published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Mycobacterium tuberculosis (MTB) is a major infective agent causing human tuberculosis (TB) in the worldwide. Although tuberculosis can be treated by a six-month course of antibiotics, the prevalence of extensively drug-resistance TB (XDR-TB) made the disease becomes a global health problem. In addition to the conventional MTB detection methods, molecular methods become significant in drug resistant MTB detection which can enhance effective drug treatment. In this study, 200 MTB respiratory specimens were collected from patients with suspected tuberculosis in Tuen Mun Hospital in Hong Kong. Based on the culture method as a gold standard for MTB detection, the presence of MTB in clinical samples was determined by IS6110single tube nested real-time PCR. In addition, by using High Resolution Melting (HRM) analysis, the presence of mutant type KatG315 gene for detecting isoniazid resistant MTB was determined. Among 66 MTB culture positive samples, 10 samples had positive acid fast bacilli (AFB) smears giving the diagnostic sensitivity 15.1%. IS6110 single tube nested PCR was amplified in 51 specimens giving 77.2% MTB detection sensitivity and 97.8% specificity. Among 51 samples positive for IS6110 PCR, 66.7% showed successful amplification in subsequent KatG-HRM assay. Two samples were confirmed to be isoniazid (INH) resistance in Public Health Laboratory Centre (PHLC). However, there was only one sample showing detectable KatG315 mutation in clinical specimen by using HRM while the other was only detected in the corresponding culture isolate. From the result of this study, single tube nested real-time PCR demonstrated MTB detection in clinical samples and INH resistant strain with KatG315mutationcan be detected by HRM analysis. Early detection of mycobacteria allow earlier treatment of the patient, thus transmission of the disease can be controlled. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Background Tuberculosis (TB) infection is a contagious disease due to infection by Mycobacterium tuberculosis(MTB) causing global health burden. There is increasing effort to develop early case detection methods and also to address the issue of multidrug resistance TB (MDR-TB). Molecular methods have been applied to provide rapid and accurate diagnosis. In addition to commercial kits being available for the detection of MTB from clinical specimens, In-house PCR assays have also been developed for the detection of MTB, and can be adjusted according to the laboratories’ own demand. Several molecular techniques like TaqMan probes and Hybridization probes may be applied to target for markers of MTB, e.g. 16s rRNA and IS6110.Detection of the mutation genes, for example, katGfor isoniazid (INH), enables determination of susceptibility of the antibiotic more rapidly than traditional culture methods, and is especially useful due to the increasing emergence of MDR-TB. A wide range of genes have been reported to be related to the resistance of INH, katG315 mutation is the most common gene among them. Therefore, genotyping katG315 allows determination of the susceptibility of INH. Objective The first objective is to evaluate the diagnostic performance of IS6110 One-tube Nested Real-Time PCR for the detection of MTB. Clinical pulmonary specimens collected from Tuen Mun Hospital were retrieved for investigation. All the specimens have already been tested for COBAS TaqMan MTB test and culture results have been obtained for all the samples. During the first stage of the study, all the specimens were tested with IS6110 One-tube Nested Real-Time PCR. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were obtained from the comparison with the gold standard of MTB detection, i.e., culture. During the second stage of the study, samples were selected to undergo katG315 HybProbe Real-Time PCR assay to determine the genotype of katG. The performance of the assay was evaluated statistically. Result In the first stage of the study, 200 samples were tested with IS6110 One-tube Nested Real-Time PCR. The assay was found to have a sensitivity of 76.92%, specificity of 98.52%, positive predictive value of 96.15%, negative predictive value of 89.86% and the diagnostic odds ratio of 221.667. In the second stage of the study, 66 samples were selected and tested for katG315 HybProbe Real-Time PCR assay, 36 samples were successfully genotyped while 30 samples failed to be genotyped. The only culture proven INH resistance specimen was not amplified at first, and culture isolate was extracted for genotyping again. The repeated test confirmed the genotype of the resistance strain to be a mutant. Conclusion katG315 HybProbe Real-Time PCR assay is a valid approach for genotyping katG. However, the sensitivity and efficiency has to be improved before application for clinical use. From the statistics obtained, COBAS TaqMan PCR assay, which is routinely used in Tuen Mun Hospital, is statistically proven to have comparatively better performance than the IS6110 One-tube Nested Real-Time PCR. Improvement on the assay is required for IS6110 One-tube Nested Real-Time PCR. However, there is great potential of applying both IS6110 One-tube Nested Real-Time PCR and katG315 HybProbe Real-Time PCR assay in clinical use with the same platform available. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

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The work presented here aims at identifying whether the genes identified in the genome of Mycobacterium tuberculosis that code for products involved in anaerobic metabolism are active or inactivated genes. The study consists of three distinct parts. In part one, serial dilutions of sputum of patients with pulmonary tuberculosis (PTB) were grown on agar surface and in high columns of un-agitated broth. The highest dilution from which mycobacteria was grown was for all patients significantly higher in the broth cultures than on the plates suggesting the presence of anaerobically metabolizing mycobacteria in the lungs of patients with PTB. Part two of the study identified gene expression by measuring the concentration of transcripts for 5 genes involved in aerobic or anaerobic pathways. This was done over a period of 15 weeks using un-agitated broth cultures (the Wayne method). Undulating patterns of gene expression were found with the genes coding for anaerobic metabolic pathway components expressed at higher levels than those coding for aerobic pathway components while the cultures grew older. Part three aimed at measuring transcription products of the same set of genes directly in sputum specimens. Although quantitation at bacterial cell level in the sputum could not be achieved, expression of all genes was established with on average larger quantities of transcripts of genes coding for the anaerobic pathway components. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2010.

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Dissertation (PhD)--University of Stellenbosch, 2007. / Bibliography. Also available on the Internet.