Centrifuge-free stool processing methods for Xpert MTB/RIF Ultra tuberculosis diagnosis in children in Uganda and Zambia: an observational, prospective, diagnostic accuracy study

Background: WHO recommends Xpert MTB/RIF Ultra (Ultra) for stool testing for tuberculosis diagnosis in children. Stool processing requires removal of debris and PCR inhibitors, frequently by using centrifugation, which can be an implementation barrier for low-income and middle-income countries (LMICs). We evaluated the diagnostic accuracy of Ultra on stool using three centrifuge-free processing methods, the simple one-step (SOS), stool processing kit (SPK), and the optimised sucrose flotation (OSF) methods against a microbiological reference standard (MRS). Methods: In this observational, prospective, multicountry, diagnostic accuracy study, we collected two respiratory samples and two stool samples in children younger than 15 years with presumptive tuberculosis in one hospital in Uganda and two hospitals in Zambia for Ultra testing and culture (on respiratory samples only). We defined positive MRS as positive culture or Ultra on respiratory sample and negative MRS as two negative respiratory samples by either culture or Ultra. We assessed the perception of the laboratory operators of test ease-of-use using a self-administered questionnaire at all sites. This study is registered with ClinicalTrials.gov (NCT04203628) and the Pan African Clinical Trial Registry (PACTR202006814433059). Findings: Of the 216 children enrolled between Jan 13, 2020, and Dec 31, 2021, 215 were included in the study and of these 104 (48·4%) were female and 211 (51·6%) were male, the median age was 1·8 years (IQR 1·1–4·8), 68 (31·6%) were HIV positive, and 38 (17·7%) were MRS positive. For one or both stool samples, depending on availability, the sensitivity of stool Ultra against MRS was 69·7% (95% CI 51·3–84·4) for SOS, 69·7% (51·3–84·4) for SPK, and 73·5% (55·6–87·1) for OSF (McNemar test p>0·6 for all), with a specificity above 96% for all methods. The SOS stool method was considered the easiest by six of seven operators because it required least manipulation and no additional reagents. Interpretation: Centrifuge-free stool processing methods could improve access to microbiological diagnosis of tuberculosis in LMICs. These results contributed to the WHO endorsement of the SOS and OSF methods. Funding: UNITAID.