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Production of TNF-alpha, IL-12(p40) and IL-17 can discriminate between active TB disease and latent infection in a West African cohort.

Sutherland JS, de Jong BC, Jeffries DJ, Adetifa IM, Ota MO - PLoS ONE (2010)

Bottom Line: We found that TNF-alpha production following EC stimulation and TNF-alpha and IL-12(p40) following TB10.4 stimulation were significantly higher from TB cases compared to TST+ HHC, while production of IFN-gamma and IL-13 were significantly higher from TST+ compared to TST- HHC following PPD or EC stimulation.We also saw a tendency for TB cases infected with M. africanum to have increased TNF-alpha and IL-10 production compared to those infected with M. tuberculosis.Our results provide further insight into the pathogenesis of tuberculosis and may enhance the specificity of the currently available diagnostic tests, particularly for diagnosis of active TB.

View Article: PubMed Central - PubMed

Affiliation: Bacterial Diseases Programme, Medical Research Council Laboratories, Banjul, The Gambia. jsutherland@mrc.gm

ABSTRACT

Background: Mycobacterium tuberculosis (MTb) infects approximately 2 billion people world-wide resulting in almost 2 million deaths per year. Determining biomarkers that distinguish different stages of tuberculosis (TB) infection and disease will provide tools for more effective diagnosis and ultimately aid in the development of new vaccine candidates. The current diagnostic kits utilising production of IFN-gamma in response to TB antigens can detect MTb infection but are unable to distinguish between infection and disease. The aim of this study was to assess if the use of a longer term assay and the analysis of multiple cytokines would enhance diagnosis of active TB in a TB-endemic population.

Methods: We compared production of multiple cytokines (TNF-alpha, IFN-gamma, IL-10, IL-12(p40), IL-13, IL-17 and IL-18) following long-term (7 days) stimulation of whole-blood with TB antigens (ESAT-6/CFP-10 (EC), PPD or TB10.4) from TB cases (n = 36) and their Mycobacterium-infected (TST+; n = 20) or uninfected (TST-; n = 19) household contacts (HHC).

Results and conclusions: We found that TNF-alpha production following EC stimulation and TNF-alpha and IL-12(p40) following TB10.4 stimulation were significantly higher from TB cases compared to TST+ HHC, while production of IFN-gamma and IL-13 were significantly higher from TST+ compared to TST- HHC following PPD or EC stimulation. Combined analysis of TNF-alpha, IL-12(p40) and IL-17 following TB10.4 stimulation resulted in 85% correct classification into TB cases or TST+ HHC. 74% correct classification into TST+ or TST- HHC was achieved with IFN-gamma alone following TB10.4 stimulation (69% following EC) and little enhancement was seen with additional cytokines. We also saw a tendency for TB cases infected with M. africanum to have increased TNF-alpha and IL-10 production compared to those infected with M. tuberculosis. Our results provide further insight into the pathogenesis of tuberculosis and may enhance the specificity of the currently available diagnostic tests, particularly for diagnosis of active TB.

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Flow chart demonstrating how cytokine production following TB10.4 stimulation will improve diagnosis of active TB.At the moment, patients with suspected TB may be confirmed by analysis of chest x-ray (CXR) plus sputum culture positivity in laboratories which have these capabilities. Analysis of TNF-α, IL-12(p40) and IL-17 production following TB10.4 stimulation should enhance classification into active disease or latent infection in laboratories without culture facilities and also for patients with suspected TB but smear/culture negative results (such as HIV-positive patients or extrapulmonary TB patients). These findings should be further validated in a short-term assay and in difficult to diagnose patient groups.
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pone-0012365-g004: Flow chart demonstrating how cytokine production following TB10.4 stimulation will improve diagnosis of active TB.At the moment, patients with suspected TB may be confirmed by analysis of chest x-ray (CXR) plus sputum culture positivity in laboratories which have these capabilities. Analysis of TNF-α, IL-12(p40) and IL-17 production following TB10.4 stimulation should enhance classification into active disease or latent infection in laboratories without culture facilities and also for patients with suspected TB but smear/culture negative results (such as HIV-positive patients or extrapulmonary TB patients). These findings should be further validated in a short-term assay and in difficult to diagnose patient groups.

Mentions: PPD stimulation resulted in differences in production of most cytokines between TB cases and TST− contacts but also a significantly higher level of IL-17 from TST+ compared to TST− contacts. IL-17 was also shown to enhance discrimination between TB cases and TST+ contacts following TB10.4 stimulation. Recent studies have suggested a role for IL-17 in protection against disease progression [8] with IFN-γ being shown to contribute to the regulation of the IL-17 producing cells [23]. We have previously shown that IL-17 is significantly decreased in TB cases compared to household contacts following overnight stimulation with both EC and PPD [24]. These differences presumably reflect the generation of a memory response to the TB antigens in the long-term culture compared to analysis of the immediate effector functions in overnight cultures. Therefore, in terms of development of a biosignature for active TB, our results suggest that long-term culture is required to distinguish between TB cases and TST+ contacts in a TB-endemic setting where the TST+ contacts have a relatively large pool of MTb-specific cells. The use of short-term assays will preferentially detect IFN-γ production by primed T cells [25], thus will show increased levels for both subjects with active disease and those individuals who have recently acquired infection thereby providing a potential explanation for lack of discrimination of active disease based on IFN-γ production following short-term antigenic stimulation. This is not to say that short-term assays won't be beneficial for detection of active disease in future, and indeed, are more clinically desirable than longer assays. Preliminary data from a recent study in South Africa did show differentiation between active disease and latent infection using short-term stimulation by analysing multiple cytokine/chemokine markers [26] but this will need to be validated in a larger cohort and at different sites in Africa due to genetic differences seen in response to TB antigens [27]. Our data shows that TB10.4 stimulation followed by multi-cytokine analysis allows discrimination between active disease and latent infection and should enhance classification of subjects, particularly those who are difficult to diagnose (ie HIV-positive or extrapulmonary TB) and at sites which lack x-ray and/or culture facilities (Fig. 4). Development of field-friendly, rapid diagnostics is essential in the fight against TB and analysis of cytokine responses following TB10.4 stimulation is promising; however it firstly needs to be validated in a short-term assay (Fig. 4). The relative use of peripheral blood IGRAs for diagnosis of active TB does appear to depend not only on the extent of pulmonary involvement but on the disease severity with low levels of cytokines (at least IFN-γ) present in more advanced disease [12]. While this may indicate less reactivity, it is more likely that the cytokine producing cells are simply required at the site of infection thus reducing the benefits of blood as a tool for diagnostics. Clearly more work is required but our results and others indicate the importance of generating an algorithm incorporating multiple factors that can accurately determine the TB status of an individual [28].


Production of TNF-alpha, IL-12(p40) and IL-17 can discriminate between active TB disease and latent infection in a West African cohort.

Sutherland JS, de Jong BC, Jeffries DJ, Adetifa IM, Ota MO - PLoS ONE (2010)

Flow chart demonstrating how cytokine production following TB10.4 stimulation will improve diagnosis of active TB.At the moment, patients with suspected TB may be confirmed by analysis of chest x-ray (CXR) plus sputum culture positivity in laboratories which have these capabilities. Analysis of TNF-α, IL-12(p40) and IL-17 production following TB10.4 stimulation should enhance classification into active disease or latent infection in laboratories without culture facilities and also for patients with suspected TB but smear/culture negative results (such as HIV-positive patients or extrapulmonary TB patients). These findings should be further validated in a short-term assay and in difficult to diagnose patient groups.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2927558&req=5

pone-0012365-g004: Flow chart demonstrating how cytokine production following TB10.4 stimulation will improve diagnosis of active TB.At the moment, patients with suspected TB may be confirmed by analysis of chest x-ray (CXR) plus sputum culture positivity in laboratories which have these capabilities. Analysis of TNF-α, IL-12(p40) and IL-17 production following TB10.4 stimulation should enhance classification into active disease or latent infection in laboratories without culture facilities and also for patients with suspected TB but smear/culture negative results (such as HIV-positive patients or extrapulmonary TB patients). These findings should be further validated in a short-term assay and in difficult to diagnose patient groups.
Mentions: PPD stimulation resulted in differences in production of most cytokines between TB cases and TST− contacts but also a significantly higher level of IL-17 from TST+ compared to TST− contacts. IL-17 was also shown to enhance discrimination between TB cases and TST+ contacts following TB10.4 stimulation. Recent studies have suggested a role for IL-17 in protection against disease progression [8] with IFN-γ being shown to contribute to the regulation of the IL-17 producing cells [23]. We have previously shown that IL-17 is significantly decreased in TB cases compared to household contacts following overnight stimulation with both EC and PPD [24]. These differences presumably reflect the generation of a memory response to the TB antigens in the long-term culture compared to analysis of the immediate effector functions in overnight cultures. Therefore, in terms of development of a biosignature for active TB, our results suggest that long-term culture is required to distinguish between TB cases and TST+ contacts in a TB-endemic setting where the TST+ contacts have a relatively large pool of MTb-specific cells. The use of short-term assays will preferentially detect IFN-γ production by primed T cells [25], thus will show increased levels for both subjects with active disease and those individuals who have recently acquired infection thereby providing a potential explanation for lack of discrimination of active disease based on IFN-γ production following short-term antigenic stimulation. This is not to say that short-term assays won't be beneficial for detection of active disease in future, and indeed, are more clinically desirable than longer assays. Preliminary data from a recent study in South Africa did show differentiation between active disease and latent infection using short-term stimulation by analysing multiple cytokine/chemokine markers [26] but this will need to be validated in a larger cohort and at different sites in Africa due to genetic differences seen in response to TB antigens [27]. Our data shows that TB10.4 stimulation followed by multi-cytokine analysis allows discrimination between active disease and latent infection and should enhance classification of subjects, particularly those who are difficult to diagnose (ie HIV-positive or extrapulmonary TB) and at sites which lack x-ray and/or culture facilities (Fig. 4). Development of field-friendly, rapid diagnostics is essential in the fight against TB and analysis of cytokine responses following TB10.4 stimulation is promising; however it firstly needs to be validated in a short-term assay (Fig. 4). The relative use of peripheral blood IGRAs for diagnosis of active TB does appear to depend not only on the extent of pulmonary involvement but on the disease severity with low levels of cytokines (at least IFN-γ) present in more advanced disease [12]. While this may indicate less reactivity, it is more likely that the cytokine producing cells are simply required at the site of infection thus reducing the benefits of blood as a tool for diagnostics. Clearly more work is required but our results and others indicate the importance of generating an algorithm incorporating multiple factors that can accurately determine the TB status of an individual [28].

Bottom Line: We found that TNF-alpha production following EC stimulation and TNF-alpha and IL-12(p40) following TB10.4 stimulation were significantly higher from TB cases compared to TST+ HHC, while production of IFN-gamma and IL-13 were significantly higher from TST+ compared to TST- HHC following PPD or EC stimulation.We also saw a tendency for TB cases infected with M. africanum to have increased TNF-alpha and IL-10 production compared to those infected with M. tuberculosis.Our results provide further insight into the pathogenesis of tuberculosis and may enhance the specificity of the currently available diagnostic tests, particularly for diagnosis of active TB.

View Article: PubMed Central - PubMed

Affiliation: Bacterial Diseases Programme, Medical Research Council Laboratories, Banjul, The Gambia. jsutherland@mrc.gm

ABSTRACT

Background: Mycobacterium tuberculosis (MTb) infects approximately 2 billion people world-wide resulting in almost 2 million deaths per year. Determining biomarkers that distinguish different stages of tuberculosis (TB) infection and disease will provide tools for more effective diagnosis and ultimately aid in the development of new vaccine candidates. The current diagnostic kits utilising production of IFN-gamma in response to TB antigens can detect MTb infection but are unable to distinguish between infection and disease. The aim of this study was to assess if the use of a longer term assay and the analysis of multiple cytokines would enhance diagnosis of active TB in a TB-endemic population.

Methods: We compared production of multiple cytokines (TNF-alpha, IFN-gamma, IL-10, IL-12(p40), IL-13, IL-17 and IL-18) following long-term (7 days) stimulation of whole-blood with TB antigens (ESAT-6/CFP-10 (EC), PPD or TB10.4) from TB cases (n = 36) and their Mycobacterium-infected (TST+; n = 20) or uninfected (TST-; n = 19) household contacts (HHC).

Results and conclusions: We found that TNF-alpha production following EC stimulation and TNF-alpha and IL-12(p40) following TB10.4 stimulation were significantly higher from TB cases compared to TST+ HHC, while production of IFN-gamma and IL-13 were significantly higher from TST+ compared to TST- HHC following PPD or EC stimulation. Combined analysis of TNF-alpha, IL-12(p40) and IL-17 following TB10.4 stimulation resulted in 85% correct classification into TB cases or TST+ HHC. 74% correct classification into TST+ or TST- HHC was achieved with IFN-gamma alone following TB10.4 stimulation (69% following EC) and little enhancement was seen with additional cytokines. We also saw a tendency for TB cases infected with M. africanum to have increased TNF-alpha and IL-10 production compared to those infected with M. tuberculosis. Our results provide further insight into the pathogenesis of tuberculosis and may enhance the specificity of the currently available diagnostic tests, particularly for diagnosis of active TB.

Show MeSH
Related in: MedlinePlus