Exeley Inc.

Bhoil, Rohit

Ahluwalia, Ajay

Chopra, Rajesh

Surya, Mukesh

Bhoil, Sabina

Signs and lines in lung ultrasound

LI, YONGDONG

CHEN, YI

NI, HONGXIA

YI, BO

ZHANG, DANDAN

ZHANG, JIANING

WANG, WENJING

LIU, YUHUI

JIAO, SULI

XU, GUOZHANG

QIAN, WEIDONG

Polish Journal of Microbiology
2021, Vol. 70, No 3, 401–404
https://doi.org/10.33073/pjm-2021-029
SHORT COMMUNICATION
* Corresponding authors: G. Xu, Ningbo Municipal Center for Disease Control and Prevention, Ningbo, P.R. China;
 e-mail: xugz@nbcdc.org.cn
 W. Qian, School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an, P.R. China;
 e-mail: qianweidong@sust.edu.cn
© 2021 Y. Li et al.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License (https://creativecommons.
org/licenses/by-nc-nd/4.0/).
The recent emergence of a novel coronavirus (SARS-
CoV-2) in China was associated with severe human 
infection and has emerged globally as a highly virulent 
human pathogen, causing severe public health con-
cerns (Holshue et al. 2020; Zhu et al. 2020). Currently, 
the virus transmission is considered the following two 
main routes: respiratory droplets and contact (To et al. 
2020). To fortify the response against the alarming levels 
of spread of the virus, many countries have tightened the 
detection of people showing symptoms and close con-
tacts and have introduced necessary and urgent actions 
to prevent its spread (Wilder-Smith et al. 2020). The 
common symptoms of SARS-CoV-2 include fever, dry 
cough, shortness of breath or difficulty breathing, and 
myalgia (Chen et al. 2020). Several confirmed cases of 
COVID-19 with recurrently positive SARS-CoV-2 ribo-
nucleic acid (RNA) in swabs and stool have been identi- 
fied in China (Wu et al. 2020). We reported the discov-
ery and complete genome characterization of SARS-
CoV-2 in the stool sample from a recovered patient.
A 26-year-old man lived in Wuhan, China, and 
denied exposure to the Huanan seafood market or wild 
animals. As shown in Fig. 1, the man got a headache 
without other evident symptoms, including fever or 
cough, on January 17, 2020. The next day, he developed 
a dry cough, nasal congestion, fatigue, muscle ache, 
and sore throat symptoms, with a body temperature of 
37.6°C. About 36 h after the onset of symptoms, he did 
not go to the local hospital for diagnosis and received 
no treatment at all. On January 20, 2020, he came to 
the fever clinic of the People’s Hospital of Yuyao City 
after he took 6 hours from Wuhan to Ningbo, Zhejiang 
province, by high-speed rail. Given his travel history, 
he was admitted immediately to an airborne isolation 
unit as a suspected case of SARS-CoV-2.
On admission, high-resolution computed tomo-
graphy of the chest was performed immediately, images 
of the patient demonstrated scattered cloud-wool 
inflammatory changes in the upper and lower lobes of 
the right lung and the lower lobe of the left lung. Mean-
while, the physical examination revealed that the patient 
was presented signs of infection; he was tachycardic, 
and his body temperature was 38.5°C, while the patient 
was breathing ambient air. Results of the blood routine 
Discovery and Full Genome Characterization of SARS-CoV-2 in Stool Specimen
from a Recovered Patient, China
YONGDONG LI1, YI CHEN1, HONGXIA NI1, BO YI1, DANDAN ZHANG1, JIANING ZHANG2,
WENJING WANG2, YUHUI LIU1, SULI JIAO1, GUOZHANG XU1* and WEIDONG QIAN2*
1 Ningbo Municipal Center for Disease Control and Prevention, Ningbo, P.R. China
2 School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an, P.R. China
Submitted 20 April 2021, revised 6 July 2021, accepted 8 July 2021
A b s t r a c t
SARS-CoV-2 was found in a recovered patient’s stool specimen by combining quantitative reverse transcription PCR (qRT-PCR) and 
genome sequencing. The patient was virus positive in stool specimens for at least an additional 15 days after he was recovered, whereas 
respiratory tract specimens were negative. The discovery of the complete genome of SARS-CoV-2 in the stool sample of the recovered 
patient demonstrates a cautionary warning that the potential mode of the virus transmission cannot be excluded through the fecal-oral 
route after viral clearance in the respiratory tract.
K e y w o r d s: COVID-19, SARS-CoV-2, genome sequencing, recovered patient
brought to you by COREView metadata, citation and similar papers at core.ac.uk
provided by Exeley Inc.
Li Y. et al. 3402
showed a leukocyte count of 6 × 109/l, a  neutrophils 
level of 65.8%, a lymphocyte percentage of 20.6%, and 
a hypersensitive-C-reactive protein level of 0.6 µg/ml.
For the quantitative RT-PCR (qRT-PCR) test, 
RNA was extracted from the samples using Magnetic 
Beads RNA Extraction Kit (Health Gene Technologies, 
Ningbo, China) according to the manufacturer’s pro-
tocol. The samples with a Ct scores less than 40 were 
considered positive. In this study, two negative con-
trols, and one positive control were randomly placed 
in samples to detect at the same time. On 21 January, 
he was confirmed positive for SARS-CoV-2 by qRT-
PCR after two sequential positive respiratory tract sam-
ple results, while the nasopharyngeal swab and stool 
specimens were negative. The results of the qRT-PCR 
test for influenza A and B, parainfluenza, respiratory 
syncytial virus, adenovirus, Mycoplasma pneumoniae, 
enterovirus, human metapneumovirus and bocavi-
rus, Chlamydia pneumoniae, and Rickettsia burneti 
were negative. According to the diagnostic criteria in 
China (General Office of National Health Commission 
2020), he was confirmed as a COVID-19 patient. The 
patient’s symptoms of diseases caused by SARS-CoV-2 
presented with progression to typical signs on day 9 of 
symptomatic treatment and antimicrobial therapy. Dur-
ing treatment, oropharyngeal swab samples of SARS-
CoV-2 RNA were performed repeatedly for surveillance 
and returned with negative results on 26 January. On 
31 January, the patient met hospital discharge criteria 
or discontinuation of quarantine and was requested to 
extend the quarantine beyond 14 days at home (Lan 
et al. 2020). However, on February 11, the stool speci-
men obtained on negative day 17 later tested positive for 
SARS-CoV-2 by qRT-PCR (the viral load of 3.2 log10 
RNA copies/ml, ORF1ab Ct 30.8; nucleoprotein gene 
Ct 31.7), including persistent high levels (Ct values, 
23 to 24), whereas the serum, nasopharyngeal, and spu-
tum specimens remained negative. The stool sample 
obtained on positive-recurrent days 10 showed a trend 
toward decreasing levels of virus. On 25 February, the 
stool specimen tested negative for SARS-CoV-2 on pos-
itive-recurrent day 14. In the hospital, a routine blood 
test performed on February 13 revealed that cell counts 
fell within reference ranges, and a computed tomogra-
phy scan on February 15 demonstrated that there were 
no identifiable abnormalities. During February 11–25, 
the man had no signs or symptoms and no detectable 
fever as well as other clinical signs or symptoms consist-
ent with SARS-CoV-2 despite the positive results for 
SARS-CoV-2 in the stool samples, while nasopharyn-
geal and sputum specimens  were negative.
Total nucleic acids were extracted from qRT-PCR-
positive samples (SARS-CoV-2-016, the oropharyngeal 
samples obtained on 20 January and SARS-CoV-2-4794, 
the stool samples obtained on February 19) using the 
QIAamp Viral RNA Mini Kit (Qiagen, Germany) and 
then employed for whole-genome next-generation 
sequencing using the Illumina MiSeq platform (Illu-
mina, USA). Double-stranded DNA for sequencing 
library preparation was performed by random prim-
ing using the Maxima H Minus Double Stranded cDNA 
Synthesis Kit (Thermo Fisher Scientific, Waltham, 
USA) with 2.5 µM random hexamers according to the 
manufacturer’s protocol. Sequence assembly was com-
pleted with the use of CLC Genomics Workbench ver-
sion 20.0.2 (Qiagen, Germany). For the examination 
of the mutation profile within the SARS-CoV-2 genome 
and phylogenetic relationship, complete genome 
sequences of SARS-CoV-2-016 and SARS-CoV-2-4794 
were aligned against publicly available SARS-CoV-2 
sequences from NCBI using Minimap2 aligner (Li 
2018). The results show that the full genome sequences 
from oropharyngeal and stool specimens were identical 
to one another and were nearly identical to other avail-
able SARS-CoV-2 sequences. The consensus sequence 
of SARS-CoV-2 was the same for two samples, suggest-
ing that the virus can spread beyond the respiratory sys-
tem to lurk in feces. In addition, there were only seven 
nucleotides and two amino acid that differed at nps3 of 
orf1ab and ORF8 between this patient’s virus and the 
Fig. 1. Periods of COVID-19 in the patient with diagnosed coronavirus disease 2019.
Running title: Full genome of SARS-CoV-2 from a recovered patient3 403
SARS-CoV-2 reference sequence (NC_045512.2). Phy-
logenetic analysis revealed that the two SARS-CoV-2 
genomes in this study are in the A clade. The genome 
sequences are accessible through GenBank (accession 
numbers MT318828 and MT318829).
Although respiratory transmission is considered 
the primary route for SARS-CoV-2 and the evidence 
is yet insufficient to establish practical measures for 
the group of patients recovered from COVID-19, who 
tested negative for respiratory tract sample results but 
positive for fecal samples. Wu et al. (2020) reported 
that a few patients’ fecal samples remained positive for 
SARS-CoV-2 RNA for a mean of 11.2 days after respira-
tory tract samples became negative by qRT-PCR. Simi-
larly, the direct evidence showed that SARS-CoV-2 was 
identified in 44 stool specimens (44 out of 153) (Wang 
et al. 2020). Here, we reported that a recovered patient 
tested positive for SARS-CoV-2 in stool specimens for 
at least an additional 15 days after he was recovered, 
whereas respiratory tract specimens were negative. 
These phenomena observed might be attributed to the 
fact that ACE2, the entry receptor for SARS-CoV-2, is 
highly expressed in the glandular cells of gastric, duo-
denal, and rectal epithelia (Hoffmann et al. 2020).
Determining whether SARS-CoV-2 exists in a com-
plete or fragmented manner by qRT-PCR is difficult. In 
addition, determining whether mutations of the virus 
that can adapt to the human host occur as long as the 
virus continues to circulate in humans by qRT-PCR 
is difficult. Here, genome sequencing was applied to 
profile the genome sequence of the virus in the sam-
ples from different dates of symptom onset. The full 
genome sequences from two samples before and after 
recovery were identical to one another, indicating 
that direct evidence was identified genetically sup-
porting the existence of the move of the virus from 
the respiratory tract into the gastrointestinal tract in 
the recovered patient, which may discharge the stool 
with the virus for a long time. Additionally, our study 
also suggests that the virus might become more adapted 
in the human body as a human disease. The genome 
data of the virus in the stool sample may contrib-
ute to further investigations of the evolution of the 
virus (Shen et al. 2020). However, the quantitative 
relationship between viral relative abundance/titer and 
infection is unclear.
Overall, our study has revealed and identified the 
full genome of SARS-CoV-2 in the stool sample of 
a recovered patient, an uncommon feature possessed 
by an RNA virus. Whether the virus, including the 
complete genome hidden in the body, can replicate and 
thereby cause the transmission via the fecal-oral route, 
awaits further investigation. However, a possible ther-
apy for this could be extended use of antiviral agents 
or in combination with traditional Chinese medicine 
(TCM) for recovered patients for further two weeks. 
Compared with Western medicine, treating patients as 
an organic whole is the prerequisite for TCM to treat all 
diseases (Zhao et al. 2021). In addition to the COVID-
19 vaccines currently in use, in the absence of specific 
drugs globally, China’s experience in the application 
of TCM can be used as a valuable reference to treat-
ing COVID-19 (Zhao et al. 2021). Meanwhile, there is 
a potential need to establish the guideline for surveil-
lance and adequate disinfection in latrines from the 
recovered patient.
Ethical statement
This study was approved by Ningbo municipal center for disease 
control and prevention ethical review committee (202001).
Author contributions
Conceptualization: Guozhang Xu, Weidong Qian; Methodology: 
Yongdong Li, Guozhang Xu; Formal analysis and investigation: Yi 
Chen; Writing – original draft preparation: Weidong Qian, Hongxia 
Ni; Writing – review and editing: Dandan Zhang, Jianing Zhang, 
Wenjing Wang; Funding acquisition: Bo Yi; Resources: Yuhui Liu, 
Yongdong Li; Supervision: Suli Jiao, Weidong Qian. All authors 
contributed to manuscript revision and approved the final version.
Funding
This study was funded by the Science and Technology Research 
Project of Ningbo Science and Technology Bureau (No: 2020C50001).
Conflict of interest
The authors do not report any financial or personal connections 
with other persons or organizations, which might negatively affect 
the contents of this publication and/or claim authorship rights to 
this publication.
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