[Show abstract][Hide abstract] ABSTRACT: The aim of this study was the molecular characterization of primary drug-resistant Mycobacterium tuberculosis strains in Thailand. We examined a group of M. tuberculosis isolates from newly registered tuberculosis (TB) cases, collected at the largest university hospital, the Siriraj Hospital, in Thailand. Of 76 selected drug-resistant M. tuberculosis strains recovered from previously untreated pulmonary TB patients whose sputum samples were sent to this hospital, 29 (38%) were single-drug resistant, 26(34%) multidrug resistant and two (2.6%) extensively drug resistant. Fifty (66%) strains belonged to Beijing genotype. The study demonstrated a severe problem of drug resistance among recently detected TB patients, and two large clusters of genetically similar strains indicated ongoing transmission of drug-resistant TB.
[Show abstract][Hide abstract] ABSTRACT: Since rifampicin resistance is a surrogate marker for multidrug-resistant Mycobacterium tuberculosis (MDRTB), the present study aimed to investigate rpoB mutations conferring rifampicin resistance in M. tuberculosis strains from Thailand, and to develop a rapid, inexpensive and simple PCR-based method for rapid detection of MDRTB. Overall, 267 M. tuberculosis isolates, including 143 MDRTB isolates, were investigated. Isolates of the Beijing strain predominated among the MDRTB isolates (79.1%), but accounted for only 45.5% of the susceptible isolates. Mutations in the rpoB gene were found most commonly at codons 531, 526 and 516 (58%, 25.2% and 9.1%, respectively). A multiplex allele-specific PCR was developed and tested with 216 clinical isolates. In comparison with the proportion method, the method showed 94.2% sensitivity and 100% specificity, and had a 100% positive predictive value and a 95% negative predictive value, which suggested that this method could be useful for screening for MDRTB, particularly in resource-limited countries.
[Show abstract][Hide abstract] ABSTRACT: Polymerase chain reaction and restriction enzyme analysis (PCR-REA) of the hsp65 gene was evaluated for use as a routine identification method for identifying mycobacteria. The accuracy, rapidity, and cost were assessed compared with the conventional biochemical method. Five hundred and forty-one mycobacterial clinical isolates obtained from the Department of Microbiology, Faculty of Medicine at Siriraj Hospital, Mahidol University, were submitted for PCR-REA and biochemical identification. PCR-REA showed high concordant result with 100, 96.2, and 94.1% for identification of Mycobacterium tuberculosis, rapid- and slow-growing mycobacteria, respectively. Discordant results were obtained from 24 (4.4%) out of 541 isolates, consisting of 9 rapid growers (6 M. chelonae, 2 M. abscessus, and 1 M. fortuitum) and 15 slow growers (9 M. scrofulaceum, 2 M. gordonae, 1 M. avium, 1 M. kansasii, 1 M. malmoense, and 1 M. terrae complex). PCR-REA demonstrated not only accurate results but was also less expensive (2.1 US dollars/sample). This method was rapid with a turn-around time of 30 hours compared with 2-4 weeks for the conventional method.
The Southeast Asian journal of tropical medicine and public health 10/2005; 36(5):1252-60. · 0.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The increasing incidence of tuberculosis and other mycobacterial infections due to AIDS epidemic resulted in the need of rapid and accurate identification of isolated mycobacteria. The correct identification result leads to the selection of an appropriate therapeutic regimen. Polymerase chain reaction and restriction enzyme analysis (PCR-REA) has been developed since 1992 and used as the rapid method for identifying mycobacteria. Several genes or sequences have been used as an amplified target for PCR-REA. The present study aims to evaluate the potential use of PCR-REA of gene-encoding heat shock protein 65 kDa (hsp65) and beta-subunit RNA polymerase (rpoB) for the identification of mycobacteria compared with conventional biochemical identification. Two hundreds clinical isolates, consisting of 50 isolates of Mycobacterium tuberculosis and 150 isolates of nontuberculous mycobacteria (NTM), were submitted for identification using PCR-REA and biochemical method. The results demonstrated that PCR-REA identified 188 isolates of both M. tuberculosis and NTM concordantly with biochemical identification. Discordant identification results obtained from 12 isolates, comprised of 8 M. scrofulaceum, 1 M. avium complex, 1 M. malmoense, 1 M. terrae complex, and 1 M. chelonae/abscessus. Overall, the concordant percentage of results obtained from PCR-REA compared with biochemical method was 100%, 98.8%, and 83.3% for M. tuberculosis complex, rapidly growing, and slowly growing mycobacteria, respectively, and the results of hsp65 PCR-REA was in agreement with those obtained from rpoB PCR-REA. From this study, PCR-REA appears to be a simple, rapid, and reliable method for identifying mycobacteria in a routine microbiology laboratory.