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Item Assessing the propensity of drug resistant tuberculosis to enter and exit the differentially culturable state(2024) Nonkula, BomikaziTuberculosis (TB), one of the oldest and most contagious infectious diseases, continues to be a global health concern. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC) which comprises of several species. These species are further subdivided into strains based on subtle genetic differences. The success of M. tuberculosis as a pathogen can be attributed to its ability to survive various stresses by adopting different growth states. Previous studies have shown that sputum from TB infected patients harbours a large proportion of drug-tolerant bacteria that are unable to form colonies on agar plates but can grow in liquid media. This population of organisms, termed differentially culturable tubercle bacilli (DCTB), could be resuscitated to grow by supplementing liquid media with cell free culture filtrates from axenic cultures of wild type M. tuberculosis H37Rv or mutant H37Rv lacking all five resuscitation promoting factors (Rpfs). Laboratory models that induce this differentially culturable state are critical for studying the physiology and metabolism of these bacteria in order to develop new TB diagnostic tests. In this study, five Beijing and five LAM drug resistant strains of M. tuberculosis were selected and used to robustly generate DCTB through an in vitro stress model using carbon starvation. The most probable number (MPN) assay and colony forming units were used to determine the amount of DCTB. Furthermore, the phenotype of these cells was studied using microscopy as well as metabolic probes that target the peptidoglycan (PG) component of the bacterial cell wall. Our findings demonstrated that applying the carbon starvation model to clinical M. tuberculosis strains (Beijing and LAM) resulted in robust levels of DCTB, as evidenced by limited growth on agar plates and enhanced growth in liquid media supplemented with culture filtrate from LAM and Beijing strains. Comparison of cell length between carbon starved cells to those grown in routine laboratory media suggested that DCTB appeared to be non-replicating and significantly shorter. The metabolic activity of the starved cultures was restored when they were supplemented with H37Rv, LAM and Beijing culture filtrate. Our results also demonstrated that Beijing strains had a higher propensity to produce DCTB compared to LAM strains and that the supplementation with Beijing culture filtrate resuscitated more DCTB. Collectively, our findings allow for the advancement of experimental systems that enable further investigation of DCTB and the properties of the Beijing strain that facilitate better adoption of the differentially culturable state.Item Biomarkers to predict Tuberculosis treatment response(2024) Boshielo, ItumelengTuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis (Mtb). Despite the implementation of multifaceted TB prevention and control efforts, a significant number of people still die from TB. Consistent with this, an uptick in TB-related mortality was recently noted, which has been ascribed to the negative effects of Coronavirus disease-2019 (COVID-19) on TB programs. The complex life cycle of Mtb is largely due to the use of immune evasion mechanisms to establish initial infection, remain dormant in the host, and reactivate pathogenicity under favourable circumstances. The prolonged TB treatment regimen is necessitated by the slow response of bacterial populations to standard TB chemotherapy, a phenomenon that may be caused by persistent, drug-tolerant bacteria. Scientific literature has provided evidence for these types of bacterial populations in the form of Differentially Culturable Tubercle Bacilli (DCTB). It has been demonstrated that DCTB represent drug tolerant bacteria that appear to be cleared at slower rate than organisms detected by routine culture methods. However, it remains unclear if DCTB populations elicit different immune responses when compared to their conventionally culturable counterparts. Herein, we address this question by optimizing a laboratory model for the generation of DCTB in vitro and test the capacity of clinical isolates of Mtb from Lineage 2 (Beijing) and Lineage 4 (LAM) to adopt the DCTB state. Using the Most probable number (MPN) assay, in the presence of culture filtrate (CF) as a source of growth factors to resuscitate DCTB, and colony forming units, the amount of DCTB in our model was quantified. As demonstrated by the limited growth on agar plates and increased growth in liquid media supplemented with CF from an axenic culture of Mtb, our findings demonstrated that carbon starvation was able to generate DCTB from clinical Mtb strains. After generating these populations, we stimulated whole blood with DCTB and conventionally culturable populations and report on the stimulation of a select set of cytokines (IFN-γ, IL-4, IL-5, IL-6, IL-12p70 and TNF-α) using a Bead Array Multiplex Immunoassay. In comparison to H37Rv-DCTB and LAM-DCTB, Beijing-DCTB induced significantly reduced levels of IL-5 and TNF-α. When comparing cytokine production between culturable and DCTB populations, within a single strain, we noted that LAMDCTB was delayed in the production of IFN-γ whilst Beijing-DCTB was not able to induce production of this cytokine when compared to conventionally culturable counterparts. These data suggest that shifting to a non-replicating DCTB state does indeed affect the ability of clinical isolates to induce immune responses. Based on these observations, we next set out to determine if DCTB affects immune responses during treatment of Mtb infected individuals. In prior work, using a prospective observational cohort, we demonstrated a substantive heterogeneity in clearance of DCTB in individuals with drug susceptible TB. We were able to classify these response patterns into three broad groups including (I) participants who were able to clear DCTB within the first two weeks of treatment (treatment-responsive); (II) those with delayed ability to clear these organisms (delayed-responsive) and (III) a group of individuals where DCTB did not change substantively during treatment (non-responders). Given these stark differences in treatment response patterns, we hypothesized that the immune responses associated with these patterns would be substantively different. In the second component of this work, we set out identify immune biomarkers that predict an effective response of DCTB to TB treatment. To quantify cytokines, chemokines and growth factors in plasma from these groups, we used a 65-plex Luminex assay, with a broad selection of targets. Statistically significant differences between these groups were analysed using the Kruskal-Wallis test with Dunn’s multiple comparisons, with p<0.05 was considered as statistically significant. When compared to patients who had TB and HIV co-infection, the number of cytokines that may possibly be used to report on the effectiveness of TB treatment was significantly higher in Mtbonly infected patients. This suggests that HIV infection significantly reduces the number of cytokines that can be used to report on TB treatment response. The ROC analysis of I-TAC, G-CSF and VEGFA showed that these cytokines have a significant discriminatory power to distinguish treatmentresponsive and non-responsive patients from HCs using DCTB as the measure of treatment response. No unifying cytokine signature that predicted DCTB response in all groups was identified. Together, our results indicate that some inflammatory markers are elevated in individuals with TB that rapidly clear bacteria during treatment. Given that these responses are based on DCTB, which represent drug tolerant populations, these select cytokines may be useful in evaluating the effectiveness of novel shorter TB treatment regimens.Item Characterisation of RSV fusion proteins from South African patients with RSV disease, 2019 to 2020(2024) Mabilo, PrinceRespiratory syncytial virus (RSV) is classified into subtypes A (RSV-A) and B (RSV-B), which are classified into different genotypes based on genetic variability of the G surface glycoprotein gene. The F surface protein gene is more conserved however variability in signal peptide, transmembrane domain, and antigenic sites have been reported. The study was conducted in the Virology laboratory, Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), South Africa. Study participants included patients of all ages from whom respiratory samples were submitted for respiratory viruses diagnoses from 2019 to 2020. The complete RSV F genes were amplified, library prep was preformed using the Nextera DNA prep kits and sequenced using amplicon-based next generation sequencing on the Illumina MiSeq sequencing platform. The Genome Detective Virus tool v2.27 was used to assemble sequencing reads and MEGA X was used for phylogenetic analysis. N-linked glycosylation and amino acid sequence variation was assessed. The overall prevalence of RSV was 5.8% (101/1 734). Seventy (69.3%; 70/101) RSV-positive samples were available for genetic characterisation of the F protein gene and thirty-one (30.7%; 31/101) were excluded due to insufficient sample volume. Only RSV-A strains were identified (91.2%; 31/34). Twenty three of thirty-one (74.2%) of the RSV F gene sequences from 2019 to 2020 clustered together with bootstrap values ranging from 64% to 99% and were NA1-like. A N-glycosylation site at position 120 gained by South African strains from 2018 is retained in strains from this study. This N-glycosylation site is present in approximately 25.8% of RSV strains from this study. The diversity of RSV-A F proteins was low, with amino acid variations observed at 30/571 (5.3%) sites. Ten mutations were detected in 4/6 antigenic sites (I, II, IV and V), with frequencies ranging from 0.3 to 100%. Antigenic changes seen exclusively among South African strains are: Y33H (0.3%) and V384T (7.3%) at site I and S275F (0.3%) at site II. Seven mutations associated with escape of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocyte (CTL) were predicted in seven epitopes. Overall amino acid mutation frequency for 2019 to 2020 RSV-A F genes is similar to that reported for South African strains from 2018 (3.3% to 6.7%). For the first time in South Africa we detected the S275F mutation which causes palivizumab resistance.Item Characterisation of variation in the CYP2C19 gene in African populations(2024) Booyse, Ross PeterBackground: CYP2C19 pharmacogenetic testing is important clinically to optimise patient response to clopidogrel and anti-depressants. This study aimed to characterise the distribution of CYP2C19 star alleles (haplotypes) across diverse African populations compared with global populations, with a view towards informing future pharmacogenetic implementations. Methods: CYP2C19 star alleles and diplotypes were called from 604 high coverage genomes from continental African populations using the StellarPGx pipeline. Results: From our analysis, CYP2C19*1 (51%), *2 (17%), and *17 (22%) were the most common star alleles across African populations in this study. We also identified 3% of African participants that had potentially novel CYP2C19 haplotypes. Over 70% of the SSA participants had either poor, intermediate, rapid, and ultrarapid metabolizer status, and would likely benefit from dosage and/or treatment alterations, especially for clopidogrel Conclusion: This study supports the necessity for CYP2C19 pharmacogenetic testing in African clinical settings and the importance of comprehensive star allele characterisation in the African context.Item Colonisation with ESKAPE organisms and Candida auris among primary caregivers and healthcare workers in a neonatal unit at a public sector tertiary South African hospital(2024) Rees, NickiBackground: Nosocomial neonatal infection remains a significant cause of mortality and morbidity, particularly in the high care and intensive care settings. Among implicated pathogens ESKAPE- C organisms are considered particularly worrisome due to their virulence, ability to gain resistance and propensity to affect multiple sites. Transmission to neonates is postulated to occur through contact with colonised adults. Objective: This study aims to describe the prevalence of colonisation of both primary caregivers and healthcare workers in contact with admitted neonates. As a secondary objective this study aims to identify the most common resistance patterns in ESKAPE-C organisms isolated from primary caregivers and healthcare workers in a neonatal unit. The overall aim of the study is to provide insight into how best to prevent hospital acquired infections in this group. Methods: This cross-sectional prevalence study describes colonisation of healthcare workers (HCW) and primary caregivers in a neonatal unit in a tertiary South African hospital. Over one week in August 2021, twenty-five primary caregivers and twenty-nine healthcare workers submitted specimens which were processed for the identification of ESKAPE-C organisms. Susceptibility was performed on identified organisms. Results: Of the healthcare worker participants 13,8% (4/29) were shown to be colonised with one or more ESKAPE-C pathogen, while 52% (13/25) of primary caregivers were shown to be colonised with one or more ESKAPE-C pathogens. Of the S. aureus organisms isolated 28,6% were MRSA, of the A. baumannii organisms isolated 66.7% were XDR and of the Enterobacteriaceae isolated 60% were ESBL producing. No CRE or VRE organisms were isolated in this study. Conclusion: This study demonstrates that the prevalence of colonisation of healthcare workers and primary caregivers is significant and reinforces the need for stringent infection prevention and control strategies to prevent transmission to vulnerable neonates.Item Combination interaction of fluconazole with efflux pump inhibitors against antifungal resistant Candida parapsilosis(2024) Alati, Marwah Omar AliIntroduction C. parapsilosis is the most common fungal pathogen recovered in neonatal intensive care units and is associated with a higher mortality rate due to its multidrug resistance. This study investigated the virulence factors and reversal of efflux activity by using a well-known antifungal drug (fluconazole) in combination with two efflux pump inhibitors (pantoprazole and omeprazole). In addition the effect of most active combination on pathogenicity markers of C. parapsilosis has studied. Materials and methods The microbroth dilution method was used to obtain antifungal susceptibility tests of conventional antifungal drugs (fluconazole and amphotericin B) against twelve clinical isolates of C. parapsilosis, as well as antifungal susceptibility tests of two efflux pump inhibitors (EPI). In addition, the combination of efflux pump inhibitors (pantoprazole and omeprazole) with fluconazole, an antifungal drug, was determined by calculating the fractional inhibitory concentration index (FICI) based on zero-interaction theory of Loewe additivity. a time-kill kinetics assay was used to study the antimicrobial activity of fluconazole and efflux pump inhibitors against C. parapsilosis. The virulence factors in C. parapsilosis isolates were determined using in vitro virulence assays. The effects of the efflux pump inhibitors on virulence factors were also studied, and comparison of percent reduction of virulence factors was determined in both fluconazole-resistant and susceptible C. parapsilosis. The effects of Pantoprazole and Omeprazole on efflux pump were also investigated using two assays (Rhodamine 6G accumulation and Hoechst 33342 accumulation). Results The efflux pump inhibitors showed enhanced antifungal activity in combination with antifungal agents against C. parapsilosis. When pantoprazole was combined with fluconazole, the median Minimum inhibitory concentration for fluconazole and pantoprazole decreased from 125 to 31.25 µg/ml and from 620 to 310 µg/ml respectively. In the time-kill kinetics assay, fungistatic activity of Fluconazole was changed to fungicidal by addition of efflux pump inhibitors (pantoprazole and omeprazole) at their ½MIC values. On the other hand, this study demonstrated that C. parapsilosis had a variety of virulence characteristics, including the ability to adhere to epithelial cells and secretion of proteinase hydrolytic enzyme. Pantoprazole and omeprazole reduced the ability of C. parapsilosis cells to adhere to epithelial cells. Adhesion was reduced in a concentration-dependent manner. The reduction was significant (p < 0.01 However, the results also showed that pantoprazole and omeprazole and their combinations was significantly reduced efflux pump activity. Conclusion Candida resistance has increased, particularly to azoles, and advent of C. parapsilosis as most prevalent non albicans Candida species poses a significant threat to the future. Combination therapy is shown to be therapeutically efficacious, and MIC values of fluconazole decreased when it was combined with omeprazole and pantoprazole. Further study is needed to understand the epidemiology, microbiology, genetics, and antibiotic susceptibility of this pathogen. The ability to express different virulence factors, such as adherence, proteinase, and phospholipase production, is strain-dependent and the widespread use of antifungal agents leads to drug resistance in C. parapsilosis.Item Combination interaction of proton pump inhibitors with fluconazole against multidrug resistant Candida auris(2024) Galane, KamogeloA significant challenge with controlling C. auris infections is its resistance to multiple antifungal agents, including azoles. Alternative strategies are required to combat the problem of antifungal resistance. Some of the suggested approaches include combination therapy and anti-virulence treatment strategies. There is growing interest in combining proton pump inhibitors (PPIs) with available antifungal drugs for better treatment outcomes. In this study, we investigated the antifungal activity of PPIs omeprazole and pantoprazole alone and in combination with fluconazole against resistant C. auris isolates. The anti-virulence activity of the synergistic combination against resistant C. auris isolates was determined. Lastly, the effect of the synergistic combination on C. auris energy-dependent efflux-activity was established. The CLSI broth micro-dilution method was used to determine the antifungal susceptibility of 25 C. auris isolates against antifungal agents (fluconazole and amphotericin B) and PPIs (pantoprazole and omeprazole). The combination interaction of fluconazole and PPIs was established by determining the fractional inhibitory concentration index (FICI) of each combination. Time-kill curves were used to determine the antifungal activity of the synergistic combination over time and to confirm the combination interaction. All 25 C. auris isolates were screened for their ability to adhere to epithelial cells and proteinase secretion using microscopy and culture technique respectively. The effect of the synergistic combination on isolates with positive pathogenicity markers was determined. Most of the C. auris isolates were resistant to fluconazole (92%) and all were sensitive to amphotericin B (100%). Omeprazole and pantoprazole had antifungal activity against C. auris at MIC values of 3125 µg/ml and 15625 µg/ml, respectively. The combination of fluconazole and pantoprazole reduced fluconazole MIC values by 4-fold, from 125 µg/ml to 31.2 µg/ml. The combination had synergistic effect against most C. auris isolates (56%), additive effect in 36% and indifference in 8%. There was no synergism in the fluconazole and omeprazole combination. However, the combination had antagonist effect against most C. auris isolates (56%). All C. auris isolates displayed some degree of adherence to epithelial cells and 96% produced proteinases. Fluconazole and pantoprazole combination significantly reduced adherence (p values < 0.05) and proteinase secretion (p values< 0.001) at inhibitory and subinhibitory concentrations. Pantoprazole inhibited fluconazole efflux at inhibitory and subinhibitory concentrations. The study showed that C. auris isolates express virulence factors such as adherence and proteinase production. The combination of pantoprazole and fluconazole has antifungal and anti-virulence activity against resistant C. auris isolates. In addition, it was shown that pantoprazole can attenuate fluconazole resistance by inhibiting the activity of energydependent efflux-pumps and it has synergistic effect with fluconazole. Therefore, pantoprazole has the potential to improve therapy outcomes when azoles are used.Item Comparison between lupus nephritis in HIV positive patients and HIV associated immune complex glomerulonephritis with “lupus–like” features: a clinicopathologic study(2024) Mathaba, Margaret MasalaBackground: Systemic lupus erythematosus (SLE) is an autoimmune disease seen commonly in black females of childbearing age. More than half of the patients present with renal disease or lupus nephritis complications. Coinfection with HIV in patients with lupus nephritis is rare. Despite Africa having the highest rate of HIV infection in the world, and there is no available data on the coexistence of HIV and Lupus nephritis. HIV is associated with a wide spectrum of renal diseases, including “lupus-like” HIV-Associated Immune Complex Kidney Disease (HIVICK). The most prevalent renal lesions in “lupus-like” HIVICK is diffuse proliferative lupus nephritis Objectives: This study aimed to compare and correlate the demographics, epidemiology, pathological and clinical findings of HIV positive patients with lupus nephritis and those with “lupus-like” HIVICK. Methods: This retrospective chart review study was conducted at the Charlotte Maxeke Johannesburg Academic Hospital in 5 years (2014-2018). We reviewed case reports that met our criteria for cases with lupus nephritis and cases with “lupus-like” HIVICK and allocated a lupus class according to the report findings. Results: Out of 2174 renal reports, 25(1.14%) patients were diagnosed with lupus nephritis and nine (0.41%) with “lupus-like” HIVICK. There were significant differences in age, serology (urea and creatinine), clinical presentation and lupus class. Conclusion: The occurrence of both HIV associated lupus nephritis and ‘lupus like’ HIVICK is rare. In our setting, the former is more common than the latter. We observed clinical and pathological differences which may be used to diagnose these disease entities.Item Defining the development of gp120-gp41 interface directed broadly neutralizing antibodies in HIV-1 infection(2024) Hlatshwayo, Vincent NkosinathiA prophylactic HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) against conserved HIV-1 envelope epitopes such as the gp120-gp41 interface which includes the FP. The isolation of gp120-gp41 interface-, and FP-directed bNAbs from chronically HIVinfected donors has made this epitope an appealing vaccine target. Moreover, promising preclinical immunogenicity animal studies have shown the possibility of eliciting such responses in animals. However, little is known about the population prevalence or kinetics of gp120-gp41 interface responses, including FP-directed responses. Lastly, few FP-directed antibodies have been isolated from people living with HIV (PLWH), limiting our understanding of common developmental pathways that can be explored for vaccine purposes. Here, we first assessed the prevalence of bNAbs in participants previously enrolled in the CAPRISA 004 Tenofovir gel trial (CAP004). We show that in this cohort, only 12% of individuals developed breadth at three years post-infection, and that high viral load and low CD4 count were associated with bNAb development, as previously reported. ELISA screening showed that only 13% of individuals developed FP-directed responses at three years postinfection. Of the 13% (n=9), only two donors had broad plasma responses, including donor CAP312, whose plasma exhibited 64% neutralization breadth at three years post-infection. In CAP312, FP binding and heterologous neutralization against a multi-clade 22 virus panel appeared simultaneously, within one year (~ 50 weeks post-infection). Taken together, our findings suggest that gp120-gp41 interface- and FP-directed responses are infrequently elicited during infection. As few FP-specific bNAbs have been isolated to date, little is known about their shared features that could be exploited for eliciting FP-specific bNAbs by vaccination. We next isolated three FP-specific mAbs from donor CAP312 at three years post-infection; AIRU-F8, AIRU-G9 and AIRU-G4 with 64, 45 and 5% breadth, respectively. We showed that our mAbs, and previously isolated bNAbs PGT151 and ACS202 share gene usage and have a similar unusually long CDRH3, as a result of a conserved motif inherited from the germline IGHJ6*02 gene. Furthermore, we showed that CAP312 mAbs have even longer CDRH3s compared to PGT151 and ACS202 despite this shared motif. We also showed, using point mutants and glycan mutants that our FP-specific mAbs have a unique neutralization profile compared to published mAbs. Overall, these results suggest that FP-specific mAbs share structural and genetic features that could be explored further for lineage vaccine development. Lastly, we delineated the ontogeny of the gp120-gp41 interface-directed nAb CAP248-2B by tracing the evolutionary pathways utilising longitudinal samples from 11-281 weeks postinfection (wpi). CAP248-2B interacts with the HIV-1 Env trimer through a long light chain CDRL3 that inserts into the viral membrane, and a heavy chain CDRH1 32ED33 motif that interacts with gp41. We showed that the unusually long light chain CDRL3 and the heavy chain 32ED33 motif are functionally redundant against heterologous viruses. We also showed that affinity maturation mutations in this lineage selected a lineage with limited heterologous neutralization breadth. In summary, these findings support further research into gp120-gp41- and FP-specific responses in multiple cohorts. Furthermore, future studies should aim to elucidate mechanisms governing the development of these responses so that productive developmental pathways can be identified and exploited for vaccine design.Item Delineating antibody responses elicited by four SARSCoV-2 variants against the Mu variant(2024) Kgagudi, PrudenceSARS-CoV-2 is the causative agent of the acute respiratory syndrome known as coronavirus disease 2019 (COVID-19). The SARS-CoV-2 spike protein plays an essential role in virus attachment, fusion and entry and is the main target of neutralizing antibodies. Continued evolution of the spike has led to the emergence of variants of concern or interest (VOC/VOIs) which pose a greater risk to the population due to augmented transmission risk, increased disease severity, antigenic escape and/or decreased effectiveness of vaccines. The Mu variant was first identified in Columbia in January 2021, however the immune escape potential of the variant remains poorly characterised. It is typified by T95I, Y144S and Y145N mutations in the N-terminal domain; R346K, E484K, and N501Y mutations in the receptor-binding domain and D614G, P681H, and D950N mutations in the S2 region. This study aimed to assess targeting of the Mu variant by neutralizing and non-neutralizing antibodies elicited by four variants during four COVID-19 waves in South Africa namely D614G, Beta, Delta and Omicron (BA.1). This was achieved by measuring antibody binding, neutralization and antibody dependent cellular cytotoxicity (ADCC) activity against Mu, and comparing these responses across each of the four variants tested. All variants elicited cross-reactive binding antibodies to Mu although the magnitude of binding to Mu was reduced. This cross-reactivity was a result of multiple binding antibody epitopes on the spike protein. We observed variable neutralization escape of Mu from antibodies triggered by infection with WT D614G and Delta. Antibodies induced by the Beta variant exhibited increased breadth towards the Mu variant when compared to those elicited by other variants. Neutralization escape of Mu from antibodies elicited by WT D614G and Delta may be caused by the 95I, 484K and 346K mutations in Mu, which are known to confer resistance in other variants. Compared to neutralization, ADCC was mostly preserved against Mu. However, the infecting variant impacted the potency of ADCC responses. WT D614G triggered significantly higher ADCC activity against Mu compared to Beta, Delta and Omicron. These data suggest that different variants trigger qualitatively different neutralizing and Fc effector responses, providing a rationale to study humoral resistance following infection by different variants. As Mu shares mutations with other VOCs, this study may aid in predicting the impact of mutations that may be common to emerging VOCsItem Delineating the ontogeny of an N332-directed anti-HIV-1 broadly neutralising antibody lineage(2024) Naidoo, ThamaraBroadly neutralising antibodies (bNAbs) develop in chronically HIV-1 infected individuals and can neutralise a variety of HIV-1 Env strains, making them an important element contributing to the design of HIV-1 vaccines. By understanding and delineating the long affinity maturation pathways that bNAb lineages follow against evolving viruses, it is hoped that this process will be able to be replicated in uninfected people with a series of vaccine immunogens. Investigating the ontogeny of a N332-directed bNAb lineage will provide insights into the key somatic hypermutations necessary for the development of neutralisation breadth. CAP255.G3 is an N332-directed bNAb isolated from CAP255, a participant in the CAPRISA 002 cohort, at 149 weeks post-infection (wpi). Six key antibody intermediates were selected from the CAP255.G3 lineage arm between 17 and 47 wpi, based on their position on a phylogenetic tree and because they contained mutations that were present in broad members of the lineage. We hypothesized that these intermediates would exhibit breadth similar to CAP255.G3. The intermediates were tested against a panel of eight autologous and six heterologous pseudoviruses using a TZM-bl neutralisation assay. The sequence identity of the first heavy chain complementarity-determining region (CDRH1) of CAP255.G3 differed from the closest intermediate and therefore, a CDRH1 chimera was constructed to determine if the CDRH1 from CAP255.G3 affected neutralisation activity. Later intermediates from 39 and 47 wpi, had moderate neutralisation activity against the autologous pseudoviruses, but only theintermediate closest to CAP255.G3 had limited neutralising activity against the heterologous pseudoviruses. Although the CDRH1 chimera had increased neutralisation activity against the heterologous viruses relative to the intermediates, it was less broad and potent than CAP255.G3. This indicates that additional affinity maturation between 47 and 149 wpi, at sites outside of the CDRH1, was required for the development of neutralisation breadth in the CAP255.G3 lineage arm..Item Development of a multi-disease targeted next generation sequencing panel to study genetic aetiology of rasopathies(2024) Mudau, Maria MabyalwaWith Next Generation Sequencing (NGS), technologies it is now possible to screen a large number of genes simultaneously through massively parallel sequencing, significantly reducing costs and time generally associated with mutation screening. After an informal survey of the diagnostic needs of the clinicians in the Division of Human Genetics, National Health Laboratory Service (NHLS), it was established that the aetiology of genetic disorders called facial dysostoses, RASopathies and Cohesinopathies (FRASC) could be understood better using NGS. These are developmental disorders that are phenotypically different and are commonly referred to our genetic clinic, currently there is limited genetic testing for these conditions in South Africa. A NGS panel targeting genes associated with the FRASC disorders was designed and optimised. Upon successful optimisation the panel was then utilised to sequence samples from patients presenting with features suggestive of RASopathies. An overall detection rate of 56.6% (34/60) was obtained for all RASopathy patients sequenced in the current study. Detection rate of 46.7% (7/15) for neurofibromatosis type 1 (NF1) patients and 60% (27/45) for patients with non-NF1 RASopathies was obtained. No clinically significant variants were identified in 26 of the 60 patients (43.3%), two of the 26 had a VUS in the MAP2K1 gene. Seven patients of the panel negatives were successfully sequenced using whole exome sequencing (WES), one patient had a pathogenic variant and the rest had variants of uncertain significance identified. This is the first report profiling mutations and clinical features in patients with RASopathies as a whole in South Africa, compared to a study focusing on NS patients only. The detection rates obtained were comparable to other international studies using NGS, except for detection rate of LZTR1 and BRAF1 gene variants observed in Noonan syndrome patients. The (35/60) 58.3% (panel and WES) of patients with a disease causing variant identified in this study now have a molecular confirmation that they previously did not have. Our results show that a targeted panel could be an effective first-line diagnostic testing for RASopathies in SA. The development of the multi-disease targeted panel in the current study has contributed to the design of the 500 gene inherited disease NGS targeted panel (IDP) that is currently being utilised in our facility for diagnostic testing of various monogenic disorders.Item Evaluating the clinical utility of a SNP-based microarray platform: a comparative study(2024) Mayisela, Minenhle PenielDevelopmental disorders make up a majority of cases seen in genetic clinics at the National Health Laboratory Service (NHLS). Chromosomal microarray analysis (CMA) is the first line testing for individuals with developmental disorders and congenital anomalies. This study compared the clinical utility of single nucleotide polymorphism (SNP) - based array (CytoScan® Optima array) and a comparative genomic hybridization (CGH) array (SurePrint G3 Unrestricted CGH ISCA v2, 8x60 kb microarray) in diagnosis of developmental disorders. This was done by looking at the differences in copy number variant calls, segment size differences and gene content between the two microarrays. The cost to run each type of microarray test for diagnosis was also compared. The copy number variant calls made by the two platforms were comparable. The SNP-based array did provide additional information that would be useful in molecular diagnosis. The final recommendation was for the CGH array and the SNP-based array to be used interchangeably based on clinical requests at the Division of Human GeneticsItem Evaluation of the genetic and metabolic determinants of postprandial glucose variability in Black South Africans(2024) Masango, BontleThis study aimed to determine the metabolic and genetic factors that account for the variation in postprandial glucose (PPG) in Black South Africans (SA). The study included 794 participants from the middle-aged Sowetan Cohort (MASC). PPG was calculated using the integrated area under the curve (iAUC) in response to an oral glucose tolerance test. Principal component analysis was applied to 31 metabolic factors to generate clusters represented by principal component variables. Polygenic risk scores (PRS) were computed for each participant using variants and weights from a validated African type 2 diabetes PRS. Linear regression models were used to evaluate the variance. The PRS did not contribute to the PPG variability in men or women. Central fat, serum lipids, and liver enzymes explained 10.8% of PPG variability in women. In men, peripheral fat, serum lipids, liver enzymes, and steroid hormones explained 10.6% of PPG variability. Our work has identified metabolic factors that predict PPG variability in Black SA.Item Exploring the interaction of host genetics and the gut microbiome in obesity in an African population(2024) Schnell, Samantha SusanObesity is a highly prevalent health concern that is on the rise in Sub-Saharan Africa. Even though genetic variation and gut microbiota have been implicated in the development of obesity independently, the interactions between these factors have not been previously explored in a South African cohort. This study aimed to identify possible associations between host genomes, body mass index as a measure of obesity, and gut microbiota composition (in the form of V3-V4 16S rRNA sequencing) in a female African cohort. Polygenic risk scores predictive of body mass index in this cohort were generated to categorise the participants into high- and low-risk groups. Subsequently, several statistical analyses were performed comparing gut microbiota between these groups. High-risk participants with high body mass index had associations identified with increased abundances of Prevotella_9 and VadinBE97. In contrast, the low polygenic risk and low body mass index sub-group was associated with greater Bacteroides levels. This study acknowledges the plausible interactions between these factors in an African cohort.Item Exploring the interplay of chemokine receptors ccr5 and cxcr6 in mechanisms of natural control in HIV-1- infected black South Africans(2024) Koor, Gemma WhitneyIn sub-Saharan Africa, HIV-1 is a significant cause of morbidity and mortality. However, research remains primarily focused on North American and European population groups, who have remarkably different genetic backgrounds to individuals from sub-Saharan Africa. HIV1 controllers represent a model of HIV-1 functional cure, with some individuals able to control viral replication, and some able to sustain immune function in the presence of high viral loads, both in the absence of antiretroviral therapy (ART). The chemokine receptors CCR5 and CXCR4 are the major coreceptors HIV-1 utilises to enter cells. The use of alternative coreceptors, such as the CXCR6 coreceptor, is thought to contribute to the lower pathogenicity exhibited by the HIV-2 and SIVsmm strains. Building on previous work conducted in our research unit on these two coreceptors in South African populations, this thesis firstly describes CCR5 genetic variants that associate with HIV-1 control or risk of progressive infection in black South Africans, and then explores constitutive expression levels of CCR5 and CXCR6 on various peripheral blood immune cell subsets in the absence of HIV-1 infection in ethnically divergent population groups. The effect of sex, age, and select CCR5 and CXCR6 single nucleotide polymorphisms (SNPs) on expression levels of these two receptors was also investigated. The CCR5 5’UTR and 3’UTR regions were PCR-amplified and sequenced from genomic DNA extracted from 145 ART-naive black South African individuals living with HIV-1 (71 HIV-1 controllers – 23 elite controllers, 37 viraemic controllers, 11 high viral load long-term nonprogressors and 74 progressors). Findings confirmed results from other studies in showing that the CCR5 HHE haplotype is deleterious for HIV-1 disease progression, and the HHA haplotype and HHA/HHC genotype associated with protection from HIV-1 disease progression. Novel haplotypes were characterised, both in the 3’UTR and spanning the CCR5 5’UTR and 3’UTR. Overall, findings suggest that two CCR5 promoter SNPs (-2459 G>A and -2135 T>C) and one CCR5 3’UTR SNP (+2919 T>G) may be key functional variants with regards to HIV-1 control in black South Africans. To gain further insight into the constitutive expression of CCR5 and CXCR6 on peripheral blood immune cells and explore the relationship between select genetic variants and expression, immunophenotyping by flow cytometry was conducted using whole blood from age- and sex-matched ethnically distinct South African HIV-uninfected individuals (17 black, 21 white). Expression levels of CCR5 and CXCR6 were assessed on CD4+ and CD8+ T cells, B cells, monocytes and NK cells, and their respective subsets. The effects of age and sex on expression levels of these two receptors was also investigated. Population-specific differences with regards to CCR5 expression on all cell types, except for B cells, were evident. Generally, black South Africans exhibited a lower expression level of CCR5 compared to white South Africans. CXCR6 expression only differed with regards to percentage of CXCR6-expressing cells, not CXCR6 density (numbers of cell surface receptors). Black individuals had a lower percentage of CXCR6-expressing CD8+ T cell subsets (naïve and effector memory) and a higher percentage of CXCR6-expressing CD14+CD16+ monocytes compared to white individuals. Overall, we found significant population-specific differences in expression levels of both CCR5 and CXCR6, multiple associations with cell activation (as measured by HLADR expression) and CCR5 and CXCR6 expression, and CCR5 and CXCR6 expression was positively significantly correlated on multiple cell subsets. Furthermore, both sex and age influenced CCR5 and CXCR6 expression, however results varied widely across the two population groups studied. Sex differences were only evident in white individuals; predominantly CXCR6 expression was increased in males compared to females. Age associations with CCR5 and CXCR6 expression were also primarily found in white individuals. Four CCR5-related SNPs that are associated with HIV-1 control in this or other studies (rs553615728 -4223 C>T SNP, rs1799987 −2459 G>A SNP, rs746492 +2919 T>G SNP and rs1015164 G>A SNP) were assessed for their potential association with CCR5 expression levels. The +2919 TG genotype significantly associated with a higher percentage of CCR5- expressing total CD8+ T cells, transitional memory and terminally differentiated CD8+ T cells compared to the GG genotype. The +2919 GG genotype associated with a lower percentage of CCR5-expressing B cells compared to the TT and TG+TT genotypes, however, only in white South Africans. The +2919 TG and TG+TT genotypes associated with significantly higher CCR5 density on all CD8+ T cell subsets, except for naïve CD8+ T cells, when compared to the GG genotype. When evaluating two CXCR6 genetic variants previously associated with HIV-1 viraemic control (rs2234355 G>A and rs2234358 G>T) in relation to CXCR6 expression, possession of the rs2234355 SNP GA genotype associated with lower CXCR6 expression on select CD4+ and CD8+ T cell subsets as well as on B cells, while possession of the rs2234358 SNP TT genotype associated with higher CXCR6 expression on multiple cell types, primarily in white South Africans. Possession of the -358TT/+355GA genotype combination associated with lower CXCR6 expression on select subsets of CD4+ T cells and monocytes. In summary, this study provides information on genetic variation in the CCR5 gene in a South African context, describes genetic variants associating with HIV-1 control in black South Africans, adds novel insight into constitutive CCR5 and CXCR6 expression levels on CD4+ and CD8+ T cells, B cells, monocytes and NK cells in HIV-1-uninfected black and white South Africans, and describes the potential associations of select genetic variants and expression. Black and white individuals differed in their baseline expression levels of CCR5 or CXCR6, which was partly driven by host genetic factors that were explored. This work highlights the importance of considering effects of ethnicity, age, and sex in any studies addressing any immune molecules in relation to differential HIV-1 outcomes of infection susceptibility/protection, disease progression, or HIV-1 virological control on antiretroviral therapy. Although conducted on small numbers of individuals, these variables clearly influenced constitutive expression of CCR5 and CXCR6, and further population-specific studies are warranted to gain further insights. Findings from this study have implications for risk of acquisition of HIV-1 infection and for disease progression in people living with HIV-1. Understanding the role of these molecules is important for informing strategies for both HIV1 prevention and HIV cure.Item Extended characterization of multi-drug resistant organisms colonising neonates at a tertiary hospital in Johannesburg, South Africa.(2024) Mntla, Nonkululeko MarciaNeonatal deaths remain high globally, particularly in sub-Saharan Africa. A third of deaths are due to infections, often secondary to multi-drug resistant (MDR) organisms. The purpose of this study was to investigate the prevalence of MDR ESKAPE+ C. auris colonisation amongst hospitalised neonates, to determine risk factors associated with MDR colonisation, and perform antimicrobial resistance characterization of these isolates. Two hundred and fifty-eight swabs were collected from 86 hospitalised neonates at a tertiary South African hospital between November and December 2020. A total of 135 ESKAPE+ C. auris isolates were identified; 68% were MDR. Majority of neonates (65%) were colonised with extended spectrum betalactamase (ESBL) producing Klebsiella pneumoniae, followed by extensivelydrug resistant (XDR) Acinetobacter baumannii. New Delhi metallo-beta-lactamase (NDM) producing A. baumannii were more prevalent than carbapenemase producing Enterobacterales (CPE). A prolonged hospital stay, median=14 days (p<.001) was identified as a risk factor for MDR organism colonisation. The high prevalence of MDR ESKAPE+ C. auris colonisation supports use of non-invasive samples to determine colonisation prevalence. More data are needed to develop improved surveillance systems which should incorporate colonisation swabs and clinical biomarkers in neonates with independently established risk factors.Item Impact of donor CYP3A5 genotype on pharmacokinetics of tacrolimus in South African paediatric liver transplant patients(2024) Wheeler, CaitlinTacrolimus is characterised by a narrow therapeutic target range and wide interpatient variability. Pharmacogenetic research attributes CYP3A5 single nucleotide polymorphisms to the variable inter-patient tacrolimus concentration dose ratios (CDR). In this study, we compared the mean tacrolimus CDR in paediatric liver transplant recipients and their living liver donors’ CYP3A5 rs776746 T>C genotypes(*1/*1, *1/*3 and *3/*3), accounting for donor and recipient characteristics. The graft-to-recipient weight ratio and the CYP3A5 donor genotypes were found to be independent factors significantly impacting the mean tacrolimus CDR. Donor CYP3A5 expressors (*1/*1 and *1/*3) were shown to have significantly lower recipient tacrolimus CDRs, therefore, higher dosages would be required in comparison to CYP3A5 non-expressors to reach the same therapeutic target range. The potential implementation of a stratified medicine dosage algorithm, combining living liver donor CYP3A5 genotyping with the calculation of the graft-to-recipient weight ratio, may predict the optimal tacrolimus dosage schedules for liver transplant recipients.Item Machine Learning on biochemical data for the prediction of mutation presence in suspected Familial Hypercholesterolaemia(2024) Hesse, ReinhardtBackground Familial hypercholesterolemia (FH) is a common monogenic disorder and, if not diagnosed and treated early, results in premature atherosclerotic cardiovascular disease. Most individuals with FH are undiagnosed due to limitations in current screening and diagnostic approaches, but the advent of machine learning (ML) offers a new prospect to identify these individuals. Our objective was to create a ML model from basic lipid profile data with better screening performance than low-density lipoprotein cholesterol (LDL-C) cut-off levels and diagnostic performance comparable to the Dutch Lipid Clinic Network (DLCN) criteria. Methods The ML model was developed using a combination of logistic regression, deep learning and random forest classification and was trained on a 70% split of an internal dataset consisting of 555 individuals clinically suspected of having FH. The performance of the model, as well as that of the LDL-C cut-off and DLCN criteria, were assessed on both the internal 30% testing dataset and a high prevalence external dataset by comparing the area under the receiver operator characteristic (AUROC) curves. All three methodologies were measured against the gold standard of FH diagnosis by mutation identification. Furthermore, the ML model was also tested on two lower prevalence datasets derived from the same external dataset. Results The ML model achieved an AUROC curve of 0.711 on the high prevalence external dataset (n=1376; FH prevalence=64%), which was superior to that of the LDL-C cut off alone (AUROC=0.642) and comparable to that of the DLCN criteria (AUROC=0.705). The model performed even better when tested on the medium prevalence (n=2655; FH prevalence=20%) and low prevalence (n=1616; FH prevalence=1%) datasets, with AUROC curve values of 0.801 and 0.856 respectively. Conclusions Despite the absence of clinical information, the ML model was better at correctly identifying genetically confirmed FH in a cohort of individuals suspected of having FH than the LDL-C cut-off tool and comparable to the DLCN criteria. The same ML model performed even better when tested on two cohorts with lower FH prevalence. The application of ML is therefore a promising tool in both the screening for, and diagnosis of, individuals with FH.Item Maternal vaccination in South Africa: timing and completeness(2024) Bourne, JuliaMaternal immunisation is an invaluable public health measure that protects not only the mother, but also the foetus and new-born infant against a host of diseases; and is recommended by both the World Health Organisation (WHO) and South African national health authorities. Pregnancy induces a heightened state of immune system vulnerability, leaving women more susceptible to severe influenza outcomes, whilst neonatal tetanus has a fatality rate of between 80-100% in the absence of medical intervention. Maternal immunisation against influenza and tetanus has been successfully utilised as a public health strategy across the globe to uphold maternal and neonatal health. Maintaining coverage is imperative for both diseases as influenza strains change seasonally and tetanus cannot be eliminated, highlighting the importance of continued maternal immunisation. This study aimed to describe the uptake of both influenza and tetanus vaccinations during pregnancy, the completion of the tetanus vaccination schedule and the timing of both influenza and tetanus immunisation within South African antenatal care facilities. In addition, this study described clinical and demographic factors affecting maternal immunisation uptake. Clinical and demographic data were collected in a parent study and were retrospectively analysed in this study using the statistical software Stata. Influenza vaccination uptake within the sampled population was found to be 16.62% (806/4851). The odds of influenza vaccination were significantly higher in women aged 21-30 years, and women with six or more ANC visits. Metro East Cape Town site in the Western Cape outperformed Gauteng sites, with significantly increased odds of influenza vaccination amongst women frequenting that site. Appropriate influenza immunisation: defined as immunisation occurring during the period of either 01/04/2017-31/07/2017 or 01/04/2018-30/06/2018, occurred in 74.86% (530/708) of the cohort. Women who were alcohol users were significantly more likely to receive an influenza vaccine – yet this may be explained by the Metro East site which had the higher influenza coverage having the highest prevalence of alcohol use during pregnancy. Of 7105 women, 7031 (98.96%) received at least one dose of tetanus toxoid vaccine (TTV). Of these women, 39.24% (2759) received one dose; 51.06% (3590) received two doses and 9.70% (682) received the recommended three doses of TTV in their index pregnancy. Tetanus schedule completion was significantly more likely in women ≤20 years, and those who presented for their booking antenatal care (ANC) visit in the first trimester. In addition, women with more than three visits had an increased likelihood of TTV schedule completion. The odds of TTV schedule completion were decreased by negatively parity and gravidity, values over one and less than six, and greater than one respectively. Women with hypertension were significantly less likely to receive three TTV doses compared to women without hypertension. Julia Bourne MSc(Vaccinology) Research Report: Version 2.0 v Tetanus immunisation schedule adherence prevalence was 0.60% (4/670) in women with three doses and 90.34% (3209/3552) in women with two. Improvements may be made in South African maternal immunisation coverage, with this study supporting the idea of targeted educational campaigns and a revision of the maternal immunisation schedule to include the tetanus, diphtheria & acellular pertussis vaccine instead of the tetanus toxoid vaccine.