Electronic Theses and Dissertations (Masters)
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Item A laboratory based retrospective study of plasma cell myeloma in the public sector of South Africa from 2017 to 2019(University of the Witwatersrand, Johannesburg, 2024) Wilding, Bradley Thomas; George, JayaBackground Plasma cell myeloma is a haematological malignancy characterized by clonal proliferation of plasma cells. This malignancy is frequently associated with the production of a monoclonal protein in either serum and / or urine, referred to as an M protein, which is used as a screening test for patients. Patients are then further investigated to assess if they meet the International Myeloma Working Group (IMWG) diagnostic criteria for plasma cell myeloma. There is limited literature describing plasma cell myeloma in South Africa, particularly in people living with HIV. Objective The primary objective of this study was to describe plasma cell myeloma in patients diagnosed in the public sector of South Africa over a three-year period. The secondary objective was to compare demographic features (age, sex) and diagnostic criteria, between the myeloma patients living with HIV and the HIV negative myeloma patients. Methods A retrospective analysis was performed on data from 4518 patients who had a positive immunofixation on serum and / or urine from public sector hospitals, between 2017 and 2019. A total of 718 of the 4518 patients met the laboratory criteria for plasma cell myeloma and were included in the analysis. Demographics (age, sex) and laboratory investigations used in the diagnostic criteria for plasma cell myeloma were analysed and statistically compared across the different HIV status of patients. Results Plasma cell myeloma patients presented at a mean age of 59.46 years with a female to male ratio of 1.2:1. In the patients that met the diagnostic criteria the most common end-organ damage present was anaemia in 77.16% patients and the most common biomarker of malignancy was a bone marrow trephine biopsy plasma cell percentage t60% in 55.71% patients. IgG isotype was the most common paraprotein detected on serum immunofixation in 58.5% of the patients. Kappa was the most common Bence-Jones protein detected in 27.16% of patients which was 1.76 times more common than lambda Bence-Jones protein. People living with HIV were younger 55.11 (±9.79) as compared to their HIV negative counterparts (p value 0.010). No other statistically significant difference was noted when comparing HIV status groups Conclusion In conclusion, this study described the demographics, laboratory investigations and diagnostic features of plasma cell myeloma patients diagnosed in the South African public sector from 2017 to 2019. We found that people living with HIV were diagnosed at younger age when compared to their HIV negative counterpartsItem A multicentre study to evaluate an in-house multiparameter immunophenotypic panel to identify precursor B-cells in the determination of measurable residual disease in paediatric B-cell acute lymphoblastic leukaemia(University of the Witwatersrand, Johannesburg, 2024) Nell, Zanre; Glencross, Deborah; Geel, JenniferBackground: Periodic assessment of measurable residual disease (MRD) is an important prognostic factor in the management of paediatric B-cell acute lymphoblastic leukaemia (ALL). Conventional polymerase chain reaction (cPCR) and multiparameter flow cytometry (MFC) are well-established in MRD determination, the latter with no current optimal immunophenotypic panel by international consensus. Objective: To determine whether an in-house immunophenotypic panel containing the discriminatory CD58-FITC (cluster of differentiation; fluorescein isothiocyanate) marker compares with cPCR in the detection of paediatric B-cell ALL MRD. Methods: This prospective descriptive validation study was performed on diagnostic and follow-up bone marrow aspirate samples, comparing an in-house immunophenotypic panel against the standardised commercial ClearLLab 10CTM B-cell/myeloid cell-2 (M2) panels in MRD assessment. These findings were then compared to cPCR to determine individual panel performance and predictive power. Results: Both immunophenotypic panels demonstrated 100% concordance in the identification of the leukaemia-associated immunophenotype (LAIP) on all diagnostic samples. The in-house immunophenotypic panel showed a higher sensitivity and specificity, and greater association with cPCR in MRD assessment in follow-up samples. In combination with shared backbone markers of the ClearLLab 10CTM B-cell/M2 panels, inclusion of CD58-FITC and CD81-APC-H7 (allophycocyanin- cyanine dye) proved most informative in accurate distinction between regenerating B-cell precursors and residual leukaemic cells. Conclusion: This work confirms the findings of previous studies, where discriminatory marker CD58- FITC in combination with backbone informative markers demonstrates both superior diagnostic and monitoring utility in paediatric B-cell ALL. The in-house immunophenotypic panel offers an attractive, comparable alternative in MRD determination in this patient population whilst awaiting cPCR results, raising the possibility of earlier clinical decision-making with potential improvement of morbidity and mortality outcomesItem A review of genetic testing for females referred to genetic counselling for a personal or family history of gynaecological cancer(University of the Witwatersrand, Johannesburg, 2023) Barnard, SebastianHereditary cancer syndromes, caused by pathogenic variants in specific genes, substantially increase an individual’s risk for cancer and are estimated to cause 10% of all uterine cancers and 20% of all ovarian cancers. However, these data are primarily based on high-income countries and to date there is no published data on the known variants and testing of cancer predisposition genes associated with gynaecological cancers in South Africa. In this study, patient records for those with either a confirmed diagnosis or family history of gynaecological cancer that were seen by the Division of Human Genetics (NHLS/Wits) were retrospectively analysed (n = 104). Associations between patient characteristics, genetic testing availability and the detection of pathogenic variants as well as the utility of risk assessment tools were investigated using statistical analysis. The majority of patients underwent diagnostic genetic testing (78/104, 75.0%), 25 (32.1%) were positive, 41 (52.6%) were negative, and 12 (15.4%) returned a variant of unknown significance. Test results were significantly different between European and non-European patients (p << 0.05) with non-European patients being 30% less likely to have a pathogenic variant detected (OR 0.7, 95% CI 0 0.22, 2.21). Patients who met genetic testing criteria according to online risk assessments were more likely to have a positive genetic test result than those who did not (p < 0.05). A disparity exists not only in genetic testing availability but also clinic attendance between public and private healthcare which is likely limiting the ability to diagnose hereditary cancer syndromes associated with gynaecological cancers in public healthcare hospitalsItem Analysis of Whole Exome Sequence Data from African Patients with HD-Like Features and No Known HDPhenocopy Syndrome(University of the Witwatersrand, Johannesburg, 2023) Naicker, Racilya; Krause, Amanda; Baine-Savanhu, FionaHuntington disease (HD) is a rare progressive neurodegenerative disorder that results from a CAG repeat expansion within the huntingtin gene (HTT). Several syndromes present with HD- like features in the absence of the HTT expansion and are termed HD phenocopies. Huntington disease-like 2 (HDL2), a known phenocopy, is most commonly observed in individuals with African, or probable African, ancestry. Therefore, previous diagnostic testing in the Division of Human Genetics, National Health Laboratory Service (Johannesburg, South Africa) screened for both HD and HDL2 in patients with HD-like phenotypes and an indication of African ancestry. Patients who tested negative for both syndromes remain undiagnosed, highlighting the need for further testing strategies. This study aimed to identify variants implicated in the HD-like phenotype of these patients. In a group of nine undiagnosed patients with Black African ancestry, a virtual gene panel was analysed through a whole exome sequencing (WES) approach. The data was filtered, and candidate variants were prioritised according to the frequency, type, and location of the variants as well as in-silico pathogenicity prediction scores. A total of 20 candidate variants in 15 genes were shortlisted and classified according to ACMG/AMP guidelines. Notably, variants in the mitochondrial DNA polymerase subunit gamma (POLG; c.2246T>C; p.Phe749Ser) and the glutaryl-CoA dehydrogenase (GCDH; c.877G>A; p.Ala293Thr) genes were classified as likely pathogenic and pathogenic, respectively. Genotype-phenotype correlation indicated a potential diagnosis of autosomal dominant progressive external ophthalmoplegia in the patient carrying the POLG variant, whereas the GCDH variant was considered an incidental finding due to a lack of correlation with the characteristics of glutaric aciduria type 1. These findings highlight the diagnostic challenges faced in the African context for patients with HD-like clinical features and call for further validation studies and re-analysis of the WES data using alternative gene panels for the patients for whom no putative pathogenic variants were identifiedItem Assessing the propensity of drug resistant tuberculosis to enter and exit the differentially culturable state(2024) Nonkula, BomikaziTuberculosis (TB), one of the oldest and most contagious infectious diseases, continues to be a global health concern. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC) which comprises of several species. These species are further subdivided into strains based on subtle genetic differences. The success of M. tuberculosis as a pathogen can be attributed to its ability to survive various stresses by adopting different growth states. Previous studies have shown that sputum from TB infected patients harbours a large proportion of drug-tolerant bacteria that are unable to form colonies on agar plates but can grow in liquid media. This population of organisms, termed differentially culturable tubercle bacilli (DCTB), could be resuscitated to grow by supplementing liquid media with cell free culture filtrates from axenic cultures of wild type M. tuberculosis H37Rv or mutant H37Rv lacking all five resuscitation promoting factors (Rpfs). Laboratory models that induce this differentially culturable state are critical for studying the physiology and metabolism of these bacteria in order to develop new TB diagnostic tests. In this study, five Beijing and five LAM drug resistant strains of M. tuberculosis were selected and used to robustly generate DCTB through an in vitro stress model using carbon starvation. The most probable number (MPN) assay and colony forming units were used to determine the amount of DCTB. Furthermore, the phenotype of these cells was studied using microscopy as well as metabolic probes that target the peptidoglycan (PG) component of the bacterial cell wall. Our findings demonstrated that applying the carbon starvation model to clinical M. tuberculosis strains (Beijing and LAM) resulted in robust levels of DCTB, as evidenced by limited growth on agar plates and enhanced growth in liquid media supplemented with culture filtrate from LAM and Beijing strains. Comparison of cell length between carbon starved cells to those grown in routine laboratory media suggested that DCTB appeared to be non-replicating and significantly shorter. The metabolic activity of the starved cultures was restored when they were supplemented with H37Rv, LAM and Beijing culture filtrate. Our results also demonstrated that Beijing strains had a higher propensity to produce DCTB compared to LAM strains and that the supplementation with Beijing culture filtrate resuscitated more DCTB. Collectively, our findings allow for the advancement of experimental systems that enable further investigation of DCTB and the properties of the Beijing strain that facilitate better adoption of the differentially culturable state.Item Audit of Lysosomal Storage Diseases Testing at the National Health Laboratory Service in Johannesburg from 2011-2020(2023) Novellie, MichaelLysosomal Storage Diseases (LSDs) are a group of Inborn Errors of Metabolism (IEM), due to the lack of a lysosomal enzyme. This results in toxic accumulation of metabolic waste products in various organs leading to neurodevelopmental regression, organ failure and premature death in the absence of treatment. Treatments for LSDs are limited. This study audited LSD diagnostic test requests received by the Division of Human Genetics, National Health Laboratory Service (NHLS) in Johannesburg from 2011 to 2020 with the aim of understanding the demand, appropriateness, and patient management of suspected LSD cases. A quantitative survey of all samples (1861 tests) referred to NHLS Johannesburg during the study period was performed. A total of 198 (13.3%) samples were rejected for testing mainly because of faulty sample collection. Of the 1663 that were accepted for testing 1457 (87.6%) tested negative, 73 (4.4%) were inconclusive and 133 (8.0%) tested positive. Fifty-five (3.1%) patients with LSD test requests, all of which were positive, were known to a Clinical Genetics unit. The most frequently requested test was for Fabry disease: 620 (33.3% of all requests), even though this disease is not the most prevalent LSD. Of the 603 accepted test requests for Fabry disease, only 6 (1.0%) tested positive. This suggests that some referring clinicians had unrealistic expectations of encountering this disease. It should be noted, however, that testing for Fabry disease is part of a broad diagnostic workup that may be applied even if the indication for testing is not specific. Access to LSD testing was unequal: private facilities were proportionally over-represented compared to public facilities; certain provinces with large referral centres (in KZN and Gauteng) were over-represented compared to smaller centres. Feedback and education of referring clinicians regarding indications for testing and importance of patient follow up, especially by clinical genetics services, are recommended. Follow up of positive MPS screening tests with specific diagnostic tests is essential. A system should be implemented where a medical geneticist phones the referring clinician and discusses further sample requirements (blood for enzyme analysis) and referral to a genetics clinic for all positive LSD screening tests. Future consideration should be given to designing a more systematic testing process, with the introduction of molecular testing to supplement biochemical testing.Item Characterisation of RSV fusion proteins from South African patients with RSV disease, 2019 to 2020(2024) Mabilo, PrinceRespiratory syncytial virus (RSV) is classified into subtypes A (RSV-A) and B (RSV-B), which are classified into different genotypes based on genetic variability of the G surface glycoprotein gene. The F surface protein gene is more conserved however variability in signal peptide, transmembrane domain, and antigenic sites have been reported. The study was conducted in the Virology laboratory, Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), South Africa. Study participants included patients of all ages from whom respiratory samples were submitted for respiratory viruses diagnoses from 2019 to 2020. The complete RSV F genes were amplified, library prep was preformed using the Nextera DNA prep kits and sequenced using amplicon-based next generation sequencing on the Illumina MiSeq sequencing platform. The Genome Detective Virus tool v2.27 was used to assemble sequencing reads and MEGA X was used for phylogenetic analysis. N-linked glycosylation and amino acid sequence variation was assessed. The overall prevalence of RSV was 5.8% (101/1 734). Seventy (69.3%; 70/101) RSV-positive samples were available for genetic characterisation of the F protein gene and thirty-one (30.7%; 31/101) were excluded due to insufficient sample volume. Only RSV-A strains were identified (91.2%; 31/34). Twenty three of thirty-one (74.2%) of the RSV F gene sequences from 2019 to 2020 clustered together with bootstrap values ranging from 64% to 99% and were NA1-like. A N-glycosylation site at position 120 gained by South African strains from 2018 is retained in strains from this study. This N-glycosylation site is present in approximately 25.8% of RSV strains from this study. The diversity of RSV-A F proteins was low, with amino acid variations observed at 30/571 (5.3%) sites. Ten mutations were detected in 4/6 antigenic sites (I, II, IV and V), with frequencies ranging from 0.3 to 100%. Antigenic changes seen exclusively among South African strains are: Y33H (0.3%) and V384T (7.3%) at site I and S275F (0.3%) at site II. Seven mutations associated with escape of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocyte (CTL) were predicted in seven epitopes. Overall amino acid mutation frequency for 2019 to 2020 RSV-A F genes is similar to that reported for South African strains from 2018 (3.3% to 6.7%). For the first time in South Africa we detected the S275F mutation which causes palivizumab resistance.Item Characterisation of the murine immune response to self-amplifying mRNA sequences encoding Hepatitis B virus surface proteins(University of the Witwatersrand, Johannesburg, 2024) Samudh, Nazia; Bloom, KristieVaccination against Hepatitis B virus (HBV) remains the most effective means of preventing infection. However, approximately 10% of vaccinated individuals fail to develop neutralising antibodies necessitating the development of improved vaccines which target the more immunogenic large HBV surface antigen (L-HBsAg) and can elicit cell-mediated immunity. Although Africa bears a high burden of HBV infections, placing many individuals at risk of contracting the disease, we rely on imported vaccines for prophylactic vaccination programmes. The COVID-19 pandemic was a stark reminder of Africa’s vaccine dependence and since then great interest has been generated in establishing vaccine manufacturing capabilities on the African continent. Herein, we explored the Alphavirus-based self-amplifying RNA (saRNA) vaccine platform to produce dose-sparing HBV vaccines which could contribute to vaccine independence. saRNAs encoding reporter proteins, small HBV surface antigen (S-HBsAg) or L-HBsAg were synthesised by optimised in vitro transcription. Expression of reporter proteins from saRNAs was achieved even at low concentrations and was observed for extended periods of time in vitro. saRNAs encoding S-HBsAg were able to trigger the interferon response in a dose-response manner in vitro, however, this did not hamper antigen expression. Expression of L-HBsAg was achieved but restricted to the intracellular space and will require sequence modification to facilitate secretion. In vivo delivery of saRNAs by electroporation or commercially available cationic liposomes was found to be unsuccessful, and further optimisation of in vivo saRNA delivery is required before determining the prophylactic potential of candidate vaccines. This preliminary study has produced promising results demonstrating the dose-sparing properties and self-adjuvanting nature of the saRNA vaccine platformItem Characterisation of variation in the CYP2C19 gene in African populations(2024) Booyse, Ross PeterBackground: CYP2C19 pharmacogenetic testing is important clinically to optimise patient response to clopidogrel and anti-depressants. This study aimed to characterise the distribution of CYP2C19 star alleles (haplotypes) across diverse African populations compared with global populations, with a view towards informing future pharmacogenetic implementations. Methods: CYP2C19 star alleles and diplotypes were called from 604 high coverage genomes from continental African populations using the StellarPGx pipeline. Results: From our analysis, CYP2C19*1 (51%), *2 (17%), and *17 (22%) were the most common star alleles across African populations in this study. We also identified 3% of African participants that had potentially novel CYP2C19 haplotypes. Over 70% of the SSA participants had either poor, intermediate, rapid, and ultrarapid metabolizer status, and would likely benefit from dosage and/or treatment alterations, especially for clopidogrel Conclusion: This study supports the necessity for CYP2C19 pharmacogenetic testing in African clinical settings and the importance of comprehensive star allele characterisation in the African context.Item Characterising the combined eects of cytochrome P450 missense variants within the star allele nomenclature(University of the Witwatersrand, Johannesburg, 2024) Khoza, Nhlamulo; Othman, Houcemeddine; Hazelhurst, ScottGenetic variations in Cytochrome P450 (CYP) enzymes shape drug responses. However, many CYP haplotypes (star alleles) lack functional annotations, posing a barrier to under- standing drug metabolism comprehensively. To address this, our study investigates combined missense variant eects on CYP enzyme structures, analyzing 261 variants across 91 CYP haplotypes. We utilized Normal Mode Analysis (NMA; FoldX and ENCoM) to explore variant impact on protein stability. Subsequently, we conducted Molecular Dynamics (MD) simulations on key alleles, CYP2D6*2 and CYP2D6*17, to reveal star allele impact on protein dynamics. Integrating NMA and MD, we uncover the interactions that collectively shape the conformation and attributes of CYP enzymes. Notably, our investigation highlights significant deviations between wild-type and CYP2D6*17 -encoded proteins in the F/G loop region, pivotal for CYP functionality. Additionally, we compare NMA results with CYP2C9 and CYP2C19 Deep Mutational Scanning (DMS) results, identifying 65% concordance. Furthermore, our NMA predictions show 80% concordance with commonly used Variant Eect Predictor tools. This alignment underscores our approach’s reliability in predicting variant eects. Our study illuminates missense variants’ nuanced impact on CYP protein structures, significant for personalized medicine and drug response prediction, as accurate drug response predictions hinge on a comprehensive understanding of CYP missense variants. Moreover, our study highlights multi-scalemodelling potential for interpreting CYP missense variants, especially in star alleles. The synergy of NMA, MD simulation, and assays like DMS is invaluable, each with distinct strengths and limitations. This research deepens our understanding of the complexity of CYP metabolism profiles, providing insights into the functional assessment of CYP star alleles and missense variants with unknown functional classifications.Item Colonisation with ESKAPE organisms and Candida auris among primary caregivers and healthcare workers in a neonatal unit at a public sector tertiary South African hospital(2024) Rees, NickiBackground: Nosocomial neonatal infection remains a significant cause of mortality and morbidity, particularly in the high care and intensive care settings. Among implicated pathogens ESKAPE- C organisms are considered particularly worrisome due to their virulence, ability to gain resistance and propensity to affect multiple sites. Transmission to neonates is postulated to occur through contact with colonised adults. Objective: This study aims to describe the prevalence of colonisation of both primary caregivers and healthcare workers in contact with admitted neonates. As a secondary objective this study aims to identify the most common resistance patterns in ESKAPE-C organisms isolated from primary caregivers and healthcare workers in a neonatal unit. The overall aim of the study is to provide insight into how best to prevent hospital acquired infections in this group. Methods: This cross-sectional prevalence study describes colonisation of healthcare workers (HCW) and primary caregivers in a neonatal unit in a tertiary South African hospital. Over one week in August 2021, twenty-five primary caregivers and twenty-nine healthcare workers submitted specimens which were processed for the identification of ESKAPE-C organisms. Susceptibility was performed on identified organisms. Results: Of the healthcare worker participants 13,8% (4/29) were shown to be colonised with one or more ESKAPE-C pathogen, while 52% (13/25) of primary caregivers were shown to be colonised with one or more ESKAPE-C pathogens. Of the S. aureus organisms isolated 28,6% were MRSA, of the A. baumannii organisms isolated 66.7% were XDR and of the Enterobacteriaceae isolated 60% were ESBL producing. No CRE or VRE organisms were isolated in this study. Conclusion: This study demonstrates that the prevalence of colonisation of healthcare workers and primary caregivers is significant and reinforces the need for stringent infection prevention and control strategies to prevent transmission to vulnerable neonates.Item Combination interaction of fluconazole with efflux pump inhibitors against antifungal resistant Candida parapsilosis(2024) Alati, Marwah Omar AliIntroduction C. parapsilosis is the most common fungal pathogen recovered in neonatal intensive care units and is associated with a higher mortality rate due to its multidrug resistance. This study investigated the virulence factors and reversal of efflux activity by using a well-known antifungal drug (fluconazole) in combination with two efflux pump inhibitors (pantoprazole and omeprazole). In addition the effect of most active combination on pathogenicity markers of C. parapsilosis has studied. Materials and methods The microbroth dilution method was used to obtain antifungal susceptibility tests of conventional antifungal drugs (fluconazole and amphotericin B) against twelve clinical isolates of C. parapsilosis, as well as antifungal susceptibility tests of two efflux pump inhibitors (EPI). In addition, the combination of efflux pump inhibitors (pantoprazole and omeprazole) with fluconazole, an antifungal drug, was determined by calculating the fractional inhibitory concentration index (FICI) based on zero-interaction theory of Loewe additivity. a time-kill kinetics assay was used to study the antimicrobial activity of fluconazole and efflux pump inhibitors against C. parapsilosis. The virulence factors in C. parapsilosis isolates were determined using in vitro virulence assays. The effects of the efflux pump inhibitors on virulence factors were also studied, and comparison of percent reduction of virulence factors was determined in both fluconazole-resistant and susceptible C. parapsilosis. The effects of Pantoprazole and Omeprazole on efflux pump were also investigated using two assays (Rhodamine 6G accumulation and Hoechst 33342 accumulation). Results The efflux pump inhibitors showed enhanced antifungal activity in combination with antifungal agents against C. parapsilosis. When pantoprazole was combined with fluconazole, the median Minimum inhibitory concentration for fluconazole and pantoprazole decreased from 125 to 31.25 µg/ml and from 620 to 310 µg/ml respectively. In the time-kill kinetics assay, fungistatic activity of Fluconazole was changed to fungicidal by addition of efflux pump inhibitors (pantoprazole and omeprazole) at their ½MIC values. On the other hand, this study demonstrated that C. parapsilosis had a variety of virulence characteristics, including the ability to adhere to epithelial cells and secretion of proteinase hydrolytic enzyme. Pantoprazole and omeprazole reduced the ability of C. parapsilosis cells to adhere to epithelial cells. Adhesion was reduced in a concentration-dependent manner. The reduction was significant (p < 0.01 However, the results also showed that pantoprazole and omeprazole and their combinations was significantly reduced efflux pump activity. Conclusion Candida resistance has increased, particularly to azoles, and advent of C. parapsilosis as most prevalent non albicans Candida species poses a significant threat to the future. Combination therapy is shown to be therapeutically efficacious, and MIC values of fluconazole decreased when it was combined with omeprazole and pantoprazole. Further study is needed to understand the epidemiology, microbiology, genetics, and antibiotic susceptibility of this pathogen. The ability to express different virulence factors, such as adherence, proteinase, and phospholipase production, is strain-dependent and the widespread use of antifungal agents leads to drug resistance in C. parapsilosis.Item Combination interaction of proton pump inhibitors with fluconazole against multidrug resistant Candida auris(2024) Galane, KamogeloA significant challenge with controlling C. auris infections is its resistance to multiple antifungal agents, including azoles. Alternative strategies are required to combat the problem of antifungal resistance. Some of the suggested approaches include combination therapy and anti-virulence treatment strategies. There is growing interest in combining proton pump inhibitors (PPIs) with available antifungal drugs for better treatment outcomes. In this study, we investigated the antifungal activity of PPIs omeprazole and pantoprazole alone and in combination with fluconazole against resistant C. auris isolates. The anti-virulence activity of the synergistic combination against resistant C. auris isolates was determined. Lastly, the effect of the synergistic combination on C. auris energy-dependent efflux-activity was established. The CLSI broth micro-dilution method was used to determine the antifungal susceptibility of 25 C. auris isolates against antifungal agents (fluconazole and amphotericin B) and PPIs (pantoprazole and omeprazole). The combination interaction of fluconazole and PPIs was established by determining the fractional inhibitory concentration index (FICI) of each combination. Time-kill curves were used to determine the antifungal activity of the synergistic combination over time and to confirm the combination interaction. All 25 C. auris isolates were screened for their ability to adhere to epithelial cells and proteinase secretion using microscopy and culture technique respectively. The effect of the synergistic combination on isolates with positive pathogenicity markers was determined. Most of the C. auris isolates were resistant to fluconazole (92%) and all were sensitive to amphotericin B (100%). Omeprazole and pantoprazole had antifungal activity against C. auris at MIC values of 3125 µg/ml and 15625 µg/ml, respectively. The combination of fluconazole and pantoprazole reduced fluconazole MIC values by 4-fold, from 125 µg/ml to 31.2 µg/ml. The combination had synergistic effect against most C. auris isolates (56%), additive effect in 36% and indifference in 8%. There was no synergism in the fluconazole and omeprazole combination. However, the combination had antagonist effect against most C. auris isolates (56%). All C. auris isolates displayed some degree of adherence to epithelial cells and 96% produced proteinases. Fluconazole and pantoprazole combination significantly reduced adherence (p values < 0.05) and proteinase secretion (p values< 0.001) at inhibitory and subinhibitory concentrations. Pantoprazole inhibited fluconazole efflux at inhibitory and subinhibitory concentrations. The study showed that C. auris isolates express virulence factors such as adherence and proteinase production. The combination of pantoprazole and fluconazole has antifungal and anti-virulence activity against resistant C. auris isolates. In addition, it was shown that pantoprazole can attenuate fluconazole resistance by inhibiting the activity of energydependent efflux-pumps and it has synergistic effect with fluconazole. Therefore, pantoprazole has the potential to improve therapy outcomes when azoles are used.Item Comparison between lupus nephritis in HIV positive patients and HIV associated immune complex glomerulonephritis with “lupus–like” features: a clinicopathologic study(2024) Mathaba, Margaret MasalaBackground: Systemic lupus erythematosus (SLE) is an autoimmune disease seen commonly in black females of childbearing age. More than half of the patients present with renal disease or lupus nephritis complications. Coinfection with HIV in patients with lupus nephritis is rare. Despite Africa having the highest rate of HIV infection in the world, and there is no available data on the coexistence of HIV and Lupus nephritis. HIV is associated with a wide spectrum of renal diseases, including “lupus-like” HIV-Associated Immune Complex Kidney Disease (HIVICK). The most prevalent renal lesions in “lupus-like” HIVICK is diffuse proliferative lupus nephritis Objectives: This study aimed to compare and correlate the demographics, epidemiology, pathological and clinical findings of HIV positive patients with lupus nephritis and those with “lupus-like” HIVICK. Methods: This retrospective chart review study was conducted at the Charlotte Maxeke Johannesburg Academic Hospital in 5 years (2014-2018). We reviewed case reports that met our criteria for cases with lupus nephritis and cases with “lupus-like” HIVICK and allocated a lupus class according to the report findings. Results: Out of 2174 renal reports, 25(1.14%) patients were diagnosed with lupus nephritis and nine (0.41%) with “lupus-like” HIVICK. There were significant differences in age, serology (urea and creatinine), clinical presentation and lupus class. Conclusion: The occurrence of both HIV associated lupus nephritis and ‘lupus like’ HIVICK is rare. In our setting, the former is more common than the latter. We observed clinical and pathological differences which may be used to diagnose these disease entities.Item Cystic fibrosis: An update on the variant profile and carrier frequency in the Black South African population(University of the Witwatersrand, Johannesburg, 2024) Smit, Ingrid; Essop, Ms F.Cystic fibrosis (CF) is an autosomal recessive disorder caused by pathogenic variants in the CFTR gene. Limited genetic research has been conducted on the Black South African population, and molecular testing is frequently the only way a diagnosis can be made. At the NHLS, testing is performed for the common 3120+1G>A (c.2988+1G>A) variant in the Black population, and other common European CF variants. Recent studies in the Division of Human Genetics show evidence of other recurrent CFTR variants. The aim of this study was to screen for these and other variants to update the CFTR variant profile and carrier frequency in the Black population. NGS data on 395 unaffected individuals was used for CFTR variant identification, annotation, prioritisation, and classification using the ACMG-AMP guidelines. The c.2988+1G>A variant accounted for 36.4% of CF alleles, which is less than previously reported (46%), suggesting that there are other common CF-causing variants in this population. The recurrent variants previously identified were not detected in this cohort, possibly due to limitations in NGS, the bioinformatic pipeline, or small sample size. Three novel likely pathogenic variants (c.3392T>C, c.3038C>G, and c.2594G>C) were identified, with carrier frequencies of 1 in 395 each, which could potentially be African-specific variants. Identifying these variants, not currently included in commercial panels, allows for targeted molecular testing in this population group. Additionally, a revised CF carrier rate of 1 in 36 was estimated which is consistent with literature, highlighting the accuracy of NGS data for carrier screening, leading to accurate risk counsellingItem Delineating antibody responses elicited by four SARSCoV-2 variants against the Mu variant(2024) Kgagudi, PrudenceSARS-CoV-2 is the causative agent of the acute respiratory syndrome known as coronavirus disease 2019 (COVID-19). The SARS-CoV-2 spike protein plays an essential role in virus attachment, fusion and entry and is the main target of neutralizing antibodies. Continued evolution of the spike has led to the emergence of variants of concern or interest (VOC/VOIs) which pose a greater risk to the population due to augmented transmission risk, increased disease severity, antigenic escape and/or decreased effectiveness of vaccines. The Mu variant was first identified in Columbia in January 2021, however the immune escape potential of the variant remains poorly characterised. It is typified by T95I, Y144S and Y145N mutations in the N-terminal domain; R346K, E484K, and N501Y mutations in the receptor-binding domain and D614G, P681H, and D950N mutations in the S2 region. This study aimed to assess targeting of the Mu variant by neutralizing and non-neutralizing antibodies elicited by four variants during four COVID-19 waves in South Africa namely D614G, Beta, Delta and Omicron (BA.1). This was achieved by measuring antibody binding, neutralization and antibody dependent cellular cytotoxicity (ADCC) activity against Mu, and comparing these responses across each of the four variants tested. All variants elicited cross-reactive binding antibodies to Mu although the magnitude of binding to Mu was reduced. This cross-reactivity was a result of multiple binding antibody epitopes on the spike protein. We observed variable neutralization escape of Mu from antibodies triggered by infection with WT D614G and Delta. Antibodies induced by the Beta variant exhibited increased breadth towards the Mu variant when compared to those elicited by other variants. Neutralization escape of Mu from antibodies elicited by WT D614G and Delta may be caused by the 95I, 484K and 346K mutations in Mu, which are known to confer resistance in other variants. Compared to neutralization, ADCC was mostly preserved against Mu. However, the infecting variant impacted the potency of ADCC responses. WT D614G triggered significantly higher ADCC activity against Mu compared to Beta, Delta and Omicron. These data suggest that different variants trigger qualitatively different neutralizing and Fc effector responses, providing a rationale to study humoral resistance following infection by different variants. As Mu shares mutations with other VOCs, this study may aid in predicting the impact of mutations that may be common to emerging VOCsItem Delineating the ontogeny of an N332-directed anti-HIV-1 broadly neutralising antibody lineage(2024) Naidoo, ThamaraBroadly neutralising antibodies (bNAbs) develop in chronically HIV-1 infected individuals and can neutralise a variety of HIV-1 Env strains, making them an important element contributing to the design of HIV-1 vaccines. By understanding and delineating the long affinity maturation pathways that bNAb lineages follow against evolving viruses, it is hoped that this process will be able to be replicated in uninfected people with a series of vaccine immunogens. Investigating the ontogeny of a N332-directed bNAb lineage will provide insights into the key somatic hypermutations necessary for the development of neutralisation breadth. CAP255.G3 is an N332-directed bNAb isolated from CAP255, a participant in the CAPRISA 002 cohort, at 149 weeks post-infection (wpi). Six key antibody intermediates were selected from the CAP255.G3 lineage arm between 17 and 47 wpi, based on their position on a phylogenetic tree and because they contained mutations that were present in broad members of the lineage. We hypothesized that these intermediates would exhibit breadth similar to CAP255.G3. The intermediates were tested against a panel of eight autologous and six heterologous pseudoviruses using a TZM-bl neutralisation assay. The sequence identity of the first heavy chain complementarity-determining region (CDRH1) of CAP255.G3 differed from the closest intermediate and therefore, a CDRH1 chimera was constructed to determine if the CDRH1 from CAP255.G3 affected neutralisation activity. Later intermediates from 39 and 47 wpi, had moderate neutralisation activity against the autologous pseudoviruses, but only theintermediate closest to CAP255.G3 had limited neutralising activity against the heterologous pseudoviruses. Although the CDRH1 chimera had increased neutralisation activity against the heterologous viruses relative to the intermediates, it was less broad and potent than CAP255.G3. This indicates that additional affinity maturation between 47 and 149 wpi, at sites outside of the CDRH1, was required for the development of neutralisation breadth in the CAP255.G3 lineage arm..Item Developing non-viral vector formulations for the delivery of synthetic self-amplifying RNA(University of the Witwatersrand, Johannesburg, 2024) Kairuz, Dylan MatthewConventional vaccines have been instrumental in reducing worldwide morbidity and mortality of infectious diseases. However, some diseases require improvements to their vaccines or do not have an effective vaccine. This has led to the development of next generation vaccines including messenger ribonucleic acid (mRNA) vaccines, a versatile platform allowing simple exchange of encoded antigen sequences for different pathogens in the mRNA. Alternatively, self-amplifying mRNA (saRNA) presents a vaccine platform which replicates in situ using Alphavirus-derived non-structural proteins, mimicking viral replication and prolonging antigen expression. This has the potential to improve immune responses and significantly lower mRNA vaccine doses. Although this is a promising platform, a vector is essential to protect saRNA from ribonuclease degradation and deliver saRNA into cells. Lipid nanoparticles (LNPs) are a safe, highly efficient delivery system, with multiple approved mRNA vaccines. However, saRNA is significantly larger than mRNA, and hence LNPs need to be optimised to achieve efficient delivery. Two main categories of LNPs have been denoted, ionisable (iLNPs) and permanently cationic LNPs (cLNPs). cLNP lipids are less expensive to synthesise and have recently been used successfully to deliver saRNA vaccines. Hence, these were predominantly examined in this study. A library of empty cLNPs were successfully produced using lipid film hydration (LFH) with sizes <150 nanometres (nm). When saRNA was complexed exteriorly to cLNPs (saRNA- Ext-cLNPs) varying sizes were observed depending on the N:P used. N:P ratio is the molar ratio of nitrogen and phosphate on the lipids and RNA respectively, hence larger sizes are expected at a lower N:P. saRNA formulated on the interior of cLNPs (saRNA-Int-cLNPs) were produced by microfluidics or a modified solvent injection (SI) method. Microfluidics formulated saRNA- Int-cLNPs had small sizes and lower polydispersity indices (PDIs) compared to the larger sizes and PDIs of SI formulated cLNPs. The library of saRNA-cLNPs were optimised by transfecting mammalian cells with saRNA encoding reporter genes at a variety of N:P ratios. Generally, the size of the LNPs did not influence the delivery efficiency and the main contributor was N:P ratio for both saRNA-Ext- and saRNA-Int-cLNPs. DOTAP (1,2-dioleoyl-3-trimethylammonium- propane) and DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) performed the best, particularly the F1 cLNPs. Although dimethyldioctadecylammonium (DDA) cLNPs showed poorer delivery efficacy at 24 hours post-transfection, interesting effects on saRNA expression kinetics were observed. A remarkable boost in expression was observed from 24 to 48 hours, and saRNA-Int DDA-cLNPs outperformed DOTAP and DOTMA cLNPs at 48 hours. High encapsulation efficiencies of >90% were observed for all cLNP candidates and the top saRNA-Ext-cLNPs candidates were not toxic. Even though unfavourable results were obtained upon intramuscular injection of saRNA-Ext-cLNPs into mice, this could be as a result of lipid film hydration (LFH) production and could be solved by microfluidics formulation. Although setbacks related to purification of saRNA-Int-cLNPs limited in vivo examination, these showed a strong potential for saRNA delivery for delivery of saRNA vaccines in the future. Overall, the work in this dissertation has developed the protocols for production and characterisation of LNPs. A library of potential cLNP candidates for saRNA vaccine delivery have been optimised in cell culture, with many showing highly efficient saRNA delivery. This will facilitate saRNA vaccine development and examination in a pre-clinical setting in South Africa, reducing reliance on the global North for equitable access of RNA vaccines and improving preparedness for potential future pandemicsItem Development of in vitro transcribed RNA interference activators for the therapeutic inhibition of the hepatitis B virus(University of the Witwatersrand, Johannesburg, 2024) Shrilall, Creanne JizellThe current mainstream treatments available for patients who are chronically infected with the hepatitis B virus (HBV) are capable of suppressing viral replication, however these therapeutic strategies cannot eradicate the virus. Thus, the development of novel therapeutic strategies capable of eradicating the virus are urgently needed. The RNA interference (RNAi) pathway can be exploited by introducing RNAi activators such as primary microRNA (pri-miRNA) mimics into cells to silence genes of interest. The exploitation of the RNAi pathway by employing DNA expression cassettes that endogenously express pri-miRNA mimics which target the hepatitis B x protein (HBx) sequence has demonstrated to be efficient in inhibiting HBV replication. However, achieving safe, efficient, and cost-effective delivery remains amajor challenge. The delivery of RNA therapeutics using non-viral vectors is safer and more cost-effective than the delivery of DNA therapeutics using viral vectors, and the use of chemically synthesised small interfering RNAs (siRNA) is expensive whereas in vitro transcription offers a cheaper alternative. Therefore, the purpose of this study was to in vitro transcribe pri-miRNA mimics that target the HBx sequence which is common to each of the viral transcripts. The rationale was that simultaneously targeting the viral transcripts may reduce viral protein production, thereby removing their immunosuppressive effects, which can potentially reactivate the host’s natural defences to clear the virus. In this study, several pri-miR-31 mimics were successfully in vitro transcribed and assessed in cultured mammalian cells. The results obtained demonstrated the mimics to modestly inhibit viral replication, however these constructs were also shown to induce non-specific silencing, activate the interferon response, and were inefficiently processed into the expected guide sequences. Nevertheless, improvements can be made to the system to promote safe and efficient gene silencing. The modest inhibition demonstrated could have been attributed to inefficient processing due to the activation of the interferon response and limited trafficking of the pri-miR-31 mimics into the nucleus. The use of chemically modified nucleotides and employing precursor miRNA (pre-miRNA) may improve the safety and efficacy of these constructs. Therefore, this study demonstrated the potential of the in vitro transcribed miRNA mimics in inhibiting HBV, however improvements to the system could increase viral replication inhibition and reduce the immunogenicity of these constructsItem Effect of Pneumococcal Conjugate Vaccine on Invasive Pneumococcal Disease in Children Hospitalized at Chris Hani Baragwanath Hospital over a 13 Year Period(University of the Witwatersrand, 2024) Eme, IfeanyiGlobally, under-5 mortality rates remain high in Africa, particularly the sub–Saharan region, where roughly 700,000 child mortalities are caused by S. pneumoniae infections. The purpose of this research project was to evaluate effectiveness of the pneumococcal conjugate vaccine (PCV) on the prevalence of invasive pneumococcal disease (IPD) in hospitalized children at the Chris Hani Baragwanath Academic Hospital (CHBAH) over a 13-year period, before and after the advent of the PCV, and its subsequent incorporation into the South African Expanded Program on Immunization (EPI). Methods: We carried out a retrospective review of demographic, laboratory, and clinical information of S. pneumoniae cultured on blood and/or cerebrospinal fluid in children below the age of 14 that were hospitalized at CHBAH between January 2006 and December 2018. The investigation was stratified into pre-PCV (2006–2008), transitional-PCV (2009–2011) and post-PCV era (2012–2018). Results: Of 867 children hospitalised for IPD over the study period, 409 (47.2%) cases occurred in pre- PCV era, 231 (26.6%) during transitional-PCV, and 227 (26.2%) in the post-PCV era. The IPD incidence (per 100,000 persons) of IPD reduced from 41.1 in 2006 to 4.6 in 2018 (p<0.001). In infants <2 years, there was 79.3% reduction in IPD incidence when comparing the pre-PCV (130.9 per 100,000 persons) and post-PCV period (27.1 per 100,000 persons). We also noted a decrease in the proportion of IPD cases in both the HIV-infected and uninfected persons, comparing pre-PCV to post-PCV period. The most prevalent vaccine serotypes were 14 (16.9%), 1 (14.0%), 6A (11.4%), 19F (11.0%), 23F (10.8%) and 6B (10.0%), whereas the predominant non-vaccine serotypes included; 15B/C (9.8%), 12F (9.3%), 16F (7.5%), NEG38 (7.5%) 35B (5.1%), and 7C (4.2%). Using the minimum inhibitory concentration (MIC), PCV introduction resulted in increased susceptibility to penicillin and cefotaxime. For the zone sizes using the Kirby Bauer disc diffusion method, vancomycin resistance was not seen in any of the isolates. All the other antibiotics showed increase in susceptibility over the period, apart from Erythromycin which initially tended to be relatively stable, but however showed increase in susceptibility across the eras as time goes on. Conclusion: The rate of IPD among children hospitalized at CHBAH has decreased markedly following the incorporation of the PCV into South African EPI programme. The study indicates a need for continued surveillance to monitor the change in resistance patterns and emergence in non- vaccine serotypes causing disease, as this is crucial for mapping out strategic intervention policies in South Africa
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