Persistent shedding of viable SARS-CoV in urine and stool of SARS patients during the convalescent phase.

Abstract

In order to further the present knowledge of the emerging severe acute respiratory syndrome-associated coronavirus (SARS-CoV), 486 different specimens from 54 patients with a clinical diagnosis of SARS were investigated for the presence of viral RNA, and 314 plasma specimens of 73 patients were examined for IgM and IgG antibodies specific against SARS-CoV using an indirect ELISA. Viral RNA was detectable in 28 of the 54 patients tested. Cumulative data showed that 67 of the 73 SARS patients demonstrated seroconversion by week 5 of illness. In contrast, only 1 of 278 healthy subjects enrolled in the study was found to be positive for the IgG antibody. Coexistence of viral RNA in plasma and specific antibodies was simultaneously observed over three consecutive weeks in two critical cases. In three convalescent patients in particular, cultivable SARS-CoV was detected in stool or urine specimens for longer than 4 weeks (29-36 days). These findings suggest that SARS-CoV may remain viable in the excretions of convalescent patients.

Full text

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Eur J Clin Microbiol Infect Dis (2005) 24: 165–171
DOI 10.1007/s10096-005-1299-5
ARTICLE
D. Xu · Z. Zhang · L. Jin · F. Chu · Y. Mao · H. Wang ·
M. Liu · M. Wang · L. Zhang · G. F. Gao · F. S. Wang
Persistent shedding of viable SARS-CoV in urine and stool of SARS
patients during the convalescent phase
Published online: 24 March 2005
C ?Springer-Verlag 2005
Abstract In order to further the present knowledge of
theemergingsevereacuterespiratorysyndrome-associated
coronavirus (SARS-CoV), 486 different specimens from
54 patients with a clinical diagnosis of SARS were in-
vestigated for the presence of viral RNA, and 314 plasma
specimens of 73 patients were examined for IgM and IgG
antibodies specific against SARS-CoV using an indirect
ELISA. Viral RNA was detectable in 28 of the 54 patients
tested. Cumulative data showed that 67 of the 73 SARS
patients demonstrated seroconversion by week 5 of illness.
In contrast, only 1 of 278 healthy subjects enrolled in the
study was found to be positive for the IgG antibody. Coex-
istence of viral RNA in plasma and specific antibodies was
simultaneously observed over three consecutive weeks in
two critical cases. In three convalescent patients in partic-
ular, cultivable SARS-CoV was detected in stool or urine
specimens for longer than 4 weeks (29–36 days). These
findings suggest that SARS-CoV may remain viable in the
excretions of convalescent patients.
Introduction
Severe acute respiratory syndrome (SARS) is a newly
emerging human infectious disease that has resulted glob-
ally in 774 deaths from 8,096 probable cases to date [1,
2]. A novel coronavirus has been identified as the etiolog-
D. Xu · Z. Zhang · L. Jin · F. Chu · Y. Mao · H. Wang ·
M. Wang · L. Zhang · F. S. Wang (?)
Beijing Institute of Infectious Diseases, Beijing 302 Hospital,
100 Xi Si Huan Middle Road,
Beijing, 100039, China
e-mail: fswang@public.bta.net.cn
Tel.: +86-10-66933332
Fax: +86-10-63831870
M. Liu
Beijing Genomics Institute, Chinese Academy of Sciences,
Beijing, China
G. F. Gao
Institute of Microbiology, Chinese Academy of Sciences,
Beijing, China
ical agent of SARS and been designated as the SARS-
associated coronavirus (SARS-CoV) [3–7]. SARS-CoV
contains a single-stranded plus-sense RNA genome ap-
proximately 30 kb in length, which typically has five ma-
jor open reading frames (ORFs) that encode the replicase
(R) polyproteins, and the spike (S), the envelope (E), the
membrane (M), and the nucleocapsid (N) proteins. SARS-
CoV can infect African green monkey kidney (Vero E6)
cells in vitro and experimentally cause a similar disease in
cynomolgus macaques (macaca fascicularis) [8].
It has been reported that around 90% (83.3–100%) of
patients with probable SARS generated antibodies against
SARS-CoV [9–11], while the figure was negligible in a
healthy population [12]. For early laboratory diagnostics,
assays based on conventional nested and/or real-time re-
verse transcription-polymerase chain reactions (RT-PCR)
have been developed to detect viral RNA in the clinical
specimens of SARS patients [3, 13, 14–16]. Even though
the viral RNA and the antibody profiles in SARS patients
have been well studied, the simultaneous detection of both
profiles in sera or plasma has rarely been documented. In
particular, there is a paucity of data concerning viral vital-
ity in the excretions of SARS patients in the convalescent
phase.
In the study presented here viral RNA was detected in
different types of clinical specimens. Dynamic profiles of
SARS-CoV RNA in plasma and antibodies against SARS-
CoV were simultaneously investigated in patients with
probable SARS. The viral RNA-positive stool and urine
samples from some patients were studied further for viral
vitality. The results obtained further the available knowl-
edge regarding the laboratory diagnostics and prophylactic
control of this emerging infection.
Materials and methods
Participants and specimens
A total of 486 clinical specimens from 54 patients with a
clinical diagnosis of SARS were tested for the presence of

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bothviralRNAandspecificantibodiesagainstSARS-CoV.
Thespecimensincluded213plasmasamples(isolatedfrom
citrate sodium-anticoagulated peripheral blood), 115 blood
cellsamples(remainingpartofthebloodfromtheplasma),
81 sputum/throat swab samples, 47 stool specimens, and
30 urine specimens. When possible, specimens were col-
lected weekly. Moreover, 101 plasma specimens from an
additional 19 patients were tested solely for the presence of
SARS-CoV antibody. The patients were all hospitalized in
ourunitfromMarchtoJune2003,theyallhadaclinicaldi-
agnosis of SARS, and all of them received ribavirin/steroid
combinationtherapy.Inaddition,plasmasamplesfrom278
healthy subjectswereanalyzed forthepresence ofantibod-
ies specific against SARS-CoV. All specimens were stored
at −70◦C until tested.
RNA extraction
RNA extraction was performed in the biosafety level 3
(P3) laboratory at the Beijing 302 Hospital. RNA was
extracted directly from plasma and urine samples. Sputum
samples were shaken for 30 min with an equal volume of
1.0% acetylcysteine and 0.9% sodium chloride, and the
supernatant was isolated by centrifugation (10,000g ×
3 min). Throat swabs and stool samples were suspended
with phosphate-buffered saline (PBS) containing 10 U/ml
RNasin(Promega,Madison, WI,USA),shakenfor10min,
and the supernatant was isolated by centrifugation as
mentioned above. Blood cells (from 1.8 ml of blood)
were treated with an equal volume of Trizol (Invitrogen,
Carlsbad, CA, USA), followed by isolating the aqueous
phase by centrifugation (12,000 g × 15 min). RNA was
extracted using the QIAmp Viral RNA Mini Kit (Qiagen,
Hilden, Germany) according to the manufacturer’s
instructions.
Nested RT-PCR
Two groups of nested primers were used for amplifying
the viral RNA. One set of BNI primers recommended by
the Word Health Organization was used to target the R
polyprotein encoding region (ORF1b) of SARS-CoV [17].
Forthis, theoutersense/antisense primerswere5?-ATGAA
TTACCAAGTCAATGGTTAC-3?/5?-CATAACCAGTCG
GTACAGCTA-3?, and the inner sense/antisense primers
were 5?-GAAGCTATTCGTCACGTTCG-3?/CTGTAGAA
AATCCTAGCTGGAG-3?, resulting in a 109-bp-long
fragment complementary to nucleotide 18182-18290 of
the BJ01 strain viral genome (GenBank accession no.
AY278488). Another set of primers was used to target
the N protein-encoding region of SARS-CoV designed
using sequence analysis software, MacVector (Accelrys,
San Diego, CA, USA). For this, the outer sense/antisense
primerswere5?-TCACTCAAGCATTTGGGAGACG-
3?/5?-TCCTTTTTAGGCTCTGTTGGTGGG-3?, and the
inner sense/antisense primers were 5?-TGCACAATTT
GCTCCAAGTGC-3?/5?TGCTTGTTCAGCAGTATGACG
TTG-3?, resulting in a 156-bp-long fragment complemen-
tary to nucleotide 29015-29170 of the BJ01 strain viral
genome. The one-step RT-PCR Kit (Qiagen) was used for
reverse transcription and the first round of PCR amplifi-
cation with outer primers. Thermal cycling consisted of
50◦C for 30 min; 95◦C for 15 min; 10 cycles at 95◦C for
15 s, at 57.5◦C for 15 s (decreasing by 1.5◦C every other
cycle), and at 72◦C for 30 s; 40 cycles at 95◦C for 15 s, at
56◦C for 15 s, and at 72◦C for 30 s. Afterwards, 2 µl of
the product was used as a template for the second round of
PCR amplification in 100-µl volume with inner primers
and Taq DNA polymerase (MBI Fermentas, Hanover, MD,
USA). Thermal cycling consisted of 30 cycles at 95◦C for
10 s, at 56◦C for 10 s, and at 72◦C for 20 s. All reactions
were carefully carried out to avoid contamination.
Positive and negative controls were included in each
reaction run. RT-PCR products were analyzed by elec-
trophoresis in 2.5% agarose gel containing 0.5 µg/ml of
ethidium bromide. To confirm the results, DNA in the elec-
trophoretic bands of the right size was recovered using the
QIAquick Gel Extraction kit (Qiagen) and cloned into a
pGEM-T vector system (Promega) for sequence analysis.
DNAsequenceswereobtainedwiththeuseofanautomated
ABI377 sequencer (Applied Biosystems, Foster City, CA,
USA). Analysis and comparison of nucleotides were car-
ried out with the DNAstar computer software (DNAstar,
Madison, WI, USA).
Antibody detection
Plasma
SARS-CoV were detected using two commercially
available ELISA kits (Jibiai Biotech, Beijing, China)
according to the manufacturer’s instructions. Indirect
assays were employed using both ELISA kits with
culture-obtained SARS-CoV antigen for the capture of
antibodies.
IgMand IgGantibodiesspecificagainst
Virus passage and isolation
These experiments were carried out in the P3 laboratory
at Beijing Genomics Institute, Chinese Academy of Sci-
ences. Stool was suspended in PBS. The supernatant was
isolated by centrifugation, sterilized by passing through
a 0.22 µm Millipore filter, and spread onto a monolayer
of Vero E6 cells in a 24-well plate. Urine was clarified
by centrifugation, sterilized, and spread onto the cells in
the same way. As a control, PBS was used instead of a
clinical specimen in triplicate wells. After incubation at
37◦C for 2 h, the infection solution was replaced with
fresh Dulbecco minimum essential medium with 5% fe-
tal calf serum (Gibco BRL, Grand Island, NY, USA).
The cultures were examined for cytopathic effect (CPE)
each day. At day 7, a blind passage was performed. The
cells were collected by vigorously blowing the monolayer
cells with the medium. The cell suspension was treated
with three rounds of freeze-thaw conditions, clarified

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167
by centrifugation, and spread onto fresh Vero E6 cells.
The blind passage was performed once each week for 3
weeks, and discarded if no CPE was observed. If CPE ap-
peared, cells were collected for electron microscopy obser-
vation and RT-PCR identification. The isolation of SARS-
CoV was confirmed by sequence analysis of the RT-PCR
products.
Results
One hundred and nine specimens from 28 of 54 SARS
patients were positive for SARS-CoV RNA by nested RT-
PCR, resulting in a 51.9% detection rate. The amplified
fragments were 109 and 156 bp in length and were con-
firmed to be parts of the SARS-CoV polymerase gene and
N gene, respectively, by sequence analysis (Fig. 1). Of 108
positive samples, 55 were double positive for both R and
N gene fragment amplification. Individual single-positive
amplification of R or N gene fragments was observed in 28
and 25 samples, respectively. Viral RNA was detectable in
42.1% (8/19) and 61.3% (19/31) of patients tested at week
1 and week 2 of illness, respectively. The detection profile
of viral RNA from different specimens is shown in Fig. 2.
Specimens obtained from the respiratory tract were more
likely than other specimens to yield viral RNA in the early
stage of illness, with seven of eight tested patients (7 of
12 tested specimens) positive at week 1. During the conva-
lescent phase of the infection (i.e., >4 weeks after disease
onset), viral RNA was detected in different specimens of
10 of 32 (31.3%) patients.
Figure 3 shows the dynamic seroconversion in cumu-
lative percentages of patients with probable SARS. From
week 1 to week 5 of illness, the rates of IgM-positive an-
tibody detection were 0% (0/22), 44.0% (22/50), 76.5%
(52/68), 81.9% (59/72), and 68.5% (50/73) per week,
respectively, while the rates for IgG antibody were 0%
(0/22), 38.0% (19/50), 72.1% (49/68), 84.7% (61/72), and
90.4% (66/73), respectively. The combined (IgM and/or
IgG) seroconversion rates were 0% (0/22), 54.0% (27/50),
82.4% (56/68), 88.9% (64/72), and 91.8% (67/73), re-
spectively. In contrast, only 1 of 278 (0.36%) healthy
subjects was positive for the SARS-CoV-specific IgG
antibody.
Fig. 1 Electrophoretic analysis of RT-PCR products for SARS-CoV
RNAamplifiedfromclinicalspecimens.Lanes1−6wereloadedwith
RT-PCR products; lane M was loaded with 100 bp DNA marker; 109
bp and 156 bp-long bands correspond to the amplified R (ORF 1b)
and N gene fragments of SARS-CoV, respectively
Fig. 2 Percent of positive results for SARS-CoV RNA detected
by RT-PCR in different clinical specimens of 54 patients with a
clinical diagnosis of SARS. Combined represents the percentage of
all patients from whom any of the specimens was positive for SARS-
CoV RNA. n represents the number of patients in whom SARS-CoV
was detected
Fig. 3 Cumulative percentage of seroconversion among 73 patients
with a clinical diagnosis of SARS. Combined represents the cumula-
tive percentage of patients demonstrating IgM and/or IgG serocon-
version
Becauseofthedifficultiesassociatedwithclinicalsample
collection, only 148 plasma samples could be collected
from25SARSpatientsduringthreeconsecutiveweeksand
tested for both SARS-CoV RNA and antibodies. Among
thesepatients,21haddetectableantibodies,and12hadone
or more specimens positive for viral RNA (Table 1). Two
patients (no. 1 and 10) were persistently positive for viral
RNA in plasma up to 5–7 weeks after disease onset. It is
notable that the coexistence of specific antibodies and viral
RNA was observed for as long as 3 weeks in these two
patients.
Twenty-one stool and urine specimens were employed
for in vitro isolation of SARS-CoV. Sputum specimens
were not employed because the use of acetylcysteine in
the viral RNA extraction process may have destroyed the

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Table 1 Serial detection of SARS-CoV RNA and antibodies against SARS-CoV in the plasma of 25 hospitalized patients with a clinical
diagnosis of SARS
Pt. no.Age (year) Sex Course (day)Manifestation
or result
Day after disease onset (IgM/IgG/RNA)
1–7 8–1415–2122–28 29–3536–42 43–49 50–56
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
20
27
42
59
39
33
51
26
32
31
19
31
42
13
45
46
33
38
21
20
48
9
45
32
18
M
M
F
M
M
M
M
M
M
F
F
M
M
F
F
F
M
F
M
M
M
M
M
M
M
77
34
73
73
41
59
59
41
30
47
59
50
49
34
31
36
41
36
67
38
38
36
30
47
46
Critical
General
Critical
Critical
General
General
Dead
General
General
Critical
General
Critical
Critical
General
General
Critical
Critical
General
Critical
General
Critical
General
General
General
General
ND
−/−/− +/+/−
ND
ND
−/−/− +/+/+
ND
ND
ND
−/−/− −/−/− −/−/− −/−/− −/−/−
−/−/− −/−/+
ND
−/−/+
ND
−/+/−
ND
−/−/− −/−/− +/+/−
−/−/− −/−/− −/−/− −/−/− −/−/−
−/−/− −/−/− +/−/−
−/−/− −/−/+
ND
−/+/−
−/−/− −/−/− +/−/−
−/−/− −/−/− +/+/−
−/−/− −/−/− −/−/− −/−/− −/−/− −/−/−
−/−/+
−/−/− −/−/+
ND
−/−/− −/+/−
−/−/+
ND+/−/−
ND
−/−/+
+/+/−
−/+/−
+/+/−
−/+/−
+/+/−
+/+/−
+/+/−
−/−/+
+/+/−
+/+/−
+/+/−
−/+/−
+/+/−
+/+/−
+/+/−
+/+/+
+/+/−
+/+/−
+/+/−
−/+/−
+/+/−
+/+/−
+/+/−
+/+/+ +/+/++/+/−
−/−/+
+/+/+
+/+/−
+/+/−
−/+/−
+/+/−
−/+/−
+/+/−
+/+/−
+/+/−
−/+/−
−/+/−
−/+/+
−/+/+
+/+/−
+/+/−
−/+/−
+/+/−
−/+/−
+/+/+
−/−/+
+/+/−
+/+/+
+/+/−
+/+/−
−/+/+
+/+/−
+/+/−
+/+/−
−/+/−
+/+/−
+/+/−
+/+/−
−/+/−
+/+/−
−/+/−
+/+/−
+/+/−
−/+/−
+/+/−
−/−/+
+/+/−
+/−/−
+/+/−
+/+/−
−/+/−
+/+/−
+/−/−
+/+/−
−/−/+
ND
+/+/−
+/+/−
−/−/−
+/+/−
+/+/−
ND
+/−/++/+/−
−/−/− −/−/+
+/+/−
+/+/−
−/−/− −/−/−
ND
+/+/−
+/+/−
+/+/−
−/+/−
+/+/−
+/+/−
+/+/++/+/−
+/−/−
+/+/−
+/+/−
ND
+/+/−
M, male; F, female; +, positive; −, negative; ND, not detected
Fig. 4 Observation of cytopathic effect (CPE) and SARS-CoV par-
ticles in Vero E6 cells infected with SARS-CoV isolated from the
stool sample of a convalescent-phase patient with SARS. a–b Light
microscopy images of Vero E6 cells without/with CPE. c Electron
microscopy image of Vero E6 cells with CPE showing the SARS-
CoV particles
protein structure of the virus. Plasma and blood specimens
were not employed because of the low viral load in these
samples. Nested RT-PCR showed that 16 of the 21 stool
and urine specimens were positive for viral RNA (10 for
both the R and N gene fragments; 6 for either the R or the
N gene fragment), and five were negative for viral RNA.
SARS-CoV was isolated from all ten of the specimens that
were positive for both the R and N gene fragments of viral
RNA after 2–3 generations of passage in culture and the
emergence of CPE and the virus in Vero E6 cells (Fig. 4);
identity was confirmed by RT-PCR followed by sequence
analysis. In contrast, the virus was not isolated from any of
the other 11 specimens (Table 2).
Discussion
As the area most affected by the SARS epidemic in the
world to date, Beijing claimed 2,521 cases and 191 deaths
from early March to late May 2003 [18]. Of these cases,
132 of the SARS patients were hospitalized and treated
in our unit at the Beijing 302 Hospital, including the
first cluster of case-patients in the area [19]. The crite-
ria for laboratory confirmation of SARS-CoV infection
are based on the following methods: SARS-CoV RNA
detection byRT-PCR, serological detection ofSARS-CoV-
relatedantibody,andisolationofSARS-CoVbycellculture
[11, 20].

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Table 2 Isolation of
SARS-CoV in urine and stool
samples by culture with Vero E6
cells
Pt. no.a
Sample no. Sampling day after
disease onset
RT-PCR results
(R gene/N gene)
Virus isolation
5 urine 94
stool 158
stool 131
urine 107
stool 159
stool 163
stool 124
urine 95
stool 154
stool 141
stool 156
urine 111
stool 126
stool 160
stool 145
urine 96
stool 142
stool 120
stool 144
urine 92
urine 89
8 d
8 d
11 d
36 d
29 d
23 d
26 d
21 d
24 d
33 d
10 d
6 d
21 d
11 d
33 d
11 d
3 d
18 d
12 d
22 d
14 d
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
−/+
+/−
−/+
+/−
+/−
+/−
−/−
−/−
−/−
−/−
−/−
+
+
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
−
6
11
24
16
15
21
9
20
2
12
22
+, positive; −, negative
aPatient numbers correspond
with those listed in Table 1
Several assays have been developed to detect viral
RNA using conventional nested RT-PCR and real-time RT-
PCR. Though real-time RT-PCR assays have a quantita-
tive advantage and are reported to have higher sensitiv-
ity/specificity insomecases,therequirementsofexpensive
reagents and special instruments are obstacles preventing
their widespread use in ordinary laboratories. During the
crucial time span of the SARS epidemic, a real-time RT-
PCR assay was still being developed. Therefore, conven-
tional nested RT-PCR still served as a practicable assay at
thetime.ThemethodusingBNIouter/innerprimerswasre-
portedtohavealowercostbutsimilarsensitivityandspeci-
ficity compared to three in-house real-time RT-PCR assays
[21]. We observed that simultaneous employment of the
primerstargetingbothR(ORF1b)andNregionsofSARS-
CoVcouldincreasethemethod’sdetectionsensitivity.This
finding is identical to that contained in a previous report by
Bressleretal.[16].TheseauthorssuggestedthattheNgene
is present in all of the sub-genomic RNAs of SARS-CoV
insteadofinonlyoneofthem(ORF1boftheRgene).How-
ever, we believe the major reason might be that most of the
SARS-CoV is not intact in clinical specimens. Thus, am-
plification of different regions of SARS-CoV will increase
the chance of detecting the disintegrated virus genome.
Nested RT-PCR may give occasional false-positive reac-
tions with stool specimens [15]. We tested 36 stool speci-
mensand24sputumspecimensfromhealthysubjectsusing
the assay. False-positive reactions were observed in four of
the stool specimens. Therefore, all positive results from the
stool specimens of the SARS patients were confirmed by
sequence analysis of the RT-PCR products. In addition, 64
of the 108 positive samples were amplified and sequenced
for the S gene of SARS-CoV, and we confirmed that all of
these samples contained S gene fragments of SARS-CoV
[22]. Employing various specimens may increase the de-
tection rate for viral RNA. In the present study, viral RNA
was detectable in 42.1% (8/19) and 61.3% (19/31) of pa-
tients with probable SARS during week 1 and week 2 of
illness, respectively. In contrast, specific antibodies against
SARS-CoV were detectable in 0% (0/22) and 50% (27/54)
of patients in the same respective weeks. Therefore, viral
RNAdetectionisparticularlyvaluableforearly-stagediag-
nostics. However,false-positiveresultshavetobecarefully
excluded, especially for stool specimens. Employment of
nasopharyngeal aspirate collected by professional physi-
ciansanduseofqualifiedreal-time RT-PCR assaysmayin-
crease the method’s sensitivity/specificity [13]. From week
2 following disease onset, serology is the gold standard for
identifying true-positive cases, since SARS-CoV is new to
humans and we commonly lack immunity against it.
In our study, 6 of 73 patients with a clinical diagno-
sis of SARS were negative for seroconversion, and two of
them were children (patients no. 14 and 22 in Table 1).
It is interesting, however, that viral RNA was detectable
in both of these patients (i.e., in 1 blood cell sample of
patient 14 and in 2 plasma samples of patient 22). Other
investigators have reported that about half of their pedi-
atric patients with a clinical diagnosis of SARS failed to
develop detectable antibodies specific against SARS-CoV
[23]. Weak humoral immune response in pediatric SARS
patients may be responsible for this unusual phenomenon.
Since our observation of four other adult patients who did
not seroconvert was terminated after 4–6 weeks of illness,
and seroconversion can still occur during weeks 7 and
8 of illness [24], one possible explanation for the nega-
tive antibody detection results may be a delayed antibody