Doctoral Degrees (Molecular Biology and Human Genetics)


Recent Submissions

Now showing 1 - 5 of 114
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    Diagnostic accuracy of novel sputum and non-sputum-based tuberculosis diagnostics in HIV-positive patients initiating anti-retroviral therapy
    (Stellenbosch : Stellenbosch University, 2023-11) Ndlangalavu, Gcobisa; Theron, Grant; Reeve, Byron; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Tuberculosis (TB) is a major health problem killing millions of people worldwide and is the leading cause of death among people living with HIV (PLHIV). PLHIV are at high risk of TB due to their weakened immune systems and 18 times more likely develop TB than people without HIV. Therefore, early detection of TB among PLHIV is fundamental and dependent on good TB diagnostics. The study addressed barriers and limitations of TB screening (triage) and diagnosis, with some limitations being facility-based active case finding, which is challenging, and delayed patient results. To highlight limitations and utility of tests in the pipeline, current and novel TB diagnostics were extensively reviewed (Chapter 2). Additionally, we tested selected PLHIV initiating antiretroviral therapy (ART), in whom TB detection is difficult due to their paucibacillary nature, are negative by the World Health Organization (WHO) recommended four-symptom screen (W4SS), and unable to produce sputum (Chapters 3 to 5). ART initiators were included irrespective of W4SS status, to challenge the existing TB testing algorithm in PLHIV. Formerly, W4SS was exclusively recommended for triage for further TB testing even in PLHIV. Due to W4SS’ suboptimal specificity and poor implementation, alternative triage tools are required, which we investigated. This includes C-reactive protein (CRP) (Chapter 3). CRP (≥10mg/l) had higher specificity than W4SS in ART initiators and reduces unnecessary downstream testing. Since CRP levels varied within groups of the same culture status, Chapter 4 investigated clinical and demographic factors associated with CRP levels, thereby informing which types of patients might be missed or falsely included by CRP-based triage algorithms. Low CD4 cell count, culture-confirmed TB, and increased TBScore II were strongly associated with elevated CRP. Sputum Xpert MTB/RIF Ultra (Ultra) is the frontline rapid pulmonary TB test in South Africa, however, supporting data PLHIV, among whom Ultra may offer sensitivity improvement over Xpert MTB/RIF (Xpert) is limited. Sputum expectoration is challenging in PLHIV, and easily accessible urine can mitigate this. Chapters 3 and 5 investigated the diagnostic accuracy of Ultra using urine versus Determine TB LAM Ag test (LF-LAM). Their performance was similar. We further investigated urine Fujifilm SILVAMP TB-LAM (FujiLAM) diagnostic accuracy versus that of LF-LAM (Chapter 5). FujiLAM had increased sensitivity and comparable specificity versus LF-LAM. However, FujiLAM’s diagnostic accuracy varied between different manufacturer lots. In summary, CRP (>10mg/l) would reduce unnecessary expensive downstream TB testing. CRP increases case detection especially in key and vulnerable populations. CRP data from this study contributed to a meta-analysis that informed the latest screening guidelines for TB, changing decades old triaging approaches. Sputum-Ultra is more sensitive than Xpert and detects ~1/2 W4SS-negative cases. Our sputum-Ultra data contributed to a systematic review evaluating the diagnostic accuracy of Xpert and Ultra, irrespective of W4SS status, and was the only study with Ultra data among PLHIV. TB detection in outpatient PLHIV can be improved with FujiLAM implementation if improved versions of the tests would be available. Non-sputum-based confirmatory tests with improved sensitivity are urgently needed to improve case detection especially in sputum-scarce and W4SS-negative people.
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    Metallophiles as sources of antimycobacterial agents
    (Stellenbosch : Stellenbosch University, 2023-12) Nyambo, Kudakwashe; Mavumengwana, Vuyo; Ngxande, Mhkuseli; Smith, Liezel; Sithole-Niang, Idah; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Mycobacterial pathogens present a significant complication to disease control globally due to their resistance to numerous antibiotics. The rise in resistant strains to current chemotherapeutic treatments has prompted the search, development and implementation of new strategies to address this challenge. Harnessing the bioactivity of natural products found in the vast chemical space by using multi-disciplinary approaches has emerged as a promising way to discover new Tuberculosis drugs. This study aimed to evaluate the potential antimycobacterial activity of secondary metabolites from bacteria, fungi, and plants in-vitro and in-silico. In addition to mining for Mycobacterium tuberculosis targets, this study went further to explore other druggable targets associated with cancer in order to fully explain exhaustive in-silico bioactivity profiles. The following experiments were conducted to satisfy the aims: (i) bacteria from gold mine tailings were isolated and identified using 16S rRNA sequencing. The crude extracts from the bacteria were screened for potential activity against Mycobacterium tuberculosis (M. tb) H37Rv, Mycobacterium smegmatis MC2155, and Mycobacterium aurum A+ in-vitro. The active extracts were tentatively identified using HPLC-qTOF, GNPS, and Ms Dial. The identified compounds were virtually screened against Mycobacterium Pks13 and PknG. The natural compound that displayed high affinity was subjected to modification through multiple synthetic routes using reaction-driven enumeration. (ii) A total of 15 fungi compounds from fungi isolated from gold mine tailings were evaluated for their potential activity against M. tb PknA, PknB, PknD, and PknE proteins using extra precision molecular docking, molecular dynamics simulations, and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculation. (iii) Genomic DNA of one bacterial colony that showed activity against M. tb, was isolated and sequenced by Illumina’s NextSeq platform. The genes responsible for producing metabolites that may have antimycobacterial activity were determined using antiSMASH and PARTIC. (iv) Predictive machine learning-based quantitative structure-activity relationship models were developed with a pIC50 as the dependable variable, while features extracted from compounds found to be active against InhA were the independent variable. Another approach in developing a multitargeted SMILES-based Long Short-term Memory (LSTM) based on pIC50, and small, skewed datasets was attempted. (v) Medicinal plant species indigenous to South Africa namely Schotia brachypetala, Rauvolfia caffra, Schinus molle, Ziziphus mucronate, and Senna petersiana were evaluated for their potential antimycobacterial activity against Mycobacterium smegmatis MC2155, Mycobacterium aurum A+, and M. tb H37Rv. Although the study was specific to mycobacteria, further exploration into cytotoxic activity against MDA-MB 231 triple-negative breast cancer cells was also attempted to see if druggable targets could also be identified in eukaryotic cells as a test of the utility and robustness of the method. The constituents of the extracts possessing antimycobacterial activity were virtually screened using a rigorous Virtual Screening Workflow. The compounds exhibiting good binding, and ADME properties were returned and subjected to molecular dynamics simulations. MM-GBSA calculations were performed to evaluate the affinity of the selected compound/s to pantothenate kinase (PanK). Crude extracts from three bacterial isolates, namely Bacillus subtilis and Bacillus licheniformis, exhibited activity against M. tb H37Rv, Mycobacterium smegmatis MC2155, and Mycobacterium aurum A+. The classes of secondary metabolites identified in this study are known to possess antibacterial activity. Virtual screening of the secondary metabolites against PknG and Pks13, returned cyclo-(L-Pro-4-OH-L-Leu) and vazabitide A with pre-MD MM-GBSA values of -42.81 kcal/mol and -47.62 kcal/mol, respectively. The modification of vazabitide A yielded a compound with a higher affinity of -85.80 kcal/mol to the Pks13, binding as revealed by the post-MD MM-GBSA. SAMN36381076 was assigned to be B. licheniformis whole genome analysis. The genome length of B. licheniformis SAMN36381076 was estimated to be 4.213156 Mb, with a G+C content of 46.08%, comprising 58 contigs and exhibiting an N50 length of 165,033 bp. The biosynthetic gene clusters identified included fengycin, butirosin A, butirosin B, schizokinen, pulcherriminic acid, bacillibactin, bacillibactin E, bacillibactin F, lichenicidin VK21 A1, Lichenicidin VK21 A2, and thermoactinoamide A. These gene clusters are known for producing secondary metabolites with antimicrobial activity. Furthermore, The B. licheniformis SAMN36381076 possesses genes that encode for six diverse antibiotic resistance mechanisms, with efflux pumps as the predominant mechanism of resistance. Metabolic analysis of B. licheniformis SAMN36381076 showed that the presence of genes involved carbohydrate degradation and assembly processes, oxyanion biogeochemical cycling, and nitrogen cycling. In-silico evaluation of fungi compounds against ser/thr kinases showed the lowest ΔGBind values of aurovertin D against PknA (-50.9 kcal/mol), aurovertin D against PknB (-50.7 kcal/mol), verticillin A against PknD (-36.8 kcal/mol), and roquefortine C against PknE (-53.4 kcal/mol). Molecular dynamics simulation showed that the PknD-verticillin A exhibited the highest stability. Furthermore, the post-MD MMGBSA ΔGBind showed that verticillin A has a high affinity for PknD -53.67 kcal/mol. The results indicated that verticillin A is a potential hit compound that can befurther optimized and modified to develop a potent antimycobacterial inhibitor. The classical machine learning models developed from logistic regression and multi-layer perceptron were identified to have significant performance metrics on the InhA dataset. The results (- R2) from the multitarget Long Short Term Memory (LSTM) model indicated the need for hyperparameter tuning. However, further external validation of the two classification models is needed. In this study, the bioactive compounds present in R. caffra and S. molle showed average activity against M. tb H37Rv (MIC 0.25-0.125 mg/mL). Norajmaline with a docking score of -7.47 kcal/mol, and pre-MM-GBSA of -37.64 kcal/mol was returned from the rigorous virtual screening. Molecular dynamics simulation and post-MD MM-GBSA revealed the stable binding of norajmaline to PanK (-58. 73 kcal/mol). Results from the Flow cytometry analysis of treated MDA-MB 231 cells revealed that the dichloromethane extracts from S. petersiana, Z. mucronate, and ethyl acetate extracts from R. caffra and S. molle induced higher levels of apoptosis than the control cisplatin. In conclusion, this study serves as a starting point for the in-silico discovery of potent antimycobacterial compounds from metallophiles (fungi and bacteria) and plants. Virtual screening accelerates the drug discovery process by identifying compounds that may possess activity, thus they can be modified to increase potency. The incorporation of a large dataset of compounds comprising different biological conditions but with the same endpoint can be used to develop robust models with exceptional generalization capabilities.
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    Novel and rapid tests for diagnosis of tuberculosis using non-sputum specimens
    (Stellenbosch : Stellenbosch University, 2023-12) Minnies, Stephanie; Theron, Grant; Reeve, Byron; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Diagnosis of TB remains a challenge, as in 2021, 60% of those who developed active TB were diagnosed. Xpert Ultra MTB/RIF (Ultra) is endorsed for TB diagnosis on sputum, but at the advent of the study, more data on the usefulness of Ultra on non-sputum specimens, particularly in HIV-endemic settings, were needed. Moreover, the impact of different sample processing such as sample concentration of non-sputum specimens by centrifugation on Ultra has not been previously investigated. Lastly, data on how Ultra on site-of-disease non-sputum specimens directly compares to other tests, either on site-of-disease or non-site-of-disease non-sputum specimens remain limited. Firstly (chapter 2), we showed that in patients with presumptive TB lymphadenitis (TBL), Ultra detects more TBL cases than Xpert MTB/RIF (Xpert), Ultra’s predecessor, and results in more people being placed on treatment. Ultra’s increased sensitivity on fine needle aspirate biopsies (FNABs) does however come with decreased specificity, and this was not significantly associated with HIV status or the use of alternate reference standards. Furthermore, we showed that study Ultra detected more TBL cases than programmatic Ultras when both tests were done, indicating that optimisation of programmatic testing of FNABs would result in improved TBL diagnosis. Moreover, we showed that FNAB Ultra false-negative results are associated with PCR inhibition. Lastly, we showed that in patients with presumptive TBL, urine-Ultra had low sensitivity. Thereafter (chapter 3), we found that in people living with HIV (PLHIV) with presumptive TB pericarditis, Ultra on unconcentrated pericardial fluid had higher sensitivity and lower specificity overall when compared to Xpert. We also found that comparing Ultra to alternate reference standards did not improve sensitivity. Exclusion of Ultra results is the superior recategorization strategy in pericardial fluid (unlike reclassifying trace results as negative). Additionally, we showed that using concentrated pericardial fluid on Ultra resulted in higher Ultra specificity but more non-actionable results. This suggests that laboratories with adequate fluid volume and capacity should concentrate pericardial fluid when possible. Furthermore, we showed that the high sensitivity of uIFN-γ on pericardial fluid is offset by its’ poor specificity, indicating that Ultra is the superior test on pericardial fluid. Lastly, Urinary Ultra and TB-LAM had low sensitivity but could reduce the need for pericardiocentesis for TB pericarditis diagnosis in 4% of patients, highlighting their potential. Thirdly (chapter 4), in patients with presumptive TB pleuritis, we showed that Ultra had similar sensitivity but higher diagnostic yield compared to Xpert, and exclusion or reclassification of trace results to negative does not increase sensitivity. Additionally, alternate reference standards and HIV status did not significantly increase Ultra’s sensitivity. Furthermore, we showed that testing Ultra with concentrated pleural fluid increases Ultra specificity, but this also increases non-actionable results, and this was also observed in pericardial fluid. Moreover, we showed that uIFN-γ on pleural fluid had high sensitivity and moderate specificity, suggesting that laboratories with sufficient funding and infrastructure should use uIFN-γ concentration for TB pleuritis diagnosis. Finally, we showed that Ultra and TB-LAM on urine could reduce the need for thoracentesis in a subset of patients for TB pleuritis diagnosis, particularly in PLHIV. Lastly (chapter 5), we showed that in bronchial fluid (BF), Ultra’s diagnostic accuracy was not significantly different between bronchoalveolar lavage fluid (BALF) and bronchial wash fluid (BWF), and thus they were not stratified in downstream analyses for TB diagnosis. We also showed that Ultra on concentrated BF had higher sensitivity and lower specificity when compared to Xpert (HIV and alternate reference standards did not significantly change Ultra’s sensitivity and specificity). Moreover, 4 in 5 Ultra “false-positives” started empirical treatment, which suggests that Ultra on BF could be detecting TB cases missed by culture. We also showed that programmatic Ultra testing on BF would benefit from optimisation as study Ultras detected more TB cases. Moreover, we showed that uIFN-γ should not be used on BF for TB diagnosis due to its’ poor sensitivity. Lastly, we showed that urinary-Ultra had low sensitivity, but still detected TB missed by tests on site-of-disease fluid, highlighting its’ usefulness. In terms of outputs, this dissertation has resulted in four first author manuscripts. One has been published in a peer reviewed journal (chapter 2) and the others’ (chapters 3, 4 and 5) are submission ready. Additionally, three ancillary publications (one of which was co-first authored) are briefly discussed in chapter 8 and can be found in the appendices. Some of this research was presented by the candidate at an international and national peer-reviewed conference. In summary, this work shows Ultra’s high sensitivity and moderate sensitivity on FNABs, pericardial fluid, pleural fluid, and BF in patients with presumptive TBL, TB pericarditis, TB pleuritis and PTB. We can therefore recommend a positive Ultra, with the inclusion of trace results, for TB diagnosis in these non-sputum specimens.
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    Detection of host and pathogen biomarkers for Mycobacterium bovis infection in African big cats (lions (Panthera leo), leopards (Panthera pardus), cheetahs (Acinonyx jubatus))
    (Stellenbosch : Stellenbosch University, 2023-12) Gumbo, Rachiel; Miller, Michele Ann; Kerr, Tanya Jane; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Animal tuberculosis (animal TB) is a chronic disease caused by infection with Mycobacterium bovis (M. bovis). In South Africa, animal TB affects many of the country’s most iconic wildlife species including leopards (Panthera pardus), African lions (Panthera leo), and cheetahs (Acinonyx jubatus), and therefore poses a risk to both ecotourism and conservation. Although the overall impact on wildlife populations is unknown, this disease has resulted in historic and recent deaths of cheetahs, lions, and leopards in South Africa. The development of speciesspecific immunological tests for diagnosis of animal TB in wild felids has so far been relatively limited. Therefore, the development of accurate antemortem tests for TB screening and diagnosis of M. bovis infection in African big cat populations is urgently required. The overall goal of this study was to identify host and pathogen biomarkers that can be used to detect M. bovis infection in cheetahs, African lions, and leopards. This was achieved by 1) using conventional mycobacterial culture and GeneXpert® MTB/RIF Ultra (GXU) qPCR for confirmation of M. bovis infection, 2) identification of potential cytokines associated with cellmediated immune (CMI) activation and detection of M. bovis sensitization using cheetah, lion, and leopard stimulated whole blood, and 3) evaluation of serological assays for M. bovis detection. Mycobacterium bovis-infected cheetahs, lions, and leopards were identified using conventional mycobacterial culture and compared with rapid results for Mycobacterium tuberculosis complex (MTBC) diagnosis that were obtained using GXU® qPCR assay. A cytokine release assay (CRA) that can distinguish between M. bovis-infected and uninfected cheetahs, leopards, and lions, using commercially available feline cytokine ELISAs with QuantiFERON® -TB Gold Plus (QFT) stimulated plasma, was developed, and partially validated in African lions. Provisional specific interferon gamma release assay (IGRA) cut-off values, for M. bovis detection in lions and cheetahs, were calculated. The findings in this study suggested that a chemokine C-X-C motif ligand 9 (CXCL9) gene expression assay (GEA), previously designed for use in African lions, showed promise as a tool for detection of both general immune activation and M. bovis immune sensitization in leopards. Although a serological test (DPP® ) used for measurement of humoral response in this study was not sensitive, CMI response-based tests, including the tuberculin skin test (TST), showed potential as antemortem screening tests for wild felids and should be further explored. The incorporation of diagnostic tools into surveillance and routine screening of wild felids for health assessment or as translocation candidates will improve TB detection and prompt management changes to prevent spread of infection and enhance disease control, especially in vulnerable populations.
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    Tools for analysis of Luminex immunoassay data: development of a robust pipeline and best practices recommendations
    (Stellenbosch : Stellenbosch University, 2023-02) Da Camara, Ncité Lima; Tromp, Gerard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Background and Scope: Immunoassays can be used to detect and measure the concentration of many antigens in a variety of specimens for the diagnosis of diseases, and for the detection of microbes and various illegal substances. In addition, they can be used to monitor and study processes, or differentiate infection, latent or active disease in a patient's immune response after infection with a pathogen by measuring the presence of specific antigens. Recent advances in the instrumentation include multiplex immuno-assays, e.g., Luminex 200 or Luminex MAGPIX®, powered by Luminex xMAP® technology. Analysis of data produced by the multiplex immunoassays is complex and current analytical approaches are highly subjective. Currently, there is no standard, robust approach for data pre-processing of multiplex immunoassay data. To overcome this knowledge gap, I developed a robust and standardized data pre-processing pipeline for multiplex immunoassay data to ensure reproducible data. In addition, provide recommendations for best practices of Luminex data generation and data pre-processing to ensure reproducible science by amending existing laboratory standard operating procedure (SOPs) and developed data pre-processing SOPs’ for the Stellenbosch University Bioinformatics Research Group. Design and Approach: I evaluated current laboratory processes, analysed existing Luminex data and drafted best practices recommendations that will ensure reliable input data for the pipeline. I implemented programmatic steps for data management, quality control, and pre-processing to provide high quality data for analyses. A variety of data preprocessing approaches were investigated, and a set of robust standardized options are provided to the user together with appropriate bioinformatics tools in the newly developed pipeline. Results: For robust and reproducible data generation and data preparation, standardization of manual laboratory best practices procedures are recommended and implemented to ensure standard file name convention, file format, file structure and standard plate layout. In addition, the development of a robust automated standardized data pre-processing pipeline using algorithms in R, freely available software will reduce variability and error introduced by humans. In addition, to ensure reliability and reproducibility of the results generated using this pipeline, the pipeline records metadata such as parameter settings, program, and package versions in the output. The methods were validated using existing de-identified Stellenbosch University iv data sets from the Stellenbosch University Immunology Research Group. In the future the pipeline can be applied to newly generated data from a variety of immunological studies. Conclusions: This pipeline will standardize and speed up data pre-processing, as well as provide consistent and reproducible results with any complex analyses. Furthermore, provide the bioinformatician or statistician with a rapid means to pre-process Luminex data for subsequent analysis. The framework developed here can be easily applied to other data analysis projects from different biomedical fields.