Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study

Abstract

Background: A nonsputum blood test capable of predicting progression of healthy individuals to active tuberculosis (TB) before clinical symptoms manifest would allow targeted treatment to curb transmission. We aimed to develop a proteomic biomarker of risk of TB progression for ultimate translation into a point-of-care diagnostic.

Methods and findings: Proteomic TB risk signatures were discovered in a longitudinal cohort of 6,363 Mycobacterium tuberculosis-infected, HIV-negative South African adolescents aged 12-18 years (68% female) who participated in the Adolescent Cohort Study (ACS) between July 6, 2005 and April 23, 2007, through either active (every 6 months) or passive follow-up over 2 years. Forty-six individuals developed microbiologically confirmed TB disease within 2 years of follow-up and were selected as progressors; 106 nonprogressors, who remained healthy, were matched to progressors. Over 3,000 human proteins were quantified in plasma with a highly multiplexed proteomic assay (SOMAscan). Three hundred sixty-one proteins of differential abundance between progressors and nonprogressors were identified. A 5-protein signature, TB Risk Model 5 (TRM5), was discovered in the ACS training set and verified by blind prediction in the ACS test set. Poor performance on samples 13-24 months before TB diagnosis motivated discovery of a second 3-protein signature, 3-protein pair-ratio (3PR) developed using an orthogonal strategy on the full ACS subcohort. Prognostic performance of both signatures was validated in an independent cohort of 1,948 HIV-negative household TB contacts from The Gambia (aged 15-60 years, 66% female), longitudinally followed up for 2 years between March 5, 2007 and October 21, 2010, sampled at baseline, month 6, and month 18. Amongst these contacts, 34 individuals progressed to microbiologically confirmed TB disease and were included as progressors, and 115 nonprogressors were included as controls. Prognostic performance of the TRM5 signature in the ACS training set was excellent within 6 months of TB diagnosis (area under the receiver operating characteristic curve [AUC] 0.96 [95% confidence interval, 0.93-0.99]) and 6-12 months (AUC 0.76 [0.65-0.87]) before TB diagnosis. TRM5 validated with an AUC of 0.66 (0.56-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. The 3PR signature yielded an AUC of 0.89 (0.84-0.95) within 6 months of TB diagnosis and 0.72 (0.64-0.81) 7-12 months before TB diagnosis in the entire South African discovery cohort and validated with an AUC of 0.65 (0.55-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. Signature validation may have been limited by a systematic shift in signal magnitudes generated by differences between the validation assay when compared to the discovery assay. Further validation, especially in cohorts from non-African countries, is necessary to determine how generalizable signature performance is.

Conclusions: Both proteomic TB risk signatures predicted progression to incident TB within a year of diagnosis. To our knowledge, these are the first validated prognostic proteomic signatures. Neither meet the minimum criteria as defined in the WHO Target Product Profile for a progression test. More work is required to develop such a test for practical identification of individuals for investigation of incipient, subclinical, or active TB disease for appropriate treatment and care.

Fig 1. Sample distribution and relative differences in protein abundance between progressors and nonprogressors.

(A) Distribution of progressor and nonprogressor samples from the discovery training and test set of South African adolescents and (B) progressor and nonprogressor samples from Gambian household contacts of TB cases used for validation. Progressor and nonprogressor samples are represented by filled and open dots, respectively. The x-axis indicates time of prospective sample collection before the diagnosis of active TB disease. Nonprogressor samples were matched to progressors, as previously described [4,5], and aligned with time to TB diagnosis. (C) Volcano plot of 2,872 proteins from a univariate KS analysis comparing all TB progressor samples and all nonprogressor controls. The negative log₁₀-transformed P values versus the log₂ of the median TB RFU value over the median control RFU value. A value of 1 on the horizontal axis corresponds to a 2-fold change in RFU. Protein abundance data are in S4 and S6 Tables, and proteins ranked according to their differential abundances are in S5 Table (training set), S7 Table (training and test set), and S2 Text. KS, Kolmogorov–Smirnov; RFU, raw fluorescence unit; TB, tuberculosis.

Fig 2

Receiver operator characteristic AUC analysis of the TRM5 signature for (A) ACS training set and (B) test set progressor and nonprogressor plasma samples, stratified by the time interval of each prospectively collected sample before the date of TB disease diagnosis. ACS, Adolescent Cohort Study; AUC, area under the curve; TB, tuberculosis; TRM5, TB Risk Model 5.

Fig 3

(A) Graphical representation of pairwise structure of the 3PR signature. Proteins that are expressed at higher levels in TB progressors, compared to nonprogressors, are shown in red. Proteins expressed at levels lower in progressors than nonprogressors are shown in blue. (B) Area under the receiver operator characteristic curve analysis of the 3PR signature for all ACS progressor and nonprogressor plasma samples, stratified by the time of each prospectively collected sample before the date of TB disease diagnosis. 3PR, 3-protein pair-ratio; ACS, Adolescent Cohort Study; TB, tuberculosis.

Fig 4. Cumulative distribution of select model proteins run on the custom validation slide arrays.

Curves demonstrate a spectrum of distributions of RFU in the ACS versus the GC6–74 cohorts. Red curves represent GC6–74 samples prior to bridging, yellow curves represent GC6–74 postbridging, and blue curves represent ACS samples. ACS, Adolescent Cohort Study; GC6–74, Grand Challenges 6–74; RFU, raw fluoresence unit.

Fig 5

ROC-AUC analysis of the TRM5 and 3PR signatures for all GC6–74 validation set plasma samples, for (A) all prospectively collected samples, and (B–E) stratified by the time interval of each prospectively collected sample before TB diagnosis. 3PR, 3-protein pair-ratio; GC6–74, Grand Challenges 6–74; ROC-AUC, area under the receiver operator characteristic curve; TB, tuberculosis; TRM5, TB Risk Model 5.