Adjunctive biomarkers for improving diagnosis of tuberculosis and monitoring therapeutic effects

SummaryObjectivesTo identify host biomarkers associated with latent tuberculosis infection (LTBI), active tuberculosis (TB), and nontuberculous mycobacteria (NTM) diseases to improve diagnosis and effective anti-TB treatment.MethodsActive TB and NTM patients at diagnosis, recent TB contacts, and normal healthy subjects were recruited. Tuberculin skin tests, QuantiFERON-TB Gold In-Tube tests, and multiplex bead arrays with 17 analytes were performed. TB patients were re-evaluated after 2 and 6 months of treatment.ResultsMycobacterium tuberculosis (M. tb) antigen-specific IFN-γ, IL-2, and CXCL10 responses were significantly higher in active TB and LTBI compared with controls (P < 0.01). Only serum VEGF levels varied between the active TB and LTBI groups (AUC = 0.7576, P < 0.001). Active TB and NTM diseases were differentiated by serum IL-2, IL-9, IL-13, IL-17, TNF-α and sCD40L levels (P < 0.05). Increased sCD40L and decreased M. tb antigen-specific IFN-γ levels correlated with sputum clearance of M. tb after 2 months of treatment (P < 0.001).ConclusionsSerum IL-2, IL-9, IL-13, IL-17, TNF-α, sCD40L and VEGF-A levels may be adjunctive biomarkers for differential diagnosis of active TB, LTBI, and NTM disease. Assessment of serum sCD40L and M. tb antigen-specific IFN-γ, TNF-α, and IL-2 levels could help predict successful anti-TB treatment in conjunction with M. tb clearance

Screening for Mycobacterium tuberculosis Infection Using Beijing/K Strain-Specific Peptides in a School Outbreak Cohort.

Background: The Beijing strain of Mycobacterium tuberculosis (M. tb) has been most frequently isolated from TB patients in South Korea, and the hyper-virulent Beijing/K genotype is associated with TB outbreaks. To examine the diagnostic potential of Beijing/K-specific peptides, we performed IFN-γ release assays (IGRA) using a MTBK antigen tube containing Beijing/K MTBK_24800, ESAT-6, and CFP-10 peptides in a cohort studied during a school TB outbreak. Methods: A total of 758 contacts were investigated for M. tb infection, and 43 contacts with latent TB infection (LTBI) and 25 active TB patients were enrolled based on serial screening with QuantiFERON-TB Gold In-Tube tests followed by clinical examinations. Blood collected in MTBK antigen tubes was utilized for IGRA and multiplex cytokine bead arrays. Immune responses were retested in 24 patients after TB treatment, and disease progression was investigated in subjects with LTBI. Results: Total proportions of active disease and LTBI during the outbreak were 3.7% (28/758) and 9.2% (70/758), respectively. All clinical isolates had a Beijing/K M. tb genotype. IFN-γ responses to the MTBK antigen identified M. tb infection and distinguished between active disease and LTBI. After anti-TB treatment, IFN-γ responses to the MTBK antigen were significantly reduced, and strong TNF-α responses at diagnosis were dramatically decreased. Conclusions: MTBK antigen-specific IFN-γ has diagnostic potential for differentiating M. tb infection from healthy controls, and between active TB and LTBI as well. In addition, TNF-α is a promising marker for monitoring therapeutic responses. These data provide informative readouts for TB diagnostics and vaccine studies in regions where the Beijing/K strain is endemic

Diagnostic Potential of a PPE Protein Derived from Mycobacterium tuberculosis Beijing/K Strain.

PURPOSE: The prevalence of Mycobacterium tuberculosis (M. tb) and the status of M. bovis BCG vaccination may affect host immune responses to M. tb antigens. Understanding of the predominant local M. tb strain and immune signatures induced by its strain-specific antigens may contribute to an improved diagnosis of tuberculosis (TB). The aim of this study was to determine immune responses to M. tb antigen which was identified from the hyper-virulent Beijing/K strain in South Korea. MATERIALS AND METHODS: Pulmonary TB patients (n=52) and healthy subjects (n=92) including individuals with latent TB infection (n=31) were recruited, and QuantiFERON-TB Gold In-Tube tests were performed. The Beijing/K-antigen specific immune signatures were examined by diluted whole blood assays and multiplex bead arrays in a setting where nationwide BCG vaccination is employed. RESULTS: Statistical analyses demonstrated that three [C-X-C motif chemokine (CXCL10), interleukin (IL)-6, interferon (IFN)-?] of 17 cytokines/chemokines distinguished active cases from healthy controls following stimulation with the Beijing/K-specific antigen. IFN-? also differentiated between active diseases and latent TB infection (p<0.01), and the detection rate of TB was dramatically increased in combination with IL-6 and CXCL10 at the highest levels of specificity (95-100%). CONCLUSION: Our data indicate that immune signatures to the M. tb Beijing/K-specific antigen can provide useful information for improved TB diagnostics. The antigen may be developed as a diagnostic marker or a vaccine candidate, particularly in regions where the M. tb Beijing/K strain is endemic

A Feasibility Study for Diagnosis of Latent Tuberculosis Infection Using an IGRA Point-of-Care Platform in South Korea.

PURPOSE: This study aimed to evaluate ichroma™ IGRA-TB, a novel point-of-care platform for assaying IFN-γ release, and to compare it with QuantiFERON-TB Gold In-Tube (QFT-GIT) for identifying Mycobacterium tuberculosis (M. tb) infection. MATERIALS AND METHODS: We recruited 60 healthy subjects, and blood samples were obtained in QFT-GIT blood collection tubes. The blood collection tubes were incubated at 37°C, and culture supernatant was harvested after 18-24 hours. IFN-γ responses were assessed by the ichroma™ IGRA-TB cartridge and the QFT-GIT IFN-γ enzyme-linked immunosorbent assay. Three active TB patients were recruited as a positive control for M. tb infection. RESULTS: The area under the receiver operating characteristic curve of the ichroma™ IGRA-TB test for differentiating between infected and non-infected individuals was 0.9706 (p<0.001). Inconsistent positivity between the two tests was found in three participants who showed weak positive IFN-γ responses (<1.0 IU/mL) with QFT-GIT. However, the two tests had excellent agreement (95.2%, κ=0.91, p<0.001), and a very strong positive correlation was observed between the IFN-γ values of both tests (r=0.91, p<0.001). CONCLUSION: The diagnostic accuracy demonstrated in this study indicates that the ichroma™ IGRA-TB test could be used as a rapid diagnostic method for detecting latent TB infection. It may be particularly beneficial in resource-limited places that require cost-effective laboratory diagnostics

Identification of immunological biomarkers which may differentiate latent tuberculosis from exposure to environmental nontuberculous mycobacteria in children.

A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic

Factors affecting immunogenicity of BCG in infants, a study in Malawi, The Gambia and the UK.

BACKGROUND: BCG immunogenicity in infants differs between populations and these differences have been attributed to various factors. In this study, the influence of geographical location, season of birth, timing of vaccination, micronutrient status (zinc) and inflammatory status (C-reactive protein, CRP) were assessed. METHODS: Immunogenicity was assessed by cytokine signature in culture supernatants from diluted whole blood samples stimulated with M. tuberculosis PPD, using a multiplex bead assay. Results were correlated with the plasma zinc and CRP concentrations at the time of sampling, and with interview and household data. BCG vaccinated infants were recruited in Malawi, The Gambia and the UK. RESULTS: In Malawi, infants vaccinated within the first week after birth showed lower production of most cytokines measured than those vaccinated later. The number of cytokines showing significant differences between Malawian and Gambian infants decreased after adjusting for season of birth. In Malawi, a proportion of infants had zinc deficiency and elevated plasma CRP (>10 mg/L), but neither zinc deficiency nor high CRP was associated with production of any of the cytokines measured. CONCLUSIONS: The cytokine/chemokine signatures observed in response to M. tuberculosis PPD in infants at 3 months post BCG vaccination were affected by geographical location, season of birth, and timing of vaccination but not associated with the concentration of plasma zinc or inflammatory status. These factors should be considered in future trials of new TB vaccines

Combination of cytokine responses indicative of latent TB and active TB in Malawian adults.

BACKGROUND: An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease. METHODS: Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested. RESULTS: Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses. CONCLUSIONS: CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB

The proportion of cytokine responders to PPD at baseline and follow-up time points.

<p>The proportion of responders producing IFN-γ (A), IL-13 (B), IL-10 (C), CXCL10 (D), IL-17 (E) and TNF-α (F) in response to PPD was analysed in 40 spouses at baseline, 49 at 6 months, 39 at 12 months and 22 at 24 months follow-up. The majority of the spouses did not produce IL-13 and IL-10 in response to PPD at baseline or 24 months later while high levels of IFN-γ, CXCL10, IL-17 and TNF-α were detected. There were no significant changes in the proportions of cytokine responders throughout the follow-up period.</p