Electronic Theses and Dissertations (Masters)

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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, Nellie
The 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 have
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, Houcemeddine
Tuberculosis (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.
Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker
(University of the Witwatersrand, Johannesburg, 2023) Tsolo, Semakaleng Theresia; Ranchod, Heena
The 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
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, Sebastian
Hereditary 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 hospitals
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, Fiona
Huntington 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 identified
Investigating the genetic cause of oculocutaneous albinism type 2 in individuals of African descent through exome sequencing
(University of the Witwatersrand, Johannesburg, 2024) Mmbi, Phophi Mmapole; Ngcungcu, Thandiswa G.
Oculocutaneous Albinism type 2 (OCA2) is a hypopigmentation disorder caused by variants in the OCA2 gene. A 2.7kb intragenic deletion is known to be the common variant that causes OCA2 in individuals of African descent. This variant accounts for 78% of OCA2-causing variant in the Southern African population. The diagnostic utility for individuals who tested either negative or heterozygous for the common 2.7kb deletion remained unsolved. This study reports on the identification of the NM 000275.3:c.1503+5G>A variant found in 3/8 (37.5%) non-2.7kb deletion OCA2 chromosomes within a small sample of OCA2 patients from the Southern African population, strongly suggesting the possibility of a second common variant, pending a larger screening study. This variant has been previously described in this population, but new variant interpretation tools have now permitted its reclassification. Furthermore, limitations encountered during the interpretation of data step in this study highlight the importance of informative clinical and phenotypic data for improved and sensible interpretation of genetic results.
Sequencing for high-risk type 1 diabetes genotypes in the South African black population using AmpliSeq Nanopore next generation sequencing
(University of the Witwatersrand, Johannesburg, 2023) Mathabela, Nomfundo Mathabela; Ali, Stuart
Introduction: Type 1 diabetes (T1D) is a chronic autoimmune disorder characterized by the destruction of the -cells of the pancreas, resulting in the inability to produce/maintain insulin. This results in an inability to maintain the blood glucose at homeostasis. Prolonged hyperglycaemia leads to micro- and macro-vascular complications. Thus, it is vital to diagnose and treat patients in a timely manner. It is important to identify individuals at increased risk of developing T1D allowing for appropriate follow ups. Numerous mutations/variants in specific genes confer an increased risk of T1D with the HLA gene accounting for approximately 40-50 % of the risk. Therefore, it is possible that by looking at genetic variation in T1D associated genes we can develop a tool that can determine the likelihood of an individual developing T1D. Study aims and objectives: The development, implementation, and validation of a Nanopore NGS method to sequence and genotype polymorphisms associated with T1D in a cohort of black South Africans which could be used to develop a genetic risk score (GRS) for T1D in this population. In addition, we aimed to compare sequencing results to two HLA genotypes obtained using PCR-RFLP. Method: Participants with T1D (n=19) and control participants (n=5) were genotyped for 12 T1D associated polymorphisms through Ampliseq Nanopore sequencing. In addition, Ion Torrent was used to confirm the Ampliseq Nanopore results. A bioinformatics pipeline that involves reference sequence generation using an in-house script, alignment of sequencing data with the reference sequence, filtering and variant calling was developed. A genetic risk score (GRS) was calculated for the participants. Participants (n=73) were genotyped by PCR-RFLP for the HLA rs2040410 and rs7454108 polymorphisms. Results: Sequencing samples individually was found to have a slightly higher Qscore than samples sequenced in multiplex (9.73 vs. 9.5). Samples sequenced individually had higher average reads (187.60 vs. 151.14 Mb), passed reads (41.47 vs. 25.99 Mb), and estimated bases (54.72 vs. 49.57 Mb) than those sequenced in multiplex. In addition, samples sequenced in a multiplex had higher average failed reads (475.58 Mb) in comparison to those sequenced individually (13.58 Mb). The average percentage difference in sequencing data generated using Ion Torrent compared to Nanopore was 5.67%. Variant calling produced average Phred-scale quality scores of 73.89 for standards (The Coriell Trio) and 89.77 for participants. The GRS calculator was not able to accurately predict which participants had T1D. Discussion and Conclusion: A next generation sequencing method and bioinformatics pipeline for the screening of participants for 645 T1D associated polymorphisms was investigated. The method combined two sequencing techniques i.e., Ion AmpliSeq and Nanopore sequencing to achieve this. The data can then be processed by in-house variant callers. With a larger sample group, this method will be useful for the investigation of genetic variants linked to T1D.
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, Scott
Genetic 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.
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, Kristie
Vaccination 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 platform
The Utilisation of Genetic Counselling Services Amongst Prenatal Healthcare Providers in Gauteng, South Africa
(University of the Witwatersrand, Johannesburg, 2024) Duvenhage, Megan
Congenital anomalies and disorders, many being genetic, continue to have high prevalence and mortality rates globally. Prenatal healthcare providers possess the necessary skills to identify these cases before birth and refer patients for genetic counselling. This study aimed to establish the utilisation of genetic counselling services and insights into the perceptions of genetic counselling amongst prenatal healthcare providers in Gauteng, South Africa. By assessing the utilisation of genetic counselling, barriers and facilitators to referrals were highlighted, and recommendations to improve service provisions in the prenatal sector were made. An electronic survey adapted from Thom and Haw (2021) was sent to prenatal healthcare providers in both the public and private healthcare sectors. A total of 54 participants were included in this study. Results show that roughly 74% of participants are able to refer to genetic counselling services, but only 57% had made use of the service. None of the participants were able to identify all appropriate reasons for referral to genetic counselling correctly, and only 24% of participants understood the responsibilities of a genetic counsellor. Misconceptions regarding the scope of practice of genetic counsellors and uncertainties surrounding the referral process were the most significant barriers to referrals. The study revealed that although prenatal healthcare providers in Gauteng are using genetic counselling services, they are not fully utilising the service due to a lack of knowledge surrounding the profession's services. Therefore, there is a need for educational resources to bridge the knowledge gap and improve prenatal healthcare in Gauteng, South Africa
Exome Sequencing of individuals with vitiligo and their relatives with and without autoimmune disorders
(University of the Witwatersrand, Johannesburg, 2023) Rabinda, Kentie Rofhiwa
Autoimmune disorders result from the immune system attacking and damaging its healthy cells because of an acquired immune system malfunction. Vitiligo is an example of an autoimmune disorders. It is a common depigmentation skin disorder with an estimated prevalence of 0.5–2% of the population worldwide. It is shown by selective loss of melanocytes causing non- scaly, chalky-white macules. Individuals with vitiligo often have other autoimmune disorders or first-degree relatives with at least one other autoimmune disorder. This study aimed to identify genetic variants in selected genes in individuals with vitiligo and their unaffected close relatives who have other autoimmune disorders. Upon DNA extraction, whole exome sequencing was performed, and five non- major histocompatibility complex (MHC) genes were analysed. Identified variants were annotated on Ensembl Variant Effect Predictor (VEP) and compared between individuals with vitiligo and their close relatives with and without autoimmune disorders in a family-specific manner. Eight variants were identified in two families, however, the three missense variants in the NLRP1 gene (rs12150220, rs11651270, and rs2301582) were observed between and across two families in individuals with autoimmune disorders. SMOC2 rs13208776, was identified as a risk locus for vitiligo in individuals from two Romanian isolate villages with vitiligo and other autoimmune disorders. None of the genotyped individuals were homozygous for the minor allele (A) and 2/12 individuals were heterozygous therefore it is unlikely that rs13208776 has any role in the development of vitiligo or any of the autoimmune disorders present in the two families. This study showed that NLRP1 gene variants segregated with vitiligo and autoimmune disorders
Expression and immunogenicity of HBV surface antigens following in vitro and in vivo gene delivery using recombinant adenoviruses
(University of the Witwatersrand, Johannesburg, 2024) Neves, Keila Dias; Maepa, Betty
Hepatitis B virus (HBV) infection poses a global health problem affecting almost 300 million people. Immunisation remains one of the most effective methods of HBV prevention. The HBV surface antigen (HBsAg) is one of the primary clinical markers for acute or chronic HBV infection. It is an important component of anti-HBV vaccines because of its presence on the viral envelope and interaction with neutralising antibodies (NAbs). Current vaccines against HBV, which deliver only the HBV small surface antigen (SHBsAg) as a purified peptide in a 3-dose regimen, are often less effective in people over the age of 40 years and in immuno compromised individuals. Additionally, these vaccines are limited to mostly humoral immunity. Recent widespread use of viruses as vectors for vaccine delivery has propelled use of adenoviruses (Ads) to the front line of vaccine research. Adenoviral vectors (AdVs) are attractive candidates for anti-HBV vaccine design because of their ability to induce both cell- mediated and humoral immune responses. Therefore, this study was conducted to assess the efficiency of AdVs in the delivery of HBV SHBsAg or large surface antigen (LHBsAg) and induction of HBV specific immune responses. Recombinant AdVs encoding either the LHBsAg (Ad5-LHBsAg), SHBsAg (Ad5-SHBsAg) or Firefly luciferase (FLuc) reporter (Ad5-FLuc) were produced. Infection of Human Embryonic Kidney 293T (HEK293T) cells with Ad5-SHBsAg showed dose-dependent expression of SHBsAg in both supernatant and lysate samples. However, as expected, significantly higher expression of LHBsAg was seen in lysates from cells infected with Ad5-LHBsAg when compared to supernatant samples, because the LHBsAg peptide is not naturally secreted. Intramuscular injection of Ad5-FLuc to Balb/c mice showed significant expression of FLuc at days 1 and 3 after injection with Ad5-FLuc, with expression decreasing by day 7, as expected. Analysis of cellular immunity showed HBV- specific IFN-γ responses in mice vaccinated with Ad5-SHBsAg, Ad5-LHBsAg and EUVAX-B control. Additionally, anti-HBsAg antibodies were detected in mice vaccinated with Ad5- SHBsAg and EUVAX-B. This study shows that AdVs are promising candidates for novel anti- HBV vaccine design and may circumvent current challenges of available anti-HBV vaccines by inducing more robust immune responses while decreasing the doses required for immunisation. This research bodes well for future work in the development of vaccine regimens involving other AdVs against a variety of infectious diseases
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 counselling
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, Ifeanyi
Globally, 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
Germline Cancer Predisposition Variants in Paediatric Rhabdomyosarcoma
(University of the Witwatersrand, Johannesburg, 2023) Pillhofer, Gabriella Peta; Lamola, Lindie; Mnika, Khuthala
Rhabdomyosarcoma (RMS) is cancer that originates from undifferentiated skeletal muscle cells. RMS is the most common tissue sarcoma in children and adolescents and has over 50% of cases occurring in individuals under the age of 10 and thus there is reason to suspect that RMS may have a hereditary predisposition. However, much of the existing research of germline predisposition in paediatric RMS is focused on ethnically European populations, and currently very little data is available from African populations. In this study, we aimed to identify RMS predisposition variants by performing whole exome sequencing (WES) on germline DNA from eight paediatric patients diagnosed with RMS. Following WES, variant annotation and filtering was performed to identify variants in genes of interest that were potentially germline causes of malignancy. Filtered variants were then classified according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. This study identified four variants of uncertain significance (VUSs) in four patients. Two of these were in genes that have previously been associated with an RMS phenotype (SDHB and BRCA1), and two were in genes that have been associated with a hereditary cancer syndrome that is not linked to RMS (CBL and CREBBP). While the highlighted variants are not of clinical significance, this study emphasises the importance of cataloguing and reporting VUSs in research in Africa. By expanding the genomic database on African patients, the analysis of variants on the continent may be made more accurate and efficient. It is the goal that the knowledge gained from this study will contribute to the information base of hereditary paediatric cancers in Africa, and that it may encourage similar research so that the field may continue to expand.
Functional consequences of novel IgG3 allelic variation CAP256-VRC26.25
(University of the Witwatersrand, Johannesburg, 2024) Khan, Fonguh Oliver
Broadly neutralizing antibodies (bNAbs) isolated from HIV-1 individuals have shown promise in prevention. We have previously demonstrated that the IgG3 isotype significantly improved the neutralization potency of CAP256-VRC26.25 over the routinely used IgG1 constant region. However, there is significant allelic diversity in immunoglobulin constant heavy G chain 3 (IGHG3), especially in African populations, of which the functional relevance is unknown compared to IgG1 version. This study assessed whether allelic variation in IgG3 could further improve neutralization potency. CAP256-VRC26.25 bNAb was engineered and expressed as 12 different IgG3 allelic variants, including two novel variants, as determined from previous IGHG3 sequencing data from the CAPRISA 002 cohort. These were tested for neutralization against an eight- virus panel and compared to the IgG1 version. Overall, the IgG3 versions of CAP256-VRC26.25 showed improved neutralization compared to the IgG1 version. Further, there were significant differences between allelic variants, IgG3*01 versus IgG3*04 (p=0.0038), IgG3*01 versus IgG3*15 (p=0.0013), IgG3*13 versus IgG3*04 (p=0.01570) and IgG3*13 versus IgG3*15 (p=0.0059). IgG3*01 showed the highest fold change (median value 8.0) relative to IgG1. IgG3*04 and IgG3*15 showed the least fold change (median value 2.2). This is due to the short hinge length of IgG3*04 and amino acid differences in IgG3*15. IgG3*01 and IgG3*13 were the most potent variants. This confirms the importance of the constant region in neutralization which has been supported by previous studies. Thus, hinge length and amino acid differences in the constant region impact neutralization. This study shows that allelic variation in the constant region can impact on neutralizationpotency and supports leveraging genetic diversity to improve antibody function
Developing non-viral vector formulations for the delivery of synthetic self-amplifying RNA
(University of the Witwatersrand, Johannesburg, 2024) Kairuz, Dylan Matthew
Conventional 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 pandemics
RB1 and Beyond: Determining genetic causes of retinoblastoma in South African patients
(University of the Witwatersrand, Johannesburg, 2024) Beukman, Danielle; Lamola, Lindie
Retinoblastoma is the most common solid tumour of the retina, affecting mainly paediatric patients. Although loss-of-function germline variants the RB1 gene is the tumour suppressor gene most often associated with heritable retinoblastoma, two contradictory research findings complicate the assumption that a pathogenic germline variant in RB1 will lead to the development of heritable retinoblastoma. Firstly, heritable retinoblastoma has been observed in cohorts with biallelic wild-type RB1 genes, secondly, biallelic loss-of-function is not always sufficient to lead to retinoblastoma tumorigenesis. By investigating germline variants in additional cancer predisposition genes to discover candidate driver genes in heritable retinoblastoma, the full germline mutational spectrum of heritable retinoblastoma can be established. In this study we investigate sequence germline variants in cancer predisposition genes beyond RB1 in South African retinoblastoma patients with an African ancestry by performing whole exome sequencing on eight patients diagnosed with retinoblastoma. Whole exome sequencing data for genes previously associated with retinoblastoma was subjected to variant annotation by means of a variant effect predictor and filtering criteria applied manually. Variant classification was performed according to guidelines proposed by The American College of Medical Genetics and Genomics and the Association of Molecular Pathology. The highest degree of variant classification was variants of unknown significance. The absence of sequence variants capable of describing the retinoblastoma phenotype in this cohort demonstrates the necessity of looking beyond RB1, combining next generation sequencing with copy number analysis pipelines and additional genome mapping technologies capable of detecting structural variants. The detection of sequence variants previously reported to be possibly damaging but now considered polymorphisms highlights the importance of revisiting variant classification. In closing, this study adds genomic information from patients with a South African ancestry to mounting genomic data, ensuring that previously underrepresented populations can also benefit from future cancer predisposition and precision medicine research.
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 Jizell
The 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 constructs
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, Jaya
Background 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 counterparts

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