To the Editor:
An outbreak caused by a newly identified coronavirus, severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China, in December 2019 (1)
and has since spread across mainland China and to other countries. The clinical spectrum
of coronavirus disease (COVID-19) ranges from asymptomatic to severe disease, and
studies in China showed that 5.0% of patients had been admitted to the ICU (2, 3).
Real-time RT-PCR assays are recommended for the diagnosis of SARS-CoV-2 infection
(4). A previous study reported SARS-CoV-2 viral loads in upper-respiratory specimens
from patients with COVID-19 (5). Here, we investigated the viral load in specimens
from multiple sites and the duration of viral shedding in respiratory-tract samples
from laboratory-confirmed critically ill patients with COVID-19 requiring ICU admission.
Methods
We conducted a retrospective, descriptive study that included 16 consecutive critically
ill patients with COVID-19 who had been admitted to the ICU of the First Affiliated
Hospital of Guangzhou Medical University. The study was approved by the Ethics Committee
of the First Affiliated Hospital of Guangzhou Medical University. The requirement
for informed consent was waived for the retrospective collection of data. A protocol
was developed for sample collection when the first patient was admitted to the ICU,
as follows: serial samples from the upper respiratory tract (throat and nasal swabs)
and lower respiratory tract (sputum or endotracheal aspirate [ETA]) were collected
daily during the first week after admission and every 2–3 days after the first week,
until two sequential negative results were obtained or the patient was discharged
from the ICU. Plasma, serum, conjunctival swabs, and urine samples were also collected
in the first week after ICU admission. Fifteen patients tested negative in these samples,
and in the remaining patient, sample collection was discontinued when two sequential
negative results were obtained. Fecal samples were collected when available, and if
unavailable, anal swabs were collected instead. Gastric fluid samples were collected
only in patients with an indwelling gastric tube. Most sampling was done according
to the designed protocol (see the online supplement). Swab samples were immediately
placed into sterile tubes containing 3 ml of viral transport medium. The specimens
were sent to the virology laboratory of our hospital for sample processing and viral
RNA extraction. We used 0.25 ml of liquid samples (viral transport medium or directly
from biological specimens) for RNA extraction. Viral RNA of SARS-CoV-2 was detected
according to the recommendations of the Chinese Center for Disease Control and Prevention
(6). Two target genes, ORF1ab (open reading frame 1ab) and N (nucleocapsid protein),
were simultaneously amplified and tested using a real-time RT-PCR assay. The viral
load was indicated as the cycle threshold (Ct) value of the N gene of SARS-CoV-2.
Positive and negative controls were included in the assay according to the manufacturer’s
protocol. A Ct value of <40 was defined as positive for SARS-CoV-2 RNA, and >40 was
defined as negative. Samples with a Ct value between 37 and 40 were retested at least
twice. The Ct values of all samples collected and tested are shown in the online supplement.
Results
A total of 16 patients (13 men and 3 women; median age, 59.5 yr; range, 26–79 yr)
who were admitted to our ICU from January 26 through February 25, 2020, were included
in this study. Twelve patients were imported cases who had recently returned from
Hubei Province, and four had been exposed to patients with confirmed SARS-CoV-2 infection.
Most patients (75%) had at least one preexisting chronic condition. All of the patients
showed evidence of pneumonia in chest radiographs, and 15 patients were diagnosed
with acute respiratory distress syndrome (ARDS) (eight with moderate ARDS and seven
with severe ARDS) upon admission. Four patients (25%) were supported with noninvasive
positive-pressure ventilation, and 12 (75%) were supported with invasive mechanical
ventilation. Extracorporeal membrane oxygenation was applied in five patients (31%)
(Table 1). As of March 20, nine patients had been discharged from the ICU and all
16 patients were alive.
Table 1.
Baseline and Clinical Characteristics, Main Interventions, and Detection of SARS-CoV-2
in Specimens from Patients with SARS-CoV-2 Infection Admitted to the ICU
Variables
All Patients (N = 16)
Age, yr, median (range)
59.5 (26–79)
Male sex, n (%)
13 (81%)
Body mass index, kg/m2, median (Q1–Q3)
24.1 (22.0–27.5)
Chronic conditions, n (%)
12 (75%)
Diabetes
6 (37%)
Chronic cardiac disease
10 (63%)
Chronic pulmonary disease
5 (31%)
Chronic neurologic disease
2 (13%)
Any malignancy
1 (6%)
Liver disease
2 (13%)
Smoker (including ex-smoker), n (%)
9 (56%)
Exposure, n (%)
Exposure to Hubei
12 (75%)
Exposure to confirmed patients
4 (25%)
Days from onset of symptoms to ICU admission, median (Q1–Q3)
12.0 (9.0–16.5)
Ratio of PaO2
to Fi
O2
on Day 1, mm Hg, mean ± SD
120.7 ± 60.8
APACHE II score on Day 1, mean ± SD
16.4 ± 7.8
SOFA score on Day 1, mean ± SD
6.9 ± 3.8
ARDS, n (%)
15 (94%)
Mild ARDS
0
Moderate ARDS
8 (50%)
Severe ARDS
7 (44%)
Mechanical ventilation during ICU stay, n (%)
Noninvasive
4 (25%)
Invasive
12 (75%)
Extracorporeal membrane oxygenation during ICU stay, n (%)
5 (31%)
Positive for SARS-CoV-2 during ICU stay, n/patients tested
Nasal swab
13/16
Throat swab
10/16
Sputum/ETA
16/16
Conjunctival swab
1/15
Blood
1/16
Urine
1/16
Gastric fluid
6/13
Feces
11/16
Anal swab
4/15
Definition of abbreviations: APACHE II = Acute Physiology and Chronic Health Evaluation
II; ARDS = acute respiratory distress syndrome; ETA = endotracheal aspirate; Q1 = quartile
1; Q3 = quartile 3; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2;
SOFA = Sequential Organ Failure Assessment.
The median number of days from the onset of symptoms to ICU admission was 12.0 days
(quartile 1–quartile 3: 9.0–16.5). During the ICU stay, nasal swab samples from 13
patients (81%) and throat swab samples from 10 patients (63%) tested positive for
SARS-CoV-2, but lower-respiratory specimens (sputum or ETA) were positive in all 16
patients (100%). Viral RNA was also detected in urine (1 patient), conjunctival swab
(1 of 15 patients; 1 patient refused to provide a conjunctival swab), and gastric
fluid (6 of 13 patients). SARS-CoV-2 viral RNA was also detected in fecal samples
from 11 patients (69%) and anal swabs (4 patients). In one patient, viral RNA was
present in all types of specimens taken, suggesting that infection in this patient
may have been systemic (Table 1).
We analyzed the viral load and duration of virus shedding in nasal and throat swabs
and lower-respiratory specimens in relation to the day of symptom onset (Figure 1A).
Surprisingly, 11 patients (69%) showed prolonged viral shedding in lower-respiratory
specimens, beyond 28 days after the onset of symptoms. As of March 20, the longest
observed period of viral shedding in lower-respiratory-tract specimens was 55 days
(patient #4; online supplement). In addition, lower-respiratory-tract specimens (sputum
or ETA) had significantly higher SARS-CoV-2 viral RNA levels (inversely related to
the Ct value) than nasal and throat swab specimens (Figure 1B). Our results indicated
that samples from the lower respiratory tract had the highest viral load but slowest
resolution of viral shedding in comparison with throat and nasal swab samples.
Figure 1.
Viral load detected in respiratory specimens obtained from critically ill patients
infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (A) Aggregated
cycle threshold (Ct) values of the nucleocapsid protein gene of SARS-CoV-2 in serial
throat swabs, nasal swabs, and sputum/endotracheal aspirate (ETA) samples from 16
patients, according to days after symptom onset. (B) Box plot of the lowest Ct values
in throat swabs, nasal swabs, and sputum/ETA samples during the entire ICU stay among
patients with coronavirus disease (COVID-19). Box-and-whiskers plot features are as
follows: the central line in the box is the median, the bottom line of the box is
the first quartile, and the top line of the box is the third quartile. Bottom of whiskers:
maximum Ct value; top of whiskers: minimum Ct value. Groups were compared using the
Kruskal-Wallis test with Dunn’s multiple comparison test. *P < 0.01.
Discussion
It is generally believed that the lung is the major target organ of SARS-CoV-2; however,
we detected viral RNA in numerous different clinical samples, including conjunctival
swabs, blood samples, gastric juices, feces, anal swabs, and urine from critically
ill patients. Wang and colleagues tested 1,070 specimens collected from 205 patients
with COVID-19 and found that the virus could be detected in different types of clinical
specimens, including respiratory-tract samples, feces, and blood (7). However, the
detection of viral RNA does not always equate with the presence of infectious virus,
and viral RNA shedding of SARS-CoV-2 does not equate with infectivity. Our colleagues
(Sun and colleagues, manuscript under review) had previously succeeded in isolating
infectious virus from a urine sample from one of our patients. This suggests that
the SARS-CoV-2 virus can replicate in extrapulmonary sites, as has been observed in
some patients with severe viral pneumonia, such as that caused by the highly pathogenic
avian influenza (8). However, the role of SARS-CoV-2 transmission via extrarespiratory
routes (e.g., fecal–oral transmission) in the spread of COVID-19 must be further investigated.
Our findings are in accord with reports showing that ACE2 (angiotensin converting
enzyme II), the putative cell entry receptor of SARS-CoV-2, is widely expressed in
a variety of epithelial cells in multiple organs (9). It is still unclear whether
replication of SARS-CoV-2 in extrapulmonary organs contributes to organ injury and
dysfunction, considering that secondary organ injury owing to hypoxia, tissue hypoperfusion,
and inflammation is common in critically ill patients.
Zou and colleagues found that SARS-CoV-2 viral RNA could be weakly detected in nasal
and throat swabs after 14 days from symptom onset (5). Pan and colleagues reported
viral loads from different types of clinical specimens collected from 82 infected
individuals within a maximum of 15 days after symptom onset (10). Zhou and colleagues
found that the median duration of viral shedding in throat swabs was 20.0 days in
COVID-19 survivors (11). Here, we found that SARS-CoV-2 viral RNA could be detected
in sputum or ETA beyond 28 days from symptom onset in 11 patients (69%), as well as
in extrapulmonary samples from these critically ill patients. These findings have
important implications for assessing transmission risk and protecting ICU staff, and
highlight the importance of effective antiviral treatment for critically ill patients
with COVID-19.
This study is limited by the small number of critically ill patients and the lack
of nonsurvivor data (there were no deaths in our ICU during the study period). In
addition, we were not able to sample consistently according to the designed protocol,
and sampling was discontinued after patients were discharged to the hospital’s isolation
ward. Longitudinal studies in a larger cohort would enhance our understanding of viral
load and shedding in patients with COVID-19.
In conclusion, critically ill patients infected with SARS-CoV-2 demonstrated higher
viral loads and prolonged shedding in lower-respiratory-tract specimens than in upper-respiratory-tract
specimens. Sampling from the lower respiratory tract may be required to assess the
true viral clearance in such patients.