3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Factors associated with World Health Organisation (WHO) clinical staging and related characteristics in HIV positive patients: an application of multistate, missing data and modelling techniques(2019) Batsirai, MurapaBackground Human Immunodeficiency Virus (HIV) remains a significant problem in sub-Saharan Africa which has the highest number (25.6 million) of people living with HIV (PLHIV). South Africa is amongst the top rank of sub-Saharan Africa countries with the highest HIV prevalence. Many studies have been done to have an in-depth understanding of HIVandAIDSdiseaseresultinginvariousinterventionsbeingimplementedtoimprove the lives of people infected by the disease. These studies are usually done using longitudinaldesignswhichhavetheadvantageofenablingresearcherstoobservepatient changes (outcomes) over time; however, they are prone to missingness due to unobserved data as patients may miss scheduled visits. This study aims to determine transition probabilities between WHO stages I, II, III and IV over time and compare Rubin’s and Bayesian methods in determine factors associated with WHO stage ailments and symptomatic conditions amongst HIV infected patients on patient level data from the Adult Wellness study. Methods The researcher conducted a secondary data analysis of the Adult Wellness study which was conducted from 2002 to 2010, to be able to quantify changes in ailments and symptomatic conditions over time, the researcher fitted the general multi-state Markov model which assumes that patients may develop more severe ailments and symptomaticconditions. ThestatesweredefinedbasedonWHOstages,thatis,stage I, II ,III and IV. The researcher also fitted three random effects ordered logistic regression models to determine factors associated with these WHO stage outcomes. The researcher employed the maximum likelihood estimation (MLE) on the first model fittedonrawdataandsecondmodelaftermultipleimputation(MI)toaccountformissing data. The last model adopted Bayesian estimation (BE) to the raw data. Finally, the researcherperformedasensitivityanalysisusingsimulateddataandfittedallthethree models described earlier. Results A total of 2,609 patients accounted for 12,102 observations were analysed. Majority of the patients were females (77.4%) antiretroviral therapy ART naïve (61.5%) having attained Grade 0-12 (77.9%). The Markov multi-state model showed that patients in WHO stage II were 1.33 times more likely to move to WHO stage III than WHO stage I whilst patients in WHO stage III were 2.26 times (0.16118/0.07124) more likely to move to WHO stage II than progressing to WHO stage IV. The probability of remaining in WHO stage I was 59% after eight year follow up period. Relative to patients with no ailments and symptomatic conditions, patients with one HIV ailments or symptomatic condition has an INCREASED RISK of progressing to advanced WHO stages, (model with raw data OR 2.07, 95%CI: 1.23-3.30). There were some unexpected results were patients on ART and cotrimoxazole (CTX) drugs showed to have increased odds of becoming worse than their counterparts. This was evident in all the three models: raw data MLE model OR 1.5828 (95% CI 1.1948,2.0969) and OR 2.0670 (1.5619,2.7355); MI MLE model OR 1.2252 (95% CI 1.0884,1.3793) and OR 1.5438 (95% CI 1.3186,1.8074); and raw data BE model OR 1.6096 (95% CI 1.2055,2.0952) and OR 2.0758 (1.5507,2.7270) for ART and CTX respectively. Both MLE and BE for the raw data gave similar estimates; however, these estimates were different from the MI MLE model which were more precise (smaller standard errors). Conclusions The results showed that patients had an increased chance of remaining in the same state than either advancing in WHO stages of ailments and symptomatic conditions or recovering. If the level of missing data is reasonable, it is recommended to apply the MI techniques. Multiple imputations and Bayesian missing data methods should be used together and determine which one produce better results per each situation. Finally simulated results showed that multiple imputation and Bayesian models become different as the percentage of missing data increasesItem Associations between genetic variation in the T-cell signalling pathways and CD4+ T-cell count recovery after ART initiation in southern African cohort(2018) Minnaar, DevinBackground: Infection by the Human Immunodeficiency Virus (HIV) can be treated by means of anti-retroviral treatment (ART), but a substantial percentage of treated individuals experienced failure to reconstitute their CD4+ T-cells to normal levels (immunological failure). The aims of this study were to determine associations between genetic variation in six candidate genes and CD4+ T-cell recovery following two years of ART in a southern African cohort. of the six candidate genes, two play roles in T cell co-activation (CD28 and ICOS), three play roles in T cell inhibition (PDCD1, PD-L1 and CTLA4) and IL7-Rα affects T cell homeostasis. We also analysed association between the same genetic variants and four plasma markers: sCD27, a marker of T cell activation; sPD-1, a marker of chronic T cell activation and/or exhaustion; sCD163, a marker of macrophage type 2 activation; lastly sCD127, a soluble form of IL7-Rα. Methods: DNA from the WRHI001 cohort was analysed with permission from the Wits HREC (Human Research Ethics Committee). We identified the demographic and clinical factors influencing CD4+ T-cell counts and these were used as covariates during multivariate analysis of associations between single nucleotide polymorphisms (SNPs) and CD4+ T-cell count. A total of 28 SNPs in CD28, CTLA4, ICOS, PDCD1, PDL1 and IL7-Rα were successfully genotyped in 262 samples using Sequenom MassARRAY. Levels of sCD27, sCD163, sPD-1 and sIL7-Rα were measured by ELISA or Luminex techniques in 76 individuals from plasma samples at two years post-ART initiation. Demographic and clinical factors influencing plasma marker levels were identified and used as covariates during multivariate analysis of associations between SNPs and plasma markers, Plink v1.07 software was used to analyse all genetic associations. Results: We found that 9% of the total cohort experienced immunological failure defined as failure to reconstitute CD4+ T-cell count >200 cells/mm3, two years after ART initiation We found that male gender, younger age, use of tenofovir, higher weight and increased CD4+ T-cell count at baseline were significantly associated with higher CD4+ T-cell count after two years of ART. Of the 28 SNPs examined three were monomorphic and one SNP, rs11568821, had a minor allele frequency (MAF) = 0.006. Of the remaining 24 SNPs, eight were significantly different in frequency from LWK (Luhya in Webuye, Kenya) frequency. Analysis of linkage disequilibrium (LD) patterns of genes on chromosome 2 (CD28, CTLA4, ICOS and PDCD1) showed strong linkages (most D’=1) within genes but not across genes on chromosome 2 despite the short distances between them. SNPs in IL7-Rα were all in strong linkage. Weak linkage was found between PDL1 SNPs. Significant associations were observed between genetic variants in the T cell co-stimulation genes ICOS and CD28, and CD4+ T-cell recovery after ART initiation. However different SNPs in ICOS were implicated in the univariate vs. multivariate analyses. During univariate analysis, ICOS rs6761201 showed significant associations with CD4+ T-cell counts in allelic, recessive, genotypic and haplotypic models, whereas in multivariate analyses, ICOS rs10183087, rs1559931 and rs4404254 showed significant associations with CD4+ T-cell counts in dominant and haplotypic models. The SNP rs3181098 in CD28 that was significantly associated with CD4+ T-cell recovery in univariate analysis was not significant during multivariate analysis. One SNP rs10815225, in PD-L1, was associated with CD4+ T-cell counts in univariate analysis only. One SNP in the homeostasis IL7-Rα gene, rs11567705, was associated with CD4+ T-cell counts in multivariate analysis only. Two of the four soluble plasma markers tested had significant associations with CD4+ T-cell counts: significantly decreased sPD-1, and higher sCD163 were detected in individuals with poor CD4+ T-cell recovery. Associations were examined between variation in the six candidate genes and the four soluble markers. The same genetic variants rs6761201 in ICOS (rs6761201) and rs3181098 in CD28 that were significantly associated with CD4+ T-cell recovery in the larger cohort (n=262), were associated with sCD27 levels, a marker of T cell activation, in the cohort subset (n=69). However we could not demonstrate significantly higher T cell activation levels in those with lower CD4+ T-cell counts in the cohort subset. In both univariate and multivariate analyses, genetic variation in the IL7-Rα gene (rs6897932) was significantly associated with sIL7Rα levels, confirming previous literature in this regard. Conclusion: Genetic variation in the ICOS, CD28, PD-L1 and IL7-Rα genes influence CD4+ Tcell counts recovery after ART in a southern African cohort. The role of these SNPs in T cell activation and CD4+ T-cell recovery during ART use should be examined using further immunological methods. SNP rs6897932 in the IL7-Rα gene was confirmed to influence sIL7-Rα plasma levels. Our observation of higher sCD163 in those with poor CD4+ T-cell recovery suggest possibly higher innate (especially macrophage type 2) activation in those with poor CD4+ T-cell recovery. The genetic variants studied here do not directly relate to innate activation and other SNPs should be studied to examine this hypothesis.Item Avoiding Truth in a Time of Knowledge: Male Youth Refusing to Test for HIV(2018) Baloyi, T EMales in KZN come to health facilities for circumcision but refuse HIV testing as a condition. As a result, they end up not circumcising. Of the population that does not want to test, male youth comprise the majority percentage. Many studies have been conducted to outline the factors leading to this minimal HCT uptake. To add, interventions have been put in place to address these factors. HIV/AIDS information, treatment and support services are available to all people, yet most still avoid testing. This is because there is a gap between the interventions to address the socio-structural factors causing the low uptake of HCT and the intended beneficiaries. This study suggests how ‘deliberate ignorance’ can clarify the gap between the socio-structural explanations of HCT avoidance and thus bridge the existing gap. This study uses the concept of deliberate ignorance to investigate the avoidance to test for HIV by young males in the Township of Naledi. The study acknowledges research on the socio-structural factors that account for avoidance to test for HIV. A qualitative approach is used to purposively select 15 male youth participants aged 18-35 years residing in Naledi. Data is thematically analyzed and reveals that even though the young males had biomedical knowledge about HIV, they still deliberately avoided testing for varied reasons that include, the lack of cure, mischievous behavior, fear of a positive diagnosis, and not thinking about testing. Based on the findings, the report argues that deliberate ignorance clarifies the socio-structural factors responsible for low HCT uptake in the sense that it taps into the individual psychological understanding of HCT. Deliberate ignorance, then bridges the gap between general HCT socio-structural interventions and the intended individual beneficiaries on the ground.Item Acquired immune deficiency syndrome: its impact on gay male lifestyles(1993) Cave, H. AnthonyResearch has revealed that many gay men continue to participate in high-risk sexual practice them at risk of expoasure to the AIDS viirus. The locus of control construct and the Health Belief Model were employed by this study in an attempt to identify those psychosocial factors which might influence gay men to adopt or neglect health protective behaviour.[Abbreviated Abstract. Open document to view full version].Item HIV-1 subtype C proteases: overexpression, structural, kinetic and thermodynamic characterisation(2016-05-10) Tomescu, Mihai-SilviuAccording to UNAIDS, there are ~36.9 million people infected with HIV-1 in the world. Of those, 25.8 million live in sub-Saharan Africa and 6.8 million in South Africa. HIV-1 subtype C accounts for over 95% of HIV infections in South Africa. HIV-1 retrovirus acquires mutations rapidly because of the viral reverse transcriptase. Naturally occurring polymorphisms distinguishing wild type C-SA PR from other proteases make it less susceptible to inhibitors. E35D↑G↑S is a C-SA PR variant with a double insertion in the flap region of the protease. The insertions and background mutations may decrease susceptibility to inhibitors as well as alter kinetic parameters due to increased flap flexibility. This study intended to characterise the effect of the mutations and insertions in E35D↑G↑S on structural, kinetic activity and drug susceptibility. Chemically-synthesised E35D↑G↑S autocatalyses rapidly, impeding further characterisation. There was no detectable overexpression of the E35D↑G↑S protease in Escherichia coli BL21 (DE3)pLysS and Rosetta 2® cells. If the protease is catalytically enhanced, attributed cytotoxicity may prevent overexpression of the protein. Increased autocatalytic activity could also prevent crystallisation. Inactive E35D↑G↑S D25A did not overexpress either, indicating that codon harmonisation with the expression host ought to be performed. C-SA PR was shown to be a predominantly beta-sheeted protein using circular dichroism spectroscopy. The KM of the fluorogenic substrate resembling the capsid/ p2 cleavage site for C-SA PR was 22.02 ±4.09 μM. The specific activity, catalytic turnover and catalytic efficiency of the wild-type C-SA PR protease were found to be 35.68 ±1.06 μmole.min-1.mg-1, 12.79 ±0.38 s-1 and 1.17 ±0.055 s-1.μM-1, respectively. The thermodynamics of binding of atazanavir, ritonavir and darunavir to C-SA PR were determined using isothermal titration calorimetry. The binding of atazanavir and ritonavir to C-SA PR is entropically driven and enthalpically opposed. However, the binding of darunavir to C-SA PR was found to be both entropically and enthalpically favourable. The dissociation constants of the inhibitors in the absence of substrate (Kd) are in the pico-molar range and increased by approximately one order of magnitude when saturating concentrations of substrate were introduced. Atazanavir, ritonavir and darunavir have dissociation constants (Kd) of 160.56 ±54.59 pM, 113.34 ±46.47 pM and 10.24 ±6.02 pM, respectively. Darunavir binds significantly tighter. Keywords: C-SA PR, E35D↑G↑S, insertion mutations, protease, autocatalysis, ITC.