Since the novel coronavirus (SARS-CoV-2) was identified in Wuhan, China, at the end
of 2019, the virus has spread to 32 countries, infecting more than 80,000 people and
causing more than 2600 deaths globally. The viral infection causes a series of respiratory
illnesses, including severe respiratory syndrome, indicating that the virus most likely
infects respiratory epithelial cells and spreads mainly via respiratory tract from
human to human. However, viral target cells and organs have not been fully determined,
impeding our understanding of the pathogenesis of the viral infection and viral transmission
routes. According to a recent case report, SARS-CoV-2 RNA was detected in a stool
specimen,
1
raising the question of viral gastrointestinal infection and a fecal-oral transmission
route. It has been proven that SARS-CoV-2 uses angiotensin-converting enzyme (ACE)
2 as a viral receptor for entry process.
2
ACE2 messenger RNA is highly expressed and stabilized by B0AT1 in gastrointestinal
system,
3
,
4
providing a prerequisite for SARS-CoV-2 infection. To further investigate the clinical
significance of SARS-CoV-2 RNA in feces, we examined the viral RNA in feces from 71
patients with SARS-CoV-2 infection during their hospitalizations. The viral RNA and
viral nucleocapsid protein were examined in gastrointestinal tissues from 1 of the
patients.
Methods
From February 1 to 14, 2020, clinical specimens, including serum, nasopharyngeal,
and oropharyngeal swabs; urine; stool; and tissues from 73 hospitalized patients infected
with SARS-CoV-2 were obtained in accordance with China Disease Control and Prevention
guidelines and tested for SARS-CoV-2 RNA by using the China Disease Control and Prevention–standardized
quantitative polymerase chain reaction assay.
5
Clinical characteristics of the 73 patients are shown in Supplementary Table 1. The
esophageal, gastric, duodenal, and rectal tissues were obtained from 1 of the patients
by using endoscopy. The patient’s clinical information is described in the Supplementary
Case Clinical Information and Supplementary Table 2. Histologic staining (H&E) as
well as viral receptor ACE2 and viral nucleocapsid staining were performed as described
in the Supplementary Methods. The images of fluorescent staining were obtained by
using laser scanning confocal microscopy (LSM880, Carl Zeiss MicroImaging, Oberkochen,
Germany) and are shown in Figure 1
. This study was approved by the Ethics Committee of The Fifth Affiliated Hospital,
Sun Yat-sen University, and all patients signed informed consent forms.
Figure 1
Images of histologic and immunofluorescent staining of gastrointestinal tissues. Shown
are images of histologic and immunofluorescent staining of esophagus, stomach, duodenum,
and rectum. The scale bar in the histologic image represents 100 μm. The scale bar
in the immunofluorescent image represents 20 μm.
Results
From February 1 to 14, 2020, among all of the 73 hospitalized patients infected with
SARS-CoV-2, 39 (53.42%), including 25 male and 14 female patients, tested positive
for SARS-CoV-2 RNA in stool, as shown in Supplementary Table 1. The age of patients
with positive results for SARS-CoV-2 RNA in stool ranged from 10 months to 78 years
old. The duration time of positive stool results ranged from 1 to 12 days. Furthermore,
17 (23.29%) patients continued to have positive results in stool after showing negative
results in respiratory samples.
Gastrointestinal endoscopy was performed on a patient as described in the Supplementary
Case Clinical Information. As shown in Figure 1, the mucous epithelium of esophagus,
stomach, duodenum, and rectum showed no significant damage with H&E staining. Infiltrate
of occasional lymphocytes was observed in esophageal squamous epithelium. In lamina
propria of the stomach, duodenum, and rectum, numerous infiltrating plasma cells and
lymphocytes with interstitial edema were seen.
Importantly, viral host receptor ACE2 stained positive mainly in the cytoplasm of
gastrointestinal epithelial cells (Figure 1). We observed that ACE2 is rarely expressed
in esophageal epithelium but is abundantly distributed in the cilia of the glandular
epithelia. Staining of viral nucleocapsid protein was visualized in the cytoplasm
of gastric, duodenal, and rectum glandular epithelial cell, but not in esophageal
epithelium. The positive staining of ACE2 and SARS-CoV-2 was also observed in gastrointestinal
epithelium from other patients who tested positive for SARS-CoV-2 RNA in feces (data
not shown).
Discussion
In this article, we provide evidence for gastrointestinal infection of SARS-CoV-2
and its possible fecal-oral transmission route. Because viruses spread from infected
to uninfected cells,
6
viral-specific target cells or organs are determinants of viral transmission routes.
Receptor-mediated viral entry into a host cell is the first step of viral infection.
Our immunofluorescent data showed that ACE2 protein, which has been proven to be a
cell receptor for SARS-CoV-2, is abundantly expressed in the glandular cells of gastric,
duodenal, and rectal epithelia, supporting the entry of SARS-CoV-2 into the host cells.
ACE2 staining is rarely seen in esophageal mucosa, probably because the esophageal
epithelium is mainly composed of squamous epithelial cells, which express less ACE2
than glandular epithelial cells.
Our results of SARS-CoV-2 RNA detection and intracellular staining of viral nucleocapsid
protein in gastric, duodenal, and rectal epithelia demonstrate that SARS-CoV-2 infects
these gastrointestinal glandular epithelial cells. Although viral RNA was also detected
in esophageal mucous tissue, absence of viral nucleocapsid protein staining in esophageal
mucosa indicates low viral infection in esophageal mucosa.
After viral entry, virus-specific RNA and proteins are synthesized in the cytoplasm
to assemble new virions,
7
which can be released to the gastrointestinal tract. The continuous positive detection
of viral RNA from feces suggests that the infectious virions are secreted from the
virus-infected gastrointestinal cells. Recently, we and others have isolated infectious
SARS-CoV-2 from stool (unpublished data), confirming the release of the infectious
virions to the gastrointestinal tract. Therefore, fecal-oral transmission could be
an additional route for viral spread. Prevention of fecal-oral transmission should
be taken into consideration to control the spread of the virus.
Our results highlight the clinical significance of testing viral RNA in feces by real-time
reverse transcriptase polymerase chain reaction (rRT-PCR) because infectious virions
released from the gastrointestinal tract can be monitored by the test. According to
the current Centers for Disease Control and Prevention guidance for the disposition
of patients with SARS-CoV-2, the decision to discontinue transmission-based precautions
for hospitalized patients with SARS-CoV-2 is based on negative results rRT-PCR testing
for SARS-CoV-2 from at least 2 sequential respiratory tract specimens collected ≥24
hours apart.
8
However, in more than 20% of patients with SARS-CoV-2, we observed that the test result
for viral RNA remained positive in feces, even after test results for viral RNA in
the respiratory tract converted to negative, indicating that the viral gastrointestinal
infection and potential fecal-oral transmission can last even after viral clearance
in the respiratory tract. Therefore, we strongly recommend that rRT-PCR testing for
SARS-CoV-2 from feces should be performed routinely in patients with SARS-CoV-2 and
that transmission-based precautions for hospitalized patients with SARS-CoV-2 should
continue if feces test results are positive by rRT-PCR testing.