Monday, August 29, 2011

Culture Methods for Mycobacterium tuberculosis

Cultivation of M. tuberculosis from clinical samples is the gold standard for the diagnosis of active TB. It can detect 100 bacilli/mL-1 of sputum in comparison with 5,000–10,000 bacilli/mL-1 needed for microscopy [18]. It also provides material for further identification and drug susceptibility testing. Conventional methods of culture have relied on egg based and agar-based media, such as the Lowenstein-Jensen (LJ) medium and Middlebrook agar [19, 20]. Following decontamination and liquefaction procedures, sputum samples are inoculated and incubated for morphological growth, which usually occurs after several weeks of incubation. Identification of M. tuberculosis is done by performing several further biochemical tests [19, 21]. However, it is laborious and time consuming requiring from 3–8 weeks to obtain the results.

The introduction of the BACTEC radiometric system (BACTECTB-460; Becton Dickinson, Sparks, MD, USA) in the 1980s wasa breakthrough since it allowed the detection of M. tuberculosisin a few days compared with weeks in the conventional culture media [22]. However, the use of radioisotopes and the cost of the equipment precluded its use on a routine basis, except in reference laboratories predominantly in developed countries. A few years ago Becton Dickinson proposed another system based on fluorescence detection of mycobacterial growth [23].

The Mycobacteria growth indicator tube (MGIT) system is based on a glass tube containing a modified Middle brook 7H9 broth together with a fluorescence quenching-based oxygen sensor embedded at the bottom of the tube. When inoculated with M. tuberculosis, consumption of the dissolved oxygen produces fluorescence when illuminated by a UV lamp. The MGIT system has been thoroughly evaluated in clinical settings for the detection and recovery of mycobacteria. BADAK et al. [24] compared the MGIT system with the BACTEC TB-460 and LJ culture medium in 1,441 clinical specimens. Out of 178 isolates recovered, 30 (17%) were M. tuberculosis with the MGIT system recovering 28 (93%) compared with 25 (83%) recovered with the LJ medium. In another multicentre study, PFYFFER et al. [25] analysed 1,500clinical specimens detecting a total of 180 mycobacterial species comprising 113 M. tuberculosis complex isolates. The combination of MGIT and BACTEC detected 171 (95%) of all isolates with a time to detection of M. tuberculosis of 9.9 days compared with 9.7 days with BACTEC and 20.2 days with solid medium proving that MGIT was a valuable alternative to the radiometric system [25]. More recently the MGIT system has been fully automated and turned into the BACTEC MGIT 960 system, which is a non radiometric, non invasive system with the tubes incubated in a compact system that reads them automatically. In a multicentre study the BACTEC MGIT 960 system was compared with the radiometric BACTEC TB-460 system and LJ medium. Analysing 2,576 specimens, the best yield was obtained with BACTEC TB-460 (201 isolates), compared with190 isolates with BACTEC MGIT 960 and 168 isolates with LJ medium [26]. In another study IDIGORAS et al. [27] compared the BACTEC MGIT 960 system for sensitivity and time to detection of mycobacteria with solid medium, and microscopy on solid media. Sensitivity of each media compared with all mediacombined for growth of M. tuberculosis was 93%, 76%, 79% and75% for MGIT 960, Middlebrook 7H11, LJ and Coletsos.The mean time to detection was 12.9 days by BACTEC MGIT960, and 15.0 days with BACTEC 460, compared with 27.0 days with Lowenstein Jensen solid medium.148 Thus, WHO recently endorsed the use of liquid tuberculosis culture and drug susceptibility testing for M. tuberculosis in low-resource settings.149 The newly developed rapid liquid culture systems have unique sensing systems to detect a small amount of bacterial growth, such as by radioactivity or oxygen concentration changes, as quickly as possible. These systems can also be used for drug susceptibility testing as well as detecting M. tuberculosis.150,151

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