Identification of M. tuberculosis Complex
The following methods are available for Mycobacterium tuberculosis complex identification:
Amplification Test (MTD)
The Mycobacterium tuberculosis complex direct test (MTD) is used to detect the presence of M. tuberculosis complex in clinical specimens. The test is a target-amplified nucleic acid probe test for the in vitro diagnostic detection of rRNA, utilizing Transcription Mediated Amplification and the Hybridization Protection Assay. In this procedure the nucleic acids are released from the bacterial cells by sonication. Heat is used to denature the nucleic acids and disrupt the secondary rRNA structure. At a constant 42°C, the rRNA target is reproduced in multiple copies by transcription of DNA intermediates. The specific sequences (amplicons) are then detected by the same hybridization principle used in the AccuProbe test manufactured by GenProbe.
The MTD is not performed on blood, feces, urine, or specimens grossly contaminated with blood. The assay can be performed on formalized tissue samples embedded in paraffin. Minimum volume of specimen required is 1.0 ml.
There can be limitations in the MTD assay such as false negative results due to the presence of inhibitors in the sample. The inhibitors can inactivate the amplification enzymes. Our laboratory tests each sample for specimen inhibitors. The laboratory report will indicate detection of inhibitors if present in the sample. In addition and with no additional fee the laboratory performs smear examination and culture isolation as an internal quality assurance control for the MTD test. Positive culture results will be reported to the submitting facility.
MTD test results for cerebral spinal fluid are reported within 24 hours from specimen receipt. Results for all other specimens are available in 72 hours from specimen receipt by the laboratory.
High Performance Liquid Chromatography (HPLC)
We consider this procedure the most reliable and cost-effective tool for the rapid differentiation between M. tuberculosis complex and non-tuberculous mycobacteria (NTM) isolated in culture. The method used is a modified version of the CDC protocol developed in the early 1990s. Cells from culture are first saponified in a strong base. The mycolic acids (from which all Mycobacteria species derive their namesake) are acidified, then dissolved and extracted in an organic solvent. Extraction effectively separates the mycolic and other fatty acids from all other cellular material present in the original sample. These fatty acids are derivatized for detection by phospholuminescence. Each sample is injected through a C-18 column on the HPLC instrument and the resulting chromatogram is examined. Isolates within the Mycobacterium tuberculosis complex have a distinct mycolic acid profile and are easily identified by HPLC. While the HPLC technique is typically unable to distinguish individual species within the M. tuberculosis complex, some strains of Mycobacterium bovis BCG can be distinguished from other members of the complex. In rare cases, M. tuberculosis complex isolates are not definitively identified by HPLC. Older cultures result in poor peak resolution, chromatograms may shift resulting in erroneous peak- naming or low-level patterns may result from insufficient biomass present in the culture. In these cases, the GenProbe method is used to identify M. tuberculosis complex isolates.
Biochemical Tests
Species identification for members within the M. tuberculosis complex is performed by conventional biochemical tests and drug susceptibility testing. These methods include niacin accumulation, nitrate reduction, detection of pyrazinimidase, susceptibility to thiacetazone, tolerance to TCH, and oxygen preference in a semi-solid medium.
GenProbe Technology (AccuProbe)
This test is a possible alternative to the HPLC procedure to identify M. tuberculosis complex in a pure culture. The AccuProbe test is based on a commercially available kit that can identify several different species of mycobacteria. Currently, the following kits are available from the GenProbe Co. (San Diego, CA): M. tuberculosis complex, M. avium-intracellulare complex, M. avium, M. intracellulare, M. kansasii, and M. gordonae. This method, unlike the MTD kit (also made by GenProbe) does not use an amplification step. The test is performed on culture. The cells are first sonicated to release nucleic acids. A chemi-luminescent labeled DNA probe is added, which hybridizes with the target ribosomal rRNA. Hybrids are detected in the luminometer during a chemical reaction that gives off light.
