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Item Determining the risk profile for chronic kidney disease (CKD) in rural South Africans using genetic risk scores and protein markers(University of the Witwatersrand, Johannesburg, 2024) Govender, Melanie Ann; Ramsay, MichèleChronic kidney disease (CKD) is a global public health concern, with disproportionate morbidity and mortality in low- and middle-income regions, including Sub-Saharan Africa. Recent advancements in multi-omics approaches have explored disease risk indicators and contributed to the understanding of the pathophysiology of CKD in high-income populations. The overall aim of this research was to assess and understand CKD in a Sub- Saharan population using genetic risk models for kidney disease and to evaluate the proteomic profile of individuals with hypertension-associated albuminuria, with a view to detecting indicators of CKD and disease progression in a Sub-Saharan cohort. The first objective was a scoping review that was undertaken to evaluate existing literature for potential biomarkers for CKD and to identify gaps in literature. Key literature gaps identified included the lack of studies that focus on HT in the context of kidney disease and only one study on African individuals residing in Africa. In this work, two research studies were developed based on existing data from the African Research Kidney Disease (ARK) study, a well characterised population-based cohort study of black individuals from Agincourt in the rural Mpumalanga Province, South Africa. The second objective was a genomics study which aimed to examine the potential of using summary statistics from three discovery datasets to assess the predictive accuracy of polygenic scores (PGSs) for CKD and kidney function markers. Limited transferability was observed, explaining <1% of the variability in kidney disease markers in this African cohort. A PGS model derived from the transethnic cohort for estimated glomerular filtration rate (eGFR) explained the highest variability (0.8%) in African individuals and was significantly associated with HT (P<0.001), diabetes (P=0.007), and HIV (P=0.001). The third objective was a proteomics study which aimed to compare proteomic profiles of cases with both HT and albuminuria to controls (neither condition) to identify proteins and pathways involved in hypertension- associated albuminuria. Pathways including immune system (q=1.4x10-45) and innate immune system (q=1.1x10-32) were linked with hypertension-associated albuminuria. Proteins including angiotensinogen, apolipoprotein L1, and uromodulin had the highest disease scores (76–100% confidence). A machine learning approach was able to identify a set of 20 proteins that contributed to classifying disease status (ie, hypertension-associated albuminuria). Page | VI The assessment of PGSs for kidney function markers contribute to understanding of CKD genetic risk prediction in Africa, while the proteomics research added new knowledge to understanding the role of proteins and pathways involved in hypertension-associated albuminuria in Africa. This research addresses the gap of a lack of ‘omics research in resident African populations. It also contributes to the understanding of risk prediction for CKD and identifies potential proteomic markers for hypertension-associated albuminuria that may inform the development of personalised treatment strategies in Africa.Item The Utility of Clinical Exome Sequencing as a First-Tier Diagnostic Tool in Critically Ill Infants in South Africa(University of the Witwatersrand, Johannesburg, 2024) Campell, Lisa; Carstens , Nadia; Krause, AmandaGenetic disorders are significant contributors to infant mortality, morbidity, hospitalisation, and the need for intensive care globally. The identification and diagnosis of genetic disorders in ill infants is challenging due to their indistinct, and often atypical disease presentations. Diagnosis is traditionally driven by a differential clinical diagnosis; however, broad genotype-first approaches are now the recommended strategy in ill infants. The use of NGS-based testing, including gene panels, whole exome sequencing and whole genome sequencing, has successfully been utilised to diagnose genetic disorders in ill infants and has begun to be implemented in global settings; however, representation of low- and middle-income countries is lacking. Local infrastructure, capacity and expertise significantly affect successful implementation in these contexts. We therefore aimed to investigate the utility and implementation of singleton virtual exome sequencing panels in a cohort of 32 ill infants in the South African State healthcare system, providing the first investigations into the use of NGS in a neonatal intensive care unit setting in Africa. Three virtual panels were used to analyse exome sequencing data: a curated panel of genes implicated in neonatal-onset conditions and enriched for variants in South African populations; a ClinVar panel; and the Developmental Disorders Genotype-to- Phenotype (DDG2P) panel. A diagnostic yield of 22% was achieved across the three virtual panels, providing a definitive molecular diagnosis in seven ill infants. These infants experienced changes in their clinical management as a result of this diagnosis, including the initiation of palliative care, familial screening and prenatal testing for future pregnancies. The adaptation of recommended implementation strategies was necessary in the South African context to address shortages in resources, infrastructure and bioinformatics capacity through the use of gene panels instead of whole exome sequencing and the use of locally available technologies; shortages in the trained genetics workforce through the reliance on primary care clinicians for referrals; and through the singleton sequencing approach to address the unavailability of parents for trio-sequencing and the additional cost factors. This pilot study demonstrated the utility of NGS for the diagnosis and management of ill infants in the South African State healthcare system and explored the challenges in implementing NGS technologies in resource-limited settings. The implementation strategy explored through this research provides a baseline from which advanced NGS diagnostic v strategies can be developed and from which scaled up investigations of utility in the South African State setting can commence.Item Molecular epidemiology and characteristics of immune adaptations across the SARS-CoV-2 spike glycoproteins from Gauteng, South Africa, 2020 to 2022(University of the Witwatersrand, Johannesburg, 2024) Subramoney, KathleenThe SARS-CoV-2 global pandemic has been fueled by several variants of concern (VOC) that have gained more efficient transmission or immune evasion properties over time. To better understand the diversity and evolutionary characteristics of SARS-CoV-2 lineages in South Africa we described the analysed the SARS-CoV-2 lineages and VOCs circulating during 2020 to 2022, as well the impact of the S protein and its potential to act as a candidate vaccine. The first objective of this study was to rapidly identify emerging VOCs based on key SARS- CoV-2 S protein mutations. The second objective was to describe the impact of intra-host immune adaptations on the evolution of SARS-CoV-2 S protein genes among individuals with SARS-CoV-2 infections. Thirdly, by timing the emergence SARS-CoV-2 dominant variants we aimed to unravel the significance and abundance of low-frequency lineages that emerged during five COVID-19 waves in South Africa. The final objective was to assess if accounting for diversity among SARS-CoV-2 S protein’s improved predicted epitope coverage of a derived immunogen. Single nucleotide polymorphism (SNP) PCR-based genotyping assays targeting specific mutations were used to detect VOCs that circulated in 2021. The allele frequencies (AF) as determined by SNP PCR analysis and variant calling from FASTQ reads using galaxy.eu were performed to describe intra-host SARS-CoV-2 S protein variants. Whole genome sequencing was performed to identify and analyse SARS-CoV-2 strains circulating in South Africa from 2020 to 2022 and detect low-frequency lineages. Mosaic vaccine suite tools were used to design an optimal S protein construct from sequences generated in this study. The construct was further tested for antigenicity, toxicity, N- and O-linked glycosylation sites and CTL predictions. A combination of P681R and L452R SNPs were detected in 73.6% (538/731) of the samples classified as Delta, while N501Y and del69/70 SNPs were detected in 3.6% (26/731) of samples classified as Alpha. The detection of the del69/70 and K417N coupled with SGTF is efficient to exclude Alpha and Beta variants and rapidly detect Omicron BA.1. SNP assays detected 5.3% of cases with Delta that displayed heterogeneity at delY144, E484Q, N501Y and P681H. However, heterogeneity was confirmed by sequencing only for the E484Q and Characterisation of SARS-CoV-2 Page 9 of 155 delY144 mutations. Variant calling from FASTQ reads identified intra-host diversity in the S protein among 9% of cases that were infected with Beta, Delta, Omicron BA.1, BA.2.15, and BA.4 lineages. Heterogeneity was primarily identified at positions 19 (1.4%) with T19IR 371 (92.3%) with S371FP, and 484 (1.9%) with E484AK, E484AQ and E484KQ. In 2020, 24 lineages were detected, with B.1 (3%; 8/278), B.1.1 (16%; 45/278), B.1.1.348 (3%; 8/278), B.1.1.52 (5%; 13/278), C.1 (13%; 37/278) and C.2 (2%; 6/278) circulating during the first wave. Beta dominating the second wave of infection in 2020. B.1 and B.1.1 continued to circulate at low frequencies in 2021 and B.1.1 re-emerged in 2022. Beta was outcompeted by Delta in 2021, which was thereafter outcompeted by Omicron sub-lineages during the 4th and 5th waves in 2022. Several significant mutations (del69-70, delY144, E484K, N501Y and D614G) identified in VOCs were also detected in low-frequency lineages. During the 5 waves of infection, B.1 and C.1/ C.2 lineages co-circulated with a dominant VOC. Following our findings of co-circulation of VOCs and other lineages and evidence of quasispecies we investigated if accounting for diversity of SARS-CoV-2 strains would render an improved S immunogen. The optimal mosaic S protein generated had predicted CTL epitope coverage of ~95% to 98% and was classified as an antigen based on a prediction score of 0.47. Reverse translation was used to generate the novel S gene for the expression construct SC2M2. The NTD and RBD regions were non-toxic, and the derived novel S protein comprised 10 additional N-linked glycosylation sites and 4 O-linked glycosylation sites when compared to the Wuhan Hu-1 strain. Our study findings have shown that (i) rapid detection of emerging VOCs was possible using SNP genotyping assays, and can be used by low to middle income countries to detect Alpha, Beta, Delta and Omicron BA.1; (ii) heterogeneity within the S protein encourages escape from neutralising antibodies and the evolution of SARS-CoV-2, which may contribute to the ongoing emergence of new variants associated with continued outbreaks globally; (iii) low frequency lineages that share mutations with VOCs could lead to convergence and recombination events that result in the next novel lineages or variants that may further increase transmissibility, infectivity and escape immunity; and lastly (iv) the novel S expression construct designed, based on previous and currently circulating VOCs and lineages, could potentially be used to develop improved SARS-CoV-2 vaccines.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.