Cell-based culture of SARS-CoV-2 informs infectivity and safe de-isolation assessments during COVID-19

Abstract

Background The detection of SARS-CoV-2 by real-time polymerase chain reaction (PCR) in respiratory samples collected from persons recovered from COVID-19 does not necessarily indicate shedding of infective virions. By contrast, 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. Here we review our experience using SARS-CoV-2 culture to determine infectivity and safe de-isolation of COVID-19 patients. Methods 195 patients with diverse severity of COVID-19 were tested (outpatients [n=178]), inpatients [n=12] and ICU [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 documented, PCR was performed on day four to confirm absence of virus replication. Cycle threshold (Ct) values of the day four PCR (Ctculture) and the PCR of the original clinical sample (Ctsample) were compared, and positive cultures were defined as a Ctsample - Ctculture value of greater than or equal to 3. Findings Of 234 samples collected, 228 (97%) were from the upper respiratory tract. SARS-CoV-2 was only successfully isolated from samples with Ctsample values <32, including in 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 0-18 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 Ctsample values. Conclusion SARS-CoV-2 culture may be used as a surrogate marker for infectivity and inform de-isolation protocols.

Nasopharyngeal Panbio COVID-19 antigen performed at point-of-care has a high sensitivity in symptomatic and asymptomatic patients with higher risk for transmission and older age

“…Although the antigen test showed an overall lower sensitivity than the RT-PCR in our study, and that reported by the manufacturer, the test was highly accurate in symptomatic patients exhibiting lower Ct values, with a sensitivity greater than 95% for Ct of 25 or lower, and at least 85% for Ct of less than 30. High SARS-CoV-2 viral load has been associated with severity of disease and mortality [9,10], and several studies support a correlation of Ct values with infectivity, as defined by growth in cell culture [11][12][13]. Although breakpoints fluctuate among different studies, a diagnostic Ct value of RT-PCR equal or greater than 32 was associated with no isolation of SARS-CoV-2 using cell-based cultures, neither with active viral replication [11].…”

The duration of infectiousness of individuals infected with SARS-CoV-2

“…32 The third large case series, Basile et al ( N = 195), reported that the latest day on which a SARS-CoV-2 was cultured was day 10 for all but 1 patient (maximum day: 18). 22 The fourth case series, Jeong et al ( N = 9) reported positive cultures for 2 patients; the patient with SARS-CoV-2 cultured in saliva on day 15 was noted to be critically ill at the time of sampling. 27 Both of the case reports which identified SARS-CoV-2 in culture beyond day 14 were of hospitalised patients with mild disease, 25 , 30 case reports described an immunocompromised patient who had recently had a heart transplantation.…”

“…Two studies each were conducted in the United States (US), 21 , 28 Taiwan, 24 , 30 the United Kingdom (UK) 31 , 34 and Germany; 25 , 33 while 1 study each was conducted in Switzerland, 29 Australia, 22 Canada, 23 Spain, 26 South Korea, 27 Hong Kong, 14 and the Netherlands. 32 Of the 13 SARS-CoV-2 virus culture studies, 11 were case series 14 , 21 -23 , 26 -29 , 31 -33 and 2 were case reports.…”

“…31 Basile et al ( N = 234 samples) concluded that any clinical sample with a Ct value of ≥37 was not indicative of replicative (or potentially transmissible) virus. 22 Bullard et al ( N = 90 samples) estimated that for every 1 unit increase in Ct value, the odds of culturing SARS-CoV-2 decreased by 32%. 23 While Folgueira et al reported culturing SARS-CoV-2 on day 32 post symptom onset in a patient with severe COVID-19, they observed a very low viral load, with a Ct value of 39.1.…”

Laboratory diagnosis of severe acute respiratory syndrome coronavirus 2

“…A negligible reduction in Ct values of the terminal cell culture supernatant compared to the original sample suggests a lack of growth. 72 Of note, the yield of cell cultures from samples with low levels of virus (inferred from Ct values of >32-34 for E and N genes) is low, 71,72 but the threshold for successful culture needs to be established in individual laboratories given the potential variability in culture methods. Similar to SARS-CoV and MERS-CoV, the addition of exogenous trypsin may improve the recovery of SARS-CoV-2 in culture by inducing the proteolytic cleavage of the S protein and promoting virus-cell membrane fusion.…”