Diagnosing pulmonary tuberculosis 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.

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

“…Consequently, minimizing the length of sequences targeted for TB urine cfDNA diagnostic assays will be critical to maximizing their sensitivity. 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 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).…”

“…First, we sequenced cfDNA only from patients living with HIV. Detection sensitivity for TB urine cfDNA is similar in HIV-positive and HIV-negative participants (7,8), but it remains unclear if there may be differences in cfDNA fragmentation patterns across these two populations. Second, owing to the requirements for high sequencing depths and attendant sequencing costs, the number of specimens analyzed in this study are necessarily limited.…”

“…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.…”