BACKGROUND: Interferon gamma release assays (IGRAs) are blood tests recommended for the diagnosis of tuberculosis (TB) infection. There is currently uncertainty about the role and clinical utility of IGRAs in the diagnostic workup of suspected active TB in routine NHS clinical practice. OBJECTIVES: To compare the diagnostic accuracy and cost-effectiveness of T-SPOT.TB® (Oxford Immunotec, Abingdon, UK) and QuantiFERON® TB GOLD In-Tube (Cellestis, Carnegie, VIC, Australia) for diagnosis of suspected active TB and to estimate the diagnostic accuracy of second-generation IGRAs. DESIGN: Prospective within-patient comparative diagnostic accuracy study. SETTING: Secondary care. PARTICIPANTS: Adults (aged ≥ 16 years) presenting as inpatients or outpatients at 12 NHS hospital trusts in London, Slough, Oxford, Leicester and Birmingham with suspected active TB. INTERVENTIONS: The index tests [T-SPOT.TB and QuantiFERON GOLD In-Tube (QFT-GIT)] and new enzyme-linked immunospot assays utilising novel Mycobacterium tuberculosis antigens (Rv3615c, Rv2654, Rv3879c and Rv3873) were verified against a composite reference standard applied by a panel of clinical experts blinded to IGRA results. MAIN OUTCOME MEASURES: Sensitivity, specificity, predictive values and likelihood ratios were calculated to determine diagnostic accuracy. A decision tree model was developed to calculate the incremental costs and incremental health utilities [quality-adjusted life-years (QALYs)] of changing from current practice to using an IGRA as an initial rule-out test. RESULTS: A total of 363 patients had active TB (culture-confirmed and highly probable TB cases), 439 had no active TB and 43 had an indeterminate final diagnosis. Comparing T-SPOT.TB and QFT-GIT, the sensitivities [95% confidence interval (CI)] were 82.3% (95% CI 77.7% to 85.9%) and 67.3% (95% CI 62.1% to 72.2%), respectively, whereas specificities were 82.6% (95% CI 78.6% to 86.1%) and 80.4% (95% CI 76.1% to 84.1%), respectively. T-SPOT.TB was more sensitive than QFT-GIT (relative sensitivity 1.22, 95% CI 1.14 to 1.31; p < 0.001), but the specificities were similar (relative specificity 1.02, 95% CI 0.97 to 1.08; p = 0.3). For both IGRAs the sensitivity was lower and the specificity was higher for human immunodeficiency virus (HIV)-positive than for HIV-negative patients. The most promising novel antigen was Rv3615c. The added value of Rv3615c to T-SPOT.TB was a 9% (95% CI 5% to 12%) relative increase in sensitivity at the expense of specificity, which had a relative decrease of 7% (95% CI 4% to 10%). The use of current IGRA tests for ruling out active TB is unlikely to be considered cost-effective if a QALY was valued at £20,000 or £30,000. For T-SPOT.TB, the probability of being cost-effective for a willingness to pay of £20,000/QALY was 26% and 21%, when patients with indeterminate test results were excluded or included, respectively. In comparison, the QFT-GIT probabilities were 8% and 6%. Although the use of IGRAs is cost saving, the health detriment is large owing to delay in diagnosing active TB, leading to prolonged illness. There was substantial between-patient variation in the tests used in the diagnostic pathway. LIMITATIONS: The recruitment target for the HIV co-infected population was not achieved. CONCLUSIONS: Although T-SPOT.TB was more sensitive than QFT-GIT for the diagnosis of active TB, the tests are insufficiently sensitive for ruling out active TB in routine clinical practice in the UK. Novel assays offer some promise. FUTURE WORK: The novel assays require evaluation in distinct clinical settings and in immunosuppressed patient groups. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and the NIHR Health Protection Research Unit in Respiratory Infections, Imperial College London, London, UK.