An ultrasensitive method for early detection of tuberculosis in asymptomatic high-risk individuals

The dynamic spectrum of tuberculosis (TB) often results in underdiagnosis warranting the need for better diagnostics to accurately detect Mycobacterium tuberculosis (Mtb) in persons with asymptomatic, paucibacillary or extrapulmonary TB, and to identify individuals at high risk of developing TB disease early. This study aimed to evaluate a dual target-based digital droplet PCR (ddPCR) assay to detect circulating cell-free Mtb DNA in plasma of individuals at high risk of developing TB disease and in those lacking a clear diagnosis of TB (asymptomatic or clinically diagnosed TB).

Forty-six healthy household contacts (HHCs) of patients with pulmonary TB who developed TB within two years of follow-up (Progressors), and 92 HHCs who did not progress to TB (Non-progressors) were included in the study. Plasma was obtained and subjected to testing using a ddPCR assay targeting two M. tuberculosis (Mtb)-specific insertion sequences, IS6110 and IS1081. Sensitivity, specificity, and ROC curves were used to assess the diagnostic performance of the test.

IS6110 and IS1081 targets were detected in 10/11 asymptomatic TB cases giving a sensitivity of 90.9% (95% CI: 62.3-99.5), and in 9/11 unconfirmed/possible TB cases giving a sensitivity of 81.8% (95% CI: 52.3-96.8). Further, the test detected Mtb-ccfDNA in 15/19 Progressors even at six months prior to TB diagnosis (79.0% sensitivity, 95% CI: 56.7-91.5; 97.8% specificity, 95% CI: 92.4-99.6). Detection rates at 12- and 18-months prior to onset of TB were 55.0% (95% CI: 34.2-74.2) and 50.0% (95% CI: 29.9-70.1), respectively. Notably, the test showed 100% sensitivity (95% CI: 43.9-100.0) in detecting extrapulmonary TB up to six months prior to clinical diagnosis.

The study highlights the potential of ddPCR-based detection of Mtb ccfDNA as a valuable tool for early identification of individuals at risk of developing active TB disease and for clarifying the diagnosis of TB in diagnostically challenging cases. Further validation in a larger cohort will confirm the findings of the study and endorse the utility of the test in clinical practice.

This work was supported by the Indian Council of Medical Research (Grant number:5/8/5/45/Adhoc/2022/ECD-1). The CTRIUMPh cohort study which provided samples for this study was supported by the NIH/DBT Indo-US Vaccine Action Programme.