Predicting infectious SARS-CoV-2 from diagnostic samples

Predicting infectious SARS-CoV-2 from diagnostic samples. Spencer EA, Heneghan C.

https://www.cebm.net/study/predicting-infectious-sars-cov-2-from-diagnostic-samples/

Published on July 27, 2020

Reference Bullard J, Dust K, Funk D, et al. Predicting infectious SARS-CoV-2 from diagnostic samples Clin Infect Dis. 2020;ciaa638. doi:10.1093/cid/ciaa638
Study type
Country Canada
Setting Public health surveillance testing among suspected cases.
Funding Details Non Reported
Transmission mode Community
Exposures

Bottom Line

SARS-CoV-2 Vero cell infectivity of respiratory samples from SARS-CoV-2 positive individuals was only observed for RT-PCR Ct < 24 and symptom onset to test of < 8 days. 

Infectivity of patients with Ct >24 and duration of symptoms >8 days may be low.

Evidence Summary

Ninety RT-PCR SARS-CoV-2 positive samples were available. The Median age of the patients sampled was 45 (30 to 59) years.

After incubation on Vero cells, 26 samples (28.9%) demonstrated viral growth. 

Median TCID50*/ml was 1,780 (282 to 8511). 

There was no growth in samples with a cycle threshold** (Ct) > 24 or symptom onset to test time (STT) > 8 days. 

Multivariate logistic regression using positive viral culture as a binary predictor variable, symptom onset to test and Ct demonstrated an OR for a positive viral culture of 0.64 (95%CI 0.49 to 0.84, p<0.001) for every one unit increase in Ct. 

This implies that for every one-unit increase in Ct, the odds of a positive culture decreased by 36%)

The area under the receiver operating characteristic (ROC) curve for Ct vs. positive culture was OR 0.91 (95%CI 0.85 to 0.97, p<0.001), with 97% specificity obtained at a Ct of >24.

The probability of obtaining a positive viral culture peaked on day 3 and decreased from that point.

*TCID50 signifies the concentration at which 50% of the cells are infected when a test tube or well plate upon which cells have been cultured is inoculated with a diluted solution of viral fluid.

** PCR assay a positive reaction is detected by accumulation of a fluorescent signal.

The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold ( exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (the lower the Ct level the greater the amount of target nucleic acid in the sample).

What did they do?

This study investigated viral culture in samples from a larger cross-sectional group of patients and compared the results with PCR testing data and time of symptom onset.

More specifically, it looked at the E gene SARS-CoV-2 RT-PCR cycle threshold (Ct) values from respiratory samples and compared these with symptom onset to test time (STT) and infectivity in cell culture as shown by the ability to infect Vero cell lines.

This was a retrospective cross-sectional study. 

  • SARS-CoV-2 RT-PCR was performed on nasopharyngeal (NP) or endotracheal (ETT) samples collected from individuals with suspected COVID-19.
  • Date of symptom onset was gathered from public health and epidemiology/surveillance and laboratory records
  • Time to symptom onset or time to test was calculated using laboratory records
  • For all positive samples, the cycle threshold (Ct) was obtained
  • Viral titres of patient samples were determined through fifty-per cent tissue culture infective dose (TCID50) assays [quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells]

Study reliability

This study aimed to examine actual viral replication (as opposed to just observing isolated virus or viral RNA). 

However, it was a moderately small study and requires repeating in a larger sample size. 

False positives in the PCR testing are unlikely but possible. Recall bias may have affected participants’ reporting of symptom onset dates. 

 

Clearly defined setting Demographic characteristics described Follow-up length was sufficient Transmission outcomes assessed Main biases are taken into consideration
Yes Yes N/A N/A Yes

What else should I consider?

Viral culture is difficult, requires elevated safety precautions and is time-consuming, but it seems we need to understand infectivity of the range of viral presence in samples from humans, so as to understand how transmission is occurring. Therefore more such studies are urgently needed.

Protocol HS23906 (H2020:211) was approved by the University of Manitoba Research Ethics Board. 

About the authors

Carl Heneghan

Carl Heneghan

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

Elizabeth Spencer

Elizabeth Spencer

Dr Elizabeth Spencer; MMedSci, PhD. Epidemiologist, Nuffield Department for Primary Care Health Sciences, University of Oxford.

Tom Jefferson

Tom Jefferson

Tom Jefferson is a senior associate tutor and honorary research fellow, Centre for Evidence-Based Medicine, University of Oxford.