Diagnostic Performance of Pooled RT-PCR Testing for SARS-CoV-2 Detection

  1. Diadem Ricarte
  2. Aubrey Gador
  3. Leomill Mendiola
  4. Ian Christian Gonzales

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Abstract

Background

With the high number of COVID-19 cases, a need to optimize testing strategy must be regarded to obtain timely diagnosis for early containment measures. With this, several studies have employed pooled RT-PCR testing for SARS-CoV-2 as this could potentially conserve laboratory resources while has the capacity to test several individuals. However, this was recommended to firstly validate the method as different laboratory reagents and equipment vary with its diagnostic performance.

Objective

The aim of this study was to determine the diagnostic performance of pooled SARS-CoV-2 nasopharyngeal/oropharyngeal swabbed samples using RT-PCR technique.

Methods

A records review of two-staged pooled RT-PCR testing data from August 10, 26, 30 and September 5, 2020 was utilized from Northern Mindanao Medical Center COVID-19 Satellite Laboratory (formerly CHDNM TB Regional Center). For the first stage, using known samples, a total of 30 pools were made for each of the pooling size, 5- and 10-pooled, on both pooling phase, pre- and post-RNA extraction. One positive individual was used to represent each of the Cycle threshold values given (<24, 25-28, 29-32, 33-36, and 37-40) while the rest of the samples were negative. For the second stage, 54 pools of five from 270 random unknown samples were used to validate the results. Target gene performance of N gene and RdRp was also determined.

Key Results

Results show that 5-pooled sample has higher sensitivity (SN), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 100% (95% confidence interval (CI) 88.97-100), 66.95% (95% CI, 60.75-72.6), 28.18% (95% CI, 20.62-37.22), and 100% (95% CI, 97.66-100) compared to 10-pooled sample that has 87.1% (95% CI, 71.15-94.87), 56.9% (50.57-63.02), 20.77% (95% CI, 14.68-28.53) and 97.14% (95% CI, 92.88-98.88). Further, these Ct values were only from the N gene, emphasizing its higher diagnostic performance as well to detect SARS-CoV-2 compared to RdRp as only a few samples were detected, thus, no analysis was made.

Conclusion

This study found out that 5-pooled sample has better diagnostic performance compared to 10-pooled samples. Specifically, all positive individual samples were detected in 5-pooled samples in pre-RNA extraction phase which these results are evident and consistent on both known and unknown samples. N gene was found out to detect more SARS-CoV-2 samples compared to RdRp.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.17.21251961: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    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:
    Further, its limitation includes no analysis on the pooling of SARS-CoV-2 swabbed samples of the symptomatic and asymptomatic individuals as only de-identified samples were utilized. Overall, this study serves as a proof of concept of the pooling method, illustrating the results of the different pooling sizes on both RNA extraction phases, of SARS-CoV-2 using RT-PCR technique. In conclusion, this study found out that 5-pooled sample has better diagnostic performance compared to 10-pooled samples as, specifically, all positive individual samples were only detected in 5-pooled samples in pre-RNA extraction phase which these results are evident and consistent on both known and unknown samples. Further, N gene was found out to detect more SARS-CoV-2 samples compared to RdRp. Based on the results of this study, the following are hereby recommended for future research and development work: For research institutes For policymakers:

    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.
    • Thank you for including a protocol registration statement.

    About SciScore

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  2. Version published to 10.1101/2021.02.17.21251961 on medRxiv