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Notice to Readers: Update: Nucleic Acid Amplification Tests for Tuberculosis

On September 30, 1999, the Food and Drug Administration approved a reformulated Amplified Mycobacterium Tuberculosis Direct Test* (MTD) (Gen-Probe®, San Diego, California) for detection of Mycobacterium tuberculosis in acid-fast bacilli (AFB) smear-positive and smear-negative respiratory specimens from patients suspected of having tuberculosis (TB). MTD and one other nucleic acid amplification (NAA) test, the Amplicor® Mycobacterium Tuberculosis Test (Amplicor) (Roche® Diagnostic Systems, Inc., Branchburg, New Jersey), previously had been approved for the direct detection of M. tuberculosis in respiratory specimens that have positive AFB smears. This notice updates the original summary published in 1996 (1) and provides suggestions for using and interpreting NAA test results for managing patients suspected of having TB.

The appropriate number of specimens to test with NAA will vary depending on the clinical situation, the prevalence of TB, the prevalence of nontuberculous mycobacteria (NTM), and laboratory proficiency (2,3). Based on available information, the following algorithm is a reasonable approach to NAA testing of respiratory specimens from patients with signs or symptoms of active pulmonary TB for whom a presumed diagnosis has not been established. 


  1. Collect sputum specimens on 3 different days for AFB smear and mycobacterial culture.
  2. Perform NAA test on the first sputum specimen collected, the first smear-positive sputum specimen, and additional sputum specimens as indicated below.
    1. If the first sputum specimen is smear-positive and NAA-positive, the patient can be presumed to have TB without additional NAA testing. However, unless concern exists about the presence of NTM, the NAA test adds little to the diagnostic work-up.
    2. If the first sputum is smear-positive and NAA-negative, a test for inhibitors should be done. The inhibitor test can be done as an option with Amplicor. To test for inhibitors of MTD, spike an aliquot of the lysated sputum sample with lysed M. tuberculosis (approximately 10 organisms per reaction, or an equivalent amount of M. tuberculosis rRNA) and repeat the test starting with amplification.
      1. If inhibitors are not detected, additional specimens (not to exceed a total of three) should be tested. The patient can be presumed to have NTM if a second sputum specimen is smear-positive, NAA-negative, and has no inhibitors detected.
      2. If inhibitors are detected, the NAA test is of no diagnostic help. Additional specimens (not to exceed a total of three) can be tested with NAA.
    3. If sputum is smear-negative and MTD-positive, additional specimens (not to exceed three) should be tested with MTD. The patient can be presumed to have TB if a subsequent specimen is MTD-positive.
    4. If sputum is smear-negative and MTD-negative, an additional specimen should be tested with MTD. The patient can be presumed not to be infectious if all smear and MTD results are negative. The clinician must rely on clinical judgement in decisions regarding the need for antituberculous therapy and further diagnostic work-up because negative NAA results do not exclude the possibility of active pulmonary TB.
  3. If the indicated repeat NAA testing fails to verify initial NAA test results, the clinician must rely on clinical judgement in decisions regarding the need for antituberculous therapy, further diagnostic work-up, and isolation.
  4. Ultimately, the patient's response to therapy and culture results are used to confirm or refute a diagnosis of TB.

NAA tests can enhance diagnostic certainty, but they do not replace AFB smear or mycobacterial culture, and they do not replace clinical judgement. Clinicians should interpret these tests based on the clinical situation, and laboratories should perform NAA testing only at the request of the physician and only on selected specimens. Laboratorians should not reserve material from clinical specimens for NAA testing if this compromises the ability to perform the other established tests that have better-defined diagnostic utility and implications. Specificity of NAA tests varies between laboratories as a result of unrecognized procedural differences and differences in cross-contamination rates (4). Multiple specimens from the same patient should not be tested together to reduce risks of methodologic errors. Laboratory directors should provide to clinicians information on the performance of NAA tests in the local setting, including sensitivity and specificity compared with culture for both smear-positive and smear-negative respiratory specimens. Substantial discrepancies can indicate problems with either culture or NAA technique. The number of NAA tests repeated because of failure of negative and positive controls also should be reported. Clinicians should understand the impact that changes in sensitivity, specificity, prevalence of TB, and prevalence of other mycobacterial diseases can have on the predictive value of the NAA test. Information is limited regarding NAA test performance for nonrespiratory specimens, or specimens from treated patients. NAA tests often remain positive after cultures become negative during therapy and can remain positive even after completion of therapy.


  1. CDC. Nucleic acid amplification tests for tuberculosis. MMWR 1996;45:950--1.
  2. Cohen RA, Muzaffar S, Schwartz D, et al. Diagnosis of pulmonary tuberculosis using PCR assays on sputum collected within 24 hours of hospital admission. Am J Respir Crit Care Med 1998;157:156--61.
  3. Jonas V, Acedo M, Clarridge JE, et al. A multi-center evaluation of MTD and culture compared to patient diagnosis [Abstract]. In: Abstracts of the 98th General Meeting of the American Society for Microbiology, 1998:358 (no. L-31).
  4. Ridderhoff J, Williams L, Legois S, Bussen M, Metchock L, Kubista R. Assessment of laboratory performance with nucleic acid amplification (NAA) tests for Mycobacterium tuberculosis (M.tb) [Abstract]. In: Abstracts of the 98th General Meeting of the American Society for Microbiology, 1998:360(no. L-43).

* Use of trade names and commercial sources is for identification only and does not constitute endorsement by CDC or the U.S. Department of Health and Human Services.

Amplicor is not approved for use with smear-negative samples. .

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