controlling M. Tuberculosis (M.tb) infection, but it is not known if specific T cell clones contribute to the outcome of M.tb infection. We recently completed a bulk T cell receptor (TCR) sequencing screen and observed that frequencies of a particular donor-unrestricted T cell (DURT) clone were higher in healthy, M.tb infected individuals who controlled infection (controllers), than individuals who progressed to tuberculosis (TB) (progressors). This clone expresses a common complementarity-determining region 3 (CDR3) α sequence, which we termed “kiif.tb”. Frequencies of kiif.tb T cells were also higher in healthy M.tb infected individuals than TB patients in another bulk T cell TCR sequencing screen in a different cohort. Objective: In my project, we aimed to confirm if healthy, M.tb infected individuals (i.e. IGRA+) have higher frequencies of kiif.tb expressing T cells compared to healthy uninfected individuals (i.e. IGRA-) or individuals with active TB. We also aimed to identify cost-effective methods for detecting kiif.tb T cells in peripheral blood mononuclear cells (pbmcs) and assess the activation phenotype of kiif.tb in IGRA-, IGRA+, and TB patients. Methods: A custom-designed digital PCR (dpcr) assay was used to quantify the frequencies of total T cells and kiif.tb T cells from IGRA-, IGRA+, and TB patients. Identification and characterisation of kiif.tb T cells by flow cytometry was performed using a custom flow cytometry-compatible RNA hybridization assay (primeflowtm) and custom monoclonal antibodies were generated from mice immunised with kiif.tb sequence. Results: Total T cell frequencies measured by dpcr in all participants correlated strongly with both bulk TCR sequencing (rho = 0.74, p-value = 0.02) and flow cytometry (rho = 0.75, p = 5x106). However, the correlation between kiif.tb T cell frequencies measured by dpcr and kiif.tb CDR3a measured using bulk TCR sequencing was modest (rho = 0.48, p-value = 0.01). Kiif.tb T cell frequencies were not significantly higher in IGRA+ individuals with recent or remote M.tb infection compared to IGRA- controls (p = 0.4 and 0.31, respectively), nor when compared to individuals with TB disease (p-value = 0.39 and 0.27, respectively). Frequencies of kiif.tb T cells, quantified by custom monoclonal antibody (clone39a9d4) staining, were not different across study groups. However, active TB patients had higher frequencies of kiif.tb T cells expressing HLA-DR compared to IGRA- controls (p-value = 0.02). We did not include IGRA+ individuals, because only a single individual in this group had sufficient kiif.tb cells for phenotyping. Frequencies of bulk T cells expressing HLA-DR were also higher TB patients than to IGRA+ individuals (p-value = 0.05). Discussion: Our results suggest that kiif.tb-specific T cell frequencies measured by dpcr, or monoclonal antibodies were not different between the clinical groups, contrary to our hypothesis. The custom dpcr assay may only accurately detect targets at higher abundances, like total T cells (30-70%), limiting accuracy for quantifying kiif.tb T cells, which occur at abundances 1000-fold lower (0-0.04%). Next steps involve validating initial findings using bulk TCR sequencing and further optimizing the monoclonal antibody staining for kiif.tb T cell quantification by flow cytometry. Notably, our HLA-DR data suggest that kiif.tb T cells are highly activated in patients with active TB, which may suggest that these DURT cells recognise M.tb antigen. With further optimization, such an antibody could useful for developing novel T-cell-based TB biomarkers
- ItemOpen AccessCharacterisation of T cell specificity, functional, activation and memory profiles associated with QuantiFERON TB Gold conversion and reversion(2021) Mpande, Cheleka Anne-Marie; Nemes, Elisa; Scriba, Thomas; Rozot, VirginieRecent acquisition of Mycobacterium tuberculosis (M.tb) infection is associated with a higher risk of tuberculosis disease, compared with remote, asymptomatic infection. M.tb infection, defined by a positive tuberculin skin test (TST) and/or IFN--release assay [IGRA e.g. QuantiFERON TB GOLD (QFT)], is commonly thought to be a chronic state. However, longitudinal studies have demonstrated the dynamic nature of M.tb infection, whereby TSTs and IGRAs revert from a positive to a negative test in some individuals, possibly an indication of bacterial clearance. Despite the first observation of discordant serial TST results over 80 years ago and the wide use of TSTs/IGRAs, there is still a limited understanding of immunological features associated with different stages of M.tb infection and discordant serial TST/IGRA results. Most studies of M.tb-specific immune responses in humans are based on cross-sectional comparisons between M.tb infection and active disease, with very few large cohort studies enabling a longitudinal assessment of different phases of infection. Thus, the main objective of this thesis was to gain a better understanding of changes in M.tbspecific CD4 T cell functional, memory and activation profiles associated with QFT conversion (acquisition of M.tb infection) and reversion (potential M.tb clearance). Our first aim was to characterise the homing, cytotoxic and functional capacity of M.tbspecific memory CD4+ T cells during recent and remote M.tb infection, with a special focus on stem cell memory T (TSCM) cells. TSCM cells play a critical role in maintaining long-lasting immunity, demonstrated by their superior longevity, proliferation and differentiation capacity compared to central memory (TCM) and effector (TE) cells. Before this study, our knowledge of TSCM cells was primarily based on virus-specific CD8+ TSCM cells. We demonstrate that M.tb-specific CD4+ TSCM cells are induced upon recent M.tb infection and maintained at steady-state during established infection. Despite being the least differentiated M.tb-specific memory subset and representing 2 years) M.tb infection, we also aimed to define an M.tb- specific T cell biomarker that can distinguish between the two infection states as current diagnostics fail to do so. Our second major finding demonstrated that recently infected individuals have lower proportions of highly differentiated IFN-+TNF+KLRG-1+ CD4+ TE cells and higher proportions of early differentiated IFN--TNF+IL2+KLRG-1- CD4+ T cells than remotely infected individuals in response to M.tb lysate but not CFP-10/ESAT-6 stimulation. Akin to their recent M.tb exposure, recently infected individuals had higher levels of T cell activation, regardless of M.tb antigen specificity, than remotely infected individuals. The degree of M.tb-specific CD4 T cell activation was identified as the best candidate biomarker for recent infection. The very same biomarker could also distinguish between progressors and non-progressors and identify individuals with active tuberculosis disease among healthy individuals with remote M.tb infection. We propose that, upon large-scale clinical validation, the T cell activation biomarker could be used as a screening test in conjunction with current tuberculosis diagnostics to guide the provision of either preventive or full tuberculosis therapy. These results have very important implications for targeting provision of preventive treatment to M.tb infected individuals at high-risk of tuberculosis, which is one of the top 10 strategies required to achieve tuberculosis elimination targets. Based on data from observational studies conducted during the pre-antibiotic era and guinea pig tuberculosis models, TST/IGRA reversion in humans is hypothesised to be associated with spontaneous (natural) clearance of infection. Similarly, individuals recently exposed to patients with tuberculosis who did not convert TST/IGRA (termed resistors) nor develop disease had M.tb-specific T cell responses that did not include IFN- production. However, clearance of M.tb infection is virtually impossible to demonstrate in healthy individuals. We clearly illustrated that acquisition infection is associated with induction of CD4+ Th1 functional and memory T cell subsets associated with increased antigen burden. We thus hypothesised that if reversion represents natural M.tb infection clearance then immune responses post-QFT reversion, if detectable, would be predominantly TSCM/TCM cells that have an IFN- independent cytokine expression profile and low T cell activation levels. Interestingly, QFT reversion was not associated with a decrease in CFP-10/ESAT-6-specific IFN-+ CD4 T cell responses detected by flow cytometry. Overall, CD4 T cell responses to CFP-10/ESAT-6 in reverters were of intermediate magnitude between non-converters and remotely infected individuals. These responses were very low in most reverters (regardless of QFT status), which may explain fluctuations around the QFT assay cut-off resulting in reversion of the test. In the reverters who had low but robustly detectable responses, CFP-10/ESAT-6-specific CD4 T cells showed low levels of M.tb-specific T cell activation, maintenance of both IFN- dependent and independent Th1 cytokine co-expressing profiles and a predominantly TTM/TE phenotype. Memory and functional profiles detected in reverters in response to M.tb lysate shared more characteristics with non-converters than persistently infected (QFT+) individuals. Based on these results, we conclude that QFT reverters represent a heterogenous population in the tuberculosis spectrum who may experience very low or no in vivo antigen exposure. Altogether, these results indicate that not everyone with a QFT+ test likely experiences ongoing in vivo M.tb exposure, as suggested by much lower T cell activation observed during remote M.tb infection and QFT reversion compared to recent M.tb infection. Whether ongoing in vivo antigen exposure is required to maintain memory responses against M.tb remains to be determined. It is possible that key features of T cell responses against M.tb, including magnitude and differentiation, are shaped by the antigen load experienced during primary infection, regardless of whether infection is subsequently cleared. Answering these questions is critical to inform the interpretation of the current immunodiagnostic assays and to determine who could be spared from preventive tuberculosis therapy. On the other hand, here we defined a biomarker of recent infection and tuberculosis disease, which could enable the provision of targeted treatment to those who would benefit the most.
- ItemOpen AccessInvestigations of mycobacteria-specific memoryy/effector T cell responses in HIV infected children receiving antiretroviral therapy(2011) Tena-Coki, Nontobeko Gwendoline; Hanekom, Willem; Scriba, Thomas; Kampmann, BeateHuman immunodeficiency virus (HIV) infected children are at greater risk of developing tuberculosis disease, and might benefit from vaccination with novel TB vaccines. However, little is known about the effect of HIV-infection on function and phenotype of T cell responses to mycobacterial antigens in children. This study compares both CD4 and CD8 T cell cytokine expression and memory phenotype in children, following in vitro stimulation with mycobacterial antigens, also contained in novel anti-TB vaccines that are currently undergoing clinical trials.
- ItemOpen AccessOptimisation of a flow cytometry antibody panel to detect BCG-induced innate responses in infants(2023) Mwambene, Temwa Dango; Scriba, ThomasThe outcome of exposure to Mycobacterium tuberculosis is highly variable ranging from clearance, latency to a wide spectrum of subclinical and clinical tuberculosis (TB) disease. The underlying basis of the variable disease outcomes to mycobacterial exposure is largely unknown and is thought to involve a complex interplay between genetic variation in both host and pathogen. This is further convoluted by factors such as age, geography, coinfections including human immunodeficiency virus (HIV) and previous Bacillus Calmette–Guérin (BCG) vaccination. As it stands, other than inborn errors in key mycobacterial susceptibility genes both animal and human studies have not found many genetic polymorphisms that are strongly and reproducibly associated with increased mycobacterial disease susceptibility and/or BCG vaccine efficacy. Given the complexity of the interplay between the variability in disease susceptibility, BCG vaccine efficacy and human exposure to genetically diverse pathogens there is a need to study both host and pathogen genetic factors that contribute to increased TB disease susceptibility. This will facilitate rational design of more broadly efficacious interventions. This MSc project is nested in a project which aims to uncover the genetic factors that result in variable responses to BCG vaccination and in so doing, expand the basis of learning for novel TB vaccine candidates. The overall aim of this MSc was to optimise a flow cytometry panel to measure the BCG-induced innate immune responses in infants. The innate response to BCG was characterised by whole blood intracellular cytokine staining of banked, previously stimulated immune cells from 25, 10-week-old, HIV-uninfected, infants. The cohort is a subset of participants of a randomised control trial conducted in Worcester, South Africa. A 13-colour flow cytometry antibody panel was successfully optimised for enumerating and measuring cytokine and cell maturation marker expression by neutrophils, monocytes, pDCs, mDCs and T cells in response to BCG stimulation. Expression of cytokines (IFNg, IL-6, TNF) and maturation markers (CD40, PD-L1) by these cell subsets to BCG stimulation were quantified, allowing identification of single nucleotide polymorphisms that associate with variability in innate responses to BCG. Results from this MSc thus inform the bigger project which aims to determine genetic determinants of innate responses to BCG and TB disease risk.
- ItemOpen AccessPerformance of host blood transcriptomic signatures for TB diagnosis and monitoring TB treatment(2025) Muwanga, Vanessa; Scriba, Thomas; Mendelsohn Simon, JenniferNon-sputum-based diagnostic tests are necessary to enable tuberculosis (TB) diagnosis and monitoring of TB treatment. This work aimed to identify which published blood transcriptomic TB signatures have the best diagnostic performance to distinguish between TB cases and other respiratory diseases (ORDs) in symptomatic adults. These signatures were also evaluated to monitor treatment in young children, to identify signatures that might indicate when treatment could be stopped. Diagnostic performance of signatures was evaluated in adults presenting for care with symptoms associated with TB, who were recruited from primary healthcare clinics in six African countries. To identify signatures that can monitor treatment response, the same set of signatures was evaluated in young children randomised to 4- or 6-month TB treatment in the SHINE trial. Twenty transcriptomic signatures were selected and measured in whole blood using multiplex, microfluidic RT-qPCR. In the adult diagnostic cohort, nine signatures achieved equivalent performance for differentiating patients with ORDs from all TB cases. Factors associated with signature scores included HIV and country. With sensitivity benchmarked at 90%, these nine signatures achieved specificities between 44%-54%. In pooled analyses, none of the signatures met the minimal target product profile criteria for a TB triage test. Country-specific analyses, however, showed that several signatures met the minimal criteria in some countries. In children from the SHINE trial, baseline scores for all signatures were highest in children with confirmed, relative to unconfirmed and unlikely TB. Baseline scores were also higher among children with disease classified as more severe on chest x-ray. Scores declined progressively during TB treatment in the confirmed and unconfirmed TB groups; no changes in scores were observed for most signatures in the unlikely TB group. Scores were higher at the end of treatment in the 4-month than the 6-month treatment arm in the confirmed TB group; no differences were observed between treatment arms in the unconfirmed and unlikely groups. These results suggest that host blood transcriptomic signatures have potential to monitor TB treatment response in children. This work supports further development of transcriptomic signatures as TB triage tests and treatment monitoring tools.