Direct detection of rifampin resistant mycobacterium tuberculosis in clinical specimens by PCR-DNA sequencing

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Direct detection of rifampin resistant mycobacterium tuberculosis in clinical specimens by PCR-DNA sequencing

 

Author: Tong, Ho-lun Simon
Title: Direct detection of rifampin resistant mycobacterium tuberculosis in clinical specimens by PCR-DNA sequencing
Degree: M.Sc.
Year: 2003
Subject: Mycobacterium tuberculosis -- Molecular diagnosis
Polymerase chain reaction -- Diagnostic use
Hong Kong Polytechnic University -- Dissertations
Department: Multi-disciplinary Studies
School of Nursing
Pages: x, 68 leaves : ill. (some col.) ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1691102
URI: http://theses.lib.polyu.edu.hk/handle/200/4173
Abstract: The emergence of multi-drug resistant Mycobacterium tuberculosis (MDR-TB) is a significant problem worldwide, the results of antibiograms for Mycobacterium tuberculosis (MTB) is severely delayed when conventional phenotypic methods are used. Rapid genotypic methods that guarantee early detection of resistant strains are required in order to avoid delay in the initiation of effective therapies and to prevent the transmission of MDR-TB strains. It will be even more advantageous if these rapid detection methods can be performed directly in clinical specimens. Rifampin (RIF) resistance is a surrogate marker of MDR-TB, this study aimed to develop a rpoB-PCR DNA sequencing method to detect rifampin resistant MTB directly from clinical samples. Altogether 290 clinical samples from Queen Mary Hospital were studied. DNA extracted from the respiratory or non-respiratory specimens were subjected to PCR for amplification of 216-bp and 157-bp products of the IS6110 and rpoB genes, respectively. If samples were positive for IS6110 gene, the PCR products of rpoB gene were sequenced. One hundred seventy three out of 181 culture-positive specimens were confirmed to be MTB by IS6110-PCR. Seven out of these 173 clinical specimens were identified as RIF resistant by both sequencing and conventional proportion method. Four types of missense mutations in codons 513, 522, 526, and 531 were identified in these RIF resistant strains. These mutations were within the core region of the rpoB gene. The rate of rifampin resistant MTB was found to be 4.0% (=7/173) in Hong Kong. Two novel mutations were identified in this study: one was a combined mutation in codons 513 and 526, and the other was in codon 522. One RIF sensitive MTB harboured point mutations in the core region of the rpoB gene. Concordance between the results of antibiotic susceptibility test and that of PCR-DNA sequencing was 99.3%. The turnaround time of detection of the MDR strains in patients was shortened from 28 days to 24 hours. The PCR-DNA sequencing method described in this study can be used as a rapid method for direct detection of RIP resistant MTB in clinical specimens.

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