Background Early and accurate diagnosis followed by timely treatment are the key prerequisites to fight tuberculosis (TB) and reduce its global burden. Despite scientific advances, the rapid and correct diagnosis of both pulmonary and extrapulmonary tuberculosis remains a challenge due to traditional reliance on detection of the elusive bacilli. Mycobacterium tuberculosis (Mtb)-specific host immune activation and cytokine production have shown significant promise as alternative means of detecting and distinguishing active disease from latent infection. We queried the diagnostic ability of phenotypic markers on Mtb-specific cytokine-producing immune cell subsets for identifying active tuberculosis. Methods Subjects belonging to the following groups were recruited – pulmonary and extrapulmonary TB, latent TB, cured TB, sick controls and healthy controls. Polychromatic flow cytometry was used to identify host immune biomarkers in an exploratory cohort comprising 56 subjects using peripheral blood mononuclear cells. Clinical performance of the identified biomarker was evaluated using whole blood in a blinded validation cohort comprising 165 individuals. Results Cytokine secreting frequencies of Mtb-specific CD4 + T cells with CD38 +CD27 – phenotype clearly distinguished infected individuals with active tuberculosis from those without disease. TNF-α secretion from CD38 +CD27 –CD4 + T cells upon stimulation with ESAT6/CFP10 peptides had the best diagnostic accuracy at a cut-off of 9.91% [exploratory: 96.67% specificity, 88.46% sensitivity; validation: 96.15% specificity, 90.16% sensitivity]. Additionally, this subset differentiated treatment-naive TB patients from individuals cured of TB following completion of anti-tuberculosis therapy. Conclusions Mtb-specific CD38 +CD27 –TNF-α +CD4 + T cell subset is a robust biomarker both for diagnosing tuberculosis and assessing cure.

RATIONALE. Early and accurate diagnosis followed by timely treatment are the key prerequisites to fight tuberculosis (TB) and reduce its global burden. Despite scientific advances, the rapid and correct diagnosis of both pulmonary and extrapulmonary tuberculosis remains a challenge due to traditional reliance on detection of the elusive bacilli. Mycobacterium tuberculosis (Mtb)-specific host immune activation and cytokine production has shown significant promise as an alternative means of detecting and distinguishing active disease from latent infection. OBJECTIVE. Phenotypic characteristics of Mtb-specific cytokine-producing immune cell subsets were investigated and queried for their diagnostic ability in identifying active tuberculosis. METHODS. Subjects belonging to the following groups were recruited - pulmonary, extrapulmonary, latent TB, cured TB, sick controls and healthy controls. Polychromatic flow cytometry was used to identify host immune biomarkers in an exploratory cohort comprising 56 subjects using peripheral blood mononuclear cells. Clinical performance of the identified biomarker was evaluated using whole blood in a blinded validation cohort comprising 165 individuals. FINDINGS. Frequencies of Mtb-specific CD4+ T cells of the phenotype CD38+CD27- clearly distinguished patients with active tuberculosis from individuals without the disease. CD38+CD27-CD4+ T cells secreting TNF-α upon stimulation with ESAT6/CFP10 peptides had the best diagnostic accuracy at a cut-off of 9.91% [exploratory: 96.67% specificity, 88.46% sensitivity; validation: 96.15% specificity, 90.16% sensitivity]. Additionally, this subset differentiated treatment-naive TB patients from individuals cured of TB following completion of anti-tuberculosis therapy. INTERPRETATION. Mtb-specific CD38+CD27-TNF-α+CD4+ T cell subset is a robust biomarker for TB diagnosis and can determine cure.

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