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 verif… Show more
“…Hence, our data are consistent with the notion that a single biomarker will not be sufficient for distinguishing TB status in various patient groups (6,32). Further consistent with prior studies by us and others, we observed qualitatively different host responses in HIV coinfected compared with HIV uninfected TB patients (32)(33)(34), as well as quantitatively different Ab levels in proportion to extent of disease (6,34).…”
Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n ؍ 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n ؍ 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n ؍ 244) and heretofore untested samples (n ؍ 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (
“…Hence, our data are consistent with the notion that a single biomarker will not be sufficient for distinguishing TB status in various patient groups (6,32). Further consistent with prior studies by us and others, we observed qualitatively different host responses in HIV coinfected compared with HIV uninfected TB patients (32)(33)(34), as well as quantitatively different Ab levels in proportion to extent of disease (6,34).…”
Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n ؍ 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n ؍ 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n ؍ 244) and heretofore untested samples (n ؍ 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (
“…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. 3Changes in plasma protein expression after TST-positive conversion.…”
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.
“…The ability to perform label-free quantification (LFQ) methods for an unbiased readout of novel targets reveals a better insight into the global cellular defects inherent in primary immune cells during disease states. These MS methods for the global quantification of proteins have allowed for the discovery of hundreds of candidate biomarkers of disease, demonstrated by a recent study of host protein biomarkers identifying active tuberculosis in HIV uninfected and co-infected individuals (52). In our study, we utilized this as a powerful discovery tool to assess the “per cell” defect of purified CD4+ T cells from HIV patients (S.…”
HIV+ patients have an increased risk for tuberculosis disease despite clinical management with ARTs. We established a culture model of Mtb-infection in PBMCs from HIV+ PPD+ donors on suppressive ART (median 6.4 years) with negligible viral loads (median <50 copies/mL) and stable CD4+ T cell counts (517 cells/mm^3). We observed that HIV+ patient lymphocytes harbored a recruitment defect to Mtb-infected macrophages. To investigate these immune defects on a per cell basis, purified CD4+ T cells from HIV patients were assessed by label-free quantification protein mass spectrometry. CD4+ T cells from HIV patients displayed diminished nucleoprotein levels – notably of histone variant H2a.Z and ribonucleoprotein A1. Only within healthy donors, transcriptional regulatory histone variant H2a.Z expression was correlated to the extent of IFN-γ induction upon Mtb-infection. Our findings may explain why HIV patients exhibit prolonged immune cell dysfunction despite suppressive ART, and implicate a per cell defect of CD4+ T cells.
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