Doctoral Degrees (Molecular Biology and Human Genetics)
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Browsing Doctoral Degrees (Molecular Biology and Human Genetics) by browse.metadata.advisor "Chegou, Novel N."
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- ItemDevelopment of immune-based TB tests suitable for resource limited settings(Stellenbosch : Stellenbosch University, 2014-12) Essone Ndong, Paulin; Walzl, Gerhard; Chegou, Novel N.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.ENGLISH ABSTRACT: - Background - Tuberculosis (TB) is still one of the leading causes of death in poor socio-economic settings. This situation is encouraged by the lack of simple and rapid tests suitable for rapid diagnosis. The newly developed Interferon-gamma Release assays (IGRAs) can detect Mycobacterium tuberculosis (M.tb) infection but fail to discriminate active TB from latently infected individuals. - Objectives - The present thesis aims to develop a rapid and simple test for the diagnosis of active TB disease. This objective was divided into four sub-objectives: 1) identification of potential M.tb antigens and host markers suitable for a TB test using a 7-day whole blood assay (WBA), 2) validate the promising results in an overnight WBA for a rapid, albeit not ex vivo, test, 3) evaluate the diagnostic utility of a two colour ELISpot test, 4) use an unbiased approach to discover multiple new host markers with diagnostic utility using mass spectrometry. - Methods and results - Participants were recruited from the Ravensmead/Uitsig community and day clinics. Stimulated and unstimulated analyte levels in 7-day and overnight WBA supernatants from active TB cases were compared to analyte levels in controls. The results of these experiments showed that Rv0081-stimulated levels of IP-10, IL-12p40, TNF-α and IL-10 were the most promising diagnostic markers in the long term assay as they could correctly classify 100% of the study participants in this assay. Acute phase proteins mainly CRP and SAA were the best diagnostic antigens in the short term assay. The diagnostic utility of these markers was greater in Quantiferon Nil supernatants compared to the stimulated samples. IFN-γ and IL-2 ELISpot was performed where it was found that single cytokine measures could not discriminate active TB to latent infection. When single and double secreting cell populations were taken into consideration, a combination model of ESAT6/CFP10-stimulated single IFN-γ, single IL-2 and IFN-γ/IL-2 double secreting cells could classify participants into their clinical groups with good accuracy. In a pilot study for future discovery of diagnostic markers by mass spectrometry, three depletion methods (ProteoSpin column, Heparin column and ProteoPrep 20) were assessed to identify the most appropriate depletion method for high abundant proteins from serum. The depleted serum samples were analysed in Orbitrap Velos. The antibody based method, ProteoPrep 20, was the best depletion method as it led to the visualisation of a larger number of proteins on Orbitrap. - Conclusion - M.tb antigen-stimulated host markers hold promises in diagnosis of active TB disease. The excellent accuracy observed in the long term assay could not be repeated in the short term assay. Acute phase proteins are the most promising but perform better in unstimulated than in stimulated supernatants and should be evaluated in ex vivo samples like serum or plasma. However, it is likely that further unbiased proteomic approaches, like mass spectrometry, will identify additional promising markers that will allow the development of ex vivo, accurate, pointof- care tests for TB.
- ItemEvaluation of host biosignatures for targeted screening for tuberculosis at the point of care, and monitoring of the response to TB treatment(Stellenbosch : Stellenbosch University, 2020, 2020-03) Mutavhatsindi, Hygon; Chegou, Novel N. ; Walzl, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Background The accurate and rapid diagnosis of TB disease remains one of the major challenges in the control of the disease. This is mainly due to limitations in the currently existing diagnostic tests. There is also an urgent need for the development of tools that may assist in monitoring the response of TB patients to treatment. This is important given the increasing burden of multi-drug resistant TB, the risk for treatment failure and of relapse after initial clinical cure and the need for early diagnosis of such poor outcomes. The work reported in this thesis investigated different biomarkers that may assist in addressing these challenges. Aims 1) To refine and validate different biomarkers selected from literature and evaluate novel biomarker combinations in a large Africa-wide study as biosignatures for the diagnosis of TB. 2) To evaluate combinations between different biomarkers selected from the literature and mycobacterial and clinical parameters (body mass index) as tools for monitoring the response to TB treatment and prediction of relapse. 3) To employ an unbiased proteomic approach in the search for new salivary biomarkers for TB. Methods Serum and saliva specimens collected through different multicentred projects including the African-European Tuberculosis Consortium (AE-TBC), the “Action TB” study and the “Tuberculosis Research Unit” (TBRU) study at different time points were evaluated. The concentrations of host inflammatory biomarkers in serum samples from all participants (aims 1 and 2 of the project) were evaluated using the Luminex multiplex platform. The investigation of new salivary biomarkers for TB was done following a discovery proteomics approach by mass spectrometry, using the Orbitrap QExacutive platform. Standard statistical analysis procedures as used in previously published studies were employed for data analysis. Main Findings 19 out of the 20 host inflammatory biomarkers that were selected from the literature on the basis of their performance in previous studies, and evaluated as diagnostic tools for TB in the AE-TBC cohort (n=1003 study participants, 277 of whom were finally diagnosed with TB and 726 of whom were diagnosed with other respiratory diseases [ORD]), showed potential in discriminating participants with TB and those with ORD individually, thereby confirming their potential as candidate TB diagnostic biomarkers. The diagnostic potential of modified versions of a previously published 7-marker signature (Apo A1, CFH, CRP, IFN-g, IP-10, SAA and transthyretin) was confirmed. However, we identified a novel 4 marker biosignature (I- 309, CRP, IP-10, and NCAM) and a novel smaller 3-protein biosignature (CRP, I-309 and NCAM) with strong potential as diagnostic biosignatures for active TB (AUCs of 0.91 and 0.90 respectively), regardless of HIV status. When the utility of host biomarkers selected from the literature was investigated for their potential to be used as biomarkers for monitoring of the response to TB treatment, it was observed that the concentrations of multiple biomarkers changed in the course of TB treatment. The levels of SAP, IP-10, and sIL-6R amongst other biomarkers increased with treatment, whereas those of Apo CIII, MMP-2 and Fas amongst others generally tended to decrease. Some of these proteins including IL-6, IP-10, IL-22 and C3 showed potential as individual biomarkers in predicting relapse both in the action tb and TBRU cohorts. However, the most important findings from this part of the work were the identification of a combination of TTP, BMI, sICAM, IL-22, IL-1β and C3 which predicted relapse at baseline, prior to the onset of TB treatment with 89% sensitivity and 94% specificity, and a combination of Apo CIII, IP-10 and sIL-6R which predicted relapse at the end of treatment with 71% sensitivity and 74% specificity. For the unbiased proteomic identification of salivary biomarkers part of the project, we identified a total of 948 unique proteins in saliva. After statistical analysis to identify the potentially most useful proteins, we identified 26 proteins with TB diagnostic potential. Importantly, two protein biosignatures including a five-protein signature comprising of P01011, Q8NCW5, P28072, Q99574 and A0A2Q2TTZ9 and a four-protein biosignature comprising of P30043, P35579, Q99574 and P31949. The five-protein signature ascertained TB with a sensitivity of 100% and specificity of 90.91% after leave-one-out cross-validation. Conclusion The usefulness of previously identified biomarkers was confirmed in large Africa-wide diagnostic study in the current thesis. Importantly, new smaller biosignatures comprising of as little as three proteins showed strong potential in TB diagnosis. Biosignatures comprising of combinations between immunological biomarkers, BMI and TTP were identified with strong potential for use as tools for monitoring of the response to TB treatment and prediction of TB relapse both prior to the initiation of treatment, early on after initiation of treatment and at the end of treatment. Results from this thesis show that the development of a rapid point-of-care diagnostic test comprising of 3 biomarkers is possible and that such a test will potentially be highly useful if used as a triage test for TB in peripheral level health centres all over Africa, which is where our study participants were recruited. We identified strong candidate biosignatures for prediction of relapse prior to the initiation of TB treatment, early on in treatment and at the end of treatment, and also novel salivary protein biomarkers with potential in the diagnosis of TB. However, the sample sizes were relatively small, and these novel findings require validation in larger studies, especially studies conducted in multiple geographical settings.
- ItemImmunological markers for active TB and early treatment response indicators(Stellenbosch : Stellenbosch University, 2017-03) Awoniyi, Dolapo Olaitan; Walzl, Gerhard; Diacon, Andreas; Chegou, Novel N.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human GeneticsBackground The difficulty in diagnosing tuberculosis (TB) and evaluating TB treatment response are two major problems that are hampering the defeat of this infectious disease. The current TB diagnostic tools have several limitations and these call for the development of a simple, rapid and accurate diagnostic test that is suitable for use in poor-resource settings. Aim and Objectives This thesis aims to identify host markers for the development of a rapid and simple test for TB diagnosis and for monitoring early TB treatment response. The objectives are: 1. To investigate the diagnostic accuracy of host markers detected in Mycobacterium tuberculosis (Mtb) antigen-stimulated overnight whole blood culture supernatant. 2. To investigate the profiles of inflammatory markers of active TB patients undergoing treatment in a 14-day EBA trial for treatment monitoring potential. 3. To investigate the combined performance of the responses of IgG, IgM and IgA to selected mycobacterial antigens for their diagnostic potential. Methodology Participants were recruited as part of the recently concluded EDCTP-funded AE-TBC study and a 14-day phase II randomised clinical trial (early bactericidal activity (EBA) study of seven treatment arms). Sputum and blood samples were collected at different time points and multiplex cytokine array analysis performed on plasma or serum samples by Luminex and anti-mycobacterial antibodies detected by ELISA. Results After overnight stimulation of whole blood with ESAT-6/CFP-10, RV0081, Rv1284 and Rv2034, the most promising diagnostic markers were CRP, Ferritin, SAA and IP-10. Unstimulated host markers yielded the best discriminatory power. A six-marker biosignature comprised mostly of unstimulated cytokine levels shows promise for active TB diagnosis. Stellenbosch University https://scholar.sun.ac.za iii There were significant changes for CRP, IL-6, VEGF, sIL-2Rα, Ferritin, and sTNFRII from baseline to end of 14 day EBA evaluation in several treatment arms. However, none of these markers mirrored the decrease in the measured bacterial load in sputum. A four-marker combination only accounted for 20% of the variation in observed in both TTP and CFU. The highest sensitivity and specificity was obtained with anti-16 kDa IgA (95%/95%) and anti-MPT64 IgA (95%/90%). A higher accuracy was obtained with a 3 or 4 antibody combination. Anti-16 kDa IgA and anti-16 kDa IgM, decreased significantly while anti-LAM IgG and anti-TB-LTBI IgG increased significantly at the end of month six anti-TB treatment. Conclusion Host biomarkers hold promise as a diagnostic tool in TB disease. In spite of the moderate accuracy of Mtb antigen-stimulated host markers, these could still have value in difficult to diagnose TB, like paediatric TB or extrapulmonary TB, and should be evaluated in future studies. Although host markers only explained a small degree of the variation in bacterial measures in early bactericidal activity studies, their potential role in overall treatment effect remains to be investigated. Serodiagnostic markers against novel Mtb antigens showed potential for future development into a simpler format for use at the point-of-care. These results should be validated in large scale studies in appropriate participant groups.