Latent Tuberculosis Infection Subjects Research Articles

A proteomics approach to the identification of plasma biomarkers for latent tuberculosis infection

A proteomic analysis was performed to screen the potential latent tuberculosis infection (LTBI) biomarkers. A training set of spectra was used to generate diagnostic models, and a blind testing set was used to determine the accuracy of the models. Candidate peptides were identified using nano-liquid chromatography-electrospray ionization–tandem mass spectrometry. Based on the training set results, 3 diagnostic models recognized LTBI subjects with good cross-validation accuracy. In the blind testing set, LTBI subjects could be identified with sensitivities and specificities of 85.20% to 88.90% and 85.7% to 100%, respectively. Additionally, 14 potential LTBI biomarkers were identified, and all proteins were identified for the first time through proteomics in the plasma of healthy, latently infected individuals. In all, proteomic pattern analyses can increase the accuracy of LTBI diagnosis, and the data presented here provide novel insights into potential mechanisms involved in LTBI.

Molecular characterization of T cell receptor beta variable in the peripheral blood T cell repertoire in subjects with active tuberculosis or latent tuberculosis infection

BackgroundT cells are closely linked to the clinical manifestations of subjects with Mycobacterium tuberculosis (MTB) infection. T cell receptor beta variable (TCRBV) is a signal and indicative molecule on the membrane of T lymphocytes, reflecting the composition and specificity of T cells. The molecular profiles of TCRBV in peripheral blood mononuclear cells (PBMCs) and their subpopulations (CD4+ and CD8+ T cells) from subjects with active tuberculosis (TB) or latent TB infection (LTBI) have not been well described.MethodsIn 42 subjects with active TB or LTBI, PMBCs and their subsets were separated and sorted. The molecular profiles of the TCRBV complementarity determining region 3 (CDR3) in the three cell populations were investigated using our recently developed gene melting spectral pattern (GMSP) assay. The TCRBV members were then cloned and sequenced when their GMSP image profiles showed a single-peak.ResultsThe average number of skewed TCRBV molecules in the CD4+ cell subset was significantly higher than that in PBMCs and CD8+ T cells. TCRBV12, BV13.1, BV13.2, and BV24 were expressed more prevalently than other TCRBV gene families in the three cell populations. In addition, relatively conserved amino acid motifs were identified in TCRBV5.1 and BV20 CDR3 in PBMCs and its subsets. The monoclonal TCRBV14 and BV23 expressed were different between active TB and LTBI subjects.ConclusionsThese results indicate that the T cell immune response is complex and multi-specific in active TB and LTBI subjects. Analysis of TCRBV expression in CD4+ T cells suggest that it could be useful in assessing the composition and status of circulating T cells. Furthermore, the expression of TCRBV14, BV23 and the sequencing of CDR3 amino acid motifs of TCRBV5.1, BV20 could be used in the differential diagnosis and treatment of subjects with active TB or LTBI.

Efficacy of inducible protein 10 as a biomarker for the diagnosis of tuberculosis

This study evaluated inducible protein 10 (IP-10) as a diagnostic biomarker for specific tuberculosis (TB) infection and evaluated the ability of IP-10 to distinguish between active TB and latent TB infection (LTBI). Forty-six patients with active pulmonary TB, 22 participants with LTBI, and 32 non-TB controls were enrolled separately. We measured IP-10 in serum and in supernatants from whole blood stimulated with TB-specific antigens. TB antigen-dependent IP-10 secretion was significantly increased in the active TB patients and LTBI subjects compared with controls, but did not differ significantly between the active TB patients and LTBI subjects. Serum IP-10 levels were higher in active TB than in LTBI (174.9 vs. 102.7pg/ml, p=0.002). The respective rates of positive responders of TB antigen-dependent IP-10 were 97.8%, 90.9%, and 12.5% in active TB, LTBI, and non-TB controls, respectively. For serum IP-10, 87.5%, 45.5%, and 9.5% of responders were positive in the respective groups. The IP-10 response to TB antigen may constitute a specific biomarker for TB infection, but does not by itself distinguish between active TB and LTBI. Serum IP-10 may enhance the diagnostic performance when used in combination with another marker.

Risk Stratification of Latent Tuberculosis Defined by Combined Interferon Gamma Release Assays

BackgroundMost individuals infected with Mycobacterium tuberculosis develop latent tuberculosis infection (LTBI). Some may progress to active disease and would benefit from preventive treatment yet no means currently exists to predict who will reactivate. Here, we provide an approach to stratify LTBI based on IFN-γ responses to two antigens, the recombinant Early-Secreted Antigen Target-6 (rESAT-6) and the latency antigen Heparin-Binding Haemagglutinin (HBHA).MethodsWe retrospectively analyzed results from in-house IFN-γ-release assays with HBHA (HBHA-IGRA) and rESAT-6 (rESAT-6-IGRA) performed during a 12-year period on serial blood samples (3 to 9) collected from 23 LTBI subjects in a low-TB incidence country. Both the kinetics of the absolute IFN-γ concentrations secreted in response to each antigen and the dynamics of HBHA/rESAT-6-induced IFN-γ concentrations ratios were examined.ResultsThis analysis allowed the identification among the LTBI subjects of three major groups. Group A featured stable HBHA and rESAT-6-IGRA profiles with an HBHA/rESAT-6 ratio persistently higher than 1, and with high HBHA- and usually negative rESAT-6-IGRA responses throughout the study. Group B had changing HBHA/rESAT-6 ratios fluctuating from 0.0001 to 10,000, with both HBHA and rESAT-6 responses varying over time at least once during the follow-up. Group C was characterized by a progressive disappearance of all responses.ConclusionsBy combining the measures of IFN-γ concentrations secreted in response to an early and a latency antigens, LTBI subjects can be stratified into different risk groups. We propose that disappearing responses indicate cure, that persistent responses to HBHA with HBHA/rESAT-6 ratios ≥1 represent stable LTBI subjects, whereas subjects with ratios varying from >1 to <1 should be closely monitored as they may represent the highest-risk group, as illustrated by a case report, and should therefore be prioritized for preventive treatment.

Dysfunction of Natural Killer T Cells in Patients with Active Mycobacterium tuberculosis Infection

Natural killer T (NKT) cells are known to play a protective role in the immune responses of mice against a variety of infectious pathogens. However, little is known about the detailed information of NKT cells in patients with Mycobacterium tuberculosis infection. The aims of this study were to examine NKT cell levels and functions in patients with active M. tuberculosis infection, to investigate relationships between NKT cell levels and clinical parameters, and to determine the mechanism responsible for the poor response to α-galactosylceramide (α-GalCer). NKT cell levels were significantly lower in the peripheral blood of pulmonary tuberculosis and extrapulmonary tuberculosis patients, and the proliferative responses of NKT cells to α-GalCer were also lower in patients, whereas NKT cell levels and responses were comparable in latent tuberculosis infection subjects and healthy controls. Furthermore, this NKT cell deficiency was found to be correlated with serum C-reactive protein levels. In addition, the poor response to α-GalCer in M. tuberculosis-infected patients was found to be due to increased NKT cell apoptosis, reduced CD1d expression, and a defect in NKT cells. Notably, M. tuberculosis infection was associated with an elevated expression of the inhibitory programmed death-1 (PD-1) receptor on NKT cells, and blockade of PD-1 signaling enhanced the response to α-GalCer. This study shows that NKT cell levels and functions are reduced in M. tuberculosis-infected patients and these deficiencies were found to reflect the presence of active tuberculosis.

Detection of immune cell response to M. tuberculosis–specific antigens by quantitative polymerase chain reaction

One third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb) and up to 10% of infected individuals develop active tuberculosis (TB) in their lifetime. Among the major challenges in the control of TB is the implementation of sensitive methods for detection of latent tuberculosis infection (LTBI). Currently, in vitro interferon gamma release assays, yielding single value readout, are used as an alternative to the traditional tuberculin skin test for the diagnosis of LTBI. More complex characterization of immune status of LTBI individuals, however, is desirable for indication of LTBI subjects for preventative chemotherapy. Here we describe a quantitative polymerase chain reaction (qPCR) for determination of expression levels of 14 genes, additional to interferon gamma, which was applied for comparison of the specific Mtb-antigen immune response of blood cells from healthy, latently infected, and TB individuals. With the use of principal component analysis and discriminant analysis, a pattern of mRNA levels of 6 genes was identified, allowing discrimination of healthy individuals from active TB and LTBI subjects. These results open the way to development of multimarker qPCR for the detection of LTBI.

Heparin-Binding-Hemagglutinin-Induced IFN-γ Release as a Diagnostic Tool for Latent Tuberculosis

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.

T-cell and serological responses to Erp, an exported Mycobacterium tuberculosisprotein, in tuberculosis patients and healthy individuals

BackgroundThe identification of antigens able to differentiate tuberculosis (TB) disease from TB infection would be valuable. Cellular and humoral immune responses to Erp (Exported repetitive protein) – a recently identified M. tuberculosis protein – have not yet been investigated in humans and may contribute to this aim.MethodsWe analyzed the cellular and humoral immune responses to Erp, ESAT-6, Ag85B and PPD in TB patients, in BCG+ individuals without infection, BCG+ individuals with latent TB infection (LTBI) and BCG- controls. We used lymphoproliferation, ELISpot IFN-γ, cytokine production assays and detection of specific human antibodies against recombinant M. tuberculosis proteins.ResultsWe included 22 TB patients, 9 BCG+ individuals without TB infection, 7 LTBI and 7 BCG- controls. Erp-specific T cell counts were higher in LTBI than in the other groups. Erp-specific T cell counts were higher in LTBI subjects than TB patients (median positive frequency of 211 SFC/106 PBMC (range 118–2000) for LTBI subjects compared to 80 SFC/106 PBMC (range 50–191), p = 0.019); responses to PPD and ESAT-6 antigens did not differ between these groups. IFN-γ secretion after Erp stimulation differed between TB patients and LTBI subjects (p = 0.02). Moreover, LTBI subjects but not TB patients or healthy subjects produced IgG3 against Erp.ConclusionThe frequencies of IFN-γ-producing specific T cells, the IFN-γ secretion and the production of IgG3 after Erp stimulation are higher in LTBI subjects than in TB patients, whereas PPD and ESAT-6 are not.