School of Pathology (ETDs)
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Item 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, LizetteSouth 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.Item 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, ClareThe 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 achievedItem 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, OliverCardiovascular 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 gapsItem Differential Gene Expression in Exfoliation Syndrome and Exfoliation Glaucoma in the Conjunctiva of Black South Africans(University of the Witwatersrand, Johannesburg, 2024) Hulley, Michaella RobynGlaucoma 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 pathogenesisItem 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, ZanéDevelopmental 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 patientsItem Development of a multiplex HIV/TB point-of-care diagnostic assay based on the microarray(University of the Witwatersrand, Johannesburg, 2023) Malatji, Kanyane BridgettHIV/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.Item Defining Fc-mediated Functions in People Living with HIV during Respiratory Viral Infection and Vaccination(University of the Witwatersrand, Johannesburg, 2024) Motsoeneng, Boitumelo Madika; Moore, PennyThere are approximately 39 million people living with human immunodeficiency virus (HIV) (PLWH) worldwide. Furthering our understanding of humoral immune responses to respiratory viral infection and vaccination in PLWH is essential for reducing the burden of these diseases, in high HIV prevalence settings, and informing vaccine implementation in this population. Influenza virus hemagglutinin (HA) stalk-specific antibodies have been associated with protection and shown to mediate Fc-mediated functions. This thesis describes HA stalk-specific antibody- dependent cellular phagocytosis (ADCP), cellular cytotoxicity (ADCC) and complement deposition (ADCD) between PLWH and people without HIV (PWOH) following immunization with a seasonal trivalent inactivated influenza vaccine (TIV). Irrespective of HIV status, ADCD was boosted while ADCC was not. ADCP was only enhanced in PWOH. The coordination of these functions differed by HIV status. Additionally, differences in the regulation of these HA stalk Fc responses by HIV infection was reported. Furthermore, ADCC was not associated with protection in this study. Pre- existing ADCP reduced the risk of influenza virus infection while TIV-induced ADCD provided protection against influenza-illness. Overall, PLWH have unique responses to TIV and HA stalk- specific ADCD correlated with protection following TIV. For SARS-CoV-2, antiretroviral treatment (ART)-naïve PLWH had reduced humoral responses to respiratory infection. The infecting variants D614G and Beta differentially triggered ADCC, ADCD and antibody-dependent cellular trogocytosis (ADCT). Regarding the kinetics, PLWH infected with D614G had delayed neutralization and ADCP while Beta infection delayed ADCT, regardless of HIV status. PLWH showed improved coordination between immune responses following respiratory infection. ChAdOx-1 nCoV-19 vaccination differed from infection in that PLWH had delayed IgG binding while neutralization and ADCP were not delayed, and ADCC was substantially enhanced than in PWOH. In conclusion, despite the delayed and differential kinetics, PLWH on ART developed equivalent responses to PWOH, supporting the rapid rollout of ART and SARS-CoV-2 vaccines to PLWH. This thesis highlights the need to include high-risk groups with different responses in future vaccination trials and also supports the assessment of novel correlates of protection for future vaccines. Overall, this thesis provided insights into the mechanisms required for protection against severe respiratory diseases and improved our understanding of vaccine-induced immunity in PLWHItem Understanding SARS-CoV-2 vaccine hesitancy among pregnant women in Soweto, South Africa: A qualitative study(University of the Witwatersrand, Johannesburg, 2024) Zungu, Zwile; Myburgh, NellieThe study focused on understanding SARS-CoV-2 vaccine hesitancy among pregnant women in Soweto, South Africa. Pregnant women are at a greater risk of experiencing COVID-19 complications during pregnancy if infected with the SARS-CoV-2 virus. Vaccination uptake remains low in the population at large. This is a qualitative exploratory study approach using key-informant interviews. A total of sixteen key informant interviews with vaccinated pregnant women, unvaccinated pregnant women, healthcare workers and alternative healers were conducted. This study took place in Soweto townships, South Africa. Thematic qualitative analysis was used to construct themes in NVivo, where the gathered data was reviewed and analysed. The study found that pregnant women experience barriers and motivations that determine their decision to get vaccinated against COVID-19. Motivators to vaccinate health concerns, monetary benefit and structural motivators such as employment, travelling and education. Barriers included vaccine related fears were the main reason for poor vaccine uptake. The lack of knowledge, healthcare system barriers, misinformation, and lack of trust in the government were some reasons for vaccine hesitancy. The study's findings show that pregnant women's decisions to get vaccinated are significantly influenced by several barriers, perceptions and the motivators they haveItem Characterisation of the genetic variation in pharmacogenes involved in anti-tuberculosis drug metabolism across African populations(University of the Witwatersrand, Johannesburg, 2024) Malinga, Thandeka Vuyiswa Bongiwe; Twesigomwe, David; Othman, HoucemeddineTuberculosis (TB) is a major health burden in Africa. Although TB is treatable, anti-TB drugs are associated with adverse drug reactions (ADRs) which are partly attributed to pharmacogenetic variation. The distribution of star alleles (haplotypes) influencing anti-TB drug metabolism, is unknown in many African populations. This presents challenges in implementing genotype-guided therapy in Africa to decrease the occurrence of ADRs and enhance the efficacy of anti-TB drugs. Therefore, this study aimed to characterise the distribution of star alleles in genes that are involved in anti-TB drug metabolism (mainly isoniazid), namely CYP2E1, NAT1, NAT2, GSTM1 and GSTT1, across diverse African populations. We used 794 high-depth whole genome sequence datasets representative of eight Sub-Saharan African (SSA) population groups. Data sources included the 1000 Genomes Project and H3Africa AWi-Gen. CYP2E1, NAT1, NAT2, GSTM1 and GSTT1 star alleles were called from the WGS data using StellarPGx. Subsequently, novel star alleles were analysed, and their allele defining variants were annotated using the Ensembl Variant Effect Predictor. We present the distribution of both common and rare star alleles influencing anti-TB drug metabolism across various SSA populations, in comparison to other global populations. Various key star alleles were identified in the SSA study populations at relatively high frequencies including NAT1*10, GSTT1*0 (>50%), GSTM1*0 (49%), and NAT2*5B (21%). Additionally, we predicted varying phenotypic proportions for NAT1 and NAT2 (acetylation) and the GST enzymes (detoxification activity) between SSA and other global populations. Fifty potentially novel haplotypes were identified computationally across the five genes. This study provides insight into the distribution of star alleles in genes relevant to isoniazid metabolism across various African populations. The high number of potentially novel star alleles exemplifies the need for pharmacogenomics studies in the African context. Overall, our analysis provides a foundation for implementing pharmacogenetic testing in Africa to reduce the risk of ADRs related to TB treatment.Item Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker(University of the Witwatersrand, Johannesburg, 2023) Tsolo, Semakaleng Theresia; Ranchod, HeenaThe World Health Organization has prioritized the development of non-sputum-based assays that are capable of detecting active Tuberculosis (TB). Tryptophan (tryp) is converted to kynurenine (kyn) by the rate-limiting enzyme indoleamine 2, 3- dioxygenase (IDO). IDO activity may serve as a biomarker for active TB. Dried blood spots (DBS) can be collected outside of medical institutions and are simple to transport. We wanted to explore the use of DBS as an alternative sample type to measure the kyn/tryp ratio and IDO mRNA gene expression in healthy people. METHODS We optimised methods for elution of dried blood spots, exploring various elution buffers. Following method optimisation, we enrolled 40 healthy participants, and collected whole blood and DBS samples. Kyn and tryp concentrations were measured using ELISA (ImmuSmol, France). IDO mRNA gene expression was determined by real-time PCR using two housekeeping genes GAPDH and BACT. Statistical analysis was performed to determine the correlation and agreement between peripheral blood samples and DBS. RESULTS For IDO activity, tryp showed good agreement between plasma and DBS with a median percentage similarity of 91.1%. In contrast, no agreement was observed for kyn with a median percentage similarity of 56.6%. The kyn/tryp ratio performed poorly due to poor detection of kyn in DBS. Percentage similarity between whole blood and DBS IDO mRNA expression using GAPDH 87.1%, while using BACT was 84.6%. Using either traditional sample types or DBS, there was no correlation between IDO gene expression and kyn/tryp ratio. CONCLUSION We showed that tryp was measurable in DBS. Tryp in DBS was 91.1% similar to values in plasma. Despite method optimization, there was poor agreement between DBS and plasma for kyn. Although IDO mRNA gene expression was detectable in DBS, method agreement with whole blood was unsatisfactory. Alternative methods for the stabilization of kyn in DBS should be explored in future studies. IDO mRNA expression should be measured from whole blood in future studies