Host markers in QuantiFERON supernatants differentiate active TB from latent TB infection: preliminary report

Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, Cape Town, Western Cape Province, Tygerberg 7505, South Africa.
BMC Pulmonary Medicine (Impact Factor: 2.4). 06/2009; 9(1):21. DOI: 10.1186/1471-2466-9-21
Source: PubMed

ABSTRACT Interferon gamma release assays, including the QuantiFERON TB Gold In Tube (QFT) have been shown to be accurate in diagnosing Mycobacterium tuberculosis infection. These assays however, do not discriminate between latent TB infection (LTBI) and active TB disease.
We recruited twenty-three pulmonary TB patients and 34 household contacts from Cape Town, South Africa and performed the QFT test. To investigate the ability of new host markers to differentiate between LTBI and active TB, levels of 29 biomarkers in QFT supernatants were evaluated using a Luminex multiplex cytokine assay.
Eight out of 29 biomarkers distinguished active TB from LTBI in a pilot study. Baseline levels of epidermal growth factor (EGF) soluble CD40 ligand (sCD40L), antigen stimulated levels of EGF, and the background corrected antigen stimulated levels of EGF and macrophage inflammatory protein (MIP)-1beta were the most informative single markers for differentiation between TB disease and LTBI, with AUCs of 0.88, 0.84, 0.87, 0.90 and 0.79 respectively. The combination of EGF and MIP-1beta predicted 96% of active TB cases and 92% of LTBIs. Combinations between EGF, sCD40L, VEGF, TGF-alpha and IL-1alpha also showed potential to differentiate between TB infection states. EGF, VEGF, TGF-alpha and sCD40L levels were higher in TB patients.
These preliminary data suggest that active TB may be accurately differentiated from LTBI utilizing adaptations of the commercial QFT test that includes measurement of EGF, sCD40L, MIP-1beta, VEGF, TGF-alpha or IL-1alpha in supernatants from QFT assays. This approach holds promise for development as a rapid diagnostic test for active TB.

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Available from: Paul van Helden, Sep 26, 2015
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    • "Besides IFN-γ, IP-10 (IFN-γ induced protein 10) was found useful for detection of M. tuberculosis infection using Mtb-specific stimulation [4] [9]. In addition , EGF (epidermal growth factor) and CCL4 (chemokine C-C motif ligand 4) also known as macrophage inflammatory protein-1β (MIP-1β) were informative markers for differentiation between TB disease and latent TB infection (LTBI) or ORD [10]. In contrast to other markers, IP-10 [11] and CCL4 [12] [13] are detected in whole blood even without antigen stimulation [9] [12]. "
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    ABSTRACT: Objective: Multi-center evaluation of a user-friendly lateral flow test for detection of IP-10 and CCL4 levels in Mycobacterium tuberculosis (Mtb) antigen-stimulated whole blood samples from tuberculosis (TB) suspects. Design and methods: A quantitative lateral flow (LF)-based assay platform was applied to detect chemokines IP-10 and CCL4. Chemokine quantitation was achieved using interference-free, fluorescent up-converting phosphor (UCP) labels. The new assays allowed worldwide shipping and storage without requiring a cold chain and were tested at seven institutes (including Ethiopia, Malawi, The Gambia, South Africa, Uganda and Namibia) employing portable lightweight readers for detection of the UCP label. At each site, clinical samples, confirmed TB and non-TB (i.e. other respiratory diseases (ORD)) cases, were collected and analyzed simultaneously with quality control (QC) human IP-10 or CCL4 standards. Results: Performance of the UCP-LF assay in Africa using QC standards indicated high robustness allowing quantitative detection between 100 and 100,000pg/mL. The optimized assays allowed successful determination of chemokine levels using 1μL whole blood sample from the locally recruited subjects with TB or ORD. Conclusion: This African multi-center trial further demonstrated the applicability of the low-tech and robust UCP-LF platform as a convenient quantitative assay for chemokine detection in whole blood. Ambient shipping and storage of all assay reagents and the availability of lightweight standalone readers were acknowledged as essential requirement for test implementation in particular in remote and resource-limited settings.
    Clinical biochemistry 09/2015; DOI:10.1016/j.clinbiochem.2015.08.013 · 2.28 Impact Factor
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    • "These tests represent indirect markers of Mycobacterium tuberculosis (M.tb) exposure and measure the cellular immune response against TB infection [5]. However, the sub-optimal sensitivity of QFT and the low specificity of TST remain critical issues in the identification of LTBI [7]. Although reports indicate interferon-gamma (IFN-␥) to be a key cytokine that is involved in the cell mediated immune response against TB infection [8], its measurement alone is not adequate for the accurate diagnosis of LTBI [9]. "
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    ABSTRACT: The tuberculin skin test (TST) and interferon-gamma release assays (IGRA), namely, the QuantiFERON-TB Gold test (QFT), remain the standard immunological diagnostic tools for latent tuberculosis (TB) infection (LTBI). However, the sub-optimal detection rates of both of these tests are major impediments in recognizing the population at risk. This study was aimed at evaluating additional cytokines besides interferon-gamma (IFN-γ) as biomarkers for improving LTBI diagnosis in the tribal population of Melghat, India. Seventy-four close TB contacts were stratified by QFT and TST results into: (i) QFT+/TST+ (n=26), (ii) QFT+/TST- (n=12), (iii) QFT-/TST- (n=35) and (iv) QFT-/TST+ (n=1) groups. A panel of cytokines (IL-6, IL-10, TNF-α and IL-2R) was then evaluated in antigen-stimulated QFT cell-free culture supernatants using IMMULITE-1000, an automated immunoassay analyzer. Cytokine estimation showed significantly higher levels of IL-6 in the QFT+/TST+ group, while significantly higher levels of IL-10 were found in the QFT-/TST- group. Correlation analysis identified a positive correlation between IL-6 and the QFT response (r=0.6723, P<0.0001), while a negative correlation was seen between QFT and IL-10 expression (r=-0.3271, P=0.0044). Similarly, IL-6 was positively correlated with TST levels (r=0.6631, P<0.0001), and conversely, a negative correlation was found between TST and IL-10 expression (r=-0.5698, P<0.0001). The positive and negative predictive values of IL-6 were found to be 92.59 and 93.33%, respectively, and the positive and negative predictive values of IL-10 were 96.55 and 91.18%, respectively. No significant impact of the demographic characteristics on cytokine positivity was observed. Our preliminary results suggest that the evaluation of additional cytokines in QFT cell-free culture supernatants may be valuable for the identification of LTBI. Combining IL-6 and IL-10 with QFT and/or TST could markedly improve the detection accuracy of LTBI. Our observations require investigation in larger well-characterized cohorts along with follow-up studies to further confirm the study outcome. Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.
    Journal of Infection and Public Health 03/2015; 8(4). DOI:10.1016/j.jiph.2015.02.003
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    • "However, IGRAs do not differentiate between active TB disease and latent infection and although useful in low incidence settings, they are only used for research purposes in high burden areas [1], [10], [11]. Alternative M.tb specific antigens [12]–[13], and alternative host markers other than IFN-γ in M.tb-specific antigen-stimulated whole blood culture assays have been investigated for TB diagnostic purposes [14], [15]. In one of these studies [15] we showed that the measurement of many host markers (including interferon-inducible protein (IP)-10, tumour necrosis factor (TNF)-α, IFN-γ, interleukin (IL)-12p40, transforming growth factor (TGF)-α, vascular endothelial growth factor (VEGF) and RANTES (CCL5)) upon stimulation with novel M.tb infection phase-dependent antigens (including Rv0081, Rv2032, Rv1737c, Rv2389c and Rv0867c) in culture supernatants showed potential in the diagnosis of TB disease [15]. "
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    ABSTRACT: Background We previously identified Mycobacterium tuberculosis (M.tb) antigen-induced host markers that showed promise as TB diagnostic candidates in 7-day whole blood culture supernatants. The aim of the present study was to evaluate the utility of these markers further, and cross-compare results with short-term antigen stimulated and unstimulated culture supernatants. Methods We recruited 15 culture confirmed TB cases and 15 non-TB cases from a high-TB endemic community in Cape Town, South Africa into a pilot case-control study from an on-going larger study. Blood samples collected from study participants were stimulated with 4 M.tb antigens that were previously identified as promising (ESAT6/CFP10 (early secreted), Rv2029c (latency), Rv2032 (latency) and Rv2389c (rpf)) in a 7-day or overnight culture assay. Supernatants were also collected form the standard QuantiFERON In Tube (QFT-IT) test. The levels of 26 host markers were evaluated in the three culture supernatants using the Luminex platform. Results The unstimulated levels of CRP, Serum amyloid P (SAP) and serum amyloid A (SAA) and ESAT-6/CFP-10 specific IP-10 and SAA were amongst the best discriminatory markers in all 3 assays, ascertaining TB with AUC of 72–84%. Four-marker models accurately classified up to 92%, 100% and 100% of study participants in the overnight, 7-day and Quantiferon culture supernatants, respectively, after leave-one-out cross validation. Conclusion Unstimulated and antigen-specific levels of CRP, SAA, IP-10, MMP-2 and sCD40L hold promise as diagnostic candidates for TB disease in short-term stimulation assays. Larger studies are required to validate these findings but the data suggest that antigen-specific cytokine production and in particular mutimarker biosignatures might contribute to future diagnostic strategies.
    PLoS ONE 07/2014; 9(7):e102584. DOI:10.1371/journal.pone.0102584 · 3.23 Impact Factor
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