3. Electronic Theses and Dissertations (ETDs) - All submissions

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    Cross-resistance among rifamycins in mycobacterium tuberculosis clinical isolates
    (2019) Mojapelo, Richard Maredi
    Introduction: Rifamycins (RFMs) are a group of antimycobacterial drugs that belong to the large family of ansamycin. RFMs inhibit Mycobacterial growth by blocking the RNA polymerase subunit B (rpoB). High level cross-resistance among RFMs in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates is commonly inferred. However, previous studies reported that the minimum inhibitory concentrations (MICs) of rifabutin (RFB) among rifampicin (RIF)-resistant M. tuberculosis carrying rpoB mutations varies depending on the mutation position. Objective: -To determine the proportion of cross-resistance among rifamycins and to assess the use of the GenoType MTBDRplus Version 2.0 assay in predicting differential susceptibility to rifamycins in M. tuberculosis isolates. Method: -A total of 300 unique baseline isolates which were collected between June 2015-April 2016 for routine laboratory based surveillance of RIF drug resistance in selected districts of South Africa were included. Drug susceptibility testing (DST) for RIF (1.0 μg/ml), RFB (0.5 μg/ml) and rifapentine (RFP) (0.5 μg/ml) was performed by the MGIT 960 system using World Health Organisation (WHO) recommended critical concentration (c.c). The MycoTB plate was used to determine MICs for RIF and RFB. To determine rpoB mutations, all the isolates were tested by Genotype MTBDRplus version 2.0 assay method and undefined isolates were sent for Sanger sequencing. Results: -The proportion of cross resistance among RFMs were: across all three (216/300;72%), between RIF and RFB (217/300;72%) and RIF and RFP (292/300;98%). The S531L mutation was the mostly associated with cross resistance to all RFMs (144/153;94%), while the D516V mutation was associated with differential susceptibility to RFB (50/52;96%). Conclusion: -The results show high levels of cross resistance across all rifamycins, however 28% of MDR/XDR-TB cases could potentially benefit from RFB as a substitute drug to the failing RIF. These findings provided additional evidence of the strong association of specific rpoB mutations with the development of RFMs cross and differential susceptibility. The use of LPA and rpoB mutations specifically S531L and D516V can be beneficial in rapidly differentiating phenotypic differential susceptibility to RFB according to this study.
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    The design and synthesis of antituberculosis peptidomimetics focusing on lassomycin derivatives
    (2019) Ngqinayo, Ntombizanele
    Tuberculosis (Tb) is a disease ranked among the top ten causes of death worldwide and is responsible for infecting around 10 million people each year. Tb is caused by the Mycobacterium Tuberculosis (M. tb) bacterial pathogen. The mycobacterium has become resistant towards currently approved drugs which mostly target the cell wall and this has led to the development of the multidrug resistant (MDR) and extremely drug resistant (XDR) M. tb strains. The resistant strains are difficult to treat and require longer treatment duration with the use of combinatory drugs that result in a number of serious side effects. These limitations have led to the search for novel anti-Tb agents and the discovery of lassomycin, an antimicrobial peptide (AMP) that utilizes a different mode of action. The peptide targets the caseinolytic protease of M. tb which is essential for cell survival and causes uncontrolled protein unfolding which results in cell death. Lassomycin is a 16amino acid long basic peptide isolated from a soil bacteria, Lentzea kentuckyensis sp. that been found to be highly selective and potent towards M. tb without affecting mammalian cells.2 The objectives of this project are (i) to modify lassomycin into drug-like derivatives by incorporating N-methylated amino acids to make the peptide more stable against enzymatic degradation; (ii) to shorten the synthetic route by replacing the lactam bridge with a disulfide bridge; (iii) to replace the arginine amino acids in the peptide sequence (difficult to couple) with lysine amino acids to investigate the role of arginine in the binding of the peptide to the acidic region of the caseinolytic enzyme; and (iv) to make the peptide more cationic to improve selectivity for the negatively charged bacterial membrane by adding lysine residues. Peptides were synthesized via the fmoc solid phase peptide synthesis strategy; purified using a semi-preparative High Performance Liquid Chromatogram (prep-HPLC) and analyzed using High Performance Liquid Chromatography Mass Spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The bactericidal activity of selected lassomycin derivatives against M. tb was determined using the Alarmar blue assay and one of the derivatives showed a bactericidal effect at a concentration of 9.87 µg/ml which is comparable to that of ethambutol. The derivatives were also found to be selective for pathogens that share a similar protease to that of the M. tb such as Bacillus Subtilis (B. subtilis) and inactive against other pathogens that do not contain the protease. The 3-dimensional structure of the active derivatives will be determined in the future using NMR spectroscopy.
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    Evaluation of new diagnostic methodology for the detection of second-line drug resistance in Mycobacterium tubercolosis clinical isolates
    (2018) Gardee, Yasmin
    The World Health Organization (WHO) Global Report, 2017, confirms that tuberculosis (TB) is the primary cause of death globally caused by an infectious agent. Drug-resistant TB (DR-TB) is reported by the WHO to affect at least 600 000 people globally and is the primary obstacle in the fight against the elimination of TB disease. The prompt identification and treatment of DR-TB is essential. Conventional culturebased diagnostic tests for TB require substantial laboratory capacity and result availability can take up to 3 - 4 months. The implementation of rapid molecular diagnostic tests such as line probe assays (LPA) and the Xpert MTB/RIF® (Cepheid, USA) have led to an improvement in reporting turnaround times. GenoType® MTBDRsl VER 2.0 LPA is designed for molecular detection of second-line drug resistance-conferring mutations in genes encoding resistance to fluoroquinolones (FLQ) (gyrA and gyrB) and second-line injectable drugs (SLID) (rrs and eis). This study evaluated the diagnostic performance of the Genotype® MTBDRsl VER 2.0 compared to phenotypic drug susceptibility testing (DST) as the gold standard, on clinical samples and Mycobacterium tuberculosis complex clinical isolates from South Africa. The performance indices (sensitivity and specificity estimates) for the assay when tested on clinical samples were as follows respectively: FLQ 77.2% (95% CI, 67.2% - 85.3%); 85.4% (95% CI, 79.0% - 90.5%) and SLID 81.9% (95% CI, 75.5% - 87.2%), 91.5% (95% CI, 86.6% - 95.5%). For clinical isolates the performance indices were as follows respectively: FLQ 100% (95% CI, 95.8% - 100%); 98.9% (95% CI, 96.1% - 99.9%) and SLID 89.2% (95% CI, 79.1% - 95.6%); 98.5% (95% CI, 95.7% - 99.7). The incorporation of the assay into the TB diagnostic algorithm will improve South Africa’s TB Control Program in prompt identification and appropriate treatment of drugresistant TB cases.
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    Antibodies to mycobacterium tuberculosis mycolic acids in patients with pulmonary tuberculosis
    (2001-09-11) Schleicher., Gunter, Klaus.
    Introduction and Aim: The waxy outer cell wall of mycobacteria consists mainly of mycolic acids (MA). The unique immuno-stimulatory properties of MA via the CD 1-restricted antigen presentation pathway have been demonstrated in humans. Purification and isolation of M.tuberculosis (MTB) MA has allowed them to be applied as an antigen in an ELISA-based sero-diagnostic assay to detect specific antibodies in the sera of humans. The aim of the study was to measure the levels of antibody to MA in the sera of patients with culture proven pulmonary tuberculosis (PTB), and in control subjects without evidence of tuberculosis.
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    Autoantibodies in pulmonary tuberculosis and leprosy in black South Africans
    (1988-12-01) Rapoport, B. L.
    Infections can cause autoantibody production. The purpose of this study was to determine the prevalence of autoantibodies in chronic mycobacterial infections in Johannesburg. Sera from 41 leprosy patients and from 49 untreated and 73 treated tuberculosis patients were tested for rheumatoid factor, antibodies against a panel of nuclear antigens, anticardiolipin antibodies and syphilis serology. The antinuclear antibody was positive in 7.3% of the leprosy group, 6.1% of the untreated TB group and 15% of the treated tuberculosis patients (p=0.0125). Antinuclear antibody positivity correlated with duration of treatment (p=0.025). The antinuclear antibody titres were low and there was no specific pattern.
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    The value of lateral chest X-rays for the diagnosis of lymphadenopathy in children with pulmonary tuberculosis
    (2018) Poyiadji, Thalia Leto
    INTRODUCTION: Tuberculosis (TB) is an important public health issue, but diagnosis in children can be challenging. The radiological hallmark of pulmonary TB (PTB) in children is mediastinal lymphadenopathy, however there is inter-observer variability in detecting this. The value of the lateral CXR in addition to the frontal view to detect lymphadenopathy has not been well studied. OBJECTIVES: To investigate the prevalence of lymphadenopathy in children with confirmed PTB detected on frontal compared to frontal-lateral CXRs. METHODS: This was a secondary analysis of a study from Red Cross Children’s Hospital in Cape Town. Children with definite TB and a control group (Lower respiratory tract infection other than TB) who had frontal and lateral CXRs were included in this study. Three radiologists independently read the CXRs in 2 separate sittings (frontal CXR and ‘combination frontallateral’ CXR). A 3 reader consensus reading was used during data analysis. Odds ratios and 95% confidence intervals were calculated to determine the presence of lymphadenopathy. Kappa statistics were calculated to determine inter reader agreement. RESULTS: Of 172 children (88 confirmed TB and 84 control children), with a median age of 29 months, lymphadenopathy was reported in 86 (50%) patients on the frontal CXR alone and in 143 (83%) on the frontal-lateral CXR combination, p= 0.00. Amongst confirmed PTB cases, 52 (60%) had lymphadenopathy on the frontal CXR alone while 72 (82%) had lymphadenopathy on the frontal-lateral CXR combination, p= 0.00. Amongst the control group, 34 (40%) had lymphadenopathy on the frontal CXR alone while 71 (85%) had lymphadenopathy on the frontal-lateral CXR combination, p= 0.00. The consensus reading using a frontal-lateral CXR combination resulted in a 5 fold increase (OR 4,9; 95% CI 2,9-8,4) in diagnosis of lymphadenopathy compared to a frontal CXR only. Overall inter reader agreement for all 3 readers was fair on both the frontal CXR (Kappa= 0,21) and the frontal-lateral CXR (Kappa= 0,23) combination. CONCLUSION: The addition of a lateral view to the frontal CXR increased detection of lymphadenopathy, however, the prevalence of lymphadenopathy was similar in children with PTB and those in the control group, with fair inter reader agreement.
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    Comparison of Chest X-ray findings in ambulatory and hospitalised children with pulmonary TB
    (2018) Baker, Gregory
    INTRODUCTION: Pulmonary TB is common in South Africa, with many children affected. Diagnosis can be challenging and chest x-ray remains fundamental for diagnosis. Interpretation is difficult and shown to have wide inter-reader variability. No study however has compared CXR findings and inter-reader agreement between ambulatory and hospitalised patients. AIM: This study compares the frequency of CXR changes, as well as inter-reader agreement in ambulatory compared to hospitalised children with suspected TB. METHOD: A pre-existing database containing CXR data of children worked up for PTB from 2008-2013 was used. Retrospective analysis of 69 ambulatory and 112 hospitalised patients, aged 0-12 years from Nolungile clinic and Red Cross Children’s Hospital respectively was done. Each sample contained 50% proven TB and 50% negative controls. Two paediatric radiologists and one paediatrician served as blinded, independent readers for the database using standardised tick sheets. RESULTS: Finding frequency; Fleiss/Free marginal Kappa (ambulatory and hospitalised respectively): Overall TB: 27.5% and 35.7%; -0.07 and 0.12 /-0.06 and 0.12 Parenchymal change: 34.8% and 67.9%; 0.24 and 0.49/0.25 and 0.60 Lymphadenopathy: 24.6% and 33.9%; 0.01 and 0.13/0.01 and 0.13 Pleural effusion: 7.3% and 18.8%; 0.27 and 0.61/0.84 and 0.80 Airway compression: 11.6 and 17.9%; 0.26 and 0.20/0.73 and 0.52 CONCLUSIONS: Our study demonstrated no significant difference in lymphadenopathy, but an increase in parenchymal change in the hospitalised group. We otherwise showed comparable results to literature regarding finding frequency, but poor inter-observer agreement. If the least expert reader were removed, results were comparable with available literature. This highlights the need for development and study of explicit CXR criteria for lymphadenopathy to improve the value of CXR for paediatric TB in all settings.
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    Tobacco smoking as a potential risk factor for pulmonary tubercolosis A meta-analysis
    (2001-12-29) Chipeta, John, Benson.
    Objective. The aim of this paper was to systematically evaluate available evidence on tobacco smoking as a risk factor for pulmonary tuberculosis. Methods. Relevant reports were identified by a systematic electronic search of Medline, Pubmed, Nioshtic, Toxline and Embasse. Methodological quality of all selected publications was assessed using a standardized checklist. Information was collected on all major study characteristics. Inter-study heterogeneity was examined qualitatively and statistically using the DerSimonian and Laird method. Results. Five case-control studies and 1 cohort study were included in the systematic review. All the 6 studies revealed a relationship between tobacco smoking and pulmonary tuberculosis. Heterogeneity across studies hampered overall statistical pooling of results, however pooled risk ratios for sub-groups were determined
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