Identification of Host Proteins Predictive of Early Stage Mycobacterium tuberculosis Infection

Abstract

The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age≥12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p<0.05) differentially expressed proteins were identified across all comparisons. Proteins associated with cellular immunity and lipid metabolism were induced early after Mtb infection. One hundred and fifty-nine proteins were selected for a targeted mass spectrometry assay. A set of longitudinal samples from 52 TST-negative subjects who converted to TST-positive or remained TST-negative were analyzed, and multivariate logistic regression was used to identify unique protein panels able to predict TST conversion with cross-validated AUC>0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy.

Keywords: Converter; LTBI; Proteomics; Tuberculosis.

Fig. 1

Cross-sectional comparison of changes in sera from non-infected (NI), baseline TST-negative future converters (CO), baseline TST-positive (LTBI), and active TB (ATB) groups. Shown are protein expression change ratios. The significant changes (p < 0.05) are color-coded red for increased and blue for decreased expression ratios, respectively.

Fig. 2

Longitudinal comparison of changes in selected* plasma proteins from baseline TST-negative subjects (N = 52) that converted to TST-positive (N = 37) or remained TST-negative (N = 15). Shown are protein expression change ratios between each individual's baseline TST-negative sample and the TST-positive conversion sample or corresponding TST-negative sample. The significant changes (p < 0.05) are color-coded red for increased and blue for decreased expression ratios, respectively. *We selected 159 proteins from the cross-sectional discovery phase using the following combination of biological and statistical criteria. All of the significantly differentially expressed proteins from the baseline converter vs NI and LTBI vs NI comparisons were selected along with the most differentially expressed proteins from comparisons to active TB. We also included the significant proteins described in the MRM-MS assay of Achkar et al. (2015).

Fig. 3

Changes in plasma protein expression after TST-positive conversion. Shown are protein expression change ratios in subjects (N = 19) with samples at baseline (D1), 3 months (M3), and 6 months (M6) that had converted to TST-positive at M3. The significant changes (p < 0.05) are color-coded red for increased and blue for decreased expression ratios, respectively.

Fig. 4

Small plasma protein biomarker panels can predict TST conversion. Combinations of up to 6 proteins using the 159 targeted proteins detected were used to define biomarker panels able to predict conversion with AUC > 0.85. The initial verification study (Verification 1: N = 52 subjects, 3 longitudinal samples per subject) defined high performing biomarker panels whose performance was confirmed using an independent set of longitudinal samples (Verification 2: N = 16 subjects, 3 longitudinal samples per subject). Shown is the performance of one 6-protein panel in prediction of TST-positive conversion in both verification studies. The AUC values shown did not differ significantly from each other.