Rapid detection of rifampin-resistant mycobacterium tuberculosis and correlation between specific mutation of RpoB gene and level of resistance

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Rapid detection of rifampin-resistant mycobacterium tuberculosis and correlation between specific mutation of RpoB gene and level of resistance

 

Author: Au, Chi-ho Anders
Title: Rapid detection of rifampin-resistant mycobacterium tuberculosis and correlation between specific mutation of RpoB gene and level of resistance
Year: 2004
Subject: Hong Kong Polytechnic University -- Dissertations
Mycobacterium tuberculosis
Rifampin
Antitubercular agents
Department: School of Nursing
Pages: xii, 84 leaves : ill. (some col.) ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1781167
URI: http://theses.lib.polyu.edu.hk/handle/200/1703
Abstract: Tuberculosis (TB) is still the major disease killer in both developing and developed countries. This problem is deteriorating in Hong Kong as the number of cases has been slowly rising in recent decades although fairly stable in recent years. In addition, the death rates are further aggravated by the increasing number of human immunodeficiency virus-positive cases. However, the most concern is the development of drug resistance in the causative agent of TB-M. tuberculosis (MTB), especially the multi-drug resistant strains (MDR-TB) which is closely-related to mutational events. MDR-TB is defined as resistance to at least isoniazid and rifampin, the two key components of the World Health Organization (WHO) DOTS (directly observed therapy, short course). Since most rifampin-resistant isolates are also resistant to isoniazid, the detection of rifampin resistance can in turn determine MDR-TB. So rifampin could act as a surrogate marker for MDR-TB which has higher study value than other anti-TB agents. This study aims to investigate the correlation between the mutation positions and resistance levels of clinical isolates of MTB in Hong Kong and to evaluate a rapid method for detection of rifampin-resistant isolates. A total of 60 rifampin-resistant clinical isolates from Ruttonjee Hospital were included in this study. Frozen stocks of these isolates were subcultured and were confirmed to be MTB by both molecular and conventional methods using IS611O-polymerase chain reaction (PCR); and niacin and nitrate tests repectively. Routine minimum inhibitory concentration to rifampin was performed on all isolates. DNA was extracted and rapid cycle real-time PCR using LightCycler (Roche) was used to detect mutations within the 81-bp rifampin resistance determining region of the rpoB gene encoding the RNA polymerase which is the target site of rifampin. Automated DNA sequencing using ABI Prism 310 genetic analyzer (Applied Biosystems) was employed to sequence the PCR products generated from real-time PCR including those with positive and negative results. The results showed correlation between susceptibility to rifampin and mutations in the rpoB gene. High levels of rifampin resistance are associated with the two most frequent mutations -codons 526 and 531. Although real-time PCR could only detect 53 mutations, all 60 mutations were detected by DNA sequencing. The combination of rapid screening by real-time PCR and subsequent DNA sequencing would be a desirable method for rapid detection of drug resistant MTB in routine clinical laboratory as the turn-around-time of susceptibility results can be greatly shortened from 28 days to within two days.

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