Advanced Techniques in Diagnostic Microbiology

23. Diagnosis of Mycobacterium tuberculosis 401
of antibiotic that inhibits growth of the agent indicates resistance (Sharma and
Mohan, 2004).
In the resistance ratio method, MIC of the isolate is shown as a multiple of the
MIC of a standard susceptible strain in order to avoid intra- and inter-laboratory
variations. Inoculum size should be strictly controlled in both tests. These tests
are not suitable for direct sensitivity testing of concentrated clinical specimens
(Sharma and Mohan, 2004).
BACTEC 460TB
In 1975, it was reported that MTB metabolic end-products could be detected by
using a 14 C-labeled substrate (Parsons et al., 2004). This system has been widely
performed in clinical laboratories in the past two decades and is considered the
gold standard in many ways (Tortoli et al., 2002). Radioactive BACTEC 460TB
system (Becton Dickinson) has been developed based on detection of 14 CO 2 produced
by consumption of a 14 C-labeled substrate. This system is used to test the
metabolism of the organism in the presence of all first-line drugs (SM, INH, RIF,
EMB, PZA) (Tortoli et al., 2000). This system is reliable and rapid for detection of
susceptibility of MTB to these drugs (Scarparo et al., 2004). Results of susceptibility
test can be available in 1 week compared with 3–4 weeks when solid media
is used (Parsons et al., 2004). BACTEC 460TB is the proposed reference method
for PZA susceptibility testing by NCCLS (Scarparo et al., 2004), but this system
uses radioactive substrates (Johansen et al., 2004).
MB/BacT Alert 3D
MB/BacT Alert 3D (Biomerieux) is a fully automated, nonradiometric system that
allows susceptibility testing of MTB to EMB, INH, PZA, RIF, and SM (Inderlied,
2004). The system is based on the detection of CO 2 released into the medium by
actively growing mycobacteria. The CO 2 generated decreases the pH, and color
change occurs in a sensor in the bottom of the vial. Color changes are detected by
a reflect metric detection unit in the instrument (Angeby et al., 2003). This system
is likely to increase biosafety profiles, and it does not contain radioactive waste
(Tortoli et al., 2000).
BACTEC MGIT 960
BACTEC MGIT 960 (Becton Dickinson) system is a nonradiometric, fully automated
continuous-monitoring system that allows susceptibility testing for INH,
RIF, SM, and EMB. Recently, the automated BACTEC MGIT 960 system was
developed to make the susceptibility testing of MTB to PZA possible (Kontos
et al., 2003). This system consists of using a modified Middlebrook 7H9 broth
in a test tube in conjunction with a fluorescence-quenching oxygen sensor. The
fluorescent compound is sensitive to the presence of oxygen in the test tube. Fluorescence
light emitted from growing mycobacteria is detected using a 365-nm UV