Transcriptomic Biomarkers for Tuberculosis: Validation of NPC2 as a Single mRNA Biomarker to Diagnose TB, Predict Disease Progression, and Monitor Treatment Response

Abstract

External validation in different cohorts is a key step in the translational development of new biomarkers. We previously described three host mRNA whose expression in peripheral blood is significantly higher (NPC2) or lower (DOCK9 and EPHA4) in individuals with TB compared to latent TB infection (LTBI) and controls. We have now conducted an independent validation of these genes by re-analyzing publicly available transcriptomic datasets from Brazil, China, Haiti, India, South Africa, and the United Kingdom. Comparisons between TB and control/LTBI showed significant differential expression of all three genes (NPC2^high&nbsp;p < 0.01, DOCK9^low&nbsp;p < 0.01, and EPHA4^low&nbsp;p < 0.05). NPC2^high had the highest mean area under the ROC curve (AUROC) for the differentiation of TB vs. controls (0.95) and LTBI (0.94). In addition, NPC2 accurately distinguished TB from the clinically similar conditions pneumonia (AUROC, 0.88), non-active sarcoidosis (0.87), and lung cancer (0.86), but not from active sarcoidosis (0.66). Interestingly, individuals progressing from LTBI to TB showed a constant increase in NPC2 expression with time when compared to non-progressors (p < 0.05), with a significant change closer to manifestation of active disease (≤3 months, p = 0.003). Moreover, NPC2 expression normalized with completion of anti-TB treatment. Taken together, these results validate NPC2 mRNA as a diagnostic host biomarker for active TB independent of host genetic background. Moreover, they reveal its potential to predict progression from latent to active infection and to indicate a response to anti-TB treatment.

Keywords: Mycobacterium tuberculosis; NPC2; Niemann–Pick disease type C2; RNA; biomarkers; diagnosis; mRNA; transcription; treatment; tuberculosis.

Figure 1

Individual normalized mRNA expression values of DOCK9, EPHA4, and NPC2 mRNA in blood. Diagnostic groups comprise controls, latent tuberculosis infection (LTBI), and active tuberculosis (TB). (A–C) DOCK9 mRNA expression in cohorts from Brazil (A), Haiti (B), and India (C). (D–F) EPHA4 mRNA expression in cohorts from Brazil (D), Haiti (E), and India (F). (G–I) NPC2 mRNA expression in cohorts from Brazil (G), Haiti (H), and India (I).The Mann–Whitney test was used to assess significance between 2 groups. The Kruskal–Wallis test was used to assess significance of differences across more than 2 groups, followed by Dunn’s multiple comparison tests correction. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.005 with respect to TB.

Figure 2

Individual normalized expression values of (A) DOCK9, (B) EPHA4, and (C) NPC2 mRNA in blood in a South African cohort (E-MTAB-8290). The diagnostic groups comprise adults with respiratory symptoms self-presenting for investigation of pulmonary TB who were ultimately diagnosed as having TB or not. S-NTB: symptomatic adults showing no laboratorial evidence of active TB disease, regardless of the history of known exposure to a TB index case. TB = active tuberculosis. The Mann–Whitney test was used to assess significance between two groups: *** p-value < 0.005, **** p-value < 0.001.

Figure 3

Individual normalized mRNA expression values of (A)DOCK9, (B) EPHA4, and (C) NPC2 mRNA in blood. Diagnostic groups comprise: controls; TB = active tuberculosis; aSARC = active sarcoidosis; naSARC = non-active sarcoidosis; LC = lung cancer; PN = pneumonia. The Kruskal–Wallis test was used to assess significance of differences across more than 2 groups, followed by Dunn’s multiple comparison tests correction. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.005 with respect to TB. ● single LC and PN patients presenting an NPC2^high profile. UK = United Kingdom.

Figure 4

Changes in DOCK9, EPHA4, and NPC2 expression during progression to active TB. Median values (interquartile ranges). The same samples were arranged according to the time from exposure to index case (A–C) or time to development of active TB (D–F). Expression values were obtained from the dataset GSE94438. The Mann–Whitney test was used to assess significance between two groups. The Kruskal–Wallis test was used to assess significance of global differences across more than two groups, followed by Dunn’s multiple comparison tests correction. Small bar: p-value comparing groups. # p-value < 0.1; * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.005.

Figure 5

Effects of anti-tuberculosis chemotherapy on the expression of DOCK9, EPHA4, and NPC2 mRNAs in blood samples from TB patients. Transcriptomic data were obtained from two publicly available datasets, China (GSE54992, A–C) and South Africa (GSE89403, D–F). Follow-up samples from TB-treated (TBtt) patients were collected before treatment initiation (day 0), at different time intervals during therapy (GSE54992: 3 months [m]; GSE89403: 7 days, and 1 month), and at the end of therapy (GSE54992: and GSE89403: 6 months). The Friedman test was used to assess significance of the longitudinal analysis among TB cases during treatment. The Mann–Whitney test was used to assess significance between two groups vs. LTBI or vs. control. The Kruskal–Wallis test was used to assess significance of global differences across more than two groups, followed by Dunn’s multiple comparison tests correction. p-values obtained comparing TBtt time intervals against non-TB cases (LTBI and control) are shown in black (●). p-values obtained comparing expression changes in TBtt patients during treatment are shown in blue (●). * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.005.