Detection of Mycobacterium tuberculosis in pediatric stool samples using TruTip technology

Background: Rapid and accurate diagnosis of childhood tuberculosis (TB) is challenging because children are often unable to produce the sputum sample required for conventional tests. Stool is an alternative sample type that is easy to collect from children, and studies investigating the use of stool for molecular detection of Mycobacterium tuberculosis ( Mtb ) have led to promising results. However, tests performed thus far are not able to examine multi-drug resistance. The TruTip workstation (Akonni Biosystems) is an automated lysis and extraction platform that can be integrated with a closed amplification system to detect both Mtb and resistance-associated mutations. Our objective here was to evaluate the use of TruTip extraction technology for Mtb detection in stool. Methods: We tested stool samples of 259 children with TB symptoms, ages 0-14 years old, in Lima, Peru. We used the TruTip workstation for sample processing and extraction, followed by IS6110 real-time PCR to detect the presence of Mtb DNA. We calculated assay sensitivity in two groups: (1) children with culture confirmed TB (N=22); and (2) children with unconfirmed, clinically diagnosed TB (N=84). We calculated specificity among children in whom TB was ruled out (N=153). Among children with TB, we examined factors associated with a positive stool test. Results: Overall assay sensitivity was 59% (95% confidence interval 39%-80%) and 1.2% (0.0%-6.5%) in children with culture-confirmed and clinically-diagnosed TB, respectively, and specificity was 97% (93%-99%). The assay detected Mtb in stool of 7/7 children with smear-positive TB [100% sensitivity; (59%-100%)], and in 6/15 [40% (16%-68%)] of children with smear-negative, culture-confirmed TB. Older age, smear positivity, culture positivity and cavitary disease were associated with a positive stool result. Conclusion: For molecular Mtb detection from stool, the TruTip workstation, in combination with IS6110 amplification, led to sensitivity and specificity estimates comparable to other tests such as Xpert. Future optimization is required to also diagnose TB disease in children who now received an unconfirmed diagnosis.