Potential role for IL-2 ELISpot in differentiating recent and remote infection in tuberculosis contact tracing

Interferon (IFN)-γ release assays (IGRA) have improved tuberculosis contact tracing, but discrimination of recent from remote Mycobacterium tuberculosis contacts is not possible by IGRA alone. We present results of a tuberculosis contact investigation with a new early-secretory-antigenic-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 specific interleukin (IL)-2 ELISpot in addition to ESAT-6 and CFP-10 specific IFN-γ ELISpot and tuberculin skin testing (TST). Results of the TST, IFN-γ ELISpot and IL-2 ELISpot were positive in 6/172 (3.4%), 7/167 (4.2%) and 6/196 (3.1%) of contacts, respectively. Close contact (≥100 hours) to the index case increased the risk of positive results in the IFN-γ ELISpot, TST, and IL-2 ELISpot by 40.8, 19.3, and 2.5 times, respectively. Individuals with a positive IFN-γ ELISpot/negative IL-2 ELISpot result had a median (IQR) duration of index case exposure of 568 hours (133_1000) compared to individuals with a positive IFN-γ ELISpot/positive IL-2 ELISpot result (median=24 hours; 20_130; p-value=0.047). Combination of a M. tuberculosis specific IFN-γ ELISpot with a M. tuberculosis specific IL-2 ELISpot significantly improved the identification of individuals with the highest risk of recent M. tuberculosis infection and is a promising method that should be explored to target tuberculosis preventive chemotherapy

TNF-α production of A) human THP-1 cells and of B) human monocyte-derived macrophages (MDM) in response to the M. tuberculosis strain of the index case (9729/07) and the laboratory strain H37Rv.

<p>PMA-differentiated human THP-1 cells and MDM were incubated with indicated multiplicities of infection (MOI) of viable <i>M. tuberculosis</i>. Supernatants were harvested 24 h after infection and TNF levels were determined by ELISA. Data from one representative experiment of three are shown (LPS: lipopolysacharide; ctrl.: medium control, n.d.: not detectable).</p

Results of <i>M. tuberculosis</i> specific enzyme linked immunospot (ELISpot), assaying interferon (IFN)- γ and interleukin (IL)-2 production in response to <i>ex vivo</i> contact of peripheral blood mononuclear cells with early antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10 in relation to the cumulative time of exposure to an infectious index case.

<p>* Differences are statistically significant.</p

Contacts with positive result in at least one of the immunodiagnostic tests.

<p>TST: Tuberculin skin test; ELISpot: enzyme-linked immunospot; BCG: Bacille Calmette-Guérin.</p

Distribution of immunodiagnostic test results.

<p>TST: tuberculin skin test; ELISpot: enzyme-linked immunospot. ^Δ In these 17 cases a skin test could not be performed. Because we did not obtain their informed consent for participation, these individuals were not tested with ELISpot in the study but tested by the city's health authorities with ELISA (<i>QuantiFERON-TB Gold</i>). $ These 2 contacts did not receive IL-2 ELISpot testing because they had only agreed on testing with test methods already approved by the health authorities. We performed IFN-γ ELISpot here. <i>§</i> In this case the amount of blood taken from the student was not sufficient to perform both ELISpot tests. This contact later received ELISA testing from the city's health authorities, in which she showed a negative result. <i>*</i> These 3 contacts or their parents did not agree on IL-2 ELISpot testing but only on test methods already approved by the health authorities. <i>^#</i> These 4 contacts showed an indeterminate IFN-γ ELISpot result. Because skin testing could not be performed, the city's health authorities then tested these 4 with ELISA, in which all of them showed a negative result.</p

Logistic regression analysis of the association of a positive interleukin (IL)-2 ELISpot assay, interferon (INF)-γ ELISpot assay and tuberculin skin test with potential explanatory factors for the presence of positive results in tests for the immunodiagnosis of tuberculosis.

<p>*OR, odds ratio; CI, confidence interval.; BCG, Bacillus Calmette-Guérin.</p