key: cord-0799353-72d16bdp
authors: Kim, Jeong-Min; Kim, Heui Man; Lee, Eun Jung; Jo, Hye Jun; Yoon, Youngsil; Lee, Nam-Joo; Son, Junseock; Lee, Ye-Ji; Kim, Mi Seon; Lee, Yong-Pyo; Chae, Su-Jin; Park, Kye Ryeong; Cho, Seung-Rye; Park, Sehee; Kim, Su Jin; Wang, Eunbyeol; Woo, SangHee; Lim, Aram; Park, Su-Jin; Jang, JunHyeong; Chung, Yoon-Seok; Chin, Bum Sik; Lee, Jin-Soo; Lim, Duko; Han, Myung-Guk; Yoo, Cheon Kwon
title: Detection and Isolation of SARS-CoV-2 in Serum, Urine, and Stool Specimens of COVID-19 Patients from the Republic of Korea
date: 2020-06-03
journal: Osong Public Health Res Perspect
DOI: 10.24171/j.phrp.2020.11.3.02
sha: c900463af32b8807478699b5878b6b0fde5d4246
doc_id: 799353
cord_uid: 72d16bdp

OBJECTIVES: Coronavirus Disease-19 (COVID-19) is a respiratory infection characterized by the main symptoms of pneumonia and fever. It is caused by the novel coronavirus severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), which is known to spread via respiratory droplets. We aimed to determine the rate and likelihood of SARS-CoV-2 transmission from COVID-19 patients through non-respiratory routes. METHODS: Serum, urine, and stool samples were collected from 74 hospitalized patients diagnosed with COVID-19 based on the detection of SARS-CoV-2 in respiratory samples. The SARS-CoV-2 RNA genome was extracted from each specimen and real-time reverse transcription polymerase chain reaction performed. CaCo-2 cells were inoculated with the specimens containing the SARS-COV-2 genome, and subcultured for virus isolation. After culturing, viral replication in the cell supernatant was assessed. RESULTS: Of the samples collected from 74 COVID-19 patients, SARS-CoV-2 was detected in 15 serum, urine, or stool samples. The virus detection rate in the serum, urine, and stool samples were 2.8% (9/323), 0.8% (2/247), and 10.1% (13/129), and the mean viral load was 1,210 ± 1,861, 79 ± 30, and 3,176 ± 7,208 copy/μL, respectively. However, the SARS-CoV-2 was not isolated by the culture method from the samples that tested positive for the SARS-CoV-2 gene. CONCLUSION: While the virus remained detectable in the respiratory samples of COVID-19 patients for several days after hospitalization, its detection in the serum, urine, and stool samples was intermittent. Since the virus could not be isolated from the SARS-COV-2-positive samples, the risk of viral transmission via stool and urine is expected to be low.

The Coronavirus Disease-19 leads to the development of Severe Acute Respiratory Syndrome (SARS), with a fever, cough, sore throat, myalgia, and headache as the most common clinical symptoms. It is a novel viral infection that first emerged in Wuhan, China in December 2019 as a pneumonia of unknown etiology [1] [2] [3] [4] . Coronaviruses infect humans and various animals, and subsequently cause respiratory, enteric, hepatic, and neurologic diseases [5, 6] . [3, 4] . The SARS-CoV and the MERS-CoV, which first emerged in 2002 and 2012, respectively, belong to the betacoronavirus genera [1, 7] . The SARS-CoV-2 is known to have a higher transmission rate and infectivity than SARS-CoV and MERS-CoV [7] [8] [9] .

Presence of a fever and a cough are the primary clinical manifestations of COVID-19 however, patients also exhibited other symptoms such as nausea, vomiting, diarrhea, and abdominal discomfort [3] . Presently, the most common method used for COVID-19 diagnosis is the detection of SARS-CoV-2 in upper and lower respiratory specimens, including nasopharyngeal swabs, oropharyngeal swabs, sputum, lower respiratory tract aspirates, and bronchoalveolar lavage. Genetic testing methods, such as real-time reverse transcription polymerase chain reaction (RT-PCR), are the standard methods of laboratory testing for COVID-19 that are currently in use in most countries.

In the present study, we investigated whether SARS-CoV-2, which infects humans and may subsequently trigger the development of various clinical symptoms, can be detected in body fluids such as serum, urine, and stool, besides respiratory specimens. In addition, we aimed to isolate the virus from SARS-CoV-2-positive samples to determine viral infectivity.

To examine viral shedding in body parts other than the respiratory tract, and the infectivity of the detected virus, respiratory specimens such as nasopharyngeal swab, oropharyngeal swab or sputum, as well as serum, urine, and stool specimens were non-periodically sampled from 74 COVID-19 patients admitted in a hospital between January 19 th and March 30 th , 2020.

Of the respiratory specimens, the upper respiratory samples 

To isolate SARS-CoV-2 from samples that tested positive for the virus in real-time RT-PCR analyses, the samples were mixed with a 1:1 nystatin (10,000 U/mL) and penicillinstreptomycin (10,000 U/mL) mixture in a 1:4 ratio, and left to react at 4°C for 1 hour. The samples were then centrifuged at 

According to the standard curve, the plasmid DNA (E and 

The study was approved by the Institutional Review Board at the Korea Centers for Disease Control and Prevention (2020-03-01-P-A). The board waived the requirement for written consent.

The presence of SARS-CoV-2 was tested in serum, urine, and There have been no published reports of SARS-CoV-2 viremia to date. The SARS-CoV-2 RNA level detected was 50% in plasma and 78% in serum samples at 7-or 14-days following hospitalization [11, 12] . In other studies, the plasma viral load of SARS-CoV-2 peaked at 4-8 days after the disease onset, and followed a pattern of transient viremia, or occasionally persisted for more than 10 days, depending on the patient [13] . In this study, the viral RNA was detected in the serum of 5 patients at 3-6 days after onset. However, owing to irregular and discontinuous blood sampling, the duration for which the RNA was detectable could not be assessed (Figure 2 ). In addition, the inhibitory effect of the antiviral drug administered during treatment after hospitalization on viral replication cannot be excluded. Blood samples were collected 1-3 times within 6 days after onset in 46% of the patients. Since SARS-CoV-2 can be transmitted during the asymptomatic stage of infection before disease onset [14, 15] , future studies should investigate viremia during the asymptomatic stage and determine the rate of viremia.

It has been reported that in SARS patients, viral shedding was detected in respiratory and stool specimens for up to 52 and 126 days, respectively [16] . However, in MERS patients, viral shedding has rarely been studied [17] . In a study on viral shedding in 37 MERS patients, the MERS-CoV RNA was detected in 93% of the lower respiratory samples for up to 3 weeks after disease confirmation, and viral RNA was detected in serum, stool, and urine samples [18] . However, virus could not be isolated from the stool or serum samples, which had the highest RNA concentration [19] . It has also been reported that MERS-CoV RNA was detected in stool samples, with the highest detection rate at Days 4-11 after onset, and was detected even at 30 days after onset in some patients [18, 19] .

Although it has been reported that SARS-CoV-2 RNA was not detectable in the upper respiratory tract at 21 days on average after confirmation [14] , there was a specific case wherein the duration of viral shedding was observed at 20 days and 37 days [20] . Apart from detection of SARS-CoV-2 RNA in the upper

Specimens Days after admission   0  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16 Figure 2 . Time points for respiratory swab and serum sample collection after admission to hospital after SARS-CoV-2 RNA detection.

Orange represents the detection of viral RNA, grey represents undetected viral RNA, and white indicates that the samples were not collected, and therefore, not tested. respiratory tract, it has been reported that SARS-CoV-2 RNA has been detected in stool samples. In a study on 28 COVID-19 patients, the most common clinical symptoms were a cough (28.6%), sore throat (28.6%), and fever (25.0%), while diarrhea (10.7%) was not a common symptom [3] . A study reported that 10% of the patients had diarrhea and experienced nausea 1-2 days before the onset of fever and respiratory symptoms however, there were no reports of viral RNA detection in the samples collected from these patients [18] . In another study, 11.6% of the hospitalized patients exhibited gastrointestinal symptoms at admittance and 49.5% exhibited such symptoms during their hospital stay, and depending on the presence of gastrointestinal symptoms, the viral RNA detection rates were 52.4% and 39.1%, respectively [21] . However, the study that reported the detection of SARS-CoV-2 RNA in stool samples did not investigate the infectivity of the detected viral RNA.

The WHO recognizes the detection of SARS-CoV-2 RNA in lower and upper respiratory samples such as nasal swab, throat swab, and sputum as the official diagnostic method for COVID-19 testing. However, in addition to respiratory samples, viral RNA was also detected in serum, urine, or stool samples, suggesting the possibility of SARS-CoV-2 transmission via such specimens [22] [23] [24] [25] . In this current study, SARS-CoV-2 RNA was detected in respiratory swabs as well as in serum, urine, and stool samples. Moreover, the virus from infected CaCo-2 cells was isolated to determine the infectivity of the viral RNA however, the presence of SARS-CoV-2 could not be confirmed in RNA-positive specimens, and the viral load was also considerably low. These results suggest that the SARS-CoV-2 is predominantly transmitted via the respiratory tract, and transmission via serum, urine, and stools is considerably limited.

The authors have no conflicts of interest to declare.

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The authors thank all those who assisted in the collection and transport of patient samples. This study was funded by Korea Centers for Disease Control and Prevention (no.: 4800-4837-301).