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Gastro Hep Advances 2022;1:844–852ORIGINAL RESEARCH—CLINICALSevere Acute Respiratory Syndrome Coronavirus 2 Is
Detected in the Gastrointestinal Tract of Asymptomatic
Endoscopy Patients but Is Unlikely to Pose a Significant Risk
to Healthcare Personnel
Michelle D. Cherne,1,* Andrew B. Gentry,2,* Anna Nemudraia,1 Artem Nemudryi,1
Jodi F. Hedges,1 Heather Walk,1 Karlin Blackwell,1 Deann T. Snyder,1
Maria Jerome,1 Wyatt Madden,1,3 Marziah Hashimi,1 T. Andrew Sebrell,1
David B. King,4 Raina K. Plowright,1 Mark A. Jutila,1 Blake Wiedenheft,1 and
Diane Bimczok11Department of Microbiology and Cell Biology, Montana State University, Bozeman, Montana; 2Department of
Gastroenterology, Bozeman Health Deaconess Hospital, Bozeman, Montana; 3Rollins School of Public Heath, Emory
University, Atlanta, Georgia; and 4Department of Clinical Research, Bozeman Health Deaconess Hospital, Bozeman, MontanaBACKGROUND AND AIMS: Recent evidence suggests that the
gut is an additional target for severe acute respiratory syn-
drome coronavirus 2 (SARS-CoV-2) infection. However,
whether SARS-CoV-2 spreads via gastrointestinal secretions
remains unclear. To determine the prevalence of gastrointes-
tinal SARS-CoV-2 infection in asymptomatic subjects, we
analyzed gastrointestinal biopsy and liquid samples from
endoscopy patients for the presence of SARS-CoV-2.
METHODS: We enrolled 100 endoscopic patients without
known SARS-CoV-2 infection (cohort A) and 12 patients with a
previous COVID-19 diagnosis (cohort B) in a cohort study
performed at a regional hospital. Gastrointestinal biopsies and
fluids were screened for SARS-CoV-2 by polymerase chain
reaction (PCR), immunohistochemistry, and virus isolation
assay, and the stability of SARS-CoV-2 in gastrointestinal liq-
uids in vitro was analyzed. RESULTS: SARS-CoV-2 ribonucleic
acid was detected by PCR in the colonic tissue of 1/100 pa-
tients in cohort A. In cohort B, 3 colonic liquid samples tested
positive for SARS-CoV-2 by PCR and viral nucleocapsid protein
was detected in the epithelium of the respective biopsy sam-
ples. However, no infectious virions were recovered from any
samples. In vitro exposure of SARS-CoV-2 to colonic liquid led
to a 4-log–fold reduction of infectious SARS-CoV-2 within
1 hour (P 
 .05). CONCLUSION: Overall, the persistent
detection of SARS-CoV-2 in endoscopy samples after resolu-
tion of COVID-19 points to the gut as a long-term reservoir for*Authors contributed equally.
Abbreviations used in this paper: COVID-19, coronavirus disease 2019; E
gene, envelope gene; GI, gastrointestinal; N gene, nucleocapsid gene; NP,
nucleocapsid protein; PFU, plaque-forming units; qRT-PCR, quantitative
reverse transcription polymerase chain reaction; SARS-CoV-2, severe
acute respiratory syndrome coronavirus 2.
Most current article
Copyright © 2022 The Authors. Published by Elsevier Inc. on behalf of the
AGA Institute. This is an open access article under the CC BY-NC-ND li-
cense (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2772-5723
https://doi.org/10.1016/j.gastha.2022.06.002SARS-CoV-2. Since no infectious virions were recovered and
SARS-CoV-2 was rapidly inactivated in the presence of colon
liquids, it is unlikely that performing endoscopic procedures is
associated with a significant infection risk due to undiagnosed
asymptomatic or persistent gastrointestinal SARS-CoV-2
infections.Keywords: Endoscopy; SARS-CoV-2; Transmission Risk; Colonic
Liquidince December 2019, the severe acute respiratoryIntroduction
Ssyndrome coronavirus 2 (SARS-CoV-2) has infected
more than 500 million people worldwide and the resulting
disease, coronavirus disease 2019 (COVID-19), has killed
more than 6.2 million individuals.1 Hospitalizations due to
the COVID-19 pandemic have stressed hospitals for
personnel and resources. Subsequently, many routine med-
ical procedures including preventive cancer screenings have
been delayed. Specifically, colorectal cancer screenings were
reduced 85%–90% in the United States in 2020 compared
to prepandemic levels2 and a study from the United
Kingdom predicted a 15%–17% increase in deaths from
colorectal cancer over 5 years due to deferred preventive
screening during the pandemic.3 To avoid such an increase
in cancer-related mortality, colonoscopy screenings must
continue but adapt to the challenges of the SARS-CoV-2
pandemic. One major challenge is to prevent transmission
of SARS-CoV-2 to healthcare personnel, which can exacer-
bate staffing shortages due to staff illness or required
quarantines.
Here, we sought to determine the prevalence of intesti-
nal SARS-CoV-2 infection in gastrointestinal endoscopy pa-
tients without active COVID-19 so that we could gain initial
2022 SARS-CoV-2 prevalence in endoscopy patients 845insights into the likelihood of SARS-CoV-2 transmission to
healthcare personnel during endoscopic procedures. Cur-
rent guidelines from the American Gastroenterological As-
sociation state that routine SARS-CoV-2 testing prior to
endoscopy is not needed to perform endoscopy safely.4
However, it is presently still unclear whether infectious
SARS-CoV-2 is shed from the gastrointestinal (GI) tract.
Acute SARS-CoV-2 infections are frequently associated with
GI symptoms including abdominal pain, diarrhea, and
vomiting,5,6 and SARS-CoV-2 ribonucleic acid (RNA) is
routinely detected in stool samples and in wastewater.5–8
Several groups have also reported replication of SARS-
CoV-2 in human gut epithelium in vitro using organoid
models9–13 and viral RNA, proteins, and virions have been
detected in small and large intestinal samples of COVID-19
patients.8,14,15 Importantly, prolonged shedding of SARS-
CoV-2 from the GI tract for >6 weeks after clinical recov-
ery in some patients has been demonstrated, suggesting that
the GI epithelium may serve as a long-term viral
reservoir.16–19
Endoscopic procedures produce copious amounts of
droplets and aerosols, which are major modes of trans-
mission for SARS-CoV-2.20,21 Coughlan et al22 measured
droplets produced by upper and lower endoscopies and
determined that endoscopic aerosols would be sufficient for
SARS-CoV-2 transmission. However, only one study de-
scribes the recovery of infectious SARS-CoV-2 virus from a
GI sample.9
We analyzed whether infectious SARS-CoV-2 was pre-
sent in GI samples of asymptomatic male and female
endoscopy patients as a basis for assessing the potential
exposure risk for healthcare personnel performing endos-
copy procedures. Our study revealed a low prevalence of
SARS-CoV-2 infection in the gut of asymptomatic patients
with no prior history of COVID-19 but a higher prevalence in
patients with a previous COVID-19 diagnosis. SARS-CoV-2
protein was identified in the colon tissue in the majority
of these patients but no infectious virus was recovered from
any SARS-CoV-2–positive GI samples. In addition, we
demonstrate the rapid inactivation of SARS-CoV-2 in the
presence of colonic liquids.Methods
Study Design and Participants
For cohort A, we enrolled 100 consecutive patients who
were undergoing upper or lower GI tract endoscopies for
diagnostic purposes between April 2020 and October 2020 at
Bozeman Health Deaconess Hospital, the major regional
healthcare provider in Bozeman, Montana. A sample size of 100
asymptomatic endoscopy patients was selected, using a simple
Bayesian estimation procedure that assumed that the number
of positives is binomially distributed with an uninformative
prior on the P parameter (no prior beliefs regarding reasonable
values of P). Male and female patients of any ethnicity aged
between 18 and 70 years, weighing at least 50 kg, and pre-
senting for gastrointestinal endoscopy were included. Exclusioncriteria were a known infection with human immunodeficiency
virus or hepatitis A, B, or C virus, ongoing pregnancy or lacta-
tion, blood clotting disorders or current medication with a
blood thinner, and presence of acute diarrhea or respiratory
symptoms. Prior COVID-19 infections were not considered for
this cohort because testing was not widely available at the time
of recruitment.
For cohort B, we enrolled 12 additional patients between
November 2020 and May 2021 who had a prior history of
COVID-19. Consecutive endoscopy patients presenting at the
Bozeman Heath Gastroenterology Clinic were recruited who
(1) fulfilled the inclusion and exclusion criteria listed above,
(2) had tested positive for COVID-19 in a polymerase chain
reaction (PCR) test between 2 weeks and 9 months prior to the
endoscopy procedure, and (3) had recovered from any clinical
symptoms at the time of the endoscopy.Community Prevalence of SARS-CoV-2 Infection
Community spread of SARS-CoV-2 during the sample
collection period was assessed based on wastewater surveil-
lance data, as described previously,7 and on confirmed COVID-
19 case numbers reported by the Gallatin City-County
Health Department (Gallatin County, Montana; https://
www.healthygallatin.org/coronavirus-covid-19/). Healthcare
personnel involved in the endoscopy procedures were tested
for SARS-CoV-2 by PCR when they displayed any COVID-19
symptoms based on Centers for Disease Control and Preven-
tion (CDC) guidelines or when they were identified as a close
contact of a person with COVID-19. Throughout this study,
none of the healthcare personnel tested positive for
SARS-CoV-2.
Collection of Gastrointestinal Tissues and Fluids
Biopsies were collected from random sites in the stomach,
duodenum, ileum, and/or colon with cold forceps during
routine outpatient endoscopic procedures by a board-certified
gastroenterologist (A.B.G.). All healthcare personnel involved
in the endoscopy procedures were equipped with N95 respi-
rators and face shields to prevent potential transmission of
infectious agents. From each patient, 6–10 biopsies were
recovered per site and were either fixed in formalin for paraffin
embedding or were minced, aliquoted, and frozen. Colon liquids
were collected using a polyp trap during lower endoscopy
procedures and then aliquoted and frozen.
Isolation of RNA
For isolation of viral RNA from colon liquids, the liquids
were first clarified by centrifugation at 5000 g for 5 minutes
and then the QiAmp Viral RNA mini kit (QIAGEN Sciences,
Germantown, Maryland) was used as instructed, with 280 mL of
clarified colon liquid. For isolation of viral RNA from tissues,
minced tissue was homogenized in 750 mL of ice-cold periph-
eral blood smear (PBS) using an OMNI THQ digital tissue
homogenizer (OMNI International) and then further broken up
using a QIAShredder column, as directed. Next, genomic
deoxyribonucleic acid was removed from the tissue lysate using
a genomic deoxyribonucleic acid exclusion column. RNA was
then extracted from the tissue lysate using the QiAmp Viral
RNA mini kit following the manufacturer’s protocol, using
846 Cherne et al Gastro Hep Advances Vol. 1, No. 5140 mL of lysate. To confirm that the sample processing pro-
tocol did not interfere with downstream SARS-CoV-2 detection,
5 mL of viral transport media from a confirmed SARS-CoV-2
positive nasal swab with N1 detected at cycle 19 were added
to the tissue homogenate (n ¼ 3) or a colon liquid sample (n ¼
1). These “spiked” samples were then immediately processed
for SARS-CoV-2 detection as described above (Figure A1).
SARS-CoV-2 N1 RNA was successfully recovered from both
samples.
Quantitative RT-PCR Detection of SARS-CoV-2
RNA isolated from tissues or colon liquids was screened
for the presence of SARS-CoV-2 using quantitative reverse
transcriptase PCR (RT-PCR) and the CDC primers (N1, N2, and
RP) and probes from the 2019-nCoV RUO Kit (IDT#
10006713). All primer and probe sequences are provided in
Table A1. SARS-CoV-2 RNA was quantified using one-step RT-
qPCR in ABI 7500 Fast Real-Time PCR System as per the CDC
protocol.23 In brief, 20 mL reactions included 8.5 mL of
nuclease-free water, 1.5 mL of primer and probe mix (IDT,
10006713), 5 mL of TaqPath 1-Step RT-qPCR Master Mix
(ThermoFisher, A15299), and 5 mL of the extracted RNA.
Nuclease-free water was used as no template control. The
2019-nCoV_N positive control and Hs_RPP30 control plasmids
(IDT# 10006625, 10006626) diluted to 200 copies/mL were
used as positive controls. amplification was performed using
the following program: 25 C for 2 minutes, 50 C for 15
minutes, 95 C for 2 minutes followed by 45 cycles of 95 C
for 3 seconds, and 55 C for 30 seconds. Data were analyzed in
SDS software v1.4 (Applied Biosystems). The no-template
control showed no amplification throughout the 40 cycles of
qualitative PCR (qPCR). Samples positive for N1, N2, and hu-
man RNaseP were considered positive for SARS-CoV-2. Sam-
ples positive for RNaseP and either N1 or N2, but not both,
were termed inconclusive, and samples negative for RNaseP
were considered invalid. SARS-CoV-2–positive samples were
reanalyzed for the presence of the N1 and envelope (E) genes
in a second laboratory to confirm the results. For detection of
the SARS-CoV-2 E gene, amplification was performed using
UltraPlex 1-step ToughMix polymerase (QuantaBio, #95166)
and the following program: 50 C for 10 minutes, 95 C for 3
minutes followed by 40 cycles of 95 C for 10 seconds, and
60 C for 60 seconds.
Viral Isolation Assay of Infected Liquids and
Tissues
To detect infectious virus in positive samples, a viral
isolation assay was performed using VeroE6 cells plated in 96-
well tissue culture–treated plates, as previously described.24
Colon liquids were centrifuged at 5000 g for 5 minutes to
sediment particulates and then the clarified colon liquids, colon
liquid sediments, and homogenized colon tissues were plated
onto VeroE6 cells in 2-fold serial dilutions, starting with a 1:2
dilution, and monitored daily for cytopathic effects.
Inactivation of SARS-CoV-2 With Colon Liquids
Six colon liquids from cohort B were centrifuged at 5000 g
for 5 minutes to remove particulates and then 250 mL of the
clarified liquid was mixed with 1 106 PFU/mL of SARS-CoV-2(strain USA-WA1/2020, BEI Resources, Manassas, Virginia).
Colon liquids with virus or a PBS control were incubated at
37 C for 10 minutes, 1 hour, or 24 hours and then serially
diluted from 101 to 106 and plated onto VeroE6 cells in
6-well plates. After 1-hour incubation at 37 C, cell layers were
coated with methylcellulose and antibiotic-rich media to pre-
vent bacterial or fungal growth from colon liquids. After 4 days,
cell layers were stained with methylene blue and plaques were
quantified.
Histology and Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues from 4 SARS-
CoV-2–positive samples and 3 negative samples were sectioned
and mounted on slides and then stained with hematoxylin and
eosin. Each tissue was assessed for histopathological alterations
by a blinded investigator. Tissue samples also were analyzed
for the presence of SARS-CoV-2 viral nucleoprotein by immu-
nohistochemistry, using a mouse monoclonal antibody (RRID
Number: AB_2827977, #40143-MM05, Sino Biological, Wayne,
Pennsylvania). Briefly, samples were deparaffinized, blocked,
and incubated with the primary antibody, followed by a sec-
ondary goat antimouse antibody conjugated to horseradish
peroxidase (HRP; #1030-05; Southern Biotechnology, Bir-
mingham, Alabama). ImmPact DAB (Vector Laboratories,
Burlingame, California) was used as a chromogen. Slides were
counterstained with Hematoxylin (#7211; Richard-Allan Sci-
entific, Kalamazoo, Minnesota) and coverslipped. For quantifi-
cation of nucleocapsid-positive cells, stained cells in the crypt
epithelium of colon tissue samples from all donors that tested
positive for SARS-CoV-2 by PCR were counted by 2 blinded
observers. Three SARS-CoV-2–negative control tissues were
analyzed in parallel.
Statistics
A statistical analysis of SARS-CoV-2 inactivation was per-
formed with a nonparametric 2-way analysis of variance and
Friedman test for multiple comparisons. Prevalence of asymp-
tomatic SARS-CoV-2 infection in the gut was determined by
Bayesian analysis.
Study Approval
The study was approved by the Institutional Review
Board of Montana State University, protocol #DB050718-FC,
and was performed under a collaboration agreement between
Montana State University and Bozeman Health Deaconess
Hospital. A written informed consent was obtained from all
participants.Results
Patient Demographics and Community Prevalence
of SARS-CoV-2
We enrolled 100 consecutive patients undergoing
routine upper or lower endoscopies (cohort A) and collected
223 biopsies from the stomach, duodenum, ileum, and colon
and 77 colon liquids (Table A2). Patient demographics are
shown in Table 1. Sample collection for cohort A was per-
formed between April 2020 and October 2020, while
2022 SARS-CoV-2 prevalence in endoscopy patients 847
Table 1. Demographics of the Endoscopy Patients in
Cohort A With No Known History of COVID-19
Total Average Median Range
(n) age (y) age (y) (y)
All patients 100 51.9 55 22–69
Female 52 51.7 54.5 24–69
Male 48 52.0 55.5 22–69moderate community spread of SARS-CoV-2 occurred
(Figure 1A). We also studied the risk of SARS-CoV-2 trans-
mission during endoscopies in a separate smaller cohort of
patients who tested positive for SARS-CoV-2 less than 9
months prior to an endoscopy procedure (cohort B).
Detailed patient demographics are shown in Table 2. Sample
collection for cohort B was performed between November
2020 and May 2021 (Figure 1A).Detection of SARS-CoV-2 in Colonic Biopsy Tis-
sue Obtained From a Colonoscopy Patient With no
History of COVID-19 (Cohort A)
Of 100 sampled patients, 1 colon tissue biopsy collected
from a 61-year-old female patient after a peak in local
community transmission (Figure 1A) tested positive for
SARS-CoV-2 RNA by qRT-PCR (1% of patients/0.45% of
tissues; Table 3, Table A2). SARS-CoV-2 detection was
independently confirmed in a second laboratory using a
backup sample. The biopsy also tested positive for the SARS-
CoV-2 E gene. Tissue samples from other regions of the GIFigure 1. SARS-CoV-2 detection in wastewater and GI samples
during the period of sample collection, with corresponding rep
(n ¼ 100) represents randomly selected individuals who had no C
with a previous diagnosis of COVID-19 within 9 months prior to t
CoV-2–positive samples were collected. (B) Detection of SARS-C
Comparison of inverse Ct values for quantitative PCR detection o
days since COVID-19 diagnosis. Boxes around data points indtract were not available from this patient and no SARS-CoV-
2 RNA was detected in the corresponding colon liquid.
Based on these results, the actual prevalence of SARS-CoV-2
infection in the GI tract of individuals without a history of
COVID-19 was estimated as 0.0118 with a 95% credible
interval of 0.0018 and 0.0384.Detection of SARS-CoV-2 in Colon Liquids of
Patients Previously Diagnosed With COVID-19
Of 12 patients in cohort B, 3 colon liquids of 10 (30%)
tested positive for SARS-CoV-2 RNA, based on the presence
of RNA for genes N1 and N2 (Figure 1B, Tables 2 and 3,
Table A3). SARS-CoV-2 detection was independently
confirmed in a second laboratory using backup samples.
Expression of the E gene was detected in 2 of the 3 colon
liquid samples that tested positive for genes N1 and N2.
Based on these results, the prevalence of SARS-CoV-2 in
asymptomatic individuals with a history of COVID-19 is
estimated as 0.2571 with a 95% credible interval of 0.0964
and 0.4838.
All SARS-CoV-2–positive colon liquids were obtained
from male patients undergoing routine screening colonos-
copy who had either an asymptomatic SARS-CoV-2 infection
(2/3) or mild respiratory disease without any GI symptoms
(1/3; Table 2). Other cohort B patients whose gastrointes-
tinal samples tested negative for SARS-CoV-2 also only had
mild to moderate COVID-19 symptoms, with no hospitali-
zations reported (Table 2). Surprisingly, one patient with
SARS-CoV-2 detection in colonic liquid had received a
COVID-19 diagnosis more than 5 months prior to the. (A) Community SARS-CoV-2 waste water surveillance data
orted case numbers in Gallatin County, Montana. Cohort A
OVID-19 symptoms and cohort B (n¼ 12) represents patients
he endoscopy procedure. Arrows indicate dates when SARS-
oV-2 RNA in colon liquid samples from 3 patients in cohort B.
f SARS-CoV-2 genes N1 and N2 in colon liquids to number of
icate data from one patient.
848 Cherne et al Gastro Hep Advances Vol. 1, No. 5
Table 2. Demographics of the Endoscopy Patients in Cohort B With Prior SARS-CoV-2 Infections
Days since
Sample Age Clinical signs SARS-CoV-2 SARS-CoV-2
Patient ID types Gender (y) BMI D.M. Hypertension Immunosuppr. of COVID-19 diagnosis detection
BDH 154 I, C, CL M 54 25.4 N N N Mild, cough 13 Y
BDH 155 C, CL M 56 23.6 N N N Loss of taste and smell 24 N
BDH 156 I, C, CL M 55 28.4 N N N Asymptomatic, close contact 40 Y
BDH 157 S, D M 44 25.3 N Y N Asymptomatic, close contact 113 N
BDH 158 I, C, CL F 67 30.8 N Y N Cough, loss of taste and smell 69 N
BDH 159 C, CL F 59 22.0 N Y N Sore throat 108 N
BDH 160 I, C, CL M 60 27.2 N N N Asymptomatic, close contact 151 Y
BDH 161 I, C, CL F 57 41.7 N N N Body aches, chills, headache, 76 N
rhinorrhrea
BDH 162 S, D M 69 27.7 N N N Cough, headache, shortness 104 N
of breath, heaviness in chest
BDH 164 I, C, CL F 22 22.0 N N N Loss of taste and smell w200–250 N
BDH 166 I, C, CL M 50 33.8 N N N Cough, diarrhea, fatigue 129 N
BDH 167 I, C, CL F 51 36.1 N N N Asymptomatic, close contact 79 N
C, colon; D, duodenum; CL, colon liquid; DM, diabetes mellitus; F, female; I, ileum; Immunosuppr., immunosuppressive therapy; M, male; N, no; S, stomach; Y, yes.colonoscopy. In the 3 SARS-CoV-2-positive patients, a
shorter time between COVID-19 diagnosis and sample
collection corresponded to a higher recovery of SARS-CoV-2
RNA (Figure 1B).Long-Term Detection of SARS-CoV-2 Protein in
the Colonic Epithelium of Patients Following a
Previous COVID-19 Diagnosis
To determine whether detection of SARS-CoV-2 RNA in
colonic liquids and tissue was associated with viral repli-
cation in the GI mucosa, we performed immunohistochem-
istry for SARS-CoV-2 nucleocapsid protein (NP). As shown
in Figure 2, viral NP was present in a small number of
epithelial cells in the colonic crypts of all 3 SARS-CoV-2 RNA
positive cohort B patients but not in control tissues from
SARS-CoV-2–negative patients. In the 3 positive tissues,
2.5  0.4 NP-positive cells per 10 crypts were identified
(Table 3). Interestingly, NP-positive material was generally
confined to a circumscribed area in the basolateral portion
of crypt epithelial cells. NP staining was not associated with
goblet cells or the surface epithelium. We did not detect any
SARS-CoV-2 NP in the corresponding ileal tissue sections of
these patients. No NP-positive cells were detected in the
colon of the cohort A patient who tested positive for the
virus.
A histopathological analysis revealed that all 3 colon
samples obtained from control patients and 2 of the SARS-
CoV-2–positive donor tissues (BDH156 and BDH160) were
histologically normal (Figure A2A). In contrast, colon tissues
from 2 patients with positive SARS-CoV-2 PCRs (BDH121
and BDH154) showed increased plasma cell density in the
lamina propria (Figure A2B and C).Colonic Tissues and Liquid Samples That Tested
Positive for SARS-CoV-2 by PCR did not Contain
Detectable Levels of Infectious Virus
We next used a viral isolation assay to detect infection-
competent virions in SARS-CoV-2 RNA positive samples.
However, no infectious virus was recovered from any of the
SARS-CoV-2–positive colon liquid or tissue samples
(Table 3). Thus, our study confirms multiple previous re-
ports that had detected SARS-CoV-2 RNA or protein in GI
tissues from COVID-19 patients but were unable to isolate
infectious virus.15,25Colonic Liquids Consistently Inactivate SARS-
CoV-2 In vitro
Zang et al26 previously reported that intestinal liquids
may inactivate SARS-CoV-2 in the lumen of the GI tract,
based on in vitro experiments with simulated GI liquids.
Therefore, we studied the inactivation of SARS-CoV-2 (USA-
WA1/2020, 106 PFU/mL) in the presence of colon liquids
that we had collected from 6 donors from cohort B,
including samples from the 3 patients who were positive for
2022 SARS-CoV-2 prevalence in endoscopy patients 849
Table 3. Detection of SARS-CoV-2 Nucleic Acids, Protein, and Infectious Virions in Gastrointestinal Samples Collected From
Asymptomatic Endoscopy Patients
Nucleocapsid
Sample type RNaseP Virus pos. cells/
Patient ID Cohort (RT-PCR) N1 (Ct) N2 (Ct) E (Ct) (Ct) isolation 10 colonic cryptsa
BDH 121 A Colon tissue 38.0 38.2 36.7 33.1 Negative 0.00  0.00
BDH 154 B Colon liquid 30.0 31.8 31.4 33.0 Negative 1.96  0.22
BDH 156 B Colon liquid 33.1 35.4 35.7 33.2 Negative 2.89  0.71
BDH 160 B Colon liquid 38.0 36.9 n.d. 31.8 Negative 2.59  0.25
aMean  SD, from 2 independent researchers; 75 or more crypts were evaluated in each biopsy sample.
n.d., not detected.SARS-CoV-2. As soon as 10 minutes postincubation, 1 of the
colon liquids from a SARS-CoV-2–negative patient had fully
inactivated the virus. After 1-hour incubation, 50% of colon
liquids had fully inactivated SARS-CoV-2 and by 24 hours
postincubation no infectious virus was detected in any colon
liquid (Figure 3A). No significant difference was seen be-
tween colon liquids with and without SARS-CoV-2 detection
(Figure 3B).Figure 2. Detection of SARS-CoV-2 protein in colonic tissue b
were immunostained for SARS-CoV-2 nucleocapsid protein (N
(A) Colon from a subject with no known previous SARS-CoV-2
samples does not contain any NP-positive cells. (B–D) Detection
cohort B patients BDH154 (B), BDH 156 (C), and BDH160 (D). Ar
equal 10 mm.Discussion
We here analyzed the prevalence of gastrointestinal
SARS-CoV-2 infection in gastrointestinal endoscopy patients
without active COVID-19 to better understand potential
transmission risks arising from endoscopy procedures. Our
study confirms previous reports that the GI tract is a site of
active SARS-CoV-2 replication and serves as a potentialy immunohistochemistry. Paraffin-embedded tissue sections
P, brown) and the counter stained with hematoxylin (purple).
infection and negative SARS-CoV-2 PCR results in intestinal
of SARS-CoV-2 nucleocapsid protein in the colonic crypts of
row heads identify infected crypt epithelial cells. All scale bars
850 Cherne et al Gastro Hep Advances Vol. 1, No. 5
Figure 3. Inactivation of SARS-CoV-2 by in vitro incubation with colon liquids. (A) To investigate inactivation of SARS-CoV-2 in
colon liquids, 106 PFU/mL SARS-CoV-2 was spiked into 6 cohort B colon liquids, and then a plaque assay using VeroE6 cells
was performed after incubation at 37 C, 5% CO2 for 10 minutes, 1 hour, or 24 hours to determine the remaining viral con-
centration. Each data point represents an individual donor; bars and error bars indicate mean  SD. Results are plotted on a
log scale, so samples where virus was fully inactivated (PFU/mL ¼ 0) are not shown. n.d., not detected. *P 
 .05, 2-way
analysis of variance with Friedman test for multiple comparisons. (B) Comparison of SARS-CoV-2 inactivation after 10 mi-
nutes incubation in the presence of colon liquids that tested negative (n ¼ 3) or positive (n ¼ 3) for SARS-CoV-2. SARS-CoV-2
was also incubated in PBS for comparison of inactivation over time. N.d., not detected.long-term reservoir for the virus.8,14,19,27 We detected SARS-
CoV-2 RNA in 30% of colon liquids collected from previ-
ously infected patients who had asymptomatic infections or
who had recovered from mild infection, with one sample
testing positive 5 months after the patient’s COVID-19
diagnosis. We also detected SARS-CoV-2 nucleoprotein in
the colonic epithelium of all 3 patients from this cohort who
had positive PCR results. All 3 of these patients were male
but no conclusions about gender-related differences could
be drawn given the small group size. The presence of viral
protein within GI tissue cells indicates active infection of the
GI tract rather than carryover contamination of the GI tract
from the upper respiratory tract, supporting previous re-
ports of SARS-CoV-2 replication in GI epithelial cells.15,27
Similar to Cheung et al14 and Livanos et al,15 we detected
SARS-CoV-2 nucleocapsid in epithelial cells at the base of
the crypts. However, we did not see any evidence of SARS-
CoV-2 infection in goblet cells in our colonic samples,
whereas Livanos et al15 demonstrated that goblet cells were
a major target for SARS-CoV-2 in the small intestine.
Intriguingly, only 1/12 of the recovered COVID-19 patients
in our study had reported any GI symptoms during the
course of their illness.
Since viral RNA and protein were detected as late as 151
days after acute SARS-CoV-2 infection, our data support the
hypothesis that the GI tract can be a long-term viral reser-
voir that sheds virus or viral RNA after respiratory infectionhas ceased. While the number of patients with previous
SARS-CoV-2 infections (cohort B) included in our study was
low, the probability for SARS-CoV-2 detection in this group
(26%) was much higher than in patients with no known
prior SARS-CoV-2 infection (1%). Given the worldwide
surges in SARS-CoV-2 infection due to the highly trans-
missible Delta and Omicron variants in summer/fall 2021
and in early 2022, the percentage of the population with a
previous infection and thus potential virus persisting in the
gut now is likely considerable. However, we recovered no
infectious virus from any positive GI tissue or liquid sample.
Notably, no infectious virus was detected from samples with
a cycle threshold value more than 30 in the SARS-CoV-2 RT-
PCR assay in several previous studies28,29 and all positive
gastrointestinal samples in our study had cycle threshold
values of >30 for all genes that were analyzed.
The detection of SARS-CoV-2 RNA in the colon liquid of
patients is indicative of active GI infection with either
shedding of SARS-CoV-2 virions or dying infected cells into
the gut lumen. However, the harsh environment of the GI
tract is expected to inactivate virus or viral RNA rapidly.30
In a previous study, Zang et al26 demonstrated that simu-
lated colon liquid led to almost complete inactivation of
SARS-CoV-2 in vitro within 24 hours. We showed that in-
cubation of infectious SARS-CoV-2 with colon liquids
collected from endoscopy patients reduced the amount of
infectious virus by approximately 4-log–fold within 1 hour
2022 SARS-CoV-2 prevalence in endoscopy patients 851and led to complete inactivation of SARS-CoV-2 within 24
hours. Considering this rapid inactivation and the relatively
low amount of SARS-CoV-2 detected in the colon liquids, any
virions shed into the GI lumen and aerosolized during
endoscopy would likely pose minimal transmission risk.
We found no evidence of significant colonic pathology in
SARS-CoV-2–positive subjects. However, some signs of
increased inflammation were present, with 2 of the positive
samples having an overabundance of plasma cells. These ob-
servations are consistent with findings from Qian et al27 who
reported abundant lymphoplasmacytic infiltrates in the rectal
lamina propria upon intestinal SARS-CoV-2 infection. Like-
wise, Livanos et al15 described significant changes in small
intestinal immune cell subsets and functions upon intestinal
SARS-CoV-2 infection. The clinical relevance and mechanism
of these changes remain a subject for further investigation.
Our study has several limitations that should be taken
into consideration. First, all samples analyzed in our study
were collected before major viral variants of concern
(Delta/B.1.617.2 and Omicron/B.1.1.529) emerged.31,32
Differential cell and tissue tropism has been reported for
certain viral variants33 but it is currently unknown whether
Delta and Omicron variants have an altered tropism for the
GI tract. Second, while our analyses clearly demonstrated
that persistence of SARS-CoV-2 in the GI tract is relatively
common, our overall sample size was small, none of the
patients in our study had severe COVID-19 symptoms, and
subjects from only one healthcare facility were recruited.
Therefore, our results may not be entirely representative
and will require further investigations.
In summary, our results highlight the multisystem path-
ogenesis of COVID-19. We uncovered asymptomatic SARS-
CoV-2 infection of the gut in the absence of respiratory
symptoms, although prevalence was low. Detection of SARS-
CoV-2 in patients previously diagnosed with COVID-19 was
considerably more common, suggesting extra precautions for
endoscopy procedures on recovered COVID-19 patients could
be necessary. Of importance, we predict these asymptomatic
infections pose minimal risk to healthcare workers per-
forming endoscopies, since no infectious virus could be
recovered from SARS-CoV-2–positive GI samples, since none
of the healthcare personnel involved in our study contracted
SARS-CoV-2 throughout the course of our study, and since we
demonstrate the rapid inactivation of infectious SARS-CoV-2
in colon liquids, which are the main sources of endoscopy
aerosols and droplets produced during the procedures. The
long-term detection of SARS-CoV-2 in GI tissue and liquids
warrants further study of SARS-CoV-2 infection in the GI tract
and its potential as a viral reservoir.Supplementary Materials
Material associated with this article can be found in the
online version at https://doi.org/10.1016/j.gastha.2022.06.
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Viruses 2021;14:23.Received March 17, 2022. Accepted June 3, 2022.
Correspondence:
Address correspondence to: Diane Bimczok, DVM, PhD, Department of
Microbiology and Cell Biology, Montana State University, 2155 Analysis Drive,
Bozeman, Montana 59718 e-mail: diane.bimczok@montana.edu.
Acknowledgments:
We would like to thank the endoscopy team at the Bozeman Health Deaconess
Hospital for their assistance with sample collection. We also would like to thank
Dr Mark Young, Montana State University, for helpful discussions and for
providing a stable plant virus for method validation.
Author Contributions:
Michelle D. Cherne: Formal analysis, investigation, methodology, validation,
visualization, writing–original draft, and writing–review and editing; Andrew B.
Gentry: Conceptualization, project administration, resources, supervision,
writing–original draft, and writing–review and editing; A. Nemudraia: Formal
analysis, investigation, methodology, and writing–review and editing; A.
Nemudryy: Formal analysis, investigation, methodology, and writing–review
and editing; Jodi F. Hedges: Formal analysis, investigation, methodology,
and writing–review and editing; Heather Walk: Investigation, methodology,
resources, and writing–review and editing; Karlin Blackwell: Investigation and
writing–review and editing; Deann T. Snyder: Investigation and writing–review
and editing; Maria Jerome: Investigation, methodology, and writing–review
and editing; Wyatt Madden: Formal analysis and writing–review and editing;
Marziah Hashimi: Investigation and writing–review and editing; T. Andrew
Sebrell: Investigation and writing–review and editing; David B. King: Concep-
tualization, supervision, and writing–review and editing; Raina K. Plowright:
Conceptualization, funding acquisition, supervision, and writing–review and
editing; Mark A. Jutila: Conceptualization, funding acquisition, resources, su-
pervision, and writing–review and editing; Blake Wiedenheft: Conceptualiza-
tion, funding acquisition, resources, supervision, and writing–review and
editing; Diane Bimczok: Conceptualization, formal analysis, funding acquisi-
tion, investigation, project administration, supervision, writing–original draft,
and writing–review & editing.
Conflicts of Interest:
The authors disclose no conflicts.
Funding:
Funding from the National Institutes of Health (U01EB029242-02S1; to D.B.
and M.J.), the Montana Agricultural Experiment Station (to D.B., B.W., and
M.J.), USDA/Hatch Multi-State project W519 (to M.J.), and the Montana State
University Office of Research, Economic Development and Graduate Educa-
tion (to D.B.) is gratefully acknowledged. R.K.P. and W.M. were funded by the
DARPA PREEMPT program Cooperative Agreement #D18AC00031. B.W. was
supported by the National Institutes of Health (R35GM134867), the M.J. Mur-
dock Charitable Trust, a young investigator award from Amgen, and a
generous gift from the Rosolowsky family.
Ethical Statement:
The corresponding author, on behalf of all authors, jointly and severally, cer-
tifies that their institution has approved the protocol for any investigation
involving humans or animals and that all experimentation was conducted in
conformity with ethical and humane principles of research.
Data Availability Statement:
The data that support the findings of this study are available from the corre-
sponding author, D.B., upon reasonable request.