Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Please see fulltext for abstract. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) affekteer die hele wêreld, maar dit is veral in ontwikkelende lande 'n groot probleem. In Suid-Afrika, soos in baie ander lande, veroorsaak die immuun-paraliserende uitwerking van HIV -koïnfeksie dat die TB-epidemie voortwoeker. Daar is bewyse dat genetiese faktore in die gasheer die uitkoms van die siekte bepaal, aangesien slegs 10% van die individue wat deur Mycobacterium tuberculosis (M tuberculosis) geïnfekteer word, uiteindelik die aktiewe siekte ontwikkel. Die aangebore immuunsisteem is die liggaam se eerste verdedigingslinie, waarna die verworwe immuunreaksie geïnisieer word. Die bakterium se lotgevalle word moontlik bepaal in hierdie vroeë stadium pas nadat dit ingeasem is. Die kollektien-molekule is veral in die long 'n belangrike deel van die aangebore immuunrespons en sluit die mannose-bindende lektien en die surfaktantproteïene A (SP-A) en D (SP-D) in. Dit is al aangetoon dat hierdie drie kollektien-molekule in die gasheer 'n rol speel in die verdediging teen M tuberculosis. In hierdie ondersoek is veral klem gelê op die surfaktantproteïene, wat voorkom asof dit belangrike en kenmerkende rolle speel in die reaksie teen die ingeasemde bakterieë. SP-A versterk die aanhegting van M tuberculosis aan die alveolêre makrofage en verhoog fagositose, terwyl SP-D die bakterieë agglutineer en so verhoed dat dit deur die makrofage gefagositeer word. Gekontroleerde assosiasiestudies in pasiënte is gedoen deur polimorfismes in hierdie gene, wat geassosieer is met TB in ander bevolkings as ons eie, te bestudeer. 'n Polimorfisme in die amino-terminaal area van die SP-D-geen is positief geassosieer met vatbaarheid vir TB. 'n Familie-gebaseerde studie is ook gedoen om die resultate van die gekontroleerde assosiasiestudie te repliseer. Verskillende resultate is verkry en word moontlik bepaal deur die familiestruktuur wat gebruik is. Die aantal families wat bestudeer is, was relatief min en daarom kan daar nie afgelei word dat die assosiasie wat voorheen waargeneem is vals is nie. 'n Groter studie sal gedoen moet word. Die impak van hierdie polimorfisme is verder ondersoek om te bepaal of dit die totale struktuur van die proteïen beïnvloed. Die effekte van hierdie polimorfisme op die konsentrasie van SP-D in die serum van aktiewe TB-pasiënte is ondersoek en vergelyk met die van kontroles. Ons kon nie vasstel watter rol, indien enige, die polimorfisme in die totale struktuur van die SP-D-molekule speel nie, maar ons het bewys dat die serumkonsentrasie van SP-D beduidend verhoog was in aktiewe TB-pasiënte in vergelyking met kontroles (p < 0.0001). Verder het ons ook gedemonstreer dat die konsentrasie van SP-D beduidend verhoog was in die IT-genotipe van die aktiewe TB pasiënte vergeleke met die kontroles (p < 0.0001). Die IT-genotipe is al voorheen positief geassosieer met vatbaarheid vir TB (T. Roos, ongepubliseerde inligting). Verskeie alleliese variante is geïdentifiseer in die SP-A-gene (SP-A1 en SP-A2) wat saam die volle funksionele SP-A-proteïen vorm. Polimorfismes wat onlangs in die kollageen-agtige area van SP-A 1 en SP-A2 gevind is (Madan et al., 2002) en een nuwe polimorfisme wat in hierdie studie geïdentifiseer is, is ondersoek in 'n Suid-Afrikaanse bevolking. Ons het beduidende positiewe assosiasie tussen 'n polimorfisme in die kollageen-agtige area van die SP-A2 geen en vatbaarheid vir TB (p = 0.007) aangetoon. Ons bevindinge bewys die belangrikheid van die bestudering van mensgenetika, wat die immuunkompetensie rig, om vatbaarheid vir infektiewe siektes te verstaan.

Thesis (MCur)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Mycobacterium Tuberculosis (TB) continues to rank among the world’s most serious problems despite biomedical achievement of effective prophylaxis and chemotherapy. In South Africa, TB is directly linked to the country’s high HIV prevalence rate and other related factors. The required knowledge, as well as people’s attitude towards a better understanding of TB are prerequisites for motivating them to seek early treatment. This study aims to explore the knowledge about and attitude towards TB among non- TB infected clinic attendees. More specifically, this study used a qualitative descriptive design to explore and describe clinic attendees’ knowledge of the cause, symptoms and treatment of TB and to explore their attitude towards the disease. A semi-structured interview technique was used to gather data. Ten clinic attendees between 20-40 years old, able to communicate in English and who had no past history of TB were conveniently sampled. Manual data analysis was done using an inductive approach. Thereafter, a deductive approach using the Health Belief Model was used to guide the discussion of the findings. Nine major themes were identified. The results confirm a gap in participants’ knowledge of the cause, symptoms and treatment of TB. Despite these gaps participants perceived that they were susceptible to TB, the dangers TB could cause and the benefits of completing the treatment. Participants indicated that they would seek medical help if they experienced TB symptoms. However, their fear was their ignorance of TB symptoms on the one hand and the fear of being stigmatized, discriminated against, as well as the quality of health service deliveries on the other hand. The findings highlight the need for on-going education about the cause of TB, transmission, symptoms and treatment at clinics and within the community. Media including radio, television, as well as schools and family should be included in TB education programmes. Immigrants should also be targeted to be included in TB education campaigns. Keys terms: Tuberculosis, Non-TB infected patients, Knowledge and attitude, Health Belief Model. / AFRIKAANSE OPSOMMING: Mycobacterium Tuberkulose (TB) word steeds gekenmerk as een van die ernstigste gesondheidsprobleme ter wệreld, ten spyte van deurbrake met betrekking tot meer doeltreffende profilakse en chemoterapie. In Suid-Afrika, word TB direk gekoppel aan die land se hoë voorkoms van MIV en ander verwante faktore. Die nodige kennis, sowel as ‘n beter begrip van mense se gesindhede teenoor TB, is voorvereistes vir die motivering wat hulle gedrag sal beïnvloed om vir vroeë behandeling te gaan. Hierdie studie het ten doel om die kennis en gesindhede teenoor tuberkulose van ongeïnfekteerde TB-pasiënte in ‘n Kaapstadse kliniek te ondersoek. In die besonder ondersoek die studie, deur ‘n kwalitatiewe beskrywende benadering, die kennis van ‘n spesifieke groep individue wat die kliniek besoek, met betrekking tot die oorsake, simptome en behandeling van TB, asook hulle houding ten opsigte van die siekte. Die studie beklemtoon ook enige vooroordele oor die siekte en identifiseer moontlike redes vir pasiënte se laat-aanmelding van TB by ‘n kliniek. ‘n Semi-gestruktureerde tegniek vir onderhoudvoering is gebruik. Tien pasiënte wat die kliniek besoek tussen die ouderdomme 20-40 jaar oud, wat in staat is om in Engels te kommunikeer, en wat geen vroeëre geskiedenis van TB het nie, is gerieflikheidshalwe per steekproef gebruik. Data is per hand versamel deur gebruik te maak van ‘n induktiewe benadering. Hierna is ‘n deduktiewe benadering gevolg, en die “Health Belief Model” is gebruik om die gesprekke te lei in die bevindinge. Nege hooftemas is geïdentifiseer. Die resultate bevestig ‘n gaping in die kennis van deelnemers oor die oorsake, simptome en behandeling van TB. Ten spyte van die gapings, was deelnemers wel bewus van die feit dat hulle blootgestel is om TB op te doen en oor wat die gevare is wat deur TB veroorsaak kan word, sowel as wat die voordele is om TB-behandeling te voltooi. Deelnemers het aangedui dat hulle mediese hulp sal vra, sou hulle die simptome van TB bespeur. Desnieteenstaande was hulle vrees enersyds oor die onkundigheid van die simptome van TB, en om gestigmatiseer teen gediskrimineer te word, asook die standaard van mediese dienste beskikbaar andersyds. Die bevindinge beklemtoon die behoefte aan voortgesette opvoeding oor die oorsake, oordrag, simptome en behandeling van TB in klinieke en binne gemeenskappe. Media soos radio en televisie, asook skole en families behoort ingesluit te word in sodanige opvoedingsprogramme. Immigrante behoort ook ingesluit te word by massa opvoedingsprojekte. Sleutelterme: Tuberkulose, ongeïnfekteerde TB-pasiënte, Kennis en gesindhede, “Health Belief Model”.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Mycobacterium tuberculosis is the etiological agent for tuberculosis, an infectious disease which is one of the leading causes of morbidity and mortality in developing countries. The emergence of drug resistant tuberculosis has negatively impacted the efficacy of current treatment regimens and threatens to undermine tuberculosis control programs worldwide. Rifampicin forms the backbone of the World Health Organization’s recommended treatment regimen for the treatment of drug susceptible tuberculosis. Resistance to rifampicin is caused by mutations in the 81 bp core region of the rpoB gene which encodes the β subunit of RNA polymerase. Numerous studies have shown that mutations at codons 531 and 526 are the most frequent in clinical isolates, yet little is known concerning the mechanistic effect of these mutations on the fidelity of RNA polymerase. In the present study, we aimed to determine the influence of rpoB mutations on the gene expression profile of M. tuberculosis cultured in vitro. To accomplish this, rifampicin resistant clinical isolates and spontaneous mutants (selected in vitro from H37Rv and a drug-sensitive clinical strain) harbouring rpoB H526Y and S531L mutations were subjected to whole genome sequencing and genome-wide transcriptional profiling. When comparing the transcription profile of H37Rv to the in vitro rpoB mutants, a large proportion of the differentially expressed genes were found to encode for proteins involved in intermediary metabolism and respiration; and cell wall and cell processes. The majority of these differentially expressed genes were downregulated. Prominent differential expression in the same functional categories was also evident when comparing the clinical isolates with these mutations; however, a greater number of genes were differentially expressed in this case. Furthermore, expression of genes that are part of the WhiB7 regulon were found to be upregulated in the rpoB526 mutants, and downregulated in the rpoB531 mutants. These findings indicate that both the position of the rpoB mutation, as well as the genetic background of the strain, play an important role in the gene expression profile of rpoB mutants. Surprisingly, transcriptional profiling of cultures that were exposed to the critical concentration of rifampicin for 24 hours did not exhibit significant differential gene expression. Whole genome sequencing, followed by bioinformatic analysis, revealed that the in vitro mutants harbour synonymous and non-synonymous single nucleotide polymorphisms in addition to the respective rpoB mutations. This suggests that the mycobacterial genome is constantly evolving, challenging previous assumptions of relatively static mycobacterial genomes. The findings from this research have provided novel insight into understanding the influence of resistance-conferring mutations on the biology of M. tuberculosis and have shown that further studies are urgently needed to better understand the complex physiology of this pathogen. This knowledge will be critical for the success of future drug development endeavours. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is die etiologiese agent vir tuberkulose, een van die grootste oorsake van morbiditeit en sterftes in ontwikkelende lande. Die verskyning van middelweerstandige tuberkulose het 'n negatiewe impak op die effektiwiteit van die huidige behandeling van tuberkulose en dreig om tuberkulose beheerprogramme wêreldwyd te ondermyn. Weerstandigheid teen rifampisien, een van die eerste-lyn anti-tuberkulose middels, word veroorsaak deur mutasies in die 81 bp kerngedeelte van die rpoB geen. Die mees algemene mutasies in kliniese isolate word gevind in kodons 531 en 526; alhoewel daar min inligting beskikbaar is oor die effek van hierdie mutasies op die funksie van RNS polimerase. Die doel van hierdie studie was om die effek van verskillende rpoB mutasies op die geen-uitdrukkingsprofiel van M. tuberculosis te bepaal. Rifampisien-weerstandige kliniese isolate en in vitro mutante (geselekteer vanaf H37Rv en 'n rifampisien-sensitiewe kliniese isolaat) met rpoB H526Y en S531L mutasies was vir hierdie doel geselekteer en gebruik om die heel genoom volgorde te bepaal en geenuitdrukking te kwantifiseer. Vergelyking van die H37Rv transkripsieprofiel met in vitro geselekteerde rpoB mutante het getoon dat 'n groot aantal gene wat differensieel uitgedruk is, vir proteïene kodeer wat betrokke is in selwand-prosesse en intermediêre metabolisme en respirasie. Die meerderheid van hierdie differensieel uitgedrukte gene was afgereguleer. ’n Soortgelyke verskynsel is ook waargeneem in kliniese isolate, met die verskil dat 'n groter aantal gene in hierdie geval differensieel-uitgedruk was. Gene wat deel vorm van die WhiB7 regulon is opgereguleer in die rpoB526 mutante, terwyl dit afgereguleer was in die rpoB531 mutante. Hierdie resultate is ’n baie sterk aanduiding dat beide die posisie van die rpoB mutasie, asook die genetiese agtergrond van die organisme, ’n belangrike rol speel in die uitdrukking van gene in rifampisin weerstandige M. tuberculosis. Kulture wat aan die kritiese konsentrasie van rifampisien vir 24 uur blootgestel is, het teenverwagting geen differensiële geen-uitdrukking getoon nie. Verder het die heel genoom volgorde bepaling van die in vitro mutante getoon dat sinonieme en nie-sinonieme enkel-nukleotied polimorfismes teenwoordig is in die onderskeie rpoB mutante. Dit dui daarop dat die mikobakteriële genoom voortdurend verander, moontlik nie so stadig as wat voorheen vermoed is nie. Die bevindinge van hierdie navorsing bied nuwe insigte om die invloed van mutasies wat middel-weerstandigheid veroorsaak op die biologie van M. tuberculosis te verstaan. Dit het ook getoon dat verdere studies dringend nodig is om die komplekse fisiologie van hierdie patogeen te verstaan. Hierdie kennis sal van kardinale belang wees vir die sukses van toekomstige ondernemings om nuwe anti-tuberkulose middels te ontwikkel.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB), a disease that affects millions of people world-wide. The species M. tuberculosis consists of a large number of different strains that can be grouped into at least 40 different known strain families. Many of the strains present with different pathogenic characteristics and host adaptations. The F11 LAM strains and Beijing strains currently have a nearly equal representation in the population of Cape Town, making up a total of 45% of all strains in this setting. The Latin-American Mediterranean (LAM) family of M. tuberculosis is proved to be the cause of a large percentage of TB cases worldwide and it is the predominant strain in high-prevalence regions such as the Western Cape and KwaZulu-Natal regions of South Africa, Zambia, Zimbabwe, and South America. This project aimed to investigate the evolution and biological characteristics of the members of the principle genetic group (PGG) 2 Latin-American Mediterranean (LAM) genotype of M. tuberculosis using a combination of whole genomic and proteomic analyses, coupled to mycobacterial molecular epidemiological techniques. The evolution of M. tuberculosis strain families from the Western Cape Province of South Africa proved to be consistent with previous evolutionary scenarios for M. tuberculosis isolated from other parts of the world. This genome-wide SNP-based phylogeny for the evolution of M. tuberculosis offers novel insight into the unique global representation of the M. tuberculosis isolates from the Western Cape, South Africa. The evolutionary scenario presented confirms six LAM sub-lineages, namely IS6110 RFLP families F9, F11, F13, F14, F15, and F26. A subset of sub-lineage defining SNPs was determined for each of the six LAM sub-lineages. The genomic changes in the LAM genotype strains observed through the SNP analysis presented here mostly occur in the genes involved in the cell wall, cell processes, intermediary metabolism and respiration. The same phenomenon was observed when the non-redundant SNPs of the non-LAM isolates were functionally annotated. The functional classification of the regulated proteins in the representative of the LAM RDRio lineage of M. tuberculosis suggests that proteins involved in the lipid metabolism, intermediary metabolism and respiration may be the key to the pathogenic effectiveness of the RDRio LAM lineage. A combination of the LAM SNP analysis and the LAM RDRio/non-RDRio comparison showed that the overall genomic- and proteomic features involved in the cell wall and cell processes of the LAM genotype differ to a large extent from what is seen in the reference strain, M. tuberculosis H37Rv. This genome wide phylogenetic study is the first of its kind in a South African context, and not only presents a robust phylogeny of the M. tuberculosis strain families, and specifically the LAM lineage, but also gives the first ever insight into the protein differences which distinguishes RDRio and non-RDRio M. tuberculosis strains from each other. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis (M. tuberculosis) is die mikrobiese agent wat tuberkulose (TB), 'n siekte wat miljoene mense wêreldwyd affekteer, veroorsaak. Die spesie M. tuberculosis bestaan uit 'n groot aantal verskillende stamme wat in ten minste 40 verskillende bekende stam-families gegroepeer word. Baie van die stamme toon verskillende patogeniese eienskappe en gasheer aanpassings. Die F11 LAM stam en Beijing stam het tans 'n byna gelyke verteenwoordiging in die bevolking van Kaapstad, wat 'n totaal opmaak van 45% van stamme wat in hierdie gebied gevind word. Die Latyns-Amerikaanse Meditereense (LAM) familie van M. tuberculosis is bewys om die oorsaak van 'n groot persentasie van TB-gevalle wêreldwyd te wees, en dit is die oorheersende stam in hoë voorkoms streke soos die Wes-Kaap en KwaZulu-Natal streke van Suid-Afrika, Zambië, Zimbabwe en Suid-Amerika. Hierdie projek het ten doel gehad om die evolusie en biologiese eienskappe van die lede van die basiese genetiese groep (BGG) 2 Latyns-Amerikaanse Meditereense (LAM) genotipe van M. tuberculosis te ondersoek deur gebruik te maak van 'n kombinasie van heel genoom en proteoom analise, gekoppel aan mikobakteriële molekulêre epidemiologiese tegnieke. Die evolusie van M. tuberculosis stam families van die Wes-Kaap Provinsie van Suid-Afrika blyk om in ooreenstemming te wees met vorige evolusionêre scenario's vir M. tuberculosis wat in ander dele van die wêreld geïsoleer is. Die genoom-wye enkelnukleotied polimorfisme-gebaseerde filogenetiese hipotese vir die evolusie van M. tuberculosis bied nuwe insig in die unieke wêreldwye verteenwoordiging van die M. tuberculosis isolate van die Wes-Kaap, Suid-Afrika. Die evolusionêre scenario wat hier aangetoon word bevestig ses LAM sub-lyne, naamlik IS6110 RFLP families F9, F11, F13, F14, F15, en F26. 'n Versameling sub-lyn definiërende enkelnukleotied polimorfismes was bepaal vir elk van die ses LAM sub-afstammelinge. Die genomiese veranderinge wat waargeneem is in die LAM-genotipe isolate deur die enkelnukleotied polimorfisme analise wat hier aangebied word, is meestal in die gene wat betrokke is in die selwand, selprosesse, intermediêre metabolisme en respirasie. Dieselfde verskynsel is waargeneem wanneer die nie-oorbodige enkelnukleotied polimorfismes van die nie-LAM isolate funksioneel geannoteer is. Die funksionele klassifikasie van die gereguleerde proteïene in die verteenwoordiger van die LAM RDRio-lyn van M. tuberculosis dui daarop dat die proteïene wat betrokke is in die lipiedmetabolisme, intermediêre metabolisme en respirasie die sleutel tot die patogene doeltreffendheid van die RDRio-LAM-lyn kan wees. 'n Kombinasie van die LAM enkelnukleotied polimorfisme analise en die LAM-RDRio/nie-RDRio vergelyking het getoon dat die totale genomiese- en proteomiese kenmerke wat verwant is aan selwand en selprosesse van die LAM genotipe tot ʼn groot mate verskil van wat gesien word in die verwysing stam, M. tuberculosis H37Rv. Hierdie genoom-wye filogenetiese studie is die eerste van sy soort in 'n Suid-Afrikaanse konteks, en bied nie net ‗n robuuste filogenie van die M. tuberculosis stam families, en spesifiek die LAM genotipe van M. tuberculosis nie, maar gee ook die eerste keer ooit insig in die proteïen verskille wat RDRio en nie-RDRio M. tuberculosis stamme van mekaar onderskei.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Medical Sciences

One third of the world’s population is affected by Tuberculosis (TB), a disease caused by infection with Mycobacterium tuberculosis (MtB). The emergence of multidrug-resistant MtB makes this disease a major public health concern. New agents are needed to treat TB infections in a manner that circumvents existing pathways of resistance. One strategy is to target the organism at the translational level by inhibiting vital modifications of RNA. One gene responsible for these modifications is the tRNA (guanosine-1)-methyltransferase, trmD, which has been shown to be essential in several bacteria. The eukaryotic and bacterial m1G methyltransferases are structurally dissimilar, making this enzyme an ideal target for selective anti-TB agents. One strategy for TrmD inhibitor design is to target the catalytic center of the enzyme. Existing inhibitors such as Sinefungin exhibit poor selectivity due to the substrate’s role, SAM, as a universal methyl donor in many biological processes. Structure/activity relationships for inhibitory compounds are sparse, impeding the design of novel antimicrobials. Crystallographic data would identify molecular features unique to TrmD, and allow design of agents complimentary to the TrmD active site with minimal differential toxicity. Presently, no crystal structure for Mycobacterium tuberculosis TrmD exists. As a first step in this direction, the MtB gene has been cloned and expressed by using a His-tagged T7 expression vector. The recombinant protein was characterized through kinetic and preliminary inhibitor assays. The native enzyme displays a mass of 50 kDa, proving this enzyme is a dimer of two identical subunits. This is similar to data found on other TrmD orthologs. Crystallization of MtB TrmD has been achieved and preliminary x-ray diffraction studies conducted.

Objetivo: Determinar los factores de riesgo para el desarrollo de tuberculosis multidrogorresistente (TBC-MDR) en pacientes del Hospital Nacional “Dos de Mayo”, de junio de 2015 a junio de 2016. Materiales y métodos: Estudio observacional, analítico, longitudinal, retrospectivo, comparativo de casos y controles. La muestra fue de 120 pacientes diagnosticados con tuberculosis en el Programa de Control de la Tuberculosis en el Hospital Nacional “Dos de Mayo”, de junio de 2015 a junio de 2016, de los cuales 40 fueron casos y 80, controles. Se realizó un análisis bivariado, usando el software IBM SPSS Statistics versión 24. En él, se cruzaron las variables edad, sexo, ocupación, contacto con TBC-MDR, antecedente de tratamiento antituberculoso, presencia de comorbilidad, co-infección con VIH e IMC; utilizando X2 y la medida de asociación odds ratio. Resultados: Se encontró que el contacto con TBC-MDR, el antecedente de tratamiento antituberculoso, la presencia de alguna comorbilidad, la co-infección con VIH y la desnutrición son factores de riesgo para el desarrollo de TBC-MDR. Por otro lado, no se pudo establecer la edad menor a 40 años, el sexo masculino o el desempleo como factores de riesgo. Conclusión: Hay una serie de factores de riesgo para el desarrollo de tuberculosis multidrogorresistente que son manejables, por lo que se podrían establecer nuevos programas o estrategias a fin de evitar, o de no ser posible, asegurar el correcto manejo de estos factores, lo que podría ayudar a frenar el incremento de casos que se ha visto en los últimos años.