<sec><title>BACKGROUND</title>Existing TB diagnostic tests rely on sputum samples, which can be difficult to collect from all patients. This study examines plasma Mycobacterium tuberculosis cell-free DNA (Mtb cfDNA) based quantitative PCR (qPCR) assay for the diagnosis of pulmonary TB (PTB).</sec><sec><title>METHODS</title>The qPCR assay targeted insertion sequence (IS6110), cyp141, and devR genes on plasma samples from 106 PTB patients and 60 controls. Sensitivity was calculated using the Xpert® MTB/RIF test, culture, and clinical diagnosis for the PTB group, while specificity was determined based on results from controls.</sec><sec><title>RESULTS</title>Among PTB cases, 92 (86.8%) were bacteriologically confirmed, with the remaining 14 (13.2%) diagnosed clinically. The sensitivity of the plasma Mtb cfDNA assay, considering all three genes, was 71.7% (95% CI 62.6-71.7) for all PTB cases, with higher sensitivity in bacteriologically confirmed cases (78.3%) than in clinically diagnosed cases (28.6%). The combined specificity was 91.7%. The combination of IS6110 and cyp141 targeted qPCR demonstrated a sensitivity of 70.8%, and IS6110 and devR showed a sensitivity of 69.8%. However, devR and cyp141 resulted in a lower sensitivity of 63.2%. IS6110 and cyp141 had sensitivities of respectively 59.4% and 60.4%, while devR had 53.8%.</sec><sec><title>CONCLUSION</title>Targeting multiple genes for plasma Mtb cfDNA-based TB diagnosis improves sensitivity and could be an important addition to current sputum-based diagnostic approaches.</sec>.