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Item Prevalence and Molecular Epidemiology of Bordetella pertussis Infection in South Africa(University of the Witwatersrand, Johannesburg, 2024) Moosa, Fahima; Wolter, Nicole; du Plessis, MignonPertussis remains a public health concern in South Africa, with increases in cases and outbreaks in recent years. We determined the incidence, transmission dynamics, serological attack rates and molecular epidemiology of B. pertussis in South Africa. Data from a longitudinal study enrolling individuals each year in 2016–2018 from two communities were used. Nasopharyngeal swabs were collected from participants twice-weekly and tested by real-time PCR. Serum was collected at 8 time points and tested using the anti-pertussis toxin IgG ELISA kit. Whole genome sequencing was performed on all available cultures (n=32) sourced from three additional surveillance programs between 2015–2019. Data were described and analyzed using univariate and multivariable regression models. Among 1684 participants, the incidence of B. pertussis was 0.21 (95% confidence interval 0.17–0.25) per 100 person-weeks. The mean duration of infection was 12 days (±standard deviation 19.1). Transmission of infection was more likely to occur from male index cases [adjusted odd ratio 12.20 (95%CI 1.57–94.96)], and individuals with ≥7 day’s infection duration [aOR 24.80 (95%CI 2.74–224.30)]. B. pertussis seroprevalence ranged from 1.8% to 5.2% across eight blood draws. The serological attack rate was 5.8% (87/1509), which was similar to the PCR attack rate (6.2%, 94/1509) (p=0.64). PCR-positive individuals aged 5–18 years (vs 19-44, aOR 6.8, 95% CI 1.3-35.1) and with episode duration of ≥7 days (vs <7 days, aOR 13.3, 95% vi CI 3.4-51.1) were more likely to seroconvert. For all individuals that seroconverted, the ≥4-fold rise in anti-PT IgG titer was detected by the next blood draw (mean: 2.9 months (range 3 weeks – 5.9 months). Using genome data, all isolates were identified as the globally-disseminated sequence type 2 and harbored the pertussis toxin promoter ptxP3. The dominant genotype was ptxP3-ptxA1-ptxB2-prn2-fimH2 (31/32, 96.9%), with no pertactin-deficient or other mutations in vaccine antigen genes identified. Within the community, despite a high incidence of B. pertussis, there was an overall low seroprevalence. Our data highlighted that increases in cases in South Africa are not likely due to evolutionary changes in the genome but potentially waning immunity due to the use of acellular vaccines and/or population immunity gaps.Item Characterisation of the dynamic gut microbiota of members of the Anopheles gambiae complex(University of the Witwatersrand, Johannesburg, 2024) Singh, Ashmika; Allam, Mushal; Oliver, Shüné V.The gut microbiota of mosquitoes plays a pivotal role in their life history. This includes insecticide resistance and immune responses, which makes it a potential target for vector control interventions. Although a growing body of research characterises the gut microbiota of various mosquito species, there is limited information on most members of the Anopheles gambiae complex. This study characterised the gut microbiota of four laboratory reared strains, namely SENN (Anopheles arabiensis- an insecticide susceptible major vector), SENN DDT (Anopheles arabiensis- an insecticide resistant major vector), MAFUS (Anopheles merus- minor vector) and SANGWE (Anopheles quadriannulatus- non-vector). The gut microbiota of fourth instar larvae, three-day old, fifteen-day old non-blood fed and fifteen-day old blood fed females was characterised and compared, bacterial identification was performed using Next-Generation Sequencing (NGS) of the bacterial 16S ribosomal RNA (rRNA) gene. The larval environment plays a role in acquiring gut microbes. This study characterised the gut microbiota of laboratory reared and wild F1 population An. arabiensis adult females and how changes in the larval environment affect the dynamics of the adult gut microbiota. Additionally, the effects of heavy metals on the gut microbiota of laboratory reared and F1 population of An. arabiensis adult females post-exposure to heavy metals in the larval breeding site were assessed. Furthermore, the effect of changes in the salt concentration in the larval environment on the gut microbiota of An. merus larvae and adults were examined. All bacterial identification of the gut microbiota was performed using NGS of the bacterial 16S rRNA. The dynamic gut mycobiota of SENN, SENN DDT and fourth instar larvae, three-day old, fifteen-day old non-blood fed and fifteen-day old blood fed laboratory reared and wild F1 population An. arabiensis females were characterised. Fungal identification was performed using NGS of the fungal internal (ITS) transcribed spacer of the rRNA cistron. Distinct differences were observed between the diversity and abundance of the gut microbiota of major malaria vectors and the minor and non-vectors, with the minor and non-vector harbouring more Plasmodium-protective bacterial genera. Pesticide-degrading bacteria were found in insecticide susceptible and insecticide resistant strains of An. arabiensis, suggesting that the gut microbiota does not contribute to insecticide resistance. However, differences between these strains indicate that the diversity of the gut microbiota is affected by the insecticide resistant phenotype. The F1 population and wild population of An. arabiensis had a greater gut microbiota diversity than laboratory reared An. arabiensis strains. Exposure to heavy metals as larvae significantly changed the abundance of gut bacteria in adults. The F1 population and SENN DDT adults had more heavy metal degrading bacterial genera present viii than SENN. Changes in the salinity of the larval environment of An. merus significantly affected larval development time, adult longevity, fecundity and deltamethrin tolerance. Additionally, An. merus adults exposed to the highest salt concentration at the larval stage had more Plasmodium-protective bacterial genera than the lower concentrations. The insecticide resistant phenotype of laboratory reared An. arabiensis affected the diversity and the differential abundance of fungi observed in the gut mycobiota. Furthermore, the gut mycobiota of the F1 population of An. arabiensis was similar to that of the laboratory reared insecticide resistant strain. In conclusion, these findings have implications for using gut bacteria as a vector control intervention in South Africa. The gut microbiota of the An. gambiae complex is a dynamic network consisting of what seems to be a core microbiota crucial to its function. The gut microbiota of the selected members of the An. gambiae complex corresponds to their capacity to transmit Plasmodium. Furthermore, the larval environment strongly influences the dynamics of the gut microbiota of members from the An. gambiae complex. Therefore, the impact of the larval environment on the adult microbiota must be considered for the development and implementation of future microbial-based strategies.Item Homologous recombination mediated insertion of anti-HBV CRISPR/Cas9 encoding sequences into the helper-dependent adenoviral vector genome(University of the Witwatersrand, Johannesburg, 2024) Farhad, Tasneem; Maepa, BettyHepatitis B is caused by the hepatitis B virus (HBV), which maintains a stable DNA genome in infected hepatocytes that prolongs infection. Current treatments serve only to reduce the effects of the infection, but do not remove the viral genome from infected cells. As such, gene therapy approaches have been explored to target and eradicate the HBV genome from infected cells. Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) has been shown to be effective against HBV, but it is difficult to efficiently deliver these sequences to targeted cells. Viral vectors like adenoviruses are often used as delivery vehicles for gene therapy. Helper-dependent adenoviral vectors (HDAdVs) are widely used, and though they are well suited to target hepatocytes, they have a large genome that is difficult to insert transgenes into. The aim of this study was to design a system to efficiently insert transgenes into the HDAdV genome through homologous recombination with an anti-HBV CRISPR/Cas9-expressing shuttle plasmid. An anti-HBV CRISPR/Cas9 sequence and a nanoLuciferase (nLuc) reporter gene was inserted into a previously constructed shuttle plasmid, that was designed to include two regions that are homologous to regions within the HDAdV genome. Each transgene-expressing shuttle was used with a plasmid expressing the HDAdV genome to co-transform recA expressing Escherichia coli (E. coli) cells, wherein homologous recombination between the two regions of homology was induced. However, restriction analysis of the resultant recombinants indicated significant instability that hindered their characterisation. A low copy ori and a 400 bp fragment of the adenovirus genome associated with increased vector stability was inserted into the shuttle and recombination between this nLuc-expressing shuttle and the HDAdV genome resulted in stable recombinants. These, however, did not include the transgene. This indicated that despite the improved stability of the recombinants, unintended recombination events still occurred that excluded the transgene from the final product. As such, further work must be done to insert the transgene elsewhere in the shuttle to ensure that recombination events include the transgene in the final recombinant. The implications of this work are that one the biggest drawbacks of HDAdV application can be improved to streamline the process and potentially expand on the future use of HDAdVs in gene therapy.Item Whole genome sequence-based characterization of group A Streptococci (GAS; *Streptococcus pyogenes*) isolates causing invasive disease in South Africa, 2019–2020(University of the Witwatersrand, Johannesburg, 2024) Zulu, Sibusisiwe Thobile ZuluInvasive group A streptococcus (iGAS) is a major cause of morbidity. Penicillin and erythromycin are recommended for treatment. A 30-valent M-protein-based vaccine is being developed. We describe the molecular epidemiology of iGAS disease in South Africa, 2019 - 2020. iGAS episodes (cultured from normally sterile sites or from necrotising fasciitis wounds, among all ages) were detected through active, national, laboratory-based surveillance. At the national reference laboratory, isolates were confirmed phenotypically based on colony morphology, β-haemolysis and sensitivity to bacitracin; antimicrobial susceptibility testing was done by disk diffusion and broth microdilution; and isolates were further characterized by whole-genome sequencing. A SNP-based phylogenetic analysis determined genetic clusters. Of 1487 iGAS cases that were reported, 618 were characterised phenotypically (viable isolates available) and 577 genotypically. Incidence was 1.7 cases/100,000 persons in 2019 and 0.9/100,000 in 2020, with peaks in the extremes of age. All isolates were susceptible to the β-lactam antibiotics. Resistance was detected against tetracycline (10.5%;65/618), erythromycin (3.6%; 22/618), chloramphenicol (0.2%;1/618) and levofloxacin (0.2%;1/618). tetM and mef genes were present among isolates resistant to tetracycline and erythromycin, respectively. Overall, 56 emm types were identified. emm76 (33/577,5.7%), emm92 (30/577,5.2%) and emm82 (29/577,5.0%) were the most prevalent. Globally, emm1 is the most frequently occurring emm type in high-income countries, yet it was only detected in 3.7% of the isolates in the present study. emm76 and emm95 types accounted for most of the tetracycline- and erythromycin-resistant isolates, respectively. emm types in the 30-valent M-protein-based vaccine accounted for 44.6% (244/547) of isolates. Phylogenetically, identical emm types clustered together. Among genetic clusters, there was no geographical and temporal aggregation of cases. iGAS treatment with β- lactams remains appropriate. The 30-valent M-protein-based vaccine offers coverage to less than half of the isolates. No unidentified outbreaks were detected.Item Investigating Knowledge, Attitudes, and Perceptions of SARS-CoV-2 Vaccine Hesitancy among Pregnant Women in Soweto, South Africa(University of the Witwatersrand, Johannesburg, 2024) Maccarthy, Samuel Oluwasegun; Myburgh, NelliePregnant women face heightened risks of severe COVID-19 consequences, making vaccination vital for their protection. In South Africa, despite government initiatives, vaccine hesitancy persists among pregnant women, hindering widespread coverage. This study delves into the knowledge, attitudes, and perceptions of COVID-19 vaccines among 60 unvaccinated pregnant women in Soweto, South Africa. It aims to identify influencers shaping their vaccine decisions, addressing a critical gap in understanding hesitancy in this vulnerable group. Data from a validated questionnaire reveal diverse information sources, with media being primary. Safety concerns emerge as the foremost hesitancy factor, and "personal decision" is a key influencer. Applying the 3C Model, the study unveils crucial factors guiding pregnant women's COVID-19 vaccination choices, providing insights for targeted public health strategies to address hesitancy in this susceptible population.Item Association of catestatin with insulin resistance disparity in normoglycaemic Black and White South African women(University of the Witwatersrand, Johannesburg, 2024) Ntuli, Philisiwe; Toman , MarketaBackground: Unexplained ethnic disparities exist in insulin resistance (IR) worldwide, affecting negatively more women of African ancestry than their white female counterparts. Insulin resistance is a pathological condition preceding the development of Type 2 diabetes (T2D), which in 2016 was the second leading cause of death in South Africa. The studies evaluating IR disparities in South Africa are very sparse, focussing mainly on obesity and fat distribution. However, IR has multifactorial aetiology and disparities have been reported irrespective of Body Mass Index (BMI) or age. Additional studies are therefore needed. From rodent-based and in-vitro studies it becomes clear that glycoprotein Chromogranin A (CgA) and its cleavage products catestatin (CST; found in pancreatic β-cells, having insulin- sensitising effects) and pancreastatin (PST; derived from pancreatic α-cells, having anti-insulin effects) are involved in the regulation of glucose metabolism. We hypothesise that women of African ancestry may have low CST levels due to dysregulated CgA cleavage, yielding in an increased proportion of deregulatory PST. Therefore, we aim to determine whether circulatory levels of CST (or CgA; CgA/CST ratio) differ between healthy normoglycaemic black and white South African women independently of obesity and whether they are significantly involved in the regulation of IR. Methods: “Apparently healthy” black (n=67) and white (n=54) women volunteers below 50 years of age from Gauteng province were enrolled in the study, following the enrolment criteria. Anthropometric measurements including weight, height, waist (WC) and hip (HC) circumferences and blood pressure (BP) were recorded. All participants underwent shortened oral glucose tolerance test (OGTT) to evaluate their glycaemic status except for externally enrolled white women (n=47). Samples were collected for measurements of glucose (fasting and 30-minutes), insulin (fasting and 30-minutes) and C-peptide (fasting) which were analysed by an automated Roche Cobas analyser. Other samples collected at fasting for CgA, CST, Interleukin 6 (IL-6), leptin, monocyte chemotactic protein-1 (MCP-1) and adiponectin were analysed by individual enzyme-linked immunosorbent assay (ELISA) kits. Surrogate indices for IR (HOMA-IR), early insulin secretion (Insulinogenic index; IGI), -cell function (Disposition index; DIo) and hepatic insulin clearance (HIC) were calculated. vi Indices of adiposity (BMI, waist to hip ratio (WHR), waist to height ratio (WHtR) and a body shape index (ABSI)) were calculated from obtained measurements. Demographic information and socio-economic status (SES) were evaluated by questionnaires. Results: Black women were younger 26 (21; 32) than white women 30 (24; 35) and had higher BMI than white women (26.9 (22.4; 31.4) kg/m2 vs 23.5 (20.8; 25.9) kg/m2 respectively). Although normoglycaemic, white women had higher basal glucose levels (4.8 ± 0.3 mmol/L vs. 4.7 ± 0.3 mmol/L). No ethnic differences were found for fasting insulin or HOMA-IR. The only difference was attributable to adiposity in obese (OB) vs non-obese (NOB) black women. Similarly, no ethnic differences were observed for DIo and IGI. However, NOB black women had reduced (p<0.001) HIC in comparison with NOB white women. Black NOB women had reduced CST and elevated CgA (and CgA/CST ratio) than white NOB women, all with p<0.001. No associations of CST, CgA or CgA/CST were observed in white women. Catestatin correlated inversely with HIC (p=0.045) and positively with leptin (p=0.010) in black women. Correction for confounders removed this significance. CgA and the CgA/CST ratio correlated negatively with HOMA-IR and fasting insulin and positively with HIC. Only after removal of the strongest determinants, positive associations for CgA (p<0.001) and the ratio (p=0.009) remained in the model for HIC. Conclusion: In the study population of normoglycaemic, normotensive and apparently healthy black and white women, we have not found elevated insulin levels or higher IR in the black ethnic group. However, we demonstrated for the first time the ethnic differences in CST with reduced levels and elevated levels of CgA and the CgA/CST ratio in black women vs white women. This difference is independent of obesity; we have not identified its reason but a genetic origin cannot be excluded. There were no direct regulatory effects of CST (CgA or CgA/CST) on IR. However, the study results indicate that HIC is the most closely associated variable of glucose- insulin dynamics with CST (CgA and CgA/CST ratio) in the black women in our study. The effects of CST on IR and glucose regulation may be indirect. The study found a positive correlation with leptin and CST-leptin interaction has been described previously in the literature. The binding of CST to insulin receptor results in reduction of obesity and this, in turn, can reduce IR and improve insulin and glucose levels.Item Optimising adult housing and insectary lighting to increase mating success in colonised Anopheles funestu(University of the Witwatersrand, Johannesburg, 2024) Mrosso, Paul CalistIn nature, malaria vectors mate in swarms that form at dusk in relation to different ground markers that either affect the amount of light or contrast light at the point of swarm formation. Specific light regimes known to stimulate mating in mosquitoes have been simulated indoors for laboratory rearing of Anopheles. These conditions have proven effective for establishing and sustaining colonies of species in the Anopheles gambiae complex, but the same has not been achieved with members of the An. funestus group. Anopheles funestus sensus stricto, a main African malaria vector species in this group, has been difficult to colonise due to insufficient mating success in laboratory cages. This study aimed to optimise adult mosquito housing conditions to increase the mating success of two An. funestus strains housed under laboratory conditions. Mating success ratios were compared in different insectaries, cage sizes, and vector densities. The light environment in adult mosquito-holding cages was manipulated with artificial horizons and visual markers to simulate natural swarm cues. Mating success ratios did not differ between insectaries, but I found that cage size and manipulating adult mosquito-rearing cage by covering them with a cloth or placing a light-contrasting marker inside the cages increased mating success of An. funestus. Furthermore, the two geographically distinct An. funestus strains were affected differently by the same cage manipulation. Covering the top half of rearing cages with black opaque cloth and placing a black light-contrasting marker on the base of the cage significantly increased the mating success of An. funestus from Mozambique (FUMOZ strain). Contrastingly, the An. funestus from Angola (FANG strain) showed increased mating success in large cage sizes and when the side of the cage was covered in black cloth. The FANG strain had consistently higher mating success ratios compared to the FUMOZ strain. These findings indicate that the mating success ratios of An. funestus in the laboratory is likely to be influenced by geographical locations or underlying genetic diversities resulting in differences in the response of colony strain to the same mating-enhancement cues. Hence, adult mosquito housing should be customised when attempting to colonise and mass- produce An. funestus.Item Assessment of the in vitro phenotypic drug susceptibility of HIV-1 subtype C drug-resistant variants to Doravirine (DOR)(University of the Witwatersrand, Johannesburg, 2024) Reddy, Nikita; Basson, AdriaanThe high prevalence of antiretroviral drug resistance warrants approval of novel antiretroviral drugs that are effective against drug resistant-variants. Doravirine (DOR) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) with an improved safety profile, requires only a single daily dose, and has shown activity against prevalent NNRTI drug resistance mutations. To understand the impact of NNRTI drug resistance mutations on DOR in South Africa, in which HIV-1 subtype C is prevalent, this study assessed the in vitro phenotypic drug susceptibility of DOR against prevalent HIV-1 subtype C drug-resistant variants. Replication-defective HIV-like pseudoviruses (PSV) containing the most prevalent NNRTI drug resistance mutation combinations (n=20), as well as the single mutations (n=15) they contain, were generated and screened in a single-cycle in vitro phenotypic assay for susceptibility to DOR. An initial screen of DOR against wild-type PSVs showed no significant differences in Islatravir (ISL) potency among subtypes B and C. Furthermore, the combination of DOR with ISL showed a significant synergistic inhibition of a wild-type HIV-1 subtype C PSV. High-level DOR resistance was observed with the V106M and Y188L single mutants, while the remaining single mutants remained susceptible or showed a low level of resistance to DOR. Combinations of mutations that included V106M, Y188L, and P225H showed a high level of resistance to DOR. Given the lower prevalence of the latter mutations in treatment-naïve and -experienced patients in South Africa, DOR would likely be an effective treatment option. However, genotypic drug resistance testing may be advised prior to initiation of DOR-containing regimens for NNRTI-experienced individuals.Item The Clinical Genomics of African Oesophageal Cancer(University of the Witwatersrand, Johannesburg, 2024) Ngundu, Nerija Lamantha; Mathew, Christopher; Penny, ClementIn South Africa, oesophageal cancer is responsible for the 6th highest cancer-related deaths, with oesophageal squamous cell carcinoma (OSCC) being the most prevalent type at an incidence rate of 8.6/cases/100,000 for males and 4.7/cases/100,000 for females. It is often diagnosed at a late stage due to its asymptomatic nature, making it too late for any form of therapeutic interventions to be introduced. Information regarding the genetics of this disease on the African continent is limited, even more so in South Africa. This makes the task of identifying molecular markers, development of diagnostic and monitoring tools quite difficult. Therefore, the aim of this study was to identify the somatic mutation profiles of African patients with OSCC, thereby assisting in the expansion of knowledge regarding the genomic landscape as well fostering the development of molecular markers that could be useful in the diagnosis and treatment of this cancer. This was done by isolating DNA from blood, saliva and tumour samples and conducting whole exome sequencing (WES) on 21 matched blood/OSCC tumour samples. Data analysis was performed using R. The WES from 21 matched blood/tumour pairs revealed that somatic single nucleotide variants (SNV) were much more prevalent in comparison to somatic insertions or deletions (indels). The tumour mutation burden (TMB) was ~2 mutations per Mb. Tumour Protein 53 (TP53) was the most commonly mutated gene with 11 of the 12 mutations occurring in the DNA binding domain of the protein. Mutations in TP53, Titin (TTN) and Mucin 19 (MUC19) suggested that these genes were involved in relatively early events in the development of the tumours. Analysis of copy number alterations revealed a high degree of complexity in the tumour genome, with frequent amplification detected on chromosomes 1p33, 11q23.3 and 21q22.2, and common regions of deletion on chromosomes 5q31.2 and 7q32.3. Three mutational signatures were identified and the molecular pathway analysis showed that the NOTCH, RTK-KAS, and TP53 pathways were the most significantly altered pathways. The alterations discovered in this study have contributed to the greater scheme of the molecular landscape of OSCC.Item An Audit of Adult Vaccination in HIV-Positive Patients Attending a Tertiary Centre HIV Clinic(University of the Witwatersrand, Johannesburg, 2024) Muchichwa, Mitchell Tanaka; Feldman, CharlesBackground: People living with HIV (PLWH) have a greater risk of acquiring vaccine preventable diseases (VPDs) and of developing severe disease due to impaired immune responses. The risk of acquiring VPDs among PLWH is greater in adults aged 15-49 years as a result of common infections found in occupational, social and travel settings. At the same time, studies have demonstrated low vaccination coverage among this population. For this reason, international and local guidelines for the vaccination of HIV-infected adults have been drawn up to address challenges in immunization in the HIV setting. However, there is little evidence on the compliance to these guidelines and on the knowledge and attitudes of PLWH to vaccination for VPDs. Aim: The aim of the study was to investigate vaccination uptake, knowledge, and acceptance of vaccination among PLWH. Methodology: In-depth interviews and retrospective file reviews among PLWH attending the Charlotte Maxeke Johannesburg Academic Hospital HIV Clinic over a 1-month period. Results: Among the 107 PLWH who were recruited and interviewed, it was found that 24.3% had received at least one vaccine following their HIV diagnosis. The majority of participants (76.6%) had a history of tuberculosis (TB), which was the most common vaccine-preventable disease among the sample. Influenza vaccination had the highest uptake rate at 76.9%. Overall, 65% of participants believed that vaccines could prevent diseases, while 41.1% thought vaccines were harmful. Interestingly, 56% did not believe that vaccines worsened their HIV condition. Furthermore, 67.2% of participants expressed their willingness to accept a vaccine if it was recommended by their healthcare provider. The most significant factors associated with vaccine uptake were awareness of the influenza vaccine and the willingness to accept a vaccine after a recommendation. Conclusion: Our study revealed poor adherence to the vaccination guidelines for PLWH in South Africa. Only four vaccines against influenza, hepatitis B, shingles and pneumococcal diseases had been taken and commonly known among this population.