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. 2016 Mar 23;11(3):e0151980.
doi: 10.1371/journal.pone.0151980. eCollection 2016.

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

Padmapriya P Banada  1 , Uvistra Naidoo  2 , Srinidhi Deshpande  1 , Farina Karim  2 , JoAnne L Flynn  3 , Melanie O'Malley  3 , Martin Jones  4 , Oliver Nanassy  5 , Prakash Jeena  2 , David Alland  1
Affiliations

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

Padmapriya P Banada et al. PLoS One. .

Abstract

Background: 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.

Methods: We 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.

Results: The 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.

Conclusion: This novel approach may permit simple and rapid detection of TB using pediatric stool samples.

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Conflict of interest statement

Competing Interests: David Alland is a member of a group of investigators who receive royalties for licensing molecular beacons such as those used in the Xpert assay. To manage this conflict of interest DA has irrevocably capped the money that he can personally receive attributable to the Xpert assay to $5000 per year. DA also reports receiving research contracts from Cepheid. Martin Jones and Oliver Nanassy are employed by Cepheid. Cepheid supplied the study with Xpert MTB/RIF assay cartridges. Xpert MTB/RIF assay cartridges and GeneXpert instruments are marketed worldwide. Other than these, there are no patents, products in development or marketed products to declare used in this study. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1
Fig 1. Flow diagram showing how stool was processed in this study.
Fig 2
Fig 2. Sample size and sensitivity.
A. M. bovis BCG was spiked at 103 CFU/ml into 0.2, 0.5 and 1 g of stool (N = 4). Columns = pressure; lines = Cycle thresholds (Cts). B. Stool samples were collected from macaques infected with M. tuberculosis and three different amounts (0.2g, 0.6g and 1g) were tested. The number in parenthesis indicates number of macaques tested at each volume. The shades of gray on the graph indicate the number of times the test was performed on the same stool sample. Light gray = 1 test only; medium gray = combined data from 2 tests; dark gray = combined data from 3 tests. All the uninfected controls tested (N = 5) were negative by this method.
Fig 3
Fig 3. Analytical limit of detection.
A. BCG and B. M. tuberculosis H37Rv (MTB) was spiked into stool at final concentrations of 10 CFU/g through 105 CFU/g, processed according to our protocol, and tested using the Xpert MTB/RIF assay.
Fig 4
Fig 4. Study flow diagram.
Xpert stool testing of 0.6g and 1.2g volume from Xpert induced sputum (IS) and gastric washing (GW) results was performed on above recruited participants. As indicated, repeat testing was performed on a limited stool number of samples that were initially negative.
See this image and copyright information in PMC

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