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

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.