The Simple One-Step (SOS) Stool Processing Method for Use with the Xpert MTB/RIF Assay for a Child-Friendly Diagnosis of Tuberculosis Closer to the Point of Care

Abstract

Young children cannot easily produce sputum for diagnosis of pulmonary tuberculosis (TB). Alternatively, Mycobacterium tuberculosis complex bacilli can be detected in stool by using the Xpert MTB/RIF (Ultra) assay (Xpert). Published stool processing methods contain somewhat complex procedures and require additional supplies. The aim of this study was to develop a simple one-step (SOS) stool processing method based on gravity sedimentation only, similar to Xpert testing of sputum samples, for the detection of M. tuberculosis in stool samples. We first assessed whether the SOS stool method could provide valid Xpert results without the need for bead-beating, dilution, and filtration steps. We concluded that this was the case, and we then validated the SOS stool method by testing spiked stool samples. By using the SOS stool method, 27 of the 29 spiked samples gave valid Xpert results, and M. tuberculosis was recovered from all 27 samples. The proof of principle of the SOS stool method was demonstrated in routine settings in Addis Ababa, Ethiopia. Nine of 123 children with presumptive TB had M. tuberculosis-positive results for nasogastric aspiration (NGA) samples, and 7 (77.8%) of those children also had M. tuberculosis-positive Xpert results for stool samples. Additionally, M. tuberculosis was detected in the stool samples but not the NGA samples from 2 children. The SOS stool processing method makes use of the standard Xpert assay kit, without the need for additional supplies or equipment. The method can potentially be rolled out to any Xpert site, bringing a bacteriologically confirmed diagnosis of TB in children closer to the point of care.

Keywords: Ethiopia; Xpert MTB/RIF; bacteriologically confirmed; children; diagnosis; point of care; simple one-step stool method; sputum; stool; tuberculosis.

FIG 1

Schematic overview of the two methods that were used in the validation phase of this study, in which spiked stool samples were tested for the presence of M. tuberculosis by using the GeneXpert Ultra system. (A) SOS stool processing method. (B) Slightly adapted TS stool processing method described previously by Andriyoko et al. (15). *, The SR (Cepheid) is an 8-ml mixture of sodium hydroxide (pH of >12.5) and isopropanol provided with every Xpert Ultra cartridge. #, After gravity sedimentation of the organic debris, carefully, without lifting the bottle and without disturbing the sediment, 2 ml of the upper layer of the debris-free supernatant was transferred. %, The PBS is pH 7.4.

FIG 2

Xpert Ultra assay results from testing of 29 spiked stool samples processed by using the SOS stool method and the TS stool method, according to the semiquantitative Xpert Ultra results. For example, the SOS method had 8 samples with M. tuberculosis (MTB) trace detected, while the results of the TS method for these 8 samples included 1 M. tuberculosis not detected, 6 M. tuberculosis trace detected, and 1 M. tuberculosis detected, very low.