Specific T-Cell Epitopes for Immunoassay-Based Diagnosis ofMycobacterium tuberculosisInfection

Abstract

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 BCGvaccinated 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 microscopyor 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%.Tuberculosis is a major cause of morbidity and mortality throughout the world. It is estimated that nearly 1% of the world population is newly infected each year and that approximately one-third of the world population is latently infected with Mycobacterium tuberculosis.On average, immunocompetent individuals infected with M. tuberculosis have a lifetime risk of developing active tuberculosis of 10%, but this risk increases to a 10% yearly risk in persons coinfected with human immunodeficiency virus. If left untreated, a patient with active pulmonary tuberculosis will transmit the infection to 10 to 15 contacts each year (World Health Organization, tuberculosis fact sheet no. 104, http: //www.who.int/mediacentre/factsheets/who104/en/, 2000). Therefore, novel tools to detect and subsequently treat infected individuals before the disease progresses to active contagious tuberculosis are an international research priority (10).In addition to chest radiographs to detect pulmonary tuberculosis, the current diagnostic assays for the detection of infection with M. tuberculosis include culture, microscopy and PCR of relevant patient material, and the tuberculin skin test. The first three me...

Identification of Specific Proteins and Peptides in Mycobacterium leprae Suitable for the Selective Diagnosis of Leprosy

“…Similarly, the findings in our present study that there were more frequent responses in the EC and TB groups to the whole recombinant proteins is perhaps an indication of possible cross-reactive responses toward peptides that show homology to as yet unidentified peptides from other mycobacteria, even though three of four whole proteins examined were supposedly "unique" to M. leprae. Other studies have shown fewer problems with cross-reactivity using peptides compared with using the whole protein, so this may be one way to avoid undesirable cross-reactivity by selecting only those peptides that stimulate responses in the targeted group (38,39).…”

Diagnosis of tuberculosis infection using interferon-γ-based assays

“…44 Results of both IFN-γ-based tests are unrelated to the BCG-vaccination status, which contributes to their high specificity. 4,7 Discordant results between these tests and TST are frequently found. 7,[45][46][47][48] In a study conducted in Barcelona (Spain), 7 among BCG-unvaccinated children, 60% and 57% of those with positive TST had negative QF-IT and T-SPOT.TB respectively.…”

“…This new antigen is encoded in RD11 and is missing from the BCG strains as well as most common environmental mycobacteria. 4 In the QFT-IT assay, the three specific M. tuberculosis antigens are already incorporated into the same tube (Fig. 1).…”

“…95 The attempts to improve IFN-γ-based tests include the study of alternative readouts to measure IFN-γ release. 95,96 the use of alternative M. tuberculosis specific antigens, 4,97 and the simultaneous measurement of chemokines 98 and interleukins. 99 The next generation of IFN-γ-based tests will significantly enhance diagnostic sensitivity without diminishing specificity, and will also reduce the rate of indeterminate results, especially in immunosuppressed patients and children.…”

Immunological Diagnosis of Active and Latent TB

“…In addition to ESAT-6 and CFP-10, QFT-GIT contains a peptide from the internal section of TB 7.7 (Rv2654), which may increase the sensitivity of the test (Aagaard et al 2004;Brock et al, 2004), though it is arguable whether specificity is improved. All three antigens are present on the wall surface of the blood collection tubes.…”