Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multi-target approach

  1. Matthew M. Hernandez
  2. Radhika Banu
  3. Ana S. Gonzalez-Reiche
  4. Adriana van de Guchte
  5. Zenab Khan
  6. Paras Shrestha
  7. Liyong Cao
  8. Feng Chen
  9. Huanzhi Shi
  10. Ayman Hanna
  11. Hala Alshammary
  12. Shelcie Fabre
  13. Angela Amoako
  14. Ajay Obla
  15. Bremy Alburquerque
  16. Luz Helena Patiño
  17. Juan David Ramírez
  18. Robert Sebra
  19. Melissa R. Gitman
  20. Michael D. Nowak
  21. Carlos Cordon-Cardo
  22. Ted E. Schutzbank
  23. Viviana Simon
  24. Harm van Bakel
  25. Emilia Mia Sordillo
  26. Alberto E. Paniz-Mondolfi

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Abstract

The COVID-19 pandemic sparked rapid development of SARS-CoV-2 diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY ® SARS-CoV-2 Panel combines RT-PCR and MALDI-TOF mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified dataset of 1,262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 through April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly-specific for the alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.09.09.21263348: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: This study was reviewed and approved by the Institutional Review Board of the Icahn School of Medicine at Mount Sinai (HS#13-00981).
    Field Sample Permit: SARS-CoV-2 specimen collection and testing: Upper respiratory tract (e.g., nasopharyngeal, anterior nares) and saliva specimens collected for SARS-CoV-2 testing underwent diagnostic testing in the MSHS Clinical Microbiology Laboratory (CML), which is certified under Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a and meets requirements to perform high-complexity tests.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: SARS-CoV-2 sequencing, assembly and phylogenetic analyses: SARS-CoV-2 viral RNA underwent reverse transcription, PCR amplification and next-generation sequencing followed by genome assembly and lineage assignment using a phylogenetic-based nomenclature as described by Rambaut et al. 27 using the PANGOLIN tool, version 2021-04-28 28 as previously described 25,26.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    The Multiple Alignment using Fast Fourier Transform (MAFFT) platform 29,30 which is publicly available for use online (https://mafft.cbrc.jp/alignment/server/add_fragments.html?frommanualnov6) was used to align each file.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Mismatches by position in PBS regions of forward/reverse primer and probe sequences were manually counted on Microsoft Excel v16.48.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    Statistical analyses: For statistical comparison of fraction of PBS with mismatches in genomes with detected targets versus those with dropout targets, normality was assessed by D’Agostino and Pearson test (GraphPad Prism 9.1.0), which indicated that all distributions were non-parametric; thus, a Mann-Whitney test (two-tailed) was performed (GraphPad).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Display Items: All figures are original and were generated using the GraphPad Prism software 9.1.0, R software package ggplot2, NCBI Multiple Sequence Alignment Viewer v.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    1.17.0 (https://www.ncbi.nlm.nih.gov/tools/msaviewer/), and finished in Adobe Illustrator 2021 (v.25.2.1).
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)

    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:
    A number of studies have described substitutions in the ORF1ab, S, E, and N genes that may interfere with specific RT-PCR targets 1,5,9,11,12,14–16,18, but these studies have inherent limitations. Several are in silico analyses that do not reflect diagnostic performance in the clinical setting 1,5,16,18, whereas others do not definitively demonstrate target dropout due to substitutions as they utilize platforms for which primer/probe sequence information is not publicly available 9,11,14,15. In addition, the later studies are based on assays that interrogate up to 3 diagnostic targets, and are limited by the number and diversity of viral sequences surveyed over finite timeframes, some prior to the emergence and expansion of VOCs. In the current study, we describe a robust evaluation of the impact of PBS mismatches on Agena MassARRAY® SARS-CoV-2 Panel target results for over 1,200 specimens over a five-month time period that corresponds with the rapid emergence of viral VOIs/VOCs (December 2020 through April 2021). This large dataset enabled direct correlation of detection of each of five different Agena diagnostic viral targets with genomic sequence data Additionally, by using publicly available primer/probe sequences to map lineage-specific substitutions, we were able to further evaluate the impact of mismatches on target results and to demonstrate an association between variation in SARS-CoV-2 PBSs and target dropout. Our analysis revealed that several mutations result in N...

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.09.09.21263348v1 on medRxiv