The role of liver fluke invasion in the pathogenesis of cholangiocarcinoma

Introduction

Human liver fluke infections occur worldwide. However, in Southeast and East Asia, environmental factors and culinary habits contribute to a higher incidence of infections in ecologically and geographically limited regions. Globalization, increasing human mobility, and environmental changes affecting the reservoir of zoonotic infections are changing the epidemiology of infectious and parasitic diseases, which until now have been limited to specific geographical regions. This may pose a challenge in diagnosis and treatment. The species Clonorchis sinensis and Opisthorchis viverrini have been recognized as biological carcinogens leading to the development of cholangiocarcinoma.

Etiology and epidemiology

Flukes are large, multicellular organisms with a characteristic leaf-like or lanceolate body shape and dorsal-ventral flattening. They belong to the phylum Platyhelminthes, class Trematoda. Over 6,000 species of these parasites have been described, and at least 100 of them are capable of infecting humans. In humans, they cause trematodiasis. The names of the pathogens are derived from their described tissue tropism and the target organ in which the adult forms lay their eggs. Thus, adults of the genera Fasciola, Clonorchis, and Opisthorchis (common liver flukes) live in the bile ducts, Paragonimus spp. (lung flukes) migrate to the lung tissue, Echinostoma and Fasciolopsis (intestinal flukes) remain in the small intestine, and Schistosoma species, known as blood flukes, parasitize the mesenteric blood vessels, portal system, or urogenital system [1-3]. It is currently estimated that 650 million people are exposed to infection with liver flukes, and the number of infected people exceeds 27 million [4].
Hermaphroditic Clonorchis flukes, which belong to the Opisthorchiidae family, similar to O. viverrini and Opisthorchis felineus, are transmitted through food and parasitize the bile ducts, gallbladder, and, less commonly, the pancreatic ducts. Their definitive hosts are carnivorous animals, both wild and domestic (dogs, pigs), and humans. The only species of the genus Clonorchis that is pathogenic to humans is C. sinensis (Chinese fluke, oriental fluke). Clonorchis sinensis infections are detected in people living in Korea, China, Taiwan, Japan, Thailand, Cambodia, Laos, the Philippines, and the Asian part of Russia, with the number of infected people estimated at over 13 million [1-4]. Opisthorchis viverrini is endemic in Southeast Asia. In 2018, the number of infected individuals was estimated at 12.39 million, with more than half of the cases occurring in Thailand, followed by Laos, Vietnam, and Cambodia [4-6].
Opisthorchis felineus flukes, not unambiguously associated with the pathogenesis of cholangiocarcinoma, are endemic in Russia (western Siberia), Ukraine, and Kazakhstan, with outbreaks also confirmed in Belarus and Italy [2, 3].
In addition to people exposed to liver fluke infection in endemic areas, travelers to regions of Asia where the parasites are present and immigrants from affected areas are also at higher risk. Cases of infection reported in other regions of the world (USA) were associated with the importation of infected fish from endemic areas. Primary infection can also occur in areas where these flukes do not live if metacercariae are imported in fish or plant products. There are also reports of liver fluke transmission through infected liver transplants [3-5].
It should be emphasized that further transmission of infection between humans is impossible.
The geographical factor influencing the location of flukes is the range of specific snail species, which act as intermediate hosts, with which asexual reproduction is associated, with the developmental stages of the parasite ranging from miracidia released from eggs, through sporocysts and rediae to cercariae. Cercariae released by snails into the water infect a second intermediate host – fish (possibly over 100 species of fish) – which are then eaten by the final host – humans.
Bithynia fuchsiana, which is an intermediate host for C. sinensis, lives in slow-flowing ponds and ditches, most commonly in China, Taiwan, and Vietnam. Another intermediate host- Alocinma longicornis – is a freshwater snail native to China and Japan, with occurrences also noted in other parts of Asia. Parafossarulus striatulus is found in freshwater environments across East Asia, with a widespread distribution in China and northern Vietnam. It has also been reported in parts of Korea, Japan, and Taiwan. These snails are often found in the local waterways, ponds, lakes, and paddy fields [7].
Bithynia snails are also known to be the sole intermediate host of O. viverrini, and are distributed throughout endemic areas of opisthorchiasis. The snails were initially reported to be native to North America, and have subsequently spread widely throughout Europe, Asia, Africa and Australia, where over 130 species from this genus have been found. The genetics of O. viverrini sensu lato and its first intermediate host Bithynia siamensis sensu lato have been predominantly characterized in Thailand and Southeast Asia [8].
Humans become infected by consuming freshwater fish containing encapsulated larvae – metacercariae. In the duodenum, the metacercariae excyst and migrate to the bile ducts. There, after about a month, upon reaching sexual maturity, individuals measuring 10-25 × 3-5 mm, located in small and medium-sized bile ducts, begin to lay 3,000 to 4,000 eggs per day, which are then excreted in the feces. The life cycle of C. sinensis is presented in Figure 1. The infection in humans can last for many years, even more than 10 [2-5]. People become infected by consuming raw, undercooked, salted, smoked, or marinated fish containing metacercariae, which can survive for many years in the fish. Therefore, fish should always undergo thermal processing before consumption.

Clinical presentation

The clinical symptoms of liver fluke infection are related to the intensity and duration of the infestation and are result directly from the involvement of the liver and bile ducts. In the early stages of the disease, they are non-specific, with patients reporting weakness, headaches, nausea, bowel movement disorders, including loose stools, and abdominal pain. Physical examination typically reveals yellowing of the white of the eyes and skin, enlargement of the liver and tenderness on palpation. Imaging studies show thickening of the gallbladder wall, its enlargement, and possibly widening of the bile ducts, periductal fibrosis, and fatty liver. Late complications include gallstones, cholecystitis, cholangitis, and extrahepatic manifestations such as pancreatitis, eosinophilic pneumonia, and renal dysfunction with a decreased glomerular filtration rate. Infection with liver flukes causes nutritional disorders and growth retardation [3, 4].
The species O. viverrini and C. sinensis were classified as biological carcinogens for humans (Group 1) in 1994 and 2009, respectively, due to the proven link between infection with these species and the development of bile duct cancer. The annual incidence of bile duct cancer (intrahepatic, cavernous, and distal extrahepatic) caused by C. sinensis infection is estimated to be 25 per 100,000 in women and 35 per 100,000 in men [4]. Bile duct cancer associated with C. sinensis infection has been shown to be diagnosed at a younger age, with a higher incidence among men, a poorer prognosis for patients under the age of 64, and, of course, a higher incidence in areas where C. sinensis is endemic [9].
For O. viverrini, on the other hand, a link between infection intensity and the development of cholangiocarcinoma has been demonstrated. In published meta-analyses, the odds ratio for cancer ranged from 4.5 (95% confidence interval [CI]: 2.6-7.7) to 6.1 (95% CI: 4.4-8.6) for C. sinensis (61, 151) and 4.4 (95% CI: 1.0-18.6) for O. viverrini, while for both species the number was 4.8 (95% CI: 2.8-8.4) [1, 4].

Diagnostics

The diagnosis of infection is established by detecting fluke eggs in stool or duodenal content under a microscope. Direct microscopy can be performed on smears prepared in saline, Lugol’s solution, using the Kato-Miura method, and using concentration techniques. Imaging tests – ultrasound, computed tomography, or magnetic resonance imaging – are helpful in detecting changes in the morphology of the bile ducts. The use of serological tests to detect specific antibodies is also recommended – the sensitivity is high using crude antigen, while specificity is usually low, especially in settings where there is cross-reactivity between different species of liver flukes (e.g., C. sinensis and O. viverrini) or with helminthiases [10]. Another weakness of these tests is that they cannot distinguish between past and active infections. On the other hand, during research in Thailand, the results from rapid and simple immunochromatographic test (ICT) kits for the diagnosis of opisthorchiasis and clonorchiasis (the detection of IgG antibodies in human infection sera) with ELISA results were compared. There was 91.7% concordance between the ICT and ELISA results [11].
An interesting proposal is to use tests that detect parasite antigens in serum, feces, or urine [12]. These may become useful in the future for diagnostics that do not require the involvement of experienced specialists.
Various molecular techniques can be used to diagnose infestation when egg detection cannot be confirmed by microscopic examination of stool samples, especially in low-intensity infestations, which are reflected in inconsistent and low egg excretion in stool, measured by the number of eggs per gram of stool sample. In addition, they enable the differentiation of species with morphologically similar eggs. They can also be used in environmental studies to detect larval stages in snails and fish [3, 4, 13]. In research conducted in Korea, the sensitivity of PCR for C. sinensis infection was explored by testing 170 preselected samples of human feces with microscopically determined densities of C. sinensis eggs. The sensitivity of the assay was 100% for the 74 samples that each had > 100 eggs/g and 91.4% for the other 70 samples found egg-positive by microcopy (i.e. those that had ≤ 100 eggs/g). Three of the 26 samples that appeared egg-negative in microscopy were found to be PCR-positive. The real-time PCR used in this study appears to be a powerful tool for both the detection and quantification of C. sinensis infections [14]. Unfortunately, another Thai study did not share the positive opinion about the real-time PCR test for O. viverrini infection; it showed that the result strongly depends on the infection intensity [15].
The stool examination is the gold standard of liver fluke infection diagnosis and should be used as the first-line diagnostic tool. It can be complemented with antigen detection if possible and available, but it is not obligatory, and this method also has its disadvantages, as described earlier in the text.

Cholangiocarcinogenesis associated with liver fluke infection

The mechanisms involved in the development of cholangiocarcinoma are better known for O. viverrini than for C. sinensis. Nevertheless, researchers agree that the basis of carcinogenesis for both species lies in (i) mechanical damage to the bile duct epithelium caused by parasitism in the bile ducts, (ii) stimulation of cell proliferation by molecules secreted by parasites (parasites’ excretory/secretory products/ESP), and (iii) immunopathology caused by inflammation induced in a situation of chronic infection [4, 16].
In the first mechanism, the presence of flukes leads to the destruction of the bile duct epithelium. In the case of C. sinensis infection, due to its larger size, it is possible for the bile ducts to become blocked, with an increase in pressure within them and further trapping of eggs, the presence of which induces granulomatous inflammation. The process associated with inflammation and healing is responsible for DNA damage and possible malignant transformation.
Studies conducted on cell lines (cholangiocytes, bile duct epithelial cancer cell lines) have shown that in O. viverrini infection, molecules secreted by parasites, undergoing endocytosis in cholangiocytes and stimulating their proliferation, were responsible for inducing the host’s immune response, increasing the expression of genes associated with epidermal growth factor and transforming growth factor β (TGF-β) pathways, triggering an inflammatory cascade with activation of Toll-like receptors (TLR) 4, activation of nuclear factor-B (NF-B), stimulation of the expression and secretion of cytokines interleukin (IL)-6 and IL-8, and induction of nitric oxide synthase cyclooxygenase. The resulting oxidative stress environment is directly responsible for DNA damage associated with cholangiocarcinogenesis [16-19]. The complex of biologically active secretory products of O. viverrini includes, among others, granulin – a growth factor that supports healing, stimulating proliferation and angiogenesis, and thioredoxin, an enzyme associated with the fluke’s defense mechanism against oxidative damage caused by the human immune response, or inhibiting apoptosis of bile duct epithelial cells, which may play a role in cholangiocarcinogenesis [18]. Experimental studies have also highlighted the importance of tetraspanins specific to O. viverrini, specific proteins expressed on cells or extracellular secretory vesicles transporting active schistosome products. Tetraspanins are involved in numerous cellular processes, both in physiology and pathophysiology, influencing the activity of localized adhesion receptors, growth factors, and the signaling pathways they initiate. Tetraspanins are implicated in the pathophysiology of cancer, particularly through their influence on the regulation of cancer cell motility and invasiveness [19]. They are also a potential target in the development of vaccines to protect against the consequences of fluke infection.
With regard to C. sinensis infection, animal models have shown that activation of TLR2 and TLR4 transcription in endothelial cells, fibroblasts, and bile duct epithelial cells caused an increase in the levels of IL-4, IL-10, tumor necrosis factor α (TNF-α), and interferon γ, and increased the amount of highly reactive and mutagenic lipid peroxidation products. The production of free radicals and the expression of pro-inflammatory cytokines IL-6 and IL-1β were intensified. Time-dependent histopathological changes appearing in infected mouse livers included bile duct epithelial hyperplasia, periductal fibrosis, edema, and inflammatory infiltration [16-19]. Clonorchis sinensis-specific secretory products have been shown to increase the expression of genes associated with carcinogenesis, including microRNAs regulating cell proliferation, and to decrease the expression of genes relevant for apoptosis regulation, including tumor suppressor microRNAs [19].
Research activities in cholangiocarcinogenesis induced by liver fluke infection also focus on changes in the microbiome of the bile ducts and intestines associated with parasitic invasion. A study in a hamster model showed that co-infection with O. viverrini and Helicobacter pylori exacerbates periductal fibrosis, cholangitis, and epithelial hyperplasia, and increases the expression of IL-1, IL-6, and TNF-α. With regard to fluke infection, a significant increase in Stenotrophomonas in tumor tissues, the dominance of Bifidobacteriaceae in the fluke microbiome, and increased production of bile acids and ammonia were noted [17, 19].
Due to the insidious onset and silent clinical symptoms in the early stage of cholangiocarcinoma, at the time of diagnosis the characteristic changes in the imaging test are already visible. If the suspicious source of infection is liver fluke infection, the diagnosis is based on those images, stool examination, and/or serology. The major challenge of antibody detection is its inability to differentiate between active and past infections because of the persistence of antibody in serum or other samples after cure, which is demonstrated by the low specificity of serological tests in those individuals from endemic areas and ingesting raw freshwater fish. In both situations, the symptoms may be similar – jaundice, weight loss, abdominal pain. As mentioned before, initial diagnosis begins with imaging tests – ultrasonography or computed tomography – which may reveal a liver mass, as well as signs of intra- or extrahepatic biliary dilation, or gallbladder distention. The main diagnosis of cholangiocarcinoma should be confirmed by tissue biopsy in ERCP (endoscopic retrograde cholangiopancreatography). If liver fluke infection is suspected, the diagnosis should be extended to specific fluke serology or stool microscopic examination.

Treatment

Praziquantel is the drug of choice for liver fluke infection. It has a good safety profile and strong antiparasitic activity. Its mechanism of action involves disrupting the functioning of cellular calcium channels, which leads to depolarization of muscle cell membranes and, as a result, spastic paralysis of the parasite’s muscles, destabilization of the outer shell, and increased sensitivity to proteolytic enzymes. The Worls Health Organization (WHO) recommends treatment with a dose of 25 mg/kg bw three times a day for 2-3 days. Another, simpler regimen with a single dose of 40 mg/kg bw is also proposed, but it has been shown to have a higher chance of treatment failure. The effectiveness of treatment is related to the intensity of the parasitic invasion and possible polymorphisms of the parasite genes.
An alternative treatment option is albendazole, a drug commonly used to combat infestations of various species of nematodes and tapeworms. Albendazole disrupts microtubule function by inhibiting β-tubulin polymerization, which in turn leads to reduced glucose uptake and glycogen deficiency in parasites. The highest treatment efficacy was demonstrated for regimens in which albendazole was administered at a dose of 8 mg/kg bw twice daily for 5 days and 10 mg/kg bw twice daily for 7 days [3, 20, 21]. Those two main treatment schedules are presented in the table below (Table 1).
Although the Chinese Food and Drug Administration approved tribendimidine as a treatment, there are reports of resistance, particularly to triclabendazole (TCBZ). It has a slightly lower cure rate than praziquantel, and non-inferiority was not shown in research in Laos [22]. However, tribendimidine has a similar egg reduction rate to praziquantel and leads to fewer adverse events and thus might be complementary to praziquantel therapy in O. viverrini infection.
As an option in other helminthic infections, artesunate has been used in vitro and in vivo for Echinostoma and Heterophyes infections, with promising results as alternative therapy. Although the exact mechanism of action for artesunate is still unknown, studies have demonstrated that parasites exposed to artesunate had slowed movement, tegument damage, and death at higher drug concentration and longer exposure time (72 hours) [23].
Steroids can be used to treat inflammation in the liver, particularly in conditions such as autoimmune hepatitis. The use of prednisolone in liver fluke inflammation should also be discussed and examined more closely. In research on hamsters, in Thailand, use of prednisolone in O. viverrini infection showed reduction of the inflammatory cells surrounding the intrahepatic bile ducts but also caused increased parasite size for all times of observation and increased eggs per worm and eggs per gram of feces. These findings suggest that prednisolone is useful in suppressing inflammation in Syrian hamster opisthorchiasis, whereas it was also beneficial for parasites by enhancing their reproductive development [24].
Long-term survival after surgical treatment of liver fluke-associated cancer is similar to that reported in patients without liver fluke infestation.

Prophylaxis

Since schistosomiasis is a serious public health problem in many countries, e.g. in China, where infection occurs through the consumption of raw or undercooked fish infected with metacercariae, prevention of infection is of great importance. Preventing fish infection with metacercariae may be crucial for controlling the disease and reducing the number of cases. Studies conducted in China have determined that a potential organism that could be used in a vaccine against C. sinensis is the non-pathogenic Gram-positive bacterium Bacillus subtilis, with lower biological activity compared to Gram-positive bacteria (such as Staphylococcus aureus). This bacterium is an effective antigen carrier for oral vaccines, and CsSerpin, which is a serine protease inhibitor, has been found to be a potential vaccine candidate [25]. This requires further research, and experiments are currently being conducted on mice.
Opisthorchis viverrini is a foodborne fluke that can cause liver and bile duct disease in people living in Southeast Asia. Infection occurs through the consumption of raw or undercooked fish containing metacercariae. In Southeast Asia, 10 million people are infected with C. sinensis, which causes cholangiocarcinoma (CCA). The development of a vaccine to prevent or minimize related pathologies will reduce the incidence of serious diseases, including CCA [25].
In general, among the current vaccine candidates, tetraspanins (TSPs) are the most promising [26].
Currently, there are no ideal commercial vaccines against Fasciola spp., Opisthorchis spp., and C. sinensis. One of the important problems associated with the development of such vaccines is the complexity of the fluke’s life cycle and its ability to evade host immunity. Another problem is the high variability of experimental parameters between different experiments. These parameters mainly include host selection, infection measurement, adjuvant or carrier used, infection time, and outcome determination. Importantly, research is mainly conducted for the prevention of disease in animals, including sheep. Finally, compared to some other vaccines that have received sufficient research and development funding, such as vaccines against HIV, COVID-19, and malaria, funding for research on a vaccine against liver flukes is also relatively insufficient. Therefore, using a bioinformatics method to simulate vaccine design and predict host-independent responses may be a more economical and practical approach, and may also reduce unnecessary animal use.

Conclusions

The development of cholangiocarcinoma in the course of chronic parasitic infection with liver flukes of the Opisthorchiidae family is a multifactorial process. In recent years, significant progress has been made in understanding the mechanisms of cholangiocarcinogenesis induced by parasitic invasion. Reducing exposure to O. viverrini and C. sinensis infection through early diagnosis and effective treatment remains a key element in the prevention of this cancer. The limited effectiveness of public health interventions, including measures to address the environmental aspects of fluke spread by reducing animal reservoirs or influencing the eating habits of vulnerable populations living mainly in specific regions of Asia or migrating from endemic areas, remains an unresolved issue [27]. It is worth bearing in mind that climate change associated with a continuous increase in global temperatures, as well as rainfall and water levels in rivers and reservoirs, will affect the habitats of snails and fish, which may potentially contribute to changes in the distribution of liver flukes.

Disclosures

This research received no external funding.
Approval of the Bioethics Committee was not required.
The authors declare no conflict of interest.

References