One-tube multiplex PCR method for rapid identification of mycobacterium tuberculosis.

A rapid, inexpensive, simple, and accurate multiplex polymerase chain reaction (PCR) was developed in a single tube for identification of Mycobacterium tuberculosis. Assessment of sensitivity and specificity of simple PCR was performed with 116 strains of M. tuberculosis complex (MTC) and 144 strains of nontuberculous mycobacteria (NTM) compared with the biochemical method. Specific amplification of KS4, MTC-specific DNA fragment, was found in 98% (114/116) of MTC and not detected in 99% (143/144) of NTM. Amplification of the mtp40 gene revealed 95% sensitivity (100/105 strains of M. tuberculosis) and 77% specificity (not found in 119/155 mycobacterial strains). A multiplex PCR method based on the combination of KS4- and mtp40-derived primers was used for identification of M. tuberculosis. Crude DNA from slow growing mycobacteria with cream rough colonies that showed both 768-bp amplified product for KS4 and 396-bp for mtp40 was identified as M. tuberculosis whereas that from MTC gave only the 768-bp product.

Evaluation of polymerase chain reaction and restriction enzyme analysis for routine identification of mycobacteria: accuracy, rapidity, and cost analysis.

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.

Six-year report of mycobacteria recovered from automated culture system at Siriraj Hospital.

An increased incidence of tuberculosis and other mycobacterial infections among immunocompromised patients has created a serious health crisis, especially in resource-poor countries. In addition, disseminated disease occurs more frequently in these patients. Rapid isolation and accurate identification of causative agents are necessary for selecting an appropriate treatment regimen. Since an isolation of Mycobacterium tuberculosis and slowly growing mycobacteria require 3-4 weeks for conventional culture, the automated system can reduce the detection time to 7-10 days. The present study demonstrated the mycobacteria recovered from hemocultures and other sterile body fluids, using the BACTEC 9000 system. Overall, 5,490 samples during the period 1998-2003 were submitted for hemocuture and the isolated mycobacteria were identified by using molecular techniques, like multiplex PCR and PCR-REA. The results demonstrated that~18-28% of hemocultures were positive for mycobacteria. Of these, M. tuberculosis appeared to be the most common species among mycobacteria isolates whereas the M. avium complex was found to be the second most common. The combined use of an automated culture system and molecular techniques as shown in this study is a useful procedure for rapid isolation and identification of mycobacteria that can reduce the time from 6-8 weeks to 2-3 weeks.