Flow chart of patients included in this study.

<p>TB = tuberculosis; LTBI = latent infection with <i>M</i>. <i>tuberculosis;</i> control = healthy individual with negative EliSpot-IGRA result; E = ESAT-6; C = CFP-10; dark grey = positive test result in the EliSpot-IGRA; light grey = negative test result in the EliSpot-IGRA.</p

Demographic characteristics of study subjects by groups.

<p>TB = tuberculosis.</p><p>LTBI = latent infection with <i>M</i>. <i>tuberculosis</i>.</p><p>m = male.</p><p>f = female.</p><p>n = number of cases.</p><p>Demographic characteristics of study subjects by groups.</p

Detection of INF-γ⁺ in EliSpot-IGRA and their concordance with INF-γ⁺ results in FluoroSpot.

<p>ESAT-6 (A) and CFP-10 (B) -induced INF-γ⁺ immune response in 200.000 PBMCs/well in participants with active tuberculosis (TB, circle, n = 18), past tuberculosis (past TB, inverted triangle, n = 10), latent infection with M. tuberculosis (LTBI, square, ESAT-6-induced n = 24, CFP-10-induced = 22) and EliSpot-negative individuals (control, triangle, ESAT-6-induced n = 17, CFP-10-induced n = 19) was analysed. LTBI and controls had been defined according to their ESAT-6 and CFP-10-induced IFN-γ EliSpot-IGRA test result and clinical data. Number of INF-γ⁺ spot-forming cells (SFC) was enumerated by EliSpot. ESAT-6 (C) and CFP-10 (D) induced- INF-γ⁺ SFC in EliSpot-IGRA (solid symbols) and FluoroSpot (open symbols) were analysed as matched pairs (connected with lines), differences were calculated using Wilcoxon signed rank test. Correlation between the number of ESAT-6 (E) and CFP-10 (F) specific INF-γ⁺ spot-forming cells (SFC) in PBMC of 69 donors detected by FluoroSpot and EliSpot-IGRA. Concordance between EliSpot-IGRA and FluoroSpot results were assessed using R² coefficient. Agreement by Bland–Altman test was expressed as mean difference (horizontal solid line) and 95% limits of agreement (dashed line) between ESAT-6 (G) and CFP-10 (H) induced- INF-γ⁺ SFC in FluoroSpot compared to EliSpot-IGRA.</p

Wild boar microsatellites genotypes

Include original genotypes for 11 microsatellites analyzed in wild boars from the Balkans, Iberia, Italy and Central-eastern Europe

ESAT-6 and CFP-10- induced cytokine response in FluoroSpot.

<p>ESAT-6 induced cytokine immune response in 200.000 PBMCs/well in participants with active tuberculosis (TB, circle, n = 18), past tuberculosis (past TB, inverted triangle, n = 10), latent infection with <i>M</i>. <i>tuberculosis</i> (LTBI, square, ESAT-6-induced n = 24, CFP-10-induced n = 22), EliSpot-negative individuals (control, triangle, ESAT-6-induced n = 17, CFP-10-induced n = 19) was analysed. Groups had been defined according to the combination of their ESAT-6 and CFP-10 induced IFN-γ EliSpot-IGRA test result and clinical data. The number of IL-2⁺ (A), INF-γ⁺ (B), IL-2⁺ INF-γ^- (C), IL-2^- INF-γ⁺ (D) and IL-2⁺ INF-γ⁺ (E) spot-forming cells (SFC) were enumerated by FluoroSpot. (F) Mean proportion of ESAT-6 (top row) and CFP-10 (bottom row) -specific cytokine secreting cells for individuals with tuberculosis, past tuberculosis and LTBI are depicted as pie charts (light grey = IL-2⁺ INF-γ^-, black = IL-2^- INF-γ⁺ and dark grey = IL-2⁺ INF-γ⁺ secreting cells). Mann-Whitney U-test for non-parametric data was used for comparative analysis. A p-value of <0.05 was considered significant.</p

BAL fluid donors and their IGRA status in either peripheral blood or in BAL cells.

<p>BAL fluid donors and their IGRA status in either peripheral blood or in BAL cells.</p

Cytokine induction in BAL cell IGRA negative and positive subjects.

<p>Induction of cytokines by activated lymphocytes and macrophages upon antigen stimulation (triangles) or infection (circles), stratified by BAL IGRA status (open symbols, BAL IGRA-negative donors; closed symbols, BAL IGRA-positive donors). The plots show the effect estimates with lines representing 95% confidence intervals (solid line, antigen stimulations; broken line, <i>M</i>. <i>tuberculosis</i> infection assays). Related p-values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187882#pone.0187882.s008" target="_blank">S3 Table</a>. ESAT-6, 6kD Early Secretory Antigenic Target; CFP-10, 10kD Culture Filtrate Protein; PPD, Purified Protein Derivative; LPS, Lipopolysaccharide; PHA, Phytohaemagglutinin; H37Rv, <i>M</i>. <i>tuberculosis</i> H37Rv; Isol2, <i>M</i>. <i>tuberculosis</i> isolate 2; Isol3, <i>M</i>. <i>tuberculosis</i> isolate 3.</p

Cellular proportions in the BAL stratified by IGRA.

<p>Proportion of the three major cell types in the BAL fluid from study participants, stratified by A) blood and B) BAL IGRA status. Notched boxplots showing five number summaries (minimum, first quartile, median, third quartile, maximum) with outliers. The lower tables indicate the number of donors in each group for which cell counts for the respective cell type were available.</p

Proinflammatory cytokine induction in blood IGRA negative vs. positive subjects.

<p>Induction of IFN-γ and IL-2 by mycobacterial antigens (triangles) and mycobacterial infection (circles), stratified by blood IGRA status (open symbols, IGRA-negative donors; closed symbols, IGRA-positive donors). Plots show the effect estimates with lines (solid line, antigen stimulations; broken line, <i>M</i>. <i>tuberculosis</i> infection assays) representing 95% confidence intervals. ESAT-6, 6kD Early Secretory Antigenic Target; CFP-10, 10kD Culture Filtrate Protein; PPD, Purified Protein Derivative; LPS, Lipopolysaccharide; PHA, Phytohaemagglutinin; H37Rv, <i>M</i>. <i>tuberculosis</i> H37Rv; Isol2, <i>M</i>. <i>tuberculosis</i> isolate 2; Isol3, <i>M</i>. <i>tuberculosis</i> isolate 3.</p

Basal cytokine production in BAL cells.

<p>Basal levels of cytokine secretion in unstimulated and uninfected BAL cells stratified by BAL IGRA results. Boxplots showing five-number summaries (minimum, first quartile, median, third quartile, maximum).</p