TB Manual

IGRAs are in vitro blood tests of cell-mediated immune response. IGRAs measure T-cell release of interferon-gamma following stimulation by antigens specific to M. tuberculosis — specifically, early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10).²² These antigens are more specific for M. tuberculosis than antigens contained in PPD because they are not shared with any BCG vaccine strains or most species of non-tuberculous mycobacteria (NTM) (other than M. marinum, M. kansasii, M. szulgai and M. flavescens).⁸ Infection with any of these specifically listed NTM strains could create a false positive IGRA.²³

A key advantage of IGRA is that, as opposed to TST, they do not require a return visit by the patient to have their result interpreted and recorded. If a patient is unable to return to have a TST read or if there is a concern a patient will not return to have a TST read, an IGRA, therefore, offers logistical advantages.

Two IGRAs are currently available in Canada: the QuantiFERON-TB Gold Plus (QFT-Plus) assay (Cellestis/Qiagen, Carnegie, Australia), and the T-SPOT.TB assay (Oxford Immunotec, Abingdon, United Kingdom). Both tests are approved by Health Canada. The WHO has recently concluded that Beijing Wantai’s TB-IGRA has comparable performance to other IGRAs and is an acceptable option, however, as of writing, this IGRA is not yet Health Canada-approved or available in Canada.²⁵

The QFT-Plus assay is an enzyme-linked immunosorbent assay (ELISA)-based, whole-blood test that uses ESAT-6 and CFP-10 antigens in an in-tube format (consisting of a TB1 and TB2 tube). The QFT-Plus assay is the fourth generation QuantiFERON assay, following initial generations QFT, QFT-Gold, and QFT-Gold-in-Tube. The major difference between QFT-Plus and the third generation QFT-Gold-in-Tube, is that QFT-Plus does not contain TB7.7 antigens and uses a second tube (TB2) that may stimulate CD8 T-cells (in addition to CD4 T-cells stimulated in the TB1 tube).^24,25 The amount of interferon-gamma detected is reported quantitatively in international units (IU) per milliliter. A positive result for M. tuberculosis infection occurs if the TB1 and/or TB2 tube has an interferon-gamma response to TB antigens that is ≥0.35 IU above the negative control.²⁶

The T-SPOT.TB assay is an enzyme-linked immunospot (ELISPOT) assay performed on separated and counted peripheral blood mononuclear cells (PBMCs); it uses the same two peptides as the QFT-Plus assay. The result is reported as number of interferon-gamma-producing T cells (spot-forming cells). A positive result for M. tuberculosis infection occurs if the spot counts in the TB antigen wells exceed a specific threshold relative to the control wells, equal to 8 spots in Canada.^27,28

Both commercial IGRAs (QFT-Plus and T-SPOT.TB) are acceptable to use; they differ, however, in terms of laboratory expertise required, cost, pre-analytical steps and ease of use. The decision on which commercial IGRA to offer is left to the discretion of provincial, commercial and hospital laboratories. In Canada, IGRAs should only be used among those older than 2 years of age (see Chapter 9: Pediatric Tuberculosis).

Indeterminate or invalid responses with IGRA occur when positive or negative control responses are too low or too high, respectively.^28,29 Causes of indeterminate or invalid IGRA responses are varied and may be due to inappropriate storage, delays in analysis or technical or patient factors. In the event of an indeterminate response, the test should be repeated. In the rare event that responses are consistently indeterminate,³⁰ consider using an alternate IGRA (ie, if consecutively indeterminate with QFT-Plus use T-SPOT.TB) or a TST.

The manufacturer-recommended cut points for interpretation were chosen to maximize sensitivity and specificity. Unlike the TST, the manufacturer has not provided a gradient of interpretation for a positive test (such as those defined in Table 1); instead, results are dichotomous. Interferon-gamma responses may be dynamic and results that are close to manufacturer-recommended cut points for a positive test should be interpreted cautiously. There is a manufacturer-defined “borderline” zone for T-SPOT.TB that is equal to 5-7 spots (a positive result according to the manufacturer is 8 spots or more), which recognizes this uncertainty. However, there is no such manufacturer-defined borderline zone for QFT. We suggest using a “borderline” zone for QFT that ranges from ≥0.2 to ≤1.0 IU/ml (a positive result according to the manufacturer is 0.35 IU/ml or greater).^31,32

Recommendation

• We strongly recommend both the tuberculin skin test and interferon-gamma release assay as acceptable alternatives for TB infection diagnosis. Either test can be used for TB infection screening in any of the situations in which testing is indicated. However, there are preferences and exceptions detailed in subsequent recommendations (good evidence).

Good practice statements

• When the initial interferon-gamma release assay result is indeterminate or invalid, the interferon-gamma release assay should be repeated or a tuberculin skin test be performed.

• When the initial interferon-gamma release assay result is borderline (equivalent to 5-7 spots with T-SPOT.TB or 0.2 to 1.0 IU/ml with QFT), the interferon-gamma release assay may be repeated or a tuberculin skin test used to help arrive at a diagnosis.

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