Discrepancy between Mycobacterium tuberculosis-specific gamma interferon release assays using short and prolonged in vitro incubation

Abstract

The sensitivities of various gamma interferon release assays (IGRAs) for the detection of past latent Mycobacterium tuberculosis infection are not known. In this study, we aimed to assess the effects of various IGRA formats and in vitro incubation periods on test outcome. The results of the tuberculin skin test (TST) were compared with those of the QuantiFERON-TB Gold in-tube (QFT-GIT) test, an overnight enzyme-linked immunospot assay (ELISPOT), and a 6-day lymphocyte stimulation test (LST) by using the same M. tuberculosis-specific peptides and samples from 27 TST-positive persons with a history of exposure to M. tuberculosis, 4 patients cured of tuberculosis (TB), and 9 TST-negative controls. Among the TST-positive persons, the LST was more frequently positive (92%; P < 0.01) than either the QFT-GIT test (33%) or ELISPOT (46%). While good agreement was observed between the QFT-GIT test and ELISPOT (kappa = 0.71) and between TST and LST (kappa = 0.78), the agreement between TST or LST, on the one hand, and the QFT-GIT test or ELISPOT, on the other, was poor. These data indicate that the QFT-GIT test and overnight ELISPOT are less sensitive for the detection of past latent TB than the 6-day LST. The observed discrepancies between these IGRAs are most likely related to differences in incubation periods. Whether TST-positive persons with positive LST results but negative QFT-GIT and ELISPOT results are at risk for the development of TB needs to be elucidated before short-incubation IGRAs can be used for the screening of individuals for latent TB before immunosuppressive treatment.

FIG. 1.

Comparison of three IGRAs and TST for detection of latent M. tuberculosis infection. TST and the QFT-GIT test (QFT) were performed with samples from 27 TST+ persons with known exposure to M. tuberculosis. ELISPOT (SPOT) and a 6-day LST were done with samples from 24 TST+ persons by using the same M. tuberculosis-specific peptides (ESAT-6, CFP-10, and TB7.7) used for QFT-GIT test. Bars indicate the percentage of positive test results. The cutoffs for positive results were an IFN-γ concentration of 0.35 IU/ml for the QFT-GIT test, 5 SFC/well above the background for ELISPOT, an IFN-γ concentration of 100 pg/ml for LST, and ≥10 mm of induration for TST. **, P < 0.01.

FIG. 2.

LST responses in persons with negative versus positive results by the QFT-GIT test and ELISPOT. A 6-day LST was performed with pools of peptides of ESAT-6, CFP-10, and TB7.7. The LST responses are indicated as the highest level of production (in pg/ml) of IFN-γ to one of the M. tuberculosis-specific peptide pools. (a) LST responses in QFT-GIT test-negative (QFT-GIT−) and QFT-GIT test-positive (QFT-GIT+) persons (cutoff, IFN-γ ≥ 0.35 IU/ml); (b) LST responses in ELISPOT-negative (ELISPOT−) and ELISPOT-positive (ELISPOT+) persons (cutoff, 5 SFCs/well above the background). Lines indicate the median level of IFN-γ production.