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Ziehl-Neelson stained MTB showing AFB w/cording morphology, courtesy of the CDC

Antimicrobial Susceptibility Testing (AST): Example of an Automated System

Approved by the Food and Drug Administration (FDA) for safety (non-radiometric broth, no needles), the Bactec™ MGIT™ 960 is continuously monitored by fluorescence detection of growth each hour. The MGIT™ 960 tubes contain 7H9 broth with a fluorescence-quenching oxygen sensor to detect growing AFB organisms. When oxygen is used (reduced) the subsequent fluorescence produced is measured in growth units (GU). When compared to the agar proportion and the radiometric BACTEC™ 460 semi-automated system, the MGIT™ 960 has proven equivalent in sensitivity and specificity, with similar time to results. Designed to replace the BACTEC™ 460 for safety and technical simplicity, the system is commonly used.
A suspension of pure inoculum (from solid or liquid medium) in the MGIT 960 tube is adjusted to a 0.5 McFarland standard then diluted 1:5 with 7H9 broth before inoculation into the drug-containing MGIT™ tubes. The MGIT™ system supplies the lyophilized antibiotics, isoniazid (INH), rifampin (RIF), ethambutol (EMB), streptomycin (SM), and the SIRE supplement (OADC enrichment), which must be added to each tube. A growth control tube is inoculated with a 1:100 dilution of the same (innoculum) suspension used in the drug-containing tubes. When the set of tubes is placed in the system cabinet, a specific drug panel is selected to match the inoculated drug-set. After growth in the control tubes reaches 400 growth units (GU), which takes 4 to 13 days, the system flags the completed test(s) and interprets susceptibility according to the following rules:
  • An isolate is resistant when the GU of the drug-containing tube is >100
  • An isolate is susceptible when the GU ≤100
If the GU of the growth control reaches 400 in less than four days, the test is invalid because of improper inoculation or contamination. If the growth control does not reach 400 in >13 days, the test is also invalid due to insufficient inoculum or improper broth support. An AFB smear should be performed on tubes giving resistant results to confirm purity and affirm typical MTBC cellular morphology (presence of cording or clumping). After testing is completed, a drug susceptibility report can be printed from the system. Questionable results should be repeated with the MGIT™ 960 or by the agar proportion method.