DETECTION OF MYCOBACTERIUM-TUBERCULOSIS DIRECTLY FROM SPUTUM BY USINGA PROTOTYPE AUTOMATED Q-BETA REPLICASE ASSAY

We have adapted an assay for the direct detection of Mycobacterium tub erculosis using a prototype automated instrument platform in which pro bes are amplified with Q-beta replicase. The assay was based on amplif ication of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. Th e assay signal was the time required for fluorescence to exceed backgr ound levels (response time [RT]). RT was inversely related to the numb er of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing greater than or equal to 10 CFU o f M. tuberculosis responded in the assay (average RT, 13.91 min), whil e most (83%) samples containing as many as 10(7) CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The sam ples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. Afte r resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 4 5 negative samples responded within 14 min. Assay sensitivity, specifi city, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.