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Title: Studies on faster methods of laboratory diagnosis of tuberculosis and analysis of phenotypic and genetic characteristics of multi-drug resistant tubercle bacilli with emphasis on their in vitro and in vivo response to isoniazid
Author: Oda, Eshetu Lemma
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1993
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Abstract:
To improve the diagnostic value of polymerase chain reaction (PCR) for the diagnosis of tuberculosis, an efficient and simple method of clinical sample pretreatment for PCR has been compared with two other methods used for this purpose and the data have shown better results with the added advantage that the method developed in this work is simpler, faster and uses safer chemicals. In addition to this, a simple absorption technique which improves the specificity of enzyme-linked immunosorbent assay (ELISA) has been developed. Serum samples from TB patients and normal subjects showed more discriminatory results leading to better diagnosis after the sera were absorbed with M vaccae antigen. Using xylene as a single major lytic agent a technique has been developed in this work which enables large scale extraction of DNA from mycobacteria in a matter of three hours. Catalase activity of drug resistant tubercle bacilli were analysed and compared with results from genetic studies using the catalase gene probe. Because all catalase negative strains (except one) gave positive hybridization signal with the catalase gene, catalase negativity in this case was shown to be due to a point mutation rather than to gene deletion. In one case, however, a unique band was demonstrated. The genetic patterns of the strains were investigated using the IS6110 insertion sequence probe also. In this thesis, 4 out of 21 isoniazid resistant tubercle bacilli were shown to grow better in the presence of isoniazid than in its absence. A highly isoniazid resistant strain which is also resistant to streptomycin and thiacetazone was catalase negative and its growth was not enhanced by isoniazid and had a diminished virulence for the guineapig. On the other hand another multi-drug-resistant strain to the above drugs and rifampicin had high catalase activity, its growth was enhanced by isoniazid and it had high virulence for the guineapig. To assess the enhancing effect of isoniazid on isoniazid resistant strains in vivo, guineapigs infected with isoniazid resistant tubercle bacilli were treated with isoniazid, and it has been shown that animals receiving treatment lost more weight than the control group. In general, the work covered in this thesis should be of value for the diagnosis of tuberculosis in situations where microscopy and culture fail to provide the desired result due to low sensitivity, in the former case, or due to delay of results as with culture. The studies on the drug-resistant tubercle bacilli could provide new insights to understand more about this field at this time when multi-drug resistant TB is becoming, as never before, an increasing concern.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.812072  DOI: Not available
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