Evaluating metagenomic sequencing as a stool-based diagnostic in children with presumptive TB in Uganda

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ID: 329052
2026
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Ranked #254 of 635 articles by views in Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

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Abstract
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 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 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 (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% CI 19%-55%), 25.7% (12%-45%), and 19.4% (13%-25%) respectively, and specificities 92.64% (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 to 25-81). mNGS utilized 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.
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Authors Carolina Agudelo, Moses Nsereko, Aggrey Ainebyona, Alfred Andama, Robert Castro, Sarrah Rose Mikhail Leung, Jascent Nakafeero, Gertrude Nannyonga, Kevin Nolan, Lucas Teran, Peter Wambi, Mark Young, Midori Kato‐Maeda, Adithya Cattamanchi, Devan Jaganath, Eric Wobudeya, Ashley R. Wolf
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Journal Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
Year 2026
DOI
10.1093/cid/ciag573
URL
https://doi.org/10.1093/cid/ciag573
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