Prolonged SARS-CoV-2 cell culture replication in respiratory samples from patients with severe COVID-19

Abstract

Objectives: This study compares the infectivity of SARS-CoV-2 in respiratory samples from patients with mild COVID-19 with those from hospitalized patients with severe bilateral pneumonia. In severe COVID-19, we also analysed the presence of neutralizing activity in paired sera.

Methods: We performed cell cultures on 193 real-time reverse transcription polymerase chain reaction respiratory samples, positive for SARS-CoV-2, obtained from 189 patients at various times, from clinical diagnosis to follow-up. Eleven samples were obtained from asymptomatic individuals, 91 samples from 91 outpatients with mild forms of COVID-19 and 91 samples from 87 inpatients with severe pneumonia. In these patients, neutralizing activity was analysed in 30 paired sera collected after symptom onset >10 days.

Results: We detected a cytopathic effect (CPE) in 91/193 (47%) samples. Viral viability was maintained for up to 10 days in patients with mild COVID-19. In patients with severe COVID-19, the virus remained viable for up to 32 days after the onset of symptoms. Patients with severe COVID-19 presented infectious virus at a significantly higher rate in the samples with moderate to low viral load (cycle threshold value ≥ 26): 32/75 (43%) versus 14/63 (22%) for mild cases (p < 0.01). We observed a positive CPE despite the presence of clear neutralizing activity (NT50 > 1:1024 in 10% (3/30) of samples.

Discussion: Patients with severe COVID-19 might shed viable virus during prolonged periods of up to 4 weeks after symptom onset, even when presenting high cycle threshold values in their respiratory samples and despite having developed high neutralizing antibody titres.

Keywords: COVID-19; Cell culture; Infection control; Neutralizing antibodies; SARS-CoV-2.

Fig. 1

Cytopathic effect (CPE) produced by SARS-CoV-2 on Vero E6 cell line. (A) Normal appearance of the cell line. (B) CPE development after 48 hours' incubation. (C) Confirmation of the specificity of the CPE observed by shell-vial technique. Microscopic visualization at 20× magnification.

Fig. 2

Distribution of samples according to symptom time to test (STT) in weeks (A) and percentage of samples with cytopathic effect (CPE) according to STT (B) in groups of patients with severe and mild COVID-19.

Fig. 3

Percentage of samples with cytopathic effect (CPE) according to the Ct amplification value (A) and the correlation of viral replication with Ct value and symptom onset (B) in groups of patients with severe and mild COVID-19. Two samples collected during the follow-up of the same patient at days 42 and 61 after symptom onset are not represented in the graph. There are statistically significant differences (p < 0.001) between the Ct values found in the samples with viral replication in both mild and severe COVID-19 compared with the samples that did not present viral replication, regardless of the severity of the disease.