Host markers in QuantiFERON supernatants differentiate active TB from latent TB infection: preliminary report

Abstract

Background: Interferon gamma release assays, including the QuantiFERON TB Gold In Tube (QFT) have been shown to be accurate in diagnosing Mycobacterium tuberculosis infection. These assays however, do not discriminate between latent TB infection (LTBI) and active TB disease.

Methods: We recruited twenty-three pulmonary TB patients and 34 household contacts from Cape Town, South Africa and performed the QFT test. To investigate the ability of new host markers to differentiate between LTBI and active TB, levels of 29 biomarkers in QFT supernatants were evaluated using a Luminex multiplex cytokine assay.

Results: Eight out of 29 biomarkers distinguished active TB from LTBI in a pilot study. Baseline levels of epidermal growth factor (EGF) soluble CD40 ligand (sCD40L), antigen stimulated levels of EGF, and the background corrected antigen stimulated levels of EGF and macrophage inflammatory protein (MIP)-1beta were the most informative single markers for differentiation between TB disease and LTBI, with AUCs of 0.88, 0.84, 0.87, 0.90 and 0.79 respectively. The combination of EGF and MIP-1beta predicted 96% of active TB cases and 92% of LTBIs. Combinations between EGF, sCD40L, VEGF, TGF-alpha and IL-1alpha also showed potential to differentiate between TB infection states. EGF, VEGF, TGF-alpha and sCD40L levels were higher in TB patients.

Conclusion: These preliminary data suggest that active TB may be accurately differentiated from LTBI utilizing adaptations of the commercial QFT test that includes measurement of EGF, sCD40L, MIP-1beta, VEGF, TGF-alpha or IL-1alpha in supernatants from QFT assays. This approach holds promise for development as a rapid diagnostic test for active TB.

Figure 1

Receiver operator characteristics curves showing the accuracies of top individual analytes in discriminating between active TB and latent TB infection. Receiver operator characteristic (ROC) curves for the accuracies of single analytes to differentiate between active TB and LTBI in QFT positive individuals. Only ROC curves for markers that differentiated between the two infection states with AUCs ≥ 0.73 are shown. AUC = Area under the curve.

Figure 2

Frequency of individual analytes in top models for discriminating between active TB and latent TB. The columns represent the number of inclusions of individual markers into the most accurate three-analyte models by general discriminant and support vector machine analysis (6 and 10 models, respectively) for discriminating between active pulmonary TB cases and LTBI in participants with positive QFT results.

Figure 3

Receiver operator characteristics curves showing the accuracies of top individual analytes in discriminating between TB disease and the absence of active TB irrespective of QFT results. Only ROC curves for markers that differentiated between groups with AUCs ≥ 0.73 are shown. AUC = Area under the curve.

Figure 4

Levels of individual analytes in all TB cases (TB) and household contacts (HHC). Each dot represents the analyte level of one participant in the study and horizontal lines represent the median values. Asterixes indicate significant differences between the TB cases (n = 23) and household contacts (n = 34). ##: p < 0.0001, #: p < 0.01, ¶¶: p = 0.01, ¶: p = 0.02. Nil: unstimulated analyte levels, Ag: Levels obtained after stimulation with Mycobacterium tuberculosis specific antigen cocktail (ESAT-6, CFP-10 and TB7.7), Ag-Nil: difference between the Mycobacterium tuberculosis specific antigen stimulated and the unstimulated levels.

Figure 5

Frequency of individual analytes in models for discriminating between active TB and no active TB. The columns represent the number of inclusions of individual markers into the most accurate three-analyte models by general discriminant and support vector machine analysis (6 and 10 models, respectively) in discriminating between active pulmonary TB cases and participants without active TB irrespective of QFT results.