Doctoral Degrees (Medical Microbiology)
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- ItemPhenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region(Stellenbosch : Stellenbosch University, 2022-12) Van Coller, Ansia; Glashoff, Richard Helmuth; Glanzmann, Brigitte; Esser, Monika; Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology.ENGLISH ABSTRACT: Background: Inborn errors of immunity (IEI) relating to increased susceptibility to severe, persistent, unusual and/or recurrent (SPUR) mycobacterial infections are of particular concern in countries such as South Africa that are hyperendemic for tuberculosis (TB). Mendelian susceptibility to mycobacterial disease (MSMD), the principal IEI relating to SPUR mycobacterial infections, was originally defined to encompass only weakly pathogenic mycobacteria such as the Bacillus Calmette–Guérin (BCG) vaccine. However, more recent studies have shown that South African MSMD patients are also likely to present with SPUR TB. There are 15 known MSMD-associated genes, which all fall within the Interleukin-12-Interferon-γ (IL-12-IFN-γ) immunological pathway, and mutations in these genes have been described to result in either reduced IFN-γ production or a lack of immune response to IFN-γ. The aim of this study was to evaluate immune dysfunction in patients that present with suspected MSMD using genetic sequencing and in vitro functional profiling assays. Additionally, T-bet, a common transcription factor that is also integral to the IL-12-IFN-γ pathway, was investigated as a potential proxy marker for MSMD. Methodology: Blood was collected from 18 patients presenting with SPUR TB for genetic sequencing and functional immune profiling. Whole genome Sequencing (WGS) was performed to identify candidate disease-associated variants. The immune profiling assays included assessment of the IL-12-IFN-γ pathway through flow cytometric detection of cytokine receptors and intracellular signalling, as well as assessment of immune cell subset distributions and Luminex®-based detection of cytokine/chemokine readouts following stimulation of patient cells with Phytohemagglutinin (PHA), BCG, and IL-12/IFN-γ. T-bet expression was measured by means of intracellular flow cytometry. Results: Through WGS, likely disease-associated novel variants were identified in 82% of the 11 patients that were successfully sequenced and 89% of the identified variants were in known MSMD-associated genes. All patients had some degree of impairment in the IL-12-IFN-γ pathway, and these readouts corresponded with the WGS findings. Further immune investigations revealed that the overall patient group had significantly reduced levels of circulating CD16+ monocytes and lymphocytes as well as reduced levels of inflammatory cytokine/chemokine production following PHA or BCG stimulation. All patients also had aberrant T-bet expression, with reduced expression in CD16+ monocytes and natural killer (NK) cells being the most prominent. There were also very strong correlations between the components of the IL-12-IFN-γ pathway, T-bet expression, CD16-expressing cells, and various cytokines/chemokines. Conclusions: The in vitro functional assessment revealed disruptions in the IL-12-IFN-γ pathway of all patients, and a lack of key immune cell subsets and the cytokines/chemokines that are typically expressed by these cells, supporting the clinical diagnosis of MSMD in these patients. While there were some commonalities for the overall patient group, each individual had a very unique phenotype, emphasising the importance of in vitro assessment in all individuals with suspected MSMD – patients with the same or different variants in the same gene had different functional readouts. T-bet was demonstrated to be a promising proxy marker for MSMD as it correlated well with the immunological deficits observed in the patients and will allow for easier detection of potential MSMD in patients with SPUR TB, which may aid in the estimation of the true prevalence of MSMD in future studies.
- ItemExploring the gut microbiome of children from Cape Town communities(Stellenbosch : Stellenbosch University, 2021-12) Van Zyl, Kristien Nel; Newton-Foot, Mae; Whitelaw, Andrew Christopher; Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology.ENGLISH ABSTRACT: Despite the increase in microbiome investigations in the last decade, descriptions of the microbiota remain limited in developing countries, particularly in children. Little is known about the gut microbiota of young children in South Africa and there is a need for investigations of the composition and diversity of microbiota and the influence of demographic, clinical, and environmental factors on the microbiota in this setting. This study formed part of the ongoing Tuberculosis child multidrug-resistant preventive therapy (TB-CHAMP) clinical trial, which aims to determine the efficacy and safety of levofloxacin preventive therapy in a cohort of children <5 years exposed to multidrug-resistant tuberculosis in the household. This sub-study explored the composition, diversity, and factors influencing the baseline bacterial and fungal gut microbiota of these children, prior to randomisation into treatment or placebo arms. A pilot study was performed to assess the effect of stool sample storage and transport conditions on the microbiota and to develop a practical DNA extraction standard operating procedure for the remainder of the sub-study and future microbiome studies in this setting. Quantitative PCR showed that the gut microbiota was not affected by storage and transport conditions common to this setting. Targeted 16S rRNA and internal transcribed spacer 1 gene sequencing was performed on the Illumina platform to characterise the bacterial and fungal microbiota. The bacterial microbiota of young children from Cape Town communities were shown to be similar to those in children from developing countries and age was the main driver of differences in both bacterial and fungal communities. While the risk for dysbiosis was shown to be low overall, recent antibiotic use and household pollution were associated with reduced bacterial diversity. The inconsistent detection and abundance of fungi even in participants of the same age, highlighted the need for expanded research, including dietary and longitudinal analysis. The need for longitudinal analysis in paediatric populations was further emphasised by a systematic review on the effects of antibiotics on the microbiome, particularly in populations who are at risk for increased antibiotic exposure. The findings also demonstrated the population and target site-specific responses of the microbiome to antibiotics, which underlined the need for individual evaluation of the effect of antibiotics on the gut microbiome. This study set the groundwork for future investigations in the TB-CHAMP cohort, including the temporal development and stability of the gut microbiome in these children, and the impact of levofioxacin preventive therapy on the microbiota and resistance reservoirs in the gut.
- ItemColistin resistance in gram-negative pathogens in the Western Cape, South Africa(Stellenbosch : Stellenbosch University, 2021-12) Snyman, Yolandi; Newton-Foot, Mae; Whitelaw, Andrew Christopher; Maloba, Motlatji Reratilwe Bonnie; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH ABSTRACT: Background Antimicrobial resistance is a public health concern and injudicious antibiotic prescribing and inadequate infection control practices have left the global community with untreatable multidrugresistant (MDR) bacteria. Colistin is a last resort antibiotic used to treat infections with MDR Gramnegative bacteria (GNB), especially carbapenem-resistant GNB. Therefore, the emergence of colistin resistance is a serious problem. This study from the Western Cape, South Africa, describes colistin resistance mechanisms in colistin-resistant GNB isolates from clinical specimens from various hospitals, stool samples from healthy children in the community, and river and storm water. Methods Colistin-resistant GNB isolates from clinical specimens from different healthcare facilities were collected from the NHLS microbiology laboratory at Tygerberg Hospital during 2016 and 2017. Fifty stool samples from healthy children (≤ 5 year of age) in the Cape Town metropolitan were collected between November 2017 and August 2018, and three surface water sources and stormwater were collected in 2019 and 2020. Selective media was used to isolate colistin-resistant GNB from the stool and water samples. Colistin resistance was confirmed using broth microdilution (BMD). The mobile colistin resistance genes, mcr-1-9, were detected by PCR and whole-genome sequencing (WGS). In selected mcr-negative isolates chromosomal colistin resistance mutations were identified by WGS. Strain typing was performed by WGS (MLST and SNP analyses) and repPCR. The functionality of mcr genes with unknown colistin resistance profiles was determined by BMD following recombinant expression or plasmid curing. Results mcr-1 was present in 55% (12/22) of Escherichia coli and 71% (5/7) of Klebsiella spp. isolates from patients at various hospitals during 2016-2017. pmrB mutations were identified in 8/10 mcrnegative E. coli and mgrB was disrupted in the two mcr-negative Klebsiella spp. isolates. Most colistin-resistant GNB isolated from hospitalised patients in 2016 and 2017 were unrelated, however, some clonal relatedness was observed in the 2017 E. coli population and a clonal expansion of an emerging colistin-resistant MDR Acinetobacter baumannii strain was noted among isolates from 2017. No previously described colistin resistance mechanism was detected in the A. baumannii isolates, but a possible novel mechanism was described. mcr-4.3 was detected in a Stellenbosch University https://scholar.sun.ac.za iii single Acinetobacter nosocomialis isolate, although recombinant mcr-4.3 did not confer colistin resistance in E. coli, plasmid curing of the mcr-4.3-containing plasmid restored colistin susceptibility. Colistin-resistant E. coli were isolated from the stools of two healthy children from the community (4%, 2/50) during 2017-2018; however, mcr genes were not detected. Colistin-resistant GNB, mainly Aeromonas spp., and mcr-5.1 and/or various mcr-3 variants were detected in the Plankenburg river, Eerste river, and Berg river and stormwater from Muizenberg and Fish Hoek in 2019 and 2020. Of the colistin-resistant Aeromonas spp. isolated from the Berg river, 25% (6/24) contained five novel mcr-3 variants, which were confirmed to confer colistin resistance. Conclusion The emergence of colistin resistance mechanisms in diverse strains obtained from hospital patients, with the limited gastrointestinal carriage of colistin-resistant Enterobacterales in community children and the disparate colistin-resistant species and mechanisms in the environment, suggest that selective pressure, and not community transmission, is the main driver of colistin resistance in clinical settings.
- ItemA phenotypic and genotypic characterisation of strain types, virulence factors and agr groups of colonising Staphylococcus aureus associated with bloodstream infection(Stellenbosch : Stellenbosch University, 2015-03) Karayem, Karayem; Hoek, Kim Gilberte Pauline; Whitelaw, Andrew Christopher; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical MicrobiologyENGLISH ABSTRACT : Several studies investigating the molecular characteristics of Staphylococcus aureus have been conducted worldwide, however, in South Africa, most of these have focused on Methicillinresistant S. aureus (MRSA). This study investigated the phenotypic and genotypic characteristics of isolates of S. aureus collected from the blood and nasal cavity of patients admitted to Tygerberg Hospital, South Africa. Investigations included determining the association between blood and nasal isolates, describing the molecular epidemiology of the population, determining the prevalence of various virulence factor genes among the different clones and descibing the accessory gene regulator (agr) functionality of S. aureus clones. Pulsed-field gel electrophoresis (PFGE), performed on 208 blood and nasal isolates from 162 patients with S. aureus bacteraemia, showed that 93 (57.4%) of the patients were colonised with the same strain type (p =0.061). MRSA was significantly associated with endogenous bacteraemia (same strain obtained from the blood and the nose) (p = 0.042). Molecular typing of the 208 blood and nasal isolates (43.3% MRSA) revealed that the majority of strains were ST239-t37-agr I (25.5%) which harboured different SCCmec types including SCCmec type III and a potentially novel type presumed to consist of ccrC/Class A mec. ST612-MRSA-IV was the second most predominant clone (10.2%). Other MRSA clones included ST5-t045 with a potentially novel variant of SCCmec type I consisting of ccrA1B1 and a ccrC/Class B mec; and ST461-MRSA-IV, reported for the first time in South Africa. All 18 (8.7%) pvl-positive isolates were MSSA except one isolate (ST612-MRSA-IV). The identification of novel MRSA clones (ST641-MRSA-IV), MSSA STs (ST2122, ST2126), and the potentially novel SCCmec type and type I variant suggest the local emergence of new clones. Twenty-one isolates (representing nine clonal complexes (CCs)) previously characterised by Multi-locus Sequence Typing (MLST) were analysed for the prevalence of 38 virulence factor genes. There was an association between different enterotoxin gene cluster (egc) gene combinations and CC5, CC22, CC30, and CC45. Both CC15 and CC97 were negative for Superantigen (SAg) genes. The intracellular adhesion locus A (icaA) gene was common (90.4%) and detected in all CCs (except CC30) and the enterotoxin I (sei) gene was significantly more widespread in MRSA isolates (77.8% in MRSA; 25.0% MSSA; p = 0.03). Accessory gene regulator dysfunction was significantly higher amongst MRSA than MSSA isolates and was more commonly associated with ST36-MRSA-II, ST239-MRSA-III and ST239-MRSA- ccrC/Class A mec. Shifting of agr in the same host was not common. Key findings in this study relate to the likely emergence of populations at Tygerberg Hospital, as evidenced by novel STs and potentially novel SCCmec types. The identification of a circulating clone within the burns unit both illustrates the potential for organisms to spread within the hospital, as well as reinforcing the value of molecular typing for infection control purposes. The association of different agr types, agr functionality, and virulence factors with typing data has shown results consistent with other studies, as well as some unusual results. However, the clinical relevance of these associations is not yet well understood, and should form the basis of further research.
- ItemSarcomeric modifiers of hypertrophy in hypertrophic cardiomyopathy (HCM)(Stellenbosch : Stellenbosch University, 2013-03) Bloem, Liezl Margaretha; Moolman-Smook, J. C.; Van der Merwe, L.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is an independent predictor of cardiovascular morbidity and allcause mortality. Significantly, it is considered a modifiable cardiovascular risk factor as its regression increases overall survival and reduces the frequency of adverse cardiac events. A clear understanding of LVH pathogenesis is thus imperative to facilitate improved risk stratification and therapeutic intervention. Hypertrophic cardiomyopathy (HCM), an inherited cardiac disorder, is a model disease for elucidating the molecular mechanisms underlying LVH development. LVH, in the absence of increased external loading conditions, is its quintessential clinical feature, resulting from mutations in genes encoding sarcomeric proteins. The LVH phenotype in HCM exhibits marked variability even amongst family members who carry the same disease-causing mutation. Phenotypic expression is thus determined by the causal mutation and additional determinants including the environment, epigenetics and modifier genes. Thus far, factors investigated as potential hypertrophy modifiers in HCM have been relatively removed from the primary stimulus for LVH; and the few studies that have been replicated yielded inconsistent results. We hypothesized that the factors that closely interact with the primary stimulus of faulty sarcomeric functioning, have a greater capacity to modulate it, and ultimately the LVH phenotype in HCM. Plausible candidate modifiers would include factors relating to the structure or function of the sarcomere, including known HCM-causal genes; and the enzymes that function in sarcomere-based energetics. Indeed, the literature highlights the relevance of sarcomeric proteins, Ca2+-handling and myocardial energetics in the development of LVH in HCM. This study, therefore, set out to evaluate the hypertrophy-modifying capacity of such factors by means of family-based genetic association testing in 27 South African HCM families in which one of three unique HCM-causing founder mutations segregates. Moreover, the single and combined effects of 76 variants within 26 candidate genes encoding sarcomeric or sarcomere-associated proteins were investigated. The study identified a modifying role in the development of hypertrophy in HCM for each of the candidate genes investigated with the exception of the metabolic protein-encoding gene, PRKAG1. More specifically, single variant association analyses identified a modifying role for variants within the genes MYH7, TPM1 and MYL2, which encode proteins of the sarcomere, as well as the genes CPT1B, CKM, ALDOA and PRKAB2, which encode metabolic proteins. Haplotype-based association analyses identified combined modifying effects for variants within the genes ACTC, TPM1, MYL2, MYL3 and MYBPC3, which encode proteins of the sarcomere, as well as the genes CD36, PDK4, CKM, PFKM, PPARA, PPARG, PGC1A, PRKAA2, PRKAG2 and PRKAG3, which encode metabolic proteins. Moreover, a number of variants and haplotypes showed statistically significant differences in effect amongst the three HCM founder mutation groups. The HCM-modifier genes identified were prioritised for future studies according to the number of significant results obtained for the four tests of association performed. The genes TPM1 and MYBPC3, which encode sarcomeric proteins, as well as the genes PFKM and PRKAG2, which encode metabolic proteins, were identified as stronger candidates for future studies as they delivered multiple significant results for various statistical tests. This study makes a novel contribution to the field of hypertrophy research as it tested the hypothesis that structural or energy-related factors located within the sarcomere may act as modifiers of cardiac hypertrophy in HCM, and succeeded in identifying a modifying role for many of the candidate genes selected. The significant results include substantial single and within-genecontext variant effects; and identified sizeable variation in the risk of developing LVH owing to the compound effect of the modifier and the individual founder mutations. Collectively, these findings enhance the current understanding of genotype/phenotype correlations and may, as consequence, improve patient risk stratification and choice of treatment. Moreover, these findings emphasize the potential for modulation of disease by further elucidation of some of the avenues identified.