Immune responses to ESAT-6 and CFP-10 by FASCIA and multiplex technology for diagnosis of M. tuberculosis infection; IP-10 is a promising marker

Abstract

Background: There is a need for reliable markers to diagnose active and latent tuberculosis (TB). The interferon gamma release assays (IGRAs) are compared to the tuberculin skin test (TST) more specific, but cannot discriminate between recent or remote TB infection. Here the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA), which quantifies expanded T-lymphoblasts by flow-cytometric analysis after long-term antigen stimulation of whole blood, is combined with cytokine/chemokine analysis in the supernatant by multiplex technology for diagnosis of Mycobacterium tuberculosis (Mtb) infection.

Methods and findings: Consecutive patients with suspected TB (n = 85), with microbiologically verified active pulmonary TB (n = 33), extra pulmonary TB (n = 21), clinical TB (n = 11), presumed latent TB infection (LTBI) (n = 23), patients negative for TB (n = 8) and 21 healthy controls were studied. Blood samples were analyzed with FASCIA and multiplex technology to determine and correlate proliferative responses and the value of 14 cytokines for diagnosis of Mtb infection: IFN- γ, IL-2, TNF-α, IP-10, IL-12, IL-6, IL-4, IL-5, IL-13, IL-17, MIP-1β, GM-CSF, IFN-α2 and IL-10. Cytokine levels for IFN-γ, IP-10, MIP-1β, IL-2, TNF-α, IL-6, IL-10, IL-13 and GM-CSF were significantly higher after stimulation with the Mtb specific antigens ESAT-6 and CFP-10 in patients with active TB compared to healthy controls (p<0.05) and correlated with proliferative responses. IP-10 was positive in all patients with verified TB, if using a combination of ESAT-6 and CFP-10 and was the only marker significantly more sensitive in detecting active TB then IFN-γ (p = 0.012). Cytokine responses in patients with active TB were more frequent and detected at higher levels than in patients with LTBI.

Conclusions: IP-10 seems to be an important marker for diagnosis of active and latent TB. Patients with active TB and LTBI responded with similar cytokine profiles against TB antigens but proliferative and cytokine responses were generally higher in patients with active TB.

Figure 1. Schematic outline of the analysis of proliferative responses and cytokine production by FASCIA and Luminex assay.

Figure 2. Cytokine results from patients with verified active TB, after stimulation with CFP-10 (2 A) and ESAT-6 (2 B), in relation to cell proliferation.

The data are presented as graphs with the number of lymphoblasts/μl on the X-axis and the level of cytokine production/cell (fg/cell) on the Y-axis. These graphs show lower levels of cytokines produced per cell, in strong responses, except for IFN-γ, where levels rise accordingly, although a few outliers influence ESAT-6 results for low levels of lymphoblasts/µl.

Figure 3. Heat maps: Cytokine levels in verified active TB patients divided by cut-off levels, after stimulation with CFP-10 (3 A) and ESAT-6 (3 B).

The individual values are represented as colours. Light colours indicate high levels of cytokines and vice versa. On the Y-axis: The different cytokines tested with the FASCIA. On the X-axis: The cytokine levels, divided by the cut-off level for each cytokine, logged and sorted by rising proliferative responses (the higher the proliferative response, the further to the right).