BackgroundThe 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-γ 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 FindingsHBHA 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.ConclusionsThe 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.
Twenty-one patients treated for active tuberculosis were examined for immune reactivity to purified protein derivative (PPD) and to a purified 32-kDa protein antigen (P32) from Mycobacterium bovis, strain BCG. Lymphoproliferation of peripheral blood leucocytes to PPD and P32 was positive in 95% and 71% of the patients respectively. A positive IFN-gamma response was detected in 62% against PPD and in 48% against P32. Low blastogenesis and IFN-gamma production were observed, especially in patients with poor general health and advanced tuberculous lesions. Twelve out of twelve (100%) of the tuberculin-positive healthy volunteers responded to PPD and P32 with mean lymphoproliferation and IFN-gamma values that were higher than in the patient group. Twelve tuberculin-negative control subjects were completely unreactive to PPD and P32 antigen. On the other hand, IgG antibodies in the serum were detected in 95% of the patients against PPD, in 77% of the patients against P32 but in none of the tuberculin-positive or negative healthy volunteers. The highest IgG levels against PPD were found in those patients with the lowest in vitro lymphoproliferation and IFN-gamma production (r = -0.54; P less than 0.05). Nonspecific interferon production following induction with Newcastle disease virus, Corynebacterium parvum, or phytohaemagglutinin was comparable in the control and patient groups. Finally, low IFN-alpha titres were detected in the serum of about 50% of the patients.
Because only 10% of individuals infected with Mycobacterium tuberculosis will eventually develop disease, antigens that are recognized differently by the immune systems of infected healthy and diseased subjects may constitute potential vaccine candidates. Here, the heparin-binding hemagglutinin adhesin (HBHA) is identified as such an antigen. Lymphocytes from 60% of healthy infected individuals (n=25) produced interferon (IFN)-gamma after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n=24). In the responders, both CD4(+) and CD8(+) cells secreted HBHA-specific IFN-gamma, and the antigen was presented by both major histocompatibility complex class I and II molecules. In contrast to the reduced ability of patients with tuberculosis to produce HBHA-specific IFN-gamma, most of them (82%) produced anti-HBHA antibodies, compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and infected healthy individuals and might provide a marker for protection against tuberculosis.
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