Resumo: De acordo com a Organização Mundial de Saúde em 2009, 9,27 milhões de novos casos de tuberculose ocorreram em 2007. Destes novos casos, 4,9% eram multidroga resistentes. Muitas pesquisas são realizadas na procura de novas drogas com atividade contra o bacilo da tuberculose, havendo então a necessidade de se entender os mecanismos de ação destes novos compostos. Este projeto objetivou propiciar ferramentas para compreender um pouco mais sobre os mecanismos de ação de novas drogas. Isolados clínicos de M. tuberculosis foram caracterizados quanto ao seu perfil de susceptibilidade aos fármacos do esquema terapêutico e foram determinadas as mutações responsáveis por estas resistências. Com os isolados caracterizados, foi construído um painel de M. tuberculosis. Pelo REMA, os isolados foram analisados quanto ao seu perfil de susceptibilidade aos fármacos (INH, RMP, STR e ETB) e avaliados quanto à presença de mutações nos genes de resistência (inhA, katG, ahpC, rpoβ, rpsL, rrs e embB) empregando a PCR-SSCP. Pelo REMA foram avaliados 80 isolados clínicos, sendo observada a resistência a INH em 74,7%, a RMP em 51,2%, a STR em 53,7% e ao ETB em 58,7%. Nos isolados resistentes, a porcentagem de mutações encontradas nos genes foi de 20,6% para inhA, 50% para katG, 6,3% para ahpC, 60% para rpoβ, 20% para rpsL e 0% para rrs e embB. Um painel com 12 isolados foi testado frente a três novos compostos, dois derivados de INH (Cu-INH1 e Cu-INH2) e um de RMP (Cu-RMP). Verificou-se que os isolados resistentes a INH foram também resistentes a Cu-INH1 e Cu-INH2. A mesma situação foi verificada em relação à RMP, com o composto Cu-RMP. Provavelmente, estes novos compostos têm os mesmos mecanismos de ação da INH e da RMP, que são os fármacos que lhes deram origem / Abstract: According to World Health Organization in 2009, 9.27 million new TB cases occurred in 2007. Among these new cases, 4.9% were multidrug resistant. Many surveys are conducted in the search for new drugs with activity against the tuberculosis bacillus, therefore there is a need to understand the action mechanism of these new compounds. This project aimed to provide tools to understand about the action mechanisms of new drugs. M. tuberculosis clinical isolates were analyzed for their susceptibility profile to drugs, mutations responsible for resistance and a panel of these characterized isolates. The isolates were analyzed for susceptibility profile to drugs (INH, RIF, STR and ETB) and evaluated for presence of mutations in the resistance genes (inhA, katG, ahpC, rpoβ, rpsL, rrs and embB) applying the PCR-SSCP. REMA evaluated 85 clinical isolates and the resistance was observed in 74.7% to INH, 51.5% to RIF, 53.7% to STR and 58.7% to ETB. In the resistant isolates, percentage of mutations found in the genes was 20.6% for inhA, 50% for katG, 6.3% for ahpC, 60% for rpoβ, 20% for rpsL and 0% for rrs and embB. A panel of 12 isolates was tested against three new compounds, two INH-derivatives (Cu-INH1 and Cu-INH2) and one RMP-derivative (Cu-RMP). The isolates resistant to INH were also resistant to Cu-INH1 and Cu-INH2 compounds. The same situation was verified in relation to the RMP with the Cu-RMP compound, indicating that probably these three new compounds have the same action mechanism of INH and RMP drugs / Orientador: Clarice Queico Fujimura Leite / Coorientador: Cleslei Fernando Zanelli / Banca: Elsa Mases Mamizuka / Banca: Daisy Nakamura Sato / Banca: Eliana Aparecida Varanda / Banca: Mario Hiroyuki Hirata / Doutor

Text in English / Botswana has witnessed highest TB rates in the southern African countries, ranking the fourth after South Africa, Swaziland and Zimbabwe. In 2012, the TB rate was on average 531/100 000 population. About 2 380 contacts out of a possible 8 110 (amounting to 29.30%) were traced nationally (Botswana 2011:8), indicating a possible gap of 5 730 which was yet to be traced in 2011. The TBCT strategies might be inadequate leading to absence of screening and treating TB contacts and reducing PTB related deaths. The purpose of this study was to describe utilisation of current TBCT and develop a strategy for a more effective TBCT in Botswana. Data was collected through a quantitative cross-sectional research design. The study further described the association between TBCT strategies and practices and determined the gaps, challenges and needs in the TBCT. Results revealed under-tracing of contacts in the number of registered and enumerated TB contacts. The results further established the risk of mixing TB contacts and the general patients. The differences in the perceptions and knowledge of the cause of TB as well as poor utilisation of the current programmes by the PTB patients denotes the need for aggressive awareness raising and health promotion strategies. The results were used to develop an alternative strategy, the IC-TBCT, which has a potential to trace all TB contacts. The strategy encourages participation, effective accountability and involvement of the beneficiaries in all efforts aiming at early contact identification and reducing the incidence of PTB. / Health Studies / D. Litt. et Phil. (Health Studies)

The study aimed to explore and describe the experiences of people treated for multidrug resistant tuberculosis (MDR-TB) in Omaheke region, Namibia in order to make relevant recommendations regarding their management. A descriptive qualitative design approach was used. Data was collected using in-depth individual interviews with six participants. The interview transcripts were analysed using thematic content analysis. Three themes emerged namely (1) Stressors related to MDR-TB diagnosis and treatment which involved nature of disease and compulsory hospitalisation (2) Impact of being treated for MDR-TB which related to emotional , social , spiritual and financial impact (3) Support structures for people treated for MDR-TB which included family members, health care professionals and friends. Systemic practical patient-centred, staff-centred and community-centred recommendations are suggested as well as recommendations for future research and an appraisal of the limitations of this study. / Health Studies / M.A. (Public Health)

Background: Pulmonary Tuberculosis (PTB) is considered a disease of the past but it remains a major cause of mortality among immune compromised patients and continues to be a significant threat to public health globally. Notably, the prevalence of diabetes mellitus (DM) has increased over the years. The biological link of TB and DM has been reported in numerous literature with DM attributed to three folds increase in the risk of TB and linked to Drug Resistant TB, especially amongst aged diabetic patients. The aim of the study was to examine the distribution of DM among TB patients and explore the risk of Drug resistant TB in Diabetics infected with TB Methods: The study employed a retrospective cross-sectional descriptive case based study involving 3638 patients diagnosed with pulmonary TB in the State of Florida, USA, 2009-2014. A comparative analysis of TB cases with DM and cases without DM adjusted for age was conducted. The risk of Drug resistant TB associated with DM was estimated through logistic regression analysis. Odds Ratios of TB/DM comorbidity were calculated and adjusted for Age using 5-year intervals from 40 years to above 70 years. Ninety-five percent (95%) confidence intervals were used and the accepted level of error was 0.05. Results: There were 3836 cases of Pulmonary TB in Florida for the period of 2009-2014. The majority of cases (65%) were males and likely unemployed (59.1%). The prevalence of DM was 12 % but when adjusted for age the prevalence of DM was 3.9% amongst patients aged below 40 years and 16.7 % in patients aged above 40 years. An estimated 469 cases had TB/DM comorbidity (12.2%). The majority of TB/DM cases were above 40 years amongst the patients with DM, 44/469 (9.4%) had drug resistant TB and a majority were resistant to Rifampin. Population density did not influence the distribution of TB in this study. Conclusion: Diabetes Mellitus, Aging, and low immunity are linked with increased rates of progressing from latent TB infection to active disease. To achieve the goal of TB elimination it is important to fully understand and identify known TB comorbidities for proper diagnosis and early initiation to care. There is a positive correlation between high DM burden and increased TB prevalence. Therefore, it is recommended that prevention of DM, hyperglycemia and comprehensive management of DM be intensified to prevent TB, improve TB treatment outcomes and reduce the risk of drug resistant TB in Florida, USA.

Masters of Science / Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). TB mainly affects lungs of patients but other parts of the body can also be affected. It kills approximately 2 million people annually. HIV/AIDS and drug resistance make TB difficult to control. Mtb CarD protein forms a physiological complex with Ribonucleic Acid Polymerase (RNAP). This complex causes Mtb to undergo dormancy rendering it difficult to control using current antibiotics. CarD and a size-reduced subunit β1 (denoted β1m for “minimized”) of Thermus thermophilus RNAP, in which the central domain has been replaced by a Gly-Gly linker, were produced and purified using affinity nickel nitrilotriaceticacid and glutathione-Stransferase (GST) affinity chromatography techniques respectively. CarD N terminal domain (CarDN) was generated from CarD by inserting a stop codon by site directed mutagenesis. CarD was stabilised by adding 5 % (v/v) glycerol to PBS pH 7.4 ensuring protein stability of up to 67 days rather than 2 days without glycerol. CarDN was stable in PBS pH 7.4 without addition of glycerol. This suggests that the CarD C terminal domain may be responsible for CarD instability. To further purify the proteins both anion exchange and gel permeation chromatography techniques were used. CarD and CarDN degrade immediately after anion exchange potentially because of the high ion concentration which partially unfolds the protein making it prone to proteolytic cleavage. GST-pull down assays were used to demonstrate complex formation between RNAP β1m and both CarD and CarDN confirming that complex formation is dependent on the N-terminal domain of CarD.

The serious threat of tuberculosis, especially XDR-TB, is a reality in Southern Africa particularly in individuals with HIV/AIDS. Therefore the importance of development of new or improved anti-TB treatment must now be emphasized more than ever. In this study, a model was created to target isoniazid (toxophore) specifically to a cholesterol rich environment where mycobacteria reside in macrophages, by making use of a sterol binding drug, Amphotericin B (haptophore). Isoniazid was covalently linked to Amphotericin B via a Schiff base to a linker molecule, terephthalaldehyde. Although this molecule showed a loss of biological activity, a discovery was made by serendipity that could have great impact in understanding how Mycobacterium tuberculosis enters and survives in the host macrophage. During the testing of the compound, it was discovered that Amphotericin B bound to mycolic acids at least as well as it binds to cholesterol, its natural ligand. This could provide proof of the structural similarity between mycolic acids and cholesterol but many more controls need to be investigated. As cholesterol was previously shown in literature to be critical for entry and survival of Mycobacterium tuberculosis in macrophages, the indication of a structural mimicry between the cell wall mycolic acids and cholesterol and the attraction of these two chemical entities to one another seems to be highly relevant. This characteristic can now be further explored to improve the understanding of the process of entry and survival of Mycobacterium tuberculosis in the macrophage host. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Benadie, Y 2006, Amphotericin B as a mycolic acid specific targeting agent in tuberculosis, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-04212008-151642 / > / Dissertation (MSc (Biochemistry))--University of Pretoria, 2008. / Biochemistry / unrestricted

Tuberculosis has re-emerged as a global health problem due to co-infection with HIV and the emergence of drug resistant strains of Mycobacterium tuberculosis. There is a need for a reliable and fast serodiagnostic assay to reduce the time required for test results from weeks to hours, in order to better control the spread of the disease. Previous studies have shown that TB patients contain antibodies against M. tuberculosis mycolic acids. In standard immunoassays such as ELISA, an unacceptable number of false positive and negative test results were obtained. This study aimed at assessing the potential of detecting anti-mycolic acids antibodies in TB patient sera on a biosensor as surrogate marker for TB infection. Mycolic acid liposomes were immobilized reproducibly on a non-derivatized biosensor cuvette and blocked with saponin. A high dilution of serum in PBS/ AE was used to calibrate the signal of the two cells, followed by binding of patient sera inhibited with either mycolic acid, cholesterol or placebo phosphatidylcholine liposomes at a lesser dilution. The inhibition was done to confirm the specificity of the binding response. There was no inhibition of binding when a sputum negative control serum (HIV-TB-) was pre-incubated with either cholesterol or mycolic acids on the biosensor coated with mycolic acid liposomes. The antibodies that are specific to mycolic acid were demonstrated in all TB positive patients on mycolic acids coated cuvette cell surfaces after pre-incubation of serum with mycolic acids. The patient sera that were false positive and false negative on ELISA tested negative and positive respectively on the biosensor. Only sera from two patients, both HIV positive, tested false positive on both ELISA and biosensor. The biosensor was able to detect anti-mycolic acids antibodies of even low affinity. In ELISA, these antibodies were washed away. No inhibition of antibody binding on cholesterol-coated cuvettes was found after pre-incubation of serum with mycolic acids or cholesterol liposomes. The cholesterol surface became unstable during pre-incubation of serum with mycolic acids. Mycolic acid appeared to be a stronger antigen than cholesterol. The anti-mycolic acids antibodies were specific and sensitive for diagnosis of TB on the biosensor. More sera should be analyzed on the biosensor to make a statistically accountable statement on whether the improved sensitivity and specificity is adequate for a simple, rapid, sensitive and accurate biosensor-based serodiagnostic assay. / Dissertation (MSc(Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted

DNA fingerprinting of Mycobacterium tuberculosis strain has been used in combination with conventional epidemiologic investigation, which has improved the understanding of tuberculosis transmission. Restriction Fragment Length Polymorphism (RFLP) based on IS6110 probe has become a standard method of fingerprinting of M tuberculosis. Since the technique is labour intensive and the discriminatory power of IS611 0 fingerprinting method for strains habouring only one to five copies is poor, other typing methods for typing M tuberculosis should be evaluated. In this regard, Amplified Fragment Length Polymorphism (AFLP) has the potential to overcome many of the RFLP problems. The first objective was to determine the suitability of the RFLP and AFLP techniques and to study the extent of transmission of tuberculosis in a referral hospital in South Africa. A total of 47 M tuberculosis isolates were differentiated using RFLP technique. The same samples were typed using the PCR- based AFLP technique and results were compared. The second objective was to determine the prevalence of isoniazid (INH) resistance and estimate the incidence of recent transmission of the disease in the Eastern-Cape (EC) and North-West province (NW) by using the best suited technique. RFLP grouped the 47 typed M. tuberculosis isolates into five families and four clusters. AFLP grouped the analyzed isolates (previously typed by RFLP) into two groups based on the banding patterns observed. As a result of the low degree of genotypic variation among the AFLP band pattern of M tuberculosis isolates, AFLP seemed less promising for individual strain differentiation of M tuberculosis. This technique can be used in future for differentiation of Mycobacterial species and The prevalence of INH resistance was found to be 6.7% in the EC and 8.4% in the NW province. The magnitude of recent transmission in the Eastern Cape studied by RFLP method, was found to be at 22% among the positive tuberculosis isolates identified. Transmission of TB in NW province was associated with reactivation rather than recent transmission due to lack of clustering of strains in that region. / Dissertation (MSc(Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Tuberculosis (TB) is a collective name for the bacterial infection, which is caused by members of the Mycobacterium tuberculosis (M. tb) complex and can infect the lungs (pulmonary) as well as the kidneys, lymph nodes, bones and joints (extra-pulmonary). The re-emergence of drug-resistant strains and the HIV epidemic are among the main reasons for the resurgence of TB and there is a need for new drugs and diagnostic assays which are rapid and sensitive. Serodiagnostic assays have the potential of being rapid, inexpensive and relatively non-invasive. The most abundant antigen in the cell wall of M. tb, which has been analysed with ELISA and resonant mirror biosensor assays for use in serodiagnosis, is mycolic acid (MA). The sensitivity previously obtained in the ELISA assay was however inadequate for serodiagnostic purposes. It was believed that MA mimicked the structure of cholesterol, thereby causing anti-cholesterol human antibodies from TB negative sera to bind to MA and result in a large number of false positives. Within this work the apparent molecular mimicry between MA and cholesterol was investigated using a competitive enzyme linked inhibition assay (CELIA) assay. The results suggested that MA in liposomes resembled the liquid ordered arrangement of cholesterol in liposomes, rather than a direct mimicry of individual molecules. The nature of the antibody from TB negative patient sera binding to MA coated onto ELISA plates was also investigated. The results obtained from this study have not disproved the hypothesis of a cross-reactive anti-cholesterol antibody, but it would appear that the MA signal from TB negative serum was partially due to the binding of anti-MA antibodies. The presence of anti-MA antibodies in TB negative serum could have been the result of prior BCG vaccination, latent infection or due to constant immune stimulation from saprophytic mycobacteria. This creates the potential of using antibodies to MA to distinguish between latent TB infection and active disease. Furthermore, in order to overcome the low sensitivity of the ELISA assay due to high background signals from TB negative serum, members of our group previously developed a resonant mirror biosensor inhibition assay based on MA contained in liposomes. The biosensor measured mass accumulation and the identity of the binding molecules were unknown. It was shown here that one of the serum components binding to the immobilised MA liposomes in the biosensor inhibition assay was immunoglobulin G antibodies. The specificity of both the ELISA and biosensor assays previously analysed using a natural mixture of MA however, remained poor, and in the search for a more specific antigen, this study investigated the potential of MA subclasses for TB serodiagnosis using ELISA. It was observed that the antibody binding signal to the MA subclasses depended on the polarity of the coating solution, for which hexane was the preferred solvent. Both the alpha- and keto-MA subclasses could better distinguish between a range of TB positive patient and TB negative sera compared with the natural mixture of MA. These results suggested that a particular subclass applied in the biosensor inhibition assay could enhance the test to reach the required sensitivity and specificity required for the serodiagnosis TB. / Dissertation (MSc)--University of Pretoria, 2011. / Biochemistry / unrestricted

Tuberculosis has become one of the world’s most devastating diseases, with more than two million deaths and eight million new cases occurring annually due to the development of drug-resistant strains of Mycobacterium tuberculosis, the breakdown of the immune system of its host by HIV, lapses in public health programmes and the fact that diagnosis of TB is not 100% reliable. Early, affordable, unsophisticated and accurate diagnosis of TB to facilitate timely and proper treatment has become of highest priority to public health. Mycolic acid (MA) is the major lipid cell wall component of Mycobacterium tuberculosis and is unique to mycobacteria and closely aligned genera. Mycolic acids have been shown to be unique antigens for TB diagnosis and have been utilized in standard serodiagnostic techniques, but sensitivity and specificity was found to be unsatisfactory. Two vastly different techniques were investigated in this study – one making use of antibodies and MA, the other, just MA and its unique physical properties of interaction with other MA using fluorescently labelled MA. In the first approach, Sepharose protein-A was employed to trap patient IgG antibodies. The anti-MA antibodies were then quantified by probing with liposomes containing fluorescently labelled MA. Although it generally worked well, a few false –positive and –negative results were obtained. This assay appeared to be more accurate than the standard ELISA immunoassay but it is more labour intensive and not even remotely as amenable to large-scale screening and automation as ELISA. The second approach is based on the release of fluorescent MA from immobilized liposomes on glass by means of the specific attraction that MA in test liposomes or TB patient serum was perceived to have on the immobilized MA. The end-point measured was the remaining fluorescent MA on the surface. Differences were observed between the control and patients’ sera at a very high dilution but not between the HIV negative, TB positive and HIV positive, TB positive patients. This was merely an exploratory investigation and more work still needs to be done before the test is ready for validation with large numbers of serum samples. If subsequent studies confirm these findings, then this concept may be converted into a simple, rapid and affordable TB diagnostic test or be used in combination with the IAsys affinity biosensor to provide a more thorough diagnosis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2009. / Biochemistry / unrestricted