Distinct clinical and immunologic patterns of interferon-γ release assays: implications for indeterminate results and sequential testing strategies.

UNLABELLED: Interferon-γ release assays (IGRAs) are widely used for tuberculosis (TB) infection testing and are often considered interchangeable. We evaluated whether differences between T-SPOT.TB (T-SPOT) and QuantiFERON-TB Gold Plus (QFT-GP) have clinically relevant implications, particularly for indeterminate results. In this prospective study, 703 patients with suspected TB infection underwent concurrent T-SPOT and QFT-GP testing. Performance was assessed across three reference strata-microbiologically confirmed disease, initiation of TB preventive treatment for presumed TB infection, and culture-confirmed nontuberculous mycobacterial disease as a specificity reference-and indeterminate results were analyzed using interferon-γ responses and host immune factors. T-SPOT showed higher positivity than QFT-GP in treated patients (77.8% vs 50.0%). Among 75 patients with indeterminate T-SPOT results (10.7%), QFT-GP provided determinate results in 89.3%, enabling clinical reclassification. Antigen-specific interferon-γ responses were significantly higher in QFT-GP-positive cases despite T-SPOT indeterminacy (P = 5.0 × 10⁻⁶). Mechanisms of indeterminacy differed: T-SPOT indeterminacy arose predominantly from near-threshold antigen responses (60.0%) and, in a minority, from failure of the assay control system, whereas QFT-GP indeterminacy was uniformly attributable to mitogen failure. T-SPOT resolved 61.9% of QFT-GP-indeterminate cases. IGRAs are not functionally interchangeable but, instead, provide complementary clinical information. Indeterminate results are mechanistically heterogeneous: near-threshold antigen responses were frequently resolved, and occasionally resolved as positive, by the alternative platform, whereas failure of the assay control system-particularly concurrent mitogen failure on both assays-marked host immune impairment. A sequential testing strategy-T-SPOT followed by QFT-GP in indeterminate cases-may improve diagnostic yield and reduce uncertainty in clinical practice. IMPORTANCE: Tuberculosis remains one of the leading infectious causes of death worldwide. Detecting people who carry Mycobacterium tuberculosis without symptoms is essential for preventing future disease, but currently available blood tests do not always provide clear answers. In this study of more than 700 patients, we found that two commonly used tuberculosis blood tests capture different aspects of the immune response and, therefore, do not always produce the same result. One test was more likely to identify infection when tuberculosis was clinically suspected, whereas the other often provided interpretable results when the first test was inconclusive. We also found that unclear results were strongly influenced by the patient's immune status. These findings suggest that the two tests can be used together rather than viewed as interchangeable and that considering immune health may improve the interpretation of tuberculosis screening results and support more effective prevention strategies.