key: cord-0713766-m8m4zix7
authors: Jeong, Hye Won; Kim, Se-Mi; Kim, Hee-Sung; Kim, Young-Il; Kim, Jun Hyoung; Cho, Jun Yeon; Kim, Sun-hyung; Kang, Hyeran; Kim, Seong-Gyu; Park, Su-Jin; Kim, Eun-Ha; Choi, Young Ki
title: Viable SARS-CoV-2 in various specimens from COVID-19 patients
date: 2020-07-23
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2020.07.020
sha: 6fc8320a5752f2ae134293ccf2e3ad3f7b104671
doc_id: 713766
cord_uid: m8m4zix7

OBJECTIVES: To determine whether various clinical specimens obtained from COVID-19 patients contain the infectious virus. METHODS: To demonstrate whether various clinical specimens contain the viable virus, we collected naso/oropharyngeal swabs, saliva, urine, and stool from five COVID-19 patients and performed a quantitative polymerase chain reaction (qPCR) to assess viral load. Specimens positive by qPCR were subjected to virus isolation in Vero cells. We also used urine and stool samples to intranasally inoculate ferrets and evaluated the virus titers in nasal washes on 2, 4, 6, and 8 days post-infection (dpi). RESULTS: SARS-CoV-2 RNAs were detected in all naso/oropharyngeal swabs, saliva, urine, and stool samples collected between days 8 to 30 of the clinical course. Notably, viral loads in urine, saliva, and stool samples were almost equal to or higher than those in naso/oropharyngeal swabs (urine 1.08±0.16 – 2.09±0.85 log(10) copies/ml, saliva 1.07±0.34 – 1.65±0.46 log(10) copies/ml, stool 1.17±0.32 log(10) copies/ml, naso/oropharyngeal swabs 1.18±0.12 – 1.34±0.30 log(10) copies/ml). Further, viable SARS-CoV-2 was isolated from naso/oropharyngeal swabs and saliva of COVID-19 patients, as well as nasal washes of ferrets inoculated with patient urine or stool. CONCLUSIONS: Viable SARS-CoV-2 was demonstrated in saliva, urine, and stool from COVID-19 patients up until days 11 to 15 of the clinical course. This result suggests that viable SARS-CoV-2 can be secreted in various clinical samples as well as respiratory specimens.

In December 2019, a novel coronavirus disease was identified in 55 Wuhan, Hubei Province of China, and a previously unknown betacoronavirus was 56 subsequently isolated from patients with pneumonia (1). The infectious agent was 57 identified as a novel coronavirus (2019-nCoV), which was named severe acute 58 respiratory syndrome coronavirus-2 (SARS-CoV-2) due to its marked similarity with 59 SARS-CoV, and could cause various levels of disease severity ranging from 60 asymptomatic infection (1%)(2, 3) to severe respiratory illness (20%) and fatality in 61 2-3% of infected patients (4). In order to effectively control the COVID-19 outbreak, 62 information regarding the period and routes of infectious virus shedding in patients is 63 essential. In general, droplets and close contact with infected respiratory secretions 64 are considered the main transmission routes of the SARS-CoV-2; however, the fecal-65 oral transmission has also been suggested since some COVID-19 patients have 66 gastrointestinal symptoms and viral RNA has been detected in oral and anal swabs 67 from patients (5-9). In addition, SARS-CoV-2 has been detected in the saliva of 68 infected patients (10) suggesting saliva may also be a source of human-to-human 69 transmission. 70 In our previous study, we established a ferret model of SARS-CoV-2 infection 71 (11) and found a relatively high amount of viral RNA in saliva, urine, and fecal 72 specimens from SARS-CoV-2 infected animals, suggesting various clinical 73 specimens from infected hosts could be transmission sources. The aim of this study 74 is to examine the viral load in various clinical specimens from acute or recovery 75 phase patients and to investigate the viability of the detected virus.

Five laboratory-confirmed COVID-19 patients hospitalized in the Chungbuk 80 National University Hospital from February 25, 2020, to March 5, 2020, were enrolled 81 in this study. We collected naso/oropharyngeal swabs, saliva, urine, and fecal 82 specimens from enrolled patients at days 8, 11, 13, 15, and 30 of the clinical course. 83 We also collected serum samples at the same time points for serologic tests. 

In order to detect SARS-CoV-2 RNA in clinical specimens, we performed a 87 quantitative reverse transcription-polymerase chain reaction (qRT-PCR) as 88 previously described (1) This study was conducted following the study protocol approved by the IgG antibody responses of all COVID-19 patients are described in Table 2 .

162 163

All clinical specimens collected from the five patients were positive for the 165 SARS-CoV-2 spike gene by qPCR ( 1.17±0.32, 2.18±0.11 log 10 copies/ml and 2.01±0.28 log 10 copies/ml, respectively.

Four symptom-recovered patients (patients 1, 2, 3, and 5) displayed viral RNA copy 172 numbers in urine and fecal samples that were almost equal to or higher than that of 173 naso/oropharyngeal swabs ( urine and fecal specimens using Vero cells, although some specimens (two urine 184 and one stool specimen) had viral loads higher than 1.30 log 10 copies/ml.

To confirm the presence of infectious SARS-CoV-2 in urine and fecal specimens, 188 two urine specimens (patient 2, days 13 of illness, viral load1.51±0.19 log 10 189 copies/ml, patient 3, days 11 of illness, viral load 2.09±0.85 log 10 copies/ml) and one 190 fecal specimen (patient 4, days 15 of illness, viral load 2.18±0.11 log 10 copies/ml) were selected for ferret infection experiments. All urine and stool-treated ferrets 192 showed moderate increases in body temperature, rhinorrhea, and decreased activity 193 at 4 dpi which persisted until 6 dpi. SARS-CoV-2 viral RNA was detected in nasal 194 washes from urine-treated ferrets at 2 dpi, although titers were as low as 0.92±0.38 195 log 10 copies/ml, and peaked at 4 dpi (3.24±1.01 log 10 copies/ml). Further, viral RNA 196 was detected in nasal wash specimens from stool-treated ferrets as early as 2 dpi 197 and persisted until 6 dpi (Table 3) . Notably, SARS-CoV-2 was isolated from the nasal 198 washes of the two urine-treated ferrets and one stool-treated ferret (Table 3) . 

Viral load kinetics 315 of SARS-CoV-2 infection in first two patients in Korea

SARS-CoV-2 RNA shedding in clinical specimens and clinical characteristics of 10 338 patients with COVID-19 in Macau

Detection of SARS-CoV-2 341 in different types of clinical specimens