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

Abstract

ABSTRACTBackgroundTuberculosis (TB) remains a significant cause of mortality worldwide. Metagenomic next-generation sequencing has the potential to reveal biomarkers of active disease, identify coinfection, and improve detection for sputum-scarce or culture-negative cases.MethodsWe conducted a large-scale comparative study of 427 plasma, urine, and oral swab samples from 334 individuals from TB endemic and non-endemic regions to evaluate the utility of a shotgun metagenomic DNA sequencing assay for tuberculosis diagnosis.FindingsWe found that the choice of a negative, non-TB control cohort had a strong impact on the measured performance of the diagnostic test: the use of a control patient cohort from a nonendemic region led to a test with nearly 100% specificity and sensitivity, whereas controls from TB endemic regions exhibited a high background of nontuberculous mycobacterial DNA, limiting the diagnostic performance of the test. Using mathematical modeling and quantitative comparisons to matched qPCR data, we found that the burden of Mycobacterium tuberculosis DNA constitutes a very small fraction (0.04 or less) of the total abundance of DNA originating from mycobacteria in samples from TB endemic regions.InterpretationOur findings suggest that the utility of a metagenomic sequencing assay for tuberculosis diagnostics is limited by the low burden of M. tuberculosis in extrapulmonary sites and an overwhelming biological background of nontuberculous mycobacterial DNA.FundingThis work was supported by the National Institutes of Health, the Rainin Foundation, the National Science Foundation, and the Bill and Melinda Gates Foundation.