Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

Abstract

Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV(-)) and co-infected (HIV(+)) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV(-) individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV(+) individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV(-) TB, 0.95 for HIV(+) TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB.

Keywords: Biomarker; Diagnostics; Host proteins; Tuberculosis.

Fig. 1

Study design outline. An initial set of HIV-samples was used to identify proteins differentially expressed in TB compared to controls (M. tuberculosis uninfected and latent M. tuberculosis infection). (A). The expression of a selected subset of 87 proteins was confirmed using an independent set of HIV⁻ and HIV⁺ samples (B). Samples were excluded from the verification analysis if their HIV status remained unidentified (N = 11) or if there were technical issues (N = 13). The remaining samples were used to verify the protein expression in sample groups stratified by HIV status (C) as well as the ability of the biomarker candidates and their combinations to discriminate between TB and other respiratory diseases (D). The classification of TB versus other respiratory diseases was established via nested cross-validation using the classification phase sample data split at random into 5 test and training sets. TB = active tuberculosis, LTBI = latent M. tuberculosis infection, NI = non-infected, ORD = other respiratory diseases.

Fig. 2

Differentially expressed proteins in sera of HIV⁻ subjects with active tuberculosis (TB) relative to controls (M. tuberculosis uninfected and latent M. tuberculosis infection). The differentially expressed proteins are indicated by dots and categorized by functional groups. Intensity values per protein were normalized and the ratios derived are displayed in logarithmic scale (y-axis). Mean and standard deviation of the expression ratio of TB versus controls are shown.

Fig. 3

Differential expression of candidate TB biomarkers in sera. Protein expression ratios expressed as fold change in subjects with active tuberculosis (TB) relative to uninfected controls (NI) and latent M. tuberculosis infection (LI), or relative to subjects with other respiratory diseases (ORD), in HIV⁺ or HIV⁻ infected groups. The numerators and denominators per comparison and the color scale are indicated. The ratio range was from 0.2 to 6.6. The HIV⁻ and HIV⁺ panel members are indicated by * and ^† respectively.

Fig. 4

Discrimination of active tuberculosis from other respiratory diseases. Area under the receiver operating curve (AUC) is shown for the biomarker panels in HIV⁻ (A) and HIV⁺ (B) subjects.