Diagnostic performance of culture filtered protein 10-specific perforin in pediatric patients with active tuberculosis

Abstract

Background: Mycobacterium tuberculosis (Mtb)-specific perforin were significantly increased in patients with tuberculosis. This study aims to evaluate the diagnosis value of Mtb-specific perforin in pediatric patients with tuberculosis.

Methods: Diagnostic performance of perforin levels induced by 6-kDa early secreted antigen target (ESAT6) or culture filtered protein 10 (CFP10) were evaluated in eighty-six samples from children participants by receiver operating characteristic curve analysis. Flow cytometry was used to detect the expression of perforin and INF-γ of CD4⁺ , CD8⁺ T cells in response to CFP10 stimulation.

Results: After ex vivo stimulation, levels of ESAT6/CFP10-specific perforin in LTBI patients were significantly higher than active TB (ATB) patients, non-tuberculosis infection (non-TB), and health control (HC) individuals. The diagnostic efficacy of CFP10-specific perforin for TB diagnosis was significantly higher than ESAT6-specific perforin and T-SPOT assay, and when 0.74 ng/mL was taken as the cutoff value, the sensitivity, specificity, and accuracy were 97.83%, 87.5%, and 93.02%. CFP10-specific perforin in both CD4⁺ and CD8⁺ T cells were significantly higher in ATB patients compared to HCs and further increased in LTBI patients. However, INF-γ was mainly secreted by CD4⁺ T cells and showed no significant difference between LTBI and ATB patients. In addition, CFP10-specific perforin can effectively distinguish between ATB and LTBI with the cutoff value of 1.80 ng/mL. Sensitivity and specificity were 88.46% and 85.62%, respectively.

Conclusions: CFP10-specific perforin may be used as a novel cellular immunity-based diagnostic marker of pediatric patients with tuberculosis, and with the potential for discriminating ATB from LTBI.

Keywords: CFP10; ESAT6; pediatric patients; perforin; tuberculosis diagnosis.

Figure 1

Levels of total perforin in PBMCs stimulated by ESAT6/CFP10. PBMCs obtained from ATB patients (n = 26), LTBI individuals (n = 20), healthy control subjects (n = 20), and non‐TB patients (n = 20) were stimulated with ESAT6 or CFP10 for 24 h. Levels of total perforin in supernatant were detected by ELISA. A, Combine ATB and LTBI as TB group, non‐TB, and HC as control group. Levels of total perforin in supernatant of ESAT6 or CFP10‐stimulated PBMCs isolated from TB group and control group. B, Levels of total perforin induced by ESAT6 in supernatant of PBMCs isolated from each indicated subgroup. C, Levels of total perforin induced by CFP10 in supernatant of PBMCs isolated from each indicated subgroup. Median values for each group of participants are represented by a horizontal bar. Statistical analysis was performed by the Mann‐Whitney U test. **P < .01, ***P < .001

Figure 2

Levels of Mtb‐specific perforin in PBMCs stimulated by ESAT6/CFP10. Mtb‐specific perforin was calculated by subtracting background. PBMCs stimulated with medium alone were used as a background control. A, Levels of Mtb‐specific perforin in supernatant of ESAT6 or CFP10‐stimulated PBMCs isolated from TB group and control group. B, Levels of Mtb‐specific perforin induced by ESAT6 in supernatant of PBMCs isolated from each indicated subgroup. C, Levels of Mtb‐specific perforin induced by CFP10 in supernatant of PBMCs isolated from each indicated subgroup. Median values for each group of participants are represented by a horizontal bar. Statistical analysis was performed by the Mann‐Whitney U test. **P < .01, ***P < .001

Figure 3

The receiver operating characteristic (ROC) curves (plotting sensitivity versus 1‐specificity) of total/Mtb‐specific perforin to discriminate patients with TB from control. The ROC analysis was performed for total/Mtb‐specific perforin to determine cutoff levels for the diagnosis of TB disease

Figure 4

Qualitative analysis of perforin and INF‐γ expression ex vivo in CD4⁺/CD8⁺ T cells from patients with ATB, LTBI, and HC individuals. Representative FACS plot of INF‐γ and perforin expression ex vivo in CD4⁺ T (A) and CD8⁺ T cells (B) after stimulation with CFP10. Each dot corresponds to the result from an individual child, and horizontal bars represent the medians of the results. *P < .05, **P < .01, ***P < .001

Figure 5

Receiver operating characteristic (ROC) for Mtb‐specific perforin as a classifier to distinguish between ATB and LTBI. The ROC analysis was performed for Mtb‐specific perforin to determine cutoff levels in distinguishing between ATB and LTBI