Diagnosing Latent Tuberculosis Infection
The 100-year Upgrade

Peter F. Barnes

Center for Pulmonary and Infectious Disease Controland Departments of Cell Biology, and Medicine, University of Texas Health Center, Tyler, Texas

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In many industrialized nations, tuberculosis case rates have declined significantly during the past decade, and elimination of tuberculosis is a realistic goal that hinges on treating latent tuberculosis infection (LTBI) to prevent development of disease. Whereas a vast array of molecular and immunologic tools is available to diagnose many infectious diseases, detection of LTBI is still based on the tuberculin skin test, a century-old test that measures the size of a bump under the skin that develops in response to a crude mixture of mycobacterial antigens. Because some antigens are shared with other mycobacteria, tuberculin reactivity can result from immunization with BCG or from exposure to environmental mycobacteria. In addition, two visits are required for administration and interpretation of the test.

The QuantiFERON-TB test (CSL Biosciences, Melbourne, Australia) is the only commercially available blood test to detect LTBI on the basis of production of higher concentrations of interferon  (IFN-) by cells in response to Mycobacterium tuberculosis than to the predominant environmental mycobacterium Mycobacterium avium complex. QuantiFERON-TB test results correlate well with tuberculin skin test reactivity (1). However, because multiple M. tuberculosis antigens are used, some of which are shared with BCG, false-positive results will probably be obtained in BCG-vaccinated persons.

During the past decade, a major scientific advance has been the identification of antigens that are expressed by M. tuberculosis, but not by BCG or by most environmental mycobacteria. The best studied of these antigens is the early secreted antigenic target 6-kD protein (ESAT-6) (2), which has multiple epitopes that are recognized by persons of many different HLA types (5). In persons with LTBI, memory T cells produce IFN- in response to stimulation in vitro with M. tuberculosis antigens, and these IFN--producing cells can be detected by the extremely sensitive enzyme-linked immunospot (ELISPOT) method (6).

In this issue of the American Journal of Respiratory and Critical Care Medicine, (pp. 824-828) Lalvani and colleagues utilized these scientific advances to develop what is likely to be the first of a new generation of tests for LTBI that are more sensitive, more specific, and more convenient than the tuberculin skin test (7). Mononuclear cells from a single blood sample were stimulated with ESAT-6 peptides, and the ELISPOT method was used to detect as few as 1 in 60,000 IFN--producing cells. This test was positive in 96% of 47 tuberculosis patients and in 85% of 26 persons presumed to have LTBI, on the basis of household contact with a patient with tuberculosis and a positive Heaf test. Because the multiple-puncture Heaf test yields more false-positive results than the intradermal Mantoux test, some of these 26 persons may not have had LTBI, and the sensitivity of the ELISPOT test may exceed 85%. The ELISPOT test was negative in 26 BCG-vaccinated control subjects, and this specificity confers a major advantage over tuberculin skin testing.

Most previous studies of the response to ESAT-6 have used an enzyme-linked immunoassay (ELISA) to measure IFN- concentrations in supernatants of ESAT-6-stimulated cells from patients with tuberculosis, with a sensitivity of 48% in the largest study of 121 patients (2). The current report suggests that the ELISPOT method is more sensitive than the ELISA for diagnosis of tuberculosis and of LTBI. In addition, ELISPOT results can be obtained after 24 h, whereas the ELISA measures IFN- production by cells cultured for 5-6 d.

Further studies are needed to establish the sensitivity and specificity of the ELISPOT test for LTBI in large populations. Because most mycobacterial epitopes are recognized in the context of specific HLA antigens, the ELISPOT test should be evaluated at multiple geographic locations among patients of different ethnicities. Although BCG vaccination does not yield false-positive ELISPOT results (4, 7), the specificity of the ELISPOT test should be studied in persons exposed to environmental mycobacteria such as M. avium complex. It may now be possible to identify such persons by skin testing with M. avium sensitin (8).

The ELISPOT test is not yet suitable for widespread use because it is costly and requires isolation of mononuclear cells, a procedure that is not performed in clinical laboratories. These problems could be overcome through technological advances, such as the use of whole blood in the assay instead of mononuclear cells (9), precoating ELISPOT plates with antibodies, reduced incubation times, and automated methods to count the number of positive spots.

Although the ELISPOT test should greatly facilitate detection of LTBI, its role in diagnosing tuberculosis is more complex. In high-incidence countries where LTBI is common, a positive ELISPOT test will not be specific for tuberculosis. In low-incidence countries, the positive predictive value of the test (probability that a patient with a positive test result has tuberculosis) will also be low, even if the test is 99% sensitive and 99% specific for LTBI and for tuberculosis. For example, in the United States, if 1,000 patients with suspected tuberculosis are tested, approximately 200 will have tuberculosis and 800 will not, and approximately 15% (120) of the patients without tuberculosis will have LTBI. This estimate is higher than the 5-10% prevalence of LTBI in the general population, as would be anticipated among patients with suspected tuberculosis. Most patients with LTBI will have positive ELISPOT tests. Therefore, among the 1,000 patients in whom the ELISPOT test is performed, 198 tuberculosis patients and 126 patients without tuberculosis will have positive tests, yielding a positive predictive value of only 61% [198/(198 + 126)].

Although a positive ELISPOT test is unlikely to confirm the diagnosis of tuberculosis, the negative predictive value of the test (probability that a patient with a negative test result does not have tuberculosis) may be extremely high. For example, using the same test characteristics and population described above, 2 patients with tuberculosis and 674 patients without tuberculosis will have negative ELISPOT tests, a negative predictive value of 99.7% [674/(674 + 2)], excluding tuberculosis with a high degree of confidence. The negative predictive value will be reduced if the sensitivity of the ELISPOT test is significantly lower in patients with tuberculosis than in those with LTBI, a realistic possibility because M. tuberculosis-induced IFN- production by blood mononuclear cells is decreased in patients with tuberculosis, particularly in those with severe disease (2, 10). Studies of larger numbers of patients with tuberculosis are needed to address this issue.

In summary, the article by Lalvani and coworkers represents a major advance in the quest for better tests to diagnose LTBI. The microbial genomics explosion will yield more M. tuberculosis-specific genes and antigens, and an ELISPOT test using peptides from multiple antigens should be more sensitive than one using ESAT-6 alone. If this test can be adapted for clinical use, I believe that it will replace tuberculin skin testing and greatly facilitate the elimination of tuberculosis in low-incidence countries.

	References

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1. Pottumarthy S, Morris AJ, Harrison AC, Wells VC. Evaluation of the tuberculin gamma interferon assay: potential to replace the Mantoux skin test. J Clin Microbiol 1999; 37: 3229-3232 [Abstract/Free Full Text].

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3. Arend SM, Andersen P, van Meijgaarden KE, Skjøt RLV, Subronto YW, van Dissel JT, Ottenhoff THM. Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6-kDa protein and culture filtrate protein 10.  J Infect Dis 2000; 181: 1850-1854 [Medline].

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5. Ulrichs T, Munk ME, Mollenkopf H, Behr-Perst S, Colangeli R, Gennaro ML, Kaufmann HE. Differential T cell responses to Mycobacterium tuberculosis ESAT6 in tuberculosis patients and healthy donors. Eur J Immunol 1998; 28: 3949-3958 [Medline].

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7. Lalvani A, Pathan AA, McShane H, Wilkinson RJ, Larif M, Conlon CP, Pasvol G, Hill AVS. Rapid detection of M. tuberculosis infection by enumeration of antigen-specific cells. Am J Respir Crit Care Med 2001;824-828.

8. Vankayalapati R, Wizel B, Samten B, Griffith DE, Shams H, Galland MR, von Reyn CF, Girard WM, Wallace RJ Jr,, Barnes PF. Cytokine profiles in immunocompetent persons infected with Mycobacterium avium complex. J Infect Dis 2001; 183: 478-484 [Medline].

9. Elliott AM, Hurst TJ, Balyeku MN, Quigley MA, Kaleebu P, French N, Biryahwaho B, Whitworth JAG, Dockrell HM, Hayes RJ. The immune response to Mycobacterium tuberculosis in HIV-infected and uninfected adults in Uganda: application of a whole blood cytokine assay in an epidemiological study. Int J Tuberc Lung Dis 1999; 3: 239-247 [Medline].

10. Sodhi A, Gong J-H, Silva C, Qian D, Barnes PF. Clinical correlates of interferon-gamma production in tuberculosis patients. Clin Infect Dis 1997; 25: 617-620 [Medline].