Thesis (MMed)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The aim of this pilot project was to determine whether mothers with laboratory confirmed or clinically suspected tuberculosis (TB) had evidence of TB in the placenta. A secondary objective was to correlate evidence of placental TB with neonatal outcome. A total of 56 placentas were examined to determine if there were any specific histopathological features predictive of tuberculosis together with Ziehl-Neelsen (ZN) staining. A total of 30 cases were positive for maternal TB and one case was a false positive maternal diagnosis of TB, whilst 25 cases were negative for maternal TB. Biopsies from these 56 placentas were collected for conventional PCR from the paraffin embedded tissue blocks. The performance of these two diagnostic modalities (histopathology and PCR) was assessed coll ectively and individually, and compared to the neonatal outcome (presence or absence of active clinical mycobacterial tuberculosis infection) and evidence of maternal pulmonary and extra pulmonary tuberculosis. The recognition of specific sites of lesions in the placenta (e.g. membranes vs. intervillous space) may lead to an understanding of the pathogenic mechanisms involved in matern alfetal transmission of tuberculosis, and thereby pave the way for further studies in understanding the pathogenesis of congenital TB. Invaluable knowledge was obtained in the diagnoses of M.tuberculosis in the placenta as it was found that micro abscesses and intervillositis were strong indicators of TB infection in the placenta, however, ZN staining still remains the gold standard for diagnosing M.tuberculosis infection in the placenta. PCR is found to have limitations, because only M.tuberculosis DNA is amplified and does not distinguish live from dead bacteria. The conclusion reached is that PCR is of limited value in the diagnosis of active M.tuberculosis infection in the placenta using FFPE tissue, while certain histological changes may be indicative of such infection; however confirmation of the organism by ZN staining is still essential. / AFRIKAANSE OPSOMMING: Die hoofdoelwit van hierdie projek was om vas te stel of moeders met bevestigde of vermoedelike TB enige indikasie van TB in die plasenta toon. ‘n Tweede doelwit was om die neonatale uitkoms teenoor die plasentale TB te korreleer. ‘n Totale getal van 56 plasentas is ondersoek om vas te stel of daar enige spesifieke histopatologiese indikasies is van tuberkulose met die hulp van die ZN spesiale kleuring. Die totale getal positiewe vir TB was 30 asook ‘n vals positiewe geval vir TB en daar was 25 TB negatiewe gevalle. Ses en vyftig biopsies is versamel van paraffien in gebedteerde weefsel vir die gebruik in PKR. Die uitvoering van hierdie twee diagnostiese modaliteite is elk individueel ondersoek asook gesamentlik om dit te vergelyk met die neonatale uitkoms (m.a.w die teenwoordigheid of aanwesigheid van mikobakteriale tuberkulose infeksie) asook die teenwoordigheid van moederlike pulmunere en ekstra-pulmunere tuberkulose. Die spesifieke ligging van die letsels in die plasenta (bv. membrane vs. intervillus spasie) kan lei tot verbeterde begrip van die patogeniese meganismes betrokke in die moeder fetale oordrag van tuberkulose en dit kan lei tot toekomstige navorsing. Waardevolle kennis is opgedoen in die diagnose van M.tuberkulose in die plasenta, want die letsels van mikro abbesses en intervillisitus gee ‘n goeie aanduiding van TB infeksie in die plasenta. Die ZN kleuring bly nog steeds die standaard metode om M.tuberculose in die plasenta te diagnoseer. PKR het baie limiete want dit kan slegs die M.tuberkulose DNA vermeningvuldig, maar dit kan nie onderskeid tref tussen lewendige en dooie bakterie nie. The slotsom in hierdie projek is dat PKR ‘n be pperkte waarde het in die diagnose van aktiewe M .tuberkulose in die plasenta, deur die gebruik van formalien gefikseerde paraffien ingebedteerde weefsel nie terwyl sekere histologiese veranderinge ‘n aa nduiding van sodanige infeksie kan wees maar dat dit deur die spesiale kleruring (ZN) bevestig moet word. / National Health Laboratory Service (NHLS)

INTRODUÇÃO: A tuberculose é um problema grave de saúde pública, acometendo indivíduos em todas as faixas etárias e em todos os estratos socioeconômicos. Apesar de estarem sob grande risco de adoecimento, as crianças carecem de meios diagnósticos sensíveis e específicos. Neste estudo avaliou-se em crianças a acurácia de um teste baseado na dosagem de interferon-gama liberado por linfócitos após estímulo com antígenos específicos do Mycobacterium tuberculosis (QuantiFERON-TB Gold In Tube® [Cellestis, Carnegie, Austrália] ).MÉTODO: Foram incluídas no estudo 184 crianças não infectadas e 11 crianças com infecção pelo Mycobacterium tuberculosis. Todas as crianças receberam previamente o BCG. Foram excluídas crianças com comprometimento do sistema imunológico. Obteve-se amostra de sangue de cada criança, e o material foi processado conforme as instruções do laboratório fabricante. O desempenho do teste foi avaliado pela construção de uma curva de características operacionais (ROC). RESULTADOS: Do total de 184 crianças sem infecção pela micobatéria, 74 (40,2%) eram do sexo feminino, e 130 (70,6%) tinham menos de quatro anos de idade. A idade média neste grupo foi de 35 meses. Seis (3,2%) crianças apresentaram resultado indeterminado do teste, uma criança (0,5%) apresentou um resultado positivo e 177 (96,2%) apresentaram resultado negativo. No grupo de 11 crianças infectadas, sete (63,0%) eram meninas, e a idade média era de 58,5 meses. Duas (18,0%) crianças neste grupo apresentaram resultado negativo do teste. A curva ROC obtida evidenciou uma área sob a curva de 0,876 (I. C 95% - 0,82 a 0,92; p<0,001), refletindo o desempenho preditivo elevado do teste. A sensibilidade do teste foi de 81,8% (IC 95% - 48,2% a 97,2%) e a especificidade de 98,8% (IC 95% - 96,0 a 99,8%), o valor preditivo positivo foi de 81,8% (IC 95%: 46,3% a 97,4%) e o valor preditivo negativo foi de 98,9% (IC 95%: 96,0% a 99,8%). CONCLUSÕES: Neste estudo o teste mostrou ter uma boa acurácia no diagnóstico da infecção pelo Mycobaterium tuberculosis em crianças previamente vacinadas com o BCG, e sua utilização rotineira pode contribuir para a melhor avaliação de crianças expostas a um doente bacilífero e na tomada de decisões sobre a introdução de quimioprofilaxia ou tratamento. / BACKGROUND: Tuberculosis is a major public health problem, affecting people from all ages and diverse socioeconomic incomes. Despite the high risk that children have to develop the disease, accurate methods for diagnosis are not yet available. In this study the accuracy of an interferon-gamma release assay (QuantiFERON-TB Gold In Tube® [Cellestis, Carnegie, Australia]) was evaluated for the diagnosis of Mycobacterium tuberculosis infection in children. METHODS: 195 children were evaluated, 184 children without mycobacterial infection, and 11 children infected by the Mycobacterium tuberculosis. All the children had been previously vaccinated with BCG. Immunocompromised children were excluded from the study. A blood sample was obtained from each child, and it was processed according the manufacturer´s instructions. The performance of the assay was evaluated by a receiver operating characteristic (ROC) curve. RESULTS: In the group of 184 noninfected children, 74 (40.2%) were female and 130 (70.6%) were younger than four years old. The mean age in this group was 35 months. Six children (3.2%) had indeterminate test result, one child (0.5%) had a positive test result, and 177 (96.2%) children had negative test results. In the group of 11 infected children, seven (63.0%) were female, and the mean age in this group was 58.5 months. Two children (18.0%) in this group had a negative test result. The ROC curve determined an area under the curve of 0.876 (95% CI 0.82 to 0.92; p< 0.001), disclosing a high positive predictive value for the test. The assay sensibility was 81.8% (95% CI, 48.2% to 97.2%) and the assay specificity was 98.8% (95% CI, 96.0% to 99.8%), the positive predictive value was 81.8% (95% CI: 46.3% to 97.4%) and the negative predictive value was 98.9% (95% CI: 96.0% to 99.8%). CONCLUSIONS: In this study, the accuracy of the assay was high for the diagnosis of Mycobacterium tuberculosis infection in children previously vaccinated with BCG. The use of this assay for the routine evaluation of children exposed to the disease may help physicians to decide on whether to start chemoprophylaxis or tuberculosis treatment.

INTRODUÇÃO: A tuberculose é um problema grave de saúde pública, acometendo indivíduos em todas as faixas etárias e em todos os estratos socioeconômicos. Apesar de estarem sob grande risco de adoecimento, as crianças carecem de meios diagnósticos sensíveis e específicos. Neste estudo avaliou-se em crianças a acurácia de um teste baseado na dosagem de interferon-gama liberado por linfócitos após estímulo com antígenos específicos do Mycobacterium tuberculosis (QuantiFERON-TB Gold In Tube® [Cellestis, Carnegie, Austrália] ).MÉTODO: Foram incluídas no estudo 184 crianças não infectadas e 11 crianças com infecção pelo Mycobacterium tuberculosis. Todas as crianças receberam previamente o BCG. Foram excluídas crianças com comprometimento do sistema imunológico. Obteve-se amostra de sangue de cada criança, e o material foi processado conforme as instruções do laboratório fabricante. O desempenho do teste foi avaliado pela construção de uma curva de características operacionais (ROC). RESULTADOS: Do total de 184 crianças sem infecção pela micobatéria, 74 (40,2%) eram do sexo feminino, e 130 (70,6%) tinham menos de quatro anos de idade. A idade média neste grupo foi de 35 meses. Seis (3,2%) crianças apresentaram resultado indeterminado do teste, uma criança (0,5%) apresentou um resultado positivo e 177 (96,2%) apresentaram resultado negativo. No grupo de 11 crianças infectadas, sete (63,0%) eram meninas, e a idade média era de 58,5 meses. Duas (18,0%) crianças neste grupo apresentaram resultado negativo do teste. A curva ROC obtida evidenciou uma área sob a curva de 0,876 (I. C 95% - 0,82 a 0,92; p<0,001), refletindo o desempenho preditivo elevado do teste. A sensibilidade do teste foi de 81,8% (IC 95% - 48,2% a 97,2%) e a especificidade de 98,8% (IC 95% - 96,0 a 99,8%), o valor preditivo positivo foi de 81,8% (IC 95%: 46,3% a 97,4%) e o valor preditivo negativo foi de 98,9% (IC 95%: 96,0% a 99,8%). CONCLUSÕES: Neste estudo o teste mostrou ter uma boa acurácia no diagnóstico da infecção pelo Mycobaterium tuberculosis em crianças previamente vacinadas com o BCG, e sua utilização rotineira pode contribuir para a melhor avaliação de crianças expostas a um doente bacilífero e na tomada de decisões sobre a introdução de quimioprofilaxia ou tratamento. / BACKGROUND: Tuberculosis is a major public health problem, affecting people from all ages and diverse socioeconomic incomes. Despite the high risk that children have to develop the disease, accurate methods for diagnosis are not yet available. In this study the accuracy of an interferon-gamma release assay (QuantiFERON-TB Gold In Tube® [Cellestis, Carnegie, Australia]) was evaluated for the diagnosis of Mycobacterium tuberculosis infection in children. METHODS: 195 children were evaluated, 184 children without mycobacterial infection, and 11 children infected by the Mycobacterium tuberculosis. All the children had been previously vaccinated with BCG. Immunocompromised children were excluded from the study. A blood sample was obtained from each child, and it was processed according the manufacturer´s instructions. The performance of the assay was evaluated by a receiver operating characteristic (ROC) curve. RESULTS: In the group of 184 noninfected children, 74 (40.2%) were female and 130 (70.6%) were younger than four years old. The mean age in this group was 35 months. Six children (3.2%) had indeterminate test result, one child (0.5%) had a positive test result, and 177 (96.2%) children had negative test results. In the group of 11 infected children, seven (63.0%) were female, and the mean age in this group was 58.5 months. Two children (18.0%) in this group had a negative test result. The ROC curve determined an area under the curve of 0.876 (95% CI 0.82 to 0.92; p< 0.001), disclosing a high positive predictive value for the test. The assay sensibility was 81.8% (95% CI, 48.2% to 97.2%) and the assay specificity was 98.8% (95% CI, 96.0% to 99.8%), the positive predictive value was 81.8% (95% CI: 46.3% to 97.4%) and the negative predictive value was 98.9% (95% CI: 96.0% to 99.8%). CONCLUSIONS: In this study, the accuracy of the assay was high for the diagnosis of Mycobacterium tuberculosis infection in children previously vaccinated with BCG. The use of this assay for the routine evaluation of children exposed to the disease may help physicians to decide on whether to start chemoprophylaxis or tuberculosis treatment.

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2017. / According to the Namibia National Tuberculosis Control Programme (NTCP) report of 2008, Namibia has one of the highest TB infection rates in the world with a case notification rate of 748/100,000. Rapid, specific and sensitive diagnosis of Mycobacterium tuberculosis (MTB) is needed for correct TB patient management. One of the aims of this study was thus to compare direct microscopy with two rapid molecular diagnostic tools (viz. GeneXpert MTB/RIF and Hain Genotype® MTBDR plus assay) for the identification of MTB from samples collected from the Khomas Region, Windhoek, Namibia. Only patients with positive TB sputum collected at the clinics and health facilities in the Khomas Region, Windhoek were eligible for the study. Three hundred and eighty-four samples were confirmed acid-fast positive by utilising the auramine staining method. The rifampicin (RIF) resistance profile detected by both molecular techniques was then compared for characterisation of the samples as drug resistant. Lastly, participants completed a survey, which included questions related to demographic and epidemiological data. Demographic data included patient age, gender, region of residence and history of treatment. The data was collected using a structured questionnaire and was captured in an Excel spreadsheet. It was then imported into Statistical Package for Social Sciences (SPSS) Version 25 for data analysis. A memorandum of understanding was also signed with the Namibia Institute of Pathology (NIP) to obtain permission to use their samples and the equipment at their site.

Les méthodes pour le diagnostic des infections à mycobactéries sont restées pratiquement inchangées pendant des décennies et n'auraient probablement pas progressé sans la réémergence inattendue de la tuberculose (TB) au cours des vingt dernières années du 20ème siècle. Le diagnostic de laboratoire de la tuberculose pulmonaire repose principalement sur la détection des organismes du complexe Mycobacterium tuberculosis (MTC) dans les prélèvements respiratoires par leur isolement et identification ou par les méthodes moléculaires. Pour les patients qui n’expectorent pas, des échantillons du tractus respiratoire peuvent être obtenus par des procédures invasives. En nous basant sur la survie connue de MTC dans le liquide gastrique, nous avons considéré que les MTC devraient également être détectables dans les selles. Nous avons recherché des MTC en parallèle dans des échantillons de tractus respiratoire et dans les selles récoltés des mêmes patients. Mycobacterium tuberculosis a été détecté après décontamination à la chlorhexidine dans des cultures sur des milieux à base d'œuf et par l'examen direct microscopique après coloration de Ziehl-Neelsen. Après la mise au point d’une technique originale d’extraction de l’ADN total à partir des selles, nous avons utilisé la détection de M. tuberculosis par PCR en temps réel en duplex utilisant le gène IS6110 comme cible moléculaire et un contrôle interne de présence d’inhibiteurs de la PCR. Ces travaux mettent en évidence l’intérêt et la sensibilité de l’analyse des selles pour le diagnostic de la tuberculose pulmonaire. Cette technique est aujourd’hui utilisée dans notre laboratoire en routine et est en cours d’évaluation par d’autres équipes.Actuellement, l'identification des mycobactéries basée sur l’analyse des caractéristiques biochimiques (test à l’uréase, test de la nitrate réductase, test de la résistance à la pyrazinamide (pza), test de la sensibilité, test de la résistance à la thiophen-2-carboxylic acid hydrazide (TCH) etc …) est le plus souvent remplacée par des méthodes de biologie moléculaire dont l’amplification par PCR d'un gène spécifique. Ces méthodes sont laborieuses et peuvent exiger un degré d'expertise élevé. Afin de simplifier l'identification précise des isolats de mycobactéries en laboratoire de routine, après avoir vérifié l’inactivation des MTC par la chaleur et l’éthanol pour pouvoir travailler hors du laboratoire P3, nous avons mis au point les conditions pour l'utilisation de la spectrométrie de masse « matrix-assisted laser desorption and ionisation-time-of-flight» (MALDI-TOF-MS) en association avec un logiciel bioinformatique spécifique, comme outil d’identification et de différenciation entre les membres du complexe MTC, du complexe Mycobacterium avium et les autres mycobactéries non-tuberculeuses. Nous avons ensuite appliqué cette technique afin d’identifier les mycobactéries à partir du milieu liquide Middlebrook 7H10 utilisé dans une étuve automatisée. L’identification des mycobactéries isolées dans notre laboratoire, est maintenant réalisée en routine par MALDI-TOF-MS. L’ensemble de ces travaux contribue à améliorer le diagnostic de routine de la tuberculose pulmonaire et les techniques mises au point sont utilisées en routine dans notre laboratoire, en particulier dans le cadre d’un « kit tuberculose ». / The diagnosis of mycobacterial infections remained practically unchanged during numerous decades and would not probably have progressed of the whole without the unexpected re-emergence of the tuberculosis (TB) during the last twenty years of the 20th century.The diagnosis of laboratory of pulmonary tuberculosis is mainly based on the detection of microorganisms of the Mycobacterium tuberculosis complex (MTC) in the respiratory specimens. For the patients who do not expectorate, samples of the respiratory tract can be only obtained according to invasive procedures. Based on the survival known for MTC organisms in the gastric liquid, we considered that MTC organisms would be detectable also in samples of stools. We compared the presence of bodies MTC in samples of respiratory tract and the specimens of stools collected from the same patients. MTC was detected in cultures on egg-based media after appropriate decontamination using the chlorhexidine and by the microscopic examination after Ziehl-Neelsen staining. After the set on of an original protocol of the total DNA extraction from stools, we were able to use by the detection with the real-time PCR of IS6110 using internal controls as PCR inhibitor control. This protocol illustrate the utility of stool analysis for the diagnosis of pulmonary tuberculosis, this technique is actually used in routine in our laboratory, it is also under investigation by other research teams.Phenotypic identification of mycobacteria based on the analysis of biochemical pattern (urase test, nitrate reductase test, resistance to pyrazinamide (pza) test, susceptibility to thiophen-2-carboxylic acid hydrazide (TCH) test, etc …) is more often replaced by molecular methods using DNA, including the development by PCR of a specific gene. These methods are generally laborious and can require a considerable degree of expertise. To simplify the identification specifies isolates of mycobacteria routinely in the laboratory, we estimated the use of the mass spectrometry "matrix-assisted laser desorption and ionization-time-of-flight" (MALDI-TOF MS) in association with a specific bioIT software, was capable of identifying and of distinguishing between the members of Mycobacterium tuberculosis complex, Mycobacterium avium complex and the non- tuberculosis mycobacteria. We verified the MTC inactivation by heating or by ethanol allowing the analysis of the inactivated MTC out of the P3 laboratory; we studied the cost-effectiveness of this method from the blood solid media by extrapolating this approach in the emergent countries. We then applied the same method to identify mycobacteria from Middlebrook 7H10 liquid media used in an automated oven. Identification of cultured Mycobacteria is now done in routine in our laboratory by MALDI-TOF-MS. These data contribute in improving the routine diagnosis of pulmonary tuberculosis and this protocol is then used routinely in our laboratory, particularly with the “kit de tuberculose” we set on.

Introduction: World Health Organization recommends screening of household contact as a key to improve detection of tuberculosis cases. Ethiopia’s current tuberculosis household contact investigation strategies rely on symptomatic contacts attending health facilities for investigation. This approach has not led to the detection of additional tuberculosis (TB) cases; alternative approaches have to be considered. The purpose of the research was to develop guidelines in endorsing the implementation of a community based household contact investigation program in Addis Ababa. Methods: A mixed method research using sequential exploratory design was conducted in Addis Ababa. In the first phase, qualitative data collection and analysis methods were used to formulate intervention approach and in the second phase, a quantitative random controlled trial was conducted, with the purpose of comparing the proposed intervention measures with routine household contact tuberculosis investigation. Frequencies and logistic regression analyses were used to determine the relative risk and associated factors. Thematic analysis was used for qualitative data analysis. Results: The in-depth interview and focus group discussion findings identified themes namely household contact investigation (HHCI) implementation, misconceptions on HHCI, challenges with HHCI Approaches, opportunities for HHCI provision, contributing factors associated with household involvement, strategies for effective HHCI service and partnerships with health bureau. In phase two, the study reported that the prevalence of TB was 7.1% among the intervention group compared to 1.9% in the control groups at the end of first year follow-up. Nine guidelines were developed to support the household contact investigation system. Conclusion: The passive case detection strategy of contact investigation did not find more cases, and tuberculosis patients and their family contacts were not satisfied with this method. However, the proposed community-based strategy shows that more TB cases can be detected by using existing medical staff. Therefore, an approach that makes the service more accessible is significant and the recommended community based TB household contact tracing approaches needs to be scaled up for its performance towards identified missed cases and enhance patient and their household contacts involvement. / Health Studies / D. Litt. et Phil. (Public Health)

Collaborative TB/HIV management is essential to prevent and treat TB among HIV-positive TB patients, and to ensure that HIV-positive TB patients are detected and treated appropriately. This quantitative, descriptive, contextual study identified problems encountered during the implementation of TB case detection among HIV-positive individuals in one Ethiopian hospital. During December 2012, 300 checklists were completed about HIV-positive patients’ TB/HIV collaborative management, as reflected in their files. Only 60.2% of HIV-positive patients, who should have received Isoniazid preventive treatment (IPT), were placed on this treatment. X-rays and laboratory examinations of sputum samples were not done according to the Ethiopian guidelines. Most TB patients’ initial screening was done by nurses, not doctors, and included only symptom screening without CD4 count considerations. Managers and healthcare personnel should improve IPT, especially for those with early HIV infection and timely effective treatment for those suffering from TB, before complications arise / Health Studies / Health Studies / M.A. (Public Health)

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The tuberculosis (TB) incidence has increased in Southern Africa and the situation is worsened by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular biological techniques have been used to understand the disease dynamics of TB. In a series of studies we describe the use of these techniques to understand the disease dynamics of TB in Southern Africa. Using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) to characterize M. tuberculosis strains from TB patients in Zimbabwe, we identified a genotype causing a disproportionate number of TB cases. The genotype belonged to the Latin American Mediterranean (LAM) lineage and we named it the Southern Africa1 (SAF1) family and later renamed it SAF1/RDRio, also reflecting its predominance in South America. To establish if this family of strains was predominant elsewhere in Southern Africa, genotypes were compared to those from Western Cape, South Africa and Zambia. The SAF1/RDRio strains were highly prevalent in Zambia but were only a minor fraction of the strains in South Africa. The geographical distribution of SAF1/RDRio strains was determined in Gweru, Zimbabwe, and was found to be spread in high incidence areas. From these two studies it was hypothesized that certain host and bacterial factors were associated with disease due to SAF1/RDRio. Subsequently potential risk factors and clinical outcomes of disease due to SAF1/RDRio strains were explored. An association was found with smoking and cavitary pulmonary disease suggesting that SAF1/RDRio caused a more severe and highly transmissible formof TB Using IS6110-RFLP, principal genetic grouping, spoligotyping, IS6110 insertion-site mapping and variable-number tandem repeats (VNTR) typing, low IS6110 copy clade (LCC) identified in Zimbabwe were characterized and compared to the strains from Cape Town, South Africa and other regions. The LCC strains from Cape Town, South Africa, were found to have close evolutionary relationship with strains from Zimbabwe and other regions and were widely distributed suggesting they play an important role in the global TB epidemic. Observations from these studies and those from other studies led to the hypothesis that specific genotypes of M. tuberculosis predominate in regions of Southern Africa. To gain an insight on the population structure of M. tuberculosis strains in Southern Africa, spoligotyping and/or IS6110-RFLP data from eight countries were compared. This is the first study to describe the M. tuberculosis population structure in Southern Africa. Distinct genotypes were associated with specific geographic regions. These findings have important implications for TB diagnostics, anti-TB drug and vaccine development. The population structure of multidrug-resistant (MDR), pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) M. tuberculosis isolates from provinces in South Africa was also determined. This is again the first study to describe the population structure of drug-resistant M. tuberculosis in South Africa. The results also showed geographic localization of genotypes and an association with resistance class. However, decreasing strain diversity was observed as the isolates evolved from MDR-TB to XDR-TB suggesting selection for the specific genotypes. These findings highlight the importance of identifying genetic markers in drug-resistant strains, to enhance early detection of those at risk of developing XDR-TB. / AFIKAANSE OPSOMMING: Die voorkoms van tuberkulose (TB) in Suider Afrika word vererger deur stamme van Mycobacterium tuberculosis wat weerstandig is teen die beskikbare anti-tuberkulose middels. Molekulêre tegnieke word gebruik om in hierdie reeks studies die dinamika van TB in Suider Afrika te ondersoek Deur spoligotipering en IS6110 restriksie fragment lengte polimorfisme (RFLP) tegnieke te gebruik om M. tuberculosis stamme van pasiente in Zimbabwe te beskryf, het ons ‘n genotipe gevind wat ‘n buitengewone aantal TB gevalle veroorsaak het. Hierdie genotipe is deel van die internasionaal beskryfde Latyns Amerikaase en Meditereense (LAM) stam familie. Ons het dit die Suider Afrikaanse Familie1 (SAF1) genoem, maar later hernoem na SAF1/RDRio, omdat dieselfde genotipe in ook volop is in Suid Amerika. Om vas te stel of hierdie familie ook oorheesend is in die res van Suider Afrika, is dit vergelyk met beskikbare databasisse van die Wes-Kaap, Suid-Afrika en Zambië. Alhoewel SAF1/RDRio in die Wes-Kaap gevind is, dra dit slegs tot ‘n mindere mate by tot die plaaslike TB epidemie. Aan die anderkant kom SAF1/RDRio baie algemeen in Zambië voor. ‘n Verdere studie wys ook dat die SAF1/RDRio familie eweredig en wyd verspreid voorkom in hoë insidensie gebiede in Gweru, Zimbabwe. Vanuit die bevindings van hierdie 2 studies, kan ons aflei dat sekere gasheer- en bakteriële eienskappe geassosieer is met SAF1/RDRio-TB-infeksie. Hierna is potensiële risiko faktore en kliniese uitkomste van siekte as gevolg van infeksie met SAF1/RDRio ondersoek. ‘n Assosiasie met rook en kaviterende pulmonale infeksie is gevind,wat daarop dui dat SAF1/RDRio erger vorm van TB veroorsaak en hoogs oordraagbaar is. Deur gebruik te maak van IS6110- (RFLP), hoof groep groepering, spoligotipering, IS6110 invoegings kaartering en veranderlike getal tandem herhaling (VNTR) tipering kon lae IS6110 invoeginsgetal (LCC) stamme van Kaapstad, Zimbabwe en ander gebiede vergelyk word. Al die LCC stamme in die studie is evolusionêr naby verwant aan mekaar en is wyd verspreid, wat dui op hulle belangrike rol in die wêreldwye TB epidemie. Waarnemings in hierdie asook ander studies het tot die hipotese gely dat spesifieke genotipes van M. tuberculosis dominant is in verskillende gebiede van Suider Afrika. Om meer insig tot die populasie samestelling van M. tuberculosis stamme in Suider Afrika in te win is spoligotipes en RFLP-data van 8 lande vergelyk. Hierdie is die eerste studie om die populasie samestelling van M. tuberculosis in Suider Afrika te beskryf en is belangrike fir toekomstige ontwikkeling van nuwe TB diagnose tegnieke, anti-TB middels en TB entstowwe. Die populasie samestelling van multiweerstandige (MDR), pre-ekstreme weerstandige (pre-XDR) en ekstreme weerstandige (XDR) M. tuberculosis van verskillende provinsies in Suid-Afrika is ook bepaal. Hierdie studie is ook die eerste wat die populasie samestelling van weerstandige M. tuberculosis in Suid-Afrika beskryf. Die resultate wys geografiese lokalisering van genotipes en ‘n assosiasie met weerstandigheidsklas. ‘n Afname in stam diversiteit soos die isolate van MDR-TB tot XDR-TB ontwikkel, dui op seleksie van spesifieke genotipes. Hierdie bevinding lê die klem op die belangrikheid van die identifisering van genetiese merkers in weerstandige stamme om die risiko vir die ontwikkeling van XDR-TB te verminder deur vroë deteksie.

Thesis (PhD)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative organism of tuberculosis (TB), is a major cause for mortality and morbidity world-wide with a death toll only second to HIV among infectious diseases. Drug resistance is widespread and cases of multiple drug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) have emerged in several countries. Drug treatment is problematic and new drugs are not developed rapidly enough to offset the rapid drug resistance mutation rate of M. tuberculosis. Simple and effective diagnostics are required to contain the spread of the disease as current routine diagnostics are not fulfilling this role. Additionally, current rapid TB diagnostics are out of reach to resource poor settings due to infrastructure, cost and skill requirements. Novel TB diagnostics are thus required that meet these requirements. Mycobacteriophages are phages that infect mycobacteria and could offer a viable and cost effective alternative rapid TB diagnostics. In this study, an affinity-tagged fluorescent reporter mycobacteriophage is described, which was engineered to act as a TB diagnostic. Its performance proved favourable and superior to current existing mycobacteriophage-based TB diagnostics. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme verantwoordelik vir tuberkulose (TB), is `n groot bron van mortaliteit en morbiditeit wêreldwyd en slegs HIV is verantwoordelik vir groter getalle sterftes as gevolg van n aansteeklike siekte. Middelweerstandigheid is algemeen en gevalle van meervoudigemiddelweerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) kom in verskeie lande voor. Antibiotika behandeling is problematies en nuwe anti-TB middels word nie vinnig genoeg ontwikkel om die antibiotika weerstandigheid mutasie spoed van M. tuberculosis te bekamp nie. Doeltreffende diagnostiese toetse word benodig om die verspreiding van die siekte te beheer en bestaande roetine diagnostiese toetse voldoen tans nie aan hierdie vereiste nie. Behalwe hiervoor, is huidige vinnige TB diagnostiese toetse buite bereik van arm instansies weens vereistes aan infrastruktuur, meegaande kostes en werknemervaardigheid. Nuwe TB diagnostiese toetse is dus nodig om aan hierdie vereistes te voldoen. Mikobacteriofaage is fage wat mikobacteria infekteer en kan moontlik 'n lewensvatbare en koste-effektiewe alternatief bied vir vinnige TB diagnostiese toetse. In hierdie studie word 'n affiniteitgekoppelde fluoreserende rapporteringsmikobakteriofaag beskryf wat ontwerp is om op te tree as `n nuwe vinnige TB diagnostiese toets. Die werking hiervan vertoon gunstige en beter resultate as die huidige, mikobacteriofaaggebaseerde TB-diagnostiese toetse.

Collaborative TB/HIV management is essential to prevent and treat TB among HIV-positive TB patients, and to ensure that HIV-positive TB patients are detected and treated appropriately. This quantitative, descriptive, contextual study identified problems encountered during the implementation of TB case detection among HIV-positive individuals in one Ethiopian hospital. During December 2012, 300 checklists were completed about HIV-positive patients’ TB/HIV collaborative management, as reflected in their files. Only 60.2% of HIV-positive patients, who should have received Isoniazid preventive treatment (IPT), were placed on this treatment. X-rays and laboratory examinations of sputum samples were not done according to the Ethiopian guidelines. Most TB patients’ initial screening was done by nurses, not doctors, and included only symptom screening without CD4 count considerations. Managers and healthcare personnel should improve IPT, especially for those with early HIV infection and timely effective treatment for those suffering from TB, before complications arise / Health Studies / Health Studies / M.A. (Public Health)