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Journal of Clinical Microbiology, June 2004, p. 2379-2387, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2379-2387.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Specific T-Cell Epitopes for Immunoassay-Based Diagnosis of Mycobacterium tuberculosis Infection

Inger Brock,1 Karin Weldingh,1 Eliane M. S. Leyten,2,3 Sandra M. Arend,2 Pernille Ravn,4 and Peter Andersen1*

Department of Infectious Disease Immunology, Statens Serum Institute,1 Department of Infectious Diseases, Hvidovre Hospital, Copenhagen, Denmark,4 Department of Infectious Diseases,2 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands3

Received 12 August 2003/ Returned for modification 12 December 2003/ Accepted 18 February 2004

The currently used method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules was evaluated by sensitive testing of the T-cell responses of peripheral blood mononuclear cells derived from M. bovis BCG-vaccinated healthy individuals to synthesized overlapping peptides. Three of the four molecules contained regions with significant specificity problems (Rv2653, Rv3873, and Rv3878). We selected and combined the specific peptide stretches from the four proteins not recognized by M. bovis BCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M. bovis BCG-vaccinated controls. The combination of the most promising stretches from this analysis showed a sensitivity level (57%) comparable to the level found with the two well-known M. tuberculosis-specific proteins ESAT-6 and CFP-10 (75 and 66%, respectively). The combination of ESAT-6, CFP-10, and the novel specific peptide stretches gave an overall sensitivity of 84% at a specificity of 97%. In a validation experiment with new experimental groups, the sensitivities obtained were 57% for the combination of peptides and 90% for the combination of the peptides, ESAT-6, and CFP-10. This combination gave a specificity of 95%.

* Corresponding author. Mailing address: Department of Infectious Disease Immunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. Phone: 45 32 68 34 62. E-mail: pa{at}ssi.dk.

Journal of Clinical Microbiology, June 2004, p. 2379-2387, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2379-2387.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
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Copyright © 2004 by the American Society for Microbiology. All rights reserved.