Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA

Abstract

Transrenal 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.

Diagnostic accuracy of Mycobacterium tuberculosis cell-free DNA for tuberculosis: A systematic review and meta-analysis

“…On the other hand, the use of urine testing for cfDNA might be an alternative diagnostic tool for PTB, but our study showed that the diagnostic efficacy of this method was limited when compared to culture. A recent study showed good efficacy of cfDNA testing using urine for the diagnosis of PTB, but the study used Xpert MTB/RIF as the reference standard [ 39 ], which might lead to inconsistent results. However, the studies examined in our meta-analysis focused on adults and lacked data on children.…”

“…We also excluded three articles that did not report sensitivity and specificity [34][35][36] and one article that reported data PLOS ONE from another included article [37]. The reference criteria used in three articles did not meet the inclusion criteria of this study and were excluded [10,38,39]. One article that simultaneously reported the efficacy of different PCR methods to detect cfDNA for PTB and EPTB diagnosis was considered as four separate studies [5], and another article that simultaneously reported the efficacy of different target gene to detect cfDNA for PTB diagnosis was considered as two separate studies [15].…”

Unbiased sequencing of Mycobacterium tuberculosis urinary cell-free DNA reveals extremely short fragment lengths

“…Decreasing the minimum detectable target length improves detection sensitivity for fragmented cfDNA (15,33,34) and has been a priority during the recent development of TB urine cfDNA assays (5)(6)(7)(8). Previously reported TB urine cfDNA assays targeted, at the shortest, amplicons of 38-40 bp (6)(7)(8). Decreasing PCR amplicon length from 49 bp to 39 bp resulted in greater than 10-fold increase in detected TB cfDNA (34).…”

“…To improve the clinical sensitivity of TB urine cfDNA assays, both sample preparation and amplification methods having maximal efficiency for very short fragments are needed. For example, recent work in our lab demonstrated that sequence-specific purification improves recovery of short cfDNA relative to conventional silica-based extraction and increases the clinical sensitivity of TB diagnosis from urine cfDNA (8,35). Ultrashort PCR using a stem-loop primer may be an attractive strategy for amplification of fragments too short for conventional PCR (15).…”

“…Even when sputum is available for testing, existing sputum-based TB assays have reduced sensitivity for diagnosis of paucibacillary, HIV-associated, pediatric, and extrapulmonary TB (1)(2)(3). Transrenal urine cell-free DNA (cfDNA) is a promising, easy-to-collect biomarker for TB with the potential to diagnose TB across these key underserved patient populations (4)(5)(6)(7)(8)(9), but it has not yet been extensively characterized or validated as a diagnostic analyte.…”