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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,
1071-412X/01/$04.00⫹0 DOI: 10.1128/CDLI.8.2.314–319.2001
Mar. 2001, p. 314–319 Vol. 8, No. 2
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Antibody Responses to Campylobacter Infections Determined by
an Enzyme-Linked Immunosorbent Assay: 2-Year
Follow-Up Study of 210 Patients
METTE AAGAARD STRID,
1
JØRGEN ENGBERG,
1
LENA BRANDT LARSEN,
1
KAMILLA BEGTRUP,
2
KÅRE MØLBAK,
1,2
AND KAREN ANGELIKI KROGFELT
1
*
Department of Gastrointestinal Infections
1
and Department of Epidemiology Research,
2
Statens Serum Institut, DK-2300 Copenhagen S, Denmark
Received 14 July 2000/Returned for modification 19 October 2000/Accepted 11 December 2000
An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM,
and IgA classes of human serum antibody to Campylobacter jejuni and Campylobacter coli. Heat-stable antigen,
a combination of C. jejuni serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA
test. A total of 631 sera from 210 patients with verified Campylobacter enteritis were examined at various
intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative re-
sults. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respec-
tively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas
within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At
follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree
of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of
infection (up to 2 months from onset of infection). The antibody response did not depend on Campylobacter
species or C. jejuni serotype, with the important exception of response to C. jejuni O:19, the serotype most
frequently associated with Guillain-Barre´ syndrome. All of the patients infected with this serotype had higher
levels of both IgM (Pⴝ0.006) and IgA (Pⴝ0.06) compared with other C. jejuni and C. coli serotypes.
Together with Salmonella serovars, Campylobacter spp. are
the most common bacterial enteric pathogens in developed
countries, and Campylobacter jejuni is now the most recognized
antecedent cause of Guillain-Barre´ syndrome (15, 16, 21). In
Denmark, the incidence of registered Campylobacter infections
has increased markedly since 1992 (from 22 cases per 100,000
inhabitants in 1992 to 78 cases per 100,000 in 1999), and a
similar emergence of Campylobacter has been observed in oth-
er industrialized countries (6). The diagnosis of Campylobacter
infections is routinely done by stool culturing on selective me-
dium, and C. jejuni and Campylobacter coli account for 94 and
6%, respectively, of Danish human isolates (17). Furthermore,
culturing of stools is not a sensitive method for detection of the
bacteria in patients treated with antibiotics or in patients with
late reactive complications such as arthritis and Guillain-Barre´
syndrome or long-lasting intestinal distress (16). In these cases
and for epidemiological studies in general, serodiagnosis is
valuable. Antibodies to C. jejuni and C. coli can be detected in
several test systems with various sensitivities using a homolo-
gous strain or selected reference strains in crude antigen prep-
arations.
Agglutination and complement fixation (24, 25) and immu-
nofluorescence (4) tests have been used for serological diag-
nosis of C. jejuni infection, but these have been limited by low
sensitivity or specificity or the need to use homologous isolates.
Few attempts on an experimental basis have been made for the
development of enzyme immunoassays for detecting antibody
response to C. jejuni (3, 9, 10, 22, 23). They all found that the
quality of a diagnostic test relies mainly on the antigen prep-
aration used.
The objective of the present study was to establish a sensitive
and specific diagnostic serologic test for the demonstration of
immunoglobulin class-specific antibodies common to the most
prevalent strains of C. jejuni and C. coli in Denmark. Various
preparations of antigens from different C. jejuni serotypes were
tested in an enzyme-linked immunosorbent assay (ELISA).
Finally, a mixture of C. jejuni heat-stable antigens O:1,44 and
O:53 (18) was found to be suitable for the diagnosis of Campy-
lobacter infections in Denmark.
MATERIALS AND METHODS
Study population and serum samples. The study included 210 stool culture-
confirmed cases of Campylobacter infection from 1996 to 1997. All patients had
gastroenteritis, were from general practice, and had a median age of 33.5 years
(range, 10 to 76 years). Each person was asked to give a blood sample at
approximately 3 weeks, 3 months, 6 months, and 2 years after onset of symptoms.
All patients gave their written acceptance, and the Danish Central Scientific
Ethical Committee approved the project. To determine the cut-off for a negative
result, we included 162 negative sera from patients submitting blood samples for
Helicobacter pylori serology testing. As control for cross-reactions, sera from
patients found positive for H. pylori (n⫽39), Yersinia enterocolitica O:3 (n⫽39),
Salmonella enterica serovar enteritidis (n⫽21), S. enterica serovar Typhimurium
(n⫽9), S. enterica serovar Typhi (n⫽5), S. enterica serovar Paratyphi B (n⫽
1), and S. enterica serovar Manhattan (n⫽1), Legionella pneumophila (n⫽21),
and Escherichia coli O:157 (n⫽4) were examined against the selected Campy-
lobacter antigen. All antisera were supplemented with 0.01% sodium azide and
stored at ⫺20°C.
Identification and serotyping of isolates. Fecal samples were cultured on
CCDA substrate (18209 SSI Diagnostica, Hillerød, Denmark) and incubated in
a microaerobic atmosphere (85% N
2
,6%O
2
,3%H
2
, and 6% CO
2
) at 37°C and
* Corresponding author. Mailing address: Department of Gastro-
intestinal Infections, Division of Diagnostics, Statens Serum Insti-
tut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. Phone: 45 3268
3745. Fax: 45 3268 8238. E-mail: kak@ssi.dk.
314
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examined after 2 to 3 days. All isolates were identified as C. jejuni or C. coli by
conventional phenotypic tests (15). C. coli was distinguished from C. jejuni by a
negative sodium hippurate test. Serotyping of C. jejuni and C. coli was under-
taken by passive hemagglutination based on heat-stable antigens in microtiter
plates against 47 C. jejuni and 19 C. coli antisera as previously described (17).
Preparation of heat-stable antigen. A number of prevalent C. jejuni serotypes
in Denmark (17) were considered candidates for the ELISA antigen, including C.
jejuni O:1,44 (SSI:8133-96), O:2 (SSI:162-96), O:4 complex (SSI:36576-95), and
O:53 (SSI:16059-96). In addition, C. jejuni O:19 (SSI:9075-96) was examined as
candidate antigen. The bacteria were kept in bovine bouillon with 10% glycerol
at ⫺80°C until use. They were grown on 10% blood agar plates supplemented
with 5% yeast (686 SSI Diagnostica) in an atmosphere of 90% N
2
,5%O
2
, and
5% CO
2
at 37°C for 2 days. All were harvested with saline, boiled for1hat
100°C, and stored at ⫺20°C.
Following the ELISA procedure described below, 40 selected acute-phase sera
from culture-confirmed Campylobacter patients and sera from 40 negative con-
trols were used in the identification of the most appropriate antigen. The com-
bination of C. jejuni O:1,44 and O:53 antigens gave the best result, exhibiting a
difference between acute-phase sera from patients and controls larger than for a
single antigen (results available on request). The combined heat-stable antigen
had a protein concentration of 1 g/ml, measured by the Pierce BCA protein
assay (reagent 23225, 0194; Pierce, Rockford, Ill.) in the ratio 1:1 between O:1,44
and O:53, and stored at ⫺80°C.
ELISA procedure. Polysorb microwell plates (17106; Nunc, Roskilde, Den-
mark) were coated overnight at 5°C with 100 l of a solution of the described
antigen in coating buffer (0.1 M sodium carbonate [pH 9.6]) with a total protein
concentration of 1 g/ml. Plates were emptied and incubated for 15 min with
blocking buffer (phosphate-buffered saline [PBS] [pH 7.4] with 5% Tween 20)
and washed four times with PBS [pH 7.4] containing 0.1% Tween 20.
All test sera were diluted 1:400 in PBS containing 0.01% (wt/vol) sodium
azide. Test and control sera were applied in duplicate for 75 min at room
temperature, followed by four cycles of washing. Horseradish peroxidase-labeled
rabbit antiserum to human IgG (Dako 216; Dako, Glostrup, Denmark), IgM
(Dako 215; Dako), or IgA (Dako 214; Dako) was diluted 1:2,000, 1:1,000, and
1:500, respectively, in washing buffer, and 100 l was added to each well, fol-
lowed by another incubation for 75 min at room temperature before washing.
Finally, 100 l of tetramethylene benzidine (4380A; Kem-En-Tec, Copenhagen,
Denmark) substrate was added and incubated for 10 min. The reaction was
stopped by adding 100 lof0.2MH
2
SO
4
. The optical density (OD) was read as
arbitrary units (a.u.) at 450 nm, with background correction at 620 nm.
Selection of reference sera. To identify suitable control sera, 40 sera from
patients positive for Campylobacter antibodies and 164 sera from patients neg-
ative for H. pylori antibodies were measured against the heat-stable combination
antigen. Sera from patients positive for Campylobacter antibodies with OD values
between 1.2 and 2.2 a.u. were pooled and used as a positive control. Sera from
patients found negative for H. pylori with OD values for Campylobacter antibod-
ies below 0.25 a.u. were pooled and used as a negative control.
Calibration system. In order to control day-to-day variation, a positive control
serum diluted 1:200, 1:400, 1:600, 1:800, and 1:1,200 was included in duplicate on
each plate along with three blind wells. The mean value of the blind wells was
subtracted from all values, and the curve was evaluated by linear regression
analysis; the test was approved only if the correlation coefficient was above 0.95.
The mean ODs of the reference sera diluted 1:400, 1:600, and 1:800 in 10 assays
performed over 10 days were: IgG, 2.353, 2.184, and 1.960 a.u.; IgM, 1.454, 1.087,
and 0.816 a.u.; and IgA, 1.421, 0.993, and 0.702 a.u., respectively. The ODs of the
positive controls were adjusted in each experiment to fit the mean slope of the
titration curve for the reference sera, and the values of the test sera were
adjusted by the same relation.
Statistical methods. The mean antibody response following Campylobacter
infection was modeled in a generalized linear mixed model tailored for the
analysis of unbalanced repeated measurements, i.e., longitudinal data with vari-
able time of follow-up and variable intervals between measurements (7). Based
on an evaluation of model fit, we decided to model square-root-transformed ODs
by a separate piecewise linear function with knots at 4.5 months after infection
for IgG, 2 months for IgM, and 2.5 months as well as 7 months for IgA. Time
since infection, Campylobacter species, C. jejuni serotype, and age were used as
explanatory variables. To account for individual variability, we used a random-
effect model, supposing that the antibody response depends on some common
level of antibodies and supposing that a linear time trend exists for each person
but with a varying interpersonal random intercept. Maximum-likelihood meth-
ods were used for the regression analyses by applying the MIXED procedure of
the SAS software (SAS Institute, Cary, N.C.), and hypothesis testing was done by
likelihood ratio tests.
Different OD values corresponding to the 0.05, 0.25, 0.50, 0.75, 0.90, and 0.95
fractiles were evaluated as potential lower cut-off values for positive results. At
each different OD value, sensitivity was defined as the percentage of samples in
the true positive group that gave a value greater than the cut-off value. The
paired sensitivity and specificity, i.e., fractiles for cut-off, estimates were graph-
ically shown in a receiver-operating characteristic (ROC) curve (20).
RESULTS
Identification and serotyping of isolates. From the 210 pa-
tients included in the study, a total of 180 Campylobacter iso-
lates were available for further analysis; 173 (96%) were C.
jejuni and 7 (4%) were C. coli. The distribution of serotypes is
shown in Table 1. Most isolates reacted in only one serum or
in a combination of sera comprising well-known complexes,
e.g., O:1,44, O:4 complex, and O:6,7. Four C. jejuni isolates
reacted with two or more antisera, which were not within well-
known complexes but were O:2,38, O:3(13,50,65), O:10,44, and
TABLE 1. Distribution of serotypes isolated from
patients during this investigation
Serotype
a
No. of samples % of total
C. jejuni
1,44 20 11.6
2 34 19.7
4 complex
b
28 16.2
19 5 2.9
53 3 1.7
1 3 1.7
2,38 1 0.6
3 7 4.0
3 (13,50,65) 2 1.2
5 6 3.5
6,7 7 4.0
9 1 0.6
10 4 2.3
10,44 2 1.2
11 3 1.7
12 7 4.0
13 1 0.6
13,65 1 0.6
15 1 0.6
17 2 1.2
18 3 1.7
21 5 2.9
23,36 2 1.2
27 1 0.6
31 2 1.2
33 1 0.6
35 2 1.2
37 3 1.7
42 1 0.6
44 4 2.3
57 4 2.3
NT 4 2.3
UK 3 1.7
Total 173 100.0
C. coli
24,47 1 14
30 1 14
46 3 44
47 1 14
54 1 14
Total 7 100
a
NT, not typeable; UK, unknown.
b
Serotypes 4, 13, 16, 43, 50, and others.
VOL. 8, 2001 DETECTION OF CAMPYLOBACTER ANTIBODIES 315
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FIG. 1. Serum antibody response to Campylobacter infection in patients. (A) IgG; (B) IgM; (C) IgA. Individual responses of 210 patients over
a 2-year period according to immunoglobulin class and the fitted population average (bold line) are shown.
316 STRID ET AL. CLIN.DIAGN.LAB.IMMUNOL.
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O:13,65, as did one of the C. coli, O:24,47. The most common
C. jejuni serotypes were O:2, O:4 complex, and O:1,44, ac-
counting for 49% of the cases, whereas the other serotypes
each represented 4.2% or less.
Antibody response by ELISA. In the 164 negative control
sera, IgG antibody values ranged from 0.08 to 2.43 a.u. (me-
dian, 0.73 a.u.), IgM ranged from 0.004 to 1.33 a.u. (median,
0.29 a.u.), and IgA ranged from 0.002 to 0.95 a.u. (median,
0.10 a.u.). The 90th percentile for IgG, IgM, and IgA was 1.49,
0.56, and 0.22 a.u., respectively. A total of 631 measurements
were taken among the 210 patients; Fig. 1 shows the antibody
response profiles at follow-up as well as the fitted population
average for each immunoglobulin class.
IgG antibody values (Fig. 1A) decreased from the acute
phase of infection up to 4.5 months after infection. The mean
value continued to decrease in the follow-up period but very
slowly around an OD of 1.0 a.u., i.e., between the 75th and 90th
percentiles of negative sera. However, the curves exhibited a
very large individual variation between patients, and some
individuals had high values throughout the follow-up period,
whereas others remained at low levels. There was no significant
effect of age for this immunoglobulin class.
In Fig. 1B, the IgM antibody values, which were raised in the
first 2 months following infection, are shown. At follow-up
more than 2 months after the infectious event, the mean re-
sponse remained stable at about 0.4 a.u. The IgM response was
highest among the youngest patients and decreased with in-
creasing age of infection (P⫽0.0001). Thus, compared with
patients older than 45 years, individuals in the age range from
10 to 25 years had 0.08 a.u. (95% confidence interval [CI], 0.04
to 0.13), 26 to 35 years had 0.04 a.u. (95% CI, 0.01 to 0.08), and
36 to 45 years had 0.03 a.u. (95% CI, 0.01 to 0.08) higher
values. The proportion of patients with an IgM value above
cut-off at first sampling by age group is presented in Table 2.
In Fig. 1C, the IgA antibody response, which declined rap-
idly from the acute phase to 2.5 months following infection, is
shown. At later follow-up, the values were almost uniformly
low. The IgA response was independent of age.
Effect of serotype. The association between antibody re-
sponse and Campylobacter species or C. jejuni serotype was
assessed by categorizing typing results in five groups, C. coli;C.
jejuni (all serotypes); common C. jejuni serotypes (O:1,44, O:2,
and O:4 complex); uncommon C. jejuni serotypes; and unclas-
sified serotypes (not typeable and unknown). The assessed
antibody response was independent on these major groups
(P⬎0.4 for all three antibody classes). However, by compar-
ing the response to C. jejuni O:19 against other classified se-
rotypes, the five O:19 patients had higher IgA values (OD,
0.03 a.u.; 95% CI, 0.00 to 0.10; P⫽0.06) and IgM values (OD,
0.07 a.u.; 95% CI, 0.01 to 0.22; P⫽0.006, adjusted for age). In
addition, patients with C. jejuni O:19 had 0.01 a.u. (95% CI,
⫺0.02 to 0.10; P⫽0.48) higher IgG values than others. The
results were essentially the same when the unclassified types
were lumped together with the group of other classified types
and included in the analyses.
Cross-reactions. Sera from patients with infection due to
other microorganisms causing gastrointestinal infections were
assayed for antibodies against C. jejuni using the ELISA. Sera
from 39 patients positive for H. pylori were tested for cross-
reactions against heat-stable combination antigen, since H.
pylori is phylogenetically the most closely related bacterium to
Campylobacter. By using the cut-offs described above for IgG,
IgM, and IgA, we found one, four, and six positive samples,
respectively. Four sera from patients with E. coli O:157 infec-
tion were all found negative. Three groups of 39 sera each from
patients with Yersinia O:3, Salmonella, and Legionella infec-
tions were assayed, and we found zero, eight, and three; two,
four, and two; and zero, zero, and three positive sera, respec-
tively, according to the cut-offs.
Determination of sensitivity. By using the 90th percentile of
the negative sera as a cut-off and thus obtaining a specificity of
at least 90%, Campylobacter infection could be detected with a
sensitivity of 71% using IgG, 60% using IgM, and 80% using
IgA within 35 days after infection. By combining all three
antibody classes, the sensitivity was 92%, and after 3 months
(90 days) from infection, the combined sensitivity was 90%
(IgG, 68%; IgM, 52%; and IgA, 76%). In Fig. 2, ROC curves
representing the diagnostic value (sensitivity and specificity) of
our test at different times after infection are presented.
DISCUSSION
The aim of this study was to establish an ELISA suitable for
a general screening of Campylobacter infections. The assay
should be sensitive for the most prevalent C. jejuni and C. coli
serotypes and have few or no cross-reactions with other gen-
era.
C. jejuni serotypes O:1,44 and O:53 used as the antigen
represent the most common serotypes (18%) and the seventh
most common (3%) in Denmark (17). The evaluation of this
antigen combination showed that the measured antibody re-
sponse was independent of the major groups of C. jejuni sero-
types and that the assay was also suitable for detecting anti-
bodies against C. coli. The serotype distribution of the 177
isolates in this study was in accordance with earlier studies in
Denmark (17). The study included five patients with C. jejuni
O:19 infections, and these patients had significantly higher
levels of IgM and IgA than others did. C. jejuni O:19 is asso-
ciated with the most serious disease caused by Campylobacter,
Guillain-Barre´ syndrome (11, 26). The high levels of IgM and
IgA in C. jejuni O:19 patients may be related to a higher affinity
of the test for antibodies against this serotype than others.
However, O:19 was not chosen for the antigen preparation,
and it is therefore more likely that the high antibody levels
reflect the high immunogenicity of C. jejuni O:19 rather than
an increased affinity of the ELISA against this serotype. This
view is in line with observations made by Rautelin et al. (19)
and warrants further studies. Allos and colleagues have re-
cently shown that C. jejuni O:19 strains, regardless of Guillain-
Barre´ syndrome association, are more serum resistant than
TABLE 2. Number of IgM-positive samples at first examination
according to patient’s age, determined 8 to 72 days after infection
a
Age (yr) No. of samples tested No. (%) positive
10–25 59 47 (79.7)
26–35 57 41 (71.9)
36–45 36 23 (63.9)
ⱖ46 58 12 (20.7)
a
Chi squared for linear trend in proportions, 42.58; P⬎0.001.
VOL. 8, 2001 DETECTION OF CAMPYLOBACTER ANTIBODIES 317
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non-O:19 strains (1). Our findings corroborate the hypothesis
that the elevated immunologic response induced by O:19
strains leads to injury of peripheral nerve structures. None of
the five patients with infections caused by C. jejuni serotype
O:19 developed Guillain-Barre´ syndrome.
It was not possible to investigate sera from infections caused
by Campylobacter species other than C. jejuni and C. coli,asthe
current method for the diagnosis of Campylobacter enteritis
is culturing of fecal samples on selective medium adjusted to
these species. Furthermore, a recent study suggests that C.
fetus, C. lari, and C. upsaliensis are minor causes of Campy-
lobacter enteritis in Denmark (8).
FIG. 2. ROC curves depicting the sensitivity and specificity of Campylobacter serodiagnosis depending on antibody class tested (IgG, IgM, and
IgA) and time since infection. The diagnostic sensitivity at different levels of specificity is shown at 0 to 2 months (solid circles), 3 to 5 months (open
circles), 6 to 11 months (open diamonds), and 12 to 23 months (open squares) after infection.
318 STRID ET AL. CLIN.DIAGN.LAB.IMMUNOL.
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The prepared crude antigen showed no cross-reactions
based on a 90th percentile towards the phylogenetically most
closely related bacterium H. pylori and the other bacteria ex-
amined. Earlier works have reported cross-reactions between
C. jejuni and H. pylori when using sonicated antigen (12).
Cross-reactions between Salmonella serovars Typhi and Para-
typhi and C. jejuni have been reported when the antigen used
was based on the flagellar protein (13). Finally, cross-reactions
of the IgM class between Legionella pneumophila and C. jejuni
based on formalin-treated antigen have been reported (5).
Class-specific antibody response profiles were determined
over a period of 2 years based on a large number of patients.
The IgA response was associated with acute infection. The
IgM response was highest in the younger age groups, and there
was a significant association between age at infection and IgM
response (Table 2). Thus, serodiagnosis based on IgM re-
sponse is a particularly useful tool for young patients. It is
likely that a considerable proportion of elderly persons have a
secondary antibody response without IgM elevation. IgG levels
were highly individually variable, and this large variability of
IgG response was also reflected by a large variation in IgG
levels among the negative controls (data not presented). At a
90% specificity, 71, 60 and 80% of acute infections could be
detected by IgG, IgM, and IgA, respectively, in a single con-
valescent-phase sample. By using a combination of all three
antibody classes, sensitivity was 92 and 90% within the first 35
days and within 3 months after infection, respectively. Further-
more, the ROC curves show that approximately 50% of the
infections can be detected serologically up to 1 year postinfec-
tion.
Blaser and coworkers (3) used a glycerol-HCl-extracted sur-
face antigen of serotypes O:1, O:2, and O:3 in their ELISA.
For IgG antibodies, the test had a specificity of 74% and 59%
sensitivity. For IgM, the specificity was 68% and the sensitivity
was 74%, and for IgA the specificity was 81% and the sensi-
tivity was 76%. Thus, the IgA ELISA was the most specific and
sensitive assay in their study to detect an acute C. jejuni infec-
tion on the basis of a single convalescent-phase serum speci-
men. This observation is confirmed in the present study, al-
though our ELISA is more specific and sensitive than those
described previously (2, 3, 10, 23).
We conclude that measurement of Campylobacter antibodies
is a useful diagnostic tool and can also be used for seroepide-
miological studies. The high IgM and IgA antibody response
against C. jejuni O:19 is of particular interest and corroborates
the notion that this C. jejuni serotype, which is frequently
associated with Guillain-Barre´ syndrome, is highly immuno-
genic.
ACKNOWLEDGMENTS
The Danish Central Scientific Ethical Committe approved the
project. We thank Eva Møller Nielsen, Danish Veterinary Laboratory,
for assistance on serotyping and for providing the sera used for sero-
typing. Special thanks to doctors in general practice and patients with-
out whose kind participation this study would not have been possible.
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