It is estimated that over nine million new tuberculosis (TB) cases worldwide occurred in 2009. However, only 63% of these cases were notified, which is lower than the 75% target set by the DOTS strategy. The lack of ideal diagnostic tests is one of the major barriers to effective TB reduction. The aim of this thesis is to evaluate the efficiency of a novel liquid culture based technique (Microscopic Observation Drug Susceptibility Assay-MODS) and a new generation fluorescence microscope (Light Emitting Diodes fluorescence microscope - LED fluorescence microscope) in diagnosis of TB and multi drug resistant tuberculosis (MDR- TB) in Viet Nam, a resource limited, high burden setting. In the studies described in this thesis, MODS had a higher sensitivity than homogenous smear microscopy, detecting an additional 13% of cases, in diagnosis of TB. Importantly, the pooled sensitivity of MODS (53.0%) was comparable to that of Mycobacteria Growth Indicator Tube technique (MGIT) (57.8%) with clinical presentation as the gold standard. In terms of multi drug resistant tuberculosis (MDR- TB) diagnosis, MODS had rather low sensitivity in detection of Isoniazid (INH) or Rifampicin (RIF) resistant and MDR isolates, 72.6%, 72.7% and 77.8%, respectively against the proportional drug susceptibility testing (DST) method. The low sensitivity of DST-MODS in this study was probably due to low bacterial load samples and use of a high INH critical concentration (O.4~g/ml). For LED microscopy, the detection rate, sensitivity, specificity and false negative of LED 40X were comparable to those of conventional fluorescence microscope and light microscope. The false positive rate • of LED 40X increased in comparison with Ziehl-Neelsen (ZN) reading in the early phases of the evaluation (2.6% and 2.7% vs 0% in the validation and implementation phases vs continuation phase, respectively), probably due to lack of experience among technicians, but no significant difference was found (P>0.05). In the last phase, no false positive result was recorded. Although the reading time of fluorescence-LED (FM-LED) and ZN reading were less than 2 minutes, ZN-light microscope (ZN-LM) reading time was shorter than that of FM-LED40X, especially in positive smears (P=0.007) in the early study phase. By the end of the study, FM-LED reading time was shorter than ZN-LM which was recorded in the early phase. With high experienced technicians there may be little or no additional benefit to case detection if a light microscope is replaced by a fluorescence microscope. In general, MODS and LED microscopy are reliable methods for use in diagnosis of TB in resource limited settings due to their accuracy, reliability and low costs. Large scale operational projects should be conducted to evaluate the feasibility and cost-effectiveness of these methods in countries where these techniques are to be implemented.

Anti-tuberculosis drug-induced liver injury is a rare but serious adverse drug reaction. This study aimed to identity specific genes conferring susceptibility to this serious adverse drug reaction, especially in relation to isoniazid treatment and to study the underlying mechanism for toxicity. Anti-tuberculosis drug-induced liver injury cases (n=26) and community controls (n=90) from Europe and South Asia were genotyped for polymorphisms in NAT2, GST genes, CYP2E1, PXR and SOD2. NAT2 slow acetylators were more susceptible to liver injury (OR=4.60; 95% CI=1.47-14.44). The GSTM1 null genotype was more common in cases than controls (OR=2.91; 95% CI=1.14-7.43). Risk of liver injury was significantly increased in subjects with combined NAT2 slow acetylator and GSTM1 null genotype (OR=3.71; 95% CI=1.48-9.31). No significant effects were seen for the other genotypes studied except that a GSTA4 haplotype was slightly more common in liver injury cases. The contribution of NAT2 genotype to isoniazid toxicity was examined using an in vitro overexpression approach. Stable expression of either NAT2*4 or NAT*5 constructs in HepG2 cells had small effects on reduced glutathione to oxidised glutathione ratio and apoptosis. These changes were consistent with higher NAT2 activity increasing isoniazid toxicity. In addition, overexpression and siRNA knockdown approaches showed protective roles for GSTA1 and A4 against isoniazid toxicity. The relevance of combinations of anti-tuberculosis drugs to overall toxicity was investigated by studies in human hepatocytes and LS180 cells. In the LS180 cells, rifampicin coadministation with isoniazid resulted in a small but significant decrease in both isoniazid and pyrazinamide toxicity. Studies on the isoniazid-rifampicin combination in human hepatocytes gave inconsistent findings but a decrease in cell toxicity due to isoniazid by pretreatment with rifampicin was seen in some donors. Increased expression of the carboxyesterase gene CES2 was seen in LS180 cells and in some hepatocytes and could represent a protective effect.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB). Mtb cells contain intracellular lipid bodies (LBs) in sputum and the levels vary between patients. Previous reports showed the essential role of nitric oxide (NO) in killing Mtb in vivo and that Mtb forms LBs in vitro upon NO treatment. These treated populations are tolerant to isoniazid and rifampicin, but the reasons for varing LB levels in vivo are not understood. The objective of this study was to determine the association between fractional exhaled nitric oxide (FeNO) concentrations with the proportion of LB[superscript +ve] tubercle bacilli in sputum. The majority of TB patients (65%) showed ≤ 20.6 ppb FeNO concentration. FeNO concentration was weak but significantly associated with the proportion of LB[superscript +ve] tubercle bacilli in sputum. FeNO concentration was also significantly associated with the proportion of LB[superscript +ve] tubercle bacilli among HIV[superscript +ve] P[superscript -] and HIV[superscript –ve] P[superscript +]/TB patients. High esosinophil count was significantly associated with FeNO concentration in both HIV[superscript –ve] and HIV[superscript +ve] /TB patients infected with intestinal parasite. The CAS and EAL Mtb spoligotypes were the dominant Mtb spoligotypes in Gondar. FeNO concentration was significantly associated with the proportion of LB[superscript +ve] tubercle bacilli among TB patients infected with the CAS Mtb spoligotype (p<0.01; r²=0.323) but not among patients infected with the EAL. The anti-microbial susceptibility pattern showed an 11.9% either mono or multi-drug resistance. The average proportion of LB[superscript +ve] tubercle bacilli among drug resistant TB patients was relatively higher than the corresponding drug sensitive TB patients. The proportion of LB[superscript +ve] tubercle bacilli was also higher among MDR-TB patients. The association between FeNO concentrations with the proportion of LB[superscript +ve] tubercle bacilli raises questions regarding L-arginine supplementation during TB treatment. The association of FeNO concentration with the proportion of LB[superscript +ve] tubercle bacilli among specific Mtb spoligotypes may reflect difference in NO tolerance.

A third of the world's population (2,000 million people) is infected with tuberculosis (TB). The two main control measures emphasised by WHO are improved case detection and cure. rates. High case detection level is of great importance to decrease personal suffering, risk of death and limit transmission in the community. TB is also a social disease and aspect such as the patients' health seeking behaviour affect TB Control programme. According to WHO, Malaysia is a country with intermediate burden of TB but Sabah, in East Malaysia has a notification rate of two and a half times that of the country's and contributes one third of the total cases. Two studies were conducted in West Malaysia on delay among TB patients but none in East Malaysia. This research was conducted in 2 phases. Phase 1 utilised a qualitative approach to get a better understanding of the health seeking behaviour of TB patients and those involved in their care (32 respondents). Phase 2 was conducted among 296 newly registered smear positive TB patients using a pre-tested questionnaire. It was found that 51.8% (95% CI: 45.9 - 57.8) of patients sought treatment after 30 days and the median patient time period was 60 days. By using multivariate analysis, those whose u'sual first treatment choice was a non-government health facility were twice as likely to have patient delay than those whose usual first choice was a government facility (Adjusted OR: 2.28, CI: 1.03 - 5.06). The median for doctor time period was 20 days and almost 43% of the respondents were put on treatment within 14 days after consultation with a doctor. In the multivariate analysis for doctor delay, those Who had never used government facility, had chest pain and loss of weight were twice more likely to be associated with doctor delay. A repeat visit to the first provider was associated with an approximately 4 times greater risk of doctor delay than those who did not (AOR: 3.88, CI: 1.79 - 8.39). Recommendations for improvement of current public health practice include ensuring greater awareness and adherence to existing guidelines and coritinuous health education on TB. The findings on delay can be used to develop a Quality Assurance Programme (QAP) to shorten doctor delay.

Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) incorporating the glycolipid trehalose 6,6-dibehenate (TDB) forms a promising liposomal vaccine adjuvant. To be exploited as effective subunit vaccine delivery systems, the physicochemical characteristics of liposomes were studied in detail and correlated with their effectiveness in vivo, in an attempt to elucidate key aspects controlling their efficacy. This research took the previously optimised DDA-TDB system as a foundation for a range of formulations incorporating additional lipids of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), by incrementally replacing the cationic content within DDA-TDB or reducing the total DDA-TDB dose upon its substitution, to ascertain the role of DDA and the effect of DDA-TDB concentration in influencing the resultant immunological performance upon delivery of the novel subunit TB vaccine, Ag85B–ESAT-6-Rv2660c (H56 vaccine). With the aim of using the DPPC based systems for pulmonary vaccine delivery and the DSPC systems for application via the intramuscular route, initial work focused on physicochemical characterisation of the systems with incorporation of DPPC or DSPC displaying comparable physical stability, morphological structure and levels of antigen retention to that of DDA-TDB. Thermodynamic analysis was also conducted to detect main phase transition temperatures and subsequent in vitro cell culture studies demonstrated a favourable reduction in cytotoxicity, stimulation of phagocytic activity and macrophage activation in response to the proposed liposomal immunoadjuvants. Immunisation of mice with H56 vaccine via the proposed liposomal adjuvants showed that DDA was an important factor in mediating resultant immune responses, with partial replacement or substitution of DDA-TDB stimulating Th1 type cellular immunity characterised by elevated levels of IgG2b antibodies and IFN-? and IL-2 cytokines, essential for providing protective efficacy against TB. Upon increased DSPC content within the formulation, either by DDA replacement or reduction of DDA and TDB, responses were skewed towards Th2 type immunity with reduced IgG2b antibody levels and elevated IL-5 and IL-10 cytokine production, as resultant immunological responses were independent of liposomal zeta potential. The role of the cationic DDA lipid and the effect of DDA-TDB concentration were appreciated as the proposed liposomal formulations elicited antigen specific antibody and cellular immune responses, demonstrating the potential of cationic liposomes to be utilised as adjuvants for subunit vaccine delivery. Furthermore, the promising capability of the novel H56 vaccine candidate in eliciting protection against TB was apparent in a mouse model.

Diagnosis of tuberculosis (TB) in developing countries relies on sputum smear microscopy, which has poor sensitivity compared to culture, particularly for immune-compromised people. Induction of sputum by inhalation of saline has the potential to increase case detection but centrifugation of the sample is necessary before microscopy. A systematic review was undertaken comparing the performance of microscopy and culture on induced sputum. A study was then undertaken in Zimbabwe on the use of magnetic beads (TB Beads) coated with chemical ligands to concentrate the mycobacteria from induced sputum, which if successful would negate the requirements for a biosafety cabinet and centrifuge, and therefore increase access to TB diagnosis in peripheral clinics. Adult TB suspects who were smear-negative on conventional microscopy underwent the induction procedure. The resulting sample was divided in two: half was processed with the TB Beads method with fluorescent and ZiehlNeelsen microscopy; the other half was subjected to centrifugation followed by fluorescent smear microscopy, and culture on Lowenstein-Jensen medium. Of the 139 patients that were enrolled, 97% produced an induced sputum sample. Mild side-effects were experienced by 13 % of patients. 26 smear-positive patients were found, of which 21 were positive on centrifugation, and 13 on TB Beads. Seven patients were culture-positive. One patient was smear-negative and culture-positive. The TB Beads elution buffer was altered by the manufacturer during enrollment, so results were analysed for both buffer types combined, as well as separately. For both buffers combined, the sensitivity and specificity of microscopy compared to culture were 43 % and 95 % respectively for ZN microscopy and 43% and 94% when using fluorescent microscopy. When both ZN and fluorescent microscopy were combined the sensitivity was 57%. However, when centrifugation was used prior to fluorescent microscopy, sensitivity increased to 86 %. In conclusion the TB Beads did not perform sufficiently well to replace centrifugation.

The main objective of the work was to develop a novel analytical tool, incorporating EVA-biosensor technology, for the diagnosis of TB, and this was achieved in three parts. The first target of the first part was the total synthesis of two novel diastereomers of α-methyl-trans-cyclopropane mycolic acids (I) and (11) present in. the cell wall of Mycobacterium avium complex and Mycobacterium Kansasii. The second target of the first part was the synthesis of four cord factors as derivatives of compounds (I) and (II), in order to understand their interaction with the immune system and for use in biosensor technologies, as well as ELISA assays, for the diagnosis of TB. The synthesized cord factors were TDM (Ill), TMM (IV), TDM (V) and TMM (VI). The second part of this project was to synthesize thiolated derivatives of CL-mycolic acids which were the disulfide (VII) and free thiol (VIII). These compounds would eventually be bound to a gold surface and their activities against tuberculosis antibodies tested. This will be used to develop a new biosensor technique for detection of TB. The third part concerned the application of the synthetic molecules in an exploratory biological investigation for the development of a rapid and accurate TB diagnostic. Three novel synthetic cord factors as synthetic antigens were used on a novel biosensor platform, the EVA -Evanescent Biosensor Technology, with TB positive and negative sera. These antigens showed significant sensitivities and specificities by this technique.

The main objective of the work was to develop a analytical method for the diagnosis of TB, and this was achieved in three parts.The first part was involved the synthesis of two oxygenated mycolic acids, a methoxy mycolic acid (I) and a keto mycolic acid (11). The successful synthesis of these mycolic acids led to the synthesis of the cord factors TDM (Ill), TMM (IV), TDM (V) and TMM (VI), which were used in modified ELISA assay as new antigens to detect TB-antibodies in serum. The second part was involved the synthesis of thiolated derivatives (VII), (VIII) and (IX) of methoxy mycolic acids. These compounds will be covalently attached to a gold surface so as to create a self assembled monolayer with antigenic properties. It is expected that this will create a stable surface for the binding of TB-antibodies in diagnosis tuberculosis tests. This will contribute to development of a new biosensor as a rapid and accurate method for detecting TB infection. The third part was concerned on analysing of TB positive and TB negative samples to detect TB-antibodies in patient serum using novel synthetic mycolic acids and their derivatives as antigens in modified ELISA assay, after determination of the optimisation conditions of the ELISA assay. Higher antibody binding signals were observed with cord factors. The sensitivity and selectivity for TDM (198) (80%, 87%), TDM (204) (75%, 90%) and TDM (254) (80%, 84%), respectively which showed good significant values in comparison with other synthetic antigens.

Tuberculosis (TB) is an immense global problem with 8;8 million new cases, leading to an estimated 1.45 million deaths from the disease in 2010 alone. Although current serological tests have been deemed inconsistent and imprecise by the World Health Organisation, they have stated that a point of care serological test for the detection TB would be of extreme value. This work sought to provide such a device. It was known that ELISA assays using trehalose di-mycolates, trehalose mono-mycolates and mycolic acids as antigens could be used to detect the presence of anti-TB antibodies in serum samples. It was hoped that by binding these antigens to a gold surface, the performance of the assays could be improved. Using information gained from ELISA assays, work was undertaken to develop a novel, reliable, easy to use point of care sensor for the detection of TB. It was decided to use gold nanopartic1es as part of the sensor, and thiol modified antigens, as well as non-thiol modified antigens (via a linker compound) were successfully bound to their surfaces. It was discovered that addition of a sodium chloride solution to the antigen coated gold nanopartic1es led to their aggregation, in turn, changing the colour of the solution from red to blue. It was also discovered that this process could be inhibited by the addition of a TB positive serum sample to the coated gold nanopartic1es prior to adding the sodium chloride solution. These observations were used to develop a novel assay for the detection of TB, which could be performed in as little as 15 minutes. Large numbers of serum samples from high burden TB populations were analysed by the developed assay, and although a number of false positive samples were detected, largely from patients with a record of infection with malaria, almost all of the positive samples could be detected with multiple antigens. The assay developed was then incorporated into a device which is fully portable and easy to use, and can be read by eye, by observing the colour of the coated gold nanopartic1e solution, and potentially, by measuring its absorbance at two different wavelengths.

Two defining characteristics of Mycobacterium tuberculosis (Mtb) in sputum are gene expression and the production of lipid bodies (LBs). Previous analysis of the Mtb sputum transcriptome demonstrated that a population of persister-like bacilli predominate in sputum. LBs are intracellular structures consisting of triacylglycerol. Their formation is environmentally regulated, and occurs in response to a number of stresses, including conditions which induce a non-replicating persistence phenotype. Based upon these findings, Mtb in sputum may express a unique, transmission-adapted phenotype. Comparison of the gene expression of Mtb in sputum against Mtb exposed to in vitro stimuli demonstrated that no obvious single growth condition fully replicated the sputum transcriptome. In contrast, Mtb cultures exposed to multiple stimuli, including phosphate-buffered saline or RPMI medium, nitric oxide, cholesterol, oleic acid and static incubation had gene expression that correlated significantly to that in sputum. However, the exact combination of stresses is yet to be defined. It was demonstrated in these studies that the presence of LBs as a single factor does not influence the transmission adaptability of Mtb bacilli as tested here. An increased proportion of LB-positive cells did not confer increased survival to transmission stresses (desiccation and ultraviolet radiation). This was confirmed using two independent methods of LB induction (triacylglycerol synthase overexpression and nitric oxide exposure). Analysis of Mtb binding to macrophages also showed that an increased proportion of LB-positive cells did not result in increased bacterial binding. However, the Mtb multi-stimulus cultures showed increased macrophage binding as compared to control cultures, although this remains to be confirmed. It was concluded that Mtb gene expression in sputum occurs secondary to multiple stimuli, and that LBs may represent an epiphenomenon of the Mtb sputum phenotype as far as transmission is concerned. If in fact Mtb in sputum are transmission-adapted, other factors must contribute to this phenotype.