Masters Degrees (Molecular Biology and Human Genetics)
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- ItemResistance to new tuberculosis drugs(Stellenbosch : Stellenbosch University,, 2024-03) Alberts, Rencia; Theron, Grant; Derendinger, Brigitta; Venter, Rouxjeane; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Introduction: Tuberculosis (TB) is a leading worldwide cause of death, further exacerbated by drug-resistant (DR)-TB. Therefore, new TB drugs are urgently needed. Bedaquiline (BDQ) is the first new TB drug in 40 years and is used for DR-TB treatment. BDQ resistance has, however, already been identified and, at the time of the study, routine phenotypic drug susceptibility testing (pDST) was not done on people initiating BDQ. Additionally, it's important to implement routine DR monitoring for BDQ and other companion drugs. Furthermore, little is known about phenotypic changes over time and whether increases in minimum inhibitory concentrations (MICs) could predict the later DR emergence. Additionally, we aimed to look at the performance of a molecular diagnostic assay for all 13 TB drugs (FLeXT) and determine its sensitivity and specificity. Methods: We determined how MICs from people who started on BDQ with sustained culture positivity changed over time from baseline (BL: closest isolate to BDQ treatment start) to follow up (FU: closest isolate to four-month sustained culture positivity) using a MIC microtitre plate from CRyPTIC. The plate included all 13 TB drugs, including new and repurposed drugs. Additionally, intercedent isolates were also tested using CRyPTIC to look at the change in MIC over time. Furthermore, we sequenced 720 sputa isolates using a manual extraction kit and the GenoXtract kit needed for the FLeXT machine. Results: BDQ MICs increased (p=0.0002) between BL [Median IQR: 0.008 (0.004-0.015)] and FU [Median IQR: 0.030 (0.015-0.019)]. The BDQ isolates had 14/17 (82.5%) isolates showing an increase in MIC over time. Isolates who had sustained culture positivity over time while on BDQ also showed larger increases in BDQ MIC over time compared to the BDQ susceptible https://scholar.sun.ac.za 6 group. Furthermore, for those isolates that indicated BDQ resistance at FU, companion drugs also indicated a higher level of variants and greater increases in CRyPTIC MIC over time. The largest increases in variants and CRyPTIC MICs were found for ethambutol (EMB), the fluoroquinolones (FQs), and ethionamide (ETH). No variants were found for the other new and repurposed drugs: clofazimine (CFZ, delamanid (DLM), or linezolid (LZD) at either timepoint, for both resistance groups. Lastly, FLeXT had a sensitivity of 87% using the manual extraction method (Fluorolyse) and aN 86% specificity for the automated extraction method (FleXT). Conclusion: This highlights the importance of doing routine testing for new drugs such as BDQ as well as companion drugs in people with DR-TB. Our results indicate that there is often a slow increase in CRyPTIC MIC that would lead to resistance over time, and this should be considered in patients with sustained culture positivity). Therefore, it is important to do resistance detection for TB drugs before initiating anyone on a regimen for the treatment to be effective. Furthermore, we proved that the FLeXT GenoXtract has great performance in the detection of DR for second-line TB drugs and serves as a much more efficient way of detecting second-line drug resistance. Molecular assays like this will improve the diagnosis pipeline and help clinicians form individualised treatment plans.
- ItemMolecular epidemiology of SARS-CoV-2 in Malawi: a comparative investigation of the COVID-19 pandemic mitigation and progression(Stellenbosch : Stellenbosch University, 2023-11) Chabuka, Lucious Bestweek; De Oliveira, Tulio; Wilkinson, Eduan; Baxter, Cheryl ; Van Wyk, Stephanie; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Nations in Eastern and Southern Africa collectively reported >23 million cases of COVID-19 from 2020 until 2023. The case numbers are, however, likely to be widely underreported. Nevertheless, these African regions have endured high caseloads, placing severe strain on already burdened healthcare systems. Genome surveillance has proven invaluable for detecting harmful variants of the SARS-CoV-2 virus in the past, allowing for informed public health responses. As Variants of Interest (VOI) and Variants of Concern (VOC) commonly emerge from these regions, effective genome surveillance remains globally important. Nearly 20 million people live in Malawi, a landlocked country in South-Eastern Africa. It has been affected by five COVID-19 pandemic waves (2020-2022), comparable to other nations in the region. Despite the strain the epidemic has placed on Malawi's health services, the impact of the genomic surveillance system during the pandemic has not yet been thoroughly examined. To allow for data-driven and informed pandemic mitigation strategies to navigate the ongoing COVID-19 pandemic and to address concerns of future pandemics, a comparative investigation of the COVID-19 pandemic progression in Southern and Eastern African regions was conducted. This epidemiological information was complemented with genome surveillance data generated for Southern and Eastern African countries. This was done to gain an in-depth understanding of the data generated and circulating variants and to determine the way SARS CoV-2 variants fuelled national and international pandemic infectious waves. Therefore, this study aimed to place the South- and East-African pandemic progression and current genome surveillance initiatives into a global context. In this study, we hypothesised that investment in the laboratory and bioinformatic expertise improved the overall pathogen surveillance capacity within these regions, and most of the SARS-CoV-2 introductions into Malawi came from South Africa. However, the data generated with the surveillance networks established within Southern African regions outcompetes those in the East African regions with enhanced capacity for so-called "in country" sequencing, allowing for reduced turnaround times. Therefore, the Southern African regions, including countries such as South Africa and Botswana, served as a model for Eastern African countries such as Malawi, Tanzania, and Kenya. Moreover, import and export virus introduction events, determined using phylogenetic inferences, indicated that more introductions occurred from Southern African regions into Malawi than from East African regions. This may reflect higher sampling and subsequent genome sequencing from Southern African regions and the economic importance of these regions that largely constitute economic and travel hubs of the African continent. This comparative investigation on COVID-19 across nations highlighted areas requiring improvement in genomic surveillance to assist nations in being better prepared for future pandemics.
- ItemDevelopment of containerized pipelines for the reproducible analysis of amplicon-, shotgun metagenomic- and metatranscriptomic data.(Stellenbosch : Stellenbosch University, 2024-03) Madzime, Ruvarashe Joylyne; Tromp, Gerard; Sanko, Tomasz; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Advances in next generation sequencing technologies have enabled the investigation of microbial genetic material directly from a biological specimen without the need for culturing. This has propelled the field of metagenomics and set techniques like amplicon-, shotgun metagenomic- and meta transcriptomic sequencing at the forefront of investigating complex microbial communities. Data from these techniques is very large, over gigabytes (GB) in size, and often needs to be analysed on high performance clusters and servers. These computational requirements introduce a problem of variation in compute environments, which leads to irreproducibility. The data are high-dimensional and compositional, and there are specific algorithms that address these qualities of the data. However, the software algorithms are updated regularly, providing multiple versions of the same software algorithm, and this too leads to irreproducibility, therefore affecting the integrity of science. This project addresses computational irreproducibility through the development of three independent computational pipelines, designed to be used on Unix/Linux-based clusters and servers. I developed a pipeline implementing QIIME2, for analysing amplicon sequence data. For meta transcriptomic data, I developed a pipeline implementing Trinity and its utility workflow for de novo assembly of transcripts and differential expression analysis. I developed a pipeline for analysing shotgun metagenomic data, implementing multiple algorithms, meta SPAdes, Maxbin2, prokka, BLAST and deep ARG. I packaged all the algorithms in separate Singularity containers for version control and consistency of execution environment. All three pipelines were developed and launched using the Next flow workflow management system. Using the respective data for each pipeline, the pipelines managed to run in an automated manner on a local university server and a PBSPro cluster. All three independent containerized pipelines were successfully implemented. Future work will include multi-stage development of containers, robust validation of the pipelines, and adding features like optional software algorithms to the pipelines.
- ItemFerrocene sulfonyl derivatives as antimycobacterials(Stellenbosch : Stellenbosch University, 2024-02) McMaster, Erin Cullen; Mavumengwana, Vuyo; Chellan, Prinessa; Niemand-Wolhuter, Nandi; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Tuberculosis (TB) remains a global health threat, necessitating innovative strategies to combat Mycobacterium tuberculosis, the causative agent. This thesis explores the multifaceted approach of developing ferrocene-based compounds as potential antimycobacterial agents. Macrophages, critical components of the host's defense against M. tuberculosis, serve as the initial battleground for M. tuberculosis infection. The bacterium adeptly manipulates host defences, thriving within macrophages and evading immune responses. Targeting mycobacteria within macrophages is crucial for successful treatment, minimizing host tissue damage, and modulating the immune response effectively. The study capitalizes on the dependence of mycobacteria on iron, a crucial element for its survival within macrophages. Ferrocene, known for its lipophilic properties, was conjugated with a selected range of sulfonamides to realise novel ferrocene sulfonamide derivatives. The latter were subjected to extensive analysis, including nuclear magnetic resonance (NMR), infrared spectroscopy (FT-IR), mass spectrometry (ESI-MS), and high-performance liquid chromatography (RP-HPLC). UV-Vis spectroscopy revealed stability concerns, prompting considerations for improving solubility and bioavailability. Turbidimetric studies highlighted poor solubility in aqueous environments, emphasizing the need for advanced delivery systemsin future studies. Microbiological assessments, including Minimum Inhibitory Concentration (MIC) experiments, shed light on the compounds' effectiveness against Mycobacterium smegmatis mc2155. The most effective derivative showed an MIC of 0.0039 – 0.00195 mg/mL compared to >0.26, >0.30, and >0.49 from derivatives ferrocene benzene sulfonyl hydrazide (P1), ferrocene ptoluene sulfonyl hydrazide (P2), and ferrocene trifluoromethyl sulfonyl hydrazide (P3) respectively. Intriguingly, certain derivatives demonstrated potential growth-promoting effects, indicating an intricate relationship between ferrocene compounds and mycobacterial growth. The ferrozine assay, assessing intracellular iron levels, suggested a dynamic interaction between mycobacteria and the ferrocene derivatives, with fluctuations in iron absorption during the growth curve. The final component of the study involved evaluating cytotoxicity on THP-1 cells, where ferrocene benzene sulfonyl hydrazide (P1), ferrocene p-toluene sulfonyl hydrazide (P2), ferrocene trifluoromethyl sulfonyl hydrazide (P3) and the ferrocene-RP-1 derivative show promise, although further replicates are essential for conclusive results. In conclusion, this thesis presents a comprehensive exploration of ferrocene-based compounds as potential antimycobacterial agents. The findings underscore the complexity of the interactions, necessitating a nuanced approach for drug development. Addressing solubility challenges and further elucidating the mechanisms underlying seeming sustenance of growth but intracellular disruptions as observed through scanning electron microscopy could pave a way for novel therapeutic strategies against tuberculosis.
- ItemThe influence of biomimetic nanoparticles on macrophage phenotype and their anti-mycobacterial efficacy on macrophages.(Stellenbosch : Stellenbosch University, 2024-02) Maaruf, Shamsuddeen Yusuf; Sampson, Samantha Leigh; Du Plessis, Nelita; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) remains a major public health concern. Despite the availability of various treatment regimens, treatment failure is common, partly attributed to the phenomenon of bacterial persisters. In addition, repeated or lengthy treatments can potentially induce drugresistant strains of M. tuberculosis which further compound TB control. New approaches to TB therapy are therefore needed. To promote the intracellular killing of M. tuberculosis through pro-inflammatory responses (M1), a promising approach is the application of nanoparticles (NPs), which include lipid-polymer hybrid (LPN), to deliver drugs or immunomodulatory compounds such as Curdlan (CN) to macrophages. This represents a novel, host-based therapeutic approach for TB. While M1 polarized macrophages are thought to be the most effective in controlling M. tuberculosis infection, it has been shown that intracellular bacilli induce a shift to the M2 phenotype. There is no evidence that the introduction of NPs such as LPN conjugated with CN can induce a shift in macrophage phenotype or that the macrophage polarization state affects the antibacterial activity of NPs. In this study, we first set out to evaluate the anti-mycobacterial efficiency of CN-functionalized NPs in vitro, which had previously shown to be promising in the inhibition of mycobacterial growth intracellularly and in vivo in a parent study. Primary cells derived from the C57BL/6 mice bone marrow called bone marrow derived macrophages (BMDMs) were used for this study. We used an attenuated M. tuberculosis H37Rv laboratory strain with a reporter plasmid (pMV306hsp+Lux) to allow real-time monitoring of luminescence as a proxy for the bacterial load. Results from the real-time luminescence monitoring confirmed that the CNfunctionalized NPs is a good anti-mycobacteria agent as previously demonstrated in the parent study. We further explored if macrophage phenotypic state and function might alter upon treatment with selected NPs, and in turn, if macrophage phenotype alters the immunological response induced by selected NPs. In our study, we examined macrophage phenotype through multiplex cytokine analysis, focusing on 11 analytes indicative of macrophage polarization status. We utilized a specific mathematical formula, termed the macrophage polarization ratio (MPR), designed for this investigation. Results demonstrated that macrophages have an attraction for CN-functionalized NPs. In addition, the NPs have an impact on macrophage immunologic and phenotypic activity. The NPs induced high production of pro-inflammatory cytokines such IFN-ɣ, IL-1β, IL-12p70, IL-18, IL-2, IL-5, TNF-α, and IL-6. In summary, our study demonstrated the bacterial strain's is consistent in terms of replication homogeneity for both liquid culture and intracellularly, confirming M. tuberculosis's suitability for TB research with a reporter plasmid (pMV306hsp+Lux). Significantly, the CN-functionalized nanoparticles demonstrated noteworthy anti-mycobacterial efficacy. Data on macrophage viability highlighted the possible importance of a lower multiplicity of infection (MOI) for BMDMs. Recognizing the need for additional research, our findings indicated that CN-functionalized nanoparticles have a discernible influence on macrophage phenotype, specifically by increasing the expression of M1 markers. This comprehensive understanding highlights the promising role of CN-functionalized nanoparticles in shaping macrophage responses, implying avenues for targeted tuberculosis therapeutic interventions. By investigating the complex area of nanoparticles' impact on macrophage phenotype during TB treatment, this study filled a knowledge gap. This study expands our understanding of the dynamics of TB treatment and emphasizes the need for additional research to fully realize the promise of nanoparticle-mediated interventions in the treatment of TB. However, further research into the concentration-dependent anti-mycobacterial activity and transcriptomic response is required.