Clinical evaluation of a fully automated, laboratory-developed multiplex RT-PCR assay integrating dual-target SARS-CoV-2 and influenza A/B detection on a high-throughput platform

  1. Dominik Nörz
  2. Armin Hoffmann
  3. Martin Aepfelbacher
  4. Susanne Pfefferle
  5. Marc Lütgehetmann

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Abstract

Introduction. Laboratories worldwide are facing high demand for molecular testing during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, which might be further aggravated by the upcoming influenza season in the northern hemisphere.

Gap Statement. Given that the symptoms of influenza are largely indistinguishable from those of coronavirus disease 2019 (COVID-19), both SARS-CoV-2 and the influenza viruses require concurrent testing by RT-PCR in patients presenting with symptoms of respiratory tract infection.

Aim. We adapted and evaluated a laboratory-developed multiplex RT-PCR assay for simultaneous detection of SARS-CoV-2 (dual target), influenza A and influenza B (SC2/InflA/InflB-UCT) on a fully automated high-throughput system (cobas6800).

Methodology. Analytical performance was assessed by serial dilution of quantified reference material and cell culture stocks in transport medium, including pretreatment for chemical inactivation. For clinical evaluation, residual portions of 164 predetermined patient samples containing SARS-CoV-2 ( n =52), influenza A ( n =43) or influenza B ( n =19), as well as a set of negative samples, were subjected to the novel multiplex assay.

Results. The assay demonstrated comparable analytical performance to currently available commercial tests, with limits of detection of 94.9 cp ml −1 for SARS-CoV-2, 14.6 cp ml −1 for influenza A and 422.3 cp ml −1 for influenza B. Clinical evaluation showed excellent agreement with the comparator assays (sensitivity of 98.1, 97.7 and 100 % for Sars-CoV-2 and influenza A and B, respectively).

Conclusion. The SC2/InflA/InflB-UCT allows for efficient high-throughput testing for all three pathogens and thus provides streamlined diagnostics while conserving resources during the influenza season.

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    SciScore for 10.1101/2020.10.25.20215285: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIRB: The use of anonymized samples was approved by the ethics committee, Freie und Hansestadt Hamburg, PV5626.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    No key resources detected.

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    However, the ability to employ inhouse assays on fully automated PCR platforms provides vastly improved scalability of testing capacity and less limitations due to availability of trained personnel. Most currently available commercial SARS-CoV-2 tests with FDA emergency authorization are designed as multi-target assays to account for emerging mutations. While the original Sarbeco-E primerset by Corman et al. (6) resides in a particularly stable region of the SARS-CoV-2 genome, single mutations have been reported within the ever-growing catalogue of available whole genome sequences (13). The RdRp/Hel assay by Chan et al. (7) was modified and adapted as a second target to provide additional security for inclusivity in SARS-CoV-2 detection. Both assays were allocated to the same channel as positivity of any single one would constitute a positive result. There was no indication that presence of Influenza-A and/or Influenza-B RNA within the reaction substantially impairs sensitivity for SARS-CoV-2. If necessary, each SARS-CoV-2 assay can be analysed separately by moving RdRp/Hel detection to channel 1, using the following probe (or comparable): 5’ Atto425-TTAAGATGT(BMN-Q535)GGTGCTTGCATACGTAGAC -BMN-Q535 3’ (see supplement figure 2). It has to be acknowledged that the SARS-CoV-2 assays used for this multiplex-setup are technically not specific for SARS-CoV-2 but for the Sarbeco-subgenus of betacoronaviruses, including SARS-CoV (from 2003) and SARS-like bat viruses. In conclusion, w...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1099/jmm.0.001295 on Access Microbiology
  3. Version published to 10.1101/2020.10.25.20215285 on medRxiv