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

Hougardy JM, Schepers K, Place S, Drowart A, Lechevin V, Verscheure V, Debrie AS, Doherty TM, Van Vooren JP, Locht C, Mascart F - PLoS ONE (2007)

Bottom Line: 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.HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Vaccinology and Mucosal Immunity, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium.

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.

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Related in: MedlinePlus

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.
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pone-0000926-g001: 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.

Mentions: Since the LTBI subjects were defined on the basis of a positive TST reaction, we first evaluated the in vitro PPD-induced IFN-γ secretion by PBMC from LTBI subjects and TB patients compared to that from CTRL. As shown in Fig. 1A, LTBI subjects and TB patients secreted significantly more IFN-γ in response to PPD than CTRL (medians 15.83 ng/ml and 6.84 ng/ml, respectively, versus 0.23 ng/ml; P<0.001), whereas the median level of IFN-γ produced was not significantly different between the LTBI and TB groups. To evaluate the sensitivity and specificity of the in vitro PPD-IFN-γ test for the discrimination of LTBI from CTRL, a ROC curve was established and the AUC determined. As shown in Fig. 1B, the AUC was 0.89 (95% IC : 0.83–0.95), indicating both a good global accuracy of the PPD-induced IFN-γ production for discriminating LTBI from CTRL, and good agreement between a positive TST reaction and elevated PPD-specific IFN-γ secretion. A similar analysis comparing the TB patients with the CTRL gave an AUC of 0.80 (95% IC: 0.73–0.87). Using the ROC analysis, a cut-off IFN-γ concentration of 0.8 ng/ml was determined as being the most discriminatory between LTBI and TB versus the CTRL.


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

Hougardy JM, Schepers K, Place S, Drowart A, Lechevin V, Verscheure V, Debrie AS, Doherty TM, Van Vooren JP, Locht C, Mascart F - PLoS ONE (2007)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1991599&req=5

pone-0000926-g001: 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.
Mentions: Since the LTBI subjects were defined on the basis of a positive TST reaction, we first evaluated the in vitro PPD-induced IFN-γ secretion by PBMC from LTBI subjects and TB patients compared to that from CTRL. As shown in Fig. 1A, LTBI subjects and TB patients secreted significantly more IFN-γ in response to PPD than CTRL (medians 15.83 ng/ml and 6.84 ng/ml, respectively, versus 0.23 ng/ml; P<0.001), whereas the median level of IFN-γ produced was not significantly different between the LTBI and TB groups. To evaluate the sensitivity and specificity of the in vitro PPD-IFN-γ test for the discrimination of LTBI from CTRL, a ROC curve was established and the AUC determined. As shown in Fig. 1B, the AUC was 0.89 (95% IC : 0.83–0.95), indicating both a good global accuracy of the PPD-induced IFN-γ production for discriminating LTBI from CTRL, and good agreement between a positive TST reaction and elevated PPD-specific IFN-γ secretion. A similar analysis comparing the TB patients with the CTRL gave an AUC of 0.80 (95% IC: 0.73–0.87). Using the ROC analysis, a cut-off IFN-γ concentration of 0.8 ng/ml was determined as being the most discriminatory between LTBI and TB versus the CTRL.

Bottom Line: 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.HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Vaccinology and Mucosal Immunity, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium.

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.

Show MeSH
Related in: MedlinePlus