Background
A group of helminth infections, caused by liver flukes of the trematode family Opisthorchiidae,
were recently the focus of discussions at a meeting where scientists from Russia,
Southeast Asia, Europe, and the United States came together in Tomsk city in Western
Siberia (Russia) to form a Tomsk OPIsthorchiasis Consortium (TOPIC). This initiative
starts a platform to raise awareness, to strengthen integrated control, and to conduct
research on a neglected infectious disease that afflicts populations not only in the
tropical regions of East Asia but also in temperate and semi-arctic areas of Europe
and Asia [1].
The Opisthorchiidae of importance to humans are Opisthorchis felineus, Opisthorchis
viverrini, and Clonorchis sinensis, each of which has a discrete, though occasionally
overlapping, geographical distribution: O. felineus is endemic in Europe and Russia;
C. sinensis in China, the Republic of Korea, and northern Vietnam; and O. viverrini
in Southeast Asia. Together they affect more than 45 million people worldwide [2].
Human infection with O. felineus results from eating raw or undercooked freshwater
fish carrying the metacercariae of the parasite. The ingested larvae develop further
and migrate to the bile ducts by chemotaxis, where adult worms feed on biliary epithelia
and contents in the bile. Adult worms shed eggs that enter the gastrointestinal tract
and are released with the faeces to the external environment. Freshwater snails of
the family Bithyniidae ingest the eggs and, following the release of the miracidia
from the eggs, several stages of development take place within the snail until cercariae
have developed. Shed cercariae can penetrate freshwater fish, where they encyst in
the skin or flesh [3].
Within the genus Opisthorchis, O. felineus is the species with the highest zoonotic
potential, which has important implications not only for veterinary medicine but also
for maintenance of transmission to humans even under high hygienic standards in which
the risk of freshwater contamination by human faeces is low [4]. The morbidities associated
with opisthorchiasis are largely hepatobiliary, specifically stemming from bile duct
fibrosis and cholangitis, and are expressed in a variety of manifestations, such as
obstructive jaundice, hepatomegaly, abdominal pain, and nausea [5]. Importantly, there
are studies in animal models supported by other epidemiologic data that indicate that
O. viverrini and C. sinensis infections can lead to cholangiocarcinoma, a generally
incurable and, hence, fatal bile duct cancer [6,7], which has resulted in the classification
of these parasites to the Group 1 carcinogens by the International Agency for Research
on Cancer [1,8,9]. Despite the unarguable public health importance of these infections,
both in terms of numbers of humans infected worldwide and clinical impact, it has
been given relatively little recognition by health authorities, grant-giving agencies,
and the pharmaceutical industry. There has been an incremental increase in awareness
following seminal work in Thailand, Korea, and, later, in China, Laos, and Cambodia,
where a number of studies have clarified the situation regarding epidemiology, pathogenesis,
and control. Notably, genome sequence information on these liver flukes is increasingly
available, for example, at www.trematode.net [10]. However, important gaps remain
and, in particular, there is a paucity of information regarding O. felineus.
Therefore, an initiative was taken to organize a meeting in Tomsk, a city located
in the region of Western Siberia that is highly endemic for O. felineus [4,11], and
bring together a multidisciplinary cadre of investigators working on the Opisthorchiidae
and infections caused by these fish-borne liver flukes. This event was welcomed by
Pfizer, a multinational pharmaceutical company that supported the meeting, and that
recognized the importance of infection with O. felineus as a neglected health threat
in Russia. The meeting aimed to highlight the ongoing public health activities and
research on diseases caused by Opisthorchiidae and, in particular, to identify the
gaps in our knowledge of the epidemiology, clinical profile, treatment, and fundamental
mechanisms of host–parasite interaction.
Scientists presenting and discussing their research findings covered a spectrum of
topics that included epidemiology and clinical aspects of these infections, as well
as aspects of host–parasite interaction and the molecular biology of these parasites.
Epidemiology, Clinical Features, and Treatment
The body of data available on the epidemiology of Opisthorchiidae comes largely from
studies conducted on infections with O. viverrini, and increasingly with C. sinensis,
in which robust data have been, and are being, collected in Southeast Asia to map
the endemic regions and to quantify the extent of the morbidity associated with the
infections [2]. Peter Odermatt from the Swiss Tropical and Public Health Institute
discussed the importance of this infection in rural areas of Thailand, Laos, Cambodia,
and Vietnam [12]. In particular, the infection is widespread in Laos, where more than
half of the population is infected; in highly endemic villages, 70% of the population
is infected. Based on microscopy, 50% of the population in some villages in Laos was
found to be infected with O. viverrini, with an increasing prevalence of infection
with age [13]. Close correlation has been found between consumption of fish and prevalence
of O. viverrini in communities in rural Laos where 23 different species of fish in
the region can be infected, and, in some villages, up to 60% of some of these fish
species carry the metacercariae [14]. Examination of cats and dogs that are often
coinhabitants of households revealed that 30% of these domestic animals were infected
with O. viverrini, and, thus, can contribute to intensifying transmission of these
parasites to humans. Furthermore, the deeply culturally rooted habit of raw fish dish
consumption is a major public health challenge [15].
Data collected on the clinical symptoms have provided a comprehensive view on the
morbidities associated with O. viverrini, reporting that increasing intensity of infection
and multiparasitic infection was associated with increasing reported symptoms of abdominal
discomfort and disease in endemic communities [16–18]. The use of ultrasonography
in the field has enabled conduct of large-scale examination of inhabitants of affected
villages to accurately delineate the abnormalities associated with the infection [19].
In the infected population examined so far, normal hepatobiliary images generally
were not observed, whereas mild and severe pathology with bile duct dilation was recorded
in a high proportion of the adult population in rural areas [20].
The association between O. viverrini and cholangiocarcinoma, or bile duct cancer,
was discussed by Banchob Sripa from Khon Kaen University, Thailand. Not only is the
highest incidence of this bile duct cancer in the world seen in Northeast Thailand,
where O. viverrini is highly endemic, but there is also compelling evidence from models
using experimental infection of laboratory rodents demonstrating the close association
between the liver fluke and cancer [21]. Whereas there is also considerable evidence
for the association of C. sinensis and bile duct cancer, so far it is unknown whether
O. felineus shares this feature, a clear area in which further research is warranted
[1].
There are ongoing efforts to understand the risk factors for the development of severe
morbidity including the bile duct cancer: coinfection with other parasitic or viral
(hepatic) infections or smoking may be important. Large multi-centre and multi-country
studies are needed to accurately map parasitic infection and risk factors for morbidity,
including biomarkers for cholangiocarcinoma. Remote sensing and spatial Bayesian statistics
that allow mapping of infection based on geophysical characteristics of the endemic
areas are important tools to bring sociodemographic and environmental factors into
the picture and, hence, prepare large-scale correlation maps of various risk factors
and morbidity, including bile duct cancer [13]. At the biological level, metabolic
phenotyping of subjects at risk of developing cholangiocarcinoma using body fluids
was explained by Oleg Mayboroda from Leiden University Medical Center, the Netherlands.
The use of enabling analytical technologies such as mass spectrometry (MS) and Nuclear
Magnetic Resonance spectroscopy (NMR), now more accessible than before, can be helpful
for early detection as well as understanding the mechanisms that lead to the development
of cancer in infected subjects. Another area for application of the technologies is
identification of metabolic profiles that are specific for Opisthorchiidae, as already
done for other parasitic infections [22–24], which might then allow the development
of simple field applicable rapid tests.
Currently, the treatment of Opisthorchiidae is based on giving praziquantel at 40
mg/kg body weight. Data presented by Peter Odermatt indicated that it might be necessary
to increase the dose of praziquantel to 75 mg/kg, but at the risk of increasing adverse
events, which is an important factor for community compliance [25]. The search for
new therapies that are more effective and have reduced adverse events has come up
with potential new drugs, such as tribendimidine [26–28].
Host–Parasite Interaction
A number of basic mechanisms of host–parasite interaction regarding pathology and
persistence were discussed. Banchob Sripa presented data on the ability of a number
of parasite-derived molecules to drive uncontrolled growth of host cells and, thus,
explain the possible association between O. viverrini and bile duct cancer. Molecules
such as parasite-derived granulin, which leads to proliferation of biliary and other
mammalian cells [29], or parasite-derived enzymes thioredoxin (TRX) and thioredoxin
peroxidase (TPX), which prevent apoptosis, could be involved in stimulating uncontrolled
growth of host cells [3]. Moreover, there are data indicating that O. viverrini extracts
are able to stimulate inflammatory cytokines, such as IL-6 and IL-8 by human cholangiocytes,
peripheral blood mononuclear cells, and that a higher level of IL-6 is seen in infected
patients with bile duct cancer compared to those without [30–32]. Together, these
data raise the question of whether the other Opisthorchiidae have strong proinflammatory
properties that would explain the increased risk of cancer upon infection with these
trematodes and open possibilities for prevention.
Linked to the inflammatory response induced by O. viverrini, Paul Brindley from George
Washington University, Washington, D.C., US, discussed the alteration in the gut and
bile microbiome of infected hosts. He presented findings that reveal that infection
with O. viverrini modified the gut microbiome in hamsters. This investigation detected
the unexpected presence of communities of very select species of bacteria in the bile
of infected hamsters. Intriguingly, a number of species of environmental bacteria,
and therefore not expected in the gut microflora, seemed to find their way into the
bile along with O. viverrini. Might these microbes play an important role in driving
chronic inflammation in the bile tract? These bacterial species were dissimilar to
endosymbiotic Neorickettsia species known to be associated with trematodes at large
[33]. Nonetheless, given that laboratory contamination can impact sequence-based microbiome
analyses, especially in analysis of biofluids discrete from gut contents and faeces
and where microbiota might be sparse [34], these findings will need to be confirmed
in other settings. If, indeed, O. viverrini metacercariae and/or developing adult
flukes vector environmental or exotic microbes into the biliary tree, persistent inflammatory
responses to this fluke-associated microbiota could be a key factor in the development
of cancer [3,19,21,35]. This report has paved the way for human studies to test whether
similar mechanisms could be at play.
The basic immunological profile in humans infected with these parasites was the subject
of the presentations by Olga Fedorova from Siberian State Medical University and Maria
Yazdanbakhsh from Leiden University Medical Center, the Netherlands. In comparison
with other parasitic helminths, relatively little has been done on the immunological
profiling of humans infected with Opisthorchiidae, especially beyond O. viverrini
infections [30]. The immunoepidemiological studies in the region of Tomsk comparing
subjects infected with and without O. felineus has indicated that there is TH2 skewing
as evident from elevated IgE in infected subjects compared to uninfected, but that
the levels are much lower than what is found in studies of helminth infected subjects
in Ghana where schistosomiasis is highly prevalent or in Indonesia in communities
with geohelminth infections. This could result from the fact that intensity of infection
is lower in O. felineus infected subjects and that there are much less coinfections
with other helminths in semi-arctic regions of Western Siberia. However, it is also
possible that the antigenic composition of the O. felineus is less capable of inducing
TH2 responses. Some evidence for this comes from in vitro studies in which human dendritic
cells cultured with O. felineus antigens lead to less TH2 skewing compared to when
dendritic cells were cultured with Schistosoma mansoni antigens [36].
The skewing of immune responses towards TH1, TH2, and regulatory T cells can be important
for understanding immunopathological processes as well as the development of cancer
associated with an infection. A number of studies of humans chronically infected with,
for example, schistosomes or geohelminths have shown the induction of regulatory T
[37,38] and B cells [39] by these parasites. These immune regulatory cells can be
involved in the suppression of responses to unrelated antigens [40,41]. A study conducted
in the Tomsk region has shown an inverse association between O. felineus and responses
to allergens [11], supporting the notion that it would be worthwhile to investigate
whether Opisthorchiidae are able to induce regulatory cells. This is of particular
importance also because of the possible link to the development of cancer. Prognosis
of cancer is poor if regulatory T cells are found in tumours and a number of studies
in experimental models have indicated that regulatory T cells are associated with
faster tumour growth [42,43]. Whether O. viverrini and C. sinensis bear molecules
that are able to induce regulatory T or B cells should be investigated. Recently,
the excretory/secretory (E/S) antigens of a nematode, Heligmosomoides polygyrus, have
been shown to drive strong regulatory T cell responses. Neutralisation of these E/S
antigens resulted in complete elimination of the worms [44]. This exciting approach
could also be considered for driving immunity to Opisthorchiidae as well as fighting
against the associated bile duct cancer.
A presentation by Thirumalaisamy Velavan, representing the Institute of Tropical Medicine,
University of Tuebingen, Germany, indicated the importance of studies regarding the
innate immune components of the complement system and its interaction with Opisthorchiidae.
O. felineus has an outer syncytial cytoplasmic layer as teguments, such as in other
trematodes. Earlier studies have demonstrated that these trematode teguments are made
up of D-mannose/D-glucose, N-acetyl-D-glucosamine/sialic acid, D-galactose, and N-acetyl-D-galactosamine
residues on the glycocalyx of the adult tegument and are expressed at all developmental
stages. These glycoconjugates serve as pathogen-associated molecular patterns (PAMPs)
for immune recognition and subsequent complement-mediated killing [45–47]. Two innate
immune recognition elements of the complement system, the mannose-binding lectin and
ficolins, were earlier shown to influence the infection outcome in S. haematobium
that causes urinary bladder cancer [48,49]. Additionally, the functional variants
of these MBL2 and FCN2 genes were established to modulate the circulating serum levels
and the binding capacity to the parasite surface, thus leading to impaired recognition
[50]. Hence, investigation of the lectin pathway proteins during Opisthorchis infection
might help in better understanding the interactions between the host and the parasite
during an establishment of active infection.
Indeed, the question of genetic regulation of susceptibility to Opisthorchiidae and
genetic control of the development of associated pathology requires attention. Maxim
Freidin from the Research Institute for Medical Genetics, Russia, and Royal Brompton
Hospital, United Kingdom, showed the results of a pilot gene–environment interaction
study in the Tomsk population that identified O. felineus infection as an important
modifier of associations between TH1/TH2-regulating genes and allergic disease. In
particular, it was shown that O. felineus infection diminishes the risk of atopic
bronchial asthma associated with the polymorphisms of the SOCS5 and IFNG genes [4].
The allele specific gene expression was found to be modified by the presence of O.
felineus antigens, thus providing a functional clue for the mechanisms of the identified
gene–environment interaction. These studies form a paradigm for assessing whether
tissue pathology/cancer development is controlled by inflammation as a result of gene–environment
interaction.
Diagnosis
Attempts to use molecular approaches to detect O. felineus in hospital-based studies
in Europe where infections are found sporadically have been described [51]. A presentation
by Vasily Petrenko from Medical Biological Union and Institute of Cytology and Genetics,
Novosibirsk, Russia, showed promising results on the detection of four Opisthorchiidae
flukes, O. felineus in particular, by Taqman-based real-time PCR with a high degree
of sensitivity and specificity when working with faeces from hamsters infected with
local strains. These results call for large-scale validation studies similar to ongoing
activities regarding the molecular diagnosis of O. viverrini and C. sinensis and reliable
discrimination of O. felineus infection [2]. Field applicability of these tests was
discussed in terms of providing point of care diagnostic assays. Thirumalaisamy Velavan
presented a new molecular diagnostic approach for rapid detection of parasites using
the Loop-mediated isothermal amplification (LAMP) method [52]. These methodologies
were established for O. viverrini targeting the internal transcribed spacer 1 (ITS1)
in ribosomal DNA for specific amplification [53]. LAMP methodologies are simple, sensitive,
specific, and faster than PCR, requiring minimal processing and instrumentation, with
results available by reading with the naked eye. The development of such a LAMP methodology
for O. felineus is being established.
Parasite Biology and Drug Targets
The research group of the Institute of Cytology and Genetics in Novosibirsk, under
the leadership of Aleksei Katokhin, has conducted phylogeographic genetics studies
of Opisthorchiidae flukes in order to compare their genetic diversities and species
histories [54]. These studies are aimed at explaining pronounced differences in capacities
of the three flukes to parasitize in various mammals and shedding light on the degree
of zoonosis of the different species [2]. These studies might help integrated control
programs and further our fundamental understanding of biological processes.
Mariya Pakharukova and Viatcheslav Mordvinov, also at the Institute of Cytology and
Genetics, Novosibirsk, presented data on the structure and functional organization
of xenobiotic biotransformation system in O. felineus [55]. The system participates,
probably, in O. felineus metabolism and in the transport of endogenous and exogenous
substrates. Importantly, cytochrome P450 is expressed differentially through the life
cycle of O. felineus and is present in O. felineus in higher amounts than in O. viverrini.
The question is whether this could have any relationship to synthesis of proinflammatory
and potentially carcinogenic compounds, e.g., oxysterol-like, catechol estrogen quinone-like,
etc., released by the flukes [56,57]. Novel data were also presented on studies on
praziquantel effects in the O. felineus hamster model and in vitro on juvenile and
adult worms, particularly, about parasite motility, viability, and tegument damage
after praziquantel treatment.
Concluding Remarks: Call for Expansion of the Consortium
The data presented and discussed led us to the conclusion that there are many groups
with overlapping interests and relevant expertise that are willing to work together
towards the common aim of controlling liver fluke infections and associated pathologies
worldwide. Most importantly, it was stressed that this is a starting point for inviting
other groups actively working in the field of liver fluke research to join the Tomsk
Opisthorchiasis Consortium by contacting Ludmila Ogorodova from Siberian State Medical
University, Tomsk, Russia (topic.consortium@gmail.com). In particular, since there
was only limited discussion of the current state of research in the area of clonorchiasis,
specialists from this field would also be especially welcome.
The Tropical Disease Research Laboratory at Khon Kaen University has a successful
control program, the so called “Lawa model,” based on using the EcoHealth approach
at the lake Lawa region of Northeast Thailand [58]. It was agreed that this control
model will be taken as a template and modified according to local health care systems
and cultural sensitivities in other endemic areas, but not differing in essence of
involving trans-disciplinary and stakeholder participation at every level.
One of the first issues to be addressed is to understand the knowledge gap on the
prevalence of O. felineus infection in Russia and the extent of the related morbidity;
in particular, whether an O. felineus infection is associated with an increased risk
of bile duct cancer. In parallel, activities that facilitate faster development of
validated diagnostic tests to accurately detect O. felineus infection will be stimulated
and treatment efficacies of currently used drugs verified, while groups will work
together in order to strengthen the scientific and technological skills needed to
understand the host–parasite interaction in terms of immune-pathogenesis and regulation.
Finally, the overarching, fundamental molecular biological efforts to identify new
drug targets or new preventive and therapeutic measures will be stimulated by combining
expertise and sharing biological material available within the Consortium.
The Consortium shall operate through, and expand towards, several parallel research
collaborative activities/work packages, depicted in the high level roadmap (Fig. 1),
such as: burden/severity and control at the community; clinical studies; biology,
and ecology of parasites; host–parasite interactions; knowledge/technology transfer.
10.1371/journal.pntd.0003563.g001
Fig 1
Consortium high level timelines/activities.
1. Siberian State Medical University, Tomsk, Russian Federation, 2. Center for Proteomics
and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands, 3. Department
of Parasitology and Leiden Parasite Immunology Group, Leiden University Medical Center,
Leiden, the Netherlands, 4. George Washington University Medical Center, United States,
5. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences,
Novosibirsk, Russian Federation, 6. Institute of Tropical Medicine, University of
Tübingen, Germany, 7. Khon Kaen University, Khon Kaen, Thailand, 8. Pfizer LLC, Moscow,
Russian Federation, 9. ReMedys Foundation, Geneva, Switzerland, 10. Royal Brompton
Hospital, United Kingdom; Research Institute for Medical Genetics, Tomsk, Russian
Federation, 11. Swiss Tropical and Public Health Institute, Basel, Switzerland.
These activities are detailed in an under-development project plan. The success of
the Consortium shall be measured upon the delivery of research results, and, in long-term,
through the applicability of these results to the society. Thus, care shall be given
to the technology transfer, through all available and feasible mechanisms, bridging
the gap between basic research and industrialization. To secure the planning, the
establishment, the coordination, the advancement, the milestones reaching, and, finally,
the knowledge/technology transfer of the results, the on-board availability of expertise
in program management and industrialization will be prioritized. This can be through
entities with industrial expertise such as Pfizer and the ReMedys Foundation, who
are both already among the founding members of the Consortium. (Pfizer strives to
positively impact the health of people around the world. Pfizer’s corporate social
investment strategy focuses on leveraging the full range of the company's resources—people,
skills, expertise, and funding—to broaden access to medicines and strengthen health
care delivery for underserved people around the world [http://www.pfizer.com/responsibility/global_health/global_health].
ReMedys is a not-for-profit entity, founded by biopharmaceutical industry experts,
intending to implement a novel, highly collaborative approach bridging the gap of
translational Research and Development [R&D]. ReMedys acts as the translational R&D
arm and hub for top research institutions, patient groups, and clinicians. Via building
and coordinating customized patient centric alliances, ReMedys brings together all
resources needed to advance, and make available to patients with high unmet need,
promising therapeutic projects [http://remedys.net].)
The founding members of the Consortium have signed a Memorandum of Understanding,
which shall be available for the signature of research groups interested to become
new members.
The overall program shall be regulated by a Consortium agreement that is under establishment,
to which new members shall be invited to join and participate. The funding for the
Consortium shall be secured through national and international grants-associated activities
by the members. As described, the Consortium aims to engage into a strategic collaborative
study on a serious, but highly neglected, infectious disease, which is responsible
for a heavy socioeconomic burden including cancer. The study shall concentrate, as
well, into sensitive populations such as children, and, hence, expects to deliver
findings with a high socioeconomic impact. Thus, the Consortium, with appropriate
tools, can develop enhanced public awareness through the patient, the doctor, the
researcher, the grant sponsor, and the government. Such an awareness shall support
its funding through, as well, public, philanthropic, international NGOs; corporate
responsibility partnerships; and so forth. Other funding mechanisms, such as crowd
funding, in which all the Consortium members can participate, shall be explored.