![The life cycle of O. felineus [15]. The eggs are shed in the biliary tree, enter the intestine, and are passed with feces. They need to be ingested by freshwater gastropod Bithynia snails, the first intermediate host, to develop into sporocysts, rediae, and free-swimming cercariae: the stage infective for the second intermediate host, cyprinid fish. Humans and other fisheating mammals and birds may serve as a definitive host after ingesting infected fish. Upon entering the host body, metacercariae infect the biliary tract of mammals, where they mature into adult worms. Pictures of cercariae and eggs are adapted from [15]; Bithynia snail, metacercaria, and adult O. felineus are original pictures.](https://www.researchgate.net/publication/334201349/figure/fig1/AS:776578707492870@1562162200641/The-life-cycle-of-O-felineus-15-The-eggs-are-shed-in-the-biliary-tree-enter-the_Q320.jpg)
Available via license: CC BY 4.0
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The liver fluke Opisthorchis felineus as a group III or group I
carcinogen
Mariya Yurievna Pakharukova
1,2
, José Manuel Correia da Costa
3,4
, and Viatcheslav Alekseevitch Mordvinov
1,*
1
Laboratory of Molecular Mechanisms of Pathological Processes, Institute of Cytology and Genetics, Siberian Branch of Russian
Academy of Sciences, 10 Lavrentiev Avenue, 630090 Novosibirsk, Russia
2
Novosibirsk State University, 2 Pirogova Street, 630090 Novosibirsk, Russia
3
Center for the Study in Animal Science, ICETA, University of Porto, Rua de D. Manuel II, Apt 55142, 4051-401 Porto, Portugal
4
Centre for Parasite Immunology and Biology, Infectious Diseases Department, INSA, Rua Alexandre Herculano 321, 4000-055
Porto, Portugal
Received 31 October 2018, Accepted 10 May 2019
Abstract
–
Opisthorchiasis caused by the liver fluke Opisthorchis felineus is one of the most common
helminthic infections in the Russian Federation. The largest area affected by opisthorchiasis felinea occupies
almost the entire territory of Western Siberia and extends to northern Kazakhstan and a part of the Ural
region. Natural endemic regions of opisthorchiasis also exist in the European part of Russia, and in the regions
of Western and Eastern Europe. According to the official statistics of the Russian Federation, up to 40 000
patients with opisthorchiasis are registered annually in the country.
Opisthorchiasis felinea affects the hepatobiliary system and causes serious liver disorders, including cancer of
the biliary tract. Other parasitoses, opisthorchiasis viverrini and clonorchiasis, are widespread in the Southeast
Asia and China. The causative agents of these diseases, liver flukes O. viverrini and Clonorchis sinensis, are
officially recognized as Group 1 biological carcinogens and are classified as the main risk factors for cholangio-
carcinoma. O. felineus is included in Group 3 of biological carcinogens and is not officially considered carcino-
genic to humans. Studies on the carcinogenic potential of this liver fluke and the epidemiology of
cholangiocarcinoma in the Russian Federation have started in earnest quite recently. Nevertheless, we have
some evidence that infection with O. felineus leads to a precancerous state of the bile duct epithelium. This
state, combined with additional risk factors, poses a real risk of cholangiocarcinoma. In our opinion, taking into
consideration the accumulated facts, the classification of the carcinogenic potential of O. felineus requires revi-
sion. In this review, we focus on the relevant characteristics of the biology and epidemiology of this helminth as
well as experimental data on opisthorchiasis felinea; this information might clarify the carcinogenicity of
O. felineus to humans.
Keywords: Cancer, Carcinogenic, Carcinogenesis, Carcinoma, Cell transition, Cercaria, Cholangiocellular
carcinoma, Clonorchis, Cyprinidae, Epidemiology, Fish, Helminthiasis, Hepatocellular carcinoma, Liver flukes,
Malignancy, Opisthorchiasis, Opisthorchis felineus, Pathogenesis, Risk, Sporocysts, Tumor
Introduction
Opisthorchis felineus (O. felineus) is a foodborne
liver fluke, a member of the genus Opisthorchis,family
Opisthorchiidae, order Digenea, class Trematoda, phylum
Platyhelminthes, and kingdom Animalia. This liver fluke
is a causative agent of opisthorchiasis felinea, a helminthia-
sis affecting mainly the hepatobiliary system and pancreas.
Infection occurs after consumption of raw or undercooked
freshwater fish infested with helminth larvae. Characteris-
tics of the human disease are: long duration, frequent
exacerbations, and possible induction of primary liver
cancer [1–6].
Liver flukes O. viverrini and Clonorchis sinensis,also
members of the family Opisthorchiidae, cause opisthorchia-
sis viverrini and clonorchiasis, the diseases that are very
similar to opisthorchiasis felinea in symptomatology and
pathophysiology [7–10]. Opisthorchiasis and clonorchiasis
are trematodiases whose natural hot spots cover a consider-
able part of Europe and Asia. Nonetheless, currently, these
diseases might be arbitrarily attributed to natural focal
infections. Many migrants from Asia live in the non-
endemic areas of opisthorchiasis and clonorchiasis, and
*Corresponding author: mordvin@bionet.nsc.ru
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
4open 2019, 2,23
Available online at:
ÓM. Pakharukova et al., Published by EDP Sciences, 2019
www.4open-sciences.org
https://doi.org/10.1051/fopen/2019016
REVIEW ARTICLEREVIEW ARTICLE
Special Issue: Parasitology and Carcinogenesis
Guest Editors: Ijaz S. Jamall and Björn L. D. M. Brücher
the tourist traffic among various countries is steadily
increasing. Consequently, patients with liver fluke infection
can be registered far from the corresponding endemic
regions [6].
The International Agency for Research on Cancer
(IARC) has classified liver flukes O. viverrini and C. sinen-
sis as Group 1 agents, i.e., the agents carcinogenic to
humans [7] and as the major causative factors of cholangio-
carcinoma (CCC) in endemic regions [7–9]. CCC with fea-
tures of cholangiocyte differentiation is one of the main
histological types of malignant tumors of biliary-tract
epithelia and is a relatively rare type of liver cancer [10].
Nevertheless, over the last 15 years, its incidence has stea-
dily increased worldwide, and nowadays, it represents the
second most common type of primary liver cancer
(15–20% of cases) after hepatocellular carcinoma [11]. The
only available therapy for CCC is surgical resection or a
liver transplant. Usually, CCC is diagnosed at advanced
stages and is regarded as an incurable and lethal cancer
with a poor survival rate of <24 months [12]. Both experi-
mental and epidemiological evidence strongly implicate
liver fluke infection in the etiology of CCC [13].
Opisthorchis felineus is still officially classified as a
Group 3 carcinogen; this group includes factors not classi-
fied as carcinogenic to humans [7]. Meanwhile, data have
been accumulating that show the carcinogenic potential of
O. felineus and support its classification as a human biolog-
ical carcinogen. This review summarizes the data indicating
that O. felineus is an underestimated parasite, and that
there is a need to revisit and revise the status of this liver
fluke.
Biology
ThecomplexlifecycleofO. felineus includes the first
and second intermediate hosts and one final host (Fig. 1)
[14,15]. By getting into water containing the feces of
humans and animals, eggs can be eaten by aquatic, gas-
tropod snail of the genus Bithynia. In their intestines,
mobile larvae (miracidia) hatch from the eggs, penetrate
through the intestinal wall into the body cavity of the mol-
lusk, and turn into sporocysts. The latter give birth to
rediae, which can then produce cercariae: free-living motile
larvae that enter water, where they actively swim. The
parthenogenetic phase in the life cycle of the helminth (from
the stage of sporocysts to the release of cercaria) lasts ~2
months. Development is hampered by the diapause of the
host during the winter period (freezing of rivers) in Western
Siberia. The first cercariae appear in May of the following
year. Mature cercariae are motile and begin to emerge from
mollusks in bright sunny weather at a rate of up to several
hundred a day [15].
Freshwater fish susceptible to opisthorchiid infestation
are Cyprinidae species: ide Leuciscus idus, dace L. leucis-
cus, roach Rutilus rutilus,andsunbleakLeucaspius delinea-
tus [14,15]. When fish and cercariae encounter each other,
cercariae attach to them, discard the tail and penetrate
through the skin into the muscles, where they form the
inner membrane, then the outer connective tissue capsule,
and turn into the next stage, the metacercariae. After 3–4
weeks, metacercariae become invasive and can infect defini-
tive hosts: domestic fish-eating animals (cats, dogs, and
pigs), wild carnivores (muskrats, water voles, foxes, bears,
and wolves), and humans [14–23]. In the acidic content of
the definitive host’s stomach, the membrane of the metac-
ercaria is destroyed, and the juveniles migrate along the bile
ducts to the liver and gall bladder, where after 25–30 days,
the helminth reaches maturation [14,15].
The human infection is most likely due to the consump-
tion of undercooked fish, because sun-dried, lightly salted,
and lightly pickled fish are traditional beer snacks
(Fig. 2). Furthermore, in the Northern regions of Western
Siberia, eating of frozen ribbons of fresh-water whitefish
served with spices, so-called stroganina, is a popular
tradition [1,23].
Many authors have demonstrated the presence of
metacercariae and adult O. felineus worms in the wild.
The complete life cycle of O. felineus,however,hasbeen
demonstrated only by Fedorov, and he has also provided
the data about the structure and timing of the development
of larval forms [15]. It has been reported that red foxes
Vulpes vulpes L. play a certain role in keeping up the natu-
ral hot spots of opisthorchiasis in Europe, caused by the
Figure 1. The life cycle of O. felineus [15].The eggs are shed in
the biliary tree, enter the intestine, and are passed with feces.
They need to be ingested by freshwater gastropod Bithynia
snails, the first intermediate host, to develop into sporocysts,
rediae, and free-swimming cercariae: the stage infective for the
second intermediate host, cyprinid fish. Humans and other fish-
eating mammals and birds may serve as a definitive host after
ingesting infected fish. Upon entering the host body, metacer-
cariae infect the biliary tract of mammals, where they mature
into adult worms. Pictures of cercariae and eggs are adapted
from [15]; Bithynia snail, metacercaria, and adult O. felineus are
original pictures.
M.Y. Pakharukova et al.: 4open 2019, 2,232
trematode O. felineus [16–21]. The natural host for O. feli-
neus in Russia is thought to be the muskrat Ondatra
zibethicus [15], which was introduced in early 1920s. In
Western Siberia, the role of muskrats is the primary contri-
bution to the natural causes of the diseases induced by
many opisthorchiids like O. felineus,O. longissimus,and
Metorchis bilis [15]. The muskrat leads a semiaquatic life-
style and settles along the banks of rivers, lakes, and fresh-
watermarshes.Itprefersshallow(1–2 m deep), ponds that
do not freeze, with banks that are covered with dense grass
[15].
Epidemiology
The trematode O. felineus is an epidemiologically
important liver fluke. This causative agent of opisthorchia-
sis has been detected for some 150 years in cats, dogs, foxes,
wild bears, and other animals (the definitive hosts) as well
as mollusks and carps (intermediate hosts) in Western and
Eastern Europe (Russia, Ukraine, Belorussia, Spain, Portu-
gal, Germany, and Italy) [14,24,25]. All these data indicate
the breadth of the geographical range of opisthorchiasis and
the undoubted participation of animals in the circulation of
the pathogen within this area, whereas the human
opisthorchiasis has a disjointed local (focal) character.
The greatest importance of humans in the circulation of
infection is seen in Western Siberia, namely in the
Ob–Irtysh basin, as evidenced by the high prevalence of
infection in this population (up to 60% in some rural
regions). Thus, most likely, in this region, the participation
of humans in the maintenance of the infection is almost cer-
tain. Toward the periphery of the basin up to the Arctic
Ocean, the percentage of the population living in the area
drops to less than one person per square kilometer. Never-
theless, prevalence of the infection among sensitive fish spe-
cies in this region is almost maximal when compared with
other territories and can be up to 100% (ide). Consequently,
the infection circulation becomes more zoonotic in the wild
(mainly with participation of domestic and wild animals).
In accordance with the Russian clinical standards, diag-
nosis of opisthorchiasis is based on the microscopic detec-
tion of eggs in stool and/or eggs and adult helminths in
bile samples after duodenal intubation [26]. The average
opisthorchiasis incidence in Russia between 1991 and 1993
and 2011–2013 was relatively constant (26.5–24.7 cases
per 100 000 people per year). The incidence of opisthorchi-
asis in Russia is unevenly distributed across regions.
Accordingly, there are hyper-endemic areas (with preva-
lence rates > 100 per 100 000 people), regions with high
prevalence (53–100), regions with average prevalence (22–
52), and those with low frequency of the disease (<22).
The Ob–Irtysh basin in Western Siberia is regarded as a
hyper-endemic area. In this region, opisthorchiasis preva-
lence rates exceed 100 per 100 000 people [1]: Khanty-
Mansiiskiy Autonomous Region (600 per 100 000 people),
Tyumen Region (201 per 100 000 people), Tomsk Region
(189 per 100 000 people), and Novosibirsk Region (127
per 100 000 people). In most regions of Russia, Eastern
Siberia, and the Far East, only low prevalence is reported
(2.5 per 100 000 population; reviewed in [1]).
On the other hand, short-term screening programs in
some rural regions in this area have revealed that the actual
frequency of O. felineus infection is much higher [5,27]. In
particular, the prevalence rate in the Tomsk Region has
been found to be 18–20% [27]; Tyumen oblast, 37%; and
Chelyabinsk oblast, 18.6%. According to autopsy data,
18.3–45.0% of individuals have been found to be infected
with opisthorchiasis [5,28,29].
The carcinogenic potential of O. felineus
1. Experimental evidence
Today, carcinogenesis is thought to be triggered by
chronic inflammation and changes in the cellular microenvi-
ronment that result in a precancerous state and mutations,
which lead to the cell proliferation and malignancy [30].
Figure 2. The Maloe lake tributary of the Ob River (Novosi-
birsk city) and intermediate host for the liver fluke Opisthorchis
felineus. (A) Maloe lake. (B) Backwater of Maloe lake, a habitat
of Bithynia snails. (C) Fresh ides (L. idus) on ice at a market.
M.Y. Pakharukova et al.: 4open 2019, 2,23 3
In subsequent sections of this paper, we present experimen-
tal data on the histopathology of opisthorchiasis felinea in
hamster liver and O. felineus metabolites that can cause
mutations in the chromosomal deoxyribonucleic acid
(DNA) of host cells. A comparative study of opisthorchiasis
felinea and viverrini in a hamster model suggests that the
histopathology of O. felineus infection involves bile duct
dysplasia, periductal fibrosis, and inflammatory infiltration
and could be considered to be more severe than that of O.
viverrini infection [31].
The main histopathological features of O. felineus infec-
tion in rodents are extensive periductal fibrosis, chronic
inflammation, proliferation of the bile duct, bile duct dys-
plasia, egg granulomas, and biliary intraepithelial neoplasia
(BilIN), with grades of 1–3. BilIN is a precursor lesion of
invasive adenocarcinoma in the biliary tract [32–34], and
represents a multistep process in carcinogenesis. Biliary
intraepithelial neoplasia with grade 1 (BilIN-1) and biliary
intraepithelial neoplasia with grade 2 (BilIN-2) lesions have
been demonstrated in O. felineus-infected hamsters. The
totality of biochemical and histopathological changes indi-
cates that O. felineus infection in this rodent model induces
precancerous lesions conducive to the development of a
malignant tumor.
Opisthorchis felineus infection results in CCC in exper-
imental animals if it is accompanied by a chemical carcino-
gen (e.g., diethylnitrosamine or dimethylnitrosamine). It
has been shown that histopathological features of CCC
appear in the liver of the hamsters at 18 weeks post-
infection [35]. In one study, after 26 weeks, in three of six
hamsters, a mass-forming CCC was found, and after 30 weeks
postinfection, CCC was detected in all the hamsters [35].
The accumulated data on histopathology in mammals
suggest that the carcinogenic potential of O. felineus in
an experimental model is very similar to the carcinogenicity
of O. viverrini and C. sinensis [31,35]. The three species of
epidemiologically important opisthorchiids are closely
related to one another in terms of the phenotype of adults,
the habitats of different life stages, and the genetics.
The liver fluke infections have similar characteristics of
the pathogenesis (i.e., mechanism). In addition, the ability
to secrete proteins and oxysterol metabolites is also charac-
teristic for liver flukes including O. felineus.
The pathogenesis of CCC resulting from the infection
with liver flukes is unknown [13,36]. Nevertheless, it can
be assumed that several important factors are involved.
In particular, one of the most important factors is mechan-
ical damage to the wall of the bile duct from suckers of the
helminths. When the tissue is damaged, a cascade of
immunological reactions is launched both against the
destroyed cells and against the helminth antigens and eggs.
Another important factor determining the parasite–host
interaction, which probably makes a significant contribu-
tion to epithelial neoplasia, is helminth secretory products,
consisting of a large number of proteins that induce cell pro-
liferation and inhibit apoptosis [36–38]. Such an effect on
the proliferation of epithelial cells of the bile ducts has been
demonstrated for O. viverrini granulin-1 [36]. The results of
CRISPR/Cas9 genome editing of O. viverrini granulin-1
proved the suggestion about its role in the hepatobiliary
morbidity of liver fluke infection [37]. Moreover, for another
O. viverrini protein, thioredoxin peroxidase (TpX), an
immunomodulatory effect has been demonstrated [36]. Par-
ticularly, thioredoxin peroxidase can inhibit T helper 1
(Th1)-type of immune response and activate the Th2 type.
A remarkable influence on the host–parasite interplay has
also been demonstrated for a defense molecule of Fasciola
hepatica helminth. This protein inhibits antigen presenta-
tion by macrophages due to its affinity for the membrane
of host macrophages [39].
The liver fluke excretory-secretory products (ESPs)
stimulate chronic inflammation and proliferation of the bile
duct epithelium. Proteomic mass spectrometry of the pro-
teins in O. felineus ESPs has identified 37 proteins [40].
Some of O. felineus ESPs are highly immunogenic [41,
42], homologs of host defense molecule (HDM), TpX, and
glutathione-S-transferase (GST) have been found in
O. felineus ESPs [40]. Moreover, O. felineus ESPs may
Figure 3. Biliary histological features in the liver biopsies from hamsters with the experimental opisthorchiasis felinea. (A) Intact
animal. (B) High-grade biliary neoplasia caused by O. felineus three months p.i. (C) Cholangiocarcinoma in the liver of hamster as a
result of a combined action of O. felineus and dimethylnitrosoamine, three months p.i. BD, bile duct; OF, O. felineus; CCC,
cholangiocarcinoma. High-grade biliary neoplasia is indicated with arrows. Pictures 3A and 3C were kindly provided by Maksimova
GA.
M.Y. Pakharukova et al.: 4open 2019, 2,234
interact with the epithelium and may accumulate inside the
cells because they are found in the gallbladder of the ham-
sters with experimental opisthorchiasis and patients with
the infection [41,42].
In addition, it can be assumed that the specific low-
molecular-weight cholesterol metabolites, which are also
found in the helminth secretory product, apparently have
oxidative and genotoxic properties, i.e., can damage the
DNA of host cells [13,32]. Such low-molecular-weight
metabolites (catechol estrogens and oxysterols) in other
species of helminths have been demonstrated, some of
which are associated with cancer initiation and are
recognized as biological carcinogens. In particular, the
trematodes O. viverrini,O. felineus,andS. haematobium
produce specific oxysterols and catechol estrogens. Oxys-
terol-like metabolites in the egg and adult worms, and in
bile, serum, and urine of O. felineus-infected hamsters have
been detected by liquid chromatography with mass spec-
troscopy [32]. Related DNA adducts suggest that infec-
tion-associated oxysterols induce chromosomal lesions in
host cells [13]. Associations between oxysterols and the ini-
tiation and progression of cancer of the colon, lungs, breast,
and bile ducts have been proposed [43,44].
The origin of these metabolites is unknown. It can be
assumed that there are two pathways for their synthesis:
enzymatic and nonenzymatic. The nonenzymatic pathway
is the reaction with the free oxygen and nitrogen species
[45]. The enzymatic pathway is the result of the liver fluke
xenobiotic metabolism network activity [46,47].
It is generally accepted that CCC tumors in this ham-
ster model occur as a result of a combined action of infec-
tion with the liver flukes, O. felineus,O. viverrini,or
C. sinensis and exposure to N-nitrosodimethylamine [35].
In contrast, a series of studies on the pathogenesis of
opisthorchiasis felinea has revealed enhanced expression of
p53, and the presence of intraepithelial bile duct neoplasia
[32,35]. Recent studies have also shown that epithelial-to-
mesenchymal transition (EMT) is involved in chronic
opisthorchiasis in rodents. At 22 weeks of chronic
opisthorchiasis in hamsters, processes of EMT intensify in
the liver [48]. The increased expression of markers of the
SMAD signaling pathway and enhanced accumulation of
a-smooth muscle actin (a-SMA) protein have been shown
by immunohistochemistry. The elevated messenger ribonu-
cleic acid (mRNA) expression of transforming growth factor
beta 1 (Tgfb1), annexin A1 (Anxa1), exostosin glycosyl-
transferase 1 (Ext1), and keratin 7 (Krt7) genes has been
demonstrated in the liver of O. felineus-infected hamsters
[48]. Taken together, these data indicate that precancerous
changes can occur in hamsters without exposure to an
exogenous carcinogen such as N-nitrosodimethylamine
(NDMA). These processes cause damage to genetic mate-
rial, with the mutations being fixed during DNA replica-
tion. Eventually, accumulation of mutations might lead to
malignant transformation of cholangiocytes and progression
to CCC [13].
1. The link between O. felineus infection and CCC in
humans
The most important step in the validation of results
obtained in a study on the carcinogenic potential of
O. felineus in an experimental model is analysis of the asso-
ciation of opisthorchiasis felinea and CCC in humans.
Given that there are no systemic epidemiological and clini-
cal programs for the control of opisthorchiasis in Russia, the
solution to this problem remains difficult. Undoubtedly, the
similarity in clinical manifestations, the disorders caused by
O. felineus and O. viverrini [28,29], and the data on the
pathogenesis of these two helminthiases acquired in experi-
mental opisthorchiasis models [1,31,32,34] are suggestive
of a rather high probability of O. felineus involvement in
the induction of these tumors. Here, we briefly discuss the
data published in the Russian medical literature on the
complications associated with opisthorchiasis as well as
the data from hospitals and morgues on autopsy results
over a long period. Unfortunately, the data on liver cancer
prevalence have certain drawbacks, in particular, the per-
formed examinations and criteria for selection of control
groups are insufficiently detailed. Nonetheless, in the
absence of other epidemiological data, the number of cases
recorded in hospitals during a given period is also valuable
information, of course, with a disclaimer that this is simple
recording of the number of cases rather than full-fledged
epidemiological studies.
In general, it is reported that the incidence of malignant
tumors, in particular, primary liver cancer is significantly
higher in regions with high prevalence of opisthorchiasis.
In a number of regions of Western Siberia in the period
1962–1971, the prevalence of primary liver cancer was 9.4
cases per 100 000 people [49,50] (reviewed in [7]). In the
period 1990–2001, liver cancer ranked seventh among the
most common cancers registered in the Tomsk region
(3.9%). The prevalence of this type of cancer in men in this
period was 11.2 cases per 100 000 people [51]. This is almost
2.5-fold more than the average prevalence rate of liver can-
cer in the Russian Federation in 2012, according to the offi-
cial statistics [51,52]. For comparison, in Moscow and
Moscow region (non-endemic areas of opisthorchiasis) the
prevalence of primary liver cancer was 1.77 and 2.3 cases
per 100 000 people, respectively [53]. The incidence of pri-
mary liver cancer in these regions is not correlated with
alcohol consumption, in particular Moscow region was in
the top three for the consumption of strong alcoholic drinks
(2016–2017), whereas Tomsk and Tyumen regions (hyper-
endemic) were with low or moderate scores [54].
In the Russian medical literature, there is information
indicating a link between O. felineus infection and liver can-
cer in the population of Western Siberia. Thus, during anal-
ysis of the results of more than 2250 autopsies conducted
during 37 years in the city morgue of Tobolsk, it was found
that in 45% of the cases, infection with opisthorchiasis was
present. Primary liver cancer has been diagnosed in 8.7% of
the infected patients [55]. According to the Tomsk City
Morgue, among persons with a confirmed diagnosis of
chronic opisthorchiasis [~350 autopsies], CCC cases have
been registered in 5% of the group [28,29]. Furthermore,
according to the information from the Tomsk Hepatology
Center, ~3% of patients with chronic opisthorchiasis have
M.Y. Pakharukova et al.: 4open 2019, 2,23 5
been operated on for biliary tract cancer and pancreatic
cancer [29].
An interesting study has been conducted in another
region of Western Siberia: the Tyumen region. There, the
prevalence rates of various types of liver cancer in
opisthorchiasis-positive (target group) and negative indi-
viduals (control group) were determined. It was established
that CCC is most common in opisthorchiasis: according to
the results of histological analysis in the target group, this
diagnosis was made in 77% of the cases. Hepatocellular car-
cinoma (HCC) in the same group of patients was detected
in 19% of cases, and 4% of patients had other types of
malignant liver tumors. In the control group, CCC occurred
in 34% of patients, hepatocellular carcinoma in 56% of
cases, and other liver tumors in 10% [49].
In agreement with these results, the data obtained in the
Tyumen region –after autopsies of 44 individuals with con-
firmed primary liver cancer –have revealed that CCC was
present in almost 80% of cases. It is important to note that
O. felineus infection was confirmed in 42 of 44 cases in that
study (reviewed in [7,56]). Thus, in endemic regions, malig-
nant tumors of the biliary tract are some of the most com-
mon types of liver cancer. It is noteworthy that in the
presence of O. felineus infection, the prevalence of CCC
exceeds that of other types of liver cancer by the factor of
four (or by 400%). Recently, two cases of CCC associated
with chronic opisthorchiasis were reported. Both cases pre-
sented with nonspecific symptoms and severe pathological
complications. Both patients were living in endemic areas
[26].
In non-endemic areas of opisthorchiasis in Russia, the
proportion of CCC does not exceed 15% of all cases of liver
cancer. For example, according to generalized data accumu-
lated at the Blokhin Russian Cancer Research Center
(Moscow), CCC is responsible for 14.9%, and hepatocellular
cancer for 85.1% of liver cancer cases [57]. In general, data
on the prevalence of CCC in the endemic and non-endemic
regions well match the general epidemiology of this type of
cancer in other endemic and non-endemic regions of the
world [9,58].
Prevention
Prevention of opisthorchiasis is based primarily on the
inactivation of O. felineus larvae in the fish by proper cook-
ing, the disease diagnostics, and effective treatment of the
patients. Preventive measures include (i) protection of
reservoirs from eggs of the parasite, (ii) the inactivation of
metacercariae in relevant species of fish, and (iii) sanita-
tion/educational programs for the population.
In Russia, where the rate of infection with opisthorchiids
among fish is high, standards have been developed to com-
bat the contamination of fish by larvae of epidemiologically
important liver flukes including O. felineus,M. bilis,and
C. sinensis [59]. These standards stipulate first and fore-
most salting, freezing, and heat treatment. Salting should
be done by means of sufficient amounts of salt at a temper-
ature of 1–20 °C, when the mass fraction of salt in fish is
14%. Duration of salting should be 10 days for bleak Albur-
nus alburnus, gudgeon Gobio gobio, minnow Phoxinus per-
cnurus,andsunbleakLeucaspius delineatus; 21 days for
roach R. rutilus, dace L. leuciscus, common rudd Scar-
dinius erythrophthalmus, small common bream Abramis
brama,andideLeuciscus idus; and 40 days for large com-
mon bream and ide [60].
Standards provide for the resistance of the metacer-
cariae to low temperatures. Fish should be frozen under
the following conditions: 40 °Cfor7h;35 °Cfor14h;
28 °C for 32 h [58]. Hot and cold smoking, dry curing,
and drying performed properly according to the official
standards disinfect all kinds of fish except ide. Unfortu-
nately, metacercariae inactivation measures are difficult
to use when fish is processed at home. Thus, it is almost
impossible to provide the necessary conditions for freezing
in home refrigerators [61]. Traditional drying of fish and
cold smoking do not inactivate parasite larvae [62]. Modern
methods have been developed to inactivate the larvae, for
example, the high efficiency of the use of microwave ovens
in the disinfection of large fish has been demonstrated as
compared with other methods [63].
It should be noted that in terms of control of
opisthorchiasis, educational programs for the population
of Western Siberia are not being actively pursued [64].
Thus, a significant part of the population of the north Tyu-
men region thinks that raw fish is healthy food (32% of the
surveyed population) and less than a half of the population
know about liver fluke infections (45.9% of the population).
The infection prevalence of the population in this region
is ~40%. It is noteworthy that newcomers also get rapidly
infected: in the first year after arrival: 2%, within
five years: 10%, and within 10 years, 25% of newcomers
become infected. During the first year after arrival, only
8% of newcomers consider raw fish a healthy food, but after
5–10 years, their number significantly increases and consti-
tutes 17%. These data point to successful adaptation of the
settlers to the peculiarities of local nutrition and their eat-
ing habits in the aboriginal style.
Only 39% of healthcare professionals in the region know
how to use the technology of pickling, which guarantees dis-
infection of fish. It should be noted that drying in this
region is one of the most common ways of processing of fish.
Only 9% of physicians, 27% of epidemiologists, and 11.5%
of other medical professionals in the region have mastery
of the proper fish drying technology that guarantees decon-
tamination [64]. Accordingly, the level of knowledge about
the invasion of fish by opisthorchiids and about the dangers
of contamination is low even among people with medical
education in this region. Apparently, all these factors in
combination lead to the high prevalence of infection with
opisthorchiids in this region.
Conclusion
The carcinogenic potential of the trematode O. felineus
appears to be poorly studied as compared to that of
closely related liver flukes O. viverrini and C. sinensis.
M.Y. Pakharukova et al.: 4open 2019, 2,236
Nevertheless, O. felineus habitat covers vast areas in Eur-
ope and Asia, and opisthorchiasis outbreaks caused by this
helminth are expectable in many countries. Thus, the
opisthorchiasis caused by O. felineus becomes a large-scale
problem, outside the scope of health care and public health
measures in individual regions.
Opisthorchiasis is a common human helminthiasis in
Russia. In Thailand and Korea, the consumption of raw fish
is traditionally widespread; in Russia, the tradition of eat-
ing raw fish is not as popular as eating sun-dried and lightly
salted fish, which are apparently infected with metacercaria
of the parasite. In contrast to Thailand (the endemic area
for O. viverrini), where CCC is a predominant type of can-
cer, primary liver cancer is not the leading cancer type in
Russia. There are probably some additional factors that
lead to carcinogenesis in humans. Apparently, one of the
key factors, as shown by experiments on model animals, is
the presence of weak carcinogens in food, such as nitrosami-
nes, which act as initiators of carcinogenesis. Hence, this
chronic infection is expected to promote the initiation of bil-
iary cancers. There are some other factors that are relative
risks and cofactors for CCC formation. In Thailand and the
Republic of Korea, the highest relative risk has been
adjusted for alcohol consumption, smoking, and physiolog-
ical characteristics such as sex and age [7]. These factors
should be taken into consideration in the areas endemic
for opisthorchiasis felinea as well.
To date, the IARC has classified O. felineus infection as
a Group 3 carcinogen, i.e. there is insufficient evidence yet
for its classification as carcinogenic, unlike the situation
with the closely related liver flukes O. viverrini and
C. sinensis,andthebloodfluke S. haematobium which
are group 1 carcinogens: definitely carcinogenic to humans.
For IARC approval O. felineus as Group 1 carcinogen, car-
cinogenic activity should be demonstrated in animal and
human studies.
The accumulated data on the histopathology in animal
models show that the carcinogenic activity of O. felineus in
an experimental model is very similar to the carcinogenicity
of O. viverrini and C. sinensis. All species of epidemiologi-
cally important opisthorchiids are similar to one another in
terms of the phenotype of adults, the habitats of different
life stages, and the genetics. The liver fluke infections share
nearly the same pathogenesis and host liver histopathology.
In addition, the ability to secrete proteins and oxysterol
metabolites is characteristic of liver flukes including
O. felineus.
Large-scale studies on the association of opisthorchiasis
felinea with CCC in humans have not been conducted, but
such information might be found at least partly in the pub-
lished literature and in hospital records. The consolidated
data show that the prevalence of primary liver cancer in
Western Siberian regions, the world’s largest endemic area
of opisthorchiasis felinea, is considerably higher as compared
with the non-endemic regions. Nevertheless, the presented
data were obtained by separate groups of researchers and
cannot present a comprehensive picture of the association
of opisthorchiasis and liver cancer. This task requires large-
scale epidemiological studies that take advantage of the
expertise gained in similar studies in other countries [9].
Abbreviations used in the text and figures
a-SMA a-smooth muscle actin
Anxa1 Annexin A1
BD Bile duct
BilIN-1 Biliary intraepithelial neoplasia with grade 1
BilIN-2 Biliary intraepithelial neoplasia with grade 2
CCC Cholangiocarcinoma
DNA Deoxyribonucleic acid
EMT Epithelial-to-mesenchymal transition
ESPs Excretory-secretory products
Ext1 Exostosin glycosyltransferase 1
GST Glutathione-S-transferase
HCC Hepatocellular carcinoma
HDM Host defense molecule
IARC International Agency for Research on Cancer
Krt7 Keratin 7
mRNA Messenger ribonucleic acid
NDMA N-nitrosodimethylamine
OF O. felineus
p.i. Postinfection
Tgfb1 Transforming growth factor beta 1
TpX Thioredoxin peroxidase
Acknowledgments
Figure 3A and C were kindly provided by Galina A
Maksimova (ICG SB RAS, Novosibirsk, Russia). Authors
acknowledge the support of members of the TOPIC
[Tomsk OPIsthorchiasis Consortium]. The English lan-
guage was corrected and certified by shevchuk-editing.
com.
Funding
This work was supported financially by the Russian
Foundation for Basic Research, https://www.rfbr.ru/rffi/
eng [No. 18-04-00417A [VAM] and the state project of
the Institute of Cytology and Genetics, the Siberian Branch
of the Russian Academy of Sciences [project No. 0324-2019-
0041] (MYP, VAM). The sponsor had no role in this
study such as study design, data collection, or decision to
publish.
Conflict of interest
The authors declare that they have no competing
interests.
M.Y. Pakharukova et al.: 4open 2019, 2,23 7
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Cite this article as:Pakharukova MY, Correia da Costa JM & Mordvinov VA, 2019. The liver fluke Opisthorchis felineus as a
group III or group I carcinogen. 4open, 2, 23.
M.Y. Pakharukova et al.: 4open 2019, 2,2310
