Susceptibility Testing of the Mycobacterium tuberculosis complex
By Kay J. Nishimura, M.T. (A.S.C.P.), March 4, 1999
Tuberculosis is the main cause of death due to a single infectious agent worldwide. In the United States, the incidence of tuberculosis declined steadily until 1985, when the downward trend reversed. A number of factors are thought to account for the resurgence of tuberculosis in the United States. These include the advent of the AIDS epidemic, immigration from areas of high endemicity, transmission in high-risk environments, and increase in the number of multidrug-resistant strains of Mycobacterium tuberculosis.
One of the most alarming aspects of the contemporary increase in tuberculosis has been the nosocomial outbreaks of multidrug-resistant (MDR) M. tuberculosis. In the United States, the Centers for Disease Control and Prevention recommends that (1) all persons with tuberculosis from whom M. tuberculosis is isolated should have drug susceptibility testing performed on their first isolate and results should be reported promptly to the health care provider and to the health department and (2) that drug susceptibility testing should be performed on additional isolates from patients whose cultures fail to convert to negative within 3 months of beginning therapy, or if there is clinical evidence of failure to respond to therapy. Primary drugs to be tested are isoniazid, streptomycin, rifampin, ethambutol, and pyrazinamide.
The method used by DLS is the Radiometric Proportion Method which is based on the original Agar Proportion Method. The BactecTM 460 instrument used for AFB cultures test specimens which have been inoculated into BactecTM 12B vials. These vials contain an enriched Middlebrook 7H9 broth base and a 14C-labeled substrate (palmitic acid). As the mycobacteria metabolize the substrate, 14CO 2 is liberated and is detected quantitatively by the BactecTM 460. The amount of 14CO 2 is translated into a growth index (GI). The radiometric technique can detect very small quantities of 14CO 2, which allows detection of mycobacterial growth at a very early stage.
The BactecTM system can be used to test all primary drugs. If an antimicrobial agent is added to the medium, the 14CO 2 production is reduced based on the degree of inhibitory action of the drug. The AFB isolate is inoculated into a 12B broth vial with and without the test drug. The number of organisms added to the control (with no drug) is 100-fold less than that added to the drug-containing medium. Susceptibility results are interpreted by comparing the daily growth (GI values) increase in the control and drug-containing media. Once the growth of the control vial reaches a certain threshold (GI), the test can be interpreted by comparing the GI of the control vial to that of the vial containing the test drug. In general, if the control vial “outgrows” the drug vial the organism is sensitive and if the reverse is true, the organism is resistant.
Limitations of the Radiometic Proportion Method
- This procedure is intended for use only with Mycobacterium tuberculosis and the primary drugs (Streptomycin, Isoniazid, Rifampin, Ethambutol, and Pyrazinamide).
- This procedure will not be used to do direct susceptibility testing (tests utilizing the actual specimen as the inoculum versus a culture of the specimen).
- This procedure will not be used for testing mixed cultures.
- The BactecTM method does not allow an estimate of the percentage of resistant bacilli.
- Antimicrobial Susceptibility Testing for Mycobacterium tuberculosis: Tentative Standard. Document M24-T, Vol. 15, No.16. National Committee for Clinical Laboratory Standards (NCCLS). December 1995.
- MMWR: Initial Therapy for tuberculosis in the era of multidrug resistance. Recommendations of the Advisory Council for the Elimination of Tuberculosis of the Centers for Disease Control and Prevention. MMWR 1993; 42:1-8.
- Manual of Clinical Microbiology. 6th ed. 1995. Patrick R. Murray, editor-in-chief. ASM Press. Washington D.C. Chapter 119; pp. 1385-1404. Antimicrobial Agents and Susceptibility Test: Mycobacteria.
- Automatable Radiometric Detection of Growth of Mycobacterium tuberculosis in Selective Media. Middlebrook, G., Z. Reggiardo, and W.D. Tigert. 1977. Am. Rev. Respir. Dis. 115: 1066-1069.