Severe Acute Respiratory Syndrome Coronavirus 2 was isolated from the feces of a deceased patient with coronavirus in China. Confirmation of infectious virus in feces confirms potential fecal-oral or fecal-respiratory infections and warrants further study.
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has recently occurred in China, causing a severe outbreak of pneumonia that has spread to more than 200 other countries.[1] As of May 5, 2020, a total of 3,517,345 cases of coronavirus disease (COVID-2019) and 243,401 deaths were reported by the World Health Organization (https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200505covid-19-sitrep-106.pdf?sfvrsn=47090f63_2). The virus is believed to spread by direct contact, vectors, respiratory droplets, and possibly aerosols.[2] Viral RNA has been detected in the feces and urine of some patients.[3–7] Infectious virus was also isolated from the urine of patients with severe COVID-19.[8] However, it is unclear whether the virus in the feces is infectious and is a source of infection.
The study was approved by the Guangdong Health Commission and the Guangzhou Medical University Ethics Committee to use sample specimens from patients and healthy donors. On January 17, 2020, a 78-year-old man with a recent travel history to Wuhan, China, was admitted to Zhongshan University’s No. 5 affiliated hospital due to a seven-day cough and intermittent fever Figure 1. Panel A https://wwwnc.cdc.gov/EID/article/26/8/20-0681-App1.pdf). Computed tomography of his chest showed multiple ground glass shadows (Appendix Figure 2). The nasopharyngeal and oropharyngeal swab samples were positive for SARS-CoV-2 RNA by quantitative reverse transcription PCR (qRT-PCR).
On January 22, the patient’s condition deteriorated and he was intubated. Ventilator assisted breathing has started. The first fecal sample was collected on January 27th and was positive for viral RNA by qRT-PCR. Serial samples of feces were taken on January 29th, February 1st and February 7th. Viral RNA was positive in all samples (Appendix Figure 1, Panel A). Viral antigens were also detected in gastrointestinal epithelial cells in biopsy samples, as reported.[9] The patient died on February 20.
A fecal sample was collected on January 29 and inoculated into Vero E6 cells. The cycle threshold for fecal samples was 23.34 for the open reading frame 1lab gene and 20.82 for the nuclear protein gene. A cytopathic effect was seen on Vero E cells 2 days after the second passage (Appendix Figure 1, Panel B). QIAamp Viral RNA Extraction Kit (QIAGEN, https://www.qiagen.com), and obtained a full-length viral genome sequence (GenBank Accession No. MT123292) using next-generation sequencing. Sequencing showed a 5 nt substitution compared to the original Wuhan strain (GenBank accession number NC045512.2) (Appendix table).
The culture supernatant was negatively stained and visualized with a transmission electron microscope. Visible virions were spherical and had clear surface spike protein predictions, consistent with previously published SARS-CoV2 images (Appendix Figure, Panel C).[1]
Viral road (logTen PFU equivalent/mL) was a clinical sample from the qRT-PCR cycle threshold and a standard curve was generated from serially diluted SARS-CoV-2 of known plaque titers. The viral load quantified using this method was at the viral RNA level rather than the infectious virus. Viral load was higher in faeces than in respiratory specimens collected at multiple time points (17-28 days post-symptom onset) (Appendix Figure, Panel D). Although virus isolation from fecal samples collected at a later time point was unsuccessful, viral RNA results remained positive and the feces of this patient collected at the time of the onset of the disease showed infectivity. Only RNA fragments are shown, not viruses.
Fecal samples were collected from 28 patients. Twelve patients, including the patients described in this report, were positive for viral RNA at one or more time points. We attempted to isolate SARS-CoV-2 virus from 3 patients with positive viral RNA. The results were successful in 2 of 3 patients, including the patients in this report, indicating that fecal infectious virus is a common symptom of COVID-19.
The patient in this report had high levels of IgG against spike proteins. The level of nucleocapsid protein-specific antibody was relatively low. The spike protein (1,274 aa) is much larger than the nuclear protein protein (420 aa), which may contain more epitopes that induce specific antibody responses.
We also used the Focus Reduction Neutralization Test to identify neutralizing antibodies. Neutralization titers (50% focus reduction neutralization test) ranged from 1:1,065 to >1:4,860 at various time points (Appendix Figure, Panel E). Testing Vero E6 cells infected with virus isolates using an indirect immunofluorescence assay and serum samples from patients and healthy donors to show that the isolated virus is infectious to susceptible cells Did. Positive reactions were obtained only with the patient’s serum (Appendix Figure 1, Panel F).
Isolation of infectious SARS-CoV-2 in feces indicates the potential for fecal-oral or fecal-respiratory infections via aerosolized feces. During the 2003 outbreak of severe acute respiratory syndrome, 329 inhabitants of Hong Kong’s private housing estates were infected. 42 people have died.[10] Upon investigating the structure of the building, it was discovered that a failure of the sewer line caused aerosolization of contaminated feces, which was thought to be the source of the infection.
Our findings indicate the need for appropriate precautions to avoid potential SARS-CoV-2 infections from feces. Discharge and hospital cleaning practices should consider this possibility in severely ill patients or in deceased patients who have a high viral load and are likely to shed infectious virus.
