PERFORMANCE OF AN AUTOMATED Q-BETA REPLICASE AMPLIFICATION ASSAY FOR MYCOBACTERIUM-TUBERCULOSIS IN A CLINICAL-TRIAL

We present data from a clinical trial study in which an automated vers ion (Galileo) of a previously described Q-Beta replicase-amplified pro be assay (J. S. Shah et al., J. Clin. Microbiol. 33:1435-1441, 1995) w as used for the direct detection of Mycobacterium tuberculosis complex in sputum. The assay was designed to target specific regions of 23S r RNA found in M. tuberculosis, Mycobacterium bovis, Mycobacterium afric anum, and Mycobacterium microti and had a sensitivity ranging from app roximately <10 to 300 CFU. The assay was tested for cross-hybridizatio n by using large numbers (e.g., 10(5) to 10(10) CFU/assay) of 133 othe r organisms commonly found in respiratory tract samples, including non -M. tuberculosis Mycobacterium spp. other bacteria, fungi, and viruses . All of these competitors tested negative by the assay. Automated ass ay results for 780 respiratory tract samples (sputum or bronchoalveola r lavage specimens) collected and tested at three trial sites in the U nited States) were compared with the results of culture and acid-fast microscopy. Aliquots of conventionally digested and decontaminated spu tum pellets were heated at 100 degrees C and mechanically disrupted pr ior to hybridization and background reduction, amplification, and dete ction in a closed disposable test pack. Pertinent elements of individu al patient histories relating to tuberculosis exposure, previous activ e disease, antituberculosis therapy status, etc., were considered in t he resolution of discrepant results for 48 (assay false-positive) samp les. Seventy-one of 90 (78.9%) culture-positive samples were positive when tested in the Galileo assay, while 7% of culture-negative samples were assay positive, corresponding to a sensitivity of 79% and a spec ificity of 93%. Following resolution of discrepant results by chart re view, the sensitivity and specificity for the Q-Beta replicase amplifi cation assay with the Galileo analyzer were 84 and 97%, respectively. A total of 69.2% of smear-negative (culture positive) samples were det ected by the assay. Ten test packs at a time were automatically proces sed by the Galileo analyzer without operator intervention following lo ading of samples. The first result was reported in approximately 3 h, and the last result was available in 6.5 h. To our knowledge, this is the first report of a clinical study with a fully automated amplificat ion probe hybridization assay for the detection of pathogens directly from a clinical specimen.