Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Thesis (MSc. (Medical Sciences)) -- University of Limpopo, 2015 / Introduction: Multidrug-resistant tuberculosis (MDR-TB) caused by resistance to at least rifampicin (RIF) and isoniazid (INH) drugs is a growing public health concern in South Africa. The detection of MDR-TB still relies on culture despite advancement in molecular diagnostic technology. Currently MTBDRplus and GeneXpert are the only available assays used in rapid diagnosis of MDR-TB using chromosomal mutations in drug target regions. Some strains are missed by these assays due to their limitation in mutational detection profile. Novel Seegene Anyplex assays simultaneously detect TB and resistance to RIF and INH using fifteen and six mutational probes, respectively within 3 hours. Limpopo Province has limited information on the circulating strains of TB. Aim: To determine drug-resistant Mycobacterium tuberculosis (M. tuberculosis) mutations using Anyplex™ MTB/NTM/MDR-TB real time assay and characterise the drug-resistant strains. Methods: We prospectively collected 204 clinical samples at Modimolle MDR-TB unit and retrospectively used 104 culture isolates from MRC laboratory in Pretoria. The MTBDRplus assay was used to screen for M. tuberculosis and drug resistant mutations to RIF and INH drugs. Anyplex™ MTB/NTM/MDR-TB assay was used for rapid detection of M. tuberculosis and drug resistance to RIF and INH within 3 hours. The discordance between phenotypic and genotypic assays was resolved by sequencing and the Anyplex™ resistant profiles were spoligotyped. Diagnostic data was collected from NHLS and MRC databases and analysed using the Microsoft excel and Epi Info version 3.5. Descriptive statistics (percentages and frequencies) were used to explain proportions. Results: The Anyplex™ MTB/NTM assay detected M. tuberculosis in 69/111(62%) and 100/104 (96%) of clinical and culture samples respectively. The sensitivities, specificity, PPV and NPV obtained for both RIF and INH resistance by Anyplex™ MDR-TB assay were 67%, 59%, 67%, 55% and 15%, 100%, 100% and 17%, respectively. Anyplex™ MTB/NTM/MDR-TB resolved 23/45 (51%) of discordant vi samples. Sequencing of remaining discordant isolates revealed L511P, L533P and D516Y mutations within rpoB gene. A novel R385W mutation within katG was also detected. Spoligotyping of Anyplex™ MDR-TB resistant clinical isolates revealed Euro American clade with 20% followed by 15% Manu2, 5% East African Indian, 5% H37Rv, 5% atypical and 50% were orphans. Conclusion: The novel Anyplex™ MTB/NTM/MDR-TB assay is a rapid and valid technique for detecting M. tuberculosis and most common mutations conferring resistance to RIF and INH. However further investigations are required, as the assay has a lower sensitivity as compared to already endorsed techniques. / National Research Foundation (NRF) and University of Limpopo TB Grant

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.

Master of Public Health - MPH / Decentralisation of multi-drug resistant tuberculosis (MDR-TB) services has resulted in improved access to care, with community-based treatment of MDR-TB shown to be more effective than centralised hospital-based care. Furthermore, increasing bed shortages resulted in the National Department of Health establishing MDR-TB policy guidelines in 2011. However, the extent to which this policy has been implemented by the decentralised MDR- TB sites and the factors influencing implementation of the policy from a management perspective were not well described. The aim of this study was therefore to explore and compare the actual and ascribed roles and responsibilities of key management-level role players at the decentralised MDR-TB sites, and to explore the factors influencing implementation of the MDR-TB decentralisation policy (2011).

Student number:0312412A Faculty of Health Sciences School of Public Health / Management of multidrug-resistant tuberculosis (MDR-TB) is more expensive, lengthy and is associated with less favourable outcomes and more adverse reactions than management of susceptible tuberculosis. The aim of this study was to review the management and treatment outcomes of registered MDR-TB patients hospitalized at Sizwe hospital during a five-year period. A cross-sectional study with both descriptive and analytic features was done on 237 MDR-TB patients hospitalized from the beginning of June 1998 to the end of May 2003. Data were analysed using SPSS version 12 Software. Main outcome measures were interim treatment outcomes at the end of hospitalization period. These outcomes comprised culture conversion rates, time to culture conversion, transfer out, interruption, and death rates. Multiple logistic regression analysis was performed to determine risk factors for poor treatment outcomes. These poor outcomes were defined as treatment interruption, failure and mortality rates. The burden of institutional care for MDR-TB patients in this setting was found to involve high numbers of MDR-TB patients for whom the allocated hospital beds were insufficient. Patients with primary MDR-TB, who had no history of nonadherence to treatment, were paradoxically more likely to be hospitalized shortly after diagnosis. Acquired MDR-TB patients were mostly managed as outpatients immediately after diagnosis only to be hospitalized later due to persistent nonadherence or disease severity. Overall, acquired MDR-TB patients were hospitalized in larger numbers than those with primary disease. This reflects the higher prevalence of acquired MDR-TB compared to primary MDR-TB. Page v Abstract Culture turnaround time was on average 19 days. The overall culture conversion rate of the hospitalized patients was low at 41.9 percent. This low culture conversion rate resulted in protracted hospitalization periods and high interim mortality rates. The mean duration of hospitalization, 3.52 months, correlated favourably with the time interval to the first culture conversion of 2.96 months. Hospitalization did not guarantee the expected adherence to treatment. Surgical interventions were done belatedly with resultant high mortality outcomes. The main reasons given by patients for refusing hospital treatment were visiting traditional healers, solving socioeconomic problems and attending to family matters. A large percentage of hospitalized patients were co-infected with HIV. HIV care and support was incomplete as antiretroviral drugs were not available at the hospital. Among the main findings of the study was the powerful influence HIV status had on poor hospitalization outcomes. Recommendations arising from the study include the need to provide ARVs at the Sizwe hospital. Admission and discharge guidelines aimed at ensuring adequate beds are reserved for deserving patients should be formulated. Continuing education for service providers must be encouraged and rewarded. Infection control procedures at both community and health institution level ought to be vigorously promoted. Patients known to be hopelessly non-adherent should at least be partially hospitalized in the interest of public health.

Doctor Pharmaceuticae - DPharm / Introduction: Drug-resistant tuberculosis remains a major world health problem and one of the leading cause of death worldwide. Despite adequate adherence to antituberculosis drugs by patients, the emergence of drug-resistance tuberculosis still occurs. This fact implies other factors leading to the emergence of resistant strains of Mycobacterium tuberculosis. A multidrug treatment regimen, which may consist of five to seven different drugs including terizidone, is used in the treatment of drugresistance tuberculosis. Terizidone is part of the multidrug regimen whose pharmacokinetics is scarce in literature and plasma concentration profile unknown. Two molecules of cycloserine joined by terephtalaldehyde moiety makes up a molecule of terizidone, which is thought to undergo complete metabolism into cycloserine in vivo. Additionally, the current literature report that terizidone and cycloserine can be used interchangeably as they are thought to be equivalent. The aim of this thesis was first to develop and validate bioanalytical methods for determination of terizidone and cycloserine in patients’ plasma samples. Secondly, to model population pharmacokinetics of terizidone and cycloserine. Thirdly, to determine the amount of cycloserine resulting from metabolism of terizidone.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Tuberculosis (TB) is an infectious respiratory disease caused by the bacterium Mycobacterium tuberculosis. TB is the second largest cause of mortality by infectious diseases and is a challenging disease to control. It spreads easily among people via droplets propagated by an infectious person. Treatment against TB has been available since the 1950s; however, various problems with treatment have led to the emergence of drug-resistance in TB bacteria, which further complicates disease control. Furthermore, TB is a disease that predominantly affects poor countries or countries with high population densities. With the generalization of travel and migration in the second half of the twentieth century, individuals infected in such countries are likely to move to or spend some time in richer countries, making TB a worldwide problem. In this thesis, we consider the role of population movement in the spread of tuberculosis by studying two different models. The first one is an extension to a spatialized context of a simple existing mathematical model for the spread of TB. We establish that, similarly to the original model, the equilibrium without disease is globally asymptotically stable when the basic reproduction number $\R_0$ is less than one. In the case that $\R_0>1$, we prove that the system is uniformly persistent. The second model considers the spread of drug-resistant TB in a population, then between connected populations. We establish that a backward bifurcation can occur and that the coupled system has more types of equilibria than the systems in isolation. Finally, we consider a general class of models including the previous two in isolation and after coupling. We investigate which dynamical properties of the isolated models are preserved when coupling the models through movement. Some new results are provided in that direction.

Tuberculosis (TB) is an infectious respiratory disease caused by the bacterium Mycobacterium tuberculosis. TB is the second largest cause of mortality by infectious diseases and is a challenging disease to control. It spreads easily among people via droplets propagated by an infectious person. Treatment against TB has been available since the 1950s; however, various problems with treatment have led to the emergence of drug-resistance in TB bacteria, which further complicates disease control. Furthermore, TB is a disease that predominantly affects poor countries or countries with high population densities. With the generalization of travel and migration in the second half of the twentieth century, individuals infected in such countries are likely to move to or spend some time in richer countries, making TB a worldwide problem. In this thesis, we consider the role of population movement in the spread of tuberculosis by studying two different models. The first one is an extension to a spatialized context of a simple existing mathematical model for the spread of TB. We establish that, similarly to the original model, the equilibrium without disease is globally asymptotically stable when the basic reproduction number $\R_0$ is less than one. In the case that $\R_0>1$, we prove that the system is uniformly persistent. The second model considers the spread of drug-resistant TB in a population, then between connected populations. We establish that a backward bifurcation can occur and that the coupled system has more types of equilibria than the systems in isolation. Finally, we consider a general class of models including the previous two in isolation and after coupling. We investigate which dynamical properties of the isolated models are preserved when coupling the models through movement. Some new results are provided in that direction.

Magister Scientiae (Occupational Therapy) - MSc(OT) / TB is a multi- faceted public health problem spurred on by the biological progression of the disease as well as the social issues associated with it. The treatment of TB is however primarily driven by the medical model where the focus is on the disease and not on a holistic view of the patient. Occupational therapy is a profession concerned with the use of occupation in the promotion of health and well being through the facilitation of the process of occupational adaptation. There is however a paucity of literature pertaining to the role that occupational therapy could play within the TB context. The aim of this study was to explore how adults with MDR- TB who undergo long-term hospitalisation at a hospital in the Western Cape experience occupational adaptation. The objectives of the study were to explore how the participants perceive their occupational identity, to explore the meaning and purpose the participants assign to their occupational engagement and to explore the how the participants perceive their occupational competence. The interpretive research paradigm employing a phenomenological qualitative research approach was utilized in this study. Purposive sampling was used to select four participants based on specific selection criteria. The data gathering methods utilized included diaries, semistructured interviews, participant observation and a focus group. Photographs taken by the researcher for the purpose of participant observation were used to elicit a rich, in depth response from the participants during the focus group discussion. All data was analysed through thematic content analysis. The study findings highlighted that the participants viewed themselves as occupational beings and that they valued the role that occupational engagement played in facilitating their occupational competence and ultimately their ability to adapt to long- term hospitalisation. The environmental demands and constraints that they experienced however infringed their engagement in meaningful occupation and hampered their ability to achieve occupational competence. It was recommended that the hospital adopt an integrative intervention approach to the management of MDR- TB patients that include principles of psychosocial rehabilitation and occupational enrichment to address occupational risk factors and institutionalisation.