Evaluating Metagenomic Sequencing as a Stool-based Diagnostic in Children With Presumptive Tuberculosis in Uganda

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Abstract
Background: Stool-based molecular tests provide noninvasive options for pediatric tuberculosis (TB) diagnosis but have lower sensitivity compared to sputum-based tests. Untargeted metagenomic next-generation sequencing (mNGS) on stool could improve sensitivity and identify new gene targets for molecular testing. Methods: We performed shotgun mNGS on DNA isolated from stool samples of children undergoing assessment for pulmonary TB in Uganda. We defined the performance of mNGS to identify Mycobacterium tuberculosis (Mtb) against a microbiological reference standard (MRS, TB if sputum Xpert Ultra or culture positive) and a composite reference standard (TB if confirmed or unconfirmed TB). We also compared the accuracy of mNGS against stool-based Xpert Ultra. Finally, we identified enriched genomic loci among Mtb-classified reads. Results: We analyzed 176 stool samples of children with a median age of 3.6 years (interquartile range [IQR], 1–6 years). Against the MRS, the sensitivities of mNGS with positive TB defined as ≥1, 2, or 5 sequence fragments were 35.5% (95% confidence interval [CI] 19%–55%), 25.7% (12%–45%), and 19.4% (7%–37%), respectively, and specificities 92.6% (87%–96%), 97% (93%–99%), and 99.3% (96%–100%). Stool Xpert Ultra had similar sensitivity (22.6%) to stool mNGS considering all samples tested. In a head-to-head comparison, stool mNGS had lower sensitivity than stool Xpert Ultra (38.5% vs 53.8%, difference −15.3%, 95% CI 14–68 vs 25–81). mNGS utilized genes encoding rRNA, virulence proteins, and membrane proteins not targeted in current PCR-based platforms. Conclusions: Metagenomic sequencing of stool DNA did not increase sensitivity of TB detection, but identified novel targets for molecular testing that may support development of more sensitive tests.
Funding Information
  • National Institutes of Health (U01AI152087)
  • National Institutes of Health (R01HL139717)
  • National Institutes of Health (R21AI176295)
  • National Institutes of Health (R35GM147512)
  • National Institutes of Health (K23HL153581)
  • National Science Foundation Graduate Research Fellowship Program
  • Hellman Foundation
  • Koret Foundation

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