Biomarkers to identify Mycobacterium tuberculosis infection among borderline QuantiFERON results

Abstract

Background: Screening for tuberculosis (TB) infection often includes QuantiFERON-TB Gold Plus (QFT) testing. Previous studies showed that two-thirds of patients with negative QFT results just below the cut-off, so-called borderline test results, nevertheless had other evidence of TB infection. This study aimed to identify a biomarker profile by which borderline QFT results due to TB infection can be distinguished from random test variation.

Methods: QFT supernatants of patients with a borderline (≥0.15 and <0.35 IU·mL^-1), low-negative (<0.15 IU·mL^-1) or positive (≥0.35 IU·mL^-1) QFT result were collected in three hospitals. Bead-based multiplex assays were used to analyse 48 different cytokines, chemokines and growth factors. A prediction model was derived using LASSO regression and applied further to discriminate QFT-positive Mycobacterium tuberculosis-infected patients from borderline QFT patients and QFT-negative patients RESULTS: QFT samples of 195 patients were collected and analysed. Global testing revealed that the levels of 10 kDa interferon (IFN)-γ-induced protein (IP-10/CXCL10), monokine induced by IFN-γ (MIG/CXCL9) and interleukin-1 receptor antagonist in the antigen-stimulated tubes were each significantly higher in patients with a positive QFT result compared with low-negative QFT individuals (p<0.001). A prediction model based on IP-10 and MIG proved highly accurate in discriminating patients with a positive QFT (TB infection) from uninfected individuals with a low-negative QFT (sensitivity 1.00 (95% CI 0.79-1.00) and specificity 0.95 (95% CI 0.74-1.00)). This same model predicted TB infection in 68% of 87 patients with a borderline QFT result.

Conclusions: This study was able to classify borderline QFT results as likely infection-related or random. These findings support additional laboratory testing for either IP-10 or MIG following a borderline QFT result for individuals at increased risk of reactivation TB.

FIGURE 1

Classification of all samples using linear discriminant analysis (LDA). QuantiFERON-TB Gold Plus results distinguished by LDA, including all cytokines (except for interferon-γ), chemokines and growth factors. All data were log₂-transformed prior to analysis. Includes all data.

FIGURE 2

Comparison of biomarker levels between positive versus low-negative QuantiFERON-TB Gold Plus (QFT) samples. A pairwise comparison of all cytokines, chemokines and growth factors between positive and low-negative QFT samples was conducted using global test analysis. The absolute correlation between biomarkers is depicted using hierarchal clustering. Uncorrected p-values are depicted on the left of the bar chart. Black lines on the clustering graph indicate significant multiplicity corrected p-values, while nonsignificant p-values are depicted with grey lines. Includes all data. See the bead-based multiplex assay section of the Methods for details of biomarkers included in the Bio-Plex Pro Human Cytokine Screening Panel.

FIGURE 3

Correlation between 10 kDa interferon (IFN)-γ-induced protein (IP-10), monokine induced by IFN-γ (MIG) and interleukin-1 receptor antagonist (IL-1ra). A positive correlation between a) IP-10 and MIG, b) IP-10 and IL-1ra, and c) MIG and IL-1ra was measured by Spearman's correlation coefficient and two-tailed p-value analysis. All data were log₂-transformed prior to analysis. Includes all data.

FIGURE 4

Accuracy of the biomarker signature. a) Biomarker signature derived from LASSO regression analysis based on patients with a positive QuantiFERON-TB Gold Plus (QFT) versus low-negative QFT result. Includes only training data. b, c) Receiver operating characteristic curve plots showing the prediction accuracy of the model allowing discrimination b) between patients with tuberculosis (TB) infection (based on a positive QFT result) and without TB infection (based on a low-negative QFT result) and c) between patients with a borderline QFT result and subjects without TB infection (based on a low-negative QFT result). Includes only test data. MIG: monokine induced by interferon (IFN)-γ; IP-10: 10 kDa IFN-γ-induced protein; IL-1ra: interleukin-1 receptor antagonist; AUC: area under the curve.

FIGURE 5

Biomarker values by QuantiFERON-TB Gold Plus categories: a) 10 kDa interferon (IFN)-γ-induced protein (IP-10), b) monokine induced by IFN-γ (MIG) and c) interleukin-1 receptor antagonist (IL-1ra). Biomarker values (medians and quartiles) are displayed as boxes, with whiskers representing 5–95% percentiles. Significant differences between groups were calculated using the Mann–Whitney U-test. Dashed lines represent the optimal cut-off (Youden index) derived from receiver operating characteristic curves (supplementary figure S3). All data were log₂-transformed prior to analysis. Includes all data. ****: p<0.0001; ns: nonsignificant.

FIGURE 6

10 kDa interferon (IFN)-γ-induced protein (IP-10) and monokine induced by IFN-γ (MIG) levels among borderline QuantiFERON-TB Gold Plus (QFT) samples. IP-10 and MIG levels of all 87 patients with a borderline QFT result. Dashed lines represent the optimal cut-offs (Youden index) derived from receiver operating characteristic curves (supplementary figure S3). All data were log₂-transformed prior to analysis. Includes all data.