Discriminating Active Tuberculosis from Latent Tuberculosis Infection by flow cytometric measurement of CD161-expressing T cells

Abstract Background The differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains challenging in clinical practice. We aimed to evaluate the diagnostic accuracy of the IFN-γ/TNF-α FluoroSpot assay for differentiating ATB from LTBI. Methods We conducted a pilot study of case-control design, using the FluoroSpot assay to simultaneously detect IFN-γ and TNF-α secretion at the single-cell level. The frequencies of antigen-specific single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ- and dual IFN-γ/TNF-α-secreting T cells were detected. The optimal cutoffs value of frequencies for differentiating ATB from LTBI were determined according to receiver operating characteristic curve analysis. The sensitivity, specificity, predictive values (PV) and likelihood ratios (LR) of the FluoroSpot assay were calculated. Results Thirty patients diagnosed microbiologically with ATB, 36 healthcare workers with LTBI and 36 healthy controls were enrolled. After stimulated by ESAT-6 or CFP-10 peptides, the median frequencies of single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ- and dual IFN-γ/TNF-α-secreting T cells in ATB patients were all significantly higher than those in LTBI and HC groups (P < 0.01). The frequencies of total IFN-γ-secreting T cells detected by FluoroSpot assay correlated significantly with those of T-SPOT.TB (r = 0.910 for ESAT-6, P < 0.001, r = 0.845 for CFP-10, P < 0.001). After stimulated by ESAT-6 peptides, with total TNF-α-secreting T cells frequencies at a cut off value of 21 iSFCs/250,000 PBMCs, the sensitivity, specificity, PLR, NLR, PPV, NPV of IFN-γ/TNF-α FluoroSpot assay in differentiating ATB from LTBI were 96.7% (95%CI, 82.8–99.9%), 94.3% (95%CI, 80.8–99.3%), 16.92 (95%CI, 4.40–65.08), 0.04 (95%CI, 0.01–0.24), 93.6% (95%CI,78.6–99.2%) and 97.1% (95%CI, 84.7–99.9%), respectively. With the frequencies of total TNF-α- and total IFN-γ-secreting T cells stimulated by ESAT-6 peptides combined, the specificity was increased to 97.1%, and the positive likelihood ratio to 31.5. The combination with CFP-10 might not improve the diagnostic accuracy of the ESAT-6 for differentiating ATB from LTBI. Conclusions IFN-γ/TNF-α FluoroSpot assay might have potential to help differentiate ATB from LTBI, but the findings need to be further verified by cross-sectional or prospective cohort studies.

Download Full-text

Mycobacterium tuberculosis latency-associated antigen Rv1733c SLP improves the accuracy of differential diagnosis of active tuberculosis and latent tuberculosis infection

10.21203/rs.3.rs-121733/v1 ◽

2020 ◽

Author(s):

Lifan Zhang ◽

Huimin Ma ◽

Shijun Wan ◽

Yueqiu Zhang ◽

Mengqiu Gao ◽

...

Keyword(s):

T Cells ◽

Differential Diagnosis ◽

Sensitivity And Specificity ◽

Latent Tuberculosis Infection ◽

Latent Tuberculosis ◽

Active Tuberculosis ◽

Tuberculosis Infection ◽

Control Group ◽

College Hospital ◽

Serial Test

Abstract Background The differential diagnosis of active tuberculosis(ATB) and latent tuberculosis infection(LTBI) is still challenging. The objective of the study was to evaluate the accuracy of the novel M. tuberculosis latency-associated antigens Rv1733c and Rv1733c SLP for differentiating ATB from LTBI. Methods A case-control study was designed to enroll pathogen-confirmed ATB cases admitted to the Peking Union Medical College Hospital and Beijing Chest Hospital, whereas those with LTBI were denoted as the control group. The Fluorescence-Immunospot (FluoroSpot) assay was used to detect the frequencies of IL-2-, IFN-γ-secreting T cells stimulated by the M. tuberculosis latency-associated antigens Rv1733c and Rv1733c SLP. The combination of the ESAT-6/CFP-10-Fluorospot test was evaluated with regard to the sensitivity, specificity, predictive value and likelihood ratio for the differential diagnosis of ATB and LTBI. Results A total of 20 pathogens-confirmed TB and 28 LTBI cases were included. The sensitivity and specificity of ESAT-6/CFP-10-FluoroSpot for the differential diagnosis of ATB and LTBI were 95% (95% CI, 75.13–99.87%) and 82.14% (95% CI, 63.11–93.94%), respectively. Following stimulation with Rv1733c and Rv1733c SLP, the maximum AUROC was 0.711 (95% CI, 0.566–0.856) as determined by the ROC curve, which was used to assess the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP. The cutoff value of 0 SFCs/2.5 × 105 PBMCs was used for the analysis. The frequency, sensitivity and specificity of Rv1733c SLP for differentiating ATB and LTBI were 75% (95% CI, 50.90–91.34%) and 60.71% (95% CI, 40.58–78.50%), respectively. The ESAT-6/cfp-10-fluorospot was combined with the frequency of single IL-2-secreting T cells, which were stimulated by Rv1733c SLP for the differential diagnosis of ATB and LTBI. This resulted in an increased sensitivity and specificity to 100% (95% CI, 83.16–100.00%), as determined by the parallel test and to 92.86% (95% CI, 71.77–97.73%) as determined by the serial test, respectively. Conclusions Rv1733c SLP has the potential to be used as a candidate antigen for T cell-based tuberculosis diagnostic tests, in combination with ESAT-6 and CFP-10, to differentiate between ATB and LTBI diagnosis.

Download Full-text

Activation Phenotype of Mycobacterium tuberculosis-Specific CD4+ T Cells Promoting the Discrimination Between Active Tuberculosis and Latent Tuberculosis Infection

Frontiers in Immunology ◽

10.3389/fimmu.2021.721013 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ying Luo ◽

Ying Xue ◽

Liyan Mao ◽

Qun Lin ◽

Guoxing Tang ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

Latent Tuberculosis Infection ◽

Latent Tuberculosis ◽

Active Tuberculosis ◽

Tuberculosis Infection ◽

Antigen Stimulation ◽

Hla Dr ◽

Tnf Α ◽

Ifn Γ

BackgroundRapid and effective discrimination between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains a challenge. There is an urgent need for developing practical and affordable approaches targeting this issue.MethodsParticipants with ATB and LTBI were recruited at Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort) based on positive T-SPOT results from June 2020 to January 2021. The expression of activation markers including HLA-DR, CD38, CD69, and CD25 was examined on Mycobacterium tuberculosis (MTB)-specific CD4+ T cells defined by IFN-γ, TNF-α, and IL-2 expression upon MTB antigen stimulation.ResultsA total of 90 (40 ATB and 50 LTBI) and another 64 (29 ATB and 35 LTBI) subjects were recruited from the Qiaokou cohort and Caidian cohort, respectively. The expression patterns of Th1 cytokines including IFN-γ, TNF-α, and IL-2 upon MTB antigen stimulation could not differentiate ATB patients from LTBI individuals well. However, both HLA-DR and CD38 on MTB-specific cells showed discriminatory value in distinguishing between ATB patients and LTBI individuals. As for developing a single candidate biomarker, HLA-DR had the advantage over CD38. Moreover, HLA-DR on TNF-α+ or IL-2+ cells had superiority over that on IFN-γ+ cells in differentiating ATB patients from LTBI individuals. Besides, HLA-DR on MTB-specific cells defined by multiple cytokine co-expression had a higher ability to discriminate patients with ATB from LTBI individuals than that of MTB-specific cells defined by one kind of cytokine expression. Specially, HLA-DR on TNF-α+IL-2+ cells produced an AUC of 0.901 (95% CI, 0.833–0.969), with a sensitivity of 93.75% (95% CI, 79.85–98.27%) and specificity of 72.97% (95% CI, 57.02–84.60%) as a threshold of 44% was used. Furthermore, the performance of HLA-DR on TNF-α+IL-2+ cells for differential diagnosis was obtained with validation cohort data: 90.91% (95% CI, 72.19–97.47%) sensitivity and 68.97% (95% CI, 50.77–82.73%) specificity.ConclusionsWe demonstrated that HLA-DR on MTB-specific cells was a potentially useful biomarker for accurate discrimination between ATB and LTBI.

Download Full-text