Trotz der weltweit 14 Millionen Erkrankten ist die Diagnostik der Tuberkulose schwierig und langwierig - eine Therapie beeinträchtigt die Patienten unter Umständen über einen langen Zeitraum. Interferon-γ-Release-Assays (IGRAs) sollten bei ihrer Einführung im Jahr 2005 diagnostische Unsicherheiten ausräumen helfen, jedoch blieb der genaue diagnostische Wert des Tests, vor allem in Kliniken mit einer hohen Prä-Test-Wahrscheinlichkeit, unklar. Die Wertigkeit dieses Testverfahrens in der Lungenfachklinik Immenhausen zu untersuchen war daher Aufgabe der vorliegenden Arbeit. Dazu wurden von 2009 bis 2012 in dieser Klinik 112 Krankheitsfälle mit Tuberkulose retrospektiv untersucht und ausgewertet. Dabei ergab sich für den QuantiFERON®-TB Gold-Test ein positiv prädiktiver Wert von 84,8% und eine Sensitivität von 88,9%, wenn man als Referenz alle zugelassenen Nachweisverfahren heranzog. Die Sensitivität des QuantiFERON®-TB Gold war signifikant höher als die des Tuberkulin-Hauttests (p=0,0008) und der Sputum-Anreicherung (p<0,0001), während sich Kultur und QuantiFERON®-TB Gold -Test nicht signifikant unterschieden (p=0,1435). Das Ergebnis des Tuberkulin-Hauttests (in mm) und die prozentuale Auswertung des QuantiFERON®-TB Gold-Tests ließen sich in eine Korrelation bringen (p=0,0828), allerdings wären für eine Einordnung dieses Zusammenhangs mehr Falldaten vonnöten. Die Analyse der falsch-negativen Quantiferon-Tests lieferte individuelle Erklärungsmöglichkeiten, jedoch keine regelhafte Ursache. Diese Ergebnisse lassen den Schluss zu, dass es sich beim QuantiFERON®-TB Gold-Test um eine gute, der Anreicherung und dem Tuberkulin-Hauttest überlegene, jedoch nicht den Goldstandard „Kultur“ verdrängende Methode zum Nachweis einer Tuberkulose handelt.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os profissionais da área da saúde formam um dos grupos mais vulneráveis à infecção pelo Mycobacterium tuberculosis (Mtb). Segundo estimativas da Organização Mundial de Saúde (OMS), 8,8 milhões de pessoas estavam infectadas pelo Mtb e ocorreram 1,4 milhão de óbitos por tuberculose (TB) em 2010. A identificação de pessoas com Infecção Latente Tuberculosa (ILTB) é considerada pela OMS como uma prioridade no controle da doença, especialmente em países em desenvolvimento em que a incidência da doença ativa tem apresentado redução. O objetivo do presente trabalho foi avaliar, no Brasil, o custo-efetividade dos testes Prova Tuberculínica (PT) e Quantiferon TB Gold-In-Tube (QTF-GIT) no diagnóstico e tratamento da ILTB em profissionais de saúde atuantes na atenção básica, sob a perspectiva do Sistema Único de Saúde (SUS), comparando cinco estratégias que incluem o QTF-GIT, distintos pontos de corte para a PT e uso sequencial dos dois testes; e analisar o impacto do tabagismo sobre o risco de ILTB entre os profissionais de saúde, destacando-se a categoria da Enfermagem. Foi realizada uma avaliação econômica completa do tipo custo-efetividade, conduzida considerando uma coorte hipotética de 10.000 profissionais de saúde atuantes na atenção básica, com horizonte temporal restrito a um ano. Um modelo analítico de decisão, caracterizado por uma árvore de probabilidades de eventos, foi desenvolvido utilizando o software TreeAge ProTM 2013 para simular os resultados clínicos e impactos econômicos em saúde da nova tecnologia diagnóstica (QTF-GIT) versus a PT tradicional. Esse modelo simulou cinco estratégias diagnósticas para detecção e tratamento da ILTB: (a) PT, usando ponto de corte de 5mm; (b) PT, usando ponto de corte de 10 mm; (c) teste QTF-GIT; (d) PT, com ponto de corte de 5mm, seguida de teste QTF-GIT quando PT positiva; (e) PT, com ponto de corte de 10mm, seguida de teste QTF-GIT quando PT positiva. Foi realizada análise de sensibilidade determinística univariada. Na determinação dos fatores associados à ILTB, foi elaborado um modelo de regressão logística múltipla com seleção hierarquizada, utilizando o software Stata. A estratégia mais custo-efetiva foi a PT no ponto de corte &#8805;10mm, considerando como medida de desfecho tanto o número de indivíduos corretamente classificados pelos testes assim como o número de casos de TB evitados. A utilização isolada do QTF-GIT revelou-se a estratégia de menor eficiência, com RCEI= R$ 343,24 por profissional corretamente classificado pelo teste. Encontrou-se risco à ILTB significantemente maior para sexo masculino [OR=1,89; IC 95%:1,11-3,20], idade &#8805; 41 anos [OR=1,56; IC 95%: 1.09-2,22], contato próximo com familiar com TB [OR=1,55; IC 95%: 1.02-2,36], status do tabagismo fumante [OR=1,75; IC 95%: 1.03-2,98] e categoria profissional da Enfermagem [OR=1,44; IC 95%: 1.02-2,03]. Concluiu-se que a PT no ponto de corte de 10mm é a estratégia diagnóstica mais custo-efetiva para ILTB entre os profissionais de saúde na atenção básica e que a ILTB está associada ao hábito do tabagismo e à categoria profissional de Enfermagem. / Health professionals form one of the groups most vulnerable to infection by Mycobacterium tuberculosis (Mtb). According to estimates by the World Health Organization (WHO), 8.8 million people were infected with Mtb and were 1.4 million deaths from TB in 2010. The identification of persons with Latent Tuberculosis Infection (LTBI) is considered by WHO as a priority in the control of disease, especially in developing countries where the incidence of active disease has shown reduction. The aim of this study was to evaluate, in Brazil, the cost-effectiveness of tests Tuberculin Skin Test (TST) and Quantiferon TB Gold-In-Tube (QFT-GIT) in the diagnosis and treatment of LTBI in health professionals working in primary care from the perspective of SUS, comparing five strategies that include the QFT -GIT, different cutoff points for TST and sequential use of two tests; and analyze the impact of smoking on the risk of LTBI among health professionals, highlighting the category of Nursing. A full economic assessment of the type cost-effectiveness was performed, conducted considering a hypothetical cohort of 10,000 health professionals working in primary care, with limited time horizont of one year. A decision analytical model, characterized by a tree of probabilities of events, was developed using the TreeAge ProTM software 2013 (TreeAge Software Inc, Williamstown, MA, USA) to simulate the clinical and economic impacts on health of new diagnostic technology (QFT -GIT) versus the traditional TST. This model simulated five diagnostic strategies for detection and treatment of LTBI (a) TST, using a cut-off of 5 mm; (B) TST, using 10 mm cut-off currently recommended by the TNP; (C) QFT-GIT test; (D) TST, with a cut-off of 5 mm, followed by QFT-GIT test when positive TST; (E) TST, with a cut-off point of 10 mm, followed by QFT-GIT test when positive TST. Univariate deterministic sensitivity analysis was performed to assess the robustness of the results. In determining the factors associated with LTBI, a multiple logistic regression model with hierarchical selection was made, using the Stata software. TST strategy at the cut-off &#8805; 10mm was the most cost-effective strategy, while the QFT-GIT alone was the most effective strategy, but showed higher cost. It was found to significantly greater risk for LTBI male [OR = 1.89; 95% CI: 1.11 to 3.20], age &#8805; 41 years [OR = 1.56; 95% CI: 1.09-2,22], close contact with a family with TB [OR = 1.55; 95% CI: 1.02-2,36], the smoker smoking status [OR = 1.75; 95% CI: 1.03-2,98] and professional nursing category [OR = 1.44; 95% CI: 1.02-2,03]. It was concluded that TST in 10mm cut-off is the diagnostic strategy more cost-effective for LTBI among health professionals in primary care and that LTBI is associated with the smoke and professional category nurse.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os profissionais da área da saúde formam um dos grupos mais vulneráveis à infecção pelo Mycobacterium tuberculosis (Mtb). Segundo estimativas da Organização Mundial de Saúde (OMS), 8,8 milhões de pessoas estavam infectadas pelo Mtb e ocorreram 1,4 milhão de óbitos por tuberculose (TB) em 2010. A identificação de pessoas com Infecção Latente Tuberculosa (ILTB) é considerada pela OMS como uma prioridade no controle da doença, especialmente em países em desenvolvimento em que a incidência da doença ativa tem apresentado redução. O objetivo do presente trabalho foi avaliar, no Brasil, o custo-efetividade dos testes Prova Tuberculínica (PT) e Quantiferon TB Gold-In-Tube (QTF-GIT) no diagnóstico e tratamento da ILTB em profissionais de saúde atuantes na atenção básica, sob a perspectiva do Sistema Único de Saúde (SUS), comparando cinco estratégias que incluem o QTF-GIT, distintos pontos de corte para a PT e uso sequencial dos dois testes; e analisar o impacto do tabagismo sobre o risco de ILTB entre os profissionais de saúde, destacando-se a categoria da Enfermagem. Foi realizada uma avaliação econômica completa do tipo custo-efetividade, conduzida considerando uma coorte hipotética de 10.000 profissionais de saúde atuantes na atenção básica, com horizonte temporal restrito a um ano. Um modelo analítico de decisão, caracterizado por uma árvore de probabilidades de eventos, foi desenvolvido utilizando o software TreeAge ProTM 2013 para simular os resultados clínicos e impactos econômicos em saúde da nova tecnologia diagnóstica (QTF-GIT) versus a PT tradicional. Esse modelo simulou cinco estratégias diagnósticas para detecção e tratamento da ILTB: (a) PT, usando ponto de corte de 5mm; (b) PT, usando ponto de corte de 10 mm; (c) teste QTF-GIT; (d) PT, com ponto de corte de 5mm, seguida de teste QTF-GIT quando PT positiva; (e) PT, com ponto de corte de 10mm, seguida de teste QTF-GIT quando PT positiva. Foi realizada análise de sensibilidade determinística univariada. Na determinação dos fatores associados à ILTB, foi elaborado um modelo de regressão logística múltipla com seleção hierarquizada, utilizando o software Stata. A estratégia mais custo-efetiva foi a PT no ponto de corte &#8805;10mm, considerando como medida de desfecho tanto o número de indivíduos corretamente classificados pelos testes assim como o número de casos de TB evitados. A utilização isolada do QTF-GIT revelou-se a estratégia de menor eficiência, com RCEI= R$ 343,24 por profissional corretamente classificado pelo teste. Encontrou-se risco à ILTB significantemente maior para sexo masculino [OR=1,89; IC 95%:1,11-3,20], idade &#8805; 41 anos [OR=1,56; IC 95%: 1.09-2,22], contato próximo com familiar com TB [OR=1,55; IC 95%: 1.02-2,36], status do tabagismo fumante [OR=1,75; IC 95%: 1.03-2,98] e categoria profissional da Enfermagem [OR=1,44; IC 95%: 1.02-2,03]. Concluiu-se que a PT no ponto de corte de 10mm é a estratégia diagnóstica mais custo-efetiva para ILTB entre os profissionais de saúde na atenção básica e que a ILTB está associada ao hábito do tabagismo e à categoria profissional de Enfermagem. / Health professionals form one of the groups most vulnerable to infection by Mycobacterium tuberculosis (Mtb). According to estimates by the World Health Organization (WHO), 8.8 million people were infected with Mtb and were 1.4 million deaths from TB in 2010. The identification of persons with Latent Tuberculosis Infection (LTBI) is considered by WHO as a priority in the control of disease, especially in developing countries where the incidence of active disease has shown reduction. The aim of this study was to evaluate, in Brazil, the cost-effectiveness of tests Tuberculin Skin Test (TST) and Quantiferon TB Gold-In-Tube (QFT-GIT) in the diagnosis and treatment of LTBI in health professionals working in primary care from the perspective of SUS, comparing five strategies that include the QFT -GIT, different cutoff points for TST and sequential use of two tests; and analyze the impact of smoking on the risk of LTBI among health professionals, highlighting the category of Nursing. A full economic assessment of the type cost-effectiveness was performed, conducted considering a hypothetical cohort of 10,000 health professionals working in primary care, with limited time horizont of one year. A decision analytical model, characterized by a tree of probabilities of events, was developed using the TreeAge ProTM software 2013 (TreeAge Software Inc, Williamstown, MA, USA) to simulate the clinical and economic impacts on health of new diagnostic technology (QFT -GIT) versus the traditional TST. This model simulated five diagnostic strategies for detection and treatment of LTBI (a) TST, using a cut-off of 5 mm; (B) TST, using 10 mm cut-off currently recommended by the TNP; (C) QFT-GIT test; (D) TST, with a cut-off of 5 mm, followed by QFT-GIT test when positive TST; (E) TST, with a cut-off point of 10 mm, followed by QFT-GIT test when positive TST. Univariate deterministic sensitivity analysis was performed to assess the robustness of the results. In determining the factors associated with LTBI, a multiple logistic regression model with hierarchical selection was made, using the Stata software. TST strategy at the cut-off &#8805; 10mm was the most cost-effective strategy, while the QFT-GIT alone was the most effective strategy, but showed higher cost. It was found to significantly greater risk for LTBI male [OR = 1.89; 95% CI: 1.11 to 3.20], age &#8805; 41 years [OR = 1.56; 95% CI: 1.09-2,22], close contact with a family with TB [OR = 1.55; 95% CI: 1.02-2,36], the smoker smoking status [OR = 1.75; 95% CI: 1.03-2,98] and professional nursing category [OR = 1.44; 95% CI: 1.02-2,03]. It was concluded that TST in 10mm cut-off is the diagnostic strategy more cost-effective for LTBI among health professionals in primary care and that LTBI is associated with the smoke and professional category nurse.

Chapter 1: Introduction to the luminescent properties of lanthanides Luminescence properties of trivalent lanthanides have been explored extensively over the past few decades owing to their unique properties. Lanthanides emission is known to be due to intra-configurational f-f transitions. Because the partially filled 4f shell is well shielded from its 26 environment by the closed 5sand 5pshells, the ligands in the first and second coordination sphere perturb the electronic configurations of the trivalent lanthanide ions only to a very limited extent. This leads to interesting properties such as long lifetimes, sharp line-like emissions etc. which in turn make lanthanides very attractive choice for commercial optical applications. Despite this, the scope of applications remained limited because of the low molar extinction coefficient values of the forbidden lanthanide f-f transitions. However, this problem has been successfully addressed by complexing the lanthanide ion with suitable ligands which can sensitize it resulting in a significant increase in the emission intensity (so called “antenna effect”). The strategy worked very well and resulted in widespread applications of lanthanides form biology to optoelectronics. This chapter discusses elementary ideas regarding the mechanism of sensitization and relevant examples that traces various applications of such lanthanide complexes from the current literature. Chapter 2: A self-assembled Europium Cholate hydrogel: a novel approach towards lanthanide sensitization Luminescent lanthanides can be of great value in a number of possible applications but their scope is limited by their intrinsic low molar absorptivities. Though this problem can be circumvented by complexing the lanthanide ion with suitable chelating ligands to improve the luminescence properties drastically, the design of such systems often involves meticulous planning and laborious synthetic steps to obtain a ligand suitable for the job. It is therefore desirable to have a simpler version of a sensitizing system that does not require the complexities of a chelating ligand but can sensitize trivalent lanthanides with comparable efficiency. It was observed in our group that divalent metal ions (Ni2+, Zn2+, Cu2+, Coetc.) form hydrogels on addition of sodium cholate. We extended to obtain hydrogels of trivalent lanthanides. Furthermore, when the gel was doped with pyrene, a ten-fold increase in the intensity of Eu(III) emission was observed (Fig 2). Thus we established a unique way to sensitize lanthanides in a hydrogel media by non-coordinating chromophores. The approach was completely modular in nature and avoids any laborious synthesis. We also tried other derivatives of pyrene as sensitizers and found that 1-pyreneboronic acid also caused similar sensitization of Eu(III). Fig 2. (a) Schematic representation of the sensitization process (the arrangement of molecules in the gel fiber is arbitrary). Eu-cholate (5 mM/15 mM) gel (a) normal light and (b) 354 nm UV excitation in the presence of 6 μM pyrene Further studies revealed, that 2,3-dihydroxynapthalene (DHN) can sensitize Tb(III) in a similar hydrogel. We also demonstrated Tb(III) to Eu(III) energy transfer process occurring in the gel when doped with DHN. This allowed us to achieve a hydrogel system with tunable luminescence properties (by varying relative ratios of Tb(III) and Eu(III) ). When the effect of divalent metal ions on such energy transfer processes were explored, it was observed that the luminescence from the composite gel of Tb(III)/ Eu(III) is tunable by Zn(II) and through proper manipulation of concentrations one can obtain white light emitting gel (Fig 3). Fig 3. Effect of Zn(II) (from left to right 0 mM, 2.8 mM, 11.3 mM) on Tb3+ (4.5 mM)/Eu3+ (0.11mM)/ sodium cholate (13.6 mM) gels. b) Tb/Eu/Zn-cholate gel (Tb3+ (4.4 mM), Eu3+ (0.11 mM), Zn2+ (7.4 mM), NaC (13.6 mM, DHN 0.2 mM) under 365 nm UV lamp (c) CIE 1931 diagram depicting the luminescence as white (black spot). Chapter 3. A “Pro-Sensitizer” based Sensing of Enzymes using Tb(III) Luminescence in a Hydrogel matrix This chapter descirbes design and realisation of a sensor system based on Tb(III) luminescnece for the detection of enzymes. The idea involved synthesizing a covalently modified DHN molecule by attaching appropriate enzyme cleavable units. We coined the term “pro-sensitizer”to describe the modified molecule which would not sensitize Tb(III) in the gel matrix but when proper enzymes are applied the free form of DHN would be released triggering a luminescence response from Tb(III). This would enable us to monitor the acitivities of the particular enzyme by examining the luminescence intensity enhancement with time (Fig 4) Fig 4. A “pro-sensitizer” based approach to detect different types of enzymes in a hydrogel matrix through Tb(III) luminescence. We applied the idea to develop a novel luminogenic gel probe for inexpensive and rapid detection of three different hydrolases, lipase, β–glucosidase and α-chymotrypsin. The corresponding “pro-sensitizer”for each enzyme were synthesized (Fig 5).The sensing technique depends on the gel matrix to provide the nessesary platform for lanthanide sensitization. Thereofore, it enjoys an edge over the contemporary techniques that typically involve specially designed and synthesized multidentate chelating ligands for this purpose. We also determined important kinetic parameters of all the enzymes, thus enabling us to have a better insight into the activity of the enzymes in the hydrogel matrix. Fig 4. Pro-sensitizers molecules for (1) lipase, (2) β-glucosidase and (3)α-chymotrypsin Chapter 4. A novel approach towards templated synthesis of lanthanide trifluoride nanoparticles Nanomaterials with excellent optical properties have been of special interest. Lanthanide derived nanoparticles, owing to their unique physical properties, provide an excellent choice for applications such as biolabels, lasers, optical amplifiers, and optical-display phosphors. Several types of lanthanide nanoparticles or nanocrystals are reported in the literature such as Nd2O3, Eu2O3, Gd2O3, Tb2O3, and Y2O3. Among them lanthanide fluoride nanoparticles have emerged as the best choice because of their low phonon energy, and thus minimum quenching of emissive Lnions thereby allowing maximum efficiency for several optical applications. In previous literature precedence, LnF3 nanoparticles were typically synthesized following conventional approaches which necessitate use of high temperatures, high pressures (hydrothermal techniques) and capping ligands. In this chapter, we demonstrated a simpler synthesis of LnF3 nanoparticles at ambient temperatures without the requirement of added capping agents. The room temperature synthesis of LnF3 was unprecedented and was achieved simply by diffusing NaF solution through the hydrogels of corresponding Ln-cholate gels. The nanoparticles were characterized by transmission electron microscopy (TEM) and by powder XRD analysis which established the presence of very small (3-4 nm) nanoparticles mono-dispersed uniformly over the the gel matrix (Fig 6). The LnF3 containing xerogels of Tb(III) and Eu(III) cholate gels were also shown to be highly emissive. Fig 6. HRTEM images of a) TbF3, b) GdF3, c) NdF3 and d) DyF3 in their corresponding gel media.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The investigation was aimed to improve the understanding of the binding interactions between DevS and DevR that are implicated in the regulation of the dormancy response in Mycobacterium tuberculosis. These binding interactions could provide good drug targets for the treatment of persistent tuberculosis, the mechanistic understanding of their binding interactions is important for the development of a validated inhibitor screen. A detailed in silico analysis of the amino acid residues that play a role in the binding of receptor DevR to both kinase DevS and the target DNA was undertaken. A reasonable approximation of the DevS structure was produced using homologous protein structures. In silico docking of DevS to DevR merely produced a set of probable candidate structures, since more than one conformation with similar docked energies was observed. The decision on which one is the more correct form can only be estimated by crystallization of this complex. Therefore, the functional expression and purification of the Dev TCS components were pursued. Denaturing HIS™-select nickel affinity gel purification in the form of matrix-assisted refolding led to the production of functional Dev TCS proteins. To understand the binding of DevR to DNA consensus sequences, as well as the nature of these interactions, a model was built of the full length DevR dimer binding to DNA consensus sequences. Based on this model, single mutations were made to DevR in vitro and their effects assessed in order to validate the model built. During Electrophoretic Mobility Shift Assay (EMSA) analysis, it was found that K179I and N183L mutants prevented the binding of DevR to the DNA consensus sequences. If DevR and DevS binding are to be used in a drug development program, it is essential to have the protocols to accurately measure their interaction, in addition to developing a fundamental understanding of how their interactions occur. The binding affinity of DevR to both DevS and the truncated soluble fragment of DevS (DevS201) were explored, using the BIAcore instrument, an SPR-based biosensor. For sufficiently strong binding between a histidine kinase and a response regulator, the KD needs to be in the nM range. The KD was calculated to be 255 nM for DevS201 and 184 nM for DevS. Therefore it can be concluded that DevS201 binds DevR strongly enough to be used in future studies, and that the BIAcore could be used to screen small-molecule inhibitors of DevR-DevS interactions. / AFRIKAANSE OPSOMMING: Die Dev twee komponent sisteem (TKS) bestaan uit ‘n histidine kinase naamlik (DevS) en ‘n reaksie reguleerder DevR. DevS en DevR is betrokke by die regulering van die dormante stadium van Mycobacterium tuberculosis. Hierdie meganisme kan ‘n deurbraak dwelm teiken vir die behandeling van sluimerende tuberkulose wees. Die meganisme van hierdie bindings interaksies is van kritieke belang, tesame met die ontwikkeling van 'n erkende inhibeerder toets. ‘n Gedetaileerde in silico analise van die aminosuur volgordes wat 'n rol speel in die binding van die reseptor DevR aan beide DevS sowel as die teiken DNS is voltooi. ‘n Model van die DevS struktuur is saamgstel met behulp van homoloë proteïen strukture. In silico mering van DevS aan DevR het `n stel van die waarskynlike kandidaat strukture verskaf, aangesien meer as een konformasie met soortgelyke merings energieë waargeneem is. Die mees waarskynlike vorm kan alleenlik geïndentifiseer word na kristallisasie van hierdie kompleks. Die funksionele uitdrukking en suiwering van die Dev TKS proteine is gevolglik uitgevoer. Funksionele Dev TKS proteïene is verkry deur denaturerende HIS-select nikkel affiniteit jel suiwering, in die vorm van matriks-geassisteerde hervouing te gebruik. Ten einde die binding te verstaan tussen DevR en DNS konsensus volgordes, sowel as die aard van hierdie interaksies, is 'n model gebou van die volle lengte DevR dimeer binding aan DNS konsensus volgordes. Hierdie model is gevalideer deur punt mutasies in DevR te skep en die gevolge daarvan te beoordeel met elektroforetiese mobiliteits verskuiwing reaksie analises. Dit is bevind dat K179I en N183L mutante, verhoed die binding van DevR aan die DNS konsensus volgordes. Die gebruik van DevR en DevS bindings in ‘n dwelm ontwikkelingsprogram, benodig die fundamentele begrip van hoe die interaksies plaasvind, sowel as akkurate protokolle om die interaksies te meet. Die BIAcore instrument, ’n SPR-gebaseerde biosensor, is ingespan om die bindings affiniteit van DevR aan beide DevS en die fragment van DevS (DevS201) te ondersoek. Om voldoende sterk binding tussen DevS en die DevR te verseker, moet die KD in die nM omgewing wees. Die KD is bepaal as 255 nM en 184 nM vir DevS201 en DevS, onderskeidelik. Die afleiding kan dus gemaak word dat DevS201 sterk genoeg aan DevR bind om in verdere studies gebruik te kan word, en dat die BIAcore gebruik kan word om klein-molekule inhibeerders van DevR-DevS interaksies te toets.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This study is the first of its kind to highlight the importance of using the latest state of the art technology available in the field of proteomics as a complementary tool to characterize the proteome of members of the Mycobacterium tuberculosis Beijing lineage which have been linked to outbreaks and drug resistance of Tuberculosis (TB). Our label-free comparative analysis of two closely related M. tuberculosis strains with different transmission patterns and levels of virulence highlighted numerous factors that may alter metabolic pathways leading to hyper-virulence whereby the strain was able to rapidly replicate in the host and cause extensive disease. This comparative analysis clearly demonstrated that both instrumentation and analysis software impacts on the number of proteins identified and thereby the interpretation of the proteomic data. These proteomes also served as substrates for the discovery of phosphorylation sites, a field of research that reflects a significant knowledge gap in the field of M. tuberculosis. By using differential separation techniques in combination with the state of the art mass spectrometry we described the phosphorylation sites on 286 proteins. This was the first study to document phosphorylation of tyrosine residues in M. tuberculosis. By this means, our data set further extend and complement previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis. Using advanced mass spectrometry methods we further investigated the impact of the in vivo evolution of rifampicin resistance on the proteome of a rifampicin-resistant strain containing a S531L rpoB mutation. We identified the presence of overabundant proteins which could provide novel insight into potential compensatory mechanisms that the bacillus uses to reduce susceptibility to anti-TB drugs. Our findings suggest that proteins involved in a stress response may relate to an altered physiology enabling the pathogen to tolerate and persist when exposed to anti-TB drugs. Together this suggests that structural changes in the RNA polymerase precipitated a cascade of events leading to alterations of metabolic pathways. In addition, we present the first comprehensive analysis of the effect of rifampicin on the proteome of a rifampicin resistant M. tuberculosis isolate suggesting that rifampicin continues to influence the biology of M. tuberculosis despite the presence of an rpoB mutation. Our analysis showed alterations in the cell envelope composition and allowing the bacterium to survive in a metabolically dormant/persistent growth state. The results presented in this study illustrate the full potential of using a proteomic approach as a complementary molecular technique to select promising candidate molecules and genes for further characterization using the tools of molecular biology. / AFRIKAANSE OPSOMMING: Die huidige studie is ‘n eerste van sy soort, deur die nuutste gevorderde tegnologie in die proteomika veld te gebruik. Die proteoom van lede van die Mycobacterium tuberculosis Beijing stam, wat die oorsaak is van tuberkulose (TB) uitbrake en ook weerstandige TB, is gekarakteriseer. Ons merkervrye vergelykende analise van twee naby verwante M. tuberculosis stamme met verskillende vlakke van oordraagbaarheid en virulensie, beklemtoon verskeie faktore wat metaboliese paaie mag verander, wat kan ly tot hiper-virulensie, wat die TB-stam in staat stel om vinniger te repliseer in die gasheer en ‘n uitgebreide siektetoestand kan veroorsaak. Die analise het duidelik gewys dat die toerusting wat gebruik word, sowel as die sagteware ‘n invloed kan hê op die hoeveelheid proteïne wat geïdentifiseer kan word en daardeur intrepretasie van proteomika data kan beïnvloed. Hierdie proteome dien as substrate vir die ondekking van fosforilasie setels, ‘n veld van navorsing wat dui op ‘n gaping in ons kennis van M. tuberculosis. Deur gebruik te maak van differensiële skeidingstegnieke en moderne spektrometrie beskryf ons fosforileringsetels in 286 proteine. Hierdie is die eerste studie wat fosforilasie van tirosien residue in M. tuberculosis beskryf. Hierdeur komplimenteer en brei ons data die huidige kennis oor gefosforileerde peptiede en fosforilasie setels in M. tuberculosis uit. Deur gebruik te maak van gevorderde massa spektrometriese tegnieke het ons verder ook die impak van in vivo evolusie van rifampicin weerstandigheid op die proteoom van ‘n rifampicin weerstandige TB-stam met die algemene S531L rpoB mutasie ondersoek. Ons het proteïne geïdentifiseer wat in groot hoeveelhede voorkom en kan nuwe insigte gee tot potensiele kompenserende meganismes wat deur die bacillus gebruik word om vatbaarheid vir anti-TB middels te verminder. Ons bevindings dui daarop dat proteïene betrokke in ‘n stresreaksie mag lei tot ‘n verandering in fisologie wat die patogeen in staat stel om anti-TB middels te verdra en te volhard in die teenwoordigheid van sulke middels. Saam impliseer dit dat ‘n ketting van gebeure wat lei tot veranderinge in metaboliese paaie, word vooraf gegaan deur strukturele veranderinge in die RNS polimerase. Tesame hiermee bied ons ook die eerste omvattende analise aan van die effek wat rifampicin op die proteoom van ‘n rifampicin weerstandige M. tuberculosis isolaat het, en wat aan die hand doen dat rifampicin voordurend die biologie van M. tuberculosis beïnvloed, ten spyte van die teenwoordigheid van ‘n rpoB mutasie. Ons analise dui op veranderinge in die samestelling van die selomhulsel wat die bakterie toelaat om te oorleef in ‘n metabolies dormante staat. Die resultate wat in hierdie studie aangebied word illustreer die volle potensiaal van ‘n proteomiese benadering as komplementêre molekulêre tegniek om belowende kandidaat molekules en gene te kies vir verdere karakterisering, deur gebruik te maak van molekulêre tegnieke. / The National Research Foundation (RSA), / Norwegian Research Council (Norway) / National Institute of Health –Forgarty (USA) / Southern Africa Consortium for Research Excellence-Welcome Trust (SACORE) (United Kingdom) / Kwazulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) (USA)

KwaZulu-Natal has been ranked as having the fourth highest incidence of transmitted Multiple Drug Resistant-Tuberculosis (MDR-TB) in sub-Saharan Africa. Substantial literature exists indicating the permanent damage that MDR-TB medication has on hearing abilities. The purpose of this study was to describe the hearing function of adults on long term MDR-TB treatment from Murchison Hospital MDR-TB unit in the Ugu District in rural KwaZulu-Natal. The primary aim of the study was to review the possible changes in hearing function in a group of adults on long-term treatment for MDR-TB. Secondly, the study aimed to estimate the number of adults who may present with changes following MDR-TB treatment and establish if relationships exist between the audiological findings and factors such as age and gender. The design of the study was a retrospective comparative data review of 68 patient records, all of which underwent audiological investigations from the start of MDR-TB treatment over a five-month period. The study made use of descriptive and inferential statistics to analyse the data. Specific inferential statistical analysis included analysis of covariance as well as regression analysis. Results from the study showed changes in hearing function in Distortion Product Otoacoustic Emissions (DPOAEs) and Pure Tone Audiometry (PTA) results at all five audiological sessions and across a range of frequencies. 84% of the total sample presented with overall refer readings for DPOAEs and 98.53% of the group of adults presented with criteria indicative of ototoxic hearing loss, specifically a bilateral mild-profound sloping SNHL on clinical PTA results. In the total sample of patient records reviewed in this study, all 68 records showed a change in hearing function, be that changes in DPOAE function and/or changes in PTA thresholds, following long-term treatment for MDR-TB. Variations in the effects of gender and ear difference were minimal and non-significant in all results. Similar presentation, to ototoxic hearing loss, of other degenerative conditions exists; however these conditions were accounted for as exclusion criteria in this study. Therefore the only remaining cause of possible hearing deficit was that of ototoxicity. The study provided valuable data regarding hearing function in a population of adults on long-term MDR-TB treatment in South Africa. Furthermore, the study has highlighted the need for the establishment of standardised audiological monitoring programmes sensitive to ototoxic hearing loss, within the South African context where the incidence of Tuberculosis (TB) and MDR-TB is reportedly high.

O estudo analisou a sustentabilidade da estratégia DOTS na visão de coordenadores de Programa de Controle Tuberculose (PCT) em sete municípios do interior do estado de São Paulo, prioritários para o controle da Tuberculose (TB). O quadro teórico está sustentado nas dimensões operacional, organizacional e política da gerência e sua articulação no contexto atual das políticas e serviços de saúde. A partir de uma abordagem quantitativa, de estudo epidemiológico descritivo por meio de levantamento de fontes secundárias, foram analisados os indicadores epidemiológicos do PCT: cura, abandono e óbito de casos novos com baciloscopia de escarro positiva, cobertura de Tratamento Supervisionado (DOT/TS) e de detecção de casos. Também optou-se pela abordagem qualitativa, análise de conteúdo-modalidade temática que utilizou como fonte a entrevista semi-estruturada com coordenadores de PCT. A Unidade Temática central foi a sustentabilidade da estratégia DOTS como um desafio para coordenadores de PCT, contendo dois sub-temas: A organização da estratégia DOTS diante da necessidade de captar/otimizar recursos e definir estratégias e A operacionalização das ações de controle da TB: raciocínio estratégico e negociação como ferramentas gerenciais. Os resultados quantitativos mostraram um coeficiente angular positivo para cura, mas a maioria não atingiu a meta de 85%; declínio no percentual de abandono, ainda aquém do esperado. A cobertura de DOT/TS apresentou aumento em todos os municípios, contudo, apenas um atingiu 95%. Nenhum município atingiu a meta de 70% de detecção de casos. Os temas que emergiram neste estudo apontaram nós críticos na captação e manutenção de incentivos financeiros; recursos humanos (RH) insuficientes e despreparados; rotatividade de pessoal; desconhecimento do destino da verba da TB e falta de autonomia para gerenciar os recursos; dificuldades de comunicação e integração com gestores; falta de priorização da doença na agenda política; priorização de agravos com repercussão política e necessidade de parcerias. Sobre a operacionalização do DOTS, o estudo apontou a cooperação de pessoas de fora do serviço como ferramenta gerencial estratégica, quando estimula a coresponsabilização da família e da comunidade como cuidadores ou multiplicadores. A avaliação, pautada em indicadores epidemiológicos, é utilizada prioritariamente para alcançar recursos, ao invés de subsidiar o planejamento. A gerência do PCT parece ser uma gerência burocrática, fundamentada no planejamento normativo. Acredita-se que a efetividade das ações gerenciais para sustentar a estratégia DOTS dependa de atores com conhecimento técnico, habilidades políticas e organizacionais, além de raciocínio estratégico para estimular e envolver todos os atores que lidam com a TB. Habilidades estas que, dificilmente são adquiridas na formação profissional. Conclui-se que os coordenadores trabalham em meio a pressões de cima e de baixo, ocupando dupla posição de transmissão, no plano hierárquico e das relações. Neste sentido, a sustentabilidade da estratégia DOTS representa desafios importantes para estes atores que utilizam poucos instrumentos gerenciais, têm pouca autonomia no processo decisório, lidam com recursos escassos e, muitas vezes, sem preparo para esta função, reforçando a necessidade de investimento na formação e capacitação contínua tanto para estes atores como para aqueles que lidam com a TB / This study analyzed the sustainability of the DOTS (Directly Observed Treatment Strategy). It shows the point of view of the TCP (Tubercolosis Control Program) coordinators of seven priority municipalities in the Sao Paulo State. The theorical framework is based on operational, organizational and political levels of the management and on its articulation in the current context of health policies and services. We carried out an epidemiological-descriptive study, using secondary sources, and through a quantitative approach. The following epidemiological TCP indicators were analyzed: cure, abandonment and death of new cases with positive sputum bacilloscopy, Supervised Treatment (DOTS/ST) and case detection coverage. We also chose to apply a qualitative approach, through a semi-structured interview with TCP Coordinators, and with the application of content analysis in the thematic modality. The main thematic unity is the sustainability of the DOTS strategy as a challenge to the TBP coordinators. It contains two sub-themes: The organization of the DOTS strategy faced with the need for resource captation/optimization and for clearly outlined strategies, and The operationalization of TB control actions: strategic thinking and the negociation as key management skills. Quantititive results show a positive angular coefficient for cure. Nonetheless, the majority did not achieve the 85% goal and the decline in the abandonment rate is still lower than expected. The ST coverage rose in all municipalities. However, only one municipality achieved coverage of 95%. None of the municipalities achieved the goal of 70% case detection rate. The themes analyzed in this study pointed to critical knots in the collection and maintenance of financial incentives; insuficient and unprepared Human Resources (HR); staff turnover; lack of knowledge concerning the destination of the TB funds, as well as lack of autonomy to manage resources; difficulties in the communication and in the integration with other managers; failure in making the disease a priority on the political agenda; priorization of aggravations with political repercussion and the need for partnerships. Concerning the DOTS operationalization, the study pointed to the cooperation of people outside the job as an important strategic management tool. It encourages family and community to take co-responsibility as caretakers and multipliers. The evaluation, which is based on epidemiological indicators, is especially used to achieve resources, instead of subsidizing the planning process. TCP management seems to be exceedingly bureaucratic and based on normative planning. We believe that the effectiveness of management actions to support the DOTS strategy needs characters with technical knowledge, political and organizational skills and strategic thinking. These skilss are important in order to encourage and get involved all those who deal with TB. These skills are hardly achieved in the professional training. We conclude that the coordinators work under pressure from the top and from the bottom of the hierarchy, and occupy a double transmission position: in the hierarchic level and in the relationship level. Thus, the sustainability of the DOTS strategy represents an important challenge for these characters, who use few management tools, have little authonomy in the decisionmaking process, have to work with scarce resources and are usually unprepared for the position they occupy. These findings highlight the need for more investments in continuing education and capacitation not only for coordinators but also for all those who have to deal with TB

Text in English / Aim: The aims of this study were to investigate the treatment outcomes of patients with MDRTB and its determinants at referral hospitals in Ethiopia. The study also aims to develop a conceptual model for enhancing treatment of patients with MDR-TB in Ethiopia. Design and methods: A concurrent mixed methods design with quantitative dominance was used to investigate treatment outcomes of patients with MDR-TB and its determinants. Results: A total of 136 (n=136) patients with MDR-TB participated in the study, 74 (54%) were male and 62 (46%) were female. Forty-one (31%) of the patients had some co-morbidity with MDR-TB at baseline, and 64% had body mass index less than 18.5kg/m2. Eight (6%) of the patients were diagnosed among household contacts. At 24 months, 76/110 (69%) of the patients had successfully completed treatment, but 30/110 (27%) were died of MDR-TB. Multivariable logistic regression revealed that the odds of unfavourable treatment outcomes were significantly higher among patients with low body mass index (BMI <18.5kg/m2) (AOR=2.734, 95% CI: 1.01-7.395; P<0.048); and those with some co-morbidity with MDR-TB at the baseline (AOR=4.260, 95%CI: 1.607-11.29; p<0.004). The majority of the patients were satisfied with the clinical care they received at hospitals. But as no doctor was exclusively dedicated for the MDR-TB centre, patients could not receive timely medical attention and this was especially the case with those with emergency medical conditions. The caring practice of caregivers at the hospitals was supportive and empathic but it was desperate and alienating at treatment follow up centres. Patients were dissatisfied with the quality and adequacy of the socio-economic support they got from the programme. Despite the high MDR-TB and HIV/AIDS co-infection rate, services for both diseases was not available under one roof. Conclusions: Low body mass index and the presence of any co-morbidity with MDR-TB at the baseline are independent predictors of death among patients with MDR-TB. Poor communication between patients and their caregivers and inadequate socio-economic support were found to determine patients’ perceived quality of care and patients’ satisfaction with care given for MDR-TB. / Health Studies / D. Litt et Phil. (Health Studies)

Multidrug-resistant tuberculosis (MDR-TB) is a public health problem in several countries such as Angola, India, China, Kenya, and Nigeria. Due to the increasing high burden of MDR-TB, most of these countries do not have adequate capacities to manage MDR-TB patients effectively. This study investigated the effect of model of care; human immunodeficiency virus comorbidity; and demographic factors such as age, gender, and marital status on the treatment outcomes of MDR-TB patients in Nigeria. The study was based on the analysis of secondary data of 402 MDR-TB patients accessed from the data systems of the National Tuberculosis, Buruli Ulcer, and Leprosy Control Program. The theoretical framework for this study was the health belief model. The results of the study showed that treatment outcomes were similar for hospital and community-based models of care. Age was the only factor found to be significantly associated with treatment outcomes; age > than 40 years was a predictor of unsuccessful treatment outcomes among MDR-TB patients at a p-value of 0.026. In the multivariate logistics regression analysis, age and model of care were found to be significantly associated with treatment outcomes at p-values of 0.043 and 0.048, respectively. Marital status, gender, and HIV comorbidity were not significantly associated with treatment outcomes. Implications of the findings of this study for social change in a health care program include opportunities to help reduce the number of patients on waiting lists for MDR-TB treatment. These strategies may ultimately help to reduce the spread of MDR-TB infection as well as the mortality associated with late treatment.