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Section III: Drug Susceptibility Testing
• X = Error – indeterminate results when certain conditions occur which may affect the
test, such as GU of the control reaches >400 in less than 4 days. In such situations, the
test should be repeated with pure, actively growing culture which is confirmed to be M.
tuberculosis complex. Certain drug resistant strains grow very slowly in the medium
and the results may not be achieved within 13 days with the standard inoculum. In such
a case, the inoculum should be increased by decreasing the dilution of the culture
suspension in order to get reportable results.
7. Reporting
Results must be reported as soon as they are available. When reporting results, it is
important to include the name of the method used, the type of drug and its level of
concentration.
In case of resistance, check the medium visually and make sure that the test culture is not
contaminated (look for turbidity and put 1 drop of the medium on an agar plate) or that the
test culture belongs to NTM. In case of unexpected results or mono-resistance against
Rifampin, PZA or Ethambutol, repeat the test to verify resistance.
8. Quality control (QC)
It is extremely important to perform a quality control of drug susceptibility testing
periodically. The minimum requirement is to test each new batch of reagents, such as SIRE
drugs or MGIT medium. If the batch QC fails, all the results obtained within that batch, as
well as the new batch of a reagent should be thoroughly reviewed and the testing should be
repeated. Use M. tuberculosis H37Rv (ATCC [American Type Culture Collection] number
27294) as a QC strain which is susceptible to all anti-tuberculosis drugs. It is not necessary
to include a resistant strain, as most of the resistant strains against a drug which are available
from ATCC and other culture collections are highly resistant and do not give any added
benefit in the quality control. The test procedure for QC organisms is the same as described
above for clinical isolates. The inoculum should be from a freshly grown culture in the
MGIT medium or on LJ slant. In case the suspension of QC bacteria is made from growth on
solid medium, follow the procedure for suspension preparation as described above. The
suspension may be stored in aliquots frozen for up to 6 months at -70ºC+10ºC (for details of
QC strain preparation see Section II-K-2).
B. Pyrazinamide (PZA) Susceptibility Testing
1. Introduction
Susceptibility testing against PZA is always carried out at a lower pH of the medium, since
pyrazinamide (PZA) is active only at the low pH in vitro. There are two methods widely
used for PZA susceptibility testing. In the proportion method, Middlebrook 7H10 agar
medium at pH 5.5 is used, with 25-50 µg/ml PZA. Colony counts, on the drug-free and drug-
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