Predicting Infectivity: Comparing Four PCR-based Assays to Detect Culturable SARS-CoV-2 in Clinical Samples

Abstract

With the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g. subgenomic RNA [sgRNA]) and replicative intermediates (e.g. negative-strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS-CoV-2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3-37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative-strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative-strand, and sgE) detected with the WHO/Charite primer-probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS-CoV-2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cut-off of >31 or specifically testing for sgRNA can serve as an effective rule-out test for viral infectivity.

Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants

“…To minimize variation resulting from sample handling, we exclusively used samples that had been frozen within two days of sample collection from the patient. Once collected, clinical samples are routinely stored and transported at 4ºC and our previous work demonstrated that infectious virus and RNA are stable at both 4ºC and room temperature for over four days 23 . While limited residual material in clinical samples makes performing traditional plaque assays technically challenging, we have successfully used a focus forming assay performed in 96-well plates to measure infectious viral titer of volumes as small as 100-200 µl 23,24 .…”

“…Once collected, clinical samples are routinely stored and transported at 4ºC and our previous work demonstrated that infectious virus and RNA are stable at both 4ºC and room temperature for over four days 23 . While limited residual material in clinical samples makes performing traditional plaque assays technically challenging, we have successfully used a focus forming assay performed in 96-well plates to measure infectious viral titer of volumes as small as 100-200 µl 23,24 . This approach enables us to measure individual infectious viral units directly from residual clinical samples, which to our knowledge are the first such measurements reported.…”

“…#4387424M), using 5 µl of extracted TNA per 25 µl reaction. RT-PCR reactions used one of two sets of primers/probes: 1) WHO-E, using the E_Sarbeco-F/R/P set 34 ; 2) Mills-sgE, using sgLeader-F2, sgE-R, and sgE-P 23 . RT-PCR was performed on an ABI 7500 real-time PCR system as previously described 35 .…”

“…A set of 165 clinical swab samples from individuals infected with the Alpha (teal circles; n=21), Epsilon (orange triangles; n=31) or Delta (purple squares; n=55+58) SARS-CoV-2 variants of concern was used to determine the relationship between viral titer and viral RNA levels for each sample. A) For each clinical sample total E RNA (RT-PCR C T detected by WHO-E primer/probe set 38 ) and B) subgenomic E RNA (RT-PCR C T detected by the Mills-sgE primer set we previously developed 23 ) is plotted on the X axis. Viral titer for each sample (FFU/mL measured by viral focus assay) is plotted on the Y axis.…”

“…R code is available upon reasonable request. and B) subgenomic E RNA (RT-PCR C T detected by the Mills-sgE primer set we previously developed 23 ) is plotted on the X axis. Viral titer for each sample (FFU/mL measured by viral focus assay) is plotted on the Y axis.…”