Cell-based culture of SARS-CoV-2 and safe de-isolation assessments during COVID-19
Cell-based culture of SARS-CoV-2 informs infectivity and safe de-isolation assessments during COVID-19. Spencer EA, Heneghan C.
Published on August 27, 2020
Transmission Dynamics of COVID-19
||Basile K, McPhie K, Carter I et al. Cell-based culture of SARS-CoV-2 informs infectivity and safe de-isolation assessments during COVID-19 medRxiv 2020.07.14.20153981; doi: https://doi.org/10.1101/2020.07.14.20153981
||Outpatients and inpatient
||Prevention Research Support Program funded by the New South Wales Ministry of Health and the National Health and Medical Research Council (APPRISE 1116530).
The threshold PCR Ct value for successful virus isolation was less than 32 for the N gene target. A Ct cut-off of ≥37 was not indicative of viable virus.
234 samples, 228 (97%) from the upper respiratory tract, were collected from 195 individuals with Covid-19.
SARS-CoV-2 was only successfully isolated from samples with Ct sample values <32
Successful virus isolation was performed for 28/181 (15%), 19/42 (45%) and 9/11 samples (82%) collected from outpatients, inpatients and ICU patients, respectively.
The mean duration from symptom onset to culture positivity was 4.5 days (range 0 to 18 days).
SARS-CoV-2 was significantly more likely to be isolated from samples collected from inpatients (p<0.001) and ICU patients (p<0001) compared with outpatients, and in samples with lower Ct sample values.
What did they do?
The isolation of SARS-CoV-2 using cell-based culture likely indicates infectivity, but there are limited data on the correlation between SARS-CoV-2 culture and PCR.
This study examined the correlation between PCR results and virus culture for SARS-CoV-2
In samples collected from patients with Covid-19 (out-patients and some in-patients with severe Covid-19). The aim was to inform the safe de-isolation of persons with persistently positive PCR tests.
195 patients with diverse severity of COVID-19 were tested (outpatients [n=178]), inpatients [n=12] and ICU [n=5]).
Samples came from either routine laboratory tests at the start of the outbreak or from patients admitted to the intensive care unit (ICU) or from a request by the attending physician (usually due to persistent PCR positivity in an individual awaiting release from quarantine) or public health units as part of outbreak investigations
SARS-CoV-2 PCR positive samples were cultured in Vero C1008 cells and inspected daily for cytopathic effect. SARS-CoV-2-induced cytopathic effect was confirmed by PCR of culture supernatant. Where no cytopathic effect was documented, PCR was performed on day four to confirm the absence of virus replication.
Cycle threshold (Ct) values of the day four PCR (Ct culture) and the PCR of the original clinical sample (Ct sample) were compared, and positive cultures were defined as a Ct sample – Ct culture value of greater than or equal to 3.
There may be analytical performance differences across different PCR tests and different laboratories.
The study was unable to investigate why some individuals shed SARS-CoV-2 RNA for longer than others or any influence of immunosuppression.
The selection of donors of samples was not clearly described and no demographic characteristics were reported.
|Clearly defined setting
||Demographic characteristics described
||Follow-up length was sufficient
||Transmission outcomes assessed
||Main biases are taken into consideration
What else should I consider?
Detection of SARS-CoV-2 RNA may not indicate the presence of viable virus with replicative capacity and by implication transmissibility
Burkardt HJ. Standardization and quality control of PCR analyses. Clin Chem Lab Med. 2000; 38(2): 87-91. Doi: 10.1515/CCLM.2000.014
‘PCR also amplifies DNA from dead organisms rendering a result diagnostically correct as positive, but clinically as false-positive. In PCR, as in any other diagnostic test, the risk of creating a false-negative result also exists. In such a case, the most probable source besides human error, low target or poor amplification and detection protocols is an inhibition caused by interfering substances in a patient’s sample. Strategies to recognize and overcome this issue are discussed in this article.’
About the authors
Carl is Professor of EBM & Director of CEBM at the University of Oxford. He is also a GP and tweets @carlheneghan. He has an active interest in discovering the truth behind health research findings
Dr Elizabeth Spencer; MMedSci, PhD. Epidemiologist, Nuffield Department for Primary Care Health Sciences, University of Oxford.