Fish and Food-Fatale: Food-borne Trematode Opisthorchis viverrini and Cholangiocarcinoma

World Health Organization (WHO) stated that Neglected Tropical Diseases (NTDs) are a diverse group of infectious diseases, consisting of 20 diseases, mainly found in 149 tropical and subtropical countries (Fig. 1) (Mitra & Mawson, 2017). NTDs affect one-sixth of the global population, with the assumption that one in every six people will be affected by at least one type of NTDs. According to the WHO, the mortality and morbidity caused by NTDs are extremely high, as measured by disability-adjusted life years (DALYs). Globally, NTDs have resulted in about 534,000 fatalities and 57 million DALYs to date (Lin et al., 2022)

Fig. 1.

Neglected Tropical Diseases affect those who lack access to proper health care, clean water, sanitary conditions, and hygiene in low-income populations and even those who are extremely poor. NTDs create losses for a nation that come from both the health and the economic sectors as well, since the costs of diagnosis, prevention, and treatment place a strain on the national purse strings (Redekop et al., 2017; Hotez, 2020). Considerable population mobility contributes significantly to the transmission of NTDs thus more than a 70 percent of the cases are found in low-income or lower-middle-income countries. As a result of the increase in international traveling and climate change arising from global warming, NTDs are currently present in a number of European nations as well. Nonetheless, when the Covid-19 outbreak spread throughout the entire world, the global goals to fight NTDs has once again been disregarded (Adepoju, 2020; Tilli et al., 2021). NTDs lead to significant illness and mortality in addition to societal ignominy and prejudice; however, NTDs are effectively managed, diminished, or eradicated through the intervention of a variety of efficient public health measure. The current most cost-effective interventions in global health are those aimed at eradicating and controlling NTDs. By 2030, point 3.3 of the Sustainable Development Goals (SDGs) on NTDs calls for the eradication of NTDs, malaria, tuberculosis (TB), and HIV/AIDS (Addisu et al., 2019).

One of the 20 different forms of NTDs on the list is food-borne trematodes. The term “food-borne trematodes” refers to a collection of illnesses brought on by a number of trematode worms, including Clonorchis, Opisthorchis, Fasciola, and Paragonimus. Two intermediary hosts serve as a broad overview of this category of trematodes. Aquatic vegetables, tiny and medium-sized fish, water snails, and crustaceans are examples of the first intermediate host and the second intermediate host, which are generally unique from one another. Humans as the definitive host will be infected if they consume food that has been contaminated by the second intermediate host (Robinson & Sotillo, 2022). The total number of people infected around the world may be more than the 2 million DALYs and deaths that the WHO estimates are lost globally. Food-borne trematodes is thought to have infected over 75 million individuals worldwide as of today, with 0.2 million new cases reported year. This information might not be accurate as there are many undetected cases (Chai & Jung, 2020).

The liver trematode worms in the Opisthorchiidae family that cause the food-borne trematode illnesses Opisthorchiasis and Clonorchiasis, which are Opisthorchis viverrini (O. viverrini), Opisthorchis felineus (O. felineus) and Clonorchis sinensis (C. sinensis). Over 700 million people globally, mostly in endemic regions of East and Southeast Asia, including Siberia, as well as numerous European countries, are at risk of acquiring one of these three tropical diseases (Suwannatrai et al., 2018). O. viverrini is mainly restricted to Southeast Asia, O. felineus is indigenous to North Asia and Eastern Europe, and C. sinensis is prevalent in Korea, sections of Russia, China, and Vietnam (Sripa et al., 2021; Pakharukova et al., 2023). According to China’s national parasite inquiry, the prevalence of Clonorchiasis has increased by about 0.37 percent since the initial examination in 1988, and a recent study found that the prevalence of C. sinensis infection is 29.3 percent (Gao et al., 2020; Jiang et al., 2021). While nearly 1.2 million people reside with O. felineus in Western Siberia and other regions of the Russian Federation (Ovchinnikov et al., 2017). In addition, it is thought that about 10 million people in Southeast Asia are affected with O. viverrini (Sripa & Echaubard, 2017; Almanfaluthi et al., 2022). This liver parasite poses serious public health issues in Thailand, Democratic People’s Republic of Lao, Cambodia, and Vietnam (Thinkhamrop et al., 2019a). There has been concern about these trematode worms in the western world due to reports of them in the United States (USA) as the surge in Asian immigrants or travelers, and the import of undercooked cyprinoid fish (Kaewkong et al., 2013; Lu et al., 2018). Additionally, some infections, like Paragonimus westermani, (P. westermani) can be acquired locally in the US where infected first and second intermediate hosts may be prevalent (Fried & Abruzzi, 2010).

The O. viverrini life cycle starts when the embryonic eggs laid by the adult worm enter the bile duct and are eliminated in the feces. These embryonated eggs do not hatch if they are not introduced to fresh water bodies (small ponds, rivers, flooded rice fields, reservoirs, or rivers), since an intermediate host snail, such as the first intermediate host O. viverrini, will ingest them. The miracidia develop into sporocysts, rediae, and cercariae after hatching in the snail’s digestive tract. Numerous thousands of cercariae are released every day as a result of snails’ asexual reproduction. From the time eggs are consumed until cercaria are released, it takes roughly two months. The free-swimming parasites then look for the cyprinoid fish, which serves as their next intermediate host. They encyst in the fins, skin, and muscles of the fish and develop into metacercaria after 21 days. The bile duct is where the metacercaria attach after swallowing and develop into adults, who lay eggs 3 to 4 weeks later (Fig. 2). The metacercaria first exist in the duodenum before ascending through the ampulla of vater into the bile duct (Khieu et al., 2019; Suwannahitatorn et al., 2019; Tangkawattana et al., 2021; Sota et al., 2022).

Fig. 2.

Opisthorchis spp. eggs are frequently indistinguishable from C. sinensis eggs, measuring 19 – 30 m long by 10 – 20 m wide. The egg has been operated on, and the opercular “shoulder” and abopercular knob are both noticeable. As they are passed through excrement, embryonic eggs (Fig. 3). Adult worms are flattened on the abdomen, approximately 7 mm (5.4 – 10.2) long by 1.5 mm (0.8 – 1.9), lanceolate, thin, and transparent. The bile ducts and pancreas of the mammalian host are where adult worms live and connect to the mucosa. They may reside in the pancreatic duct, gallbladder, or larger bile duct in cases of serious illness. They produce 2000 to 4200 eggs per day (3160 eggs in average) (Rachprakhon & Purivirojkul, 2021a, Charoensuk et al., 2022). The exact life span of O. viverrini in human is not clearly known, however, it may be over than 25 years (Kaewkes, 2003; Petney et al., 2018)

Fig. 3.

There had formerly been some investigations of the genetic diversity of O. viverrini, and the first publication utilizing the isoenzyme analysis method appeared in 1993 (Sueblinvong et al., 1993). Since then, a number of molecular and genetic indicators studies have been carried out on O. viverrini using multilocus enzyme electrophoresis (MEE), nuclear and mitochondrial DNA sequencing, and microsatellite marker fragment analysis (Saijuntha, 2007; Kiatsopit et al., 2014; Pitaksakulrat et al., 2018; Namsanor et al., 2020).

According to a thorough molecular study in 2007, O. viverrini sensu lato (s.l.) is a species complex consisting of numerous cryptic species (genetically different species that share a similar morphology) found in different wetland systems in Thailand and Laos (Saijuntha, 2007). Bithynia snails serve as first intermediate vector, The first intermediate hosts in the life cycle are, a complex species that includes at least 11 cryptic species that are related to O. viverrini sl. As a result, both species complexes are highly genetically variable in relation to geography (Hughes et al., 2017).

Despite this, a later study in 2010 that used AMOVA to assess the populations rejected O. viverrini s.l. as a species complex since a study didn’t find any differences that were significant. Six populations of O. viverrini from Thailand, Laos, and Cambodia were examined in this genetic study using a single marker and a portion of the ND1 sequence. The limited variation that has been noted in a number of prior research also makes it crucial to emphasize that mitochondrial DNA appears to be inappropriate and untrustworthy for systematic and population genetic studies of O. viverrini (Thaenkham et al., 2010). Numerous research looking at genetic diversity and population structures have employed microsatellite DNA markers to bolster the idea that O. viverrini was a species complex (Pitaksakulrat et al., 2017; Namsanor et al., 2020). In Bangkok, Thailand, most of the Opisthorchis viverrini s.l. cercariae were detected in the snail vector, Bithynia siamensis siamensis (Rachprakhon & Purivirojkul, 2021b). O. viverrini s.l. can be recognized from C. sinensis and O. felineus by differences in morphology, genetics, and geographic distribution (Saijuntha et al., 2014; Pakharukova & Mordvinov, 2022).

O. viverrini can cause hepatobiliary abnormalities, such as cholangitis, cholecystitis, cholelithiasis, advanced periductal fibrosis, and lastly, cholangiocarcinoma as the end point. Due to its connection to recurrent neglected tropical diseases whose effects on public health are advised to be underestimated, food-borne trematodes have come under increased scrutiny. The greatest incidence of CCA in the world and a hotspot for O. viverrini transmission can both be found in northeast Thailand. In this region, the annual incidence of CCA ranges from 93.8 to 317.6 per 100,000 people, with the majority of cases ending in death within one to two years of diagnosis (Steele et al., 2018). The widespread nature of helminth infection is a significant epidemiological trait. This means that while most infected people have relatively low worm burdens, some have substantial worm burdens. The risk of sickness is typically higher in those with the most worms, who also contribute disproportionately to environmental pollution. In O. viverrini, the usual pattern of overspread has a long history. It has been hypothesized that environmental exposure (host behavior shows considerable age dependence), individual vulnerability (genetic and immunogenic variables), and social factors are to blame for the overabundance of helminths (Echaubard et al., 2016).

The O. viverrini’s complex life cycle, which involves a number of hosts and surroundings, makes it challenging to control using traditional techniques. Poor hygiene and sanitation may serve as the one of main factor resulting in stool contamination to water bodies, hence the reservoir hosts such as stray or domestic dogs and cats may also contribute to the transmission of this fluke. (Sripa et al., 2017; Tangkawattana & Sripa, 2018).

The persistence of O. viverrini infection in endemic regions is influenced by a diverse array of risk factors (Suwannatrai et al., 2018). Age may be a factor in the increased burden of infection among working-age individuals, as well as the higher incidence of infection among men and those with lower levels of education. Cultural activities related to fishing, food preparation, and eating raw fish, which are deeply ingrained as parts of the region’s native rice fish culture, are the major factors contributing to the high rate of O. viverrini infection in the endemic area (Prakobwong & Suwannatrai, 2020). Consuming freshwater fish that has already been salted, or smoked that is either raw or undercooked and has metacercaria on it can infect humans or other fish-eating animals. It is just the consumption of raw fish which caused the infection of O. viverrini, meanwhile the consumption of fish sauce doesn’t have any impact as the fish sauce has undergone further process from the raw fish into fish sauce as the end product (Thaewnongiew et al., 2014).

Food sharing among household and neighbor is a common practice in communities of local inhabitants (Isaan people) in Northeast Thailand, as this province bear the heaviest burden of O. viverrini infection (Phimpraphai et al., 2018). Sharing raw fish dishes among neighbors may increase the prevalence of infection but also raise the danger of inflammation-related diseases because the average per-person infection intensity will be lower. Food sharing was tightly and favorably correlated with eating raw fish frequency and the dishes consumed variation (Saenna et al., 2017). This implies that raw fish dishes are more likely to be accepted, offered, and consumed in households with higher food sharing propensities. Comparatively to homes that do not exchange food or do so only to a limited extent, this raises the risk of transmission. The smaller, less common raw fish is typically used in these communal dishes. The type of fish that is potentially infected with “pla khao noi” (Isan-Lao for small cyprinid fish) which is widely used in processing raw fish is usually found in local villages to be sold cheaply or distributed free of charge to fishermen’s families and friends. Other factors that promoting the everlasting transmission of O. viverrini including occupation factor where rice farmers and fishermen are more likely to consume raw fish dishes and defecate in or near wetlands and rice fields due to their daily activities. As praziquantel (PZQ) is easily obtained through pharmacy in Thailand, local people are taking this drug by themselves without proper medical supervision every time they get reinfection with O. viverrini, and this phenomenon eventually leading to drug resistance. Saengsawang et al., revealed that the vicious cycle of O. viverrini reinfection and repeated usage of anthelmintic PZQ obviously increasing the risk of CCA (Saengsawang et al., 2016; Suwannatrai et al., 2018; Thinkhamrop et al., 2019b).

To overcome the continuous transmission of O. viverrini, a novel control method known as the “Lawa model” employing the Eco-Health / One Health approach was implemented to the Khon Kaen Province’s in Lake Lawa area, Thailand where liver flukes are endemic. The program comprises anthelmintic medicine therapy, innovative approaches to intense health education in the community and in schools, ecosystem monitoring, and engaged community involvement. Due to its success stories, this technique began in 2007 and has continued for more than ten years, as this control method has been adopted by WHO (Sripa et al., 2017).

Fish-borne trematode infection and CCA have a well-established link. This infection can result in hepatobiliary diseases such as hepatomegaly, chronic cholangitis, cholecystitis, cholelithiasis, periportal system fibrosis, gallstones, and bile duct cancer or cholangiocarcinoma (CCA) (Sripa et al., 2021). The World Health Organization’s International Agency for Research on Cancer (WHO) categorized C. sinensis and O. viverrini as a Group 1A carcinogen (a metazoan parasite harmful to humans). It is estimated that 100 per 100,000 in men and 40 per 100,000 in women or approximately 1 % of the invidual with opisthorchiasis would develop into CCA and the disability-adjusted life years (DALYS) caused by the infection of O. viverrini and CCA as high as 150 – 235 × 103 DALYs (Saijuntha et al., 2019b, Brindley et al., 2021a, Liau et al., 2023).

Despite not being officially recognized as a human carcinogen, O. felineus is a member of Group III has potentially carcinogenic (Banks et al., 2012; Saltykova et al., 2018; Tantrawatpan et al., 2020). In recent publication by Fedorova et al, they stated that a positive relationship between infection of O. felineus with the risk for CCA in the hospital-based study in Westen Siberia, as the CCA patients had the history of O. felineus infection. This study revealed that this species might hold the possibility to be the Group 1A carcinogen, as the O. viverrini and C. sinesis (Fedorova et al., 2023).

In comparison to O. felineus area, long-term observation study indicated that CCA is more frequently documented in O. viverrini and C. sinensis endemic regions. Even though the carcinogenesis pathology caused by parasitic infection may be driven by a variety of events known as chronic inflammatory infectious processes, helminthiases related to malignancy remain mainly unknown. Additional pathways involve genomic instability, induction of invasion, angiogenesis, and cancer metastases, deregulation of tumor suppressor genes, prolonged cell growth, cell death prevention, energy metabolism regulation, and immune evasion (Machicado & Marcos, 2016; Pakharukova et al., 2019; Fedorova et al., 2020). The three liver trematode species differ in how they become carcinogens, but there have been no comparative research studies, thus the information that is currently accessible is not sufficiently clear.

Depending on its cellular source, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two subtypes of primary malignant liver cancer that are distinguished histologically (El Jabbour et al., 2019). Hepatocellular carcinoma responsible for nearly 75 and 80 percent of cases worldwide. The rarest type of cancer is by far CCA constituting 10 to 25 percent of primary hepatic cancers. However, liver cancer incidence reveals 15 times higher in East Asian regions where Opisthorchiasis and/or Clonorchiasis are endemic. The fatality and morbidity of CCA are directly proportional and therefore have a shabby prognostic. The most prevalent kind of CCA is extrahepatic estimated 80 – 90 % and about 50 % of all CCA are perihilar (affecting the main branches of the ducts). The 5-year survival rate for CCA patients after diagnosis has not increased is not good, as low as 5 % to 17 % of total cases (Chansitthichok et al., 2020).

Cholangiocarcinoma can develop based on the anatomical location into intrahepatic (iCCA) or extrahepatic, while extrahepatic CCA are divided into perihilar (pCCA), and distal (dCCA); those may appear differently in terms of clinical findings, pathology, and epidemiology (Fig. 4) (Pratap et al., 2018; Khan et al., 2019; Singal et al., 2020).

Fig. 4.

Various iCCA-specific risk factors are associated, although it is unclear how they cause iCCA to evolve. The prevalence of CCA varies globally due to variations in genetic and environmental risk factors. The liver fluke infestation is pathogenically linked to CCA throughout Asia, especially in regions where O. viverrini, Clonorchis sinensis and kindred flukes is endemic (Saijuntha et al., 2019a). A sizable previous amount of epidemiologic evidence supports the involvement of liver flukes in the emergence of CCA, particularly Clonorchis sinensis (Chinese liver fluke) and O. viverrini, both are significantly related to malignancy. Opisthorchiasis infection has been linked to an elevated risk of 2 to 5 times developing intra and extra-hepatic CCA as a result of adult flukes residing in the biliary tract over years and triggering an immunological response from the host and inducing prolonged inflammation (Massarweh & El-Serag, 2017).

The mechanism of O. viverrini induced CCA is uncertain, but chronic inflammation is thought to play a role as results from the flat worm infecting the host’s biliary tract. (Chaiyadet et al., 2015; Zimmermann, 2019). The most frequent cause of CCA is malignant transformation of cholangiocyte cells, while it is also possible for peribiliary gland epithelial cells and/or biliary stem cells to evolve into cancer. Moreover, hepatic stem/progenitor cells are confirmed to be the source of some subgroups of CCA and combined HCC/CCA (Nakagawa et al., 2018). There are at least three different pathways embroiled in the opisthorchiasis driven carcinogenesis. First, once the parasites mechanically injured the biliary epithelium, the tissue healing has begun. In order to build a cellular covering for the injury, tissue healing reactions cause the release of enzymes that breakdown the extracellular matrix, impede with epithelium adhesion, and stimulate cell proliferation. Normally, a sum of released substances reacts to cease the process after the injury has healed. Healing and recovery from injury is hampered by chronic epithelial breakage caused by the grazing and passage activity of the liver fluke. DNA damage and oncogenesis are presumed to be caused by the continuous cell division involved with tissue injury that is mechanically driven in the presence of external co-factors. The wound - healing reaction apparently causes cell proliferation, which, as in existence of co-factors, considerably aids in the growth of cancer (Sripa et al., 2018b, Sawanyawisuth et al., 2021).

Chronic immunological responses are the second pathway that can increase the risk of malignancy. During cholestasis and inflammation, it is discovered that the quantities of growth factors, cytokines, and bile acids are enhanced. These substances contribute to these molecular changes and promote the proliferation and viability of changed cells. The immune reaction is initiated when Nitric Oxide (NO) and oxygen radical spurt from activated phagocytes often eradicate various antigens. This reaction may have unfavorable harmful side effects as NO is carcinogenic and induces endogenous carcinogenic nitrosamine production (CCA primary catalyst) (Smout et al., 2011; Chaiyadet et al., 2015).

The immune reaction in those with opisthorchis infection unaffecting the invading worms resulting in a protracted reaction and NO buildup. Inducible nitric oxide synthase (iNOS) is upregulated in inflammatory cholangiopathies and CCA, where cytokines promote iNOS production in epithelial cells. Plasma and urine nitrite contents increase although nitrosamine and their precursors’ contents from diet are not rising in opisthorchis infection. As the excretory metabolite of nitrosamines, nitrites are a good indicator of significant endogenous nitrosamine synthesis. The eradication of the parasite, which is easily achieved by using the anthelmintic medication praziquantel, should make this fluke infection outcome reversible (Jaiswal et al., 2001; Smout et al., 2011; Sripa et al., 2018a).

Further research established that the areas of fluke-induced tumorigenesis contain DNA breakage caused by nitrite generation ((Kawanishi et al., 2006). Oxidative DNA breakage and nitrates including 8-oxo-7,8-hydro-2′-deoxiguanine (8-oxodG) and 8-nitroguanine (8-NG), both have been connected to inflammation-related tumorigenesis. Nitric oxide (NO) and reactive nitrogen oxide species (RNOS) synthesis are known to interact with cytoplasmic DNA and proteins, both of which are the results of increased inducible nitric oxide synthase (iNOS) activity. Additionally, interaction between reactive nitrogen oxide species (RNOS) and cellular genome trigger in DNA strands breakage and transmutation. Nitric oxide (NO) and RNOS as well interact with DNA repair enzymes which is promptly rendered inactive by S-nitrosylation of the cysteine residue in its active site, to further enhance mutagenesis. During opisthorchiasis elicited an inflammatory response and released ROS/RNS with high oxidative potential. Afterward, biomolecules in the inflammatory region, including O. viverrine in the bile duct, will react with ROS/RNS. Opisthorchiasis has also been linked to higher rates of nitrite and nitrate which are indicators of endogenous NO production. A study linking urinary nitrite levels and infection severity with a higher risk of CCA, demonstrated that nitrosamines are involved in the pathogenesis of opisthorchiasis. Nevertheless, the source of the rise in nitrite content is unknown, which may be related to modifications in the host’s endogenous nitrite synthesis or a rising in external nitrites from fermentation products (Jaiswal et al., 2000; Vale et al., 2013) Long-lasting inflammation can also come from the eggs that the flukes lay and potential for cancer, even has not been thoroughly investigated, it may considerably impact toward disease as observed in the other primary cancer-causing fluke.

Finally, various soluble proteins and other mediators that fulfill numerous of function at the host-parasite boundary are excreted and secreted by liver fluke to endure for an extended of time in a hostile environment (Drurey & Maizels, 2021). It has long been assumed that this interaction affects host cellular homeostasis and aids in the development of cancer. In vitro studies demonstrated that the ES product of O. viverrini to mammalian cell lines causes morphological changes because the ES product is mitogenic and can be absorbed by host bile cells. This finding is supported by in vivo studies, the growth of CCA in a hamster opisthorchiasis model was faster when the model was given ES products before being infected with O. viverrini indicating that immunological memory can speed up carcinogenesis (Pakharukova et al., 2022).