Interferon Gamma Release Assays for Latent Tuberculosis: What Are the Sources of Variability?

Abstract

Interferon gamma release assays (IGRAs) are blood-based tests intended for diagnosis of latent tuberculosis infection (LTBI).IGRAs offer logistical advantages and are supposed to offer improved specificity over the tuberculin skin test (TST). However, recent serial testing studies of low-risk individuals have revealed higher false conversion rates with IGRAs than with TST. Reproducibility studies have identified various sources of variability that contribute to nonreproducible results. Sources of variability can be broadly classified as preanalytical, analytical, postanalytical, manufacturing, and immunological. In this minireview, we summarize known sources of variability and their impact on IGRA results. We also provide recommendations on how to minimize sources of IGRA variability. Interferon gamma (IFN-␥) release assays (IGRAs) are laboratory alternatives to the tuberculin skin test (TST) for diagnosis of latent tuberculosis infection (LTBI) (1). IGRAs are ex vivo assays that measure T-cell response after overnight stimulation with antigens that are relatively specific for Mycobacterium tuberculosis. IGRAs are increasingly replacing the TST for annual screening of U.S. health care workers and are also utilized in student/employee health and public health programs and in screening of patients prior to immunosuppression (1). In addition, the IGRA conversion rate is now being used as a measure of vaccine efficacy in TB vaccine trials (2).IGRAs are supposed to offer improved specificity over the TST (1). By and large, this advantage holds true in populations that receive Mycobacterium bovis BCG vaccination after infancy (1 year of age) or receive multiple doses; the specificity of TST is compromised in such settings (1). However, in practice, IGRAs have proved less specific in low-risk North American health care workers and college students (1). Furthermore, several studies have raised concerns over high rates of IGRA reversions (1). Given the growing use of IGRAs, it is imperative that we identify the underlying sources of variability and understand their impact on IGRA accuracy.The two most widely used IGRAs include the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay (Cellestis/Qiagen, Carnegie, Australia) and the T-SPOT.TB (T-SPOT) assay (Oxford Immunotec, Abingdon, United Kingdom). Although a new, four-tube version of QFT (called QFT-Plus) has been launched by Qiagen, this version is currently not available in the United States. The QFT-GIT assay, the FDA-approved version, is an enzyme-linked immunosorbent assay (ELISA)-based, whole-blood test that uses peptides from the two RD1 antigens (ESAT-6 and CFP-10) as well as peptides from one additional antigen (TB7.7 [Rv2654c]) in an in-tube format. The QFT-GIT assay consists of three tubes: the negative-control (nil) tube that measures background IFN-␥ response, the antigen tube that measures antigen-specific response, and the positive-control (mitogen) tube that measures nonspecific T-cell response. The qualitative result (negative, positive, or indeterminate) is in...

Evaluation of QuantiFERON-TB Gold-Plus in Health Care Workers in a Low-Incidence Setting

“…We also observed a high degree of correlation between quantitative QFT-Plus (TB1 and TB2) and QFT results. For the small number of patients with discordant results, the discordance was mostly due to quantitative results bordering the assay cutoff (0.2 to 0.7 IU/ml) (19)(20)(21)(22). The latter finding is consistent with patterns of discordant results reported in prior IGRA reproducibility studies and suggests that sources of variability previously described for QFT also are acting on QFT-Plus (22).…”

“…For the small number of patients with discordant results, the discordance was mostly due to quantitative results bordering the assay cutoff (0.2 to 0.7 IU/ml) (19)(20)(21)(22). The latter finding is consistent with patterns of discordant results reported in prior IGRA reproducibility studies and suggests that sources of variability previously described for QFT also are acting on QFT-Plus (22). Importantly, in this study we found that 2.1% and 3.0% of 626 HCWs with no identifiable risk factors for LTBI had positive QFT and QFT-Plus results, respectively.…”

Latent Mycobacterium tuberculosis Infection and Interferon-Gamma Release Assays

“…As reviewed by Banaei and colleagues, sources of variability can be broadly classified as preanalytical, analytical, postanalytical, manufacturing, and immunological (40). Figure 4 provides an overview of the major sources of variation with the QFT assay (15,40). Similar challenges also affect the reproducibility of the T-SPOT.TB assay.…”

“…Reproducibility studies have identified various sources of variability that contribute to nonreproducible results, and these studies have been systematically reviewed elsewhere (39). As reviewed by Banaei and colleagues, sources of variability can be broadly classified as preanalytical, analytical, postanalytical, manufacturing, and immunological (40). Figure 4 provides an overview of the major sources of variation with the QFT assay (15,40).…”

“…Further, extensive efforts need to be made to ensure adequate training and standardization. Table 2 lists suggestions to better standardize IGRAs and reduce the amount of test variability (40). When IGRAs are used for serial testing of health care workers, simplistic cut-offs (e.g., change from negative to positive) should not be used, because this results in very high conversion rates (41).…”