ABSTRACT
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.
IMPACT OF THIS RESEARCH We identified and validated CD38+CD27−TNF-α+ as a robust biomarker with diagnostic accuracies >90% in both PBMCs and whole blood that can be translated into a reliable and cost-effective in vitro diagnostic test with ease. By not removing samples with insignificant immune response and instead classifying them as negative, our study represents a truly realistic assessment of the diagnostic accuracy of the identified biomarker in a clinical setting.
Competing Interest Statement
VS received grants from Department of Biotechnology, Government of India (BT/PR23695/MED/29/1195/2017 Dated 28/12/2017) and Beckman Coulter India Private Limited (SP/BECO-16-0001) outside the submitted work. MPA received salary from Beckman Coulter India Private Limited outside the submitted work.
Funding Statement
This work was funded by DBT-IISc Partnership Program Phase-II (BT/PR27952/INF/22/212/2018). The funding agency had no role in the study. Beckman Coulter India Private Limited provided flow cytometer, antibodies, reagents and salary for MPA.
Author Declarations
I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.
Yes
The details of the IRB/oversight body that provided approval or exemption for the research described are given below:
This study was carried out in accordance with the Declaration of Helsinki. Ethical clearance and necessary approvals were obtained from the Institutional Review Board of Indian Institute of Science (IISc), Bengaluru (5-14032018) and Employees State Insurance Corporation Medical College & Post Graduate Institute of Medical Sciences & Research (ESIC MC & PGIMSR), Bengaluru (532/L/11/12/Ethics/ESICMC&PGIMSR/Estt. Vol..III).
All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.
Yes
I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).
Yes
I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.
Yes
Data Availability
Study protocols, exported flow cytometry data and de-identified FCS files that underlie the results reported are available from the corresponding author upon reasonable request.