Abstract
BackgroundRapid 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. Our objective was to evaluate stool as an alternative specimen to sputum for Mtb detection in children. We did so using the TruTip workstation (Akonni Biosystems), a novel automated lysis and extraction platform.MethodsWe tested stool samples from 259 children aged 0–14 years old, in Lima, Peru who presented with TB symptoms. Following extraction with TruTip, we detected the presence of Mtb DNA by IS6110 real-time PCR. We calculated assay sensitivity in two groups: (1) children with culture confirmed TB (N = 22); and (2) children with clinically-diagnosed unconfirmed TB (N = 84). We calculated specificity among children in whom TB was ruled out (N = 153). Among children who were diagnosed with TB, we examined factors associated with a positive stool test.ResultsAssay sensitivity was 59% (95% confidence interval [CI]: 39–80%) and 1.2% (95% CI: 0.0–6.5%) in children with culture-confirmed and clinically-diagnosed unconfirmed TB, respectively, and specificity was 97% (95% CI: 93–99%). The assay detected Mtb in stool of 7/7 children with smear-positive TB (100% sensitivity; 95% CI: 59–100%), and in 6/15 of children with smear-negative, culture-confirmed TB (40% sensitivity; 95% CI: 16–68%). Older age, smear positivity, culture positivity, ability to produce sputum and cavitary disease were associated with a positive stool result.ConclusionTesting of stool samples with the TruTip workstation and IS6110 amplification yielded sensitivity and specificity estimates comparable to other tests such as Xpert. Future work should include detection of resistance using the TruTip closed amplification system and assay optimization to improve sensitivity in children with low bacillary loads.
Highlights
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 can be obtained from most children and Mycobacterium tuberculosis (Mtb) can be detected in stool using Xpert [5,6,7,8,9,10] or other laboratory-developed Polymerase Chain Reaction (PCR) assays [11,12,13]
Study population From 628 children enrolled in the study, we selected 259 children based on stool sample availability (22 children with culture confirmed TB, 84 children with clinicallydiagnosed unconfirmed TB, and 153 children in whom TB had been ruled out (Fig. 1))
Summary
Rapid and accurate diagnosis of childhood tuberculosis (TB) is challenging because children are often unable to produce the sputum sample required for conventional tests. We did so using the TruTip workstation (Akonni Biosystems), a novel automated lysis and extraction platform. Sputum induction and gastric aspiration can be used to obtain respiratory specimens from children unable to expectorate sputum; gastric aspiration is invasive and neither procedure is widely implemented in resource-constrained settings. Due to these diagnostic challenges, bacteriologic confirmation of TB is obtained in only a small minority of children diagnosed with TB [2,3,4]. The TruTip workstation is an automated platform including lysis and homogenization with TruTip nucleic acid extraction and purification (Akonni Biosystems, Frederick, MD, USA) [15,16,17]. The aim of the present study was to estimate sensitivity and specificity of Mtb detection in stool from children with symptoms compatible with intrathoracic TB in Lima, Peru, using this novel technology with IS6110 real-time PCR
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