Heparin-binding-hemagglutinin-induced IFN-gamma release as a diagnostic tool for latent tuberculosis

Abstract

Background: The detection of latent tuberculosis infection (LTBI) is a major component of tuberculosis (TB) control strategies. In addition to the tuberculosis skin test (TST), novel blood tests, based on in vitro release of IFN-gamma in response to Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 (IGRAs), are used for TB diagnosis. However, neither IGRAs nor the TST can separate acute TB from LTBI, and there is concern that responses in IGRAs may decline with time after infection. We have therefore evaluated the potential of the novel antigen heparin-binding hemagglutinin (HBHA) for in vitro detection of LTBI.

Methodology and principal findings: HBHA was compared to purified protein derivative (PPD) and ESAT-6 in IGRAs on lymphocytes drawn from 205 individuals living in Belgium, a country with low TB prevalence, where BCG vaccination is not routinely used. Among these subjects, 89 had active TB, 65 had LTBI, based on well-standardized TST reactions and 51 were negative controls. HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI. PPD-based tests yielded 90.00% sensitivity and 70.00% specificity for the detection of LTBI, whereas the sensitivity and specificity for the ESAT-6-based tests were 40.74% and 90.91%, and those for the HBHA-based tests were 92.06% and 93.88%, respectively. The QuantiFERON-TB Gold In-Tube (QFT-IT) test applied on 20 LTBI subjects yielded 50% sensitivity. The HBHA IGRA was not influenced by prior BCG vaccination, and, in contrast to the QFT-IT test, remote (>2 years) infections were detected as well as recent (<2 years) infections by the HBHA-specific test.

Conclusions: The use of ESAT-6- and CFP-10-based IGRAs may underestimate the incidence of LTBI, whereas the use of HBHA may combine the operational advantages of IGRAs with high sensitivity and specificity for latent infection.

Figure 1. PPD, ESAT-6, and HBHA-induced IFN-γ concentrations and ROC analysis of the results.

Results of the IFN-γ ELISA obtained for three different groups of subjects: non-infected controls (CTRL), subjects with latent M. tuberculosis infection (LTBI) and patients with active tuberculosis (TB) are represented on the left part of the figure. PBMC were in vitro stimulated with different antigens, PPD (A), ESAT-6 (C) and HBHA (E) and the IFN-γ concentrations were measured in 96 h cell culture supernatants. The horizontal dotted lines represent the cut-off for positive values as determined by ROC curves analysis, whereas the horizontal filled lines represent the medians of the results. ROC curves established for each antigen are represented on the right panels (PPD, B; ESAT-6, D; HBHA, F). The filled lines represent the curve established for LTBI compared to CTRL, whereas the dotted lines are those established for TB compared to CTRL. *, P<0.05; ***, P<0.001.

Figure 2. Comparison between the HBHA-induced IFN-γ concentrations in recently and in remote M. tuberculosis infected LTBI subjects.

Blood samples were collected from LTBI subjects during the first two years (recent) or after two years (remote) of their TST conversion. Their PBMC were in vitro stimulated with HBHA, and the IFN-γ concentrations were measured in 96 h cell culture supernatants. No significant difference in the IFN-γ concentrations measured in the two groups was noted. The horizontal dotted line indicates the positivity cut-off of the test, and the horizontal filled lines indicate the medians.

Figure 3. Correlations between the IFN-γ concentrations induced by different mycobacterial antigens.

Results obtained for LTBI are represented on the left panels (A, C), and those obtained for TB are on the right panels (B, D). The degree of correlation between the in vitro-induced IFN-γ concentrations by the different mycobacterial antigens is represented for PPD and HBHA (A and B) and for ESAT-6 and HBHA (C and D). The dotted lines indicate the positivity cut-off for each test.