Test variability of the QuantiFERON-TB gold in-tube assay in clinical practice

Abstract

Rationale: Although IFN-γ release assays (IGRAs) are widely used to screen for Mycobacterium tuberculosis infection in high-income countries, published data on repeatability are limited.

Objectives: To determine IGRA repeatability.

Methods: The study population included consecutive patients referred to The Methodist Hospital (Houston, TX) between August 1, 2010 and July 31, 2011 for latent tuberculosis (TB) infection screening with an IGRA (QuantiFERON-TB Gold In-Tube; Cellestis, Carnegie, Australia). We performed multiple IGRA tests using leftover stimulated plasma according to a prospectively formulated quality control protocol. We analyzed agreement in interpretation of test results classified according to manufacturer-recommended criteria and repeatability of quantitative TB response.

Measurements and main results: During the study period, 1,086 test results were obtained from 543 subjects. Per the manufacturer's cut-point, the result of the second test was discordant from that of the first in 28 (8%) of 366 patients with valid test results, including 13 with an initial negative result and 15 with an initial positive result. Although agreement between repeat test results was high (κ = 0.84; 95% confidence interval, 0.79-0.90), the normal expected range of within-subject variability in TB response on retesting included differences of ± 0.60 IU/ml for all individuals (coefficient of variation, 14%), and ± 0.24 IU/ml (coefficient of variation, 27%) for individuals whose initial TB response was between 0.25 and 0.80 IU/ml.

Conclusions: There is substantial variability in TB response when IGRAs are repeated using the same patient sample. IGRA results should be interpreted cautiously when TB response is near interpretation cut-points.

Figure 1.

Study flow diagram. Positive, negative, and indeterminate tests were classified according to manufacturer’s recommended criteria (27). Analyzed QuantiFERON Gold-In Tube (QFT-GIT) negative test results were negative less than 0.25 IU/ml with nil control greater than or equal to 0.10 IU/ml, or negative 0.25–0.35 IU/ml, regardless of nil control response. Only the first and second tests for each subject were included in the main analysis. *Very low TB response was defined as TB response less than −0.35 IU/mL and less than −0.5 times nil control (30). TB = tuberculosis.

Figure 2.

Normal expected variability of borderline tuberculosis (TB) response. True mean TB response (square, defined as the average value of an unlimited number of measurements taken under the same conditions) and associated 95% variability (± 1.96 SD; the solid line to each side of the square represents 1.96 SD). Given an estimated true negative mean TB response of 0.349 IU/ml (just below test cut point for positivity) and a normal expected range of within-subject variability of ± 0.24 IU/ml, 95% of subjects demonstrate variability between 0.11 and 0.59 IU/ml. Thus, the low positive zone (gray shading) is defined as the interval (0.35–0.59 IU/ml) within which a positive result could be expected to revert to negative on retest based solely on the inherent variability of the test. Measured TB response greater than 0.59 IU/ml is unlikely to be associated with an estimated true mean TB response less than 0.35 IU/ml, and thus should be considered to indicate a true positive value. The repeatability of QuantiFERON Gold-In Tube (QFT-GIT) assays was assessed using a linear mixed effects model fit to the numerical IFN-γ values. Borderline TB response was defined as IFN-γ concentration 0.25–0.80 IU/ml; the manufacturer-recommended cut point (0.35 IU/ml) is demonstrated as a dashed line.