Screening and targeted sequencing of stool for microbiologic confirmation and drug resistance determination in paucibacillary tuberculosis

In 2023, an estimated 10.8 million people developed tuberculosis, and 1.25 million people died from this disease, including 161,000 deaths in people with HIV (PWH) in whom tuberculosis remains the leading cause of death. Detecting Mycobacterium tuberculosis drug resistance remains a challenge among patients with paucibacillary tuberculosis; since there is such low bacterial load in their sputum it's unable to be detected via microscopy, there is also not enough bacteria for other sputum-based tests which could provide resistance testing. At an outpatient clinic in Eswatini from 2020-2023, stool and sputum samples were provided by a subset of children, adolescents, and adults prospectively enrolled in a tuberculosis diagnostic study. In addition to standard diagnostic testing available in country (direct sputum Xpert, stool Xpert, and phenotypic drug susceptibility testing of sputum culture), stool samples underwent extraction and sequencing using targeted next generation sequencing (tNGS), using both the Oxford Nanopore Technologies (ONT) TB Custom Kit (on an ONT MinION Mk1b) and the Deeplex Myc-TB kit (on an Illumina iSeq 100). From 250 participants with pulmonary tuberculosis diagnosed in Eswatini during our study period, 85 (34%) were smear negative on sputum microscopy. Of these, 21/85 (24.7%) participants had adequate M. tuberculosis DNA shed in their stool for attempting tNGS. Targeted sequencing on stool detected M. tuberculosis DNA in 14-19% (n = 12/85-16/85) and provided a full report of mutations associated with drug resistance in 12-14% (n = 10/85-12/85) of patients with paucibacillary (smear-negative) tuberculosis, expanding drug resistance detection beyond other methods. Targeted sequencing of stool, even when applied to patients with paucibacillary disease, can provide case confirmation and expanded drug resistance information.