COVID-19 testing and correlation with infectious virus, cycle thresholds, and analytical sensitivity

COVID-19 testing and correlation with infectious virus, cycle thresholds, and analytical sensitivity. Spencer EA, Jefferson T, Heneghan C.

https://www.cebm.net/study/covid-19-testing-and-correlation-with-infectious-virus-cycle-thresholds-and-analytical-sensitivity/

Published on August 27, 2020

Reference Gniazdowski V, Morris P, Wohl S et al. Repeat COVID-19 molecular testing: correlation with recovery of infectious virus, molecular assay cycle thresholds, and analytical sensitivity. medRxiv 2020.08.05.20168963; doi: https://doi.org/10.1101/2020.08.05.20168963
Study type
Country USA
Setting Hospital based SARS-CoV-2 testing facility
Funding Details The NIH Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C and T32A1007417 Molecular and Cellular Basis of Infectious Diseases . DdPCR was performed in collaboration with Bio-Rad Laboratories
Transmission mode Viral Load
Exposures

Bottom Line

Molecular detection of SARS-CoV-2 RNA did not mean infectious virus was present.   The use of Ct values and clinical symptoms in combination with PCR testing for SARS-CoV-2 provides a more accurate assessment of the potential for infectious virus shedding.

Evidence Summary

The cultured specimens (from 161 cases with positive PCR) spanned a wide range of cycle threshold values reflecting different viral loads.

  • Infectious virus was associated with mean Ct values of 18.8 ± 3.4 (median 18.7)
  • Not obtaining infectious virus was associated with mean Ct values 27.1 ± 5.7 (median 27.5) (see the figure)

Samples with a Ct value below 23 yielded 91.5% of virus isolates. 

However, 29% of specimens that were negative for viral growth on VeroE6 cells were in the same Ct value range and 11.9% were below a Ct value of 20

Prolonged viral RNA shedding was associated with recovery of infectious virus in specimens collected up to 20 days after the first positive result in patients who were symptomatic at the time of specimen collection. 

The observed general trend was an increase in the Ct values over time indicating a reduction in the viral RNA load, and further correlating, in the majority of the patients, with failure to recover infectious virus on cell culture. 

Four patients had infectious virus recovered from specimens collected up to 22 days after the first positive result, however, infectious virus shedding was not associated with a specific outcome as one patient was never admitted, one was hospitalized with no oxygen requirements, and two had more severe disease. Recovery of infectious virus was associated with persistence of symptoms in all but one patient

Many patients who tested negative for SARS-COV-2 showed a subsequent positive result. The follow up positive testing on previously negative patients produced Ct values higher than 29.5. Attempted recovery of infectious virus from these specimens was negative.

Correlation between recovery of SARS-CoV-2 infectious virus on cell culture and Ct values. Nasopharyngeal specimens were cultured on VeroE6 cells and the recovery of virus and the development of cytopathic effect were monitored for up to 4 days post-infection. Viral growth was confirmed by antigen staining or PCR. *** paired t-test, P<0.0001.

Gniazdowski V, Morris P, Wohl S et al. Repeat COVID-19 molecular testing: correlation with recovery of infectious virus, molecular assay cycle thresholds, and analytical sensitivity.medRxiv 2020.08.05.20168963; doi: https://doi.org/10.1101/2020.08.05.20168963

What did they do?

Consecutively collected specimens from a retrospective cohort of COVID-19 patients who were assessed for RNA and infectious virus shedding. 

The researchers analyzed the molecular diagnostics data from Johns Hopkins Hospital in the time frame March 11th to May 11th 2020.

Whole genome sequencing confirmed the virus genotype in patients with prolonged viral RNA shedding, and droplet digital PCR (ddPCR) was used to assess the rate of false negative standard of care PCR results.

Clinical samples for 161 patients’ specimens that were positive by molecular testing were cultured with VeroE6 cells, and indirect immunofluorescence microscopy used to assess recovery of infectious virus. 

To assess the correlation between the repeated positivity, viral loads, and laboratory recovery of infectious virus, a randomly selected subset of 29 patients was evaluated. The authors examined the Ct values of all test results, days between testing, as well as viral growth on cell culture (if performed)

Study reliability

Details of the clinical symptomatology of all cases were not available. 

Clearly defined setting Demographic characteristics described Follow-up length was sufficient Transmission outcomes assessed Main biases are taken into consideration
Yes No N/A N/A Unclear

What else should I consider?

The paper additionally reports details of genome sequence comparisons.

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.