Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA

Abstract

ABSTRACTTransrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 mL urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0– 91.5%) and 100% specificity (95% CI: 86.2–100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14–2804 copies/mL (median 14.6 copies/mL) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was non-significantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine LAM-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.