Rapid ID
Rapid methods are available for directly detecting mycobacterial organisms in a specimen concentrate and for rapid methods performed on colonies grown in culture (either on a slant or from a positive broth).
Molecular methods for direct detection from smear-positive specimens
Several molecular methods are recommended as rapid (results available within two hours) and highly specific (85–97%) for smear-positive specimens. However, sensitivity ranges from 36% to 100% and specificity from 54% to 100% when data from smear-negative and smear-positive specimens are combined.
- As few as 10–100 organisms/mL of the specimen can be detected with nucleic acid amplification tests (NAAT), compared with the 104 CFU/mL sensitivity exhibited by a positive acid-fast bacillus (AFB) smear.
- Two commonly used NAATs, approved by the Food and Drug Administration (FDA) for respiratory specimens, may be used. First is the Gen-Probe Amplified MTD® (M. tuberculosis Direct) system (Gen-Probe Inc., San Diego, CA), which provides results in two hours for M. tuberculosis (MTB). Second is the Xpert®MTB/RIF test (Cepheid), designed to provide rapid molecular detection (less than two hours) of Mycobacterium tuberculosis DNA in specimens. When positive, it also detects rifampin resistance mutations.
- The next generation of Cepheid testing, the Xpert®MTB/RIF Ultra, was endorsed by the WHO for use in pulmonary, extra-pulmonary, and pediatric samples. Other NAAT systems are commercially available, but no others have been approved for direct detection from extra-pulmonary specimens.
- In 2018, the WHO began field-testing the GeneXpert® Omni, a small, lightweight, and durable instrument designed for use with the Xpert® MTB/RIF cartridges. The system provides portability with built-in batteries and superior communication capabilities for tracking results, with a near-field communication chip allowing for cellular data transfer.
Molecular methods require specialized equipment performed with technical expertise and are expensive unless there is a significant test volume. Other rapid methods that employ broth to identify MTB include DNA sequencing and high-performance liquid chromatography (HPLC) analysis of mycolic acids. These techniques are usually performed in reference or public health laboratories rather than clinical laboratories.
Regulatory Guidance
- Both the Clinical Laboratory Standards Institute (CLSI) and the Centers for Disease Control and Prevention (CDC) publish guidelines for the performance of molecular methods.
- The CDC recommends that at least one of a series of respiratory specimens from patients with pulmonary symptoms has an NAAT.
- Note: Culture remains the reference standard for confirming M. tuberculosis complex (MTBC), susceptibility testing, and genotyping.
Standard ID
- Phenotypic confirmation (e.g., growth rate, pigment, and colony morphology from growth on solid media) is essential to confirm the ID of molecular, as well as standard methods of mycobacteria ID.
- Growth rate is defined as “slow” after at least seven days of incubation for M. tuberculosis, with optimal temperature between 35° and 37°C.
- Colony morphology is described as rough and non-pigmented on Lowenstein-Jensen (LJ) but varies on different media types.
- Biochemically, M. tuberculosis accumulates niacin and reduces nitrate when utilized in tubed media or paper-strip assays.
