Dataset Information

Application of digital PCR to determine the reliability of Xpert Xpress SARS-CoV-2 assay with envelope (E) gene negative and nucleocapsid (N2) gene positive results.

ABSTRACT: This study used digital polymerase chain reaction (dPCR) to determine whether envelope (E) gene-negative and nucleocapsid (N2) gene-positive (E-N+) results obtained with the Cepheid Xpert Xpress SARS-CoV-2 assay are reliable. Using droplet digital PCR results as a reference, 18 of 22 E-N+ samples with a low viral load (81.8%) were identified as true positives.

SUBMITTER: Wong RC

PROVIDER: S-EPMC9119709 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Application of digital PCR to determine the reliability of Xpert Xpress SARS-CoV-2 assay with envelope (E) gene negative and nucleocapsid (N2) gene positive results.

Wong River Chun-Wai RC Wong Ann Han AH Ho Yolanda Iok-Ieng YI Siu Gilman Kit-Hang GK Lee Lam-Kwong LK Leung Eddie Chi-Man EC Lai Raymond Wai-Man RW

Diagnostic microbiology and infectious disease 20220520 4

This study used digital polymerase chain reaction (dPCR) to determine whether envelope (E) gene-negative and nucleocapsid (N2) gene-positive (E-N+) results obtained with the Cepheid Xpert Xpress SARS-CoV-2 assay are reliable. Using droplet digital PCR results as a reference, 18 of 22 E-N+ samples with a low viral load (81.8%) were identified as true positives. ...[more]

PMID: 35691105

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Project description:The Xpert® Xpress SARS-CoV-2 and Xpert® Xpress SARS-CoV-2/Flu/RSV tests were rapidly developed and widely used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In response to emerging genetic variability, a new SARS-CoV-2 target (RNA-dependent RNA-polymerase) has been added to both tests: Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus test. A rapid evaluation of both tests was performed in South Africa, using residual respiratory specimens. Residual respiratory specimens (n = 125) were used to evaluate the Xpert® Xpress CoV-2 plus test and included 50 genotyped specimens. The Xpert® Xpress CoV-2/Flu/RSV plus test was assessed using 45 genotyped SARS-CoV-2 specimens, 10 influenza A, 10 influenza B and 20 respiratory syncytial virus specimens. Results were compared to in-country standard-of-care tests. Genotyped specimens tested the performance of the test under pressure from circulating SARS-CoV-2 variants of concern. Reference material was included to assess the test limits and linearity. The Xpert® Xpress CoV-2 plus test performance compared to reference results across residual respiratory specimens was good (positive percentage agreement (PPA) = 95.2%, negative percentage agreement (NPA) = 95.0%) The Xpert® Xpress CoV-2/Flu/RSV plus test showed good performance across all residual respiratory specimens (PPA = 100%, NPA = 98.3%). All genotyped variants of concern were detected by both tests. The Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus tests can be used to diagnose SARS-CoV-2, and to diagnose and differentiate SARS-CoV-2, influenza A, influenza B and respiratory syncytial virus, respectively. The NPA was lower than the recommended 99%, but was influenced by the low number of negative specimens tested. The variants of concern assessed did not affect test performance. It is recommended that sites perform their own assessments compared to in-country standard-of-care tests.

Project description:BackgroundDigital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation.ResultsWe reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably.ConclusionsLike any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.

Dataset Information

Application of digital PCR to determine the reliability of Xpert Xpress SARS-CoV-2 assay with envelope (E) gene negative and nucleocapsid (N2) gene positive results.

Publications

Application of digital PCR to determine the reliability of Xpert Xpress SARS-CoV-2 assay with envelope (E) gene negative and nucleocapsid (N2) gene positive results.

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