ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 14, No. 3 Copyright © 1984, Institute for Clinical Science, Inc.

Pathology and Immunology of of the TSIEH SUN, M.D. Department of Laboratories, North Shore University Hospital, Manhasset, NY 11030 and Department of Pathology, Cornell University Medical College, New York, NY 10021

ABSTRACT The existence of cases among the Asian immigrants and the major clinical and pathologic features encountered in those patients is emphasized. Owing to the longevity of the parasite, clinical symptoms may occur long after endemic exposure. The common manifestations among the immigrants are recurrent pyogenic cholangitis and pancreatitis. The relationship between clonorchiasis and and the possible pathogenesis of this tumor are discussed. The specific immuno­ logic phenomenon in clonorchiasis is described.

Epidemiology takes about one month for the metacer- caria to mature, when Clonorchis eggs Clonorchiasis is endemic in the Far can be detected in stools. Eggs hatch East, mainly Hong Kong, , , only after ingestion by a suitable Japan, , and .6 It was es­ . In the snail host, the hatched mir­ timated that 19 million people were in­ acidium passes through stages of sporo- fected in endemic areas.22 However, the cyst, redia, and cercaria. The cercariae incidence of clonorchiasis dropped mark­ invade the flesh of a suitable fish host edly owing to environmental changes in and become encysted metacercariae. Ap­ recent years. In Japan, for instance, proximately 100 species of fresh water water pollution eliminates most of the fish have been incriminated as the inter­ snail hosts of Clonorchis sinensis.22 The mediate host of C. sinensis. On the other composition of manure before use in hand, the snail host of C. sinenis is more China kills Clonorchis ova in stools, thus specific: only of the genus Parafos- interrupting the life cycle.22 sarulus, Bulimus, Alocimua and Melan- The infection is caused by consump­ oides can act as intermediate host. Since tion of raw, infected fish. The metacer- these snails have not been found in cariae in fish flesh are released in the North America, clonorchiasis will not be­ whence the parasite migrates come endemic in the United States in along the common duct and finally spite of the influx of Asian refugees. stays in the intrahepatic bile ducts.23 It However, Americans may obtain clon- 208 0091-7370/84/0500-0208 $01.20 © Institute for Clinical Science, Inc. CLONORCHIASIS 209 orchiasis through long-term and short­ Pathology term visits to endemic areas.21 The long life span of C. sinensis (20 to The major pathologic findings are seen 30 years) creates a problem for the Asian in the liver. The mechanical injury immigrants. There are well-documented caused by the suckers of the parasite and cases in which the patients developed chemical stimulation by its metabolic clinical symptoms many years after products may contribute to the changes leaving the endemic area.16 Most of the of the epithelium of the bile ducts.2' The patients reported are from Hong Kong. two basic morphologic changes are ade­ Less frequently seen are patients from nomatous proliferation and goblet cell China and Korea. Laotian refugees usu­ metaplasia (figure I).6,7’8 In chronic in­ ally carry viverrini rather fections, the adenomatous tissue is grad­ than C. sinensis. However, the ova of ually replaced by fibrous tissue, thus these two parasites are practically indis­ causing thickening of bile ducts which tinguishable. Accordingly, cases of clon- can be visible grossly. orchiasis are most frequently seen in The most common complication in large cities where there are many clonorchiasis is recurrent pyogenic chol­ Chinese immigrants, such as New York angitis (RPC) or oriental cholangiohepa- City,10 Los Angeles,28 and Montreal.15 titis (figure 2).6-22 This is also the most Even in South Carolina, seven to 43 important clinical finding in immigrants cases of clonorchiasis were reported an­ in North America, leading to the diag­ nually since 1978.13 nosis of clonorchiasis.1-2,717’28 Owing to

F ig u r e 1. An adult C. sinensis in an intrahepatic . The sucking force of the ventral sucker (V) obviously causes mechanical injury to the ductal epithelium with resultant desquamation and superficial ulceration. Adenomatous proliferation of epithelium, as shown here, is the consequence of such damage. Hematoxylin and eosin. (X200). From Sun22 with permission. 210 SUN

iâ JfWBW iBF&k.'*'' JP iBu5r M HS^aW «■ F ig u r e 2 . A case of recurrent pyogenic cholangitis showing heavy cellular infiltration of a denuded bile duct. The lumen contains inspissated bile mixed with Clonorchis ova. Hematoxylin and eosin. ( X 160). From Sun22 with permission. goblet cell metaplasia of the bile duct ep­ beyond the bile duct, causing perichol­ ithelium, the bile contains high contents angitis and periductal fibrosis. Liver ab­ of mucous secretion. This mucinous bile scess may occur if the parenchyma is in­ and the existence of the parasite and its volved by the infectious process. A ova in the bile duct cause cholestasis and postnecrotic or biliary may de­ furnish a favorable environment for sec­ velop on this basis; however, unlike ondary bacterial infection. The causative , “pipe stem” cirrhosis is agent is usually Escherichia coli as­ never seen in uncomplicated cases. An­ cending from the intestine. The ova and other complication of RPC is stricture of bacterial colonies may thus form the the left hepatic duct which may or may nidus of an intrahepatic which not be accompanied by atrophy of the left is usually muddy. The lack of primary hepatic lobe.28 The most severe sequela stone in the is characteristic is hepatic coma owing to extensive liver of this situation. Owing to cholangitis and damage. partial biliary obstruction, the patients Pancreatitis is another complication usually show repeated episodes of ab­ which may present as a major clinical dominal pain, , , and he­ manifestation in Asian immigrants.16 A patomegaly, which are typical for RPC. Chinese immigrant in New York devel­ This clinical entity can be diagnosed by oped 25 years after en­ a cholangiogram demonstrating the demic exposure.16 The patient did not re­ markedly dilated intrahepatic bile ducts spond to symptomatic therapy and with partial filling defects representing recovered only after anthelminthic stones or a mass of flukes.7 treatment. In Hong Kong, over one- The consequence of RPC is multiple. third of clonorchiasis cases had pan­ Most frequently, the infection spreads creatic involvement.4 However, the pa­ CLONORCHIASIS 211 tient may not have any clinical evidence epidemiologic statistics, i.e., cholangio­ of pancreatitis. On the other hand, clon- carcinoma is more frequently seen in orchiasis is the causative agent in 83 per­ clonorchiasis endemic areas than non-en- cent of patients with pancreatitis of un­ demic areas, and Clonorchis infection is known etiology in Hong Kong.12 This seen in 94.7 percent of cholangiocarci­ Clonorchis-related pancreatitis usually noma cases as compared to 31.7 percent develops one to three hours after a rich in a control population in Hong Kong.3 meal, which may have stimulated excess Cholangiocarcinoma has also been exper­ flow of pancreatic juice in a partially ob­ imentally produced in infected structed pancreatic duct, leading to au­ with Clonorchis.89 The fact that cholan­ todigestion.12 The pathology of pan­ giocarcinoma usually develops in the creatic clonorchiasis is similar to the second order intrahepatic bile duct hepatic lesion, namely, adenomatous where Clonorchis is located and that hyperplasia of ductal epithelium (figure adenomatous proliferation of ductal epi- 3).6 Squamous metaplasia is also fre­ thelim can be seen side by side with ma­ quently demonstrated and is character­ lignant changes (figure 4) is also suppor­ istic of pancreatic clonorchiasis.22 When tive of the relationship between these acute pancreatitis occurs, features of two entities. acute inflammation are present. The exact pathogenesis of cholangio­ The most grave consequence of clon­ carcinoma is still unknown. It seems orchiasis is cholangiocarcinoma.3,5,14 The likely that Clonorchis infection is only a causative role of clonorchiasis in the de­ predisposing factor by inducing prolifer­ velopment of this tumor is supported by ation of the epithelium of bile ducts.5

F ig u r e 3 . Two sections of an adult C. sinensis in a pancreatic duct which is partly denuded with maked adenomatous hyperplasia. Hematoxylin and eosin. (X50). From Sun22 with permission. 212 sun­

s' -- - * V >v> «;; ■ #:/& ■. >*• #«f F ig u r e 4 . A case of cholangiocarcinoma showing a cross section of an adult C. sinensis in the lumen of a cancerous bile duct. Note the coexistence of normal epithelium and malignant cells. Hematoxylin and eosin. (X50). From Sun22 with permission.

The proliferating epithelium may be sus- which are found commonly in preserved ceptable to the action of (s) foodstuffs in the Far East.5,14 In southern present at levels insufficient to induce China and Hong Kong, it has been found cholangiocarcinoma in non-infected in­ in salted fish and dried shrimps. Al­ dividuals. Hou has suggested that the though the pathogenesis of cholangio­ metabolic or degenerative products of carcinoma is still inconclusive, it is rea­ the parasite and/or a bile component al­ sonable to assume that the etiology is tered chemically by the parasite can be multifactorial in origin. the origin of carcinogenic substances.8,9 CZcmorc/i/s-associatedcholangiocarci- However, Flavell argues that if this is noma is characterized by prominent true, the carcinogen(s) should be accu­ mucin secretion. It is also frequently ac­ mulated in the gallbladder and induce companied by extensive fibrosis. Al­ tumor at this site.5 Another possible car­ though the tumor can be multicentric, it cinogen is aflatoxin, a mycotoxin com­ is usually located near the hilum of the monly found in fungus infected food­ liver. The parasite is frequently found in stuffs.3 Although aflatoxins can produce the bile duct which is surrounded by the cholangiocarcinoma in primates experi­ tumor. mentally, they are more frequently Unlike schistosomiasis in which the linked to .5 ova are the major cause of pathologic le­ More recently, attention has been drawn sions, Clonorchis eggs are inert as they to the carcinogenic effect of nitrosamines are impermeable and the antigens se­ CLONORCHIASIS 213 creted by the miracidium do not reach blood vessels, leading to pulmonary hy­ the host tisue. In a study by Sun of 525 pertension. Clonorchis-infected cases, only three cases showed egg-induced lesions: eosin­ Immunology ophilic granuloma in the gallbladder (figure 5), giant cell reaction in the liver, The immunologic response in Clon­ and pulmonary embolism.19 An addi­ orchis infected patients is an interesting tional case of egg-granuloma in the portal phenomenon. Humoral antibody can be areas of the liver was reported recently detected by different techniques such as by Sun.21 In these cases, eggs were immunofluorescent technique, gel-dif- partly damaged by the acute inflamma­ fusion, indirect hemagglutination, and tory process; thus, the antigens released circum-adult precipitation.18 These anti­ by the miracidia induced tissue reac­ bodies may show detrimental effects on tions. the adult worms in vitro,20 but they are A rare complication of clonorchiasis is not protective to the host in vivo.24 pulmonary hypertension.11 This condi­ Therefore, patients may contract re­ tion is usually seen in patients with RPC peated , and hundreds or thou­ in whom infected necrotic tissue from sands of flukes can be recovered from the liver is carried by the blood stream one patient. The non-protective nature to the lung and causes repeated mi­ of these antibodies is also evidenced by croembolization of small pulmonary comparing the worm-burden in artifi-

F ig u r e 5. A case of eosinophilic granuloma of gallbladder caused by Clonorchis infection. Note Clonorchis ova are seen inside and outside multinucleated foreign body giant cells. The background is infiltrated by eosinophils and histiocytes. Hematoxylin and eosin. (x800). 214 SUN

T A B L E I

Postulated Mechanism of Antibody Production in Clonorchiasis

Antibody Forming Site Blood Circulation ^ Regurgitate , B ile ,

P rim a ry " M ain Immunological Antigenic Stimulation Antigenic Stimulation Antigenically Inert Role ?Metabolic Products Metabolic Products from from Digestive System Digestive and Reproductive Systems Developmental M etacercaria- A d u lt ------► Egg S tag e T e s ts Fluorescent antibody Fluorescent antibody Fluorescent antibody (Positive) (Positive.) (Negative) Gel-diffusion Gel-diffusion (Positive) (Negative) I n d ir e c t I n d ir e c t Haemagglutination Haemagglutination (Positive) (Negative) Circum-adult Circum-oval Precipitation Precipitation (Positive) (Negative) Inoculation No granuloma formation cially immunized and non-immunized carbohydrase, and esterase.26 The meta­ animals, which showed no statistically sig­ bolic antigens are mainly from mature nificant difference between these two adult worms, but metacercariae also groups.24 show weak reaction to infected human or The antibodies detected in patients animal sera.25 Since antibodies are de­ and experimentally infected animals are tected four weeks after infection in rab­ mainly induced by the metabolic prod­ bits, guinea , and , i.e., soon after ucts of the parasite.24 Somatic antigens the metacercariae have developed into are not effective antigens, and egg anti­ the adult form, the initial antigenic stim­ gens are not responsible for antibody ulation is probably coming from meta­ production in patients and experimental cercariae. The relationship between the animals. By immunofluorescent studies, various developmental stages and anti­ the gastrointestinal tract and the repro­ body production is illustrated in table I. ductive organs of the parasite react with antibodies from patients and infected an­ Summary imals, thus identifying the sources of metabolic products that elicit antibody Clonorchis sinensis is still an impor­ formation.25 The exact effective fraction tant in Asia, affecting millions in metabolic products of the parasite was of patients. Because of its long life span, not identified, but they contain protease, clonorchiasis can be seen in Asian im­ CLONORCHIASIS 215 migrants in North America long after Microembolic pulmonary hypertension in pyo­ their endemic exposure. When patients genic cholangitis. Brit. Med. J. 1:22-24, 1968. 12. M cFadjean, A. J. S., and Y e u n g , R. T. T .: from endemic areas show symptoms of Acute pancreatitis due to Clonorchis sinensis. recurrent pyogenic cholangitis, biliary Trans. Roy. Soc. Trop. Med. Hyg. 60:466-470, obstruction, or acute pancreatitis, clon- 1966. 13. N o b l e t , G. P. and H u n t e r , W. H .: Clonor- orchiasis should be included in the dif­ chiasis/ in South Carolina. J. ferential diagnosis. A diagnosis can be South Carolina Med. Assoc. 79:75- 79, 1983. 14. S c h w a r tz , D. A.: Helminths in the induction of easily established by stool examination : , Clonorchis si­ for eggs, but sometimes duodenal nensis and cholangiocarcinoma. Trop. Geogr. drainage is necessary for the detection of Med. 32:95-100, 1980. eggs. In the case of RPC, a cholangio- 15. S e a h , S . K. K.: Intestinal parasites in Chinese immigrants in a Canadian city. J. Trop. Med. gram showing dilated intrahepatic bile Hyg. 76:291-293, 1973. ducts with partial filling defects is char­ 16. Shungar, R. A. and R y a n J. J.: Clonorchis si­ nensis and pancreatitis. Am. J. Gastroenterol acteristic and diagnostic for those from 64:400-403, 1976. the endemic areas. In chronic cases, pra­ 17. S u lliv a n , W. G. and K o e p , L. J.: Common bile ziquantel can be used to eliminate the duct obstruction and cholangiohepatitis in clon­ orchiasis. J. Am. Med. Assoc. 243:2060-2061, flukes.13 In acute cases, however, sur­ 1980. gical intervention should be consid­ 18. S u n , T.: Clonorchiasis in Hong Kong. Proceed­ ered.28 ings of the IVth Southeast Asian Seminar on Par­ asitology and Tropical Medicine. Schistosomiasis and Other Snail-Transmitted . References Harinasuta, C., ed. SEAMEC Central Coordi­ nating Board for Tropical Medicine and Public 1. A m e r e s , J. P., L e v i n e , M. P., and D e B lasi, Health, Bangkok, 1969, pp. 243-252. H. P.: Acalculous clonorchiasis obstructing the 19. S u n , T.: The non-antigenicity of intact ova of common bile duct. A case report and review of Clonorchis sinensis. J. Med. Microbiol. 2:358— the literature. Am. Surg. 42: 170-172, 1976. 360, 1969. 2. B a k e r , M. S., B a k e r , B. H., and Woo, R.: Bil­ 20. S u n , T.: The in vitro action of antisera on the iary clonorchiasis. Arch. Surg. 114:748, 1979. adults of Clonorchis sinensis. Trans. Roy. Soc. 3. B a l a m a r i c , J.: Intrahepatic bile duct carcinoma Trop. Med. Hyg. 63:582-590, 1969. and C. sinensis infection in Hong Kong. Cancer 21. S u n , T.: Clonorchiasis: A report of four cases and 31:468-473, 1973. discussion of unusual manifestations. Am. J. 4. C h a n , P. N. and T e o h , T. B.: The pathology of Trop. Med. Hyg. 29:1223-1227, 1980. Clonorchis sinensis of the pancreas. 22. S u n , T.: Clonorchiasis and opisthorchiasis. Pa­ J. Pathol. Bacteriol. 93:185-189, 1967. thology and Clinical Features of Parasitic Dis­ 5 . F l a v e l l , D. J.: Liver-fluke infection as an ae- eases. Sun, T., ed., New York, Masson, 1982, tiological factor in bile-duct carcinoma of man. pp. 243-252. Tran. Roy. Soc. Trop. Med. Hyg. 75:814-824, 23. Sun, T., C h o u , S.T., and G ib s o n , J. B.: Route of 1 9 8 1 . Clonorchis sinensis to the mammalian liver. Exp. 6. G i b s o n , J. B. and Su n , T.: Clonorchiasis. Pa­ Parasit. 22:346-351, 1968. thology of Protozoal and Helminthic Diseases. 24. S u n , T. and G ib s o n , J. B.: Antigens of Clonor­ Marcial-Rojas, R. A., ed. Baltimore, Williams & chis sinensis in experimental and human infec­ Wilkins, 1971, pp. 546-566. tions, an analysis by gel-diffusion technique. 7. Ho, C. S. and W e s s o n , D. E.: Recurrent pyo­ Am. J. Trop. Med. Hyg. J8:241-252, 1969. genic cholangitis in Chinese immigrants. Am. J. 25. S u n , T. and G ib s o n , J. B.: The sites of antigen Roentgenol. Rad. Ther. Nucl. Med. 122:368- formation in different developmental stages of 374, 1974. Clonorchis sinensis. Jpn. J. Med. Sei. Biol. 8. Hou, P. C.: Primary carcinoma of bile duct of 22:263-271, 1969. the liver of the (Felis catus) infected with 26. S u n , T. and G ib s o n , J. B.: Metabolic products of Clonorchis sinensis. J. Pathol. Bacteriol. 37:239- adult Clonorchis sinensis: Their composition and 244, 1964. antigenic potential. J. Helminthol. 43:395-402, 9. Hou, P. C.: Hepatic clonorchiasis and carcinoma 1969. of the bile duct in a . J. Pathol. Bacteriol. 27. S u n , T. and T u n g - M a , L.: Ultrastructural 89:365-367, 1965. changes in the biliary tracts of guinea pigs in­ 10. K a m m e r e r , W. S., V a n d e r D e c k e r , J. D., fected with Clonorchis sinensis. J. Pathol. K e i t h , T. B., and M o t t , K. E.: Clonorchiasis in 109:291-293, 1973. New York City Chinese. Trop. Doctor 7:105- 28. Y e l l i n , A. E. and D o n o v a n , A. J.: Biliary li- 106, 1977. thiasis and helminthiasis. Am. J. Surg. 142:128- 11. L ai, K. S., M c F a d j e a n , A. J. S., and Y e u n g , R.: 136, 1981.