Ability of PCR assay to identify Mycobacterium tuberculosis in BACTEC 12B vials

Abstract

Introduction of PCR to directly detect Mycobacterium tuberculosis in clinical specimens has shown promise; however, interfering substances in clinical material have contributed to lowered assay sensitivities. We evaluated the ability of a PCR assay to detect M. tuberculosis in BACTEC 12B broth cultures. Clinical specimens were processed and inoculated into BACTEC 12B vials. Evaluation was approached in two phases, starting with an initial evaluation in which an aliquot of 12B broth was removed when the growth index (GI) was > or = 10 and stored at 4 degrees C until assayed by PCR. Of the 290 specimens initially assayed, 129 were culture negative for mycobacteria as well as PCR negative for M. tuberculosis. Except for one, cultures (n = 102) which grew mycobacteria other than M. tuberculosis were all PCR negative. The remaining 59 broths were all culture and PCR positive for M. tuberculosis; 39% (n = 23) of these cultures when assayed by PCR had GIs of < or = 50. Following initial evaluation, 200 12B BACTEC vials with GIs of > or = 10 were assayed in a similar manner except that specimens were amplified twice weekly to determine PCR's impact on the length of time to identification of M. tuberculosis as compared with standard laboratory practices. Utilization of PCR resulted in a mean time to detection of M. tuberculosis of 14 days, compared with 29 days by using commercially available nucleic acid probes to identify M. tuberculosis complex from growth of BACTEC 12B subcultures on solid media. In light of an overall sensitivity and specificity of 100 and 99.7%, respectively, coupled with the ability to identify M. tuberculosis days or weeks before other methods can be applied, we conclude that PCR might prove to be a rapid alternative for identification of M. tuberculosis in culture and allow for earlier setup of susceptibility testing.