Electronic Theses and Dissertations (PhDs)

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    Exploring the Interplay of Chemokine Receptors CCRS and CXCR6 in Mechanisms of Natural Control in HIV-1-lnfected Black South Africans
    (University of the Witwatersrand, Johannesburg, 2023-06) Koor, Gemma Whitney; Tiemessen, Caroline T.; Paximadis, Maria; Shalekoff, Sharon
    In sub-Saharan Africa, HIV-1 is a significant cause of morbidity and mortality. However, research remains primarily focused on North American and European population groups, who have remarkably different genetic backgrounds to individuals from sub-Saharan Africa. HIV-1 controllers represent a model of HIV-1 functional cure, with some individuals able to control viral replication, and some able to sustain immune function in the presence of high viral loads, both in the absence of antiretroviral therapy (ART). The chemokine receptors CCR5 and CXCR4 are the major coreceptors HIV-1 utilises to enter cells. The use of alternative coreceptors, such as the CXCR6 coreceptor, is thought to contribute to the lower pathogenicity exhibited by the HIV-2 and SIVsmm strains. Building on previous work conducted in our research unit on these two coreceptors in South African populations, this thesis firstly describes CCR5 genetic variants that associate with HIV-1 control or risk of progressive infection in black South Africans, and then explores constitutive expression levels of CCR5 and CXCR6 on various peripheral blood immune cell subsets in the absence of HIV-1 infection in ethnically divergent population groups. The effect of sex, age, and select CCR5 and CXCR6 single nucleotide polymorphisms (SNPs) on expression levels of these two receptors was also investigated. The CCR5 5’UTR and 3’UTR regions were PCR-amplified and sequenced from genomic DNA extracted from 145 ART-naive black South African individuals living with HIV-1 (71 HIV-1 controllers – 23 elite controllers, 37 viraemic controllers, 11 high viral load long-term non-progressors and 74 progressors). Findings confirmed results from other studies in showing that the CCR5 HHE haplotype is deleterious for HIV-1 disease progression, and the HHA haplotype and HHA/HHC genotype associated with protection from HIV-1 disease progression. Novel haplotypes were characterised, both in the 3’UTR and spanning the CCR5 5’UTR and 3’UTR. Overall, findings suggest that two CCR5 promoter SNPs (-2459 G>A and -2135 T>C) and one CCR5 3’UTR SNP (+2919 T>G) may be key functional variants with regards to HIV-1 control in black South Africans. To gain further insight into the constitutive expression of CCR5 and CXCR6 on peripheral blood immune cells and explore the relationship between select genetic variants and expression, immunophenotyping by flow cytometry was conducted using whole blood from age- and sex-matched ethnically distinct South African HIV-uninfected individuals (17 black, 21 white). Expression levels of CCR5 and CXCR6 were assessed on CD4+ and CD8+ T cells, B cells, monocytes and NK cells, and their respective subsets. The effects of age and sex on expression levels of these two receptors was also investigated. Population-specific differences with regards to CCR5 expression on all cell types, except for B cells, were evident. Generally, black South Africans exhibited a lower expression level of CCR5 compared to white South Africans. CXCR6 expression only differed with regards to percentage of CXCR6-expressing cells, not CXCR6 density (numbers of cell surface receptors). Black individuals had a lower percentage of CXCR6-expressing CD8+ T cell subsets (naïve and effector memory) and a higher percentage of CXCR6-expressing CD14+CD16+ monocytes compared to white individuals. Overall, we found significant population-specific differences in expression levels of both CCR5 and CXCR6, multiple associations with cell activation (as measured by HLA-DR expression) and CCR5 and CXCR6 expression, and CCR5 and CXCR6 expression was positively significantly correlated on multiple cell subsets. Furthermore, both sex and age influenced CCR5 and CXCR6 expression, however results varied widely across the two population groups studied. Sex differences were only evident in white individuals; predominantly CXCR6 expression was increased in males compared to females. Age associations with CCR5 and CXCR6 expression were also primarily found in white individuals. Four CCR5-related SNPs that are associated with HIV-1 control in this or other studies (rs553615728 -4223 C>T SNP, rs1799987 −2459 G>A SNP, rs746492 +2919 T>G SNP and rs1015164 G>A SNP) were assessed for their potential association with CCR5 expression levels. The +2919 TG genotype significantly associated with a higher percentage of CCR5-expressing total CD8+ T cells, transitional memory and terminally differentiated CD8+ T cells compared to the GG genotype. The +2919 GG genotype associated with a lower percentage of CCR5-expressing B cells compared to the TT and TG+TT genotypes, however, only in white South Africans. The +2919 TG and TG+TT genotypes associated with significantly higher CCR5 density on all CD8+ T cell subsets, except for naïve CD8+ T cells, when compared to the GG genotype. When evaluating two CXCR6 genetic variants previously associated with HIV-1 viraemic control (rs2234355 G>A and rs2234358 G>T) in relation to CXCR6 expression, possession of the rs2234355 SNP GA genotype associated with lower CXCR6 expression on select CD4+ and CD8+ T cell subsets as well as on B cells, while possession of the rs2234358 SNP TT genotype associated with higher CXCR6 expression on multiple cell types, primarily in white South Africans. Possession of the -358TT/+355GA genotype combination associated with lower CXCR6 expression on select subsets of CD4+ T cells and monocytes. In summary, this study provides information on genetic variation in the CCR5 gene in a South African context, describes genetic variants associating with HIV-1 control in black South Africans, adds novel insight into constitutive CCR5 and CXCR6 expression levels on CD4+ and CD8+ T cells, B cells, monocytes and NK cells in HIV-1-uninfected black and white South Africans, and describes the potential associations of select genetic variants and expression. Black and white individuals differed in their baseline expression levels of CCR5 or CXCR6, which was partly driven by host genetic factors that were explored. This work highlights the importance of considering effects of ethnicity, age, and sex in any studies addressing any immune molecules in relation to differential HIV-1 outcomes of infection susceptibility/protection, disease progression, or HIV-1 virological control on antiretroviral therapy. Although conducted on small numbers of individuals, these variables clearly influenced constitutive expression of CCR5 and CXCR6, and further population-specific studies are warranted to gain further insights. Findings from this study have implications for risk of acquisition of HIV-1 infection and for disease progression in people living with HIV-1. Understanding the role of these molecules is important for informing strategies for both HIV-1 prevention and HIV cure.
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    The role of the 20-hydroxyecdysone (20E) signaling pathway in modulating Anopheles arabiensis reproduction, gut microbiome and anti-bacterial immunity
    (University of the Witwatersrand, Johannesburg, 2023-05) Ekoka, Elodie; Dahan-Moss, Yael; Koekemoer, Lizette
    The 20-hydroxyecdysone (20E) signaling pathway, which is activated when 20E binds to its ecdysone receptor, EcR, is a promising target to reduce Anopheles mosquitoes’ ability to transmit malaria. The function of this pathway is typically assessed by altering the pathway and assessing how this manipulation affects a phenotype of interest. Two ways to alter this pathway include injecting mosquitoes with 20E or reducing EcR transcript levels with RNA interference (RNAi). Whether the 20E signaling pathway regulates An. arabiensis fecundity, fertility, gut bacteria, and immunity has never been investigated. These questions were addressed in this study by using a South African An. arabiensis strain. First, RNAi was used to investigate whether EcR silencing affects An. arabiensis reproductive output. While EcR depletion did not affect the mosquito fecundity, both vitellogenesis and egg fertility were impaired, as indicated by adecrease in the expression of some yolk genes and the number of eggs that hatched into larvae. Next, a link between the gut bacteria and EcR expression was established, by showing that antibiotic-fed (i.e., with less gut bacteria) mosquitoes displayed fewer EcR transcripts. To investigate whether the relationship between An. arabiensis gut microbiome and EcR expression was mediated by the mosquito innate immune defenses, the expression of ten selected anti-bacterial immune genes was measured in the gut and the whole mosquito after disturbing the 20E signaling pathway. This experiment uncovered that the 20E signaling pathway down-regulates the mosquito anti-bacterial immune defenses, which may favour bacterial proliferation post feeding. Finally, the effect of Gram-negative and Gram-positive bacteria on EcR expression was assessed by injecting mosquitoes with each type of bacteria and quantifying EcR transcripts. The results suggested that only Gram-negative bacteria influenced EcR expression. Altogether, these results demonstrated that An. arabiensis reproduction, gut microbiome, and antimicrobial peptides are regulated by 20E.
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    Synthetic cytology image generation to augment teaching and quality assurance in pathology
    (University of the Witwatersrand, Johannesburg, 2023-05) McAlpine, Ewen David; Michelow, Pamela; Celik, Turgay
    INTRODUCTION: Urine cytology offers rapid and relatively inexpensive screening for the detection of high-grade urothelial neoplasia in patients with haematuria. In our setting of a public sector laboratory in South Africa, however, there is a paucity of such specimens with which to train cytotechnologists and cytopathologists. Advancements in Generative Adversarial Networks present a potential solution to this problem by allowing for the generation of synthetic urine cytology images to supplement teaching and training. We illustrate an end-to-end machine learning model – from dataset creation to testing synthetic images in pathology personnel – to assess this technology in a real-world setting. METHODS: Two hundred and fourteen urine cytology slides were digitised and processed to construct a morphologically balanced dataset containing examples of benign, atypical and malignant urine cytology images. This dataset was used to train a StyleGAN3 model to generate synthetic urine cytology images. These synthetically generated images were then tested in a group of pathology personnel – both pathologists and trainees – to assess whether a difference between real and synthetic urine cytology images exists. Diagnostic error rate and subject image assessment were tested. RESULTS: StyleGAN3 was able to generate a wide morphological diversity of realistically appearing benign, atypical and malignant urine cytology images. When testing how these synthetic images were perceived by pathology personnel, there was no significant difference in diagnostic error rate, subjective image quality or inclusion of synthetic images in a cytology teaching set. DISCUSSION: This work presents a proof-of-concept illustration of the feasibility of the use of synthetic cytology images to supplement pathology teaching when real examples may be difficult to obtain. Furthermore, this work presents important insights into the dynamics of pathology dataset creation and discusses the use of synthetic data in health education and the ethical and legal issues that arise with the use of synthetic patient data. CONCLUSION: Our work demonstrates that realistic, morphologically diverse urine cytology images can be generated using existing GANs technology and that human observers find such synthetic data visually acceptable. Additionally, our data indicate that there is no significant difference in synthetic data in terms of subjective image quality or diagnostic classification as determined by pathology personnel.
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    The contribution of common genetic variants to breast cancer risk in South African black populations
    (University of the Witwatersrand, Johannesburg, 2023-08) Hayat, Mahtaab; Brandenburg, Jean-Tristan; Ramsay, Michèle; Mathew, Christopher
    Breast cancer is the second most common cancer in South African black women. The contribution of common genetic variants to breast cancer risk is well studied in non-African populations, but little is known about their role in resident African populations, and there are no published genome-wide association studies (GWAS) on breast cancer in Africa. This PhD thesis aimed to determine the contribution of common genetic variants to breast cancer in a South African black population. A GWAS was carried out in 2,573 black female breast cancer patients from the Johannesburg Cancer Study and 744 population-matched, female controls from the AWI-Gen study. All participants were from Soweto, Johannesburg, South Africa. Samples were genotyped on the H3Africa SNP array. Replication testing was done of existing loci from European and African American (AA) populations in the resident African data, and loci from the resident African data in European and AA populations. A meta-analysis was carried out with an AA population. Finally, existing polygenic risk scores (PRSs) were tested in the resident African dataset. Three variants at two loci were strongly associated with breast cancer in this study. Two variants (rs77422433, p-value=2.89x10-08, odds ratio (OR):0.46, 95% confidence interval (95%CI): 0.40-0.52 and rs112410019, p-value=3.01x10-08, OR: 0.47, 95%CI: 0.41-0.53) were located within the DNA repair gene XRCC5. These variants were not previously associated with breast cancer, suggesting that it may be an African specific risk locus. The second locus is on chromosome 16 in CES5A (rs3859109, p-value=4.54x10-08, OR=0.70, 95%CI: 0.68-0.73), and had not previously been associated with breast cancer. None of these SNPs were replicated in European and AA populations. The meta-analysis with AA data revealed strong association of an intergenic SNP with breast cancer (rs139299680, pmeta=7.25x10-08) on chromosome 3. A polygenic risk score (PRS) developed in European populations demonstrated poor transferability to this African dataset. This GWAS is the first to be conducted in a resident black African population. This study suggests that there may be African-specific genetic risk factors for African breast cancer, and that large genome-wide studies in African populations are essential to develop a comprehensive understanding of the genetics of breast cancer in Africa.
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    Development of an Anopheles arabiensis sex separation strain and optimisation of mosquito handling, packaging and transport conditions for the South African Mosquito Sterile Insect Technique programme
    (University of the Witwatersrand, Johannesburg, 2023) Mashatola, Thabo; Munhenga, Givemore; Koekemoer, Lizette
    South Africa is taking significant strides towards eliminating malaria transmission within its borders. However, existing vector control strategies that focus on indoor mosquito management face challenges with Anopheles arabiensis, the primary malaria vector. To bolster these efforts, the sterile insect technique (SIT) is being considered as an additional vector control strategy. SIT involves mass-rearing and sterilising specific pest insects, which are then released to mate with wild insects, effectively reducing the pest population. The South African SIT project faces a crucial challenge of efficiently separating female mosquitoes from the production line. This is because female mosquitoes are capable of transmitting the malaria parasite, making their elimination vital. Current methods like manual separation and resistance-based sorting have operational limitations and require further optimisation for field trials. To address this challenge, this thesis conducted optimisation and acclimatisation experiments on adult Anopheles arabiensis females, aiming to transition them to an artificial membrane feeding technique. Comparative assessments demonstrated that artificial blood-feeding methods utilising a Hemotek® membrane feeding system and hog casing could effectively replace conventional methods without significant detriment to reproductive fitness. Subsequently, the study explored the use of ivermectin, a toxicant, to spike blood during artificial feeding to target and eliminate females. An optimal concentration of ivermectin (7.5 ppm) was identified, showing potential for segregating females from males during laboratory rearing. However, complete female elimination within the desired timeframe was not achieved, indicating that this method serves as a secondary phase for female elimination. The study also investigated the use of genetic sexing strains (GSSs) to selectively eliminate females. Although efforts to induce temperature-sensitive lethal mutations temperature sensitive lethality mutations and random morphological variations were unsuccessful, further cross mating studies and insights from previous studies on thermosensitive strains from Cameroon informed future research aimed at developing GSSs tailored to the South African genetic background Another challenge addressed was the optimal temperature and compaction conditions for chilling and immobilising sterile males during handling and transport. This is crucial for maintaining the quality of sterile males. Optimal knockdown temperature ranges (4°C – 8°C for 20 minutes) and packaging conditions (1000 sterile, marked adult males per 27000 cm³ Bugdorm-1® cage) were identified for laboratory handling and transport, facilitating recent small-scale pilot trials with successful packaging and transportation of sterile males to field sites. These advancements signify significant strides in South Africa's malaria elimination endeavours, while also playing a pivotal role in shaping the development of efficient operational protocols for SIT. Furthermore, as the country remains steadfast in its commitment to eliminating malaria, these innovative approaches offer a promising trajectory forward and establish a robust framework for orchestrating the SIT operational phase.
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    Equitable access to vaccines: exploring the role of accessability, acceptability, affordabilityand availability with a focus on COVID-19
    (University of the Witwatersrand, Johannesburg, 2024) Schwalbe, Nina; Cutland, Clare
    The coronavirus disease (COVID-19) crisis brought to light many challenges, including “vaccine equity”. In other words, it raised the question: was the distribution of vaccines “fair”? While, on the one hand, there have been unprecedented advances in the science and technologies associated with vaccines, including extraordinary speed and scale-up of manufacturing, there were also significant barriers related to rollout and reaching those most at risk of severe COVID-19. These challenges have disproportionately affected low- and middle-income countries and low-income populations in high-income countries. Building on evidence from other vaccine preventable diseases, this thesis describes the challenges and opportunities concerning vaccine access with a focus on production and distribution (the “supply side”). It explores access using a “4A's” framework to conceptualise the components of access to medicines: affordability, availability, acceptability, and accessibility. The research identifies a range of access policy levers across the end-to-end process of vaccine research, development, and rollout (affordability, acceptability, accessibility, acceptability); reviews these levers as they apply to vaccine manufacturing (affordability, availability); explores the lever of financial incentives to increase coverage (acceptability); and explores the potential of using precision public health to improve vaccine impact by targeting vaccine distribution to groups most risk (accessibility). This thesis identifies several policy and program interventions ranging from regulatory harmonisation and intellectual property sharing, to using precision public health to target the delivery of vaccines to those most at risk. It also shows that while financial incentives may help, governments cannot “buy” coverage. It proposes that in future, vaccine development and deployment should start and end with a “4A’s” strategy and provides practical recommendations on how that can be achieved
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    The development and value assessment of an integrated cardiovascular disease risk score in an African setting
    (University of the Witwatersrand, Johannesburg, 2024) Kamp, Michelle; Ramsay, Michѐle; Lewis, Cathryn; Pain, Oliver
    Cardiovascular diseases (CVD) are a significant health threat in Africa and are a leading cause of mortality and morbidity in the region. Risk stratification is the preferred approach to disease prevention but is challenging to apply due to scarce data and current scores not being validated in African populations. CVD risk is influenced by genetic and environmental factors, with the latter mostly considered in risk calculators. Precision medicine (PM) aims to tailor medical diagnostics and therapeutics by leveraging the genetics, environment, and lifestyle of individual patients. Polygenic scores (PGS) can quantify an individual’s genetic burden for CVD and improve the predictive value of risk tools when included with conventional risk factors. PGS for European ancestry populations are reaching the point where they may be useful in clinical care. However, transferability of European-derived PGS to African populations is limited and may hamper application in clinical practice. The overarching aim of the study was to develop and evaluate an integrated CVD risk score incorporating both genetic and non-genetic factors for continental African populations. This comprised of developing ancestry-aligned PGS for various cardiometabolic traits, and then deriving, evaluating, and comparing the predictive utility of genetic, non-genetic, and integrated (genetic + non-genetic) CVD risk prediction models using elastic net regression with nested 10-fold cross validation. Furthermore, we aimed to contextualise the potential utility of such PM-based tools in Africa through an assessment of the current landscape of translational genomics on the continent and by gauging clinician’s perceptions on implementing the derived CVD-risk stratification tool in South Africa’s public health setting. This study demonstrated the potential of genetic information to enhance disease risk stratification among continental African populations. Results revealed PGS provide both independent and complementary information in predicting dyslipidaemia, hypertension, and obesity - integrated scores improved prediction by at least 2.5% compared to models consisting of non-genetic factors alone. The study also highlights the challenges limiting the advancement of PM in Africa – 1. the paucity of genetic and phenotypic data within continental African populations limits the development and validation of robust and clinically useful models. This challenge is exacerbated by Euro-centric genomic and computational tools and echoes the call for more inclusive methodological approaches. 2. Despite positive perceptions of PM, there is an urgent need to address complex structural and operational barriers, including insufficient funding, lack of political will, healthcare infrastructure deficits, and training and education gaps
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    Differential Gene Expression in Exfoliation Syndrome and Exfoliation Glaucoma in the Conjunctiva of Black South Africans
    (University of the Witwatersrand, Johannesburg, 2024) Hulley, Michaella Robyn
    Glaucoma is a heterogeneous group of clinically and genetically complex optic neuropathies. The most prevalent identifiable secondary cause of glaucoma is the ocular manifestation of exfoliation syndrome (XFS), known as exfoliation glaucoma (XFG), a severe form of glaucoma characterized by rapid progression. While XFS is systemic, XFG is defined by fibrillar extracellular matrix (ECM) deposits in the eye. XFG has a recognised genetic aetiology, specifically with contributions from LOXL1 variants, however the molecular pathogenesis remains unclear. Conjunctival cells have not previously been used for whole transcriptome sequencing, and gene expression studies on XFG are lacking, particularly in South Africa. The aim of this study was to validate the use of conjunctival cells for whole transcriptome sequencing and interrogate the gene expression profiles of individuals with XFS and XFG. Conjunctival cells were collected from 19 cases and 15 control participants for RNA extraction, cDNA library construction and sequencing. Differential gene expression was assessed between cells from cases and controls and 81 genes were found to be differentially expressed, with 80 upregulated in cases. Interestingly, LOXL1, which has shown contradictory expression results in previous studies, was not identified as differentially expressed. The most significant overexpression was of the CCN2 gene, which encodes a matricellular protein that interacts with structural ECM molecules. A potential novel pathway involving megakaryocytes and the activation of neutrophils was, however, identified. Megakaryocytes and neutrophils are both involved with interstitial fibrosis, with neutrophils also responding to chemotactic signals to induce inflammation. The inflammatory pathway has long been associated with XFS, XFG and other fibrotic disorders, such as Alzheimer's disease and rheumatoid arthritis. Genes involved in the inflammatory process were overexpressed in XFG, including TGF-β, IL-1β, IL- 33, EGR1 and EGR3. EGR1 and EGR3 are regulated by fibrotic signals and therefore play an important role in fibrosis, which may contribute to XFS fibrillar deposits. They are also regulated by TGF-β, thus supporting a complex interplay of the inflammatory process and fibrosis in XFS pathogenesis
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    The role of small genetic variants in the aetiology of developmental disorders in South Africa - a whole exome sequencing study
    (University of the Witwatersrand, Johannesburg, 2024) Molatoli, Mhlekazi Cathrine; Lombard, Zané
    Developmental disorders (DD) are a diverse group of chronic conditions characterized by significant limitations to both mental and physical development. Genetic variants have been identified as the underlying aetiology in about 40-50% of DD cases. Whole exome sequencing (WES) is the recommended first-line genetic test in this group of patients and is associated with diagnostic yields of 16-45%. However, in South Africa and other resource-poor settings, karyotype testing and MLPA analysis (offering low diagnostic rates of 3% and ~9% respectively) are still being utilized for genetic testing. Thus, a higher proportion of patients remain with unexplained DD due to the limitations of these diagnostic tools and limited genetic services. The main challenge facing the clinical implementation of WES in African settings is the complex data analysis and interpretation associated with the large amount of variant data produced. This is especially challenging as African ancestry individuals have been demonstrated to have a high level of genetic diversity resulting in a higher number of novel variants reported compared to European ancestry individuals. This study seeks to investigate whether the clinical utility of WES can be replicated in an African setting. Additionally, we seek to make recommendations for variant filtering and prioritization, thus making the process of WES data analysis for DD patients more efficient. To achieve these, WES was performed in 117 patients with unexplained DD and their 180 unrelated parents. Variant data was filtered and prioritized using two in-house semi-automated pipelines. The first pipeline, prioritized variants overlapping known DD genes, as identified using the G2P-DDG2P bioinformatics analysis tool. The second pipeline identified de novo variants in trio families using the trio-dnm bioinformatics analysis tool. Sanger sequencing was used to validate low-quality prioritized variants prior to in-house interpretation and curation, and all subsequently identified putative disease-causing variants prior to reporting. Of the 117 patients from 115 families analysed, a positive molecular diagnosis was achieved for 29 families, resulting in a diagnostic yield of 25.2% (29/115). Leveraging currently available DD data, our findings demonstrate the diagnostic and clinical utility of WES which resulted in recommendations for improving patient clinical management and surveillance. This study has also developed and made recommendations for variant filtering and prioritization strategies, which can be implemented in both research and diagnostic settings to streamline and aid in the identification of putative disease-causing variants in DD patients
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    Development of a multiplex HIV/TB point-of-care diagnostic assay based on the microarray
    (University of the Witwatersrand, Johannesburg, 2023) Malatji, Kanyane Bridgett
    HIV/AIDS mortality is caused by opportunistic illnesses/infections that take advantage ofthe weakened immune system in infected individuals. In Africa, the most common of these opportunistic illnesses include infection by Mycobacterium tuberculosis (M.tb) responsible for tuberculosis (TB). HIV co-infection with M.tb has negative implications for disease management given that each pathogen accelerates the morbidity caused by the other. The effective management of patients infected with both pathogens is restricted by the fact that their diagnosis is done separately. The situation is more difficult in remote areas where patients must wait for much longer to obtain their TB diagnostic results. In addition, the current diagnostic tests for the detection of TB such as chest X-ray and bacterial culture have a long turnaround time, are expensive to perform, and require sophisticated equipment and trained personnel. It is in this context that this project sought to develop an HIV and TB multiplex microarray-based assay for the detection of the two diseases using one test. The project used a 2.5 x 7.6 cm epoxy-coated glass slide as well as high- binding 96 well plates to which HIV-1 p24 and M.tb CFP10, ESAT6 and pstS1 antigens, known to be markers of active TB, were immobilized. The immobilized antigens were then incubated with anti-p24, anti-CFP10, anti-ESAT6 and anti-pstS1 primary antibodies diluted in human serum to mimic physiological conditions where the antibodies would exist in the presence of other proteins. Detection of binding between the antigens and primary antibodies was achieved by means of secondary antibodies conjugated to either a fluorescence dye or horseradish peroxidase (HRP). In chapter two of the study, the immobilization of the HIV and TB antigens on the epoxy-coated glass slides as capture molecules of the HIV and TB antibodies diluted in human serum was performed. The antigen-antibody reactions detection were achieved by means of fluorescence dye conjugated secondary antibodies. This chapter also covered the sensitivity and specificity of the technology where the epoxy-coated glass slides were compared to the gold standard 96 well high-binding plates. Data showed that the HIV and TB antigen-antibody reactions were specific, and the slides were more sensitive relative to the 96 well high-binding plates with limits of detection many folds lower. To be specific, the limit of detection from the slides averaged 0.954 ng/ml compared to 4474.6 ng/ml for the plates. The detection limit concentrations of the slides were lower than the reported physiological concentrations of HIV and TB antibodies in infected individuals. Chapter two also focused on the evaluation of the antigens’ stability on the epoxy-coated glass slides by determining the optimal experimental pH buffer, temperature, storage condition (dry or wet), as well as the shelf-life. Data showed that the optimal pH and temperature for the HIV and TB antigens immobilized on the slides were pH 7.4 and 25 ˚C. Moreover, the antigens could be stored dry for at least 90 days without losing their function. Overall, this chapter showed that the epoxy-coated microarray slides performed better than the gold standard 96 well high-binding plates in terms of sensitivity; and that the immobilized antigens could remain stable for a long period, and do not require specialized storage conditions; thus, making the microarray technology a potential diagnostic tool for the multiplex detection of HIV and TB in the case of co-infection. Chapter three of the study focused on the proof-of-concept of the technology using human serum samples infected with HIV. The chapter showed that the technology could detect p24 antibodies in six out of seven samples infected with HIV, i.e., it detected p24 antibodies in 85.7% of samples known to be HIV positive. Furthermore, HIV negative samples also proved to be negative with this technology, thus no false positives were observed. Moreover, the technology was specific for HIV detection as no binding was observed on TB antigens. Therefore, these data support what was observed in the previous chapter when the HIV antibodies were spiked in normal human serum. Chapter four explored the application of the diagnostic technology for the point-of-care (POC) detection of HIV and TB antigen- antibody reaction, using HRP conjugated secondary antibodies, as well as the 2,2′-azino- bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and 3,3',5,5'-tetramethyl Benzidine (TMB) substrates for colour change based endpoint. This chapter also covered the sensitivity and specificity of the immunoassay in the high-binding 96 well plates and on epoxy-coated glass slides. Similar to what was observed in the previous chapter, the HIV and TB antigen-antibody interactions were specific, and the epoxy-coated microarray slides were more sensitive than the 96 well high-binding plates with limits of detection averaging 815-folds lower than the plates. Nevertheless, both platforms were found to be sensitive enough to be used for the POC detection of HIV and TB co infection using visual inspection. Furthermore, the stability of the antigens in the 96 well high-binding plates using colour change detection was also evaluated. The antigens were found to be stable in the high-binding plates at different pH and temperature conditions; however, pH 7.4 and 25 ˚C were optimal. In addition, the antigens were stable when stored dry in the plates for a period of three months. In addition, between the two HRP substrates used, TMB was faster and more sensitive to the HIV and TB antigen-antibody reactions than the ABTS substrate, and the difference was statistically significant (p<0.05). The importance of this chapter is that it eliminated the need for sophisticated equipment to detect the presence of HIV and TB antibodies, as the detection could be achieved by visual inspection. Overall, data in this chapter supported further development of the microarray technology for the POC HIV and TB co-infection diagnosis. Chapter five attempted to produce the CFP10, ESAT6, and pstS1 TB antigens in plants to reduce the cost associated with the current commercially available bacteria-produced antigens.