Gamma Interferon-Based Immunodiagnosis of Tuberculosis: Comparison between Whole-Blood and Enzyme-Linked Immunospot Methods

Abstract

The gamma interferon secretion levels of 52 adults whose T cells had been stimulated with the Mycobacterium tuberculosis antigens CFP-10 and ESAT-6 were measured by enzyme-linked immunospot methods and a whole-blood-based assay. The test results correlated well (r ‫؍‬ 0.689, P < 0.0001). Its simple format and use of only small volumes of blood make the whole-blood assay suitable for pediatric practice and field trials.Mycobacterium tuberculosis is a significant threat to global health (11). The skin test reagent used to aid diagnosis of both active and latent tuberculosis, purified protein derivative (PPD), is impaired in specificity and sensitivity (22), and the skin reaction may cause discomfort. Recent interest in the field of immunodiagnosis has therefore concentrated on antigens encoded by the RD1 genomic segment of M. tuberculosis, which is deleted from all BCG strains (5,15,19). In addition, the idea of replacing the tuberculin skin test (TST) by a method that assays the in vitro production of gamma interferon (IFN-␥) in response to M. tuberculosis antigens has gained popularity (2, 6, 9, 17). The most promising approach combines the recognized high sensitivity of ELISPOT analysis with the use of peptides covering the sequence of the RD1-encoded ESAT-6 molecule into a diagnostic test of greater sensitivity and specificity than the TST (4, 13, 18, 21). The enzyme-linked immunospot (ELISPOT) technique requires separation of cells from blood, the use of multiple wells of an expensive precoated plate, and an automated ELISPOT counter. Some have therefore wondered whether and how these advances could be applied to medically underserved environments (3). A kit based on whole-blood production of IFN-␥ detected by enzyme-linked immunosorbent assay (ELISA) is available and has the advantage of relative simplicity, but being based on PPD, it does not ameliorate the poor specificity of skin testing (6). In addition, this test requires 10 ml of blood, a large amount if one considers its application in pediatric practice.We were interested to determine, by the use of a sensitive IFN-␥ ELISA, whether a form of the whole blood stimulation assay that is used by many groups to assay antigen specific responses to RD1 encoded antigens (1,3,7,9,16) could match the performance of the ELISPOT assay. Ethical permission for this study was provided by the Harrow local research ethics committee (Harrow LREC 1646). To perform the whole-blood assay, heparinized blood was diluted 1:10 in RPMI and 180 l of the diluted blood was stimulated with 2.5 g/ml ESAT-6 prepared as described previously (20) or 5 g/ml CFP-10 (encoded by the gene adjacent to ESAT-6, obtained commercially from Lionex, Braunschweig, Germany) or no stimulus in duplicate on a 96-well plate. Diluted blood was cultured at 37°C in a CO 2 incubator for 72 h, at which point the supernatants were aspirated for determination of IFN-␥ level by ELISA (Antibody pair from BD Pharmingen catalogue no. 554548 and 554550). This ELISA has a working sensitivity of Ͻ10 pg/ml. Initially...

Effect of HIV-1 Infection on T-Cell–based and Skin Test Detection of Tuberculosis Infection

“…As both IFNGRAs depend predominantly on the CD4 recognition of M. tuberculosis antigens (27), a variable that will affect test performance in the HIV-infected group is the absolute CD4 ϩ cell count. Data were therefore explored by stratification by CD4 ϩ cell count: Ͻ 350, Ͻ 250, and Ͻ 200/mm 3 .…”

Comparison of two commercial interferon-γ assays for diagnosingMycobacterium tuberculosisinfection

“…Although previous studies have compared ELISA-and ELISPOT-based IFN-c assays [18,19], no other work has stringently compared the two commercially available assays considered here. In the present study, when the manufacturer-recommended cut-offs were adopted, the sensitivity of SPOT was significantly higher than those of TST or QFT-G, especially in immunocompromised subjects, and the specificity of QFT-G was significantly higher than that of TST.…”

Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment

“…Antigen-stimulated whole-blood assays were performed as previously described (42). Concentrations of IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17, G-CSF, GM-CSF, IFN-α, IFN-γ, TNF, CXCL8, CXCL9, CXCL10, CCL2, CCL3, CCL4, CCL5, CCL11, EGF, FGF-β, HGF, and VEGF were quantified using a human 30-plex bead immunoassay panel (Invitrogen).…”