An In-House RD1-Based Enzyme-Linked Immunospot-Gamma Interferon Assay Instead of the Tuberculin Skin Test for Diagnosis of LatentMycobacterium tuberculosisInfection

Abstract

Identification of individuals infected with Mycobacterium tuberculosis is essential for the control of tuberculosis (TB). The specificity of the currently used tuberculin skin test (TST) is poor because of the broad antigenic cross-reactivity of purified protein derivative (PPD) with BCG vaccine strains and environmental mycobacteria. Both ESAT-6 and CFP-10, two secretory proteins that are highly specific for M. tuberculosis complex, elicit strong T-cell responses in subjects with TB. Using an enzyme-linked immunospot (ELISPOT)-IFN-␥ assay and a restricted pool of peptides derived from ESAT-6 and CFP-10, we have previously demonstrated a high degree of specificity and sensitivity of the test for the diagnosis of TB. Here, 119 contacts of individuals with contagious TB who underwent TST and the ELISPOT-IFN-␥ assay were consecutively recruited. We compared the efficacy of the two tests in detecting latent TB infection and defined a more appropriate TST cutoff point. There was little agreement between the tests (k ‫؍‬ 0.33, P < 0.0001): 53% of the contacts with a positive TST were ELISPOT negative, and 7% with a negative TST were ELISPOT positive. Furthermore, respectively 76 and 59% of the ELISPOT-negative contacts responded in vitro to BCG and PPD, suggesting that most of them were BCG vaccinated or infected with nontuberculous mycobacteria. The number of spot-forming cells significantly correlated with TST induration (P < 0.0001). Our in-house ELISPOT assay based on a restricted pool of highly selected peptides is more accurate than TST for identifying individuals with latent TB infection and could improve chemoprophylaxis for the control of TB.One-third of the world's population is believed to be latently infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), whose global resurgence, supported by the human immunodeficiency virus type 1 (HIV-1) pandemic and emerging multidrug resistance, underlines the need for new control measures and strategies to make a specific diagnosis and prevent transmission (19). Moreover, continued vigilance against active and latent TB is essential in industrialized countries because of the increased immigration from areas with endemic infection.The compulsory screening of immigrant populations for TB has been proposed as a means of controlling this communicable disease (14, 15) but, although the tuberculin skin test (TST) is the method of choice for detecting latent M. tuberculosis infection (LTBI), it cannot be considered a gold standard because of the number of false-positive and negative reactions, and the variability of their interpretation (1, 2). In particular, the purified protein derivative (PPD) antigen used for the test has broad cross-reactivity with antigens derived from various mycobacterial species, including the vaccine strains of Bacillus Calmette-Guérin (BCG) (1, 2), and false-negative results occur in patients with advanced TB or severe immunodeficiency (10,25). Another operational drawback of TST is the need for a return visit to allo...

Comparison of an In-house and a Commercial RD1-based ELISPOT-IFN- Assay for the Diagnosis of Mycobacterium tuberculosis Infection

“…Our assay is equivalent to the commercial T-SPOT.TB assay, is cheaper, and it is probably not confounded, unlike the TST, by BCG vaccination in our setting. Thus, RD1-based ELISPOT assays are more accurate than TST in identifying LTBI as recently reported [20][21][22][23] and may help to avoid unnecessary treatments. …”

Enzyme-Linked Immunospot: An Alternative Method for the Detection of Interferon Gamma in Johne's Disease

“…Research carried out on TB has shown that specific antigens such as region of difference 1 (RD1) including culture filtrate protein 10, early secreted target 6, and other proteins such as Rv2031c (also known as HspX, alpha crystalline, or the 16-kDa antigen) used in an IFN-c ELISPOT tests can differentiate between active and latent TB patients and people who have been vaccinated with Bacillus Calmette-Guérin. 10,11,14,22 However, the use of a single RD1 antigen resulted in a high degree of specificity but low sensitivity. Improved results are seen when multiple RD1 antigens are used.…”

“…38 In addition, the ELISPOT has been developed as an alternative technology for diagnosing TB, as it has the advantage of being able to discriminate between different forms of TB, such as latent or active infections. 10 As these factors would also be important in the diagnosis of JD, an IFN-c ELISPOT was developed for the detection of MAP infection in sheep. The ELISPOT was then compared with IFN-c ELISA methods for the detection of MAP-infected sheep.…”

Assessment ofMycobacterium tuberculosisOmpATb as a Novel Antigen for the Diagnosis of Bovine Tuberculosis

“…Based on a genomic approach, a great variety of M. tuberculosis complex antigens have been screened for immunogenicity. ESAT-6 and CFP-10 have been shown to be outstanding diagnostic target proteins in the whole-blood IFN-␥ assay for cattle (1,2,10,14,18,29,40,41,45) and for humans (3,8,15,17). These proteins are considered particularly interesting because their genes are absent from M. bovis BCG, thus allowing differentiation between infected and vaccinated individuals.…”