Cholangiocarcinoma and Clonorchis sinensis infection: A case control study in Korea

Transcription

1 Journal of Hepatology 44 (2006) Cholangiocarcinoma and Clonorchis sinensis infection: A case control study in Korea Dongil Choi 1, Jae Hoon Lim 1, *, Kyu Taek Lee 2, Jong Kyun Lee 2, Seong Ho Choi 3, Jin Seok Heo 3, Kee-Taek Jang 4, Nam Yong Lee 5, Seonwoo Kim 6, Sung-Tae Hong 7 1 Department of Radiology and Center for Imaging Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Ilwon-Dong, Kangnam-Ku, Seoul , South Korea 2 Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Ilwon-Dong, Kangnam-Ku, Seoul , South Korea 3 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Ilwon-Dong, Kangnam-Ku, Seoul , South Korea 4 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Ilwon-Dong, Kangnam-Ku, Seoul , South Korea 5 Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Ilwon-Dong, Kangnam-Ku, Seoul , South Korea 6 Biostatistics Unit, Samsung Biomedical Research Institute, Samsung Medical Center, 50, Ilwon-Dong, Kangnam-Ku, Seoul , South Korea 7 Department of Parasitology and Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul , 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]Z ), ORZ2.385 (95% CIZ ), and ORZ2.272 (95% CIZ ), respectively. The risk factors for distal extrahepatic CC were radiologic evidence of C. sinensis (ORZ6.571; 95% CIZ ) and history of eating raw freshwater fish (ORZ2.6; 95% CIZ ). 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 Received 6 July 2005; received in revised form 15 November 2005; accepted 16 November 2005; available online 27 December 2005 * Corresponding author. Tel.: C x2518; fax: C address: jhlim@smc.samsung.co.kr (J.H. Lim). There is a wide variation in the incidence of cholangiocarcinoma (CC) in different parts of the world, which is much higher in East Asia than in Western Europe and America. This variation is believed to be associated with the distribution of the risk factors for this disease. There are several documented risk factors for CC, including primary /$32.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi: /j.jhep

2 D. Choi et al. / Journal of Hepatology 44 (2006) 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 epidemiological 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, 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, years) admitted for alcoholinduced 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 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 histopathological 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, Characteristics Cases (nz185) no. (%) Controls (nz185) no. (%) Odds ratio a (95%CI) Stool microscopy for C. sinensis b Positive 3 (2.5) 5 (4.1) 0.6 ( ) Negative 119 (97.5) 117 (95.9) Reference Pathologic examinations for C. sinensis b Positive 13 (17.6) 8 (10.8) ( ) Negative 61 (82.4) 66 (89.2) Reference Serologic test for C. sinensis b,c Positive 25 (15.2) 11 (6.7) ( ) Negative 139 (84.8) 153 (93.3) Reference Skin test for C. sinensis b Positive 19 (13.8) 12 (8.7) 1.7 ( ) Negative 119 (86.2) 126 (91.3) Reference Radiologic examinations for C. sinensis Positive 156 (84.3) 57 (30.8) ( ) Negative 29 (15.7) 128 (69.2) Reference History of eating raw freshwater fish Ever 94 (50.8) 58 (31.4) ( ) Never 91 (49.2) 127 (68.6) Reference Any evidence for C. sinensis d Positive 167 (90.3) 92 (49.7) ( ) 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.

3 1068 D. Choi et al. / Journal of Hepatology 44 (2006) :5000. Diaminobenzidine was used as the substrate for the color reaction and the absorbance was read at 490 nm using an ELISA reader (Emax w Precision Microplate Reader, Molecular Devices Corp., Calf, USA). The presence of surface antigens of hepatitis B and antibodies (IgG) of hepatitis C was examined in order to detect liver diseases related to viral hepatitis B and C, respectively. Among the cases and controls, 321 patients (86.8%) underwent a skin 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 mm 2 or greater on the forearm was considered as a positive result. All of the patients underwent contrast-enhanced helical CT. Cholangiographic 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]. CIZ ) (Figs. 1 and 2), a history of eating raw freshwater fish (ORZ2.385; 95% CIZ ) and a positive serologic result for C. sinensis (ORZ2.272; 95% CIZ ). The numbers of cases with pathological evidence of C. sinensis and a positive skin test for 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 were considered to indicate poor agreement; k values of , fair agreement; k values of moderate agreement; k values of , good agreement; and k values of , excellent agreement [13]. 3. Results Table 1 presents the distribution of the 185 cases of CC, and of the matched control group. The risk factors for CC were radiologic evidence of C. sinensis (ORZ8.615; 95% Fig. 1. Cholangiocarcinoma (CC) in the mid common duct, with clonorchiasis, in a 60-year-old man. Pathology reveals a 2.4-cm welldifferentiated tubular adenocarcinoma after segmental resection of the 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, 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 represents an extrahepatic CC. (C) Endoscopic retrograde cholangiography shows complete obstruction of the common duct (arrow) and diffuse dilated intrahepatic ducts (arrowheads).

4 D. Choi et al. / Journal of Hepatology 44 (2006) 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 (arrowheads), 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% CIZ ). 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% CIZ ). 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 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 intrahepatic CC (ORZ4.000; 95% CIZ ). 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% CIZ ), a history of eating raw freshwater fish (ORZ3.143; 95% CIZ ), a positive serologic result for C. sinensis (ORZ4.999; 95% CIZ ) and a positive skin test for C. sinensis (ORZ4.5; 95% CIZ ). 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 ). 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% CIZ ) and a history of eating raw freshwater fish (ORZ2.6; 95% CIZ ). 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% CIZ ). 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, ) [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

5 1070 D. Choi et al. / Journal of Hepatology 44 (2006) 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, Characteristics Cases (nz51) no. (%) Controls (nz51) no. (%) Odds ratio a (95%CI) Stool microscopy for C. sinensis b Positive 0 3 (9.1) Not available c Negative 33 (100) 30 (90.9) Reference Pathologic examinations for C. sinensis b Positive 0 1 (5.3) Not available c Negative 19 (100) 18 (94.7) Reference Serologic test for C. sinensis b Positive 7 (15.6) 4 (8.9) 1.75 ( ) Negative 38 (84.4) 41 (91.1) Reference Skin test for C. sinensis b Positive 5 (12.8) 6 (15.4) 0.8 ( ) Negative 34 (87.2) 33 (84.6) Reference Radiologic examinations for C. sinensis Positive 36 (70.6) 16 (31.4) ( ) Negative 15 (29.4) 35 (68.6) Reference History of eating raw freshwater fish Ever 22 (43.1) 17 (33.3) ( ) Never 29 (56.9) 34 (66.7) Reference Any evidence for C. sinensis Positive 42 (82.4) 27 (52.9) ( ) Negative 9 (17.6) 24 (47.1) Reference Surface antigen of hepatitis B virus Positive 4 (7.8) 5 (9.8) 0.8 ( ) Negative 47 (92.2) 46 (90.2) Reference Antibody of hepatitis C virus Positive 1 (2.0) 1 (2.0) 1.00 ( ) 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 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% CIZ ) [7]. Several prior epidemiological reports in Hong Kong and Korea reported an association between C. sinensis infection and intrahepatic CC, but not extrahepatic CC [5 7,14]. We believe that the present epidemiological 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 is still unknown [12,18]. The carcinogenesis associated with 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-nitrosodimethylamine, and subsequently epithelial proliferation (promotion) 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 carcinogenesis, 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, clonorchiasis has been principally diagnosed by stool microscopy

6 D. Choi et al. / Journal of Hepatology 44 (2006) 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, Characteristics Cases (nz53) no. (%) Controls (nz53) no. (%) Odds ratio a (95%CI) Stool microscopy for C. sinensis b Positive 2 (5.3) 0 Not available c Negative 36 (94.7) 38 (100) Reference Pathologic examinations for C. sinensis b Positive 2 (11.8) 2 (11.8) 1.0 ( ) Negative 15 (88.2) 15 (88.2) Reference Serologic test for C. sinensis b Positive 10 (20.8) 2 (4.2) ( ) Negative 38 (79.2) 46 (95.8) Reference Skin test for C. sinensis b Positive 10 (25.0) 3 (7.5) 4.5 ( ) Negative 30 (75.0) 37 (92.5) Reference Radiologic examinations for C. sinensis Positive 49 (92.5) 9 (17.0) 41.0 ( ) Negative 4 (7.5) 44 (83.0) Reference History of eating raw freshwater fish Ever 29 (54.7) 14 (26.4) ( ) Never 24 (45.3) 39 (73.6) Reference Any evidence for C. sinensis Positive 50 (94.3) 20 (37.7) ( ) 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, Characteristics Cases (nz81) no. (%) Controls (nz81) no. (%) Odds ratio a (95%CI) Stool microscopy for C. sinensis b Positive 1 (2.0) 2 (3.9) 0.5 ( ) Negative 50 (98.0) 49 (96.1) Reference Pathologic examinations for C. sinensis b Positive 11 (28.9) 5 (13.2) ( ) Negative 27 (71.1) 33 (86.8) Reference Serologic test for C. sinensis b Positive 8 (11.3) 5 (7.0) 1.6 ( ) Negative 63 (88.7) 66 (93.0) Reference Skin test for C. sinensis b Positive 4 (6.8) 3 (5.1) ( ) Negative 55 (93.2) 56 (94.9) Reference Radiologic examinations for C. sinensis Positive 71 (87.7) 32 (39.5) ( ) Negative 10 (12.3) 49 (60.5) Reference History of eating raw freshwater fish Ever 43 (53.1) 27 (33.3) 2.6 ( ) Never 38 (46.9) 54 (66.7) Reference Any evidence for C. sinensis Positive 75 (92.6) 45 (55.6) ( ) 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.

7 1072 D. Choi et al. / Journal of Hepatology 44 (2006) [26]. However, sporadic routine stool microscopic examinations 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 histopathological 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, ultrasonography, or cholangiogram have been regarded as characteristic, 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 clonorchiasis 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 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 longstanding 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-hepatobiliary) 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

8 D. Choi et al. / Journal of Hepatology 44 (2006) associated with the development of CC, including extrahepatic 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-A and C-A We thank Seung-Yull Cho at Sungkyunkwan University School of Medicine for consultations regarding parasitology. References [1] Chapman RW. Risk factors for biliary tract carcinogenesis. Ann Oncol 1999;10: [2] Shaib Y, El-Serag HB. The epidemiology of cholangiocarcinoma. Semin Liver Dis 2004;24: [3] Pinyosophon A, Wiwanitkit V. The prevalence of hepatitis B seropositivity among patients with cholangiocarcinoma. Viral Immunol 2002;15: [4] Yamamoto S, Kubo S, Hai S, Uenishi T, Yamamoto T, Shuto T, et al. Hepatitis C virus infection as a likely etiology of intrahepatic cholangiocarcinoma. Cancer Sci 2004;95: [5] Kim YI, Yang DH, Chang KR. Relationship between Clonorchis sinensis infestation and cholangiocarcinoma of the liver in Koreaepidemiological and pathologic reappraisal of 495 consecutive primary carcinoma of the liver on Seoul and Busan areas. Seoul J Med 1974;15: [6] Chung CS, Lee SK. An epidemiological study of primary liver carcinomas in Busan area with special reference to clonorchiasis. Korean J Pathol 1976;10: [7] Shin HR, Lee CU, Park HJ, Seol SY, Chung JM, Choi HC, et al. Hepatitis B and C virus, Clonorchis sinensis for the risk of liver cancer: a case control study in Pusan. Korea Int J Epidemiol 1996;25: [8] Patel AH, Harnois DM, Klee GG, LaRusso NF, Gores GJ. The utility of CA 19-9 in the diagnoses of cholangiocarcinoma in patients without primary sclerosing cholangitis. Am J Gastroenterol 2000;95: [9] Choi MH, Park IC, Li S, Hong ST. Excretory secretory antigen is better than crude antigen for the serodiagnosis of clonorchiasis by ELISA. Korean J Parasitol 2003;41: [10] Min DY. Remarks on the diagnosis of Clonorchis sinensis infection. Arzneimittelforschung 1984;34: [11] Lim JH. Radiologic findings of clonorchiasis. AJR Am J Roentgenol 1990;155: [12] Hong ST. Clonorchis sinensis. In: Miliotis MD, Bier JW, editors. International handbook of foodborne pathogens. New York: Marcel Dekker; p [13] Fleiss JL. The measurement of interrater agreement. In: Fleiss JL, editor. Statistical methods for rates and proportions. 2nd ed. New York: Wiley; p [14] Hou PC. The relationship between primary carcinoma of the liver and infestation with Clonorchis sinensis. J Pathol Bacteriol 1956;72: [15] Schwartz DA. Helminths in the induction of cancer: Opisthorchis viverrini, Clonorchis sinensis and cholangiocarcinoma. Trop Geogr Med 1980;32: [16] Belamaric J. Intrahepatic bile duct carcinoma and C. sinensis infection in Hong Kong. Cancer 1973;31: [17] Choi BI, Kim HJ, Han MC, Do YS, Han MH, Lee SH. CT findings of clonorchiasis. AJR Am J Roentgenol 1989;152: [18] Infection with liver flukes (Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis). IARC Monogr Eval Carcinog Risks Hum 1994;61: [19] Flavell DJ. Liver-fluke infection as an aetiological factor in bile-duct carcinoma of man. Trans R Soc Trop Med Hyg 1981;75: [20] Watanapa P, Watanapa WB. Liver fluke-associated cholangiocarcinoma. Br J Surg 2002;89: [21] Holzinger F, Z Graggen K, Buchler MW. Mechanisms of biliary carcinogenesis: a pathogenetic multi-stage cascade towards cholangiocarcinoma. Ann Oncol 1999;10: [22] Lee JH, Rim HJ, Sell S. Heterogeneity of the oval-cell response in the hamster liver during cholangiocarcinogenesis following Clonorchis sinensis infection and dimethylnitrosamine treatment. J Hepatol 1997;26: [23] Kakar S, Burgart LJ. Tumours of the biliary system. Curr Diagn Pathol 2005;11: [24] Schwartz DA. Cholangiocarcinoma associated with liver fluke infection: a preventable source of morbidity in Asian immigrants. Am J Gastroenterol 1986;81: [25] Sher L, Iwatsuki S, Lebeau G, Zajko AB. Hilar cholangiocarcinoma associated with clonorchiasis. Dig Dis Sci 1989;34: [26] Hong ST, Choi MH, Kim CH, Chung BS, Ji Z. The Kato Katz method is reliable for diagnosis of Clonorchis sinensis infection. Diagn Microbiol Infect Dis 2003;47: [27] Chen M, Lu Y, Hua X, Mott KE. Progress in assessment of morbidity due to Clonorchis sinensis infection: a review of recent literature. Trop Dis Bull 1994;91:R7 R65. [28] Lee SH, Hong ST, Kim CS, Sohn WM, Chai JY, Lee YS. Histopathological changes of the liver after praziquantel treatment in Clonorchis sinensis infected rabbits. Korean J Parasitol 1987;25: [29] Choi BI, Park JH, Kim YI, Yu ES, Kim SH, Kim WH, et al. Peripheral cholangiocarcinoma and clonorchiasis: CT findings. Radiology 1988; 169: [30] Ryu KN, Lim JH, Cho YJ, Yang MH. Comparative study of radiologic pathological findings of experimental clonorchiasis in rabbits. J Korean Radiol Soc 1993;29:1 8. [31] Choi BI, Han JK. Other parasitic diseases. In: Okuda K, Mitchell DG, Itai Y, Ariyama J, editors. Hepatobiliary disease pathophysiology and imaging. London, UK: Blackwell Science; p [32] Choi D, Lim JH, Kim SK, Kim EY, Lee M, Hong ST. Long-lasting sonographic and histopathological findings in cured clonorchiasis of rabbits. Korean J Parasitol 1999;37: [33] Hong ST, Park KH, Seo M, Choi BI, Chai JY, Lee SH. Correlation of sonographic findings with histopathological changes of the bile ducts in rabbits infected with Clonorchis sinensis. Korean J Parasitol 1994; 32: [34] Choi D, Lim JH, Hong ST. Relation of cholangiocarcinomas to clonorchiasis and bile duct stones. Abdom Imaging 2004;29: [35] Hong ST. Changes of anti-clonorchis sinensis IgG antibody in serum after praziquantel treatment in human clonorchiasis. Korean J Parasitol 1988;26:1 8. [36] Donato F, Gelatti U, Tagger A, Favret M, Ribero ML, Callea F, et al. Intrahepatic cholangiocarcinoma and hepatitis C and B virus infection, alcohol intake, and hepatolithiasis: a case control study in Italy. Cancer Causes Control 2001;12: [37] Kobayashi M, Ikeda K, Saitoh S, Suzuki F, Tsubota A, Suzuki Y, et al. Incidence of primary cholangiocellular carcinoma of the liver in Japanese patients with hepatitis C virus-related cirrhosis. Cancer 2000;88: [38] Shaib YH, El-Serag HB, Davila JA, Morgan R, McGlynn KA. Risk factors of intrahepatic cholangiocarcinoma in the United States: a case control study. Gastroenterology 2005;128: