Multigene <i>Mycobacterium tuberculosis</i> cell-free DNA assay

&lt;sec&gt;&lt;title&gt;BACKGROUND&lt;/title&gt;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).&lt;/sec&gt;&lt;sec&gt;&lt;title&gt;METHODS&lt;/title&gt;The qPCR assay targeted insertion sequence (IS 6110 ), cyp141, and dev R 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.&lt;/sec&gt;&lt;sec&gt;&lt;title&gt;RESULTS&lt;/title&gt;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 IS 6110 and cyp141 targeted qPCR demonstrated a sensitivity of 70.8%, and IS 6110 and dev R showed a sensitivity of 69.8%. However, dev R and cyp141 resulted in a lower sensitivity of 63.2%. IS 6110 and cyp141 had sensitivities of respectively 59.4% and 60.4%, while dev R had 53.8%.&lt;/sec&gt;&lt;sec&gt;&lt;title&gt;CONCLUSION&lt;/title&gt;Targeting multiple genes for plasma Mtb cfDNA-based TB diagnosis improves sensitivity and could be an important addition to current sputum-based diagnostic approaches.&lt;/sec&gt;