TB susceptibility agar proportion test, courtesy of CDC.
State-of-the-art AFB susceptibility testing is the FDA-approved automated system, including the BACTEC MIGIT 960 (Becton-Dickinson) and the ESP® Culture System II (Trek Diagnostic Systems) for first-line antibiotics, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA). These antibiotics have been recommended by the Clinical and Laboratory Standards Institute (CLSI) for testing.
Preferences for automated systems include decreased time for the indirect method (utilizes a pure culture of the test isolate) and the time from media inoculation to reporting is 2 to 4 weeks. The standard agar proportion method is more labor intensive and time-consuming than the commercial broth automated methods. Greater laboratory safety is maintained when bottles growing AFB are contained inside the automated system cabinet. A recent study demonstrates second-line (as well as first-line) drug testing on the BACTEC MGIT 960 also provides acceptable results in a distinctly shorter time. Second-line drugs usually include 4 or more of the following: Increased dosages of INH and EMB, capreomycin, ethionimide, amikacin, p-aminosalicyclic acid (PAS), rifabutin, streptomycin and levofloxacin.
Manual methods: Agar proportion
The agar proportion method for susceptibility testing of M. tuberculosis complex (MTBC) became the gold standard for testing in the U.S. and Europe from its development in the 1960s. Susceptibility testing of MTBC includes M. tuberculosis, M. bovis and M. africanum. Either Middlebrook 7H10 medium, to which drugs made from reference powders are added or agar diffusion with drug-impregnated discs placed after the agar solidifies, may be employed. The 7H10 medium is supplemented with oleic acid-albumin-dextrose-catalase (OADC) as standard procedure. However, when drug-resistant strains are present, growth may not be adequate unless 7H11 agar, with higher concentrations of INH, RIF, PZA and EMB, is used. Quality control strains from the American Type Culture Collection (ATCC) are recommended by the CLSI as essential to successful performance of the method. Resistant strains of MTB (INH and RIF or other drugs) are available from ATCC.
Using the indirect method, a pure culture of MTBC is suspended in 7H9 broth and incubated at 35⁰ to 37⁰C until the turbidity matches a 1.0 McFarland standard. The number of CFU/mL varies from 10-2 - 10-4. In the direct method, the inoculum from a processed specimen or an untreated sterile specimen is diluted (according to number of AFB observed on a smear) to the correct concentration. Because the concentration of tubercle bacilli is presumably greater, the direct method has the advantage of a shorter time to reporting results. However, the contamination rate (in non-sterile specimens) with this method may be as high as ≥ 15%, rendering the test a failure. The contamination must be ≤ 5%, which is more easily obtained by the indirect method.
Agar proportion testing is often performed on quadrant drug plates with 0.1 ml of diluted inoculum added to each quadrant; test specimen is added to one quadrant and control to the next. After drying, the plates are enclosed in CO2 permeable polyethylene bags or individually sealed and incubated at 35⁰ to 37⁰ C in 5-10% CO2 in air. Plates are checked for contamination after one week when the procedure may be repeated by the indirect method, if necessary. Interpretation of the test requires that the proportion of (mutant) colonies resistant to a drug is greater than 1% (the concentration of drug in the test quadrant). The per cent resistance is derived by dividing the colony count on the drug-containing quadrant by the count on the control quadrant, which must grow ≥ 50 colonies for a valid test. Three weeks are required to determine susceptibility, though resistance may be determined earlier.