Journal of Hepatology 44 (2006) 1066–1073

www.elsevier.com/locate/jhep

Cholangiocarcinoma and Clonorchis sinensis

infection: A case–control study in Korea
Dongil Choi1, Jae Hoon Lim1,*, Kyu Taek Lee2, Jong Kyun Lee2, Seong Ho Choi3,
Jin Seok Heo3, Kee-Taek Jang4, Nam Yong Lee5, Seonwoo Kim6, Sung-Tae Hong7
1
Department of Radiology and Center for Imaging Science, Samsung Medical Center, Sungkyunkwan
University School of Medicine, 50, Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea
2
Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan
University School of Medicine, 50, Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea
3
Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine,
50, Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea
4
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine,
50, Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea
5
Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine,
50, Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea
6
Biostatistics Unit, Samsung Biomedical Research Institute, Samsung Medical Center, 50, Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea
7
Department of Parasitology and Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul 110-799, South Korea

Background/Aims: The authors conducted a hospital-based case–control study to evaluate the role of Clonorchis
sinensis infection as a risk factor for the development of cholangiocarcinoma (CC), including extrahepatic CC, in
Korea.
Methods: Cases of 185 patients with CC (intrahepatic, 51; hilar, 53; and distal extrahepatic, 81) and matched controls
underwent stool microscopy, pathological examinations, serologic test for C. sinensis using ELISA, skin test for
C. sinensis, radiologic examinations, and interview concerning history of eating raw freshwater fish.
Results: Radiologic evidence of C. sinensis, history of eating raw freshwater fish, and positive serologic result for
C. sinensis were found to be related to an increased risk of CC, with the odds ratios (OR)Z8.615 (95% confidence
interval [CI]Z5.045–16.062), ORZ2.385 (95% CIZ1.527–3.832), and ORZ2.272 (95% CIZ1.147–4.811), respect-
ively. The risk factors for distal extrahepatic CC were radiologic evidence of C. sinensis (ORZ6.571; 95% CIZ3.170–
15.943) and history of eating raw freshwater fish (ORZ2.6; 95% CIZ1.294–5.66).
Conclusions: Radiologic evidence of C. sinensis, history of eating raw freshwater fish and positive serologic result for
C. sinensis were significantly associated with CC, including extrahepatic CC.
q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Cholangiocarcinoma; Clonorchis sinensis; Risk factors; Case–control studies

1. Introduction

There is a wide variation in the incidence of cholangio-

carcinoma (CC) in different parts of the world, which is
Received 6 July 2005; received in revised form 15 November 2005; much higher in East Asia than in Western Europe and
accepted 16 November 2005; available online 27 December 2005
* Corresponding author. Tel.: C82 2 3410 2501x2518; fax: C82 2 3410 America. This variation is believed to be associated with the
2559. distribution of the risk factors for this disease. There are
E-mail address: jhlim@smc.samsung.co.kr (J.H. Lim). several documented risk factors for CC, including primary
0168-8278/$32.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jhep.2005.11.040
 D. Choi et al. / Journal of Hepatology 44 (2006) 1066–1073 1067

sclerosing cholangitis, liver fluke (Opisthorchis viverrini
and Clonorchis sinensis) infection, bile duct stones,
thoratrast exposure, and choledochal cysts [1,2]. Some
researchers have also suggested that chronic liver disease
due to viral hepatitis was a potential risk factor for
intrahepatic CC [2–4]. A few reports recorded epidemiolo-
gical evidences of a correlation between CC and C. sinensis
infection, but they only dealt with intrahepatic CC and were
not well organized [2,5–7].
In order to assess the role of C. sinensis in the risk of
developing CC, including extrahepatic CC, we analyzed the
data from a hospital-based case–control study conducted in
Korea, a country, which still has multiple endemic regions.
2. Materials and methods
2.1. Subjects
This case–control study was conducted in a major teaching hospital
located in Seoul, Korea. The patients in this hospital were referred from
other hospitals located throughout Korea. One hundred and ninety eight
patients with CC visiting for the first time between 2003 January and 2004
December were identified. Thirteen of these patients were excluded
because of coincident stones in the bile ducts (nZ6), very severe illness
(nZ5), and combined CC and hepatocellular carcinoma (nZ2), so that 185
patients with CC were finally included in this study. Of these 185 patients,
136 were verified by histopathology (nZ106) and cytology (nZ30). The
remaining 49 patients were considered to have CC on the basis of their
radiologic findings and elevated serum tumor marker (CA 19-9O100 U/L),
and the absence of both cholangitis and significant biliary obstruction [8].
There were 51 patients with intrahepatic CC, 53 patients with hilar CC, and
81 patients with distal extrahepatic CC. There were 115 men and 70 women
(median age, 64 years; range, 32–79 years).
The controls were selected from patients admitted to the same network
for non-hepatobiliary diseases in the Department of Gastroenterology of the
same hospital. They were matched to the cases by age (G3 years), sex,
admission date (G2 months), and geographic area (i.e. residing in a rural
area over the past 10 years). The controls consisted of 115 men and 70
women (median age, 64 years; range, 31–79 years) admitted for alcohol-
induced pancreatitis (nZ79, 42.7%), pancreatic tumor including cancer
(nZ52, 28.1%), infection or inflammation in the gastrointestinal tract (nZ
24, 13.0%) tumor of the gastrointestinal tract (nZ20, 10.8%), and other
miscellaneous illnesses (nZ10, 5.4%). Patients who had been admitted for
hepatic tumors, liver cirrhosis, chronic or acute hepatitis, biliary stones,
cholangitis, cholecystitis, gall bladder cancer, or ampulla of Vater cancer
were excluded from the control group. Patients were excluded if there were
no available images of contrast-enhanced CT including the liver. Eligible
participants had to be in good physical and mental condition in order to
provide reliable answers. The institutional review board approved this
study, and we obtained informed consent from each patient.
2.2. Diagnostic methods for C. sinensis infection
Among the cases and controls, a total of 304 patients (82.2%)
underwent a stool microscopy to detect C. sinensis eggs using the
formalin–ether sedimentation technique. Pathological specimens or bile
juice were obtained from 150 patients (81.1%) with CC and 102 controls
(55.1%). The pathological specimens were used for routine histopatholo-
gical examinations. When we observed either eggs or worms in the
pathological specimens or bile juice, we considered that the patients had
pathological evidence of C. sinensis.
Among the cases and controls, 349 patients (94.3%) underwent a
serologic test. Specific serum antibodies to C. sinensis were screened using
an enzyme-linked immunosorbent assay (ELISA) [9]. The antigen was
diluted 1:400, the sera were diluted 1:100, and the conjugate (IgG Whole
Molecule, Anti-Human; MP Biomedicals, Aurora, Ohio, USA) was diluted

Table 1
Distribution of the 185 cases of cholangiocarcinoma and 185 matched controls and corresponding ORs with 95% CI, according to diagnostic methods
for Clonorchis sinensis: Korea, 2003–2004

Characteristics Cases (nZ185) no. (%) Controls (nZ185) no. (%) Odds ratioa (95%CI)
b
Stool microscopy for C. sinensis
Positive 3 (2.5) 5 (4.1) 0.6 (0.123–2.445)
Negative 119 (97.5) 117 (95.9) Reference
Pathologic examinations for C. sinensisb
Positive 13 (17.6) 8 (10.8) 1.625 (0.685–4.105)
Negative 61 (82.4) 66 (89.2) Reference
Serologic test for C. sinensisb,c
Positive 25 (15.2) 11 (6.7) 2.272 (1.147–4.811)
Negative 139 (84.8) 153 (93.3) Reference
Skin test for C. sinensisb
Positive 19 (13.8) 12 (8.7) 1.7 (0.791–3.852)
Negative 119 (86.2) 126 (91.3) Reference
Radiologic examinations for C. sinensis
Positive 156 (84.3) 57 (30.8) 8.615 (5.045–16.062)
Negative 29 (15.7) 128 (69.2) Reference
History of eating raw freshwater fish
Ever 94 (50.8) 58 (31.4) 2.385 (1.527–3.832)
Never 91 (49.2) 127 (68.6) Reference
Any evidence for C. sinensisd
Positive 167 (90.3) 92 (49.7) 7.250 (3.965–13.257)
Negative 18 (9.7) 93 (50.3) Reference

OR, odds ratio; 95% CI, 95% confidence intervals.

a
Estimates from multiple conditional logistic regression equations.
b
The sum does not add up to the total because of some missing values.
c
Serologic test performed by using enzyme-linked immunosorbent assay (ELISA) technique.
d
If at least one of the six examinations for C. sinensis is positive.
1068 D. Choi et al. / Journal of Hepatology 44 (2006) 1066–1073

1:5000. Diaminobenzidine was used as the substrate for the color reaction CIZ5.045–16.062) (Figs. 1 and 2), a history of eating raw
and the absorbance was read at 490 nm using an ELISA reader
(EmaxwPrecision Microplate Reader, Molecular Devices Corp., Calf,
freshwater fish (ORZ2.385; 95% CIZ1.527–3.832) and a
USA). The presence of surface antigens of hepatitis B and antibodies (IgG) positive serologic result for C. sinensis (ORZ2.272; 95%
of hepatitis C was examined in order to detect liver diseases related to viral CIZ1.147–4.811). The numbers of cases with pathological
hepatitis B and C, respectively.
Among the cases and controls, 321 patients (86.8%) underwent a skin evidence of C. sinensis and a positive skin test for
test with an intradermal injection of 0.02 mL of the 1:10,000 diluted crude
antigen of C. sinensis in veronal buffered saline (C. sinensis antigen for skin
test; Shinpoong Co. Ltd, Seoul, Korea) into their forearm [10]. Twenty
minutes after the injection, the presence of wheals with an average diameter
of 60 mm2 or greater on the forearm was considered as a positive result.
All of the patients underwent contrast-enhanced helical CT. Cholangio-
graphic imaging, including magnetic resonance cholangiography, was
obtained in 132 patients (71.4%), and ultrasonography was performed in
112 patients (60.5%). Three experienced, abdominal radiologists who were
blinded to the results of other diagnostic methods, determined the evidence
of C. sinensis infection and made their diagnosis by consensus for each
patient. On the basis of the radiologic finding of diffuse dilatation of the
peripheral intrahepatic bile ducts, C. sinensis infection was considered to be
present if there was either mild or moderate dilatation of the intrahepatic
bile ducts up to the periphery of the liver without dilatation of the segmental
or lobar intrahepatic ducts, or disproportionately more severe dilatation of
the peripheral intrahepatic ducts proximal to the bile duct obstruction
by CC [11]. The pattern of biliary tree dilation was judged with CT,
cholangiography and ultrasonography, or a combination of these findings.
During the admission period of cases and controls, a trained interviewer
used a structured questionnaire to collect data regarding the cases and
controls, concerning their social characteristics, including areas and
duration of residence in the past, and their history of eating raw freshwater
fish, including the time and frequency, the number of occasions and the
amount they ate, and the species of freshwater fish consumed. The study
coordinator and medical doctors made every effort to keep the interviewer
blind to the clinical diagnosis of the participants. After a thorough review of
the questionnaire filled out by the interviewer, an experienced physician
with no knowledge of any other information concerning the patient,
determined whether or not he or she had a significant history of eating raw
freshwater fish. The patient was considered to have a significant history
of eating raw freshwater fish if he or she met all of the following criteria:
(1) one or more definite experiences of eating raw freshwater fish, (2) the
amount consumed was more than a single mouthful, and (3) one or more
of the freshwater fish consumed were known to be intermediate hosts of
C. sinensis [12].

2.3. Statistical analysis

Odds ratios (ORs) of CC, together with their corresponding 95%

confidence intervals (95% CIs), in relation to stool microscopy,
pathological evidence, serologic test for C. sinensis, skin test for
C. sinensis, radiologic evidence, and a significant history of eating raw
freshwater fish were derived using a matched conditional multiple logistic
regression analysis of the complete pairs with the use of SAS software
(version 8.2 for Windows; SAS Institute, Cary, NC, USA). We also
obtained the ORs of intrahepatic CC, hilar CC, and distal extrahepatic CC.
For intrahepatic CC, the ORs associated with the serologic evidence of viral
hepatitis B and C were assessed. By using Fisher’s exact test with the
permutation method for multiple testing, we evaluated the differences in the
positive rates of the examinations among the three kinds of CC. The k
statistics were used to assess the correlations (agreements) for C. sinensis
among the six different examinations. The degree of agreement was
categorized as follows: k values of 0.00–0.20 were considered to indicate Fig. 1. Cholangiocarcinoma (CC) in the mid common duct, with
poor agreement; k values of 0.21–0.40, fair agreement; k values of 0.41– clonorchiasis, in a 60-year-old man. Pathology reveals a 2.4-cm well-
0.60 moderate agreement; k values of 0.61–0.80, good agreement; and k differentiated tubular adenocarcinoma after segmental resection of the
values of 0.81–1.00, excellent agreement [13].
common duct. Clonorchis sinensis eggs were found in both the stool and
bile juice of the patient. He had many episodes of eating raw freshwater
fish about 30 years ago. (A) Contrast-enhanced CT shows a diffuse,
3. Results mild intrahepatic ductal dilatation (arrows) caused by C. sinensis
infection. (B) CT at a level lower than (A) shows the enhancing,
segmental thickened wall (arrows) of the mid common duct, which
Table 1 presents the distribution of the 185 cases of CC, represents an extrahepatic CC. (C) Endoscopic retrograde cholangio-
and of the matched control group. The risk factors for CC graphy shows complete obstruction of the common duct (arrow) and
were radiologic evidence of C. sinensis (ORZ8.615; 95% diffuse dilated intrahepatic ducts (arrowheads).
 D. Choi et al. / Journal of Hepatology 44 (2006) 1066–1073
Fig. 2. Cholangiocarcinoma (CC) in the mid common duct, without
clonorchiasis, in a 64-year-old woman. Pathology reveals a 1.5-cm
papillary adenocarcinoma after segmental resection of common duct.
She had no evidence of C. sinensis infection. (A) Contrast-enhanced
CT shows mild dilatation of the central intrahepatic duct (arrow-
heads), but the peripheral intrahepatic ducts are not dilated. (B) CT
at a level lower than (A) shows the enhancing, segmental thickened
wall (arrows) of the mid common duct and a suspicious intraluminal
mass, which represents an extrahepatic CC. (C) MR cholangiography
shows an intraluminal tumor (arrows) at the level of the mid
common duct.
C. sinensis were slightly higher than the corresponding
numbers of controls, however these differences were not
statistically significant. Using as an evidence of C. sinensis
when at least one of the six examinations was positive,
C. sinensis infection was a risk factor for CC (ORZ7.250;
95% CIZ3.965–13.257).
1069
Table 2 presents the distribution of the 51 cases of
intrahepatic CC, and of the matched control group.
Radiologic evidence of C. sinensis was the only risk
factor for intrahepatic CC (ORZ4.999; 95% CIZ1.05–
3.82). The numbers of cases with a positive serologic test
results for C. sinensis and a history of eating raw
freshwater fish were slightly higher than the correspond-
ing numbers of controls, however, these differences were
not statistically significant. Using as an evidence of
C. sinensis when at least one of the six examinations was
positive, C. sinensis infection was a risk factor for
intrahepatic CC (ORZ4.000; 95% CIZ1.501–10.657).
With regard to the serologic tests, neither viral hepatitis B
(ORZ0.8) nor viral hepatitis C (ORZ1.0) was found to
be related to the risk of intrahepatic CC.
Table 3 presents the distribution of the 53 cases of
hilar CC, and of the matched control group. The risk
factors for hilar CC were radiologic evidence of
C. sinensis (ORZ41.0; 95% CIZ8.933–726.8), a history
of eating raw freshwater fish (ORZ3.143; 95% CIZ
1.411–7.950), a positive serologic result for C. sinensis
(ORZ4.999; 95% CIZ1.318–32.526) and a positive skin
test for C. sinensis (ORZ4.5; 95% CIZ1.160–29.514).
Using as an evidence of C. sinensis when at least one of
the six examinations was positive, C. sinensis infection
was a risk factor for hilar CC (ORZ16.000; 95% CIZ
3.834–66.763).
Table 4 presents the distribution of the 81 cases of distal
extrahepatic CC, and of the matched control group. The risk
factors for distal extrahepatic CC were radiologic evidence
of C. sinensis (ORZ6.571; 95% CIZ3.170–15.943) and a
history of eating raw freshwater fish (ORZ2.6; 95% CIZ
1.294–5.66). The numbers of cases with pathological
evidence of C. sinensis, a positive serologic test result for
C. sinensis and a positive skin test for C. sinensis were
slightly higher than the corresponding numbers of controls,
however these differences were not statistically significant.
Using as an evidence of C. sinensis when at least one of the
six examinations was positive, C. sinensis infection was a
risk factor for distal extrahepatic CC (ORZ7.000; 95%
CIZ2.742–17.867).
Among the three types of CC, viz. intrahepatic, hilar, and
distal extrahepatic, there were no significant differences in
the positive rates of stool microscopy, pathological
examinations, serologic test for C. sinensis, skin test for
C. sinensis, radiologic examinations, or a history of eating
raw freshwater fish.
Most of the k values for agreements among the six
different examinations showed fair, moderate and good
agreements (range, 0.212–0.669) [13]. However, three k
values showed poor agreements: k value of stool
microscopy and radiologic examinations in the cases was
0.125, k value of stool microscopy and history of eating raw
freshwater fish in the cases was 0.073, and k value of
pathological examinations and radiologic examinations in
the controls was 0.188.
1070 D. Choi et al. / Journal of Hepatology 44 (2006) 1066–1073

Table 2
Distribution of the 51 cases of intrahepatic cholangiocarcinoma and 51 matched controls and corresponding ORs with 95% CI, according to
diagnostic methods for C. sinensis, hepatitis B virus and hepatitis C virus: Korea, 2003–2004

Characteristics Cases (nZ51) no. (%) Controls (nZ51) no. (%) Odds ratioa (95%CI)
Stool microscopy for C. sinensisb
Positive 0 3 (9.1) Not availablec
Negative 33 (100) 30 (90.9) Reference
Pathologic examinations for C. sinensisb
Positive 0 1 (5.3) Not availablec
Negative 19 (100) 18 (94.7) Reference
Serologic test for C. sinensisb
Positive 7 (15.6) 4 (8.9) 1.75 (0.529–6.680)
Negative 38 (84.4) 41 (91.1) Reference
Skin test for C. sinensisb
Positive 5 (12.8) 6 (15.4) 0.8 (0.198–3.023)
Negative 34 (87.2) 33 (84.6) Reference
Radiologic examinations for C. sinensis
Positive 36 (70.6) 16 (31.4) 4.999 (2.080–14.814)
Negative 15 (29.4) 35 (68.6) Reference
History of eating raw freshwater fish
Ever 22 (43.1) 17 (33.3) 1.556 (0.682–3.734)
Never 29 (56.9) 34 (66.7) Reference
Any evidence for C. sinensis
Positive 42 (82.4) 27 (52.9) 4.000 (1.501–10.657)
Negative 9 (17.6) 24 (47.1) Reference
Surface antigen of hepatitis B virus
Positive 4 (7.8) 5 (9.8) 0.8 (0.198–3.023)
Negative 47 (92.2) 46 (90.2) Reference
Antibody of hepatitis C virus
Positive 1 (2.0) 1 (2.0) 1.00 (0.040–25.264)
Negative 50 (98.0) 50 (98.0) Reference

OR, odds ratio; 95% CI, 95% confidence intervals.

a
Estimates from multiple conditional logistic regression equations.
b
The sum does not add up to the total because of some missing values.
c
Not available to perform statistics (OR calculation).

4. Discussion is still unknown [12,18]. The carcinogenesis associated with

C. sinensis infects more than 20 million people in East
Asia, including China, east Russia, Korea, and Vietnam [12].
In 1956, Hou reported that C. sinensis flukes were found in
58% of patients with intrahepatic CC autopsied in Hong
Kong [14]. Some other studies in endemic areas have also
reported high incidences of concurrent intrahepatic CC and
clonorchiasis (C. sinensis infection) [15–17]. The incidence
of CC is significantly higher in endemic areas, which are
generally areas situated along the banks of a river, than in
non-endemic areas, even within the same country [5,6,12]. A
case–control study in Busan (a city surrounded with highly
endemic regions) in Korea reported a significant relationship
between the presence of C. sinensis eggs in the stool and
intrahepatic CC (41 patients with CC; relative riskZ2.7;
95% CIZ1.1–6.3) [7]. Several prior epidemiological reports
in Hong Kong and Korea reported an association between
C. sinensis infection and intrahepatic CC, but not extra-
hepatic CC [5–7,14]. We believe that the present epidemio-
logical study is the first to find evidence of an association
between C. sinensis infection and extrahepatic CC.
Though C. sinensis is believed to be a probable
carcinogen for CC, its precise mechanism of carcinogenesis
clonorchiasis may be a cumulative result arising from
multiple factors [19,20]. Chronic irritation and bile
contamination in the bile ducts due to C. sinensis worms
themselves and their secretion is considered an essential
factor in carcinogenesis [20,21]. An experimental study
suggested that activation (initiation) by N-nitrosodimethy-
lamine, and subsequently epithelial proliferation (pro-
motion) by C. sinensis, stimulated the primitive oval cells
to transform into carcinomas [22]. The authors of this study
observed a high incidence of CCs in hamsters treated with
N-nitrosodimethylamine and then infected with C. sinensis
[22]. Some investigators have hypothesized that the
epithelium of the bile ducts, if persistently exposed to
such carcinogens, might undergo the following sequence;
mucosal adenomatous hyperplasia–metaplasia–dysplasia–
carcinoma [20–23]. Owing to the long life span (over 20
years) of C. sinensis and the long sequence of carcinogen-
esis, some cases of CC associated with clonorchiasis in
immigrants from endemic areas have been reported in
western countries [24,25].
Detection of eggs in the stool or bile can lead to the
definite diagnosis of C. sinensis infection, thus, clonorch-
iasis has been principally diagnosed by stool microscopy
 D. Choi et al. / Journal of Hepatology 44 (2006) 1066–1073 1071

Table 3
Distribution of the 53 cases of hilar cholangiocarcinoma and 53 matched controls and corresponding ORs with 95% CI, according to diagnostic
methods for C. sinensis: Korea, 2003–2004

Characteristics Cases (nZ53) no. (%) Controls (nZ53) no. (%) Odds ratioa (95%CI)
Stool microscopy for C. sinensisb
Positive 2 (5.3) 0 Not availablec
Negative 36 (94.7) 38 (100) Reference
Pathologic examinations for C. sinensisb
Positive 2 (11.8) 2 (11.8) 1.0 (0.120–8.331)
Negative 15 (88.2) 15 (88.2) Reference
Serologic test for C. sinensisb
Positive 10 (20.8) 2 (4.2) 4.999 (1.318–32.526)
Negative 38 (79.2) 46 (95.8) Reference
Skin test for C. sinensisb
Positive 10 (25.0) 3 (7.5) 4.5 (1.160–29.514)
Negative 30 (75.0) 37 (92.5) Reference
Radiologic examinations for C. sinensis
Positive 49 (92.5) 9 (17.0) 41.0 (8.933–726.8)
Negative 4 (7.5) 44 (83.0) Reference
History of eating raw freshwater fish
Ever 29 (54.7) 14 (26.4) 3.143 (1.411–7.950)
Never 24 (45.3) 39 (73.6) Reference
Any evidence for C. sinensis
Positive 50 (94.3) 20 (37.7) 16.000 (3.834–66.763)
Negative 3 (5.7) 33 (62.3) Reference

OR, odds ratio; 95% CI, 95% confidence intervals.

a
Estimates from multiple conditional logistic regression equations.
b
The sum does not add up to the total because of some missing values.
c
Not available to perform statistics (OR calculation).

Table 4
Distribution of the 81 cases of distal extrahepatic cholangiocarcinoma and 81 matched controls and corresponding OR with 95% CI, according to
diagnostic methods for C. sinensis: Korea, 2003–2004

Characteristics Cases (nZ81) no. (%) Controls (nZ81) no. (%) Odds ratioa (95%CI)
b
Stool microscopy for C. sinensis
Positive 1 (2.0) 2 (3.9) 0.5 (0.023–5.218)
Negative 50 (98.0) 49 (96.1) Reference
Pathologic examinations for C. sinensisb
Positive 11 (28.9) 5 (13.2) 2.199 (0.8–6.981)
Negative 27 (71.1) 33 (86.8) Reference
Serologic test for C. sinensisb
Positive 8 (11.3) 5 (7.0) 1.6 (0.534–5.294)
Negative 63 (88.7) 66 (93.0) Reference
Skin test for C. sinensisb
Positive 4 (6.8) 3 (5.1) 1.333 (0.294–6.767)
Negative 55 (93.2) 56 (94.9) Reference
Radiologic examinations for C. sinensis
Positive 71 (87.7) 32 (39.5) 6.571 (3.170–15.943)
Negative 10 (12.3) 49 (60.5) Reference
History of eating raw freshwater fish
Ever 43 (53.1) 27 (33.3) 2.6 (1.294–5.66)
Never 38 (46.9) 54 (66.7) Reference
Any evidence for C. sinensis
Positive 75 (92.6) 45 (55.6) 7.000 (2.742–17.867)
Negative 6 (7.4) 36 (44.4) Reference

OR, odds ratio; 95% CI, 95% confidence intervals.

a
Estimates from multiple conditional logistic regression equations.
b
The sum does not add up to the total because of some missing values.
1072 D. Choi et al. / Journal of Hepatology 44 (2006) 1066–1073
[26]. However, sporadic routine stool microscopic examin-
ations performed in general hospitals (without a specialized
laboratory for parasites) are insufficient to detect a few
C. sinensis eggs in the stool of patients with a light infection.
It is also impossible to detect C. sinensis eggs in the stool of
patients with CC who have bile duct obstruction. Some
other diagnostic tools such as skin tests, serologic tests and
radiologic examinations have been introduced. Skin test is
known to be sensitive but not specific, and ELISA is widely
used for serodiagnosis with moderate sensitivity and
specificity but cross-reactions occur with other parasitic
fluke infections [12,27].
The majority of the cases of infection are of light burden
with low EPG (number of eggs per gram in feces) counts
[12]. Any diagnostic method may not detect all of the lightly
infected patients, but moderate or heavy infections can be
easily diagnosed. Since many patients are lightly infected,
the diagnostic limitations described above should be
overcome by using multiple tests, including fecal, serologic
and skin tests, and radiologic examinations. Thus, we
evaluated all of these tests in our study.
The radiologic findings of clonorchiasis are diffuse
dilatation of the intrahepatic bile ducts up to the peripheral
margin of the liver, but larger intrahepatic and extrahepatic
bile ducts are not dilated or minimally dilated [11]. These
findings reflect pathophysiology of bile ducts and
C. sinensis. Adult worms of C. sinensis usually reside in
the medium-sized or small intrahepatic bile ducts, and
occasionally in the extrahepatic bile ducts. The histopatho-
logical changes of bile ducts due to C. sinensis are mucosal
hyperplasia and periductal fibrosis with persistent ductal
dilation [12,28–30]. Radiologic findings on CT, ultrasono-
graphy, or cholangiogram have been regarded as character-
istic, even pathognomonic, of clonorchiasis in endemic
areas [11,29,31]. Because of the persistent dilatation of the
bile ducts after the treatment or death of C. sinensis, past
infection of C. sinensis can be diagnosed as well [12,32].
Some experimental studies reported that dilated bile ducts
were not completely normalized after cure [32,33].
The radiologic findings of CC associated with clonorch-
iasis results from a combination of the findings of two
diseases, namely diffuse dilatation of the peripheral
intrahepatic ducts is caused by changes that are secondary
to C. sinensis infection, and segmental and severe dilatation
proximal to the tumor results from obstruction caused by
CC [29]. Clonorchiasis-associated extrahepatic CC shows
diffuse dilatation of the peripheral and central intrahepatic
bile ducts (Fig. 1). If distal obstruction develops due to
extrahepatic CC, preexistent dilatation of intrahepatic bile
ducts associated with clonorchiasis becomes aggravated
[34]. On the other hand, in those cases where an extrahepatic
CC is not associated with clonorchiasis, the dilatation of the
peripheral intrahepatic bile ducts is not conspicuous (Fig. 2).
A positive stool microscopy or pathological examination
definitely indicates active infection of C. sinensis. Positive
results of serologic and skin tests have been reported to
persist for approximately 6 months and 10 years,
respectively, after treatment [12,35]. Radiologic evidence
and a history of eating raw freshwater fish may be a
reflection of a long period of past infection as well as active
infection. Considering that carcinogenesis is a multi-step
process spanning a long period of time (presumably 10–40
years) [19–23], radiologic evidence and a history of eating
raw freshwater fish would seem to provide more important
information as to whether C. sinensis infection is a risk
factor for CC. In this context, the results of our study
indicated the existence of a strong correlation between a
long period of infection of C. sinensis and CC.
The relationship between viral hepatic diseases and
intrahepatic CC has been the subject of considerable debate
[2–4,36–38]. We investigated the role of the viral markers
of hepatitis B and C in the risk of intrahepatic CC in 51
patients. The results showed no significant association,
however this might be related to the small number of cases.
The effects of bile duct stones were not analyzed, because of
the difficulty in obtaining accurate historic evidence of long-
standing disease. With regard to the possible sources of bias,
we excluded patients with biliary diseases, such as bile duct
stones and cholangitis, which are known to be possible risk
factors.
This study has several potential limitations. The first is
the limitation inherent in all such case–control studies,
namely recall and reporting bias. In our study, such potential
recall biases included that associated with the subject’s
history of eating raw freshwater fish. The subject’s recall
may have been affected by severe illness. We attempted to
minimize this type of recall bias by not including very ill
individuals. The second limitation is the missing data. For
stool microscopy, pathologic examination and skin test, in
particular, the numbers of missing data were not small.
Many patients must maintain fasting before or immediately
after operations, and thus it is difficult to obtain sufficient
amount of feces. And, some patients could not move to the
laboratory for skin tests due to poor physical condition.
Missing data could make the analyses to be correspondingly
weaker. We used the complete pairs of the cases and control
in the statistical analysis. Thirdly, the controls were selected
from patients admitted for gastrointestinal (non-hepatobili-
ary) diseases. There have been no definite evidences that
C. sinensis infection was not associated with these
gastrointestinal diseases of the control patients. Finally,
for the stool microscopy, we used the formalin–ether
sedimentation technique, which is widely used
in hospitals, but is not the best fecal examination for
C. sinensis [12,26]. More than half of the patients with CC
showed obstruction of the common duct. These factors
might have led to the low rate of positive stool microscopy
and the lack of association between C. sinensis in the stool
and CC in this study.
In conclusion, this study showed that radiologic evidence
of C. sinensis, a history of eating raw freshwater fish and a
positive serologic result for C. sinensis were significantly
 D. Choi et al. / Journal of Hepatology 44 (2006) 1066–1073
associated with the development of CC, including extra-
hepatic CC. In particular, radiologic evidence of C. sinensis,
which was the only risk factor for all three kinds of CC, may
be indicative of a long-standing infection by C. sinensis, and
was considered more objective than a history of eating raw
freshwater fish.
Acknowledgements
This work was supported by the Samsung Biomedical
Research Institute grants, #SBRI C-A3-110-1 and C-A3-
110-2. We thank Seung-Yull Cho at Sungkyunkwan
University School of Medicine for consultations regarding
parasitology.
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