Cell-based Culture Informs Infectivity and Safe De-Isolation Assessments in Patients with Coronavirus Disease 2019
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Abstract
Background
The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA by reverse-transcription polymerase chain reaction (PCR) does not necessarily indicate shedding of infective virions. There are limited data on the correlation between the isolation of SARS-CoV-2, which likely indicates infectivity, and PCR.
Methods
A total of 195 patients with Coronavirus disease 2019 were tested (outpatients, n = 178; inpatients, n = 12; and critically unwell patients admitted to the intensive care unit [ICU] patients, n = 5). SARS-CoV-2 PCR-positive samples were cultured in Vero C1008 cells and inspected daily for cytopathic effect (CPE). SARS-CoV-2–induced CPE was confirmed by PCR of culture supernatant. Where no CPE was observed, PCR was performed on day 4 to confirm absence of virus replication. The cycle thresholds (Cts) of the day 4 PCR (Ctculture) and the PCR of the original clinical sample (Ctsample) were compared, and positive cultures were defined where Ctsample − Ctculture was ≥3.
Results
Of 234 samples collected, 228 (97%) were from the upper respiratory tract. SARS-CoV-2 was isolated from 56 (24%), including in 28 of 181 (15%), 19 of 42 (45%), and 9 of 11 samples (82%) collected from outpatients, inpatients, and ICU patients, respectively. All 56 samples had Ctsample ≤32; CPE was observed in 46 (20%). The mean duration from symptom onset to culture positivity was 4.5 days (range, 0–18). SARS-CoV-2 was significantly more likely to be isolated from samples collected from inpatients (P < .001) and ICU patients (P < .0001) compared with outpatients, and in samples with lower Ctsample.
Conclusions
SARS-CoV-2 culture may be used as a surrogate marker for infectivity and inform de-isolation protocols.
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SciScore for 10.1101/2020.07.14.20153981: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding In addition, no viral replication was identified with blind passaging of the first 20 cultures without CPE and Ctsample - Ctculture <3, indicating the absence of viable virus. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: Routine mycoplasma testing was performed to exclude mycoplasma contamination of the cell line and all culture work was undertaken in a physical containment laboratory level 3 (PC3) biosafety conditions. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Determination of TCID50: A primary isolate, SARS-CoV-2 /01/Human/NSW … SciScore for 10.1101/2020.07.14.20153981: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding In addition, no viral replication was identified with blind passaging of the first 20 cultures without CPE and Ctsample - Ctculture <3, indicating the absence of viable virus. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: Routine mycoplasma testing was performed to exclude mycoplasma contamination of the cell line and all culture work was undertaken in a physical containment laboratory level 3 (PC3) biosafety conditions. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Determination of TCID50: A primary isolate, SARS-CoV-2 /01/Human/NSW Australia|EPI_ISL_407893|2020-01-24 (GISAID), was titrated in serial one log dilutions (from 10−1 to 10−8) to obtain a 50% tissue culture infective dose (TCID50) using 24-well culture plates of Vero E6 cells. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources Plates were sealed with AeraSeal™ Film (Excel Scientific, Inc., Victorville, CA, USA) to minimise evaporation, spillage, and well-to-well cross-contamination. Excel Scientificsuggested: NoneResults 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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study 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.
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