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.
Highlights
The World Health Organization (WHO) estimates that over half a million new cases of childhood tuberculosis (TB) occur every year, with an annual mortality of up to 80,000 [1, 2]
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)
Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% and 84% for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% and 94%, respectively
Summary
Tuberculosis (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. Data Availability Statement: All relevant data are within the paper. Cepheid provided support in the form of salaries for authors MJ and ON and supplied the study with Xpert MTB/RIF cartridges, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript
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