Interferon-Gamma Release Assays and Tuberculin Skin Test

Tests for tuberculosis infection detect the immune response to Mycobacterium tuberculosis antigens. The tuberculin skin test (TST, Mantoux) measures delayed type hypersensitivity 48 to 72 hours after intradermal injection of 5 tuberculin units of purified protein derivative.

Interferon gamma release assays (IGRAs: QuantiFERON-TB Gold Plus and T-SPOT.TB) measure interferon gamma released by T cells in blood when stimulated by ESAT-6 and CFP-10 antigens (specific to M. tuberculosis and few non-tuberculous species, not present in BCG). Both tests show sensitisation to the organism, not active disease, and cannot separate latent infection from active tuberculosis.

Tests are used to identify latent tuberculosis infection (LTBI) in people at risk of progression, so that preventive treatment can be given. About a quarter of the world population (about 2 billion people) has latent infection, and 5 to 10% develop active disease during their lifetime, half in the first 2 years. The risk is 10% a year in people with HIV not on treatment.

Indications. Contacts of infectious tuberculosis, people starting immunosuppressive treatment (anti-TNF drugs, high dose corticosteroids, transplant, chemotherapy). Other indications: HIV infection, silicosis, diabetes, chronic kidney disease and dialysis, healthcare workers, migrants from high burden countries, people in prisons and shelters, children under 5 years in contact with a case, and abnormal chest radiograph suggestive of old tuberculosis. Not used to diagnose active tuberculosis in adults.

Specimen. TST: intradermal injection on the forearm, read at 48 to 72 hours as millimetres of induration (not erythema). IGRA: heparinised or specialised tube blood, 1 mL in each of four tubes (QuantiFERON) or a heparin tube (T-SPOT), delivered to the laboratory within 16 hours (QuantiFERON) or 32 hours (T-SPOT with enhancement), kept at room temperature, never refrigerated.

Tuberculin skin test. Cut offs depend on risk.

• 5 mm or more: HIV infection, recent contact with active tuberculosis, immunosuppression (equivalent of prednisolone 15 mg a day for a month or more, anti-TNF therapy, transplant), fibrotic changes on chest radiograph.
• 10 mm or more: recent arrivals from high prevalence countries, people who inject drugs, residents and staff in congregate settings, healthcare workers, children under 4 years, medical conditions that raise risk (diabetes, kidney failure, silicosis, malignancy, low body weight).
• 15 mm or more: people with no risk factors.
• BCG vaccination can cause false positives, especially with vaccination after infancy or repeated doses, but its effect wanes after 10 years, and large reactions (over 15 mm) are likely due to infection.

IGRA.

• QuantiFERON-TB Gold Plus: positive if the TB antigen minus nil (negative control) is 0.35 IU/mL or more (and 25% or more over nil). Indeterminate if nil is high (over 8 IU/mL) or the mitogen (positive control) response is low (under 0.5 IU/mL). Negative otherwise.
• T-SPOT.TB: spot count (ESAT-6 and CFP-10 panels) minus nil. Positive if 8 or more spots. Borderline 5 to 7 (repeat). Negative 4 or fewer.
• Sensitivity of both tests is 80 to 90% for active tuberculosis (60 to 75% in HIV-infected patients), and specificity is 95 to 99% (lower for the TST at 70 to 90% in BCG-vaccinated individuals).

Choice of test.

• IGRA is preferred in BCG-vaccinated people and those unlikely to return for a TST reading.
• TST is preferred in children under 2 to 5 years (limited IGRA data), where IGRA is not available, and for repeat testing in some settings.
• Either test may be used, and a second test increases sensitivity in high risk, immunosuppressed patients when the first is negative.
• Neither test excludes active tuberculosis. Active disease is investigated by symptoms, chest radiograph, and microbiology.

Reversion and conversion. A conversion from negative to positive within 2 years (increase of 10 mm on TST) indicates recent infection and a higher progression risk. IGRA reversions and conversions near the cut off are common and variable (as many as 20% of uncertain results), and serial IGRA is not used alone for healthcare worker surveillance without confirmation.

Predictive value. Positive predictive value for progression to active disease is low (about 2 to 3% over 2 years for IGRA-positive people), and higher in children, HIV patients and recent contacts (up to 10% or more). Negative predictive value is high (over 99%) in low risk groups.

Critical values and findings needing immediate action.

• A positive test in a person with symptoms (cough over 2 weeks, fever, night sweats, weight loss) or an abnormal chest radiograph: investigate for active tuberculosis (sputum for Xpert and culture) before starting preventive treatment.
• A positive test in a child under 5 years in contact with an infectious case, or in a person with HIV: start preventive treatment after excluding active disease.
• A positive test before anti-TNF therapy or transplant: preventive treatment for at least 1 month before starting biological therapy, where possible.
• Indeterminate IGRA in an immunosuppressed patient: repeat the test, or TST, or treat if risk is high.
• A negative test in an immunosuppressed patient with high risk: does not exclude infection, and is interpreted with the clinical context.

Interferences and pitfalls.

• Neither test can separate latent from active tuberculosis, and a positive test does not equal disease.
• False negative results: HIV with low CD4 count, immunosuppressive medicines (corticosteroids, anti-TNF), malnutrition, severe disseminated tuberculosis, recent viral infection or live vaccine (measles, varicella), sarcoidosis, uraemia, lymphoma, extremes of age, and poor injection technique.
• False positive TST: BCG, non-tuberculous mycobacteria, and boosting after repeated testing (two step testing is used in healthcare worker baselines).
• IGRA false positives: Mycobacterium kansasii, szulgai and marinum infection, and laboratory error. False negatives follow pre-analytic delays.
• IGRA indeterminate results occur in 2 to 10%, more in immunosuppressed patients and children.
• Tests do not identify drug resistance, and preventive treatment is chosen by contact source susceptibility (rifampicin based short regimens).
• Positive test results should not be repeated to check whether they revert.
• Tests do not indicate the time of infection.

Special groups.

• Children: TST and IGRA have lower sensitivity under 5 years, and a negative test in a contact is repeated 8 to 10 weeks after last exposure. Preventive treatment is started immediately in young contacts while waiting.
• HIV-infected patients: IGRA and TST sensitivity is lower at low CD4, and preventive treatment is given irrespective of the test result in high burden countries.
• Pregnancy: tests are valid, and preventive treatment is deferred until after delivery unless risk is high.
• Older adults: waning immunity gives false negatives, and booster effects confuse repeat TST.
• Regions: in high burden countries, tests are not used in adults because most are infected, and preventive treatment is targeted to contacts, people with HIV and young children.

Follow up tests. Chest radiograph, sputum for Xpert MTB/RIF and culture, HIV test, liver function tests and hepatitis B and C serology before preventive treatment. Also: full blood count, glucose or HbA1c, baseline visual acuity (if ethambutol is considered), repeat IGRA or TST if the first is indeterminate or discordant, and clinical review for symptoms during preventive treatment.