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

Interferon-gamma Release Assays (IGRAs) significantly increases the possibility for early diagnosis of tuberculosis, but IGRAs alone cannot discriminate active TB from LTBI. Therefore, fast and reliable discrimination of active tuberculosis, especially bacteriology negative tuberculosis, from LTBI is a great necessity. Here we established an assay based on flow cytometric multiparameter assay assessing expression of CD161 along with CD3, CD4, and CD8, whereby a set of indices formulated by the percentages of CD3(+)CD161(+), CD3(+)CD4(+)CD161(+) and CD3(+)CD8(+)CD161(+) T cells multiplied with lymphocyte/monocyte ratio were established. Application of the CD3(+)CD8(+)CD161(+) index to compare a cohort of active tuberculosis with a cohort of LTBI or health control yielded 0.7662 (95% confidence interval [CI] 0.6559-0.8552) or 0.7922 (95% CI 0.6846-0.8763) for sensitivity and 0.9048 (95% CI 0.8209-0.9580) or 0.8939 (95% CI 0.8392-0.9349) for specificity when the TB cohort was AFB(+); the corresponding results were 0.7481 (95% CI 0.6648-0.8198) or 0.7557 (95% CI 0.6730-0.8265) for sensitivity and 0.8571 (95% CI 0.7637-0.9239) or 0.8603 (95% CI 0.8008-0.9075) for specificity when the TB cohort was AFB(-). Our results reveal that in combination with IGRAs, CD161-based indices provide a novel, fast diagnostic solution addressing the limitation of current tuberculosis diagnostics

Increased complement C1q level marks active disease in human tuberculosis.

BACKGROUND: Complement functions as an important host defense system and complement C5 and C7 have been implicated in immunopathology of tuberculosis. However, little is known about the role of other complement components in tuberculosis. METHODS: Complement gene expression in peripheral blood mononuclear cells of tuberculosis patients and controls were determined using whole genome transcriptional microarray assays. The mRNA and protein levels of three C1q components, C1qA, C1qB, and C1qC, were further validated by qRT-PCR and enzyme-linked immunosorbent assay, respectively. The percentages of C1q expression in CD14 positive cells were determined by flow cytometry. Finally, C1qC protein level was quantified in the pleural fluid of tuberculosis and non-tuberculosis pleurisy. RESULTS: C1q expression increases significantly in the peripheral blood of patients with active tuberculosis compared to healthy controls and individuals with latent TB infection. The percentage of C1q-expressing CD14 positive cells is significantly increased in active TB patients. C1q expression in the peripheral blood correlates with sputum smear positivity in tuberculosis patients and is reduced after anti-tuberculosis chemotherapy. Notably, receiver operating characteristic analysis showed that C1qC mRNA levels in peripheral blood efficiently discriminate active from latent tuberculosis infection and healthy controls. Additionally, C1qC protein level in pleural effusion shows improved power in discriminating tuberculosis from non-tuberculosis pleurisy when compared to other inflammatory markers, such as IL-6 and TNF-α. CONCLUSIONS: C1q expression correlates with active disease in human tuberculosis. C1q could be a potential diagnostic marker to discriminate active tuberculosis from latent tuberculosis infection as well as tuberculosis pleurisy from non-tuberculosis pleurisy

The demographic characteristics of the study populations.

a<p>These patients were follow-up to 3, 6 months after anti-TB chemotherapy, and gene expression profile were performed by microarrays.</p>b<p>“ND” indicated not done.</p>c<p>“NA” indicated not applicable.</p>d<p>14 patients were follow-up to 0, 3, 6, 12 months after anti-TB chemotherapy for examine the dynamic change of ClqC level in plasma.</p>e<p>PBMC and PFMC from the same patient (n = 15) were collected to compared the C1q expression in different tissues.</p

Increased C1q expression in the pleural fluid and BALF compared to matched PBMC from TB patients.

<p>A) qRT-PCR assay of C1q A/B/C mRNA levels in PFMC and PBMC from the same TB patient (n = 15). B) qRT-PCR assay of C1q A/B/C mRNA levels in BALF cells and PBMC from the same TB patient (n = 7). C) ELISA of C1qC concentration in pleural fluid and plasma from the same TB patient (n = 15). D) ELISA of C1qC concentrations in BALF and plasma from the same TB patient (n = 7). Differences between groups were compared with paired <i>t</i>-test, <i>P</i> values are indicated. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001.</p

Increased C1qA/B/C expressions in active TB patients.

<p>A) Comparison of the transcriptional signature of the complement genes as determined by microarray assays between HC (n = 6), LBTI (n = 6), and TB (n = 9) PBMC samples. Pseudocolors indicate differential expression (red, up-regulation; green, down-regulation; black, no change in expression). B) Expression of C1qA/B/C genes in PBMC from validation cohort 1 consisting of HC (n = 45), LTBI (n = 142), and TB (n = 164) was determined by qRT-PCR. Relative gene expression was normalized to GADPH. C) ELISA of C1qC concentration in the plasma from HC (n = 20), LTBI (n = 32), and TB (n = 32). The means of C1qC concentration in HC LTBI and TB were 583.8±134.3, 525.8±124.0 and 751.4±166.1 ng/ml, respectively. Differences between groups were compared with the one-way analysis of variance followed by Newman-Keuls multiple comparison test; <i>P</i> values are indicated. ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Active TB patients exhibited increase in percentage of C1q-expressing CD14 positive cells.

<p>A) Expression of C1q in different cell types. Peripheral blood was stained with anti- CD3, CD14, C1q and CD68 antibody and C1q expression is determined by flow cytometry. B) The percentages of C1q expressing on CD14+ cells from active TB (n = 30), LTBI (n = 30) and HC (n = 30). Differences between groups were compared with the one-way analysis of variance followed by Newman-Keuls multiple comparison test; <i>P</i> values are indicated. ** <i>P</i><0.01, *** <i>P</i><0.001. C) Immunofluorescence and D) Immunohistochemistry analysis of C1q expressing in TB patient's lung tissue.</p

C1q is a useful marker to distinguish active TB patients from HC and LTBI.

<p>ROC analysis of the power of C1qA, C1q B, and C1qC mRNA to distinguish A) active TB from HC, and B) active TB from LTBI. C) ROC analysis of the power of C1qC protein levels in plasma from HC, LTBI and TB. AUC, area under curve. 95%CI, 95% confidence interval.</p

Increased C1qC level in the pleural effusion distinguishes TB from Non-TB pleurisy.

<p>A) The levels of C1qC, ADA, IL-6, IL-1α, IL-1β, and TNF-α in pleural fluid from TP patients (TB, n = 36) and non-TB patients (non-TB, n = 34) were determined by Luminex liquid array-based multiplexed immunoassays. Differences between groups were compared with Un-paired t-test, P values are indicated. * P<0.05; ** P<0.01; *** P<0.001. B) ROC analysis of the power of ADA, C1qC, IL-6, and TNF-α in pleural fluid to distinguish TP from non-TB. AUC, area under curve; 95%CI, 95% confidence interval.</p

C1q Expression in PBMC is associated with sputum smear AFB positivity and reduced after anti-TB treatment in PTB patients.

<p>A) qRT-PCR assay of C1qA/B/C gene expression in PBMCs from sputum smear AFB negative (AFB-, n = 21) and positive PTB patients (AFB+, n = 143). B) Microarray and C) qRT-PCR assay of C1qA/B/C gene expression in PBMC from active PTB patients (n = 9) at 0, 3 and 6 months after initiation of anti-TB treatment. D) ELISA of C1qC concentration in the plasma of PTB patients (n = 14) at 0, 3, 6, and 12 months after initiation of anti-TB treatment. The means of C1qC concentration at 0, 3, 6, and 12 months after anti-TB treatment were 781.2±144.5, 626.3±177.8, 522.1±223.1 and 513.3±220.2 ng/ml, respectively. Differences between AFB- and AFB+ patients were compared with Unpaired t-test; C1q expression at different time points were compared with the one-way analysis of variance followed by Newman-Keuls multiple comparison test; <i>P</i> values are indicated. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p