A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay

Abstract

BackgroundTuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available.MethodsWe developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa.ResultsThe assay’s analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6–0.9) and 84% (95% CI 0.6–0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77–1) and 94% (95% CI 0.7–0.99), respectively.ConclusionThis novel approach may permit simple and rapid detection of TB using pediatric stool samples.

Xpert MTB/RIF on Stool Is Useful for the Rapid Diagnosis of Tuberculosis in Young Children With Severe Pulmonary Disease

“…In our current study, stool Xpert was positive only in children with radiologically severe TB, with a sensitivity of 82% for smear-positive TB and 45% for bacteriologically confirmed cases with severe disease. This is consistent with a laboratory study where the level of detection for Xpert on Macaque stools was determined to be approximately 1000 colony forming units (CFU)/mL (36), whereas in respiratory samples it is approximately 100 CFU/mL(37). …”

“…The different protocols used in published studies could partly explain the variable results observed. For molecular analysis, stool samples require careful processing to remove PCR inhibitors and particulate matter that can interfere with assay performance (36, 39-41). Stool processing currently remains relatively labor intensive, but work is ongoing to develop centrifugation-free protocols applicable to primary/ district level healthcare facilities (36).…”

Stool Xpert® MTB/RIF test for the diagnosis of childhood pulmonary tuberculosis at primary clinics in Zimbabwe

“…Emerging evidence suggests that using larger sample volumes, of 0.6 g25 and 2 cm 3 ,26 and pre-treatment with a stool-processing buffer to inactivate PCR inhibitors,25 achieves greater diagnostic yield of the stool Xpert test.…”

Tuberculosis in Children

“…75,76 Detecting the organism in stool may be an option where GeneXpert MTB/RIF testing is available. 77,78 …”