TB Reference Laboratory
Identification of Mycobacteria
High Performance Liquid Chromatography (HPLC)
This test is the primary identification test at TDSHS for all mycobacteria. Identification of mycobacteria by HPLC is species specific and is based on the separation of mycolic acids contained in the cell wall of the bacteria. The range of speciation and the sensitivity of the test are greater than that of genetic probe (without amplification) and conventional biochemical testing. HPLC testing also can be performed directly on a clinical specimen with a positive smear regardless of the source of the specimen. For culture confirmation, pure viable cultures on solid or in liquid media should be submitted for testing.
A limited number of Mycobacterium species can be identified by the use of commercially available DNA probes. The GenProbe AccuProbe system is a nucleic acid hybridization test that utilizes a highly specific and sensitive, single stranded DNA probe. The probe is labeled with a chemiluminescent acridinium ester and is complimentary to the ribosomal RNA (rRNA) of the organism. During cell lysis, the rRNA is released and the labeled DNA probe combines with the target rRNA to form a stable DNA:RNA hybrid. A selection reagent destroys all unbound probe. The hybrids are detected and measured in a GenProbe luminometer. A positive result is a numerical reading that is equal to or greater than an established cut-off value.
TDSHS currently uses DNA probes specific for M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae. The use of DNA probes provides a rapid identification for these mycobacterial species in a few hours versus weeks when conventional methods are employed. Pure, viable cultures on solid media should be submitted for testing and should be less than four weeks old.
Conventional Biochemical Tests
Once the isolate has been placed in a subgroup based on rate of growth and pigmentation, identification to the species or complex level may be initiated. Key biochemical tests useful for identifying the suspected species are selected and the organism’s biochemical profile is elucidated. The profile is compared to the reaction patterns of known species. If an unknown organism matches any one pattern exactly, the identification may be considered definitive for that species.
Susceptibility Testing of Mycobacteria
The BACTEC procedure for susceptibility testing of M. tuberculosis is based on mycobacterial utilization of 14C - labeled palmitic acid as a sole carbon source in a liquid medium. During respiration, the organism produces 14CO2. The amount of 14CO2 is a reflection of the rate and amount of growth occurring in the medium-containing vial and is numerically expressed as the "Growth Index"(GI). When an antituberculous drug is added to the medium, inhibition of growth occurs if the test organism is susceptible. This suppression is detected by either a decrease or a very small increase in the daily GI relative to the drug-free control. However, if the organism is resistant, little or no suppression of growth occurs.
Three primary antituberculous drugs are tested by TDSHS in BACTEC: isoniazid, rifampin, and ethambutol. If a strain of M. tuberculosis is resistant to a primary drug, then these secondary drugs are tested in BACTEC: PZA, streptomycin, ethionamide, kanamycin, rifabutin, and ofloxacin. The BACTEC method of susceptibility testing offers an advantage over conventional testing because results are reportable generally in 4-6 days versus 3 weeks. Acceptable cultures for susceptibility testing are pure, viable cultures in liquid or on solid media that have not been subcultured excessively.
Agar Proportion Method
The agar proportion susceptibility test is based on a comparison of the number of microbial colonies growing on the surface of drug-containing agar relative to the number of colonies growing on the drug-free control. Antituberculous drugs are incorporated into cooled Middlebrook 7H10 agar. The agar is then poured into quadrant petri plates. One quadrant in each plate is reserved for the control. The organism is reported as resistant to a particular drug if the number of colonies on the drug quadrant is equal to or greater than 1% of the number of colonies growing on the drug-free control.
The TDSHS panel of drugs for agar proportion susceptibility testing of M. tuberculosis contains isoniazid, rifampin, ethambutol, streptomycin, rifabutin, ethionamide, kanamycin, capreomycin, and ofloxacin. Rifampin susceptibility for M. kansasii is also performed by TDSHS.