MAGANAARACHCHI, DN2011-02-142011-02-1420012001MAGANAARACHCHI, DN, PCR based detection techniques and DNA finger printing by restriction fragment analysis of mycobacterium tuberculosis, University of Colombo UC(MED), 2001: 343p.http://imsear.searo.who.int/handle/123456789/129638Dissertation: PhD, University of Colombo: UC(MED), 2001.In Sri Lanka definitive diagnosis of tuberculosis depends on the culture of mycobacteria, but the slow growth of the organism delays the diagnosis. Examination of direct smears for acid fast bacilli is the widely used method for the detection of mycobacteria, but lacks the desired specificity and relatively large number of bacteria is needed for detection. The objective of the study was to develop a rapid method to detect Mycobacterium tuberculosis from clinical samples and the applicability of DNA amplification techniques to a developing country like Sri Lanka. In this study, the potential use of PCR was investigated using the primers Pt8-Pt9 based on the insertion sequence IS 6110 of the Mycobacterium tuberculosis complex (Kolk etal, 1992). The study focused mainly on extrapulmonary tuberculosis where a definitive diagnosis was difficult. In this study 465 clinical samples were tested using PCR and the results obtained indicates that this approach offers may advantages over conventional methods and could be used to detect Mycobacterium tuberculosis in clinical samples. The sensitivity of the PCR method is largely dependent on the efficiency of the DNA extraction procedure which, could probably be improved by modifications of the lysis technique. For the DNA extraction and purification from the clinical samples two methods, the standard phenol extraction procedure desctibed by Sambrook etal (1982) and the guanidinium thiocyanate method described by Boom et al(1990) were used. The Boom's method was preferred to phenol/chloroform extraction method as most or all of the inhibitory substances in clinical samples were effectively removed by this method. Based on this study PCR was found to be more sensitive than culture or microscopy in those with a final diagnosis of extra pulmonary tuberculosis. In conclution, DNA amplification is a rapid, reliable and accurate method with a high degree of sensitivity and specificity for the detection of Mycobacterium tuberculosis DNA sequences and it can replace the conventional culture method in the diagnosis of extra pulmonary tuberculosis and tuberculosis meningitis except in the situation when antibiotic sensitivity results are required. Since PCR is too expensive to use in routine laboratories in Sri Lanka, one center could function as a reference laboratory where clinical samples can be tested using Polymerase Chain Reaction. Typing of Mycobacterium tuberculosis isolates is of great potential value for basic and epidemiological studies on tuberculosis. Results obtained from Restriction fragment length polymorphism typing show that the mahority of circulating Mycobacterium tuberculosis strains in Sri Lanka belong to a limited number of families, but the degree of IS6160 DNA polymorphism among strains were high. Of the 20 strains isolated from prisoners, none of the strains displayed identical fingerprints. In bacterial isolates of prisoners and ex-prisoners from the general population, there were 2 strains, which had identical banding patterns, while there were clear similarities between several isolates. From the general population 5 sets of identical banding patterns were observed. More than 68 percent had less than 5 copies of the IS sequence suggesting that our local M.tuberculosis strains have a fewer number of copies compared to data shown in most countries. Among the strains tested there were two strains that lacked the IS 6110 element. Drug resistant M. tuberculosis strains were examined by RFLP typing to determine whether a significant association between specific RFLP types and drug resistance is present. There were no specific RFLP types that could be associated with a particular type of drug resistance. Incidentlly the acquired drug resistance was 51.2 percent and multi drug resistance (MDR-TB) was 3.5 percent. The prevalence of acquired drug resistance to individual drugs was comparatively lower in Sri Lanka compared to other countries except ethambutol resistance. Furthermore there were strains that were resistant to second line drugs without showing resistance to first line drugs. Wide variation in drugs sensitivity patterns in the study indicates the necessity to have an antibiotic sensitivity test before instituting treatment for recurrent TB patients.en-USUniversity of Colombo, UC(MED): Sri Lanka HELLIS NetworkMycobacterium tuberculosisPolymerase Chain ReactionMycobacterium tuberculosis-diagnosisThesis