key: cord-0700640-34wg9xqg
authors: Folgueira, Maria Dolores; Luczkowiak, Joanna; Lasala, Fátima; Pérez-Rivilla, Alfredo; Delgado, Rafael
title: Prolonged SARS-CoV-2 cell culture replication in respiratory samples from patients with severe COVID-19
date: 2021-02-22
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2021.02.014
sha: ebcc38dfdf4d5514b14f02178d7e901f7b406eb9
doc_id: 700640
cord_uid: 34wg9xqg

OBJECTIVES: This study compares the infectivity of SARS-CoV-2 in respiratory samples from patients with mild COVID-19 with those from hospitalised patients with severe bilateral pneumonia. In severe COVID-19, we also analysed the presence of neutralising 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, neutralising activity was analysed in 30 paired sera collected after symptom onset >10 days. RESULTS: We detected a cytopathic effect (CPE) in 91 (91/193, 47%) samples. Viral viability was maintained for up to 10 days in the patients with mild COVID-19. In the 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 neutralising activity (NT50 >1:1024 in 10% (3/30) of samples. CONCLUSIONS: 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 neutralising antibody titres.

J o u r n a l P r e -p r o o f INTRODUCTION 48 SARS-CoV-2, a novel human coronavirus that emerged in Wuhan (China) in late 49 2019, 1,2 has been responsible for the largest pandemic in a century. 50 The use of real-time reverse transcription polymerase (rRT-PCR) 3 as a diagnostic and 51 follow-up tool for SARS-CoV-2 infection has led to hypotheses regarding infectivity 52 duration, the possibility of reactivation, and even reinfection. 4 Although rRT-PCR is the 53 gold standard diagnostic method, it is less useful as a follow-up technique, because 54 samples from patients who have overcome either mild or severe SARS-CoV-2 infection 55 still have detectable viral RNA for variable periods of time. [5] [6] [7] In the absence of 56 diagnostic methods with reliable quantification, the cycle threshold (Ct) value obtained 57 in amplification has been employed as a semiquantitative measure and has been 58 proposed as a parameter for elaborating approaches to removing patients from 59 isolation. 8 Establishing a reliable cut-off Ct value is difficult, given the large number of 60 available rRT-PCR-based diagnostic tests; the need to use more than 1 molecular test 61 in most clinical laboratories to meet growing demand, and the use of different types of 62 samples during patient follow-up. Hence, the importance of establishing the duration 63 of virus viability in various clinical situations. The assessment of SARS-CoV-2 viability 64 will help establish criteria for isolating patients.

This study compared viral detection by rRT-PCR and the infectivity of SARS-CoV-2 in 71 respiratory samples from patients with mild COVID-19 with those from hospitalised 72 patients with severe bilateral pneumonia. In those patients with severe COVID-19, we 73 also analysed the presence of anti-SARS-CoV-2 immunoglobulin G (IgG) and the 74 neutralising activity in paired sera with respiratory samples, as well as the correlation 75 between its presence and viral viability. The percentage of samples that presented viral replication for each of the patient 179 groups is shown in Table 1 , along with other sample data and patient demographics. Patients with severe COVID-19 presented infective virus at a significantly higher rate 204 (47%, 24/51) than outpatients (18%, 7/38) (P < 0.01). 205 In this regard, it is noteworthy that 2 of 7 bronchial aspirates presented CPE despite 206 the fact that the median Ct value for this type of sample was 35.0 (IQR 32.6-38.9). 208 Of the 30 sera collected with STT >10 days, 12 were paired with a CPE-positive 209 respiratory sample, and 18 were paired with a CPE-negative respiratory sample. 

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 287 We would like to express our gratitude to Dra. Estela Paz and the Immunology 288 Department for their help in providing residual serum samples. 289 The professional editing service ServingMed.com provided technical editing of the 290 manuscript prior to submission. 291