medRxiv : the preprint server for health sciences · 2026
Evaluating metagenomic sequencing as a stool-based diagnostic in children with presumptive TB in Uganda.
СтатьяПрепринтНазвание и аннотация: Перевод в очереди
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Agudelo Carolina, Nsereko Moses, Ainebyona Aggrey, Andama Alfred, Castro Robert, Leung Sarrah Rose Mikhail, Nakafeero Jascent, Nannyonga Gertrude, Nolan Kevin, Teran Lucas, Wambi Peter, Young Mark G, Kato-Maeda Midori, Cattamanchi Adithya, Jaganath Devan, Wobudeya Eric, Wolf Ashley R
Аннотация
BACKGROUND: Stool-based molecular tests are a noninvasive option 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 the stool-based Xpert Ultra test. 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). !"#$%&'(')*(+,-. (')*(&*%&$'$/$'$*&(01(234-(5$')(60&$'$/*(78(9*1$%*9(as ≥ 1, 2, or 5 sequence fragments were 35.5% (95% CI 19%:;;<=.(>;?@<(AB>< : 45%), and 19.4% (13%-25%) respectively, and specificities 92.64% (87%-96%), 97% (93%-99%), and 99.3% (96%-100%). Stool Xpert Ultra had similar sensitivity (22.6%) to stool mNGS considering all samples tested. In a head-to-head comparison, stool mNGS had lower sensitivity than stool Xpert Ultra (38.5% vs. 53.8%, difference -15.3%, 95% CI 14-68 to 25-81). mNGS utilized rRNA, virulence proteins and membrane proteins not targeted in current PCR-based platforms.
CONCLUSIONS: Metagenomic sequencing of stool DNA did not increase sensitivity of TB detection, but identified novel targets for molecular testing that may support development of more sensitive tests.
METHODS: We performed shotgun mNGS on DNA isolated from stool samples of children undergoing assessment for pulmonary TB in Uganda. We defined the performance of mNGS to identify Mycobacterium tuberculosis ( Mtb ) against a microbiological reference standard (MRS, TB if sputum Xpert Ultra or culture positive) and a composite reference standard (TB if confirmed or unconfirmed TB). We also compared accuracy of mNGS against the stool-based Xpert Ultra test. Finally, we identified enriched genomic loci among Mtb classified reads.
RESULTS: We analyzed 176 stool samples of children with a median age of 3.6 years (IQR, 1-6 years). !"#$%&'(')*(+,-. (')*(&*%&$'$/$'$*&(01(234-(5$')(60&$'$/*(78(9*1$%*9(as ≥ 1, 2, or 5 sequence fragments were 35.5% (95% CI 19%:;;<=.(>;?@<(AB>< : 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.