Molecular Detection of Rifampicin Resistance in Mycobacterium Tuberculosis.

Priya, Santharam (2010) Molecular Detection of Rifampicin Resistance in Mycobacterium Tuberculosis. Masters thesis, PSG Institute of Medical Sciences and Research, Coimbatore.

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Abstract

INTRODUCTION : Tuberculosis (TB) is as old as the mankind. TB is the most common cause of death Due to a single infectious agent worldwide in adults. According to the recent estimates, one third of the human population (2 billion people) is infected with mycobacterium tuberculosis worldwide. Of these, more than half the cases occur in five South-East Asian Countries. India accounts for nearly one third of the global TB burden. In 2007, nearly 1.77 Million people died of TB and the global case fatality rate is estimated to be 45% in hivpositive TB-patients and 35% in HIV-negative TB-patients. Nearly 500,000 people die from TB in India every year - more than 1,000 every day, 1 every minute. In India, prevalence of primary MDR-TB in newly diagnosed cases has been observed to be 3.4 per cent or less. Data meticulously collected at the Tuberculosis Research Centre (TRC), Chennai over the last three decades suggest that MDR-TB levels in newly diagnosed patients have been one per cent or less7. Prevalence of MDR-TB among previously treated patients has been observed to be higher. In view of the emerging resistant mutants in the global and Indian scenario, the need for prompt detection of Mycobacterium tuberculosis in suspected cases, susceptibility pattern and genetic basis of drug resistance becomes paramount importance. In most laboratories in developing countries, smear examination and culture remains the only available choice. Though culture is the gold standard, it is time consuming due to the long replication time of mycobacteria and results are available only by 4-8 weeks. Good, reliable laboratory support is seldom available in developing nations. When facilities for culture and sensitivity testing are not available, therapeutic decisions are most often made by Standard Guidelines such as those published by the WHO. Although the DOTS strategy is the basis of good TB control, the strategy should be modified in some settings to identify drug-resistant cases sooner, and to make use of second-line drugs in appropriate treatment regimens. AIM : To detect multidrug resistance in Mycobacterium tuberculosis. OBJECTIVES : Isolation and characterization of Mycobacterium tuberculosis in respiratory specimens collected from patients attending the RNTCP outpatient clinic and from inpatients at PSG Hospitals by conventional methods. To detect drug resistance for Isoniazid and Rifampicin by two phenotypic methods namely Absolute concentration method and MTT assay. To detect mutations in rpob gene from the above Rifampicin resistant isolates of Mycobacterium tuberculosis by genotypic method using PCR and DNA Sequencing. MATERIALS AND METHODS ; A total of 117 respiratory specimens from patients attending the RNTCP outpatient clinic and from inpatients at PSG Hospitals during the period between June 2008 and June 2009 were included in the study. Human ethical clearance for the study was obtained from the Institutional ethical committee prior to collection of samples. A copy of the certificate is attached. The respiratory samples included were sputum, bronchoalveoaler lavage and pleural fluid. RESULTS : Out of the 117 respiratory samples collected for the identification and drug susceptibility testing of Mycobacterium tuberculosis, 104 (88%) were sputum, 11(9%) were bronchoalveolar lavage, and 2 (2%) were pleural fluid as shown in Fig 1. The RNTCP sputum grading ranged from scanty to 3+. The distribution of various grades and the percentage of sputum samples for each grading are shown in Fig 2 Of the 117 specimens processed, 13 had no growth and 3 samples showed growth on Lowenstein Jensen medium with p-nitro benzoic acid (PNB). Out of the 104 positive cultures, 3 which were resistant to PNB were excluded. SUMMARY : The study was taken up mainly to find out the Multi drug resistance in Mycobacterium tuberculosis in this part of Tamil Nadu. A total of 117 respiratory specimens were collected from the RNTCP outpatient clinic and in-patients from PSG hospitals. RNTCP criteria was followed for sample collection , staining and grading of sputum smears. Among the samples, sputum grading ranged from scanty, 1+, 2+ and 3+. Out of 117 specimens collected, 104 (89%) were culture positive and 3 more isolates were excluded from the study as they were resistant to p-nitro benzoic acid suggesting Non-tuberculous mycobacteria. A total of 101 culture positive isolates were further subjected for characterization using biochemical tests namely Niacin production, Nitrate reduction and Heat stable catalase. Among the study group there was male preponderance with a male to female ratio of 3:1. The peak age distribution was found to be 45 – 54 years (27%). All the 101 isolates were found to belong to M.tuberculosis complex of which 25% were found to be M.bovis.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Molecular Detection ; Rifampicin Resistance ; Mycobacterium Tuberculosis.
Subjects: MEDICAL > Microbiology
Depositing User: Subramani R
Date Deposited: 11 Aug 2017 02:33
Last Modified: 11 Aug 2017 02:33
URI: http://repository-tnmgrmu.ac.in/id/eprint/1318

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