Evaluation of T-Cell Responses to Novel RD1- and RD2-EncodedMycobacterium tuberculosisGene Products for Specific Detection of Human Tuberculosis Infection

Abstract

The tuberculin skin test for diagnosing Mycobacterium tuberculosis infection suffers from antigenic crossreactivity of purified protein derivative with BCG, resulting in poor specificity in BCG-vaccinated populations. Comparative genomics has identified several genetic regions in M. tuberculosis and M. bovis that are deleted in M. bovis BCG. Proteins encoded in these regions will form the basis of new specific T-cell-based blood tests that do not cross-react with BCG, but only two, early secretory antigen target 6 and culture filtrate protein 10, have been studied in detail in humans. We investigated four novel gene products, encoded by RD2 (Rv1989c) and RD1 (Rv3873, Rv3878, and Rv3879c), that are absent from most or all of the vaccine strains of BCG, respectively. Sixty-seven overlapping peptides were tested in ex vivo gamma interferon enzyme-linked immunospot assays in 49 patients with culture-confirmed tuberculosis and 38 healthy BCG-vaccinated donors. Forty-five percent (95% confidence interval [CI], 31 to 57%) and 53% (95% CI, 39 to 67%) of the tuberculosis patients responded to Rv3879c and Rv3873, respectively, identifying these proteins as major M. tuberculosis T-cell antigens in humans, while 35 and 25% of the patients responded to Rv3878 and Rv1989c, respectively. Of the 38 BCG-vaccinated donors, 1 (2.6%) responded to peptides from Rv3878 and Rv3879c, 3 (7.9%) responded to Rv3873, and none responded to Rv1989c. Exclusion of cross-reactive peptides encoded in conserved motifs of Rv3873, a PPE family member, increased its specificity to 97.4%. The high specificity of Rv3879c peptides and nonconserved Rv3873 sequences, together with their moderate sensitivity in tuberculosis patients, identifies these peptides as candidates for inclusion in new T-cell-based tests for M. tuberculosis infection.Accurate diagnosis of tuberculosis (TB) infection is essential for the treatment, prevention, and control of this resurgent disease (1). Since Mycobacterium tuberculosis is often difficult to culture from patients with active TB, and impossible to culture from healthy latently infected people, an immunebased diagnostic test indicating the presence or absence of M. tuberculosis infection would be very useful for the diagnosis of active TB and screening for latent M. tuberculosis infection. The only widely used test is the century-old tuberculin skin test (TST), which has many drawbacks. Foremost among these is its poor specificity (21). This results from the broad antigenic cross-reactivity of purified protein derivative (PPD), a crude mixture of more than 200 M. tuberculosis proteins widely shared among M. tuberculosis, M. bovis Bacillus CalmetteGuérin (BCG), and most environmental mycobacteria (22). Hence, false-positive results are common in people with environmental mycobacterial exposure and previous BCG vaccination. Since most of the world's population is BCG vaccinated and since the confounding effect of BCG persists for up to 15 years after vaccination (41), this is a significant problem. Thus, there has been a...

Diagnosis of Tuberculosis in Dialysis Patients

“…Four case-control studies on 127 BCG-vaccinated low-risk individuals in aggregate showed that T-SPOT.TB has a specificity of 100% (26,27,(32)(33)(34). A case-control study on 216 Japanese BCG-vaccinated adults showed a 98% specificity of QFT-G, much higher than for TST (35%) (29).…”

A systematic review of rapid diagnostic tests for the detection of tuberculosis infection

“…163,164 Proteins encoded in these regions have formed the basis of new specific T cell-based blood tests that do not cross-react with BCG, but only two antigens, ESAT-6 and CFP 10, have been studied in detail in humans. 165 ESAT-6 is a secreted antigen that is expressed in the M. TB complex, but is absent from BCG and most atypical mycobacteria. 163,166 ESAT-6 and CFP-10 share the same messenger RNA transcript, which suggest that they may interact with one another and serve a common function in detection of M. TB.…”

Diagnosis of tuberculosis infection using interferon-γ-based assays

“…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.…”