Identification of Mycobacterium tuberculosis and Mycobacterium avium-M. intracellulare directly from primary BACTEC cultures by using acridinium-ester-labeled DNA probes

Abstract

Identification of members of the Mycobacterium tuberculosis complex and the M. avium-M. intracellulare complex (MAC) directly from primary BACTEC cultures was evaluated by using acridinium-ester-labeled DNA probes (AccuProbe; GenProbe, Inc., San Diego, Calif.). In preliminary experiments, blood present in samples was found to interfere with the assay because of nonspecific chemiluminescence, which was measured in relative light units (RLUs). There was a direct relationship between the age of the culture and the number of nonspecific RLUs. A protocol using 1% sodium dodecyl sulfate-5 mM EDTA to treat BACTEC broth cultures which, with specimens containing blood, gave on the average a ninefold reduction in nonspecific chemiluminescence was developed. By using this treatment protocol, 120 specimens were tested directly from BACTEC broth cultures with an AccuProbe for the M. tuberculosis complex and/or the MAC. In order to establish the background of the specimen, the patient sample was assayed without probe. The criteria for the inclusion of BACTEC cultures in the evaluation were a growth index of greater than or equal to 100 and a positive smear for acid-fast bacilli directly from the BACTEC broth. For the 120 cultures tested, if a hybridization result of greater than or equal to 30,000 RLUs was considered positive, the sensitivities for detecting the M. tuberculosis complex and the MAC were 47 and 90%, respectively, with a specificity of 100% for both. However, if a ratio of the RLUs obtained with the MAC or the M. tuberculosis complex probe to those obtained with the specimen background of >/= 20 was considered positive, this gave 77% sensitivity and 100% specificity for BACTEC cultures containing M. tuberculosis complex isolates and 96% sensitivity and 100% specificity for those growing MAC isolates.