Detecting Mycobacterium tuberculosis in Bactec MGIT 960 Cultures by Inhouse IS 6110-based PCR Assay in Routine Clinical Practice

Abstract

Diagnosis of tuberculosis is challenging because the current methods are time-consuming and laborious. We have developed a method combining the Bactec MGIT 960 rapid culture system with the IS6110-based PCR for rapid diagnosis of tuberculosis. A total of 1745 samples from 712 patients treated at the Tri-Service General Hospital, Taipei, Taiwan between June and August 2005 were tested. An aliquot of positive Bactec MGIT 960 culture fluids was Kinyoun stained, and the samples positive for Kinyoun staining were directly assayed by the IS6110-based PCR. The same samples were also examined by the conventional methods for identification of Mycobacterium tuberculosis complex. One hundred and four samples from 62 patients were positive according to the Bactec MGIT 960 system. Among these, 59 (56.7%) were positive and 45 (43.3%) were negative according to the IS6110-based PCR. Compared with the conventional identification methods, the IS6110-based PCR assay correctly identified all 59 M. tuberculosis isolates and gave negative results for all nontuberculosis mycobacteria. The mean turnaround time for M. tuberculosis identification by this combined method was 6.41 days for smear-positive and 14.33 days for smear-negative specimens. The sensitivity of the IS6110-based PCR assay was determined to be 5 cells or 0.1 pg of mycobacterial DNA. The combined use of the automated Bactec MGIT 960 system and the IS6110-based PCR assay is sensitive and rapid for the detection of M. tuberculosis complex, and we recommend that this method be used routinely for identification of mycobacteria in clinical laboratories.