Our results show that accurate susceptibility testing of M. tuberculosis can be accomplished by the enumeration of mycobacterial cells with a flow cytometer. Results of tests were available 72 h after M. tuberculosis organisms were incubated with EMB, INH, and RIF. Most importantly, the method is safe. Mycobacteria were killed by exposure to paraformaldehyde before susceptibility samples were analyzed with the flow cytometer.
The flow cytometric susceptibility test depends on multiplication of M. tuberculosis
organisms. Although M. tuberculosis
grows slowly, with cell division occurring every 15 to 24 h, small but significant increases in the numbers of mycobacteria can be accurately detected and quantified by flow cytometric analysis. Within 72 h after the initiation of testing procedures, the number of mycobacteria in the drug-free and drug-containing assays could be compared. A decrease in the number of M. tuberculosis
cells of 45, 50, or 25% in suspensions containing INH, RIF, or EMB, respectively, was required to accurately predict the same results as those obtained by the agar proportion method. The proportion method is the “gold standard” for susceptibility testing of M. tuberculosis
We tested 17 isolates of M. tuberculosis
for susceptibility or resistance to EMB, INH, and RIF by the flow cytometric and proportion methods. Agreement between the results from the two methods was 97, 100, and 100% for INH, EMB, and RIF, respectively. Overall, agreement was 98%. Two discrepancies were detected among the 85 tests performed. These isolates were susceptible to 0.2 μg of INH/ml by the flow cytometric test but resistant to this concentration of INH by the proportion method. When higher concentrations (1.0 and 5.0 μg/ml) of INH were tested, identical susceptibility results were obtained by both the flow cytometric and proportion methods. A likely explanation for these discrepancies is the cutoff value. We arbitrarily set the cutoff value for defining susceptibility to INH at 45%. This required the detection of 45% less M. tuberculosis
cells in the drug-containing assay compared to the number of M. tuberculosis
cells detected in the drug-free controls. The value could have been 30, 35, or 40% or any numerical value within these numbers. Determination of the best-fit cutoff value will require evaluation of multiple isolates of M. tuberculosis
by several laboratories. Another possible explanation is the bactericidal activity of INH. The flow cytometric results were obtained during an interval (72 h) of high bactericidal activity for INH. It has been reported that INH bactericidal activity decreases rapidly in medium after 7 days of incubation due to formation of isonicotinic acid and other metabolites (14
). Therefore, mycobacteria that survived inactivation at a low concentration (0.2 μg/ml) of INH would be considered resistant to INH by the proportion method after the recommended incubation period of 3 weeks. When higher concentrations of INH (1.0 and 5.0 μg/ml) were tested by the flow cytometric and proportion methods, the same susceptibility results were obtained.
A major concern with use of the flow cytometer for susceptibility testing of M. tuberculosis
is biosafety. A single tubercle bacillus is infectious for humans. Because the flow cytometer operates under a pressurized system, generation of a small number of infectious particles (5 μm) is possible. Previously, we showed that susceptibility testing of M. tuberculosis
could be accomplished by flow cytometry in 24 h. The rapidity of the method was dependent upon addition of FDA to differentiate viable from nonviable mycobacteria after treatment with antimycobacterial agents (7
). Unfortunately, attempts to inactivate or kill the mycobacteria and still maintain the differential effects of FDA failed. Therefore, viable mycobacteria with or without exposure to antimycobacterial agents were processed with the flow cytometer. Although this FDA-dependent assay is accurate, rapid (24 h), and reproducible, most clinical laboratories do not have the facilities or expertise to carry out the procedure safely.
In this study, we developed a flow cytometric procedure that greatly reduced the risk of infection to the health care worker using the flow cytometer. All assay suspensions were treated with 1.0% paraformaldehyde before processing with the instrument. Viable tubercle bacilli were not recovered from the drug-containing or drug-free suspensions 40 min after treatment with paraformaldehyde. Increasing the duration of exposure of suspensions of M. tuberculosis organisms to paraformaldehyde also did not alter the results of the susceptibility tests. Accurate susceptibility results could still be obtained if paraformaldehyde treated samples were analyzed 24 to 72 h after exposure to paraformaldehyde. This step further augments the biosafety of the procedure. However, tubercle bacilli trapped in the lips or caps of the susceptibility tubes could have escaped treatment with paraformaldehyde. Therefore, processing samples outside a biosafety level 3 tuberculosis facility is not recommended unless exquisite care is taken to assure that samples are treated properly.
The flow cytometric susceptibility assay also has another advantage besides increased biosafety. The assay does not depend on use of FDA (7
) for obtaining results. Instead, the numbers of mycobacteria in the assay suspensions with or without exposure to antimycobacterial agents are determined 72 h after initiation of testing. Although the requirement for enumeration of mycobacteria delays the reporting of results by 48 h compared to the FDA-dependent flow cytometric susceptibility assay, this biologically safer procedure is still more rapid than the BACTEC-460 system (6
Other advantages of the flow cytometric assay are its simplicity and low cost. The materials required to perform the test include 7H9 broth, microtubes, and the antimycobacterial agents. In addition, samples can be rapidly processed by the flow cytometer (under 60 s). If results are indeterminate, the flow cytometric susceptibility tests can be reanalyzed after incubation for 24 h. Technician time for performing the flow cytometric susceptibility test is similar to that required by the radiometric proportion method. Furthermore, the cost of the flow cytometric test is considerably less than the cost incurred by the radiometric proportion method. The actual test components cost less than 3 dollars. Although the cost of the flow cytometer is high, when the high cost of supplies for performing susceptibility testing by the radiometric instrument is considered, the cost of a flow cytometer is justifiable, especially if a refurbished instrument is obtained.
In conclusion, the flow cytometric method is safe for performing susceptibility testing on clinical isolates of M. tuberculosis. The assay is extremely simple to perform and can be completed in 72 h after the initiation of testing.