Annex 6. Answers to frequently asked questions on IGRAs

What are IGRAs?

Interferon gamma release assays (IGRAs) are whole-blood tests that can aid in diagnosing M. tuberculosis infection (43). Like TST, IGRAs cannot differentiate between TB infection and TB disease. WHO reviewed available evidence around the two most widely used IGRAs in 2017, QuantiFERON®-TB Gold In-Tube test (QFT-GIT) and T-SPOT.TB test (T-Spot), to facilitate recommendation on their use to identify individuals having TB infection.

How do IGRAs work?

White blood cells from individuals infected with M. tuberculosis release interferon-gamma (IFN-g) when mixed with antigens derived from M. tuberculosis. Recognition that interferon gamma plays a critical role in regulating cell-mediated immune responses to M. tuberculosis infection led to development of IGRAs (149–151). IGRAs detect sensitization to M. tuberculosis by measuring IFN-g release in response to antigens representing M. tuberculosis. IGRAs assess response to synthetic overlapping peptides that represent specific M. tuberculosis proteins, such as early secretory antigenic target-6 (ESAT-6) and culture filtrate protein 10 (CFP-10). These proteins are present in all M. tuberculosis complex species and stimulate measurable release of IFN-g in most infected persons but are absent from BCG vaccine strains and from most nontuberculous mycobacteria. For IGRAs to measure IFN-g response accurately, a fresh blood specimen that contains viable white blood cells is needed.

QFT-GIT¹³: In QFT-GIT the control materials and antigens are contained in special tubes used to collect blood for the test, thus allowing more direct testing of fresh blood. One of the tubes contain heparin and the test antigens (single mixture of 14 peptides representing the entire amino acid sequences of ESAT-6 and CFP-10 and part of the sequence of TB7.7), while two accompanying tubes serve as negative and positive controls. The negative/control tube contains heparin alone, and the positivecontrol tube contains heparin, dextrose and phytohaemagglutinin. Blood (1 ml) is collected into each of the three tubes, mixed with the reagents already in the tubes and incubated for 16 to 24 hours. Plasma is separated, and the IFN-g concentration in the plasma is determined using a sensitive ELISA. To interpret QFT-GIT, the response is calculated as difference in IFN-g concentration in plasma from blood stimulated with antigen (i.e. the single cocktail of peptides representing ESAT-6, CFP-10 and TB7.7) minus the IFN-g concentration in plasma from blood incubated without antigen (i.e. nil).

T-SPOT: Peripheral blood mononuclear cells (PBMCs) are incubated with control materials and two mixtures of peptides, one representing overlapping sequences of the entire amino acid sequence of ESAT-6 and the other representing the entire amino acid sequence of CFP-10. It identifies M. tuberculosis sensitized effector TB cells, activated by the presence of CFP 10 and ESAT-6 antigens. Effector T cells have a short life cycle; however, their continuing presence indicates that the individual’s immune response is currently encountering and fighting a pathogen somewhere in the body. Measuring the presence of effector T cells in a blood specimen therefore indicates ongoing TB infection. The blood specimen is centrifuged, and the sample is diluted to ensure that a standard number of PBMCs get added to each of the four test wells (nil control, panel with CFP 10 antigen, panel with ESAT-6 antigen and positive control), which are precoated with antibodies to IFN-g and incubated for 16–20 hours. In the presence of activated effector T cells (infected individuals), the TB specific antigens, ESAT-6 and CFP 10, stimulate the release of IFN-g which binds the antibodies to IFN-g on the base of the well. Wells are washed and a secondary antibody to IFN-g is added. Following another incubation and washing step, the substrate is added. This produces spots on the well floor, where the IFN-g was secreted by T Cells. The spots are enumerated in each of the test wells to provide the test results and the difference between antigen panels and nil is compared. Reading is performed manually or by an Elispot reader.


What are the advantages of IGRAs?

• Requires a single visit to conduct the test. But results can be available only within 24 hours which requires a second visit.

• Prior BCG vaccination does not cause a false-positive IGRA test result.

• For serial and periodic screening of people who might have occupational exposure to TB (such as surveillance programmes for health care workers), IGRAs offer technical, logistic and possible economic advantages compared with TSTs. Two-step testing is not required for IGRAs, because IGRA testing does not boost subsequent test results.

• American Academy of Paediatrics revised its guideline to include use of IGRA down to children two years of age, suggesting use of IGRA in children two years and older (152).

What are the disadvantages and limitations of IGRAs?

• Blood samples must be processed within 8–30 hours after collection while white blood cells are still viable.

• Errors in collection or transportation of blood specimens or running the test can decrease accuracy.

• Tests are expensive.

• A greater risk of test conversion due to false-positive IGRA results with follow-up testing of low-risk health care workers who have tested negative at prior screening. An IGRA conversion is defined as a change from negative to positive within two years. Association between IGRA conversion and subsequent disease risk has not been demonstrated.

• Limited data on the use of IGRAs to predict who will progress to TB disease in the future.

• Limited data on the use of IGRAs for children younger than two years of age (higher risk of indeterminate results), persons recently exposed to M. tuberculosis, immunocompromised persons, and serial testing.

What are the steps in conducting an IGRA test?

• IGRAs should be performed and interpreted according to established protocols.

• Arrangement for IGRA testing should be made prior to blood collection to ensure that the blood specimen is collected in the proper tubes.

• Draw a blood sample from the person according to the test manufacturer’s instructions.

• IGRAs should be performed and interpreted according to established national protocol.

• Arrange for delivery of the blood sample to the laboratory in the time the laboratory specifies to ensure testing of samples while the blood cells are still viable.

• Schedule a follow-up appointment for the person to receive test results.

• Based on test results, provide follow-up evaluation and treatment as needed.

Nurses and other health care workers who have been trained in phlebotomy can request an IGRA and deliver the result to the person tested, explaining its significance.

How to interpret IGRA test results?

IGRA interpretations are based on the amount of IFN-g released or on the number of cells that release IFN-g. Both the standard qualitative test interpretation (positive, negative, or indeterminate) and the quantitative assay measurements (nil, TB and mitogen concentrations or spot counts) should be reported. This will permit a more refined assessment of results and promote better understanding of the test result. Like TSTs, IGRAs should be used as an aid in diagnosing M. tuberculosis infection. A positive test result suggests that M. tuberculosis infection is likely, and a negative result means that infection is unlikely. An indeterminate result indicates uncertain likelihood of M. tuberculosis infection. A borderline test result (T-Spot only) also indicates an uncertain likelihood of M. tuberculosis infection.

For healthy persons with a low likelihood both of TB infection and progression to TB disease, a single positive IGRA or TST result should not be taken as reliable evidence of M. tuberculosis infection. Because of the low probability of infection, a false-positive result is more likely. In such situations, the likelihood of M. tuberculosis infection and of disease progression should be reassessed, and the initial test results should be confirmed. Repeat testing, with either the initial test or a different test, may be considered on a case-by-case basis or alternatively assume that the initial result is a false-positive, without additional testing.

¹³  Please note that the guidance covers only currently recommended IGRA tests in the 2020 WHO guidelines for programmatic management of TPT, which are based on reviews done in 2017. The manufacturers of QFT-GIT plan to phase this test out and substitute with the 4 tube QFT plus test.

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