Host Cytokine Responses Induced after Overnight Stimulation with Novel M. tuberculosis Infection Phase-Dependent Antigens Show Promise as Diagnostic Candidates for TB Disease

Abstract

BackgroundWe 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.MethodsWe 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.ResultsThe 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.ConclusionUnstimulated 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.

New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles

“…For comparative purposes we also included ten additional Mtb antigens identified previously (Rv0867, Rv1009, Rv1733, Rv1737, Rv2029, Rv2032, Rv2034, Rv2389, Rv2450, and Rv3353)2324414748495051525354. Diluted whole blood samples were stimulated for six days and the cytokine levels were assessed by a seven-plex assay (data shown only for IFN-γ, IL-17, TNF-α and IP-10 Fig.…”

Evaluation of cytokine responses against novel Mtb antigens as diagnostic markers for TB disease

“…The reconstituted antigens were then diluted in sterile 1Â PBS, mixed with undiluted whole blood from each study participant at a final concentration of 10 mg/ml, and incubated overnight (20e24 h) in 24-well tissue culture plates (Corning Ò Corstar Ò , Sigma) as previously described. 18 Sterile 1Â PBS (Lonza, Cat #: 17-517Q) was used as the negative control.…”

“…In a follow up to these studies, we evaluated the potential of some of these promising antigens to elicit a host response in a short term (overnight) whole blood assay compared to the long term (7-day) whole blood assay. 18 This study also evaluated the accuracy of some of these previously reported novel candidate antigens but in a larger study employing a short term (overnight), more field-friendly whole blood assay.…”

Regulation network of serum cytokines induced by tuberculosis-specific antigens reveals biomarkers for tuberculosis diagnosis

“…As such, the following methods are currently being used to search fornew TB biomarkers: cytokine array analyses of the sera from patients with active or latent TB infections compared to those from healthy individuals (Weiner et al, 2012;Liu et al, 2013); microarray analyses of the supernatants of centrifuged peripheral blood after stimulation with TB-specific antigens from patients with active or latent TB infections compared to that from healthy individuals (Frahm et al, 2011;Kellar et al, 2011;Murthy et al, 2011;Borgstrom et al, 2012;Skogstrand et al, 2012;Anbarasu et al, 2013;Armand et al, 2014;Essone et al, 2014); and extraction and culture of leukocytes from the peripheral blood of patients with active or latent TB infection and healthy individuals, followed by analysis of the differences insecreted cytokines before and after stimulation with TB-specific antigens (Rivera-Ordaz et al, 2012). However, consistent results have not been obtained using the aforementioned methods because the number of cytokines analyzed is often small and the types of assays usedto detect them vary between studies; thus, a more systematic approach for analyzing all cytokines is needed.…”