Chronic Opisthorchis viverrini Infection and Associated Hepatobiliary Disease Is Associated with Iron Loaded M2-like Macrophages

Chronic Opisthorchis viverrini-induced hepatobiliary disease is associated with significant leukocyte infiltration, including activated macrophages; however, the polarization of infiltrating macrophages remains to be fully characterized. In this study, we characterized macrophage polarization and phenotype in chronic O. viverrini-induced hepatobiliary disease in humans and hamsters using gene expression and histochemical analysis. Chronic O. viverrini infection and associated hepatobiliary diseases were associated with iron loaded M2-like macrophages in both humans and hamsters. This study provides suggestive evidence that iron loaded M2-like macrophages promote hepatobiliary disease in chronic O. viverrini infection.

Liver Flukes: the Malady Neglected

Liver fluke disease is a chronic parasitic inflammatory disease of the bile ducts. Infection occurs through ingestion of fluke-infested, fresh-water raw fish. The most well-known species that cause human infection are Clonorchis sinensis, Opisthorchis viverrini and Opisthorchis felineus. Adult flukes settle in the small intrahepatic bile ducts and then they live there for 20-30 years. The long-lived flukes cause long-lasting chronic inflammation of the bile ducts and this produces epithelial hyperplasia, periductal fibrosis and bile duct dilatation. The vast majority of patients are asymptomatic, but the patients with heavy infection suffer from lassitude and nonspecific abdominal complaints. The complications are stone formation, recurrent pyogenic cholangitis and cholangiocarcinoma. Approximately 35 million people are infected with liver flukes throughout the world and the exceptionally high incidence of cholangiocarcinoma in some endemic areas is closely related with a high prevalence of liver fluke infection. Considering the impact of this food-borne malady on public health and the severe possible clinical consequences, liver fluke infection should not be forgotten or neglected.

Association of parasitic infections and cancers.

Recent advances in the fields of molecular biology, epidemiology and infectious diseases have led to significant revelations to clarify the relationship between cancer and infective agents. This article reviews the relationship between parasitic infections and carcinogenesis and the possible mechanisms involved. Few parasites, e.g., Schistosoma haematobium and Opisthorchis viverrini have been found to be strongly associated with bladder cancer and cholangiocarcinoma respectively. The evidence for the association of several other parasites and cancers has also been postulated.

Experimental investigation of opisthorchiasis-associated cholangiocarcinoma induction in the Syrian hamster - pointers for control of the human disease.

Appropriate animal models for specific diseases in man can facilitate elucidation of mechanisms underlying tumour development and allow potential interventions and therapeutic regimens to be tested in vivo before consideration for use in the human situation. In the North-east of Thailand exceptionally high levels of cholangiocellular carcinomas (CCCs) are encountered, related to infestation with Opisthorchis viverrini liver flukes. The Syrian hamster can also be infected with metacercariae of the fluke and heavy loads of parasites cause the development of cirrhotic livers. While the presence of flukes alone does not give rise to neoplasms, large yields of cholangiofibrotic lesions and CCCs can be readily induced with additional carcinogenic insult. While removal of the parasite with the antihelminthic drug Praziquantel can protect against carcinogenesis, this is dependent on the timing of the drug administration and the efficacy of application to the human situation remains to be confirmed. The available information would suggest that interest needs to be concentrated on potential chemopreventive agents which could be administered to individuals at high risk. Furthermore, understanding of the genesis of CCCs and the characteristics of preneoplastic lesions, again as assessed in the animal model, might allow novel approaches to identification of early stage cases and effective surgical intervention.

Prevalence of Opisthorchis viverrini infection among villagers harboring Opisthorchis-like eggs.

The precise occurrence of Opisthochis viverrini infection in humans, who were positive for Opisthorchis-like eggs in a stool examination, was determined using the potassium permanganate staining method. In the 68 specimens examined, there were more individual O. viverrini eggs (38.24%) than singular Haplorchis taichui eggs (29.41%). One-fourth of the total specimens contained a mixed infection of O. viverrini and H. taichi eggs. The median ratio of O. viverrini: H. taichui eggs in mixed infection cases was 2.29 (min = 1, max = 17.5). It is suggested that chemotherapy with praziquentel treatment should be given to patients who are positive for O. viverrini-like eggs.

Establishment and characterization of cell lines from liver fluke-associated cholangiocarcinoma induced in a hamster model.

Cholangiocarcinoma (CCA) is a relatively rare tumor that occurs primarily in tropical countries and particularly in those with a high incidence of liver fluke infection. A hamster model for a liver fluke-associated CCA has been described previously. In the present study, hamster cholangiocarcinoma cell lines were established and characterized in order to obtain information regarding diagnostically useful tumor marker which could shed light for a future investigation for human cholangiocarcinoma. Two related cell lines, one from the original intrahepatic bile duct tumor and one from an allotransplanted tumor, were established. The established cell lines were found to have population doubling times of 31 and 26 hours respectively, and were maintained in Ham's F12 medium supplemented with 10% fetal bovine serum for over 80 passages. The cell monolayers were subjected to scanning and transmission electron microscopic study and found to have ultrastructural characteristics, including cytoplasmic lumens, consistent with those of adenocarcinoma cells of epithelial origin. An immunoperoxidase study using monoclonal antibodies (MAbs) specific for tumor antigens showed the cytoplasm and membrane of both cell lines to be positive. These antigens were also secreted in soluble form into the culture medium, judging from polyacrylamide gel electrophoresis in the presence of SDS and from immunoblot analyses. Different lines of evidence presented suggested that a 200 kDa glycoprotein produced and secreted by the tumor cell lines could be considered a cholangiocarcinoma-associated marker which has diagnostic potential.

Development of immune-complex glomerulonephritis and amyloidosis in Syrian golden hamsters infected with Opisthorchis viverrini.

Renal disease associated with Opisthorchis viverrini infection was investigated in Syrian golden hamsters. On the fourth week after infection with 100 viable metacercariae; anti-tegumental membrane antibodies were detected in the sera by immunofluorescence antibody technic and by enzyme-linked immunosorbent assay. Six weeks after infection tegumental and anti-tegumental membrane immune-complex and amyloid fibrils were found in the glomeruli. Amyloid was characterized to be AA protein. Acute proliferative glomerulonephritis associated with the brightest immune-complex deposits developed in week 8 after infection. Intensity of immune-complexes in all glomeruli were reduce gradually thereafter and replaced by amyloid. Progressive obsolescence of the glomeruli, tubular atrophy, interstitial inflammation and fibrosis associated with massive proteinuria and deterioration of renal function appeared in week 10 after infection toward the end of the experiment in week 38 after infection.

A histopathologic study of 61 cases of peripheral intrahepatic cholangiocarcinoma.

The histopathological study of 61 cases of peripheral intrahepatic cholangiocarcinoma was reported. Of the 5 autopsied livers and 13 surgically resected livers, 89 per cent showed massive gross appearance, 94 per cent contained Opisthorchis viverrini or showed evidence of previous opisthorchiasis. Gall stones were found in 7 cases (11.5%), common bile duct stones were found in one case. The tumors were classified into well differentiated, less differentiated and uncommon types (adenosquamous, anaplastic and mucinous) which were found in 14.66, 70 and 13.66 per cent respectively. Of the 61 liver specimens, 92 per cent produced mucin and 53 per cent had more than one histological types. The important routes of intrahepatic spreadings were lymphatic involvement (90%) and permeation into the portal connective tissue (80%). One case of combined hepato-cholangiocarcinoma was observed in a cirrhotic liver containing Opisthorchis viverrini and positive HBsAg.

Cholangiocarcinoma masquerading as liver abscess.

A 60-year-old man from Eastern Thailand was admitted to hospital because of right upper quadrant abdominal pain and fever. Ultrasonographic examination revealed two cavitary lesions in the right lobe of the liver. Needle aspiration obtained 110 ml of anchovy sauce-like pus which showed no bacteria on gram stain and routine culture. Serological test for E. histolytica antibody was negative. Initially, the patient responded well to metronidazole. Two weeks later, the symptoms recurred and sonography revealed one large cavitary lesion with three adjacent locules in the right lobe of the liver. Repeated needle aspiration again showed anchovy sauce-like pus which grew Enterobacter agglomerans. O. viverrini ova were detected in the stool. Laparotomy revealed histologically proven cholangiocarcinoma. This report indicates that O. viverrini infection associated with CCC can masquerade as liver abscess.

Opisthorchis viverrini eggs and adult flukes as nidus and composition of gallstones.

Three cases of opisthorchiasis with calculous cholecystitis was presented. The diagnosis of gallstones was made before praziquantel therapy in 2 cases, and 4 years after treatment in one case. The patients underwent cholecystectomy. Numerous O. viverrini eggs and one dead fluke were found in the bile in one case. A single stone was recovered from each gallbladder. All stones contained O. viverrini eggs. Fragments of flukes were also present in two cases. The stones consisted of a mixture of bilirubin pigment and calcium salts.

Incidence of bacteremia in patients with opisthorchiasis during recurrent cholangitis.

A total of 257 haemocultures were performed in 50 patients with opisthorchiasis when they presented signs and symptoms of biliary tract infection. 19 patients showed positive haemoculture. There are no significant relationship between the age of the patient and the incidence of positive haemoculture. Septic shock occurred in 5 patients, one patient died. Out of 221 aerobic cultures, 14% were positive and of the 36 anaerobic cultures 11% were positive. The most common organism was Staphylococcus followed by Klebsiella and Bacillus spp. Anaerobic bacterias were Streptococcus spp. Clostridia spp. was not found in this study. Most organisms were sensitive to cefotaxime, cephalothin, kanamycin and chloramphenicol, and the least sensitive to ampicillin.

Clinical features in severe opisthorchiasis viverrini.

Clinical features of severe opisthorchiasis were studied in 88 patients. The ratio of males to females was 6.3 : 1; 75% were over 40 years old. The presenting symptoms were obstructive jaundice, 25% associated with secondary infection of biliary system; cholangitis and cholecystitis; intraabdominal mass, which was enlarged liver; 18% had palpable gallbladder; 18% had adenocarcinoma of the bile duct. High bilirubin was found in 46% of cases, high alkaline phosphatase in 80%; elevation of serum transaminase in 78% and low serum albumin in 62% of patients. There was no correlation between severity of the disease and the faecal egg output. The obstructive jaundice patients had low or no egg output. Two patients had no eggs in stool, but numerous Opisthorchis viverrini eggs and flukes were found in the gallbladder and bile ducts at operation.

Enzyme-linked immunosorbent assay for detection of Opisthorchis viverrini infection.

Antibodies to O. viverrini in the sera of people from endemic and non-endemic areas were investigated using indirect ELISA technique. For the patients from the endemic area, 92.8% who passed eggs in the stool were found to be positive for O. viverrini antibody. In addition, 46.5% of the people who did not pass eggs in the stool were also found to have low titer of O. viverrini antibody. On the other hand only 2.4% of the people from the non-endemic area with other intestinal parasite infections were found to have O. viverrini antibody in their sera. It was concluded that positive reaction of O. viverrini antibody is not cause by cross-reaction with other parasites but low liter of antibody is probably due to low-level or past infection. There is a positive correlation between the titer of O. viverrini antibody and intensity of infection as indicated by number of eggs excreted per milligram of feces. Patients with a few O. viverrini eggs in feces, but biopsy-proved-cholangiocarcinoma had very high titer of antibody.

Albendazole in the treatment of opisthorchiasis and concomitant intestinal helminthic infections.

A total of 52 adult patients with opisthorchiasis with or without concomitant intestinal helminthic infections were treated with albendazole at dosage regimens of 400 mg twice daily for 3 days (group I with 25 patients) and 7 days (group II 27 patients). By concentration method with four examinations from two faecal specimens of each patient the cure rates and percentage egg reduction on day 30 in group I and group II were 12% and 33%, 94 and 95 respectively; by Stoll method the cure rates and percentage egg reduction were 40% and 63%, 92 and 92 in group I and group II, respectively. There were no statistically significant differences between the results of the two regimens. Twenty six patients with hookworm and 9 patients with S. stercolaris infections were cured. Opisthorchis viverrini, N. americanus, A. duodenale, A. ceylanicum, E. malayanum, E. ilocanum, S. stercolaris, E. vermicularis, and T. saginata segments were expelled in stools, mostly on days 1 to 4. Most of the patients felt relieved from symptoms of pain in the right hypochondrium and epigastrium. The side effects were mild and transient. There were no evidence of toxic effects on the bone marrow, heart, liver or kidneys. Albendazole was shown to be effective against Opisthorchis viverrini infection as well as other concomitant intestinal helminthic infections; but the optimal dosage and duration of treatment have not yet been achieved.

Ultrastructure of cholangiocarcinoma associated with opisthorchiasis.

An electron microscopic study was carried out on eleven surgical liver biopsy specimens obtained from patients with cholangiocarcinoma associated with opisthorchiasis. The tumor cells of histologically well differentiated cholangiocarcinoma had few cytoplasmic organelles. They contained relatively large nuclei, abundant free ribosomes and numerous groups of fine fibrils. Each cell was surrounded by a basement membrane. Numerous long microvilli were seen projecting into the glandular lumen. The moderately differentiated cholangiocarcinomatous cells exhibited increased organelle content, marked variation in the shape of the nuclei with deep cytoplasmic invagination into the nuclear membrane; there were small intranuclear pseudoinclusions, and shorter microvilli. The tumor cells showed intracellular microvillus-lined spaces, abundant free ribosomes, many fine fibrils and their surrounding basement membranes were incomplete. The ultrastructure of the poorly differentiated cholangiocarcinoma was similar to that of the moderately differentiated tumor, except for fewer microvilli, abundant cytoplasmic organelles, and ill-defined or absent basement membrane.

Hepatic carcinoma with opisthorchiasis.

Eighty-seven cases of primary hepatic carcinoma associated with opisthorchiasis were reviewed. The prevalence of liver carcinoma among 154 cases of liver fluke infection was 56.6%. The tumours were classified histologically as cholangiocarcinoma in 67 cases (77%), hepatocellular carcinoma in 9 (10.3%), mixed hepatocholangiocarcinoma in 4, squamous carcinoma in 2, and undifferentiated carcinoma in 5. Metastases, found in 71 cases, occurred most frequently in the regional lymph nodes and lungs. There were numerous or moderate numbers of liver flukes in most of these cases.