Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples
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Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples. / Eigner, Ulrich; Nörz, Dominik; Reucher, Svenja; Furrer, Jan; Sun, Jingtao; Chu, Kristina; Kolb, Melissa; Hefner, Nadine; Pfefferle, Susanne; Lütgehetmann, Marc.
In: J MICROBIOL METH, Vol. 172, 05.2020, p. 105882.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples
AU - Eigner, Ulrich
AU - Nörz, Dominik
AU - Reucher, Svenja
AU - Furrer, Jan
AU - Sun, Jingtao
AU - Chu, Kristina
AU - Kolb, Melissa
AU - Hefner, Nadine
AU - Pfefferle, Susanne
AU - Lütgehetmann, Marc
N1 - Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.
PY - 2020/5
Y1 - 2020/5
N2 - BACKGROUND: The cobas® omni Utility Channel enables users to integrate lab-developed tests (LDTs) on the cobas® 6800 System to perform molecular diagnostics with high-throughput capacity and full automation. At present, there are no CE- or FDA-approved tests for stool pathogens on this system. To assess the performance of stool as a matrix, we evaluated the analytical and clinical performance of an LDT for detection of Clostridioides difficile (C. difficile) toxin B using the Utility Channel (C.diff_UTC).METHODS: A 10% stool suspension prepared from liquid stool samples diluted in phosphate buffered saline was used for analysis. Limit of detection (LoD) was determined in six dilutions with 126 replicates/dilution. Clinical evaluation was performed using 514 predetermined patient stool samples from two study sites in Germany. The C.diff_UTC was compared with LC 480 amplification and an LDT or the R-BioPharm C. difficile assay. Discrepant results were further analyzed using the GeneXpert C. difficile assay.RESULTS: Limit of detection was 23.48 cfu/mL (95% Confidence Interval [CI]: 19.14-31.01) with inter-run variation of <2 cycle thresholds at 3 × and 10 × LoD. No cross-reactivity was observed with a panel of fecal organisms and pathogens. Bioinformatic analysis showed coverage of the major C. difficile toxinotypes by the primer/probe set. Clinical evaluation revealed sensitivity of 96.7% (95% CI: 88.7-99.6) and specificity of 99.3% (95% CI: 98.0-99.9) compared with the reference method; inhibition rate was 3.5% (18/514).CONCLUSION: Using a predesigned primer/probe set, the C.diff_UTC assay features analytical performance and clinical sensitivity and specificity comparable to currently available nucleic acid amplification tests (NAATs) and is suitable for high-throughput testing. This was a proof-of-concept study, indicating the cobas Utility Channel could likely be adapted for other clinically relevant stool pathogens in outbreak scenarios.
AB - BACKGROUND: The cobas® omni Utility Channel enables users to integrate lab-developed tests (LDTs) on the cobas® 6800 System to perform molecular diagnostics with high-throughput capacity and full automation. At present, there are no CE- or FDA-approved tests for stool pathogens on this system. To assess the performance of stool as a matrix, we evaluated the analytical and clinical performance of an LDT for detection of Clostridioides difficile (C. difficile) toxin B using the Utility Channel (C.diff_UTC).METHODS: A 10% stool suspension prepared from liquid stool samples diluted in phosphate buffered saline was used for analysis. Limit of detection (LoD) was determined in six dilutions with 126 replicates/dilution. Clinical evaluation was performed using 514 predetermined patient stool samples from two study sites in Germany. The C.diff_UTC was compared with LC 480 amplification and an LDT or the R-BioPharm C. difficile assay. Discrepant results were further analyzed using the GeneXpert C. difficile assay.RESULTS: Limit of detection was 23.48 cfu/mL (95% Confidence Interval [CI]: 19.14-31.01) with inter-run variation of <2 cycle thresholds at 3 × and 10 × LoD. No cross-reactivity was observed with a panel of fecal organisms and pathogens. Bioinformatic analysis showed coverage of the major C. difficile toxinotypes by the primer/probe set. Clinical evaluation revealed sensitivity of 96.7% (95% CI: 88.7-99.6) and specificity of 99.3% (95% CI: 98.0-99.9) compared with the reference method; inhibition rate was 3.5% (18/514).CONCLUSION: Using a predesigned primer/probe set, the C.diff_UTC assay features analytical performance and clinical sensitivity and specificity comparable to currently available nucleic acid amplification tests (NAATs) and is suitable for high-throughput testing. This was a proof-of-concept study, indicating the cobas Utility Channel could likely be adapted for other clinically relevant stool pathogens in outbreak scenarios.
U2 - 10.1016/j.mimet.2020.105882
DO - 10.1016/j.mimet.2020.105882
M3 - SCORING: Journal article
C2 - 32119956
VL - 172
SP - 105882
JO - J MICROBIOL METH
JF - J MICROBIOL METH
SN - 0167-7012
ER -
