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. 2022 Oct 10;12(1):16972.
doi: 10.1038/s41598-022-21244-x.

Metagenomic DNA sequencing to quantify Mycobacterium tuberculosis DNA and diagnose tuberculosis

Adrienne Chang  1 , Omary Mzava  1 , Liz-Audrey Kounatse Djomnang  1 , Joan Sesing Lenz  1 , Philip Burnham  1 , Peter Kaplinsky  1 , Alfred Andama  2 , John Connelly  3 , Christine M Bachman  3 , Adithya Cattamanchi  4 , Amy Steadman  3 , Iwijn De Vlaminck  5
Affiliations

Metagenomic DNA sequencing to quantify Mycobacterium tuberculosis DNA and diagnose tuberculosis

Adrienne Chang et al. Sci Rep. .

Abstract

Tuberculosis (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. We conducted a large-scale comparative study of 428 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. We found that the composition of the control population had a strong impact on the measured performance of the diagnostic test: the use of a control population composed of individuals from a TB non-endemic region led to a test with nearly 100% specificity and sensitivity, whereas a control group composed of individuals 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. Our findings suggest that the utility of a minimally invasive metagenomic sequencing assay for pulmonary tuberculosis diagnostics is limited by the low burden of M. tuberculosis and an overwhelming biological background of nontuberculous mycobacterial DNA.

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Conflict of interest statement

I.D.V. has received research grants from the Bill and Melinda Gates Foundation, the National Institutes of Health, and the Rainin Foundation. P.B. has received research grants from the National Science Foundation. I.D.V. and P.B. are inventors on the patent US-2020-0048713-A1 titled “Methods of Detecting Cell-Free DNA in Biological Samples”. The rights to the patent were licensed by Eurofins through Cornell University. I.D.V. is a Member of the Advisory Board and has stock in Karius Inc. A.C., O.M., L.D.K., J.L., P.K., A.A., J.C., C.M.B., A.C., and A.S. declare no potential conflict of interest.

Figures

Figure 1
Figure 1
Geographic distribution of samples included in this study. High tuberculosis burden countries are shaded in red.
Figure 2
Figure 2
(A) The fragment length distributions of M. tuberculosis DNA in plasma (red) and urine (blue). (B) The fragment length distribution of M. tuberculosis DNA (purple) and host chromosomal DNA (green) in oral swabs are similar, suggesting that the microbial DNA is genomic and the fragmentation profile is not an intrinsic property but rather the consequence of sample preparation steps. (C) The abundance of M. tuberculosis DNA across all cohorts and biofluids. The majority of non-endemic samples have little to no detectable M. tuberculosis DNA, while the abundance of M. tuberculosis DNA in endemic and tuberculosis samples increases from plasma to oral swab. Dashed line indicates a limit of detection cutoff of 0.1 RPM.
Figure 3
Figure 3
The performance of the metagenomic assay in discriminating (A) sputum positive versus sputum negative samples, (B) tuberculosis versus endemic cohorts, and (C) tuberculosis versus non-endemic cohorts (AUC = area under the curve). (D) Evaluating the effects of incorporating non-endemic samples (blue) and endemic samples (red) on the diagnostic performance via simulation show that the inclusion of non-endemic samples skews the assay’s sensitivity.
Figure 4
Figure 4
(A) Classification precision of synthetic M. tuberculosis reads spiked into non-endemic urine samples (n = 29, 5 replicates per sample), endemic urine samples (n = 23, 5 replicates per sample), and tuberculosis urine samples (n = 66, 5 replicates per sample). (B) The classification precision is correlated with the relative coverage of M. tuberculosis to total Mycobacterium in the sample.
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