- 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.
- ItemThe relevance of apoptosis in the pathogenesis of human immunodeficiency virus-1 disease(Stellenbosch : Stellenbosch University, 2004-12) Cotton, Mark F.; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.ENGLISH ABSTRACT: A simple and rapid scatter-based flow cytometric assay was developed to detect apoptosis in CD4+ and CD8+ T cells from a mixed population of cells. The assay was suitable for children. Apoptotic PBMCs were confirmed by morphologic assessment in clinical samples ex vivo and after overnight culture. The scatter-based assay was validated in a number of ways. Firstly, PBMCs were irradiated with 500 rads and cultured overnight to induce apoptosis. Thereafter, PBMCs were labeled with a CD4 MAb. CD4+ cells were sorted into apoptotic and viable populations by scatter characteristics (diminished forward and increased side scatter). Morphology was assessed by fluorescence microscopy. The majority of cells with apoptotic scatter characteristics had apoptotic morphology (chromatin condensation) (80.6%). Ninety-two percent of cells from the viable region had normal morphology. CD4+ T cell apoptosis measured by scatter was then correlated with the TdT assay for DNA fragmentation. Lastly, CD4+ T cell apoptosis by scatter and annexin V uptake were also shown to correlate. In the latter experiments, PBMC morphology and cell death by trypan blue uptake were studied simultaneously and confirmed the two flow cytometric assays. Apoptosis of CD4+ and CD8+ T cells has been shown in PBMCs from HIV infected adults analyzed after overnight culture. Since cell death may be an artifact of in vitro culture, and because there is little information on apoptosis in paediatric HIV disease, I undertook a cross-sectional analysis in PBMCs analyzed immediately ex vivo from HIV infected children and adults. Patients were studied in Denver, CO, USA. PBMCs from 21 children, 4 adolescents and 9 adults and seronegative age-matched controls were stained for CD4 and CD8 surface markers. Apoptotic cells were detected in a newly characterized flow cytometric assay by diminished forward and increased side scatter. For the scatter assay, PBMCs had been labeled initially by an indirect method involving an intermediary incubation in the presence of biotinylated MAbs at 37°C for 30 minutes prior to incubating with streptavidin-FITC at 4°C for 20 minutes. Thereafter, the intermediary incubation step was removed and PBMCs were incubated with PE-conjugated CD4+ and CD8+ MAbs. Both CD4+ and CD8+ T cell apoptosis appeared enhanced in the indirect method. The significant differences were abolished after subtraction of data from simultaneously studied time-matched controls. CD4+ and CD8+ T cell apoptosis were significantly higher in HIV-infected study subjects than in simultaneously studied seronegative controls. PBMCs were assayed immediately ex vivo and after overnight culture after stimulation by an anti-TCR MAb as well as spontaneously. There was a direct correlation between CD4+ and CD8+ T cell apoptosis and CD4+ T cell depletion. A significant correlation was also shown between apoptosis immediately ex vivo and after overnight culture. I then studied apoptosis in a South African population comprising 18 symptomatic children and 4 seroreverters. CD4+ and CD8+ T cell apoptosis were significantly higher in symptomatic HIV-1-infected children than in seroreverters and seronegative controls. CD4+ T cell apoptosis correlated with depletion of CD4+ T cell percentage in symptomatic HIV-1-infected children. I also noted elevated CD4+ T cell apoptosis in patients recovering from intercurrent disease in comparison to those who were either acutely ill or relatively asymptomatic outpatient attendees. Lastly, I compared CD4+ and CD8+ T cell apoptosis in cohorts from Denver, CO and Tygerberg Children’s Hospital, South Africa. I selected only patients with moderate or severe HIV infection from both centers. South African patients were significantly younger, more malnourished, had higher gamma globulin levels and were less likely to receive ART. CD8+ T cell apoptosis was higher in North American patients suggesting a possible impairment in CD8+ activity in the South African study subjects.
- ItemDetermination of the permeability of biological membranes to various chemical markers, including anti-HIV drugs(Stellenbosch : University of Stellenbosch, 2009-12) Pretorius, Erina; Bouic, Patrick J. D.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds are produced by parallel synthesis and combinatorial chemistry. The pharmaceutical industry therefore requires rapid and accurate methods to screen new drugs leads for membrane permeability potential in the early stages of drug discovery. Around 50 % of all investigational new drugs fail in pre-clinical and clinical phases of development due to inadequate absorption/permeation, distribution, metabolism, excretion and/or unacceptable toxicity. This may be decreased by applying in vitro screening methods early in the discovery process. Reliable in vitro models can be applied to determine permeation of the test compounds, which will help avoid the wasting of valuable resources for the development of drugs that are destined to fail in preclinical and clinical phases due to insufficient permeability properties. It is important to decide as early as possible on the most promising compound and physical formulation for the intended route of administration. With awareness of the increasing importance of in vitro models in the investigations of the permeability properties of drug compounds, this research project was specifically devoted to determine the suitability of our in vitro model to evaluate and predict drug permeability. A continuous flow-through diffusion system was employed to evaluate the permeability of nine different compounds/drugs with different chemical properties, across three biological membranes. The biological membranes chosen for the present study were human vaginal mucosa, human skin tissue and human small intestine mucosa. The continuous flow-through diffusion system was furthermore utilised to investigate the effects of de-epithelialisation of mucosal surfaces, chemical enhancers, temperature, permeant concentration and formulation on the permeability of the test compounds/drugs. The in vitro permeability information and data from the flow-through diffusion model were compared to in vitro and in vivo literature studies and drug profile. An in vitro model that is able to reliably predict in vivo data will shorten the drug development period, economise resources and may potentially lead to improved product quality. In this thesis research results are reported on the permeability of the mentioned biological membranes to the various chemical markers, including anti-HIV (human immunodeficiency virus) drugs. The permeability studies will be discussed in three sections: vaginal mucosa, skin tissue, small intestine mucosa. The results of the vaginal permeability studies showed that the three peptides (MEA- 5, MDY-19 and PCI) readily penetrated the vaginal mucosa. MDY-19 had a higher flux rate than MEA-5, commensurate with its smaller molecular size (weight). The surfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreased the lag time of the peptide. Removal of the vaginal epithelium increased the flux rates of the peptides across the mucosa and may have implications for a more rapid uptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19 and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCs of the overall mean flux values of MDY-19 and PCI increased with concentration according to well-established diffusion theory. The experiments on the permeability of different terbinafine hydrochloride formulations through human skin demonstrated that the terbinafine hydrochloride formulations used in this study, readily diffused into the skin tissue. However, no flux values for any of the terbinafine hydrochloride formulations through the skin into the acceptor fluid were found. The mean terbinafine concentrations in the skin after 24 h exposure to the three commercial, terbinafine hydrochloride formulations were 3.589, 1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skin exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the three commercial formulations. The results of the temperature study demonstrated that an increase of 5 ºC caused a significant increase in flux values of tritiated water across skin. The flux values for tritiated water across skin at 37 ºC were on average double those at a temperature of 32 ºC. The permeability of excised human small intestine mucosa to different oral dosage drugs was investigated over a 24 h period. The four drugs selected were zidovudine, propranolol hydrochloride, didanosine and enalapril maleate. They were selected as representative model compounds of drug classes 1 (high solubility, high permeability) and 3 (high solubility, low permeability) according to the Biopharmaceutics Classification System. The flux rates of the four chosen test drugs were influenced by the length of the experiment. Between the time periods 2-4 h and 4-6 h, zidovudine’s mean flux values across small intestine tissue were respectively 1.8 and 2.0 times higher than didanosine and 2.3 and 2.2 times higher than enalapril. Propranolol’s mean flux values were respectively 1.2 and 1.4 times higher than didanosine and 1.6 higher than enalapril during both the 2-4 and 4-6 h time periods. Between both the time periods 2-4 and 4-6 h AZT’s mean flux values were 1.4 times higher than propranolol and didanosine’s mean flux values were respectively 1.3 and 1.1 times higher than enalapril during the mentioned time periods. Class 1 drugs showed a significantly higher flux rate across the jejunal mucosa compared to the class 3 drugs and these results are in line with their Biopharmaceutics Classification System classification. The in vitro model has proved to be reliable to predict permeability of class 1 and 3 drugs and also showed correlation with human in vivo data. It seems that the in vitro flow-through diffusion model used in the present study have the potential to overcome some of the problems and limitations demonstrated by other in vitro techniques and may potentially serve as a future tool for pharmaceutical companies to predict the diffusion characteristics of new drugs and different formulations, across different biological membranes. Furthermore, it may serve as a prospective method for assessing the bioequivalence of alternative (generic) vehicles or formulations containing the same drug/compound.
- ItemGenetic investigations of pneumocystis jirovecii : detection, cotrimoxazole resistance and population structure(Stellenbosch : University of Stellenbosch, 2005-12) Robberts, Frans Jacob Lourens; Liebowitz, L. D.; Chalkley, L. J.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.Pneumocystis jirovecii is a significant contributor to the burden of disease in immunocompromised patients. The polymerase chain reaction (PCR) is more sensitive and specific than microscopy. Cotrimoxazole prophylactic breakthrough and treatment failures have been reported, and associated with mutations at codons 55 and 57 of P. jirovecii dihydropteroate synthase (DHPS). No phylogenetic or population genetic models have been successful in elucidating P. jirovecii intraspecies strain relatedness. Aims: 1) Compare detection rates of nine PCR techniques and immunofluorescence microscopy (IF); 2) Determine the extent of co-infecting pathogens associated with Pneumocystis Pneumonia (PcP); 3) Determine local P. jirovecii ITS1-5.8S-ITS2 rDNA strain types, and model lineage evolution employing a coalescent-theory based statistical parsimony network analysis; 4) Investigate the possible emergence of cotrimoxazole-resistant strains Methods: PCR was evaluated on clinical specimens employing: ITS nested; DHPS single and nested; DHFR nested; major surface glycoprotein (MSG) heminested; mitochondrial large subunit rRNA (mtLSUrRNA) single and nested; 18S rRNA onetube nested, and real-time 5S rRNA PCR. Retrospective analysis of co-infecting pathogens seen in PcP patients was conducted. ITS regions were amplified, cloned and sequenced. Statistical parsimony was applied for coalescence based network genotype analysis. DHPS genome walking was attempted and DHPS and DHFR primer annealing sites explored. Amplified DHPS and DHFR genes were cloned and sequenced. Results: Most sensitive PCR technique was mtLSUrRNA nested followed by 5S realtime PCR. A poor correlation exist between mtLSUrRNA PCR and IF. Review of clinical records suggested a high rate of false-positive IF results. P. jirovecii was detected in 4.3% M. tuberculosis-positive HIV-positive, and 2.5% M. tuberculosispositive HIV-negative patients. P. jirovecii was detected in 45% HIV-negative patients. The most prevalent ITS type was Eg. Four new combinations: Eo, Je, Ge, No; 11 new ITS1 and 13 new ITS2 sequences were identified. A new ITS2 type was detected in three patients and designated u. More than one strain type was detected in 15/19 patients. Analysis of 5.8SrDNA region revealed 13 clones containing 1-2 nucleotide polymorphisms. Of 85 mtLSUrRNA PCR-positive specimens, currently employed primers amplified DHPS and DHFR genes from 53 and 27 specimens, respectively. Newly designed DHPS primers increased detection in 3 / 28 previously DHPS-negative mtLSUrRNA-positive specimens. Of 56 DHPS genes amplified and sequenced, one contained the double mutation (Thr55Aa; Pro57Ser). DHFR Ala67Val was detected in three specimens and a new DHFR genotype (Arg59Gly; C278T) was demonstrated. Conclusions: The study emphasises the need to evaluate PCR primers against local strains. It is recommended that mtLSUrRNA PCR be performed in parallel to IF and discordant results resolved with clinical evaluation. Co-infection with P. jirovecii and M. tuberculosis occurs in South Africa, and treatment for both pathogens is recommended when demonstrated by the laboratory. ITS genotyping employing statistical parsimony network analysis suggests type Eg as major ancestral haplotype, and supports recombination contributing to strain diversity worldwide. DHPS mutations may signal emergence of resistance to cotrimoxazole in South Africa, however, low sensitivity of primers limits surveillance efforts.