Background. Centrifuge-free processing methods support stool Xpert Ultra testing for childhood tuberculosis (TB), but there are limited data on their accuracy, acceptability and usability. Methods. We conducted a prospective evaluation of stool Xpert Ultra in India, South Africa, and Uganda with three methods: Stool Processing Kit (SPK), Simple One-Step (SOS), and Optimized Sucrose Flotation (OSF). Children <15 years old with presumptive TB had respiratory specimen testing with Xpert Ultra and culture. Stool was tested using Xpert Ultra after processing with each method. We compared the accuracy of each method to a microbiological reference standard (MRS) and a composite reference standard (CRS). We surveyed the laboratory staff to assess acceptability and usability of the methods. Results. We included 607 children, of whom the median age was 3.5 years (IQR 1.3-7), 48% were female, and 15.5% were HIV positive. Against the MRS, the sensitivities of SPK, SOS and OSF were 36.9% (95% CI 28.6-45.8), 38.6% (95% CI 17.2-51), and 31.3% (95% CI 20.2-44.1), respectively. The specificities of SPK, SOS and OSF were 98.2% (95% CI 96.4-99.3), 97.3% (95% CI 93.7-99.1) and 97.1% (95% CI 93.3-99), respectively. Laboratory staff reported that the methods were acceptable and usable, but SOS was most feasible to implement in a peripheral facility. Sensitivity increased among children who were culture-positive (55-77.3%) and was low (13-16.7%) against the CRS. Conclusions. Stool processing methods for Xpert Ultra were acceptable, usable, and performed similarly, with highest sensitivity among children with culture-positive TB.

MPN, DA, PB and FIND were involved in the development of SPK, PD and ET for SOS, and MB and ML for OSF. DA holds patents related to tuberculosis detection and drug treatment, and receives royalty payments for one or more of these patents that have been licensed by Rutgers University to Cepheid. The other authors declare no conflicts of interest.

The work was supported by the National Institutes of Health, through the National Institute of Allergy and Infectious Diseases [U01AI152087 to AC and CMD; and R01AI131617 to DA], and the National Heart, Lung, and Blood Institute [K23HL153581 to DJ]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. FIND provided funding to all the sites and the SPK supplies. Cepheid provided in-kind support of Xpert Ultra cartridges and GeneXpert machines, but had no role in the study design, analysis, interpretation and decision to publish.

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