Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Drug resistance in Mycobacterium tuberculosis is an increasing global problem. Drug resistance is mostly caused by single nucleotide polymorphisms (SNPs) within the bacterial genome. This observed increase in global incidence of drug resistant tuberculosis (TB) has sparked the search for new anti-TB drugs and the repurposing of drugs that are currently used against other organisms or species of mycobacteria. One such repurposed drug, clofazimine (CFZ), is currently used for the treatment of leprosy, caused by Mycobacterium leprae. The mechanism of action of CFZ is not clear, but it is hypothesized that CFZ is reduced by a mycobacterial type II NADH oxidoreductase (NDH-2). The reduction of CFZ drives the production of reactive oxygen species (ROS) which is toxic to the pathogen. The aim of this study was to elucidate the mechanism of CFZ resistance. Towards this aim, spontaneous in vitro CFZ resistant mutants were selected, characterized and whole genome was used identify SNPs which may cause CFZ resistance. Mutations were identified in a transcriptional regulator encoded by Rv0678, fatty-acid-AMP ligase, or FadD28 (Rv2941) and glycerol kinase or GlpK (Rv3696c). Mutations in Rv0678 have previously been shown to play a role in both CFZ resistance and bedaquiline (BDQ) cross-resistance, while no link has been found between CFZ resistance and mutations in fadD28 and glpK. The novel, non-synonymous SNP identified in Rv0678 resulted in the replacement of an alanine residue with threonine at codon 84, which is located in the DNA binding domain. Virtual modelling of the mutated Rv0678 protein showed that the A84T mutation may influence DNA binding, possibly due to its proximity to the DNA binding domain. This mutation caused a change in hydrophobicity, which may influence binding to DNA. Previous studies showed that mutations in Rv0678 resulted in the upregulation of mmpL5, a putative efflux pump. However, the mechanism whereby CFZ resistance occurs via increased abundance of this efflux pump in the cell wall is not clear and needs further investigation. The cross-resistance between CFZ and BDQ, caused by mutations in Rv0678, is of concern and may influence the planning of anti-TB drug regimens for the future. The roles of the other two mutations identified in this study in CFZ resistance is also not clear and requires further investigation. Finally, the findings of this study support the role of Rv0678 in CFZ resistance thereby suggesting that this gene could be useful as a diagnostic marker to test for CFZ resistance in clinical isolates. / AFRIKAANSE OPSOMMING: Middelweerstandigheid in Mycobacterium tuberculosis is 'n wêreldwye toenemende probleem. Middelweerstandigheid word meestal veroorsaak deur enkel nukleotied polimorfismes (SNPs) in die bakteriële genoom. Hierdie toename in middelweerstandige tuberkulose (TB) het gelei tot die soektog na nuwe anti-TB-middels en die alternatiewe aanwending van middels wat tans teen ander organismes of spesies van mikobakterieë gebruik word. Een so 'n alternatiewe middel, clofazimine (CFZ), word tans gebruik vir die behandeling van melaatsheid wat veroorsaak word deur Mycobacterium leprae. CFZ se meganisme van werking is nie duidelik nie, maar dit word vermoed dat CFZ gereduseer word deur 'n mikobakteriële tipe II NADH oksidoreduktase (NDH-2). Die reduksie van CFZ dryf die produksie van reaktiewe suurstof spesies wat giftig is vir die patogeen. Die doel van hierdie studie was om die meganisme van CFZ weerstandigheid te ondersoek. Om hierdie doel te bereik was spontane in vitro CFZ weerstandige mutante gekies, gekarakteriseer en heel genoom volgorde bepaling is gebruik om SNPs te identifiseer wat CFZ weerstandigheid veroorsaak. Mutasies in Rv0678, 'n transkripsie reguleerder, vetsuur-AMP ligase, of FadD28 (Rv2941) en gliserol kinase of GlpK (Rv3696c) geïdentifiseer. Dit is al voorheen gevind dat mutasies in Rv0678 ‘n rol speel in beide CFZ weerstandigheid en bedaquiline (BDQ) kruis-weerstandigheid, terwyl geen verband gevind is tussen CFZ weerstandigheid en mutasies in fadD28 en glpK nie. Die nuwe, nie-sinonieme SNP, geïdentifiseer in Rv0678 het gelei to die vervanging van 'n alanien aminosuur met treonien by kodon 84, wat geleë is in die DNS bindings domein. Virtuele modellering van die gemuteerde Rv0678 proteïen het getoon dat die A84T mutasie DNS binding moontlik kan beïnvloed, as gevolg van sy nabyheid aan die DNS bindings domein. Hierdie mutasie veroorsaak 'n verandering in die hidrofobiese natuur, wat DNS binding kan beïnvloed. Vorige studies het getoon dat mutasies in Rv0678 lei tot die opregulering van mmpL5, 'n waarskynlike uitvloei pomp. Die meganisme waardeur CFZ weerstandigheid veroorsaak, deur ‘n groot aantal van hierdie uitvloei pompe in die selwand, is nie duidelik nie en moet verder ondersoek word. Die kruis-weerstandigheid tussen CFZ en BDQ, wat veroorsaak word deur mutasies in Rv0678, is van belang en kan die beplanning van anti-TB middel behandeling vir die toekoms beïnvloed. Die rolle van die ander twee mutasies, wat in hierdie studie geïdentifiseer is, in CFZ weerstandigheid is ook nie duidelik nie en vereis verdere ondersoek. Ten slotte, die bevindinge van hierdie studie steun die rol van Rv0678 in CFZ weerstandigheid en dit dui daarop dat hierdie geen gebruik kan word as 'n diagnostiese merker om vir CFZ weerstandigheid te toets in kliniese isolate.

Submitted in fulfillment of the requirements for the degree of Master’s of Technology: Radiography, Durban University of Technology, Durban, South Africa, 2015. / Introduction Tuberculosis remains a leading cause of death, second to the Human Immunodeficiency Virus. The risk of latent tuberculosis infection and active tuberculosis disease is a known occupational hazard. In South Africa, a high tuberculosis burden country, the potential of Mycobacterium tuberculosis transmission to health care workers is high. This includes diagnostic radiographers and other radiology staff working in radiology departments. Purpose of the Study This study aimed to investigate the association of demographic and occupational factors with latent tuberculosis infection in radiology staff in public sector hospitals of the eThekwini Health District. Methodology This cross-sectional study was conducted from 26 February 2013 to 07 June 2013. Quantitative methods were used to test for associations of demographic and occupational factors with latent tuberculosis infection in participants. A sample size of 181 participants for an estimated population of 340 radiology staff was recommended at the proposal stage. The study consisted of two phases; the questionnaire survey (phase one) and the administration of a two-step tuberculin skin test (phase two). Data was obtained with regard to demographics, occupational history, social behaviours, medical history; and family and home histories. Demographic and occupational associations with latent tuberculosis infection were made in relation to the size of the first tuberculin skin test induration. Frequency distributions were developed to describe data categories. Pearson’s and Spearman rho’ correlation coefficients were used to test for correlations between the independent variables. The chi-square test was used to determine associations between the categorical independent variables and the dependent variable. Bivariate analyses were performed using these tests. The multivariate analysis was performed using logistic and linear regression on the dependent variable. Results A total of 182 questionnaires were returned from approximately 280 radiology staff. At the outset, all doctors working in the radiology department had to be excluded due to numerous failed attempts to enlist their participation. Fifty-three (29.12 percent) participants were excluded from phase one of the study and a further thirteen participants were excluded from phase two. The total sample was 116 participants. Of the 116 participants, 86.2 percent tested positive for latent tuberculosis infection at the first step of the two-step testing method used. One (0.86 percent) participant went on to convert at the second step, testing positive at this level. Demographic associations with latent tuberculosis infection included age (older) as an associated factor. A significant demographic association with latent tuberculosis infection was the use of alcohol (p-value 0.033 on the multivariate analysis). Occupational associations with latent tuberculosis infection included longer durations of employment. The annual income (higher income earners) displayed significant associations with latent tuberculosis infection (p-value 0.048 on the multivariate analysis). It is necessary in this study to note that participants include support personnel (lower income earners) making up 37.8 percent of the study, diagnostic radiographers making up 48.3 percent; and radiography managers/assistant managers (highest income earners) making up 13.8 percent of the study. Conclusion and recommendations The risk of transmission of Mycobacterium Tuberculosis to health care workers is a known occupational hazard. This study has described the prevalence of latent tuberculosis infection in radiology staff, at district and regional hospitals within the eThekwini Health District. With 23.62 percent of all participants already having active TB disease and 86.2 percent of the tested group displaying positive results for latent tuberculosis infection, using the tuberculin skin tests, the need for tuberculosis screening is essential. The findings of this study will be used as a health improvement mechanism for stakeholders, having identified potential gaps in medical screening in healthcare in Kwa-Zulu Natal. This study makes recommendations for the early detection of active tuberculosis infection and the monitoring of health care workers that are latently infected, thus assisting in reducing the rate of conversion of latent tuberculosis infection to active tuberculosis disease in radiology staff. This reduces long-term exorbitant costs related to health care associated infections, such as tuberculosis. It also reduces rates of transmission and cross infection to both co-workers and already immunocompromised patients, helping to curb the overall epidemic in South Africa.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Setting Study conducted in Tygerberg, Cape Town in South Africa. Hypothesis Host biomarkers associated with the antimycobacterial immune response during active infection with M. tuberculosis and during anti-tuberculosis chemotherapy are indicative of bacterial killing in the host and can be used in models to predict eventual treatment outcome. Objectives 1. To investigate immune parameters that were selected in a biological context as biomarkers of the extent of disease and early response to anti-tuberculosis treatment. 2. To use selected immune parameters to characterise fast and slow responders to anti-tuberculosis therapy. Findings Evaluation of cytokine multiplex fluorescent bead-based immunoassays as a screening tool in the search for biomarkers The data showed that cytokine multiplex fluorescent bead-based immunoassays achieved acceptable recoveries to detect antigen-specific IFN- responses in whole blood supernatant making it attractive for biomarker screening. However, proper optimisation needs to be done and proper controls included when using these kits. Markers of extent of disease High levels of CRP at diagnosis were found to be associated with the presence of multiple cavities on chest X-rays. A high level of suPAR and sICAM-1 at diagnosis were associated with the extent of alveolar disease. Also significant were the associations between the level of granzyme B, LAG-3 at diagnosis and the size of the cavities. No significant associations were observed between sTNFRs or DR5 with the chest X-ray grading of tuberculosis disease. Early classification of fast and slow responders to anti-tuberculosis treatment After cross-validation classification, discriminant analysis (DA) and support vector machine (SVM) analysis of selected immune parameters (sICAM-1 CRP, granzyme B, suPAR, sTNFRs, LAG-3 and CD3dim/CD56+ (% of CD45+) resulted in a 75% to 100% correct classification of the fast responders and a 82% to 100% correct classification of the slow responders when using DA. For SVM, the correct classification of the fast responders ranged from 88% to 100%, and that for the slow responders ranged from 95% to 100%. Differential gene expression in fast and slow responders to treatment Direct comparison of fast and slow responders showed that IL-4 transcripts were significantly higher in the fast responders at week one after initiation of treatment when compared to slow responders. IL-42 was also differentially expressed. Although IL- was significantly up-regulated in both fast and slow responders after one week of treatment compared to diagnosis, IL- expression was more than two folds higher in slow responders than in fast responders. No significant differences between the fast and slow responders were observed in the expression of TGF-, TGF-RII, Foxp3 and GATA-3. Conclusion Predictive models for differential anti-tuberculous treatment responses combining host proteins are promising and should be included in larger prospective studies to find the optimal markers for inclusion into clinical trials of new drugs and for implementation into clinical practice. / AFRIKAANSE OPSOMMING: Ligging Studie onderneem in Tygerberg, Kaapstad, Suid-Afrika. Hipotese Gasheerbiomerkers wat verband hou met die antimikobakteriële immuunrespons tydens aktiewe infeksie deur M. tuberculosis en tydens teentuberkulose chemoterapie dui op bakteriële doding in die gasheer en kan in modelle gebruik word om die uiteindelike uitkoms van die behandeling te voorspel. Doelwitte 1. Om gekose immuunparameters in ’n biologiese konteks as biomerkers van die omvang van siekte en vroeë reaksie op behandeling te ondersoek. 2. Om gekose immuunparameters te gebruik om vinnige en stadige reageerders op teentuberkulosebehandeling te karakteriseer. Bevindings Evaluering van die sitokien veelvuldige fluoresseer-pêrelbaseerde immuuntoets (cytokine multiplex fluorescent bead-based immunoassays) as ’n siftingsinstrument in die soeke na biomerkers Die data het getoon dat die sitokien veelvuldige fluoresseer-pêrelgebaseerde immuuntoets in staat was om antigeenspesifieke IFN--respons te meet wat dit aanloklik maak vir biomerkersifting. Sorgvuldige optimering moet egter gedoen word en behoorlike beheer moet ingesluit word wanneer hierdie stelle gebruik word. Merkers van omvang van siekte Hoë vlakke van CRP by diagnose is getoon om verband te hou met die teenwoordigheid van veelvoudige holtes op die pasiënte se borskas x-strale. Hoë vlakke van suPAR en sICAM-1 by diagnose was assosieer met die omvang van alveolêre siekte. Die assosiasie tussen die vlakke van granzyme B, LAG-3 by diagnose en die grootte van die holtes was ook betekenisvol. Daar was geen betekenisvolle assosiasies toe sTNFRs of DR5 en die borskas x-straalgradering van tuberkulosesiekte nie. Vroeë klassifikasie van vinnige en stadige reageerders op teentuberkulosebehandeling Ná klassifikasie op grond van kruisstawing het diskriminant-analise (DA) en ondersteuningsvektormasjiene (SVM) van geselekteerde immuunparameters (sICAM-1 CRP, gransiem B, suPAR, sTNFRs, LAG-3 en CD3dim/CD56+ (% van CD45+)) gelei tot ’n 75% tot 100% korrekte klassifikasie van die vinnige reageerders met DA en ’n 82% tot 100% korrekte klassifikasie van stadige reageerders. Vir SVM het die korrekte klassifikasie van vinnige reageerders gewissel van 88% tot 100%, en vir stadige reageerders het dit gewissel van 95% tot 100%. Differensiële geenuitdrukking in vinnige en stadige reageerders op behandeling In vergelyking met die vlak by diagnose is die uitdrukkingsvlak van IL-4 in die vinnige reageerders betekenisvol opgereguleer met ’n faktor van 9.2 teen die eerste week ná die aanvang van behandeling, in kontras met die stadige reageerders. Daar was geen verskille tussen die vinnige en die stadige reageerders met betrekking tot die uitdrukking van TGF-, TGF-RII, Foxp3 en GATA-3 nie. Gevolgtrekking Voorspellende modelle vir differensiële tuberkulose behandelingsresponse wat gasheerproteïene kombineer, hou belofte in en behoort in groter prospektiewe studies ingesluit te word om die mees geskikte merkers te vind vir insluiting in kliniese proewe van nuwe middels en vir implementasie in kliniese praktyk.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008. / This series of studies includes both methodological analyses, aimed at furthering our understanding of, and improving the tools used in molecular epidemiology, and investigative projects which have used these tools to add to our knowledge of the M. tuberculosis epidemic. Using serial isolates from tuberculosis patients, we have investigated the evolutionary rate of the IS6110 RFLP pattern. In accordance with other studies, we determined a ½-life for this epidemiological marker of 10.69 years, confirming its appropriateness for this purpose. We also identified an initial, much higher apparent rate which we proposed was the result of pre-diagnostic evolution. In support of this, our investigations in the context of household transmission of M. tuberculosis revealed that IS6110-based evolution is closely associated with transmission of the organism, resulting in a strain population rate of change of 2.9% per annum. To accommodate evolution within estimates of transmission, we proposed that calculations incorporate the concept of Nearest Genetic Distance (cases most similar in RFLP pattern and most closely associated in time). We used this to create transmission chains which allowed for limited evolution of the IS6110 marker. As a result, in our study community, the estimated level of disease attributable to ongoing transmission was increased to between 73 and 88% depending on the Genetic Distance allowed. We identified the duration of a study as a further source of under-estimation of transmission. This results from the artefactual abridgement of transmission chains caused by the loss of cases at the temporal boundaries of a study. Using both real and simulated data, we showed that viewing a 12-year study through shorter window periods dramatically lowered estimates of transmission. This effect was negatively correlated with the size of a cluster. Various combinations of MIRU-VNTR loci have been proposed as an alternative epidemiological marker. Our investigations showed that, while this method yielded estimates of transmission similar to those of IS6110, there was discordance between the two markers in the epidemiological linking of cases as a result of their independent evolution. Attempting to compensate for this by allowing for evolution during transmission improved the performance of IS6110, but generally had a deleterious effect of that of MIRU-VNTR. However, this marker remains a valuable tool for higher phylogenetic analysis and we used it to demonstrate a correlation between sublineages of the Beijing clade and the regions in which they are found. We proposed that, either the host population had selected for a particular sublineage, or that specific sublineages had adapted to be more successful in particular human populations. We further explored the dynamics of the epidemic over a 12-year period in terms of the five predominant M. tuberculosis clades. We found that, while four of these clades remained relatively stable, the incidence of cases from the Beijing clade increased exponentially. This growth was attributed to drug-sensitive cases although drug-resistant Beijing cases also appeared to be more successful than their non-Beijing counterparts. Possible factors contributing to this clade’s success were a greater proportion of positive sputum smears and a lower rate of successful treatment.