Evaluation of the relationship between quantitative PCR results and cell culturing of SARS2-CoV with respect to symptoms onset and Viral load – a systematic review

  1. Rozenberg Gilad
  2. Erster Oran
  3. Ghersin Itai
  4. Mandelboim Michal
  5. Neuberger Ami
  6. Schwartz Eli

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Abstract

Background

Viral culture is currently the most accurate method to demonstrate viability and infectivity of Severe acute respiratory syndrome Coronavirus (SARS-2 CoV). Routine clinical diagnosis, however, is mostly performed by PCR – based assays that do not discriminate between infectious and non-virus. Herein, we aimed to determine the correlation between positive viral cultures and either PCR positivity, the Cycle Threshold (Ct) or the number of viral copies.

Methods

A systematic electronic literature search was performed and studies that reported both viral SARS-CoV-2 culture and PCR–based assays were included. A separate search for samples from blood, urine, stool, breast milk and tears were performed. To convert Ct values reported in the reviewed studies were to viral genomic copies, calibration experiments with four different reaction performed, using quantified RNA molecules.

Results

A total 540 articles were reviewed, and 38 studies were included in this review. Out of 276 positive-culture of non-severe patients, 272 (98.55%) were negative ten days after symptoms onset, while PCR assays remained positive for up to 67 days. In severely ill or immunocompromised patients positive-culture was obtained up to 32 days and out of 168 cultures, 31 (18.45%) stayed positive after day 10. In non-severe patients, in Ct value greater than 30 only 10.8% were still culture-positive while in Ct >35 it was nearly universally negative. The minimal calculated number of viral genome copies in culture-positive sample was 2.5 × 10 3 copies / mL. These findings were similar in immunocompromised patients. Recovering positive culture from non-respiratory samples was sporadically obtained in stool or urine samples. Conversion of Ct values to viral genome copies showed variability between different PCR assays and highlighted the need to standardize reports to correctly compare results obtained in different laboratories.

Conclusion

During the pandemic phase, non-severe COVID-19 patients who are recovering and are not immuno-suppressed, can be regarded as non-infectious, within 10 days from symptom onset, or with Ct value greater than 35 (or a calculated viral load lower than 1.2×10 3 copies / mL). These findings have important implications for recovering patients and asymptomatic patients, with respect to isolation criteria. The conversion of Cq values to viral genome copies described herein may be useful in future work, enabling a more standardized comparison between results reported in different studies from different laboratories.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.23.21262162: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    RandomizationWe have included randomized controlled trials (RCTs), non-randomized comparative cohorts and case series studies until August 15, 2020.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Outcome measures: Search methods for identification of studies: We conducted a systematic electronic literature search with the PubMed and MedRxiv search engines.
    PubMed
    suggested: (PubMed, RRID:SCR_004846)
    MedRxiv
    suggested: (medRxiv, RRID:SCR_018222)

    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 limitations of this review include lack of larger cohorts of COVID-19 with serial viral cultures, lack of standardization of laboratory techniques, and a reporting bias that probably leads to overestimation of cases with prolonged viral shedding. Another fundamental limitation is the need to draw practical conclusions from cell culture data. Due to obvious safety limitations, it is very complicated to perform SARS-CoV-2 infection experiments with animal model. Therefore, cell tissue culture (T/C) is the best practical alternative. However, inferring human viral infectivity based on T/C experiments needs to be done carefully, due to multiple stages, which the sample undergoes, before the test is performed. Sampling method, sampling media, transport timeline and storage conditions, may all affect the test results. Additionally, factors such as different susceptibility and presence/absence of effective immune response may also affect the quantity of infectious virus in the sample. A second limitation concerning T/C results is the different practices between different laboratories, different conditions of the cells used and different sample processing methods. Each of these factors separately can have a profound effect on the outcome. Combined, they render the comparison of different studies performed under different conditions very complicated. The reasons for unsuccessful culturing may stem from technical issues, and should therefore be interpreted carefully, when trying to...

    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

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  2. Version published to 10.1101/2021.08.23.21262162v1 on medRxiv