[Comparative evaluation of two different formulae of Middlebrook 7H9 broth in a fully automated mycobacteria culture system, MB/BacT; the effect of Tween 80].

Two different formulae of Middlebrook 7H9 broth, one containing Tween 80 [Tween (+) broth] and the other containing vancomycin but not Tween 80 [Tween (-) broth], were evaluated in parallel for a fully automated mycobacteria culture system, MB/BacT(Organon Teknika, Durham, NC, U.S.A.). A total of 586 clinical sputum specimens were digested and decontaminated by the semi-alkaline protease-N-acetyl-L-cysteine-NaOH (SAP-NALC-NaOH). Each part of sample treated was inoculated into the MB/BacT Process Bottle containing the respective Middlebrook 7H9 broth. Culture bottles were incubated in the MB/BacT at 37 degrees C for up to 56 days. Of 586 samples, 110 isolates of Mycobacterium tuberculosis complex and 77 of nontuberculous mycobacteria (NTM) were isolated. The occurrence of false alarm due to breakthrough contamination was 3.2 in Tween (+) broth and 2.9% in Tween (-) broth. Also, the positivities of mycobacteria by the respective culture media were comparable. However, Tween (-) broth could detect positive cultures for mycobacteria, particularly for M. tuberculosis complex at the earlier incubation cycle when compared to Tween (+) broth. The time to detect 50% positive cultures for M. tuberculosis complex was 20.5 days for Tween (-) broth and 34.3 days for Tween (+) broth, respectively. With the results, it was concluded that; Tween (+) broth produced homogeneous mycobacterial growth in culture media, and thus, it was easy to prepare the inoculum directly adjusted to McFarland turbidity to the susceptibility test. However, the present formula of Middlebrook 7H9 broth supplemented with Tween 80 was not enough suitable for the rapid detection of positive cultures and needs some revisions to improve.

Use of nucleic acid probes for identification of Mycobacterium tuberculosis directly from MB/BacT bottles.

The feasibility of using nucleic acid probes directly from positive MB/BacT broth to identify mycobacteria was determined in this study. A total number of 2,727 specimens were cultured into the MB/BacT (Organon Teknika) automated system and on conventional Loweinstein-Jensen (LJ) slants. The Gen-Probe AccuProbe culture identification tests (DNA probes) were used on samples from bottles which were identified as positive for mycobacteria by MB/BacT. Samples of positive MB/BacT broth (0.1 ml) were used directly in the broth culture method for the DNA probes as published by Gen-Probe. Centrifugation of the contents of the bottle was not done prior to probe testing. The number of mycobacteria detected by MB/BacT and LJ was 253 (221 isolates of M. tuberculosis and 32 isolates of mycobacteria other than M. tuberculosis [MOTT]). A total of 96.4% (213 of 221) of the bottles growing M. tuberculosis produced a positive direct DNA probe result for M. tuberculosis complex. One hundred percent (16 of 16) of the bottles growing M. gordonae produced a positive direct DNA probe result for M. gordonae. A total of 3.6% (8 of 221) of the bottles growing M. tuberculosis did not yield a positive direct DNA probe result for M. tuberculosis complex. The testing of subcultures made onto solid media from the positive bottles by AccuProbe identified six of these eight M. tuberculosis isolates. Two (0.9%) M. tuberculosis isolates gave a negative result for the M. tuberculosis probe test applied on the MB/BacT broth and its subculture. The rest of the positive MB/BacT bottles growing MOTT (16 of 32) were negative for M. gordonae, M. avium, M. intracellulare, and M. kansasii probes. The sensitivity and specificity of AccuProbe for the identification of M. tuberculosis and M. gordonae directly from MB/BacT broth were 96.4 and 100% for M. tuberculosis and 100 and 100% for M. gordonae, respectively. The direct testing of positive MB/BacT broth by AccuProbe, without prior centrifugation, allows for the accurate and rapid identification of M. tuberculosis and M. gordonae.

Evaluation of BacT/Alert 3D liquid culture system for recovery of mycobacteria from clinical specimens using sodium dodecyl (lauryl) sulfate-NaOH decontamination.

A total of 52 mycobacterial isolates were recovered from 1,197 clinical specimens decontaminated by a sodium dodecyl (lauryl) sulfate (SDS)-NaOH protocol. Of these, 94% were recovered with the BacT/Alert 3D system (Organon Teknika, Durham, N.C.) and 79% were recovered on Löwenstein-Jensen (LJ) medium. Mean times to detection of organisms of the Mycobacterium tuberculosis complex (n = 47) were 22.8 days with LJ medium and 16.2 days with the system. The BacT/Alert 3D system is a rapid and efficient detection system which can be used with an SDS-NaOH decontamination procedure.

Comparison of a nonradiometric system with Bactec 12B and culture on egg-based media for recovery of mycobacteria from clinical specimens.

The MB/BacT (Organon-Teknika, USA) is a fully automated, rapid, nonradiometric system for the culture of mycobacteria from clinical samples. The rate of recovery of mycobacteria and the time to detection obtained with the MB/ BacT were compared with those obtained with Löwenstein-Jensen and Coletsos solid media and Bactec 7H12 (12B) (Becton-Dickinson, USA) broth when 600 processed specimens were inoculated into all media in parallel. Specimens included 383 respiratory samples, 20 urine samples, 23 purulent exudates, 13 stool samples, 103 blood samples, 14 bone marrow aspirates, and 44 body fluid samples or aspirates. Overall, 106 mycobacterial isolates comprising six species were recovered, of which 100 (94.3%) were detected with MB/BacT, 98 (92.5%) with egg-based media, and 96 (90.2%) with Bactec 12B. The recovery rates of Mycobacterium tuberculosis complex with MB/BacT, egg-based media, and Bactec 12B were 98.7%, 93.7, and 89.9%, respectively. The average number of days to detection of single mycobacterial isolates was 14.2 days for MB/BacT, 26.1 days for egg-based media, and 11.7 days for Bactec 12B. The contamination rates were higher in MB/BacT (5%) than in Bactec 12B (1.8%) or egg-based media (1.5%). MB/BacT is a reliable, nonradiometric, less labor-intensive alternative to Bactec 12B for recovery of mycobacteria, but, as with other liquid culture methods, MB/BacT should be used in combination with a solid medium, not on its own.