Role of interferon gamma release assay in the diagnosis and management of Mycobacterium tuberculosis-associated uveitis: a review

QuantiFERON-TB assay (QFT)

The first version of the QuantiFERON-TB test (QIFN) was approved by the FDA as a diagnostic tool for latent TB infection in 2001.10 It is an ELISA-based whole blood test that uses peptides from the M. tuberculosis-specific antigens. The result is reported as quantification of IFN-γ in international units (IU) per millilitre. The patient is considered positive for latent TB infection if the reported value is above the cut-off specified by the manufacturer. The QFT assay has four versions: QIFN, QFT-Gold, QFT-GIT and QFT-Gold Plus. The mechanisms of action of tests are summarised in table 1.

T-SPOT.TB assay

The T-SPOT.TB is an enzyme-linked immunosorbent spot (ELISpot) assay in which peripheral blood mononuclear cells are processed.31 It was licensed in the European Union in July 2004 and received FDA premarket approval in July 2008.32 These cells react with synthetic peptides representing ESAT-6 and CFP-10, and IFN-γ antibodies are used to bind to the IFN-γ as the cells release it. A secondary antibody, which is enzyme-labelled, is added which attaches to the bound IFN-γ. A detection reagent is introduced which reacts with the enzyme-labelled antibody. This reaction produces IFN-γ producing spots, which are then counted. The patient is considered to be positive for TB infection if the number of spots in the TB antigen well exceeds the provided threshold value.33

Prevalence of disease (endemic versus non-endemic)

Spectrum bias or effect refers to the phenomenon in which there is a variance in the performance of a test in different clinical settings. This may arise due to a variation in prevalence of the disease in different studies. Because of this effect, tests may not be as accurate in populations different from the ones they were developed in.38 Leeflang et al39 analysed data from 23 meta-analyses and found there to be a statistically significant (p<0.05) negative correlation between specificity and prevalence. They theorised that this association was caused by clinical variability (range in patient spectrum, different referral pathways and reader expectations) and artefactual differences (distorted inclusion of patients, verification bias and imperfect reference standard). WHO recommends that IGRAs should not replace microbiological methods of diagnosis of pulmonary and extrapulmonary TB in low-income and middle-income countries (which may have a higher incidence than high-income countries).40

Babu et al41 stated that in countries where TB is endemic immune-based tests might not be very useful as they do not directly detect the organism but show that an immune response has taken place. In a study of 687 contacts of patients with culture-confirmed systemic TB in France, QFT-GIT’s positive predictive value for progression to TB was 1.96%, while the negative predictive value was 99.8%. This study supports the utility of QFT-GIT in a low prevalence setting.42 Kang et al43 conducted a multicentre study to investigate the clinical usefulness of the T-SPOT.TB in diagnosing active TB in China, a high burden country. They found that there was a high number of false positives when using this test (43.6%, 95% CI 40.9% to 46.3%) and did not recommend its use in a high burden country.

Immune suppression

Immune suppression secondary to corticosteroids reduces the release of IFN-γ and therefore leads to the test being less accurate in patients under treatment. Edwards et al44 demonstrated this in an ex vivo model study. They also stated that in these patients, inducible protein 10 (IP-10) might serve as a more reliable biomarker. Glucocorticoids are noted to have a significant influence on IGRAs in children if treated for more than a week.45 Bao et al found glucocorticoids to affect the mitogen-stimulated response and therefore negatively affect the IGRA results in both adults and children.46 ,47 QFT-Plus sensitivity was noted to be lower among people with HIV/AIDS with severe immunosuppression.48

An older study evaluating the clinical usefulness of T-SPOT.TB in immunocompromised patients (53 with malignancy, 29 patients with diabetes mellitus, 23 taking immunosuppressive drugs and 14 with end-stage renal disease) found T-SPOT.TB cannot be used alone to rule in TB due to its poor specificity in these groups. They found the usefulness to be particularly less in the group taking immunosuppressive drugs.49 T-SPOT.TB test was found not to be affected by CD4+ T lymphocyte count (p=0.289), and it was stated that in HIV-infected patients with low CD4+ T lymphocyte counts, T-SPOT.TB test may be considered for latent TB infection diagnosis.50

Antituberculous treatment

In a study by Bao et al,46 44.4% of patients with active (pulmonary or extrapulmonary) TB converted to a negative reading with the QFT-GIT after less than 3 months of ATT. This is thought to be due to the bactericidal effect of ATT on M. Tuberculosis, which reduces antigen production and hence reduces antigen-stimulated IFN-γ production.

However, another study reported that the effects of therapeutic concentrations of ATT (isoniazid, rifampicin and ciprofloxacin) on IFN-γ and other cytokines were comparatively small, but found that dexamethasone caused a marked change in IFN-γ concentrations. The authors hypothesised that large changes in IFN-γ were due to effects of the treatment, rather than the immunomodulatory effects of these drugs.51

Factors unrelated to patients

Sources of variability unrelated to patients (eg, due to manufacturing or analytical sources) can also reduce the reliability of IGRA tests. The presence of variability means that the changes in the results may not have an immunological basis. A dichotomous cut-off value leads to greater ambiguity in the interpretation of results. Pai et al9 categorised the known sources of variability into manufacturing sources, preanalytical sources, analytical sources (random errors causing fluctuations in measurements) and immunological sources (boosting by PPD and modulation by Pathogen Associated Molecular patterns (PAMP)). Preanalytical sources represent the main component of causes of variability and are the ones most extensively researched. Gaur et al52 found that blood volume variability affected the results, and standardising the blood volume to 0.8 mL may increase assay sensitivity. They also found that shaking the nil and TB antigen tubes separately at different strengths contributed to variability in results. Multiple authors have reported that removing the dichotomous cut-off value and instead introducing a range would increase the reliability and validity of the QFT-GIT.53 54 Jonsson et al53 found that retesting of results that fell in either the borderline negative or positive ranges resulted in reversions or conversions that would otherwise have been classified as false positives or negatives, leading to possible undertreatment or overtreatment. The T-SPOT.TB uses a borderline category to indicate that results in that range should be retested. Rego and colleagues55 found that this range, rather than a cut-off value, resulted in less false positives, as most of those in the borderline range that were retested turned out to be negative.

Utility of IGRA in the diagnosis of TBU

Diagnostic test results should be interpreted carefully based on the patient’s ocular signs and symptoms, age, and comorbidities. Gupta et al56 defined this as the pretest likelihood of a patient having OTB. If the pretest likelihood is determined to be low, a positive test may not determine that the patient has TB. Ang et al57 evaluated the usefulness of IGRA in the diagnosis of TBU. They reported low sensitivity to exclude OTB. A Bayesian latent class study by Agrawal et al58 also concluded that QFT-GIT alone could not separate TBU from non-TBU. Gineys et al59 state that the cut-off value for QFT, that is, 0.35 IU/mL, is too low for the diagnosis for uveitis, and that raising this value might prevent unnecessary anti-TB treatment. In another study testing 50 patients suspected with TBU with QFT-Gold in India, Sudharshan et al60 found that the percentage of positive results was higher in patients with conditions affecting the posterior uveitis, especially serpiginous-like choroiditis. Testing in 181 patients in a hospital in Korea led to similar results, with posterior uveitis being a finding in 44.6% of patients with a positive IGRA result.61 In a retrospective cohort study conducted in Singapore, a mid-level burden country, including cases from August 2006 to February 2007, Ang and colleagues62 found that QFT-GIT was not superior to TST in sensitivity and was only slightly more specific. They suggested that since QFT-GIT was only slightly superior to TST in the diagnosis of TBU, a combination of both tests should be used.

Utility of IGRA in screening for latent TB before TNF treatment

Antitumour necrosis factor (TNF) agents are used to treat immune-mediated inflammatory disorders, including uveitis. The use of these therapies leads to immune suppression and may precipitate infections. One concern is the conversion of a latent TB infection to an active one. This concern has led many countries to make latent TB screening mandatory before the commencement of anti-TNF treatment. In contrast to what has been reported about the effects of corticosteroid use on QFT-Gold, Sargın et al63 found QFT-Gold and T-SPOT.TB to be good alternatives to TST in rhuematoid arthritis (RA) patients for latent TB detection as they were not significantly affected by prior vaccinations or by corticosteroid use. Other studies have also found QFT-GIT to be appropriate for screening latent TB.64 65 Due to poor agreement between TST and QFT, researchers in Spain recommended using both tests.66