MTB colony morphology on LJ medium, courtesy CDC.
There are rapid methods available for both the direct detection of mycobacterial organisms in a specimen concentrate, as well as rapid methods performed on colonies grown in culture (either colonies on a slant or from a positive broth).
Molecular methods for direct detection from smear-positive specimens
There are several molecular methods, recommended as rapid (results available within two hours) and highly specific (85%-97%) for smear-positive specimens. However, sensitivity ranges from 36%-100% and specificity from 54%-100% when data from smear-negative and smear-positive specimens are combined. As few as 10-100 organisms/mL of 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. Currently, there are only two NAATs approved by the Food and Drug Administration (FDA) for respiratory specimens. 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), which received US market approval by the FDA in July 2013. It is designed to provide rapid molecular detection (less than two hours) of Mycobacterium tuberculosis DNA in specimens and, when positive, it also detects rifampin resistance mutations. There are other NAAT systems that are commercially available, but none have been approved for direct detection from extra-pulmonary specimens.
Molecular methods require specialized equipment performed with technical expertise and are expensive unless there is a large 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, however, are usually performed in reference or public health laboratories, rather than clinical laboratories. 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 a NAAT. It is important to note that culture continues as the reference standard for confirmation of M. tuberculosis complex (MTBC), for susceptibility testing, and genotyping.
Phenotypic confirmation (eg, 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 incubation for M. tuberculosis, with optimal temperature between 35º and 37º 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 tubed media or paper-strip assay are utilized.