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Experimental Clonorchiasis in Dogs: CT Findings before and after Treatment¹

¹ From the Department of Radiology and the Institute of Radiation Medicine, Clinical Research Institute (K.H.L., J.K.H., C.J.Y., S.H.K., B.I.C.); Department of Parasitology (S.T.H.); and Department of Pathology (S.L.); Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. From the 2001 RSNA scientific assembly. Received January 4, 2002; revision requested March 1; final revision received October 22; accepted December 10. Address correspondence to S.T.H. (e-mail: hst@snu.ac.kr).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To determine whether disease activity in clonorchiasis can be assessed with dynamic computed tomography (CT).

MATERIALS AND METHODS: Ten dogs infected with metacercariae of Clonorchis sinensis underwent serial dynamic CT examinations. Five dogs were sacrificed 14 weeks after infection, and another five infected dogs were treated with praziquantel at the 14th week and continued to undergo CT examinations until death or sacrifice at the 25th (n = 1) or 40th week (n = 4). CT images were evaluated for dilatation of the bile ducts, contrast enhancement of bile duct walls and adjacent hepatic parenchyma, visualization of flukes within the bile ducts, and presence of calcifications. Changes in CT findings were analyzed statistically by using a mixed linear model and a generalized estimating equations model.

RESULTS: Dilatation of the bile ducts, contrast enhancement of the ductal walls, transient hepatic attenuation differences, and flukes per se were observed at CT from the 2nd through the 5th week, were most apparent between the 5th and 13th weeks, and disappeared or decreased markedly in degree after treatment in all dogs (P < .05). Nodular calcifications were observed at CT after treatment in two dogs. Histopathologic examination revealed proliferation and enlargement of arteries in the periductal area and the portal tracts and congestion of hepatic sinusoids during the acute phase. Treatment was associated with periductal hyalinization, degeneration of the periductal arteries, and calcification of the ductal epithelium.

CONCLUSION: Disease activity in experimental canine clonorchiasis can be assessed with dynamic CT.

© RSNA, 2003

Index terms: Animals • Bile ducts, CT, 76.12113 • Experimental study • Liver, CT, 761.12113 • Parasites

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Clonorchiasis is a fish-transmitted trematodiasis in East Asia (1–3) caused by Clonorchis sinensis, which lives in the bile ducts of humans and other mammals. It induces dilatation of the bile duct, hyperplasia of the mucosal epithelium, and periductal inflammation (4–8).

Despite a gradual decrease in the incidence of human clonorchiasis infections in recent decades, it is still estimated that about 15 million people in the world are currently infected (3). A national survey in Korea in 1997 revealed that the prevalence of clonorchiasis was 1.4% (9). This difficulty of controlling clonorchiasis has been attributed mainly to the difficulty of detecting cases of infection, although other contributory factors, including reinfection after treatment, have been discussed (10).

The diagnosis of clonorchiasis is easily made by detecting eggs in the feces; however, mass screening with fecal examination has become more difficult because of poor voluntary cooperation (11,12). Other supplementary diagnostic methods, including an intradermal test, enzyme-linked immunosorbent assays, ultrasonography (US), computed tomography (CT), and cholangiography (13–15), have been used. However, none of these methods has been reported to surpass fecal examination because of their limited sensitivity, specificity, or applicability (11). Nevertheless, US is considered to be an accurate and feasible diagnostic method for clonorchiasis (10,13,16,17). US findings (eg, diffuse dilatation of the small intrahepatic bile ducts) have been regarded as pathognomonic for clonorchiasis (10,13). At present, clonorchiasis is commonly diagnosed incidentally during US screening of the abdomen for other purposes, because symptoms of clonorchiasis are vague and nonspecific (13,17,18).

However, results of more recent studies (11,19,20) have indicated that US has low diagnostic accuracy in clonorchiasis. According to the results of a study in an endemic area (11), the sensitivity of US was 52% and the specificity was 51%; the low sensitivity was attributed to the presence of false-negative cases of mild infection, and the low specificity was attributed to false-positive cases with residual abnormalities after cure. This low specificity is of particular interest because the number of cases cured has been continuously increasing over recent decades owing to nationwide control of clonorchiasis and to ecologic changes (9,10). US is less useful in the differentiation of cured clonorchiasis from active infection because it depicts pathologic changes in the bile ducts (13) that may persist for years after cure (5,6,10,12,16,17,21,22) rather than the presence of the worm itself.

Therefore, a more elaborate method is necessary for the diagnosis of active clonorchiasis (11,18). In addition to offering a high sensitivity, any new diagnostic method should reflect the activity of the disease and enable the avoidance of false-positive diagnoses in cured cases. Dynamic CT is one of the most effective diagnostic methods in various biliary diseases and focal hepatic lesions and theoretically may satisfy these requirements. The purpose of this experimental study was to determine whether disease activity in clonorchiasis can be assessed with dynamic CT.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study Design
The experimental protocols used in this study were approved by the committee on animal research at our institution. Ten mongrel dogs (12–16 months in age, 16–20 kg, male and female) were infected with the metacercariae of C sinensis and followed up with serial CT examinations. The dogs were randomly separated into one of two groups (groups A and B); each group consisted of five dogs. Dogs in group A were sacrificed 14 weeks after infection. Dogs in group B were treated with 100 mg/kg of praziquantel (Distocide; Shinpoong Pharmaceutical, Seoul, Korea) for 3 days during the 14th week and continued to undergo serial CT examinations until sacrifice at the 40th week (Fig 1). One dog in group B accidentally expired, probably due to a side effect of general anesthesia, immediately after CT on the 25th week. During the follow-up period, all dogs were kept in laboratory cages and fed a commercial pellet diet and tap water.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Graph depicts the experimental design. Dogs were separated into two groups (groups A and B); each group consisted of five dogs. Dogs in group A were sacrificed 14 weeks after infection, and dogs in group B were treated with praziquantel and continued to undergo serial CT examinations until sacrifice at the 40th week. The numbers in the box and the vertical bars represent the times (in weeks after infection) at which CT examinations were performed; asterisks indicate the times at which fecal examinations were performed.

Preparation of Dogs for CT Scanning
Dogs were not fed for 12 hours before CT scanning to ensure CT image quality and to minimize the side effects of general anesthesia; however, water was provided to avoid dehydration. Anesthesia for CT scanning was induced with an intramuscular injection of 10 mg per kilogram of body weight of ketamine hydrochloride (Ketalar; Yuhan Yanghang, Seoul, Korea) and 4 mg/kg of xylazine hydrochloride (Rompun 2%; Bayer Korea, Seoul, Korea) and maintained with intravenous injection of 10 mg/kg of zolazepam (Zoletil; Virbac, Carros, France). The antecubital vein was catheterized with an 18-gauge plastic cannula for contrast material injection. CT scans were acquired during quiet self-breathing after endotracheal intubation.

CT Protocol
Two-phase helical CT (Somatom Plus 4; Siemens Medical Systems, Erlangen, Germany) was performed with the dogs in the supine position with the following scanning parameters: 120 kVp; 200 mA; table feed, 5 mm; collimation, 3 mm; and reconstruction interval, 2 mm. After the injection of 2.5 mL/kg of contrast material (iopromide, Ultravist 370; Schering, Berlin, Germany) at a rate of 2 mL/sec, arterial phase scanning was initiated by using a bolus tracking method 5 seconds after aortic attenuation exceeded 100 HU. Portal phase scanning was initiated 15 seconds after arterial phase scanning. This CT protocol was chosen on the basis of results of preliminary experiments with single-level dynamic scanning.

Fecal Examination
So that its results could be compared with the CT findings, a fecal examination was performed immediately and at the 14th and 40th weeks after infection. The stools were examined microscopically for the presence of C sinensis eggs by using the formalin-ether concentration method and the Stoll dilution method for egg counting.

CT-Histopathologic Correlation
After the scheduled CT examinations were completed, dogs were sacrificed with an intravenous injection of a lethal amount of thiopental sodium (Pentothal; Choongwae Pharm, Seoul, Korea). Autopsies were performed by a parasitologist (S.T.H.) and observed by a radiologist (C.J.Y.). So that we could strictly correlate CT findings with histopathologic findings, representative areas at CT (five to 10 areas in each dog) were located in the livers by using reference CT images and surface US. After we recorded these locations, portions of the livers were carefully excised and prepared for microscopic examination. The liver and hepatobiliary apparatus, including the pancreas, were carefully harvested, and any pathologic findings were photographed. The remaining entire liver specimens were carefully sliced to a thickness of 3–5 mm in anatomic axial planes corresponding to those in which the CT images were obtained.

Each slice was carefully examined, the caudal surface of each slice was photographed, and the flukes within the bile ducts were counted. Contact radiographs (Faxitron 43805N; Hewlett-Packard, Sunnyvale, Calif and X-Omat V; Eastman Kodak, Rochester, NY) were obtained of representative slices that contained calcifications. Representative areas of pathologic and normal-appearing liver were selected for slide preparation by the two examiners (S.T.H., C.J.Y.) in consensus. After the locations of the areas that were sampled had been recorded, the selected tissues were prepared for fixation, paraffin embedding, cutting, and staining with hematoxylin-eosin in the usual manner.

Image Analysis
CT images were evaluated for: (a) dilatation of bile ducts, (b) contrast enhancement of bile duct walls, (c) the contrast enhancement pattern of hepatic parenchyma adjacent to areas in which findings a and b were observed, (d) visualization of flukes within bile ducts, and (e) the presence of calcifications at precontrast CT. These findings were chosen for evaluation on the basis of a review of previous reports of CT and US findings of clonorchiasis (10–12,17–25). The calibers of the three largest intrahepatic bile ducts on each portal phase scan were recorded by averaging three measurements for each duct. Each measurement was performed with magnification by using a digital caliper provided by a picture archiving and communications system (Marotech, Seoul, Korea). Findings b and c were evaluated with a semiquantitative scale as absent (-) or minimal (+), mild (++), or severe (+++) in degree by three abdominal radiologists (K.H.L., J.K.H., B.I.C.) in consensus. The presence of flukes per se and calcifications was also determined by the three observers in consensus. A pathologist (S.L.) who is an expert in hepatic pathology, together with a parasitologist (S.T.H.) and a radiologist (K.H.L.), assessed histopathologic specimens for findings that might correspond to and help explain the CT findings.

Changes in finding a were analyzed statistically by using a mixed linear model (SAS 8.1; SAS Institute, Cary, NC). Changes in findings b, c, and d were analyzed statistically by using a generalized estimating equations model. Findings b and c were analyzed after binary collapse of the rated responses (ie, ratings of - and + were grouped into one binary category, and ratings of ++ and +++ were grouped into another). For statistical analysis, the follow-up period of 40 weeks was divided into three phases: the 0–4th-week period, the 5th–13th-week period, and the 17th–40th-week period. For finding d, the 0–4th weeks and 17th–40th weeks were considered to be a single phase to be compared with the second phase (5th–13th weeks).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CT findings evaluated in this study were (a) dilatation of intrahepatic bile ducts, (b) contrast enhancement of bile duct walls, (c) transient hepatic attenuation difference (THAD) in parenchyma adjacent to dilated ducts, (d) visualization of flukes per se within the bile ducts, and (e) the appearance of tiny dotlike calcifications after treatment. Findings a–d were observed from the 2nd–5th week after infection, were most apparent at the 5th–13th weeks, and disappeared or decreased in degree after treatment in all dogs (Fig 2).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 2a. Graphs illustrate changes in CT findings during the follow-up period of 40 weeks in dogs infected with metacercariae of C sinensis. Ten dogs were followed up until the 13th week, five dogs were followed up through the 17th-25th weeks, and four dogs were followed up through the 30th-40th weeks. (a) Graph illustrates the calibers of the intrahepatic bile ducts. The connecting line indicates the change in the averaged calibers. Error bars represent standard errors of the mean. (b) Graph illustrates the degree of contrast enhancement of the walls of the bile ducts (finding b). (c) Graph illustrates the degree of THAD in the parenchyma adjacent to the dilated bile ducts (finding c). (d) Graph illustrates the identification of flukes at CT. Findings b and c were evaluated with a semiquantitative scale as absent (-) or minimal (+), mild (++), or severe (+++) in degree. After treatment (arrow), contrast enhancement of the ductal walls and THAD remained to a minimal or mild degree in two dogs, and flukes were identified at CT in one of these two dogs. # = dog with persistent ductal wall enhancement and THAD, * = dog with persistent ductal wall enhancement, THAD, and flukes.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Figure 2b. Graphs illustrate changes in CT findings during the follow-up period of 40 weeks in dogs infected with metacercariae of C sinensis. Ten dogs were followed up until the 13th week, five dogs were followed up through the 17th-25th weeks, and four dogs were followed up through the 30th-40th weeks. (a) Graph illustrates the calibers of the intrahepatic bile ducts. The connecting line indicates the change in the averaged calibers. Error bars represent standard errors of the mean. (b) Graph illustrates the degree of contrast enhancement of the walls of the bile ducts (finding b). (c) Graph illustrates the degree of THAD in the parenchyma adjacent to the dilated bile ducts (finding c). (d) Graph illustrates the identification of flukes at CT. Findings b and c were evaluated with a semiquantitative scale as absent (-) or minimal (+), mild (++), or severe (+++) in degree. After treatment (arrow), contrast enhancement of the ductal walls and THAD remained to a minimal or mild degree in two dogs, and flukes were identified at CT in one of these two dogs. # = dog with persistent ductal wall enhancement and THAD, * = dog with persistent ductal wall enhancement, THAD, and flukes.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 2c. Graphs illustrate changes in CT findings during the follow-up period of 40 weeks in dogs infected with metacercariae of C sinensis. Ten dogs were followed up until the 13th week, five dogs were followed up through the 17th-25th weeks, and four dogs were followed up through the 30th-40th weeks. (a) Graph illustrates the calibers of the intrahepatic bile ducts. The connecting line indicates the change in the averaged calibers. Error bars represent standard errors of the mean. (b) Graph illustrates the degree of contrast enhancement of the walls of the bile ducts (finding b). (c) Graph illustrates the degree of THAD in the parenchyma adjacent to the dilated bile ducts (finding c). (d) Graph illustrates the identification of flukes at CT. Findings b and c were evaluated with a semiquantitative scale as absent (-) or minimal (+), mild (++), or severe (+++) in degree. After treatment (arrow), contrast enhancement of the ductal walls and THAD remained to a minimal or mild degree in two dogs, and flukes were identified at CT in one of these two dogs. # = dog with persistent ductal wall enhancement and THAD, * = dog with persistent ductal wall enhancement, THAD, and flukes.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 2d. Graphs illustrate changes in CT findings during the follow-up period of 40 weeks in dogs infected with metacercariae of C sinensis. Ten dogs were followed up until the 13th week, five dogs were followed up through the 17th-25th weeks, and four dogs were followed up through the 30th-40th weeks. (a) Graph illustrates the calibers of the intrahepatic bile ducts. The connecting line indicates the change in the averaged calibers. Error bars represent standard errors of the mean. (b) Graph illustrates the degree of contrast enhancement of the walls of the bile ducts (finding b). (c) Graph illustrates the degree of THAD in the parenchyma adjacent to the dilated bile ducts (finding c). (d) Graph illustrates the identification of flukes at CT. Findings b and c were evaluated with a semiquantitative scale as absent (-) or minimal (+), mild (++), or severe (+++) in degree. After treatment (arrow), contrast enhancement of the ductal walls and THAD remained to a minimal or mild degree in two dogs, and flukes were identified at CT in one of these two dogs. # = dog with persistent ductal wall enhancement and THAD, * = dog with persistent ductal wall enhancement, THAD, and flukes.

View larger version (138K): [in this window] [in a new window] [Download PPT slide]   Figure 3a. Transverse CT scans obtained in a dog in group A during (a) arterial and (b) portal phases at the 5th week after infection, and (c) photomicrograph of a liver specimen harvested at the 14th week. Note the dilatation of the peripheral intrahepatic bile ducts and their clublike blunt tips (arrow in a and b). Ringlike or tubular contrast enhancement of ductal walls and a wedge-shaped area of contrast enhancement in the adjacent parenchyma at the periphery of the liver are more visible on a. (c) In a section of the dilated bile ducts, periductal proliferation of arteries (arrows) and veins (arrowheads), as well as papillary proliferation of the bile duct epithelium and periductal inflammation, are seen. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (137K): [in this window] [in a new window] [Download PPT slide]   Figure 3b. Transverse CT scans obtained in a dog in group A during (a) arterial and (b) portal phases at the 5th week after infection, and (c) photomicrograph of a liver specimen harvested at the 14th week. Note the dilatation of the peripheral intrahepatic bile ducts and their clublike blunt tips (arrow in a and b). Ringlike or tubular contrast enhancement of ductal walls and a wedge-shaped area of contrast enhancement in the adjacent parenchyma at the periphery of the liver are more visible on a. (c) In a section of the dilated bile ducts, periductal proliferation of arteries (arrows) and veins (arrowheads), as well as papillary proliferation of the bile duct epithelium and periductal inflammation, are seen. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (162K): [in this window] [in a new window] [Download PPT slide]   Figure 3c. Transverse CT scans obtained in a dog in group A during (a) arterial and (b) portal phases at the 5th week after infection, and (c) photomicrograph of a liver specimen harvested at the 14th week. Note the dilatation of the peripheral intrahepatic bile ducts and their clublike blunt tips (arrow in a and b). Ringlike or tubular contrast enhancement of ductal walls and a wedge-shaped area of contrast enhancement in the adjacent parenchyma at the periphery of the liver are more visible on a. (c) In a section of the dilated bile ducts, periductal proliferation of arteries (arrows) and veins (arrowheads), as well as papillary proliferation of the bile duct epithelium and periductal inflammation, are seen. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (166K): [in this window] [in a new window] [Download PPT slide]   Figure 4a. Transverse CT scans obtained during the arterial phase in a dog in group B (a) 3, (b) 13, (c) 17, and (d) 40 weeks after infection, and (e) photomicrograph of liver specimen harvested at the 40th week. (a-d) Note gradual changes in the calibers of the dilated bile ducts (arrowheads in b-d), the degree of contrast enhancement of the ductal walls, and the THAD in the adjacent parenchyma during the follow-up period. Note that the THAD precedes the other findings at the 3rd week. (e) Histopathologic examination at the 40th week revealed the degeneration of periductal arteries (arrows), as well as the denudation of the ductal epithelium and decreased periductal inflammation. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (162K): [in this window] [in a new window] [Download PPT slide]   Figure 4b. Transverse CT scans obtained during the arterial phase in a dog in group B (a) 3, (b) 13, (c) 17, and (d) 40 weeks after infection, and (e) photomicrograph of liver specimen harvested at the 40th week. (a-d) Note gradual changes in the calibers of the dilated bile ducts (arrowheads in b-d), the degree of contrast enhancement of the ductal walls, and the THAD in the adjacent parenchyma during the follow-up period. Note that the THAD precedes the other findings at the 3rd week. (e) Histopathologic examination at the 40th week revealed the degeneration of periductal arteries (arrows), as well as the denudation of the ductal epithelium and decreased periductal inflammation. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (163K): [in this window] [in a new window] [Download PPT slide]   Figure 4c. Transverse CT scans obtained during the arterial phase in a dog in group B (a) 3, (b) 13, (c) 17, and (d) 40 weeks after infection, and (e) photomicrograph of liver specimen harvested at the 40th week. (a-d) Note gradual changes in the calibers of the dilated bile ducts (arrowheads in b-d), the degree of contrast enhancement of the ductal walls, and the THAD in the adjacent parenchyma during the follow-up period. Note that the THAD precedes the other findings at the 3rd week. (e) Histopathologic examination at the 40th week revealed the degeneration of periductal arteries (arrows), as well as the denudation of the ductal epithelium and decreased periductal inflammation. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (161K): [in this window] [in a new window] [Download PPT slide]   Figure 4d. Transverse CT scans obtained during the arterial phase in a dog in group B (a) 3, (b) 13, (c) 17, and (d) 40 weeks after infection, and (e) photomicrograph of liver specimen harvested at the 40th week. (a-d) Note gradual changes in the calibers of the dilated bile ducts (arrowheads in b-d), the degree of contrast enhancement of the ductal walls, and the THAD in the adjacent parenchyma during the follow-up period. Note that the THAD precedes the other findings at the 3rd week. (e) Histopathologic examination at the 40th week revealed the degeneration of periductal arteries (arrows), as well as the denudation of the ductal epithelium and decreased periductal inflammation. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (182K): [in this window] [in a new window] [Download PPT slide]   Figure 4e. Transverse CT scans obtained during the arterial phase in a dog in group B (a) 3, (b) 13, (c) 17, and (d) 40 weeks after infection, and (e) photomicrograph of liver specimen harvested at the 40th week. (a-d) Note gradual changes in the calibers of the dilated bile ducts (arrowheads in b-d), the degree of contrast enhancement of the ductal walls, and the THAD in the adjacent parenchyma during the follow-up period. Note that the THAD precedes the other findings at the 3rd week. (e) Histopathologic examination at the 40th week revealed the degeneration of periductal arteries (arrows), as well as the denudation of the ductal epithelium and decreased periductal inflammation. (Hematoxylin-eosin stain; original magnification, x40.)

Contrast Enhancement Pattern of Adjacent Parenchyma
Hyperenhancement in the parenchyma adjacent to the dilated bile ducts (finding c) was first observed on arterial phase CT scans at the 2nd (n = 6) or 3rd (n = 4) week of infection in all dogs. Contrast enhancement of the parenchyma appeared as a wedge-shaped area with well-defined straight borders in the peripheral portions of the liver (the typical appearance of THAD). Dilated bile ducts were usually observed at the apices of triangular areas of contrast enhancement. This finding became apparent (ie, was assigned a mild or severe degree score) at the 5th week in all dogs. Until the 13th week, this finding decreased in degree; however, it remained to a mild or severe degree in seven dogs (Fig 3). In group B dogs, this finding completely disappeared in three dogs and decreased in degree in two dogs 3 weeks after treatment (Fig 4). The odds of observing this finding to a mild (++) or severe (+++) degree during the 5th through 13th weeks were 92.33 (95% CI: 8.12, 1,049.7) times higher than the odds during other periods (P < .001).

Visualization of Flukes per Se at CT
Small nodular or linear intraductal materials with higher attenuation than that of bile were noted in all dogs on CT scans obtained during the 5th (n = 5) or 7th (n = 5) week. This finding (finding d) remained until treatment (ie, the 13th week) in nine dogs. These intraductal materials ranged from 2 to 6 mm in width and varied in length to up to 20 mm. They often appeared as U-shaped or double lines at CT; this appearance probably represented the posture of flukes folded in the bile ducts. These lesions corresponded to intraductal nonshadowing echogenic foci or casts observed at surface US performed during sacrifice. Dissection and histopathologic examination revealed that these lesions were flukes or their aggregates (Fig 5). In group B dogs, this finding completely disappeared 3 weeks after treatment in four dogs. The odds of the presence of this finding during the 5th through 13th weeks were 68.80 (95% CI: 6.76, 700.5) times higher than the odds during other periods (P < .001).

View larger version (136K): [in this window] [in a new window] [Download PPT slide]   Figure 5a. Demonstration of flukes at CT in a dog in group A. (a) Transverse CT scan obtained during the portal phase at the 13th week shows nodular intraductal material (arrows) within the bile ducts. Note the inverted U shape (arrowheads) of one such collection of material. (b) Surface US image shows echogenic intraductal material (arrows) in the same area. (c) Section of the portal tract shows the enfolded posture of flukes (arrowheads) within the bile ducts. Note the enlargement of the arteries (solid arrows) as compared with the accompanying portal veins (open arrows). (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (136K): [in this window] [in a new window] [Download PPT slide]   Figure 5b. Demonstration of flukes at CT in a dog in group A. (a) Transverse CT scan obtained during the portal phase at the 13th week shows nodular intraductal material (arrows) within the bile ducts. Note the inverted U shape (arrowheads) of one such collection of material. (b) Surface US image shows echogenic intraductal material (arrows) in the same area. (c) Section of the portal tract shows the enfolded posture of flukes (arrowheads) within the bile ducts. Note the enlargement of the arteries (solid arrows) as compared with the accompanying portal veins (open arrows). (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (152K): [in this window] [in a new window] [Download PPT slide]   Figure 5c. Demonstration of flukes at CT in a dog in group A. (a) Transverse CT scan obtained during the portal phase at the 13th week shows nodular intraductal material (arrows) within the bile ducts. Note the inverted U shape (arrowheads) of one such collection of material. (b) Surface US image shows echogenic intraductal material (arrows) in the same area. (c) Section of the portal tract shows the enfolded posture of flukes (arrowheads) within the bile ducts. Note the enlargement of the arteries (solid arrows) as compared with the accompanying portal veins (open arrows). (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (173K): [in this window] [in a new window] [Download PPT slide]   Figure 6a. (a) Transverse CT scan obtained during the arterial phase 40 weeks after infection and (b) photomicrograph of liver specimen harvested at the 40th week from a dog in group B (the dog represented by the asterisks in Fig 2). (a) In this dog, unlike the dog in Figure 4, CT findings of infection in the acute stage remain to a mild degree. Note the dilatation of intrahepatic bile ducts (arrowheads), contrast enhancement of bile duct walls (arrow), and hyperattenuation in the parenchyma adjacent to the dilated ducts. (b) Histopathologic features of the active stage are seen in the corresponding areas. Note the periductal proliferation of arteries (arrowheads) and the papillary proliferation of the bile duct epithelium, as well as the partial denudation of the ductal epithelium (arrows). (Hematoxylin-eosin stain; original magnification, x40.) Pretreatment fecal examination and worm recovery during autopsy showed positive results (not shown) in this dog.

View larger version (168K): [in this window] [in a new window] [Download PPT slide]   Figure 6b. (a) Transverse CT scan obtained during the arterial phase 40 weeks after infection and (b) photomicrograph of liver specimen harvested at the 40th week from a dog in group B (the dog represented by the asterisks in Fig 2). (a) In this dog, unlike the dog in Figure 4, CT findings of infection in the acute stage remain to a mild degree. Note the dilatation of intrahepatic bile ducts (arrowheads), contrast enhancement of bile duct walls (arrow), and hyperattenuation in the parenchyma adjacent to the dilated ducts. (b) Histopathologic features of the active stage are seen in the corresponding areas. Note the periductal proliferation of arteries (arrowheads) and the papillary proliferation of the bile duct epithelium, as well as the partial denudation of the ductal epithelium (arrows). (Hematoxylin-eosin stain; original magnification, x40.) Pretreatment fecal examination and worm recovery during autopsy showed positive results (not shown) in this dog.

View larger version (154K): [in this window] [in a new window] [Download PPT slide]   Figure 7a. The formation of calcifications in a dog in group B. (a) Transverse CT scan obtained during the portal phase at the 13th week shows linear intraductal materials (arrowheads) within the bile ducts that represent the aggregates of flukes. (b) Transverse unenhanced CT scan obtained at the 34th week shows nodular or rod-shaped calcifications (arrows) scattered along the portal tracts. (c) Contact radiograph of the corresponding area shows microcalcifications. (d) Histopathologic examination revealed the formation of calcific spherules (arrows) along the walls of the bile ducts. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (139K): [in this window] [in a new window] [Download PPT slide]   Figure 7b. The formation of calcifications in a dog in group B. (a) Transverse CT scan obtained during the portal phase at the 13th week shows linear intraductal materials (arrowheads) within the bile ducts that represent the aggregates of flukes. (b) Transverse unenhanced CT scan obtained at the 34th week shows nodular or rod-shaped calcifications (arrows) scattered along the portal tracts. (c) Contact radiograph of the corresponding area shows microcalcifications. (d) Histopathologic examination revealed the formation of calcific spherules (arrows) along the walls of the bile ducts. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (117K): [in this window] [in a new window] [Download PPT slide]   Figure 7c. The formation of calcifications in a dog in group B. (a) Transverse CT scan obtained during the portal phase at the 13th week shows linear intraductal materials (arrowheads) within the bile ducts that represent the aggregates of flukes. (b) Transverse unenhanced CT scan obtained at the 34th week shows nodular or rod-shaped calcifications (arrows) scattered along the portal tracts. (c) Contact radiograph of the corresponding area shows microcalcifications. (d) Histopathologic examination revealed the formation of calcific spherules (arrows) along the walls of the bile ducts. (Hematoxylin-eosin stain; original magnification, x40.)

View larger version (141K): [in this window] [in a new window] [Download PPT slide]   Figure 7d. The formation of calcifications in a dog in group B. (a) Transverse CT scan obtained during the portal phase at the 13th week shows linear intraductal materials (arrowheads) within the bile ducts that represent the aggregates of flukes. (b) Transverse unenhanced CT scan obtained at the 34th week shows nodular or rod-shaped calcifications (arrows) scattered along the portal tracts. (c) Contact radiograph of the corresponding area shows microcalcifications. (d) Histopathologic examination revealed the formation of calcific spherules (arrows) along the walls of the bile ducts. (Hematoxylin-eosin stain; original magnification, x40.)

Histopathologic Examination
In group A dogs, 181–470 (mean, 325) worms were found in the slices of each liver specimen. In group B dogs, no worm was identified in four dogs, and three worms were recovered in the remaining dog (one of the two dogs in which CT findings b–d remained after treatment).

At microscopy, the hematoxylin-eosin–stained microsections of the livers of group A dogs revealed marked changes in the intrahepatic bile ducts and surrounding tissues, including dilatation of the bile ducts, papillary proliferation of bile duct epithelium, periductal infiltration of inflammatory cells, periductal fibrosis, and the presence of adult flukes with a folded posture in the bile ducts. In the areas adjacent to the dilated bile ducts, the proliferation of arteries was observed in the periductal area and the portal tracts; these arteries were relatively larger than the accompanying portal veins. Congestion of hepatic sinusoids was also noted in the same areas (Figs 3, 5).

In group B dogs, histopathologic examination revealed denudation of the ductal epithelium, decreased periductal inflammation, hyalinization of the periductal stroma with decreased numbers of fibroblasts, and degeneration of the periductal arteries (Fig 4). Hypertrophied arteries were not observed in the portal tracts of group B dogs. In two dogs with calcification at CT, there were transitional calcification areas in degenerating ductal epithelium, and the formation of solid calcific spherules was noted along the walls of the bile ducts (Fig 7). In one dog, histopathologic features of the active stage of infection remained in areas corresponding to the regions at which CT findings b–d remained after treatment (Fig 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CT yields comprehensive diagnostic information about the entire biliary tree, and CT findings such as diffuse uniform dilatation of the small intrahepatic bile ducts without a focal obstructing lesion have been considered to be pathognomonic of clonorchiasis (18,22,24,25). Dilatation of the bile ducts extends up to the periphery of the liver, and sometimes the peripheral portions of the bile ducts dilate to a greater degree, resulting in a clublike appearance.

Regarding the changes in ductal caliber during the follow-up period, our results are in agreement with those of previous experimental studies involving US that revealed that ductal dilatation is detected as early as the 4th week after infection (10), that ductal dilatation is most apparent in the 5th week of infection (26), and that the bile ducts remain dilated or slightly decompressed until the 9th to 22nd week (10,17,26) and decompressed to some degree for 18 months after treatment (12).

Contrast enhancement of the ductal walls has been reported in a small number of cases and is believed to reflect marked periductal inflammation (22). However, Choi et al (25) reported that CT failed to depict this finding in their study. This inconsistent observation has been attributed to differences in the severity of infection (22). On the basis of the results of the present study, there may be two other explanations for this inconsistency. First, the activity of the disease (ie, whether it runs a natural course or is affected by treatment) is an important factor in contrast enhancement of the ductal walls. Contrast enhancement of the ductal walls was seen only in the acute phase of the disease in our study and disappeared or markedly decreased after treatment. Second, the discrepancy in previous studies may be attributable to the use of conventional CT scanning, because contrast enhancement of the ductal walls was pronounced only on the arterial phase CT scans in the present study. In our study, histopathologic examination revealed marked proliferation of the periductal arteries, as well as periductal inflammation, in group A dogs and degeneration of periductal arteries and decreased periductal inflammation in group B dogs. These findings suggest that arterial proliferation could be the mechanism of ductal wall contrast enhancement.

Although several possible mechanisms of THAD have been described in various malignant diseases, including hepatocellular carcinoma (27) and inflammatory diseases such as hepatic abscess (28), it has not been reported that THAD can appear in a parasitic disease such as clonorchiasis. Our study findings revealed histopathologic changes in the intrahepatic vasculature in clonorchiasis, including the proliferation and enlargement of arteries in the portal tracts and periductal areas and the congestion of hepatic sinusoids. Together with the histopathologic vascular changes, CT findings of THAD and increased arterial flow at the ductal walls might represent a possible mechanism for the altered intrahepatic hemodynamics in clonorchiasis. However, the actual mechanism of the THAD observed in this study is not clear despite pathologic correlation, and its clinical importance has yet to be determined. Nevertheless, results of our study indicate that intrahepatic hemodynamic alteration begins during the early acute phase of infection and then changes depending on the disease course, probably reflecting the degree of acute inflammation. In addition, these hemodynamic alterations can be objectively imaged with dynamic CT.

In general, CT has been considered to be of little value in the delineation of flukes (18,25). To the best of our knowledge, the visualization of flukes per se at CT has not previously been reported except by Lim (29). Lim (29) found that CT performed in some patients depicted high-attenuating foci scattered within the bile ducts and presumed that these aggregates were flukes. In the present study, small nodular or linear intraductal lesions were identified on most CT scans during active infection and were confirmed to be flukes per se. The worms ranged from 2 to 6 mm in width and varied in length to up to 20 mm at CT, as described in previous cholangiographic studies (18,30–33); this contradicts the descriptions of much larger worm sizes in earlier reports in the parasitology literature (3).

The formation of calcification in cured clonorchiasis has not yet been reported to our knowledge. In our experience, so-called benign calcifications or calcified granulomas with a branching distribution are occasionally observed at US or CT in human cases of clonorchiasis, and our results suggest a possible mechanism for the formation of these calcifications. Calcification seems to be a degenerative process of the proliferating ductal epithelium after treatment.

Although our study results demonstrated that dynamic CT findings reflect the disease activity of clonorchiasis in dogs, it is not clear whether these CT findings can enable accurate discrimination of active cases of human clonorchiasis from cured ones, and the diagnostic accuracy of these findings has yet to be determined. It is not clear that contrast enhancement of the ductal walls and THAD will be consistently observed in human cases, which typically involve more chronic and milder infections, because in our study the aforementioned CT findings decreased after the 7th week even before treatment, as illustrated in Figure 2. These CT findings might reflect acute inflammation in the initial phase of infection and do not necessarily suggest the presence of live worms. In addition, contrast enhancement of the ductal walls and THAD might have been exaggerated in this study because a relatively higher dose of intravenous contrast material was injected compared with the usual human dose.

It is also not clear whether the demonstration of calcifications is direct evidence of cured cases, although calcifications appeared exclusively in cured dogs in our study. To determine if dynamic CT can enable discrimination between active clonorchiasis cases and cured cases in humans, further research is necessary, including a comparison of CT findings between active human cases and cured cases and experimental studies with longer periods of follow-up.

It is encouraging that in the present study CT findings were positive in the acute phase as early as the 2nd week after infection, as well as in minimal infection after partial treatment. This rate of detection with CT is better than that offered with fecal examination (because eggs can only be found in stool about 25 days after infection [3]) and is comparable to the detection rate with high-resolution US, which can reveal minimally infected cases in the 3rd week after infection (26). However, early detection has little clinical importance, because most human infections are chronic (3,18).

In conclusion, dynamic CT findings of experimentally induced acute clonorchiasis in dogs consist of (a) dilatation of the peripheral intrahepatic bile ducts, (b) contrast enhancement of the ductal walls, (c) THAD in the parenchyma adjacent to the dilated ducts, and (d) visualization of flukes within the bile ducts. After treatment, these findings disappear or decrease in degree, and (e) tiny nodular calcifications appear at CT. In our study, these dynamic CT findings correlated with histopathologic findings and fecal examination results. Disease activity in experimental canine clonorchiasis can be assessed with dynamic CT.

Practical applications: The results of this study demonstrate that dynamic CT not only reveals the disease activity in clonorchiasis but also enables the detection of mild infection in partially treated cases. Therefore, it may be possible to discriminate actively infected cases from cured cases with dynamic CT. Although dynamic CT will not replace fecal examination as the primary diagnostic examination for clonorchiasis, the diagnosis of active clonorchiasis at CT is important because clonorchiasis is so prevalent in eastern Asia (1–3,9) that it is commonly diagnosed incidentally at radiologic examinations and because clonorchiasis is closely associated with cholangiocarcinoma and recurrent pyogenic cholangitis (1,24,29). Moreover, the results of this study give answers to the long debates in the literature about CT findings and clearly demonstrate the histopathologic basis of these findings. The results of this study also indicate that the THAD observed on dynamic CT images may be associated with arterial proliferation at the portal tracts and is reversible in inflammatory disease of the liver.

	ACKNOWLEDGMENTS

 
We thank Hyuk Jae Choi, Myoung Soo Kim, RT, Hyun Jung Lee, RT, and Shunyu Li, MD, for technical assistance in animal preparation and fecal examination. We are grateful to YunHee Choi, PhD, Department of Preventive Medicine, Seoul National University College of Medicine, for her help with the statistical analysis.

	FOOTNOTES

 
Abbreviation: THAD = transient hepatic attenuation difference

Author contributions: Guarantor of integrity of entire study, B.I.C.; study concepts, K.H.L., J.K.H.; study design, K.H.L., S.T.H.; literature research, S.H.K., K.H.L.; experimental studies, C.J.Y., K.H.L.; data acquisition, C.J.Y., K.H.L.; data analysis/interpretation, K.H.L., S.L., J.K.H., S.T.H., S.H.K.; statistical analysis, K.H.L.; manuscript preparation, K.H.L., C.J.Y., S.L.; manuscript definition of intellectual content, K.H.L., S.T.H., J.K.H.; manuscript editing, K.H.L., S.H.K.; manuscript revision/review, S.T.H., J.K.H., B.I.C.; manuscript final version approval, B.I.C., S.T.H.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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