RNA-extraction-free diagnostic method to detect SARS-CoV-2: an assessment from two States, India

  1. Madhumathi Jayaprakasam
  2. Sumit Aggarwal
  3. Arati Mane
  4. Vandana Saxena
  5. Amrita Rao
  6. Bhaswati Bandopadhyay
  7. Banya Chakraborty
  8. Subhasish Kamal Guha
  9. Mekhala Taraphdar
  10. Alisha Acharya
  11. Bishal Gupta
  12. Sonia Deb
  13. Aparna Chowdhury
  14. Kh Jitenkumar Singh
  15. Prashant Tapase
  16. Ravindra M Pandey
  17. Balram Bhargava
  18. Samiran Panda

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Abstract

With increasing demand for large numbers of testing during COVID-19 pandemic, came alternative protocols with shortened turn-around time. We evaluated the performance of such an approach wherein 1138 consecutive clinic attendees were enrolled; 584 and 554 respectively from two independent study sites in the cities of Pune and Kolkata. Paired nasopharyngeal and oropharyngeal swabs were tested by using both reference and index methods in blinded fashion. Prior to conducting RT-PCR, swabs collected in viral transport medium (VTM) were processed for RNA extraction (reference method) and swabs collected in dry tube without VTM were incubated in Tris-EDTA-Proteinase K buffer for 30 minutes and heat inactivated at 98°C for 6 minutes (index method). Overall sensitivity and specificity of the index method were 78.9% (95% CI 71% to 86%) and 99 % (95% CI 98% to 99.6%) respectively. Agreement between the index and reference method was 96.8 % (k = 0.83, SE=0.030). The reference method exhibited enhanced detection of viral genes (E, N and RdRP) with lower Ct values compared to the index method. The index method can be used for detecting SARS-CoV-2 infection with appropriately chosen primer-probe set and heat treatment approach in pressing time; low sensitivity constrains its potential wider use.

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  1. ScreenIT

    SciScore for 10.1101/2021.09.19.21263807: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The proposal for evaluation was developed in early October 2020 and approval was obtained from the Central Ethics Committee for Human Research (CECHR) of Indian Council of Medical Research (ICMR) on 30th October 2020.
    Consent: Written informed consent was obtained from individuals consenting to participate in this study.
    Sex as a biological variablenot detected.
    RandomizationIn order to ensure blinding, a different set of unique codes were randomly generated from the ICMR-headquarter, New Delhi for each study site using Excel based tool for labelling the corresponding swabs collected and placed in dry tubes.
    BlindingIn order to ensure blinding, a different set of unique codes were randomly generated from the ICMR-headquarter, New Delhi for each study site using Excel based tool for labelling the corresponding swabs collected and placed in dry tubes.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    In order to ensure blinding, a different set of unique codes were randomly generated from the ICMR-headquarter, New Delhi for each study site using Excel based tool for labelling the corresponding swabs collected and placed in dry tubes.
    Excel
    suggested: None
    The sensitivity, specificity, concordance, discordance, positive predictive value, negative predictive value and agreement between the tests and their 95% confidence interval (CI) were computed using Stata version 13.1 (StataCorp LP, College Station, TX, USA).
    StataCorp
    suggested: (Stata, RRID:SCR_012763)
    Graphpad Prism (version 5) and R-CRAN (version 4.0.3) with ggplot2 library was used for graphical representations.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    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:
    The lower sensitivity of this RNA-extraction-free RT-PCR method in real world setting appears to be one of its limitations.

    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 10.1101/2021.09.19.21263807v1 on medRxiv
  3. Version published to 10.1017/s0950268821002302