Background. Accurate assessment of treatment efficacy would facilitate clinical trials of new antituberculosis drugs. We hypothesized that early alterations in peripheral immunity could be measured ...by gene expression profiling in tuberculosis patients undergoing successful conventional combination treatment. Methods. Ex vivo blood samples from 27 pulmonary tuberculosis patients were assayed at diagnosis and during treatment. RNA was processed and hybridized to Affymetrix GeneChips, to determine expression of over 47 000 transcripts. Results. There were significant ≥2-fold changes in expression of >4000 genes during treatment. Rapid, largescale changes were detected, with down-regulated expression of 1261 genes within the first week, including inflammatory markers such as complement components C1q and C2. This was followed by slower changes in expression of different networks of genes, including a later increase in expression of B-cell markers, transcription factors, and signaling molecules. Conclusions. The fast initial down-regulation of expression of inflammatory mediators coincided with rapid killing of actively dividing bacilli, whereas slower delayed changes occurred as drugs acted on dormant bacilli and coincided with lung pathology resolution. Measurement of biosignatures during clinical trials of new drugs could be useful predictors of rapid bactericidal or sterilizing drug activity, and would expedite the licensing of new treatment regimens.
Although tuberculosis (TB) causes more deaths than any other pathogen, most infected individuals harbor the pathogen without signs of disease. We explored the metabolome of >400 small molecules in ...serum of uninfected individuals, latently infected healthy individuals and patients with active TB. We identified changes in amino acid, lipid and nucleotide metabolism pathways, providing evidence for anti-inflammatory metabolomic changes in TB. Metabolic profiles indicate increased activity of indoleamine 2,3 dioxygenase 1 (IDO1), decreased phospholipase activity, increased abundance of adenosine metabolism products, as well as indicators of fibrotic lesions in active disease as compared to latent infection. Consistent with our predictions, we experimentally demonstrate TB-induced IDO1 activity. Furthermore, we demonstrate a link between metabolic profiles and cytokine signaling. Finally, we show that 20 metabolites are sufficient for robust discrimination of TB patients from healthy individuals. Our results provide specific insights into the biology of TB and pave the way for the rational development of metabolic biomarkers for TB.
Persistent antigen stimulation in chronic infections has been associated with antigen-specific T cell dysfunction and upregulation of inhibitory receptors, including programmed cell death protein 1 ...(PD-1). Pulmonary tuberculosis (TB) disease is characterized by high levels of
(Mtb), yet the relationship between bacterial load, PD-1 expression, and Mtb-specific T cell function in human TB has not been well-defined. Using peripheral blood samples from adults with LTBI and with pulmonary TB disease, we tested the hypothesis that PD-1 expression is associated with bacterial load and functional capacity of Mtb-specific T cell responses. We found that PD-1 was expressed at significantly higher levels on Th1 cytokine-producing Mtb-specific CD4 T cells from patients with smear-positive TB, compared with smear-negative TB and LTBI, which decreased after completion of anti-TB treatment. By contrast, expression of PD-1 on Mtb-specific CD8 T cells was significantly lower than on Mtb-specific CD4 T cells and did not differ by Mtb infection and disease status.
stimulation of PBMC with Mtb antigens demonstrated that PD-1 is induced on proliferating Mtb-specific CD4 T cells and that Th1 cytokine production capacity is preferentially maintained within PD-1
proliferating CD4 T cells, compared with proliferating Mtb-specific CD4 T cells that lack PD-1 expression. Together, these data indicate that expression of PD-1 on Mtb-specific CD4 T cells is indicative of mycobacterial antigen exposure and identifies a population of effector cells with Th1 cytokine production capacity. These studies provide novel insights into the role of the PD-1 pathway in regulating CD4 and CD8 T cell responses in Mtb infection and provide rationale for future studies to evaluate PD-1 expression on antigen-specific CD4 T cells as a potential biomarker for bacterial load and treatment response in human TB.
...in South Africa, an estimated 30% of patients with tuberculosis do not initiate treatment.2 In 2020, the COVID-19 pandemic has further decreased the number of people diagnosed with, and treated ...for, tuberculosis by up to 50% in some of the highest-burden countries because of reallocation of resources from tuberculosis to COVID-19.1 The failure of tuberculosis diagnostic practices has come at the cost of lost lives, ongoing transmission, and exacerbated organ destruction. A WHO meeting in 20143 aimed to build consensus around four target product profiles for new active tuberculosis diagnostic tests: (1) a point-of-care non-sputum biomarker test for all forms of tuberculosis; (2) a point-of-care first contact test to triage patients requiring confirmatory testing; (3) a point-of-care sputum-based smear replacement test; and (4) a rapid drug susceptibility test for microscopy centres. Since publication of the WHO 2014 consensus paper, several promising whole-blood host mRNA signatures have been reported that need further prospective evaluation in appropriate clinical settings.4–7 In The Lancet Infectious Diseases, Long T Hoang and colleagues8 investigate six novel and three previously published transcriptomic signatures from whole blood from patients in secondary care clinical practice in England by use of microarray. Overly rigid application of target product profiles might do a disservice to improvement of the repertoire of diagnostic tests for tuberculosis, because they should be guidelines rather than strict decision points.
Type 2 diabetes mellitus (DM) is a major risk factor for developing tuberculosis (TB). TB-DM comorbidity is expected to pose a serious future health problem due to the alarming rise in global DM ...incidence. At present, the causal underlying mechanisms linking DM and TB remain unclear. DM is associated with elevated levels of oxidized low-density lipoprotein (oxLDL), a pathologically modified lipoprotein which plays a key role during atherosclerosis development through the formation of lipid-loaded foamy macrophages, an event which also occurs during progression of the TB granuloma. We therefore hypothesized that oxLDL could be a common factor connecting DM to TB. To study this, we measured oxLDL levels in plasma samples of healthy controls, TB, DM and TB-DM patients, and subsequently investigated the effect of oxLDL treatment on human macrophage infection with Mycobacterium tuberculosis (Mtb). Plasma oxLDL levels were significantly elevated in DM patients and associated with high triglyceride levels in TB-DM. Strikingly, incubation with oxLDL strongly increased macrophage Mtb load compared to native or acetylated LDL (acLDL). Mechanistically, oxLDL -but not acLDL- treatment induced macrophage lysosomal cholesterol accumulation and increased protein levels of lysosomal and autophagy markers, while reducing Mtb colocalization with lysosomes. Importantly, combined treatment of acLDL and intracellular cholesterol transport inhibitor (U18666A) mimicked the oxLDL-induced lysosomal phenotype and impaired macrophage Mtb control, illustrating that the localization of lipid accumulation is critical. Collectively, these results demonstrate that oxLDL could be an important DM-associated TB-risk factor by causing lysosomal dysfunction and impaired control of Mtb infection in human macrophages.
There is an urgent need for new tools for the rapid diagnosis of tuberculosis disease. We evaluated the potentials of 74 host markers as biomarkers for the immunological diagnosis of tuberculosis and ...monitoring of treatment response. Fifty-five individuals that presented with signs and symptoms requiring investigation for tuberculosis disease were prospectively recruited prior to clinical diagnosis, at a health centre in Cape Town, South Africa. Patients were later classified as having tuberculosis disease or other respiratory diseases (ORD) using a combination of clinical, radiological and laboratory findings. Out of 74 host markers that were evaluated in plasma samples from study participants using a multiplex platform, 18 showed potential as tuberculosis diagnostic candidates with the most promising being NCAM, CRP, SAP, IP-10, ferritin, TPA, I-309, and MIG, which diagnosed tuberculosis disease individually, with area under the ROC curve ≥0.80. Six-marker biosignatures containing NCAM diagnosed tuberculosis disease with a sensitivity of 100% (95%CI, 86.3-100%) and specificity of 89.3% (95%CI, 67.6-97.3%) irrespective of HIV status, and 100% accuracy in the absence of HIV infection. Furthermore, the concentrations of 11 of these proteins changed with treatment, thereby indicating that they may be useful in monitoring of the response to tuberculosis treatment. Our findings have potential to be translated into a point-of-care screening test for tuberculosis, after future validation studies.
•Initial treatment of tuberculosis (TB) leads to cellular stress and a metabolic shift.•Levels of binding immunoglobulin protein (BiP) are higher after 1 week of TB treatment compared to levels at ...diagnosis.•BiP levels decrease after successful TB treatment.•End of TB treatment BiP levels are similar to those of healthy controls.
An increased Mycobacterium tuberculosis burden inside the host leads to higher demand of response proteins. This in turn results in metabolic shift and cellular stress, which is caused by the accumulation and trafficking of these proteins within the endoplasmic reticulum (ER). To resolve this, cells trigger the unfolded protein response (UPR), which is mainly mediated by binding immunoglobulin protein (BiP)/glucose-regulated protein 78 (GRP78) chaperone, and this in turn upregulates its transcription. This chaperone protein facilitates proper protein folding within the ER; however, it can also be passively secreted into the extracellular environment or be expressed on cell surfaces attached to anchor proteins and transmembrane proteins. This notion has been shown in studies on chronic inflammation, including cancer and arthritis, with the detection of BiP-specific antibodies from different sample types. The present study analysed secreted BiP from plasma samples collected from healthy participants and patients with newly diagnosed tuberculosis (TBdx), seen over the course of TB treatment at week 1 (W1), month 2 (M2), and month 6 (M6). The results revealed that during the initial TB disease and treatment period, cells are subjected to stress conditions resulting in metabolic shifts, which lead to the secretion of the intracellular UPR-mediating chaperone protein, BiP. This was indicated by mean differences between TBdx (mean 40.88ng/ml) and W1 (68.57ng/ml) in the TB participant groups. However, no difference was observed between the healthy group (mean 42.64ng/ml) and TBdx group (mean 40.88ng/ml). Analysis of paired time-point visits revealed increased BiP secretion during early TB treatment. The detection of BiP in plasma samples was found to decrease after successful TB treatment to levels comparable to those in the healthy controls. Evaluation of BiP levels in larger TB treatment studies may lead to the identification of a new target for early TB diagnosis and host-directed immunotherapy.
There is an urgent need for new tools for the early diagnosis of TB disease and monitoring of the response to treatment, especially in resource-constrained settings. We investigated the usefulness of ...host markers detected in saliva as candidate biomarkers for the immunological diagnosis of TB disease and monitoring of treatment response.
We prospectively collected saliva samples from 51 individuals that presented with signs and symptoms suggestive of TB disease at a health centre in Cape Town, South Africa, prior to the establishment of a clinical diagnosis. Patients were later classified as having TB disease or other respiratory disease (ORD), using a combination of clinical, radiological and laboratory findings. We evaluated the concentrations of 69 host markers in saliva samples using a multiplex cytokine platform, and assessed the diagnostic potentials of these markers by receiver operator characteristics (ROC) curve analysis, and general discriminant analysis.
Out of the 51 study participants, 18 (35.4%) were diagnosed with TB disease and 12 (23.5%) were HIV infected. Only two of the 69 host markers that were evaluated (IL-16 and IL-23) diagnosed TB disease individually with area under the ROC curve ≥0.70. A five-marker biosignature comprising of IL-1β, IL-23, ECM-1, HCC1 and fibrinogen diagnosed TB disease with a sensitivity of 88.9% (95% CI,76.7-99.9%) and specificity of 89.7% (95% CI, 60.4-96.6%) after leave-one-out cross validation, regardless of HIV infection status. Eight-marker biosignatures performed with a sensitivity of 100% (95% CI, 83.2-100%) and specificity of 95% (95% CI, 68.1-99.9%) in the absence of HIV infection. Furthermore, the concentrations of 11 of the markers changed during treatment, indicating that they may be useful in monitoring of TB treatment response.
We have identified novel salivary biosignatures which may be useful in the diagnosis of TB disease and monitoring of the response to TB treatment. Our findings require further validation in larger studies before these biosignatures could be considered for point-of-care screening test development.
Stroke is a common complication in children with tuberculous meningitis (TBM). Host proteins may give us insight into the mechanisms of stroke in TBM and serve as biomarkers for detection of stroke, ...however, they have not been widely explored. In this study, we compared the concentrations of cerebrospinal fluid (CSF) and serum proteins between children who had TBM-related stroke and children with TBM without stroke.
We collected CSF and serum from 47 children consecutively admitted to the Tygerberg Academic Hospital in Cape Town, South Africa between November 2016, and November 2017, on suspicion of having TBM. A multiplex platform was used to measure the concentrations of 69 host proteins in CSF and serum from all study participants.
After classification of study participants, 23 (48.9%) out of the 47 study participants were diagnosed with TBM, of which 14 (60.9%) demonstrated radiological arterial ischemic infarction. The levels of lipocalin-2, sRAGE, IP-10/ CXCL10, sVCAM-1, MMP-1, and PDGF-AA in CSF samples and the levels of D-dimer, ADAMTS13, SAA, ferritin, MCP-1/ CCL2, GDF-15 and IL-13 in serum samples were statistically different between children who had TBM-related stroke and children with TBM without stroke. After correcting for multiple testing, only the levels of sVCAM-1, MMP-1, sRAGE, and IP-10/ CXCL10 in CSF were statistically different between the two groups. CSF and serum protein biosignatures indicated stroke in children diagnosed with TBM with up to 100% sensitivity and 88.9% specificity.
Serum and CSF proteins may serve as biomarkers for identifying individuals with stroke amongst children diagnosed with TBM at admission and may guide us to understand the biology of stroke in TBM. This was a pilot study, and thus further investigations in larger studies are needed.
•33 biomarkers were evaluated in saliva samples from patients with presumed TB disease.•A seven-marker saliva biosignature has potential in the diagnosis TB disease.•Cytokines detected in saliva may ...be useful in monitoring of TB treatment response.
There is an urgent need for new tools for the rapid diagnosis of tuberculosis (TB) disease in resource-constrained settings. Tests based on host immunological biomarkers maybe useful, especially if based on easily available samples. We investigated host biomarkers detected in saliva samples from individuals with suspected pulmonary TB disease, as tools for the diagnosis of TB disease and monitoring of the response to treatment.
We collected saliva samples from 104 individuals that presented with symptoms requiring investigation for TB disease at a primary health care clinic in the outskirts of Cape Town, South Africa, prior to assessment for TB disease. We evaluated the concentrations of 33 host markers in stored saliva samples using a multiplex cytokine platform. Using a combination of clinical, radiological and laboratory results and a pre-established diagnostic algorithm, participants were later classified as having TB disease or other respiratory diseases (ORD). The diagnostic potentials of individual analytes were analysed by the receiver operator characteristics curve approach while the predictive abilities of combinations of analytes for TB disease were analysed by general discriminant analysis, with leave-one-out cross validation.
Of the 104 individuals enrolled, 32 were pulmonary TB cases. There were significant differences in the levels of 10 of the markers investigated between the patients with TB disease and those with ORDs. However, the optimal diagnostic biosignature was a seven-marker combination of salivary CRP, ferritin, serum amyloid P, MCP-1, alpha-2-macroglobulin, fibrinogen and tissue plasminogen activator. This biosignature diagnosed TB disease with a sensitivity of 78.1% (95% CI, 59.6–90.1%) and specificity of 83.3% (95% CI, 72.3–90.7%) after leave-one-out cross validation. When compared to baseline levels, the concentrations of 9 markers including granzyme A, MCP-1, IL-1β, IL-9, IL-10, IL-15, MIP-1β, ferritin and serum amyloid A changed significantly by months 2 or 6 after initiation of TB treatment, thereby indicating that they might be useful in monitoring the response to TB treatment.
We have identified candidate biomarkers in saliva, which may be useful in the diagnosis of TB disease and monitoring of the response to TB treatment. These results require further validation in larger studies.
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