Masters Degrees (Molecular Biology and Human Genetics)

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Analysis of ex vivo host biomarkers in sputum samples for diagnosis of pulmonary tuberculosis
(Stellenbosch : Stellenbosch University, 2019-12) Mendy, Joseph F.; Chegou, Novel N.; Sutherland, Jayne; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
Background Despite all the interventions deployed to control tuberculosis (TB), the disease still continues to be the principal cause of death from a single infectious agent in resource constrained settings. An estimated 60% of suspected TB patients do not have access to TB diagnostic tests. With the limitations of the current diagnostic tests and the importance of early diagnosis and initiation of treatment, biomarker diagnosis of TB would be an optimal option. For biomarkers are indicators of immune activity and state. Therefore, host or pathogen biomarker of TB disease would be ideal. Hence, the aim of this project was to profile a broad array of host markers for development of optimal signatures for detection of pulmonary tuberculosis from other respiratory disorders using ex vivo sputum samples Methods We recruited patients who were seeking medical attention at the MRCG at LSHTM outpatients department and Tuberculosis clinic with symptoms suggestive of TB, prior to clinical or microbiological diagnosis. All age groups were recruited. Sputa were collected at baseline from all participants and at 1 and 2 months from the confirmed TB cases. The sputa were digested with Sputolysin and the supernatant analysed using Luminex arrays while RNA extracted from the pellet were analysed with RT-qPCR. Statistical analyses and graphs were generated using R programming Language and GraphPad Prism, with a q value ≤ 0.05 considered significant. A receiver operating curve (ROC) was used to assess the diagnostic performance of individual and combination markers. Results Confirmed TB (428) and ORD (313) patients were analysed, 70 markers were assessed for diagnostic potential and treatment response. Of these, 37 were significantly different between TB and ORD. The best single marker was MMP-2 with an AUC of 0.73. An eight-marker signature (IFNү, IL-1β, IL-8, IL-10, IL-12p70, MIP-1β, RANTES and VEGF) was able to diagnose smear and culture positive TB from ORD with an AUC of 0.77, sensitivity of 78% and specificity of 70%, while a threemarker signature (IL-1β, IL-7 and VEGF) classified smear negative but culture positive TB from ORD with an AUC of 0.74, sensitivity of 86% and specificity of 60%. Among children who had TB, a fourmarker signature (FGF, IL-4, MIP-1a and RANTES) differentiated those with TB from ORD, with an AUC of 0.87, sensitivity of 82% and specificity of 87% and a five-marker signature consisting of BAFF, C3L1, IL-22, MMP-3 and sTNFR1 was able to discriminate TB and HIV co-infected from ORD with an AUC of 0.90, sensitivity of 88% and specificity of 85%. We also found a four-marker signature consisting of EGF, IL-15, MIP-1β and TNF-β that could predict slow versus fast treatment responders at baseline with an AUC of 0.74, sensitivity of 75% and specificity of 80%. Conclusion We have discovered novel sputum host biomarkers and biosignatures for screening of tuberculosis and treatment response. The data is promising for potential translation into a user friendly device as a rapid screening test for pulmonary TB. However, this markers and signatures require further investigations to authenticate their usefulness.
Chemokine and cytokine measurements using Luminex and Selected Reaction Monitoring (SRM): comparing technologies
(Stellenbosch : Stellenbosch University, 2020-12) Nkonyane, Tshepiso Ruth; Chegou, Novel N.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: Background: There is an urgent need for tools for the rapid and accurate diagnosis of tuberculosis (TB) disease in resource-constrained settings. Tests based on host immunological biomarkers may be useful, especially if based on easily available samples. Objectives: To evaluate the usefulness of plasma proteins as biomarkers for diagnosing TB, to validate previously identified biosignatures [4-marker biosignature (CCL1+CRP+IP10+NCAM), 2-marker biosignature (CCL1+CRP) and 3-marker biosignatures; [(CCL1+NCAM+SAA), (CRP+IP-10+SAA), (CCL1+CRP+NCAM), (CCL1+CRP+SAP), (CRP+IP-10+MIG) and (CRP+SAP+MIG)] in a new cohort of adults suspected of having TB, and to develop a Selected Reaction Monitoring (SRM) for detecting TB protein biomarkers. Methods and Materials: We collected plasma samples from 151 individuals that presented with symptoms requiring investigation for TB disease at a primary health care clinic in the outskirts of Cape Town, South Africa, prior to assessment for TB disease. We evaluated the concentrations of 18 host markers in stored plasma samples using a multiplex platform. Using a combination of clinical, radiological and laboratory diagnosis, patients were later classified as having TB disease or other respiratory diseases (ORD). The diagnostic potential of individual analytes was analyzed using the receiver operating characteristic curve while the predictive abilities of a combination of analytes for TB disease were analyzed using the general discriminant analysis. Selected Reaction Monitoring (SRM) was used to develop an assay for the diagnosis of TB. Skyline software was used for the development of the SRM assay and analysis of the generated data. Results: Out of the 151 individuals that enrolled, 56 were pulmonary TB cases. Out of the 18 host markers that were evaluated in Chapter 3, there were significant differences in the levels of 9 host markers between patients with TB disease and those with ORDs. The concentrations of VEGF-A, IP-10, MMP-3, ferritin, SAA, CFH and CCL 1 were significantly higher in the TB cases than individuals with ORD, whereas NCAM and Apo A1 levels were significantly higher in the ORD group. When the diagnostic accuracies of individual host markers were investigated by ROC curve analysis, two markers (IP-10 and CCL 1) showed strong potential with AUC ≥0.85. When the data obtained from all study participants were fitted into GDA models regardless of HIV status, combinations between up to seven different host markers showed potential in the diagnosis of TB disease. However, an optimal diagnostic biosignature comprising of five proteins (transthyretin+MMP-3+IP-10+CFH+CCL1) diagnosed TB with an AUC of 0.90 (0.83- 0.97). Eight (8) previously identified biosignatures were assessed in study participants regardless of HIV status. A 4-marker biosignature (CCL1+CRP+IP-10+NCAM), 2-marker biosignature (CCL1+CRP) and 3-marker biosignatures [(CCL1+NCAM+SAA), (CRP+IP10+SAA), (CCL1+CRP+NCAM), (CCL1+CRP+SAP) and (CRP+IP-10+MIG)] all diagnosed TB with AUC ≥0.85, while one 3-marker signature (CRP+SAP+MIG) only diagnosed TB with AUC of 0.62. The most optimal diagnostic biosignature in all participants regardless of HIV status was the 4-marker (CCL1+CRP+IP-10+NCAM) signature which diagnosed TB with a sensitivity of 0.76(0.50-0.92) and specificity of 0.85(0.65-0.95). We further evaluated these biosignatures in HIV negative participants only and these biosignatures showed promise with areas under the ROC curves (AUC) ≥0.85 for all, except one 3-marker (CRP+SAP+MIG) biosignature. The most optimal diagnostic biosignature in HIV negative participants only was the 3-marker biosignature (CCL1+NCAM+SAA) that diagnosed TB with a sensitivity of 0.79(0.49-0.94) and specificity of 0.84(0.63-0.95). An SRM assay consisting of 83 proteins was developed, based on cytokines and chemokines that were previously investigated in diagnosing TB and reported in chapter 4. Conclusion We have shown that different candidate plasma biosignatures have potential in the diagnosis of TB disease. The observed results require further validation in a larger study. The previously identified biosignatures showed promise when validated in the current study. The biosignatures both identified and validated in this thesis hold promise as good candidate biomarkers/ biosignatures to be considered for the future development of point of-care TB tests. The developed SRM- based assay may be a useful alternative tool to diagnose TB. The assay requires further validation and optimization.
Evaluation of host biomarkers for early diagnosis of tuberculosis disease in children
(Stellenbosch : Stellenbosch University, 2018-12) Manyelo, Masilo Charles; Chegou, Novel N.; Walzl, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
Background: The diagnosis of tuberculosis (TB) remains a challenge in children. There is an urgent need for new tools for early diagnosis of TB disease in children Objectives: To evaluate the usefulness of a previously described 3-marker cerebrospinal fluid (CSF) biosignature (VEGF, IL-13 and cathelicidin LL-37) and other CSF biomarkers for diagnosis of tuberculous meningitis (TBM), and evaluate the utility of a previously identified adult 7-marker serum protein biosignature (CRP, IFN-γ, IP-10, CFH, Apo-AI, SAA and transthyretin) and other blood biomarkers for diagnosis of pulmonary TB (PTB) and TBM in children. Methods: CSF and serum samples were collected from children with suspected meningitis, whereas serum samples were collected from children with suspected PTB for investigation of biomarkers for the diagnosis of childhood TBM and PTB, respectively. Children in the TBM project were enrolled at the Tygerberg Academic Hospital, whereas those in the PTB study were enrolled at the Red Cross War Memorial Children’s Hospital in Cape Town, South Africa. Children were classified as TBM or no-TBM and PTB or no-PTB, using combination of clinical, radiological and laboratory findings. Using a multiplex platform, the concentrations of 69 host biomarkers were evaluated in CSF and serum samples from children in the TBM study whereas 40 host markers were evaluated in serum samples from children in the PTB study. The diagnostic accuracies of individual biomarkers were assessed by receiver operator characteristics (ROC) curve, whereas the General Discriminant Analysis (GDA) was used to assess the accuracies of combinations between different host biomarkers. Results: Of the 69 host biomarkers evaluated in CSF and serum samples from children in the TBM study, multiple individual host biomarkers showed potential as diagnostic candidates for TBM as ascertained by area under the ROC curve (AUC). The previously described 3-marker CSF biosignature was validated in the project. However, refinement of the biosignature by substitution of IL-13 and cathelicidin LL-37 with two new proteins (MPO and IFN-γ) resulted in a new biosignature with improved accuracy (AUC of 0.97). Furthermore, we identified a 4- marker CSF biosignature (sICAM-1, MPO, CXCL8 and IFN-γ), which also diagnosed TBM with AUC of 0.97. The adult 7-marker serum biosignature, modified by the replacement of transthyretin with NCAM1, diagnosed TBM with AUC of 0.80. However, a childhood TBM-specific serum biosignature (adipsin, Aβ42 and IL-10) diagnosed TBM with AUC of 0.84. The adult signature performed with an AUC of 0.79 in children with PTB, showing no significant difference in the diagnosis of childhood PTB or TBM. However, novel childhood PTB-specific biosignatures performed better than the adult 7-marker signature. Conclusion: The adult 7-marker signature showed potential in the diagnosis of both PTB and TBM in children recruited from a high TB incidence area. We validated a previously established 3- marker CSF biosignature, but a refined signature showed much improved accuracy. The biosignatures identified in this thesis hold potential for development of new diagnostic tools for PTB and TBM in children for possible use at the point-of-care. Our findings require further validation in larger and multi-site studies.
Evaluation of host transcriptional biomarkers in the diagnosis of TB disease and optimization of multi-colour immunophenotyping assay from the QuantiFERON® TB GOLD Plus cell sediments
(2020-11) Ndou, Sedzani; Chegou, Novel N. ; Gutschmidt, Andrea; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Background QuantiFERON-TB Gold-Plus (QFT-Plus) test are designed for the diagnosis of Mycobacterium tuberculosis (M. tb) sensitization, hence it’s low specificity in the diagnosis of active TB. It remains unknown if the cell sediments left-over after performing the QFT-Plus ELISA can be useful for the diagnosis of active TB. Objectives To assess the expression of selected genes that have been shown in the literature as TB diagnostic or prognostic candidate biomarkers, in the cell sediments left-over after performance of the QFT-Plus test, and ascertain whether such genes when detected in QFT-Plus sediments, have any potential in the diagnosis of active TB. Methods We analysed the expression level of 13 genes namely: STAT1, GBP1, GBP5, TRAFD1, SERPING1, DUSP3, BATF2, TAP1, ETV7, SCARF1, FCGR1B, KLF2 and, GBP2, from the QFT-Plus left-over sediments using Taqman-probe qRT-PCR arrays in a total of 44 patients who were enrolled after presenting with the signs and symptoms of TB. The diagnostic accuracy of genes was evaluated using receiver operator characteristics (ROC) curves analysis whereas multi-biomarker analysis techniques were used to assess the usefulness of combinations between different genes in the diagnosis of TB. Some of the sediments from the QFT-Plus were fixed and stored for Flow Cytometry analysis in the future. The Flow Cytomery panel was designed to include the T cell subsets as well as other immune biomarkers such as IL-1B, CX3CR1, CD154 and CD56 which have been previously shown to have potential in the diagnosis of TB disease using Luminex platform Results Of the 13 individual genes evaluated in this study, the expression of 5 genes namely, BATF2, KLF2, DUSP3, GBP1 and GBP5 was significantly different (P<0.05) between the TB patients and individuals with other respiratory diseases (ORD). Individually, these genes diagnosed TB with area under the ROC curve (AUC) ≥0.89 in both the unstimulated and M. tb antigen-stimulated samples. When the genes detected in the unstimulated (Nil) sediments were fitted into general discriminant analysis (GDA) models, a 3-marker biosignature consisting of BATF2, GBP1 and GBP5 was found to be the most optimal in diagnosing TB and performed with an AUC of 0.99. However, when the Nil-derived 3-marker signature was applied on TB1 stimulated samples, the AUC reduced slightly to 0.98 and when applied to TB2 stimulated samples, the AUC remained the same as what was observed for unstimulated samples. Overall, stimulation of blood with different antigens led to different expression patterns for different genes in the specimens. Conclusion In conclusion, the results presented in this thesis show that sediments left-over after supernatants are harvested for performance of the QFT-Plus test, and which are often discarded, may be valuable samples for evaluation of both unspecific and M. tb antigen-specific gene expression. We furthermore showed that the detection of such genes may be a valuable method for the diagnosis of active TB and for future implementation purposes especially where limited blood volumes are available. Our sample size was small; hence these findings require further validation in larger studies.
Evaluation of multiple cytokine levels to improve our understanding of protective immune responses against Tuberculosis and to develop novel diagnostic methods
(Stellenbosch : Stellenbosch University, 2013-03) Phalane, Khutso Gemina; Walzl, Gerhard; Chegou, Novel N.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Important steps towards the global control of Tuberculosis include the improvement of diagnosis, the development of effective vaccines and the identification of correlates of protection/protective immunity to Mycobacterium tuberculosis. This study has of three objectives: 1. To validate the findings of a previous study that showed increased levels of IL-1β and decreased levels of IL-17 in children who are exposed to tuberculosis but remain uninfected compared to those who are exposed/infected and unexposed/uninfected. 2. To define the protective immunological phenotype in children with negative IGRA’s and TST following exposure to Mycobacterium tuberculosis. 3. To evaluate a number of cytokines in both serum and saliva samples of identified tuberculosis cases and controls for their diagnostic potential and to evaluate saliva as a possible new diagnostic sample type. The study designs were as follows: Objectives1, and 2: Children with documented tuberculosis exposure and with Mycobacterium tuberculosis infection as assessed through interferon gamma release assays, children with exposure but no infection and a control group with no exposure nor infection were investigated. These participants were selected according to their exposure and infection phenotypes from a larger TB household contact study that was conducted in communities in Cape Town. Whole blood was stimulated in QuantiFeron tubes overnight and ten cytokines were measured in antigen stimulated and unstimulated supernatants by Luminex multiplex Immunoassay. Differential production of cytokines in the three groups was evaluated. Objective 3. Saliva and serum samples were collected from thirty eight adults with suspected tuberculosis who were recruited from a community health centre in Cape Town, after which the levels of thirty three host markers were evaluated in the samples using the Luminex platform. The main findings of the studies included: 1. Increased levels of IL-1β and decreased levels of IL-17 in children who are tuberculosis exposed but remain uninfected compared to those who are exposed/infected and unexposed/uninfected could not be confirmed. 2. Immune responses other than IFN-γ are different in children with different exposure and infection phenotypes. Higher IL-23 and IL-33 levels in children with tuberculosis exposure without subsequent Mycobacterium tuberculosis infection compared to children with no exposure were shown. 3. In both the tuberculosis cases and controls, the levels of most markers were above the minimum detectable limit in both serum and saliva, but marker levels were not consistently higher in one sample type. The levels of fractalkine , IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF and IL-5 in saliva, and those of IL-6, IL-2, SAP and SAA in serum, were significantly higher in tuberculosis patients, in comparison to the levels obtained in those without active tuberculosis (p<0.05). The area under the ROC curve was ≥ 0.70 for most of these markers, thereby confirming their diagnostic potential for TB disease. The work presented in this thesis has identified markers that may grant an improved understanding on the mechanisms that are associated with protection against Mycobacterium tuberculosis in children. The preliminary results presented show that the identification of host markers in saliva is possible and the utility of saliva for the development of rapid immune-based tests for active tuberculosis is promising.
Evaluation of novel host markers detected in plasma and saliva as biosignatures for the rapid diagnosis of TB disease and monitoring of the response to TB treatment
(Stellenbosch : Stellenbosch University, 2016-12) Jacobs, Ruschca; Chegou, Novel N.; Walzl, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH SUMMARY: BACKGROUND: There is an urgent need for new tools for the rapid diagnosis of tuberculosis (TB) disease and monitoring of the response to treatment. OBJECTIVES: To investigate the usefulness of host markers detected in plasma and saliva, as well as antibodies against M. tuberculosis (M.tb) antigens, as biomarkers for the diagnosis of TB disease and monitoring of the response to treatment. To investigate the usefulness of a diagnostic approach involving the combination of antibodies and cytokines as a tool for diagnosing TB disease. METHODS: We prospectively collected plasma and saliva samples from individuals that presented with symptoms requiring investigation for TB disease at a health centre in Cape Town, South Africa, prior to the establishment of a clinical diagnosis. Patients were later classified as having TB disease or other respiratory diseases (ORD), using a combination of clinical, radiological and laboratory findings. The concentrations of host inflammatory biomarkers were investigated in plasma and saliva samples from all study participants using a multiplex platform, whereas antibody responses against seven M.tb antigens, were investigated by ELISA. The diagnostic accuracies of individual biomarkers were assessed by receiver operator characteristics (ROC) curve analysis, whereas the accuracies of combinations between different biomarkers were assessed by General Discriminant Analysis (GDA). RESULTS: Of the 74 host markers evaluated in plasma, 18 showed diagnostic potential as determined by area under the ROC curve (AUC), with the most promising being NCAM, CRP, SAP, IP-10, ferritin, TPA, I-309, and MIG, which diagnosed TB disease individually with AUC ≥0.80. A six-marker plasma protein biosignature comprising of NCAM, SAP, IL-1β, sCD40L, IL-13 and Apo A-1 diagnosed TB disease with a sensitivity of 100% (95% CI, 86.3-100%) and specificity of 89.3% (95% CI, 67.6-97.3%), irrespective of HIV status, whereas six-marker plasma protein biosignatures diagnosed TB disease with 100% accuracy in the absence of HIV. Of the 69 host markers that were investigated in saliva, only two (IL-16 and IL-23) showed diagnostic potential with AUC ≥0.70. A five-marker salivary biosignature comprising of IL-1β, IL-23, ECM-1, HCC1 and fibrinogen diagnosed TB disease with a sensitivity of 88.9% (95% CI,76.7-99.9%) and specificity of 89.7% (95% CI, 60.4-96.6%), regardless of HIV infection status, whereas eight-marker salivary biosignatures performed with a sensitivity of 100% (95% CI, 83.2-100%) and specificity of 95% (95% CI, 68.1-99.9%) in the absence of HIV infection. IgA responses against four M.tb antigens (NarL, Rv3019c, “Kit1” and “Kit2”) were significantly different between TB patients and individuals with ORD, with combinations between different antibodies diagnosing TB disease with an AUC of 0.80. The diagnostic accuracy of the antibodies increased when used in combination with patient’s symptoms or cytokines. Finally, the concentrations of biomarkers detected in plasma and saliva changed during TB treatment, thereby indicating that they may be useful in monitoring of the response to TB treatment. CONCLUSIONS: We have identified novel plasma and salivary biosignatures which may be useful in the diagnosis of TB disease and monitoring of the response to TB treatment. Our findings require further validation in larger studies.
Evaluation of the potential of Mycobacterium tuberculosis antigen-specific host biomarkers detected in QuantiFERON® TB GOLD Plus supernatants in the diagnosis of TB disease
(Stellenbosch : Stellenbosch University, 2018-12) Manngo, Makhadzi Portia; Chegou, Novel N.; Gutschmidt, Andrea; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: BACKGROUND The diagnosis of tuberculosis (TB) disease remains a challenge. This is mainly due to limitations with the current TB diagnostic tests including unavailability of rapid pointof-care tests. New TB diagnostic tests are therefore urgently needed. The QuantiFERON-TB® Gold (QFT) Plus test is a recently introduced test for the diagnosis of M. tb infection, and disease in some patient groups. As this is a relatively new test which is currently in use worldwide, it is important that its performance be evaluated, especially in high TB burden settings. Furthermore, it is not known whether measurement of host markers other than Interferon-gamma in culture supernatants of individuals with active TB or other respiratory diseases (ORD), has potential in the diagnosis of TB disease. OBJECTIVES 1) To evaluate the usefulness of the QFT Plus test in the diagnosis of TB disease, and assess the utility of the test, when used in combination with symptoms, as a tool for diagnosis of TB disease in people suspected of having active TB in a high burden setting. 2) To evaluate alternative host biomarkers detected in QFT Plus supernatants, other than IFN-γ as biosignatures for the diagnosis of active TB METHODS We recruited 120 participants presenting at a primary health care clinics in Cape Town, South Africa with symptoms requiring investigation for TB disease. These participants formed part of a larger ongoing biomarker project known as the ‘ScreenTB’ study. Participants were later classified as TB or ORD based on the results of clinical and laboratory tests. After performing the standard QFT Plus test in study participants, the concentrations of 37 host biomarkers were evaluated in culture supernatants using a multiplex immunoassay. RESULTS Out of 120 individuals included in the study, 35 (29.2%) were diagnosed with active TB and were culture positive. The QFT Plus test diagnosed TB disease in all study participants with sensitivity and specificity >70%. A combination of symptoms including cough, fever and weight loss diagnosed TB disease with sensitivity and specificity >70% with an area under the receiver operator characteristics curve of 0.81. Multiple host biomarkers detected in the unstimulated and antigen-stimulated QFT Plus tubes showed potential as diagnostic markers for TB. Individual markers which diagnosed TB disease with sensitivities and specificities >60% included ITAC-1, IL-3, I-309, MIG, and EGF, P-selectin. Combinations between host biomarkers showed potential in the diagnosis of TB disease with a six-marker biosignature derived from unstimulated supernatants (APO-CII, ITAC-1, MIG, MCP-2, I-309, and NCAM-1) diagnosing TB disease with a sensitivity and specificity >78%, a four-marker TB1 and TB2 antigenspecific biosignature (TNFα, LIGHT, MIG and P-selectin ) which diagnosed TB disease with sensitivity and specificity >73%, after leave-one-out cross validation. CONCLUSION The sensitivity of the QFT Plus test for active TB was inferior to the published >80% mentioned in the package insert by the manufacturer. Host biomarkers detected in QFT Plus supernatants showed potential in the diagnosis of active TB disease. Further validation studies are needed before such markers may be considered as candidate biomarkers for a blood-based diagnostic test for active TB.

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Browsing Masters Degrees (Molecular Biology and Human Genetics) by browse.metadata.advisor "Chegou, Novel N."

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