July - September 2007: 
Volume 20, Issue 3

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Novel tests for diagnosing latent TB infection. IFN-γ release assays
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Targeted tuberculin skin testing (TST) for latent tuberculosis infection (LTBI) is an essential component of public health efforts to eliminate tuberculosis (TB). A relevant meta-analysis showed that the identification and treatment of persons infected by M. tuberculosis diminishes the risk of subsequent development of active TB by about 90%1. However, this strategy is limited by the poor diagnostic accuracy of TST.

TST may give a false-negative result in the presence of immunosuppression, often due to HIV infection, or other high risk factors for progression of latent to active TB. The poor sensitivity of TST in these cases has a negative impact on the management of those individuals who would benefit the most for targeted testing and preventive therapy. TST uses tuberculin purified protein derivative (PPD), which is a crude mixture of more than 200 M. tuberculosis proteins, which are also present in all Bacille Calmette Guèrin (BCG) vaccine strains, and in other non-tuberculous mycobacteria (NTM).

As a result, false-positive reactions can occur because of previous BCG vaccination or sensitization to NTM. BCG is the most widely used vaccine in the world. In a recent review the duration of the effect on TST from BCG vaccination was estimated at around 15 years2,3. In addition, NTM are still endemic in several areas of the world. The low specificity of TST due to these two factors could lead to unnecessary long-term preventive therapy of persons without M. tuberculosis infection.

In order to overcome these problems, extensive research concerning new diagnostic tools for LTBI has been conducted. In the past decade, two T-cell based tests for LTBI, dependent on the release of interferon-γ (IFN- γ) and known as IFN-γ release assays (IGRAs), have been developed and licensed for commercial distribution in many countries. One of these tests, the QuantiFERON (QFT)-TB Gold (Cellestis, Australia), uses enzyme-linked immunosorbent assay to measure antigen-specific production of IFN-γ by circulating T-cells in whole blood. The other test, the T-SPOT.TB (Oxford Immunotec, United Kingdom), uses the Elispot technique to measure peripheral blood mononuclear cells that produce IFN-γ. The readout of the two tests is different. QFT-ΤΒ Gold measures the level of IFN-γ in the supernatant of the stimulated whole blood sample. T-SPOT.TB enumerates individual Tcells producing IFN-γ after stimulation. T-SPOT.TB should be more sensitive, because it detects IFN-γ in the immediate vicinity of the T-cell from which it is released, and this superiority should be more marked in cases with lownumbers or dysfunction of T cells.

Current versions of both IGRAs use more specific M. tuberculosis antigens such as Early Secreted Antigen Target- 6 (ESAT-6), and Culture Filtrate Protein-10 CFP-10). The genes encoding these antigens are found in the region of difference 1 (RD1) of the M. tuberculosis genome, which is absent from the genome of all BCG vaccine strains and from commonly encountered NTM, with the exception of M. kansasii, M. szulgai, and M. marinum4.

IGRAs have an internal positive control that is due to T-cell stimulation with a potent non-specific stimulator. This controls the results of IGRAs for technical errors, including failure to add viable functioning cells to the well. Furthermore, the failure of the positive control signifies that the test result cannot be reliably interpreted (indeterminate result) and may reflect underlying in vivo immunosuppression4,5.

Because there is no gold standard for testing LTBI, sensitivity has been estimated from studies of: 1) patients with active TB, and 2) persons in contact with patients with active TB, categorized into gradients of exposure. Persons with active TB are not suffering from LTBI but are definitely infected by M. tuberculosis, and the results obtained could be extrapolated to LTBI. Thus in a recent meta-analysis of TST and IGRAs studies in patients with active tuberculosis, pooled average sensitivity of TST was 71% (65% -74%), of QFT-ΤΒ Gold 76% (7Τ - 83%), and T-.SPOT. TB 88% (81% - 95%). In paediatric populations the pooled estimates of sensitivity of all tests were much lower.6 Thus, IGRAs appear to be of value in diagnosing active TB, but they should not replace appropriate bacteriological/molecular confirmation. They would be expected to have the greatest potential benefit in excluding TB diagnosis or in difficult to diagnose TB cases, such as extra-pulmonary TB and especially TB meningitis.

In meta-analysis of studies of persons in contact with active TB, the pooled sensitivity and diagnostic accuracy of IGRAs and TST were similar (ROR 1.4, 95% CI: 0.66-2.34). This might be due to the heterogeneity of the studies, which came from both high and low prevalence countries4,6. Compared to TST, IGRAs were more strongly associated with exposure in low TB prevalence countries (ROR 2.07; 95% CI: 95 - 4.53), but less closely associated with exposure in a high prevalence country (ROR 0.66; 95% CI: 0.47 - 0.92). Furthermore, in low TB prevalence countries IGRAs were found to be more closely associated than TST with the duration and the degree of exposure. These results are not easily explained6,7.

Specificity of IGRAs was estimated in BCG vaccinated individuals who were at low risk of LTBI in case control studies, which assumed that the low-risk control subjects did not have LTBI. In four such studies on a total of 127 persons, the specificity of T-.SPOT.TB was 100%. In two other studies on a total of 215 persons, the specificity of QFT-ΤΒ Gold was 96-98%, while the specificity of TST was 36-51%5. In studies of BCG-vaccinated exposed contacts, the specificity of IGRAs was unaffected. IGRAs showed a less strong association with BCG vaccination than TST (ROR 0.23, 95% CI: 0.05-1.12)4-7. According to these results, IGRAs are more specific than TST for the diagnosis of LTBI in BCG-vaccinated populations. Studies for the estimation of the specificity of IGRAs in persons sensitized to NTM are lacking.

Studies concerning the performance of IGRAs in immunosuppression are scarce. In a study of 39 HIV-coinfected patients with TB, the sensitivity of T-SPOT.TB was 92% and of TST 50%8. In 293 children from Africa, the respective sensitivity to these tests was 83% and 63%, respectively. In children aged younger than 3 years with malnutrition and HIV co-infection the sensitivities for T-SPOT.TB were 85%, 78%, 73%, respectively and for TST 51%, 44% και 36%, respectively9. In a study on 590 HIV+ individuals the QFT-ΤΒ Gold in Tube (IT) was positive in 27 (4.6%), indicating the presence of LTBI. Among QFT-IT positive patients, 78% had risk factors such as long-term residency in an area highly endemic for TB, known TB exposure or previous TB disease10.

Studies assessing the efficacy of IGRAs in infants, children and teenagers are rare. In infants and older children T-SPOT.TB results correlated better than TST results with TB exposure. In older teenagers without BCG vaccination, QFT-ΤΒ Gold performed similarly TST, and in younger children less well than TST7.

The reliability of IGRAs in immunosuppressed individuals and at extreme ages is varied. Indeterminate results on QFT-ΤΒ Gold testing range from 12% to 21% and are associated with young age (<5 years), old age (>80 years), and immunosuppression, mainly due to HIV infection with low CD4 counts. In contrast, indeterminate results with T.SPOT.TB in the presence of the same factors are rare7.

The findings presented support the application of IGRAs in routine clinical practice. Current national guidelines recommend the use of IGRAs for the diagnosis of LTBI and as an adjunct in the evaluation of patients with suspected TB, but national guidelines vary, according to several considerations. The current European guidelines recommend the use of IGRAs for: 1) individuals at risk of LTBI who have tested positive by TST, and 2) individuals for whom the TST is unreliable (i.e., those with immune suppression) In contrast, the current USA guidelines suggest the use of IGRAs for all groups, as a direct replacement for the TST12.

Both sets of guidelines have limitations, mainly due to the absence of a gold standard for asymptomatic M tuberculosis infection. In Europe, if the guidelines were followed, TST-negative, IGRAs-positive individuals would only be identified among immunosuppressed populations. However, an unknown number of immunocompetent individuals with negative TST results, who might have tested positive by IGRAs would not receive treatment. In the USA, persons with positive IGRAs only would be treated for LTBI, but it is not known if such persons are truly infected, whether they have a significant risk of progression to active TB and thus, whether they stand to benefit from treatment. Another reason for the differing recommendations between USA and Europe lies in considerations of health economics12.

The conclusions from the studies published to date are that IGRAs are more specific than TST, because they are not confounded by prior BCG or sensitization to NTM. In immunocompetent individuals the performance of both IGRAs is similar, but T-SPOT.TB is probably more sensitive for the diagnosis of LTBI in immunosuppressed patients. Indeterminate results are common with QTF-GOLD and when present are strongly associated with immunosuppression and very young and very old age, while indeterminate results with T-SPOT.TB are rare in all risk groups studied to date. IGRAs could have great potential benefit in excluding the diagnosis of TB or in TB cases that are difficult to diagnose, such as extra-pulmonary TB and especially TB meningitis.


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