I= PCR BASED IDENTIFICATION AND GENETIC RELATEDNESS AMONG STRAINS OF MYCOBACTERIUM TUBERCULOSIS IN PAKISTAN
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Title of Thesis
PCR BASED IDENTIFICATION AND GENETIC RELATEDNESS AMONG STRAINS OF MYCOBACTERIUM TUBERCULOSIS IN PAKISTAN

Author(s)
Rubina Tabassum
Institute/University/Department Details
Institute of Chemistry/ University of the Punjab
Session
2003
Subject
Chemistry
Number of Pages
235
Keywords (Extracted from title, table of contents and abstract of thesis)
mycobacterium tuberculosis, tb, polymerase chain reaction, is6110, tuberculosis, m. tuberculosis, anti-tuberculous

Abstract
Thesis deals with three major issues related to the management of TB in Pakistan namely diagnosis, diversity of M. tuberculosis isolates and drug resistance. Molecular techniques were used along with conventional methods to evaluate the work.

Among various target sequences used for the detection of M. tuberculosis by Polymerase chain reaction (PCR), the insertion sequence IS6110 is the most commonly used target sequence. The presence and the copy number of this target sequence. The presence and the copy number of this target sequence were confirmed in M. tuberculosis isolates collected from various geographical locations in Pakistan. None of the M. tuberculosis isolate from Pakistan was found lacking this insertion sequence. The copy number of IS6110 was found to vary from 3-20. The percentage of isolates having upto five copies of IS6110 was found to be 6.52, 21.7% isolates have a copy number between 5 and 10 while those with more 10 copies were 32 out of 46 (69.5%). PCR based detection of M. tuberculosis was carried out to know the sensitivity and specificity this test. After validation of the technique with known cultures and known concentration of M. tuberculosis DNA(Dexyribonucleic acid), the technique was applied to clinical specimens and was compared either with microscopy staining or culture. These studies established superiority of PCR over culture for early and rapid diagnosis of M. tuberculosis. Two target sequences reported for the detection of M. tuberculosis were compared and IS6110 was found to be the best target sequences for the detection of M. tuberculosis in clinical specimens.

In pulmonary TB patients, sputum is the specimen of choice and in extra-pulmonary cases, body fluid or tissue from the affected area is preferred. This requires various invasive procedures and sometimes it is not possible to have the proper specimen. In the present study, peripheral blood was tested as an alternative specimen both in pulmonary and extra-pulmonary cases. It was found that peripheral blood offers a reliable specimen for the detection of M. tuberculosis in general and for the diagnosis of extra-pulmonary TB in particular.

The diversity of M. tuberculosis isolates in Pakistan was studied by standard IS6110 based restriction fragment length polymorphism (RFLP) analysis. All the isolates were found to be highly polymorphic. Cultures originating from certain areas were found to be more related than isolates collected from distant areas. The data showed that M. tuberculosis that TB was never controlled in this region.

The current standard for M. tuberculosis typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Due to advantages of being highly reproducible and comparison of the data with the available worldwide, Variable Number Tandem repeat (VNTR) analysis was used to fingerprint M. tuberculosis from different regions of Pakistan. The standard strain H37Rv was included as control to assess the authenticity of the results. A total of 50 fingerprints were found in 70 samples analyzed by VNTR indicating again a high diversity of M. tuberculosis isolates. Some of the isolates has the same VNTR pattern but differed in RFLP analysis. Some of the isolates has the same RFLP pattern were but different in VNTR typing.

Development of multiple drug resistance (MDR) is the most important factor that has compounded the spread of the disease. Mutations associated with the drug resistance against drugs commonly used for the treatment of TB were determined. Resistance to isoniazid is more complex and mutations in a least five different genes are associated with resistance development. Four mutations in Kat G genes were screened in M. tuberculosis isolates form Pakistan and Kat G 315 mutation was detected in about 60% of MDR isolates hydroxidperoxide (AhpC) and inhA which encodes an enoyl-acyl carrier protein reductase required for mycolic acid biosynthesis and is a major target for isoniazid (INH) in mycobacterial species are also involved in isoniazid resistance. Hence mutation in both these genes were also screened. Two mutations in rpoB gene associated with resistance to rifampicin were screened in M. tuberculosis isolates from Pakistan. The common mutation 531 reported from other parts of the world was found in 28 isolates out of 41 (68.29%) samples where as mutation526 was found in 6 sample. Thus, mutation in these codon has high predicative value. M. tuberculosis isolates from Pakistan were screened for alternations in codon 306 of the embB gene as mutation in this codon are reported to confer resistance to ethambutol. Mutations in rpsL gene associated with resistance to streptomycin were also screened and was found to have good predictive value for drug resistance. The identification of mutation associated with drug resistance in M. tuberculosis isolates from Pakistan will provide basis for understanding factors responsible for the development of multiple drug resistance and used of genotyping for rapid screening of resistance/ susceptibility to drug commonly used for the treatment of TB.

Download Full Thesis
4741.93 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
131.45 KB
2 1 Introduction 1-9
142.65 KB
  1.1 Tuberculosis 1
  1.2 The Global Threat Of Tuberculosis 2
  1.3 Global TB Control Programme And Causes Of Its Inability To Eliminate The Disease 5
  1.4 Tuberculosis Incidence, Related Programme And Outcome Indicators For Pakistan 5
  1.5 The Aims Of Research Project 9
3 2 Literature Review 10-63
813.94 KB
  2.1 History Of Tuberculosis 10
  2.2 Epidemiology Of Tuberculosis 12
  2.3 Infection And Disease 12
  2.4 Clinical Presentation 13
  2.5 Morphology And Growth Of M. Tuberculosis 14
  2.6 Molecular Biology Of M. Tuberculosis 16
  2.7 The Diagnosis Of Tuberculosis 27
  2.8 PCR Based Methods For The Detection Of M. Tuberculosis 36
  2.9 Strain Typing Of M. Tuberculosis 38
  2.10 Activity Of Drugs Against M. Tuberculosis 48
  2.11 Resistance Against Anti- Tuberculous Drugs 51
  2.12 Mechansim Of Action Of Drugs Used For Tuberculosis Treatment And Mutations Associated With Drugs Resistance 59
4 3 Materials And Methods 64-87
405.21 KB
  3.1 Collection Of M. Tuberculosis Cultures And Clinical Specimens 64
  3.2 Extraction Of DNA From Clinical Specimens Of PCR 68
  3.3 DNA Extraction From M. Tuberculosis Culture 70
  3.4 PCR For The Detection Of M. Tuberculosis 71
  3.5 Methods To Avoid False Positive Or False Negative PCR 74
  3.6 Analysis Of PCR Products By Agarose Gel Electrophoresis 74
  3.7 Detection Of Amplified DNA By Slot/ Dot-Blot Hybridization 75
  3.8 Culture M. Tuberculosis On LJ Medium 76
  3.9 Acid Fast Microcopy 76
  3.10 Rep PCR-Based Estimation Copy Number Of Is6110 In M. Tuberculosis Isolates Form Pakistan 76
  3.11 RFLP Fingerprinting Of M. Tuberculosis 78
  3.12 VNTR Analysis Of M. Tuberculosis Isolates 79
  3.13 Characterization Of Mutation In Genus Associated With Drug Resistance In M. Tuberculosis 82
5 4 Results 88-177
2782.3 KB
  4.1 The Presence And Copy Number Of Insertion Sequence IS6110 In M. Tuberculosis Isolates From Pakistan 88
  4.2 The Presence Of High Copy Number Of IS6110 In M. Tuberculosis Isolates From Pakistan Determined By RFLP Analysis 95
  4.3 Comparison Of Conventional And IS6110 Based PCR Method For Detection Of M. Tuberculosis In Clinical Samples 105
  4.4 Comparison Of PCR Sensitivity For Detection Of M. Tuberculosis In Clinical Specimens Using Two Different Target Sequences 110
  4.5 Evaluation Of Peripheral Blood As A Specimen PCR Based Diagnosis Of Pulmonary Tuberculosis 113
  4.6 Clinical Experience In Application Of PCR Based Detection Of M. Tuberculosis In Patients Suspected For Tuberculosis
 
  4.7 Finger Printing Of M. Tuberculosis Isolates By Vntr 125
  4.8 Identification Of Mutations Associated With Drug Resistance In M. Tuberculosis 138
6 5 Discussion 178-192
432.04 KB
  5.1 References 193-208