Electronic Theses and Dissertations (Masters)

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A multicentre study to evaluate an in-house multiparameter immunophenotypic panel to identify precursor B-cells in the determination of measurable residual disease in paediatric B-cell acute lymphoblastic leukaemia
(University of the Witwatersrand, Johannesburg, 2024) Nell, Zanre; Glencross, Deborah; Geel, Jennifer
Background: Periodic assessment of measurable residual disease (MRD) is an important prognostic factor in the management of paediatric B-cell acute lymphoblastic leukaemia (ALL). Conventional polymerase chain reaction (cPCR) and multiparameter flow cytometry (MFC) are well-established in MRD determination, the latter with no current optimal immunophenotypic panel by international consensus. Objective: To determine whether an in-house immunophenotypic panel containing the discriminatory CD58-FITC (cluster of differentiation; fluorescein isothiocyanate) marker compares with cPCR in the detection of paediatric B-cell ALL MRD. Methods: This prospective descriptive validation study was performed on diagnostic and follow-up bone marrow aspirate samples, comparing an in-house immunophenotypic panel against the standardised commercial ClearLLab 10CTM B-cell/myeloid cell-2 (M2) panels in MRD assessment. These findings were then compared to cPCR to determine individual panel performance and predictive power. Results: Both immunophenotypic panels demonstrated 100% concordance in the identification of the leukaemia-associated immunophenotype (LAIP) on all diagnostic samples. The in-house immunophenotypic panel showed a higher sensitivity and specificity, and greater association with cPCR in MRD assessment in follow-up samples. In combination with shared backbone markers of the ClearLLab 10CTM B-cell/M2 panels, inclusion of CD58-FITC and CD81-APC-H7 (allophycocyanin- cyanine dye) proved most informative in accurate distinction between regenerating B-cell precursors and residual leukaemic cells. Conclusion: This work confirms the findings of previous studies, where discriminatory marker CD58- FITC in combination with backbone informative markers demonstrates both superior diagnostic and monitoring utility in paediatric B-cell ALL. The in-house immunophenotypic panel offers an attractive, comparable alternative in MRD determination in this patient population whilst awaiting cPCR results, raising the possibility of earlier clinical decision-making with potential improvement of morbidity and mortality outcomes
Assessing the propensity of drug resistant tuberculosis to enter and exit the differentially culturable state
(2024) Nonkula, Bomikazi
Tuberculosis (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.
Audit of Lysosomal Storage Diseases Testing at the National Health Laboratory Service in Johannesburg from 2011-2020
(2023) Novellie, Michael
Lysosomal Storage Diseases (LSDs) are a group of Inborn Errors of Metabolism (IEM), due to the lack of a lysosomal enzyme. This results in toxic accumulation of metabolic waste products in various organs leading to neurodevelopmental regression, organ failure and premature death in the absence of treatment. Treatments for LSDs are limited. This study audited LSD diagnostic test requests received by the Division of Human Genetics, National Health Laboratory Service (NHLS) in Johannesburg from 2011 to 2020 with the aim of understanding the demand, appropriateness, and patient management of suspected LSD cases. A quantitative survey of all samples (1861 tests) referred to NHLS Johannesburg during the study period was performed. A total of 198 (13.3%) samples were rejected for testing mainly because of faulty sample collection. Of the 1663 that were accepted for testing 1457 (87.6%) tested negative, 73 (4.4%) were inconclusive and 133 (8.0%) tested positive. Fifty-five (3.1%) patients with LSD test requests, all of which were positive, were known to a Clinical Genetics unit. The most frequently requested test was for Fabry disease: 620 (33.3% of all requests), even though this disease is not the most prevalent LSD. Of the 603 accepted test requests for Fabry disease, only 6 (1.0%) tested positive. This suggests that some referring clinicians had unrealistic expectations of encountering this disease. It should be noted, however, that testing for Fabry disease is part of a broad diagnostic workup that may be applied even if the indication for testing is not specific. Access to LSD testing was unequal: private facilities were proportionally over-represented compared to public facilities; certain provinces with large referral centres (in KZN and Gauteng) were over-represented compared to smaller centres. Feedback and education of referring clinicians regarding indications for testing and importance of patient follow up, especially by clinical genetics services, are recommended. Follow up of positive MPS screening tests with specific diagnostic tests is essential. A system should be implemented where a medical geneticist phones the referring clinician and discusses further sample requirements (blood for enzyme analysis) and referral to a genetics clinic for all positive LSD screening tests. Future consideration should be given to designing a more systematic testing process, with the introduction of molecular testing to supplement biochemical testing.
Characterisation of RSV fusion proteins from South African patients with RSV disease, 2019 to 2020
(2024) Mabilo, Prince
Respiratory 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.
Characterisation of variation in the CYP2C19 gene in African populations
(2024) Booyse, Ross Peter
Background: 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.
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, Nicki
Background: 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.
Combination interaction of fluconazole with efflux pump inhibitors against antifungal resistant Candida parapsilosis
(2024) Alati, Marwah Omar Ali
Introduction 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.
Combination interaction of proton pump inhibitors with fluconazole against multidrug resistant Candida auris
(2024) Galane, Kamogelo
A 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.
Comparison between lupus nephritis in HIV positive patients and HIV associated immune complex glomerulonephritis with “lupus–like” features: a clinicopathologic study
(2024) Mathaba, Margaret Masala
Background: 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.
Delineating antibody responses elicited by four SARSCoV-2 variants against the Mu variant
(2024) Kgagudi, Prudence
SARS-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 VOCs
Delineating the ontogeny of an N332-directed anti-HIV-1 broadly neutralising antibody lineage
(2024) Naidoo, Thamara
Broadly 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..
Evaluating the clinical utility of a SNP-based microarray platform: a comparative study
(2024) Mayisela, Minenhle Peniel
Developmental 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 Genetics
Evaluation of the genetic and metabolic determinants of postprandial glucose variability in Black South Africans
(2024) Masango, Bontle
This 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.
Exploring the interaction of host genetics and the gut microbiome in obesity in an African population
(2024) Schnell, Samantha Susan
Obesity 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.
Extended characterization of multi-drug resistant organisms colonising neonates at a tertiary hospital in Johannesburg, South Africa.
(2024) Mntla, Nonkululeko Marcia
Neonatal 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.
Genetic characterisation of epidermolysis bullosa in South African patients
(University of the Witwatersrand, Johannesburg, 2024) Vania, Ashira; Dillon, Bronwyn
Background: Epidermolysis bullosa (EB) is a clinically and genetically heterogeneous inherited skin condition, characterised by the formation of blistering skin lesions in response to minimal abrasive skin trauma, with variable additional clinical complications. EB is divided into four subtypes based on histological characteristics: simplex EB (EBS), junctional EB (JEB), dystrophic EB (DEB) and Kindler syndrome (KS). EB is diagnosed on the basis of family history, and clinical signs and symptoms, in conjunction with histological examination, immunofluorescence mapping and transmission electron microscopy to determine subtype. Where available, molecular genetic testing can identify the causative genetic pathogenic variant/s. There is little recently published research on EB and its genetic aetiology in South African cohorts. The aim of this study was to design a multigene EB panel to investigate the pathogenic genetic variant/s responsible for EB in a group of South African patients, thereby providing information for targeted symptomatic treatment, medical surveillance, and to allow for more accurate genetic counselling and future reproductive options. Methods: Thirteen South African patients with clinically-diagnosed EB were recruited from the genetic clinic in four local State hospitals in Gauteng, South Africa. They were phenotypically characterised in terms of family history of the disorder, clinical features, and histological subtype. Whole exome sequencing was performed and a virtual panel of 11 of the commonly implicated EB-associated genes to screen for pathogenic variants. Genetic variants detected were analysed and classified according to American College of Medical Genetics and Genomics guidelines. Results: The 13 patients all had similar clinical characteristics of generalised skin blistering from birth. Skin biopsy with histopathology examination was available for 7/13 (54%). Histological and electron microscopy investigations correlated with molecular findings in only three cases. A genetic result that either confirmed the clinical diagnosis or supported the clinical diagnosis of EB was found in over half of the study cohort (8/12 (67%)). Three recurring variants were identified; COL7A1 (c.3265C>T), LAMB3 (c.958_1034dup) and LAMB3 (c.1034_1035insGGG; previously unreported). Of the eight patients with a confirmed/supported genetic diagnosis, 50% had JEB and 13% had EBS Conclusion: Of the 13 clinically diagnosed EB patients, 8/13 (67%) could be genetically characterised and three parents had confirmed carrier status; allowing for accurate genetic counselling, in terms of inheritance and recurrence risk information. JEB was seen in a higher frequency and EBS in a lower frequency than would be expected, reflecting a possible ascertainment bias. This study validates the clinical utility of an EB multigene panel for South African patients with EB, with particular focus on COL7A1 and LAMB3
Impact of donor CYP3A5 genotype on pharmacokinetics of tacrolimus in South African paediatric liver transplant patients
(2024) Wheeler, Caitlin
Tacrolimus 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.
Laboratory Evaluation of Aspergillus Galactomannan Lateral Flow Assays
(University of the Witwatersrand, Johannesburg, 2023) Ubbink, Anja; Chibabhai, Vindana
Invasive aspergillosis diagnosis is based on a combination of clinical, radiological, and mycological factors, including the detection of Aspergillus galactomannan antigen in serum and bronchoalveolar lavage fluid (BALF). Lateral flow assays (LFA) introduced for rapid detection of galactomannan in serum and BALF include the IMMY sōna Aspergillus LFA (IMMY LFA) and the Dynamiker QuicGMTM Aspergillus Galactomannan Ag LFA (QuicGM LFA). Objective To evaluate the performance of the IMMY LFA and QuicGM LFA in South Africa. Methods Serum and BALF samples previously tested by Platelia BioRad Aspergillus GM-EIA were analysed using the two different LFAs. Percentage agreement and precision was assessed. Results Forty-six serum- and 13 BALF samples were tested using the IMMY GM LFA and 48 serum- and 6 BALF samples were tested using the QuicGM LFA. Using an optical density ≥0.5 as positive, results were compared to the BioRad Aspergillus GM-EIA. For the IMMY LFA, serum samples had a positive percent agreement (PPA) of 0% (0/1); negative percent agreement (NPA) of 91% (41/45) and overall percent agreement (OPA) of 89% (41/46). BALF samples had a PPA of 75% (3/4), NPA 50% (5/10) and OPA of 57% (8/14). For the QuicGM LFA, serum samples had a PPA 0% (0/3), NPA of 96% (43/45) and OPA of 90% (43/48). BALF samples had a PPA of 100% (1/1), NPA of 100% (5/5) and OPA of 100% (6/6). For the IMMY LFA, between-day reproducibility for 72% (13/18) and 63% (5/8) for serum and BALF samples, respectively. Between-batch reproducibility was 89% (16/18) and 50% (4/8), respectively for serum and BALF samples. For the QuicGM LFA, between-day reproducibility was 75% (9/12) and 75% (3/4) for the serum and BALF samples, respectively. The between-batch reproducibility was 100% (8/8) for serum and 100% (3/3) for BALF. Conclusion A follow-up evaluation with a larger sample size utilizing clinical, radiological, and laboratory data is warranted to determine the assays’ clinical utility. What this study adds Invasive aspergillosis is a life-threatening disease, where a prompt diagnosis improves outcome. Currently there is no Aspergillus galactomannan assay available in the South African state sector. This study evaluates two lateral flow assays for the detection of Galactomannan in South Africa
Machine Learning on biochemical data for the prediction of mutation presence in suspected Familial Hypercholesterolaemia
(2024) Hesse, Reinhardt
Background 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.
Molecular characterization of invasive Streptococcus agalactiae in South Africa, 2019 – 2020
(2024) Ntozini, Buhle
Background: Group B streptococcus (GBS) is a leading cause of neonatal meningitis and sepsis. Capsular polysaccharide and protein-based GBS vaccines are currently in development. Therefore, it is important to understand the serotype and antigen distribution of GBS to assess potential vaccine coverage. We aimed to use whole genome sequencing (WGS) and phenotypic methods to assess the serotype and antigen distribution, antimicrobial susceptibility patterns, and the phylogeny of invasive GBS isolates in South Africa (SA). Methods: As part of national laboratory-based surveillance, 661 invasive GBS isolates cultured from normally sterile-site specimens (e.g. blood and cerebrospinal fluid) from patients of all ages, were collected from 2019-2020. Phenotypic identification was based on colony morphology and β-hemolysis. Serotyping was performed using latex agglutination. Antimicrobial susceptibility testing was based on disc diffusion and broth microdilution methods. Whole genome sequencing (WGS) was performed using the NextSeq 550 System and the 2 x 100-bp paired-end mode. WGS based prediction of serotypes, surface protein genes, and resistance gene detection was performed using a validated GBS bioinformatics pipeline developed by the US Centers for Disease Control and Prevention (CDC). Results: A total of 658/661 isolates yielded good quality sequences from WGS and three isolates poor quality sequences. The isolates belonged to 6 major clonal complexes (CCs): CC1 (37/658, 5.6%), CC8/10 (69/658, 10.5%), CC17 (272/658, 41.3%), CC19 (45/658, 6.8%), CC23 (188/658, 28.6%) and CC24 (37/658, 5.6%). Five singletons (10/658, 1.5%) were identified. Excluding phenotypically and genotypically non-typeable isolates, concordance for serotyping using latex agglutination and WGS was 99.7% (647/649). Overall, 6 serotypes were detected: III (281/656, 42.8%), Ia (183/656, 27.9%), V (78/656, 11.9%), II (955/656, 8.4%), Ib (44/656, 6.7%), and IV (15/656, 2.3%). Three percent (20/658) of the isolates were non-susceptible to penicillin. Erythromycin and clindamycin resistance was detected in 16.1% (106/658) and 3.8% (25/657) of the isolates respectively. Majority (91.5%, 625/657) of the isolates were resistant to tetracycline. Fifty-five percent (11/20) of penicillin non-susceptible isolates had mutations in the PBP2x gene known to confer resistance, while no PBP2x resistance mutation were detected in the remaining resistant isolates. ermTR (34.9%, 37/106) and mefA/E (29.2%, 31/106) resistance genes were the most common determinants of erythromycin resistance. Clindamycin resistance was mediated by the erm (ermB, T, and TR) genes. Tetracycline resistance was driven by the presence of the tet genes (tetM, tetO, and tetL), with tetM being the most common (95.8%, 599/625). Nearly all the isolates carried at least one of the 3 main pilus gene clusters (657/658, 99.8%), 1 of the 4 homologous alpha/Rib family determinants (656/658, 99.7%) and 1 of the serine-rich repeat (Srr) protein genes (626/658, 95.1%). The hvgA virulence gene was found exclusively in CC17 isolates. Conclusion: Our results show that the majority of isolates circulating in SA belong to the 5 major GBS CCs (CC1, CC8/10, CC17, CC19, and CC23). This study suggests that vaccines currently under development should provide good coverage in our setting. We show that the GBS6 vaccine that targets serotypes Ia, Ib, II, III, IV, and V has the potential to cover 100% of invasive GBS disease in SA. A pilus protein-based vaccine has a potential coverage of 99.8% and the GBS-NN and the latch peptide vaccines have a potential coverage of 68.5% and 95.3% respectively. Βeta-lactams remain appropriate for treatment and intrapartum antibiotic prophylaxis (IAP). However, detecting the emergence of non-susceptibility requires ongoing surveillance.

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