Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing

Abstract

Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing,

Metagenomic DNA sequencing to quantify Mycobacterium tuberculosis DNA and diagnose tuberculosis

“…Detection of tuberculosis using a metagenomic sequencing assay for tuberculosis diagnostics is thus akin to looking for a needle in a haystack: M. tuberculosis DNA constituted less than 4.4% of the total abundance of Mycobacterium in samples from TB endemic regions included in this study. Our work suggests that the median abundance of M. tuberculosis is lower than 0.06 and 0.42 copies/mL in blood and urine, respectively, and lower than 284 genome copies/µg of DNA collected by oral swab, an estimate that is in agreement with previous reports quantifying the abundance of M. tuberculosis DNA through sequence-specific amplification 8 , 27 . Improvements to a metagenomic sequencing assay for tuberculosis diagnostics could be made by increasing the volume of input biofluid 28 , choosing a sample preparation workflow with improved DNA extraction and short read amplification 12 , 27 , or enriching for M. tuberculosis- specific sequences using ultrashort PCR amplicons 8 , 29 .…”

“…For example, circulating plasma DNA has been used to monitor infection and rejection after lung transplantation 5 . Numerous studies have demonstrated that DNA from Mycobacterium tuberculosis , the causative agent of tuberculosis, can be detected in plasma, urine, and oral fluids 6 – 8 . However, its diagnostic performance in distinguishing positive sputum culture from negative sputum culture samples has fallen short of the performance standards set by the WHO (98% specificity, 80% sensitivity) 9 .…”

A needle in a haystack: metagenomic DNA sequencing to quantify Mycobacterium tuberculosis DNA and diagnose tuberculosis

“…Detection of tuberculosis using a metagenomic sequencing assay for tuberculosis diagnostics is thus akin to looking for a needle in a haystack: M. tuberculosis DNA constituted less than 4.4% of the total abundance of Mycobacterium in samples from TB endemic regions included in this study. Our work suggests that the median abundance of M. tuberculosis is lower than 0.06 and 0.42 copies/mL in blood and urine, respectively, and lower than 284 genome copies/µg of DNA collected by oral swab, an estimate that is in agreement with previous reports quantifying the abundance of M. tuberculosis DNA through sequence-specific amplification 7,22 . Improvements to a metagenomic sequencing assay for tuberculosis diagnostics could be made by increasing the volume of input biofluid 23 , choosing a sample preparation workflow with improved DNA extraction and short read amplification 10,22 , or enriching for M. tuberculosis- specific sequences using ultrashort PCR amplicons 7,24 .…”

“…Our work suggests that the median abundance of M. tuberculosis is lower than 0.06 and 0.42 copies/mL in blood and urine, respectively, and lower than 284 genome copies/µg of DNA collected by oral swab, an estimate that is in agreement with previous reports quantifying the abundance of M. tuberculosis DNA through sequence-specific amplification 7,22 . Improvements to a metagenomic sequencing assay for tuberculosis diagnostics could be made by increasing the volume of input biofluid 23 , choosing a sample preparation workflow with improved DNA extraction and short read amplification 10,22 , or enriching for M. tuberculosis- specific sequences using ultrashort PCR amplicons 7,24 . These approaches would minimize noise from nontuberculous mycobacteria and increase the sequencing budget allocated to M. tuberculosis .…”

“…Metagenomics DNA sequencing has the capacity to detect all potential infectious pathogens in a clinical sample using an unbiased approach. Numerous studies have demonstrated that DNA from Mycobacterium tuberculosis, the causative agent of tuberculosis, can be detected in plasma, urine, and oral fluids [5][6][7] . However, its diagnostic performance in distinguishing positive sputum culture from negative sputum culture samples has fallen short of the performance standards set by the WHO (98% specificity, 80% sensitivity) 8 .…”