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Simple Summary: The zoonotic liver flukes Opisthorchis viverrini and Clonorchis sinensis infect small mammals, such as cats, dogs, pigs, rodents, and rabbits, as well as humans. Human infection subsequently develops into bile duct malignancy, also referred to as cholangiocarcinoma (CCA). Understanding the molecular systematics and population genetics of these liver flukes has an important role in prevention and control, and is important in comprehending their roles in zoonotic transmission. Different molecular markers have varying evolution rates and contain different genetic information. Polymorphic genetic markers are necessary and more suitable for such investigations. Therefore, we screened seven intron regions of the taurocyamine kinase gene (TK) to determine their potential as genetic markers for population genetic investigations of the liver flukes O. viverrini and C. sinensis which were collected from a range of geographical isolates and animal hosts. We identified a suitable intron region of TK, i.e., intron 5 of domain 1 (TkD1Int5) as having the most potential as a polymorphic marker. Results showed that TkD1Int5 is effective in examining the genetic variation and heterozygosity of O. viverrini and C. sinensis, but further studies are required to better understand the role of different species of animals as reservoir hosts of these zoonotic liver flukes. Opisthorchiasis and clonorchiasis are prevalent in Southeast and Far-East Asia, which are caused by the group 1 carcinogenic liver flukes Opisthorchis viverrini sensu lato and Clonorchis sinensis infection. There have been comprehensive investigations of systematics and genetic variation of these liver flukes. Previous studies have shown that O. viverrini is a species complex, called "O. viverrini sensu lato". More comprehensive investigations of molecular systematics and population genetics of each of the species that make up the species complex are required. Thus, other polymorphic genetic markers need to be developed. Therefore, this study aimed to characterize the intron regions of taurocyamine kinase gene (TK) to examine the genetic variation and population genetics of O. viverrini and C. sinensis collected from different geographical isolates and from a range of animal hosts. We screened seven intron regions embedded in TK. Of these, we selected an intron 5 of domain 1 (TkD1Int5) region to investigate the genetic variation and population genetics of theses liver flukes. The high nucleotide and haplotype diversity of TkD1Int5 was detected in O. viverrine. Heterozygosity with several insertion/deletion (indel) regions were detected in TkD1Int5 of the O. viverrine samples, whereas only an indel nucleotide was detected in one C. sinensis sample. Several O. viverrine samples contained three different haplotypes within a particular heterozygous sample. There were no genetic differences between C. sinensis isolated from various animal host. Heterozygous patterns specifically detected in humans was observed in C. sinensis. Thus, TkD1Int5 is a high polymorphic genetic marker, which could be an alternative marker for further population genetic investigations of these carcinogenic liver flukes and other related species from a wide geographical distribution and variety of animal hosts. [ABSTRACT FROM AUTHOR]

Background: The direct reliance of humans on and their interactions with freshwater ecosystems in the Lower Mekong Basin have given rise to parasitic infections, which is particularly prevalent in Northeast Thailand where raw fish consumption is practiced. This study examined the interactions between environments, ecosystem (dis-)services, human raw fish consumption habits, and raw fish dish sharing on liver fluke infection risk. Method: Water fecal contents and the first intermediate snail host were sampled between June and September of 2019. One hundred twenty questionnaires were surveyed in two villages of different environmental surroundings, one next to a river and the other located inland, in Northeast Thailand. Multivariate regression analyses using linear mixed effect models assessed the influence of social, behavioral and perceptual factors on raw fish consumption frequency, willingness to avoid consumption and liver fluke infection status. Social network analysis compared the degree of raw fish dish sharing between the villages and assessed the probable influence of connections to fish procurement locations and sharing activities on liver fluke infection risk. Results: High abundance of the first intermediate snail host and presence of fecal contamination in water could endanger both villages to ecosystem disservices of parasitic transmission. The river-side village relied more on provisioning ecosystem services than the inland village (29.7% vs. 16.1% of villages) to consume raw fish as their main source of protein. Males in both villages (64.5 and 40.4 days/year for the respective villages) are also likely to consume koi pla and pla som, higher risk fish dishes, more frequently than females (4.1 and 4.3 days/year for the respective villages). The consumption habits of both villages were driven mostly by deriving cultural ecosystem services. Participation in raw fish dish sharing activities significantly reduced the odds of an individual being willing to avoid the consumption (Odds ratio = 0.19). Network analysis suggested that river-side villagers had a more direct raw fish dish sharing interaction and they procured fish from multiple locations; these characteristics might potentially account for more liver fluke infected households in the village. Conclusion: Villagers' raw fish consumption is driven by deriving cultural ecosystem services, and the geographic settings of the villages potentially affect villagers' fish procurement locations and infection risk. The findings underscore the linkages between villagers and their surrounding ecosystem environments as pertinent determinants for foodborne parasitic disease risk. [ABSTRACT FROM AUTHOR]

Global liquid chromatography mass spectrometry (LC-MS) profiling in a Thai population has previously identified a urinary metabolic signature in Opisthorchis viverrini -induced cholangiocarcinoma (CCA), primarily characterised by disturbance in acylcarnitine, bile acid, steroid, and purine metabolism. However, the detection of thousands of analytes by LC-MS in a biological sample in a single experiment potentially introduces false discovery errors. To verify these observed metabolic perturbations, a second validation dataset from the same population was profiled in a similar fashion. Reverse-phase ultra-performance liquid-chromatography mass spectrometry was utilised to acquire the global spectral profile of 98 spot urine samples (from 46 healthy volunteers and 52 CCA patients) recruited from Khon Kaen, northeast Thailand (the highest incidence of CCA globally). Metabolites were differentially expressed in the urinary profiles from CCA patients. High urinary elimination of bile acids was affected by the presence of obstructive jaundice. The urine metabolome associated with non-jaundiced CCA patients showed a distinctive pattern, similar but not identical to published studies. A panel of 10 metabolites achieved a diagnostic accuracy of 93.4% and area under the curve value of 98.8% (CI = 96.3%–100%) for the presence of CCA. Global characterisation of the CCA urinary metabolome identified several metabolites of biological interest in this validation study. Analyses of the diagnostic utility of the discriminant metabolites showed excellent diagnostic potential. Further larger scale studies are required to confirm these findings internationally, particularly in comparison to sporadic CCA, not associated with liver fluke infestation. [ABSTRACT FROM AUTHOR]

Helminth transmission and morbidity are dependent on the number of mature parasites within a host; however, observing adult worms is impossible for many natural infections. An outstanding challenge is therefore relating routine diagnostics, such as faecal egg counts, to the underlying worm burden. This relationship is complicated by density-dependent fecundity (egg output per worm reduces due to crowding at high burdens) and the skewed distribution of parasites (majority of helminths aggregated in a small fraction of hosts). We address these questions for the carcinogenic liver fluke Opisthorchis viverrini, which infects approximately 10 million people across Southeast Asia, by analysing five epidemiological surveys (n = 641) where adult flukes were recovered. Using a mechanistic model, we show that parasite fecundity varies between populations, with surveys from Thailand and Laos demonstrating distinct patterns of egg output and density-dependence. As the probability of observing faecal eggs increases with the number of mature parasites within a host, we quantify diagnostic sensitivity as a function of the worm burden and find that greater than 50% of cases are misdiagnosed as false negative in communities close to elimination. Finally, we demonstrate that the relationship between observed prevalence from routine diagnostics and true prevalence is nonlinear and strongly influenced by parasite aggregation. [ABSTRACT FROM AUTHOR]

Background: Control and elimination of the liver fluke (Opisthorchis viverrini) is a primary preventive strategy against cholangiocarcinoma in Southeast Asia. A sensitive parasitological diagnostic method is required to facilitate a surveillance and control program. In this study, we evaluated the performance of Mini Parasep® SF stool concentrator kit (stool kit) compared with Kato-Katz (KK) and the quantitative formalin-ethyl acetate concentration technique (FECT) for detection of O. viverrini and co-endemic parasitic infections. Methods: A cross-sectional survey for parasitic infection in residents aged > 15 years in a community in Kalasin province, Northeast Thailand, was conducted in 2018. Fecal samples were collected and screened by KK method, and a subset of samples was further examined by the stool kit and FECT methods. The results were analyzed for prevalence of parasitic infections in addition to the diagnostic performance of the methods for qualitative and quantitative detection of helminthiases. Results: The initial survey of parasitic infection determined by the KK method (n = 567) showed the prevalence of O. viverrini was 32.63%, followed by Taenia 2.65%, echinostomes 1.76%, hookworms 1.41%, Trichuris trichiura 0.53% and Strongyloides stercoralis 0.53%. Within a subset of samples tested with multiple diagnostics (n = 150), the detection rates of O. viverrini by the stool kit, FECT and KK methods were 27.3%, 30.7% and 28.7%, respectively. The diagnostic sensitivity for opisthorchiasis was similar for FECT (75.5%), KK(66.0%) and the stool kit (67.3%). For other parasitic infections, FECT and stool kit methods performed better than KK, particularly in detecting minute intestinal flukes (MIF), S. stercoralis and coinfections. When measuring the intensity of O. viverrini infection (fecal egg counts), the stool kit results showed a significant positive correlation with KK and FECT (P < 0.05). Conclusions: As the stool kit is simple to use and shows a comparable performance to FECT, it may serve as an alternative method of fecal examination for screening of helminthiasis including opisthorchiasis. [ABSTRACT FROM AUTHOR]

Transmission of the carcinogenic liver fluke Opisthorchis viverrini is ongoing across Southeast Asia. Endemic countries within the region are in different stages of achieving control. However, evidence on which interventions are the most effective for reducing parasite transmission, and the resulting liver cancer, is currently lacking. Quantitative modelling can be used to evaluate different control measures against O. viverrini and assist the design of clinical trials. In this article we evaluate the epidemiological parameters that underpin models of O. viverrini and the data necessary for their estimation, with the aim of developing evidence-based strategies for parasite control at a national or regional level. Halting the transmission of O. viverrini is crucial to preventing cases of cholangiocarcinoma, yet there is no consensus on the best strategies for large-scale parasite control. Quantitative modelling can provide insights and guide control programmes more rapidly and using fewer resources than clinical trials. To give reliable output, models depend on making accurate assumptions and using parameters inferred from relevant datasets. Mathematical models of O. viverrini dynamics have recently been published and provide a valuable starting point. However, many key epidemiological processes remain poorly understood, including the links between parasite exposure, anthelmintic treatment, and carcinogenesis. We highlight areas where new studies are required to fill knowledge gaps. Recent developments in statistical modelling and parasite genomics provide opportunities to make rapid gains. [ABSTRACT FROM AUTHOR]

The freshwater snail Bithynia siamensis goniomphalos serves as the first intermediate host of liver fluke Opisthorchis viverrini, a foodborne parasite, of which human infection has persisted in Southeast Asia for decades. The snail Filopaludina martensi martensi has been proposed as a biological control agent against B.s. goniomphalos, but knowledge on the snail ecology and population dynamics between the two species remains rudimentary. This study investigated selected abiotic and biotic factors influencing the distribution and abundance of B.s. goniomphalos and F.m. martensi. Water quality, soils, and snails were collected from 34 localities in Northeast Thailand. Soil properties and snail soft tissue elemental concentrations were analyzed. Experiments were performed to examine interspecific competition. Statistical analysis was conducted to explore the associations between water and soil properties and soft tissue elemental concentrations. The results showed that B.s. goniomphalos had the highest mean dominance in streams and red-yellow podzolic soils, while F.m. martensi snails preferred ponds and latosol soils. Negative correlation in species abundances was found between the two species. Interspecific competition was detected, with B.s. goniomphalos growth rates hampered by the presence of F.m. martensi. Despite the possibility of using F.m. martensi to control B.s. goniomphalos, B.s. goniomphalos exhibited a greater adaptability to different water and soil properties, suggesting that the species could colonize a wide range of environmental conditions. This study provides further insights into the ecology of the two snail species, underscoring the importance of considering abiotic factors when assessing the possible biological control agent to control O. viverrini transmission. [ABSTRACT FROM AUTHOR]

Background: Like many trematodes of human health significance, the carcinogenic liver fluke, Opisthorchis viverrini, is spread via fecal contamination of snail habitat. Methods for assessing snail exposure to fecal waste can improve our ability to identify snail infection hotspots and potential sources of snail infections. We evaluated the feasibility of culturing fecal indicator bacteria from Bithynia snail intestinal tubes as a method for assessing snail exposure to fecal waste. Snails and water samples were collected from a site with a historically high prevalence of O. viverrini infected snails ("hotspot" site) and a site with historically no infected snails ("non-hotspot" site) on two sampling days. Snails were tested for O. viverrini and a stratified random sample of snails from each site was selected for intestinal tube removal and culture of gut contents for the fecal indicator bacteria, Escherichia coli. Water samples were tested for E. coli and nearby households were surveyed to assess sources of fecal contamination. Results: At the hotspot site, 26 of 2833 Bithynia siamensis goniomphalos snails were infected with O. viverrini compared to 0 of 1421 snails at the non-hotspot site. A total of 186 snails were dissected and cultured. Escherichia coli were detected in the guts of 20% of uninfected snails, 4% of O. viverrini-positive snails and 8% of snails not examined for cercarial infection at the hotspot site. Only one of 75 snails from the non-hotspot site was positive for E. coli. Accounting for sampling weights, snails at the hotspot site were more likely to have gut E. coli than snails from the non-hotspot site. The concentration of fecal indicator bacteria in surface water was higher at the hotspot vs non-hotspot site on only the first sampling day. Conclusions: Fecal indicator bacteria can be detected in the intestinal tubes of Bithynia snails. The presence of fecal indicator bacteria in Bithynia snail guts may indicate risk of O. viverrini infection in snail populations. This method has the potential to aid in identifying locations and time windows of peak snail infection risk and may be applicable to other trematodes of human-health significance. [ABSTRACT FROM AUTHOR]

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Abstract: Bithynia snails are important hosts of the human liver fluke, Opisthorchis viverrini. Allozyme markers were used to examine genetic variation and the systematics of Bithynia snails from different geographical localities in Thailand and Lao PDR. Our results show that Bithynia siamensis goniomphalos contains two major evolutionary clades with at least nine morphologically similar but genetically distinct “cryptic” species (taxa). Clade A consists of snails from the Chi and the Mun River wetlands, Thailand and clade B consists of snails from the Nam Ngum River wetland, Lao PDR, as well as snails from some localities in the Songkram River wetland, Thailand. There is an association between O. viverrini cryptic taxa with B. s. goniomphalos cryptic taxa within the defined wetlands. Bithynia species are the critical amplifying component of O. viverrini and they control transmission from humans to fish second intermediate hosts. These findings are particularly important for understanding the transmission dynamics of O. viverrini. They are, in addition, a link in the life cycle of O. viverrini which can be used for developing and implementing programs to eliminate and control opisthorchiasis and its associated cholangiocarcinoma. [Copyright &y& Elsevier]

Khon Kaen Province in northeast Thailand is known as a hot spot for opisthorchiasis in Southeast Asia. Preliminary allozyme and mitochondrial DNA haplotype data from within one endemic district in this Province (Ban Phai), indicated substantial genetic variability within Opisthorchis viverrini. Here, we used microsatellite DNA analyses to examine the genetic diversity and population structure of O. viverrini from four geographically close localities in Khon Kaen Province. Genotyping based on 12 microsatellite loci yielded a mean number of alleles per locus that ranged from 2.83 to 3.7 with an expected heterozygosity in Hardy–Weinberg equilibrium of 0.44–0.56. Assessment of population structure by pairwise FST analysis showed inter-population differentiation (P<0.05) which indicates population substructuring between these localities. Unique alleles were found in three of four localities with the highest number observed per locality being three. Our results highlight the existence of genetic diversity and population substructuring in O. viverrini over a small spatial scale which is similar to that found at a larger scale. This provides the basis for the investigation of the role of parasite genetic diversity and differentiation in transmission dynamics and control of O. viverrini. Author Summary: Infection with the liver fluke (Opisthorchis viverrini) is a risk factor for cholangiocarcinoma (CCA), which is highly prevalent in Khon Kaen Province, Thailand. Within this Province, there is considerable variation in the epidemiology of opisthorchiasis among districts. Preliminary allozyme and mitochondrial DNA data indicate that genetic variation in O. viverrini occurs even over a small endemic area within the province. Here, we used microsatellite DNA analyses to examine the population genetic structure of O. viverrini from four geographically close localities. Analyses of adult worms based on 12 microsatellite loci revealed varying levels of genetic diversity and population substructuring between the four localities. Worms originating from one locality (Phu Wiang) had significantly higher genetic diversity than the other three localities. Data on the population genetic structure observed in these localities are similar to those found at a larger geographic scale. This provides background data to further investigate the biological and epidemiological significance of these genetic variants. [ABSTRACT FROM AUTHOR]

Opisthorchiasis caused by Opisthorchis viverrini induces periductal fibrosis via host immune/inflammatory responses. Plasma protein alteration during host-parasite interaction-mediated inflammation may provide potential diagnostic and/or prognostic biomarkers. To search for target protein changes in O. viverrini-infected hamsters, a 1-D PAGE gel band was trypsin-digested and analyzed by a LC-MS/MS-based proteomics approach in the plasma profile of infected hamsters, and applied to humans. Sixty seven proteins were selected for further analysis based on at least two unique tryptic peptides with protein ID score .10 and increased expression at least two times across time points. These proteins have not been previously identified in O. viverrini-associated infection. Among those, proteins involved in structural (19%), immune response (13%), cell cycle (10%) and transcription (10%) were highly expressed. Western blots revealed an expression level of protein tyrosine phosphatase alpha (PTPα) which reached a peak at 1 month and subsequently tended to decrease. Fibronectin significantly increased at 1 month and tended to increase with time, supporting proteomic analysis. PTPa was expressed in the cytoplasm of inflammatory cells, while fibronectin was observed mainly in the cytoplasm of fibroblasts and the extracellular matrix at periductal fibrosis areas. In addition, these protein levels significantly increased in the plasma of O. viverrini-infected patients compared to healthy individuals, and significantly decreased at 2-months post-treatment, indicating their potential as disease markers. In conclusion, our results suggest that plasma PTPα and fibronectin may be associated with opisthorchiasis and the hamster model provides the basis for development of novel diagnostic markers in the future. [ABSTRACT FROM AUTHOR]

Re-infection with liver fluke, Opisthorchis viverrini, increases proinflammatory molecules involved in inflammation-mediated disease and carcinogenesis in an animal model. To clarify whether these genes respond to parasite antigen in peripheral blood leukocytes (PBL) of opisthorchiasis patients, we examined the transcriptional level of oxidant-generating (toll-like receptor 2 (TLR-2), nuclear factor-kappa B (NF-KB), and cyclooxygenase 2 (COX-2)), anti-oxidant-generating (manganese superoxide dismutase 2 (SOD-2) and catalase (CAT)), proinflammatory cytokine (interleukin (IL)-1β), and anti-inflammatory cytokine (IL-10), in PBL exposed to parasite antigen in O. viverrini-infected patients compared with healthy individuals in an in vitro experiment. After O. viverrini antigen-treated PBL, quantitative RT-PCR analysis revealed that increased expression of cytokines and oxidant-generating genes in PBL was similar between O. viverrini-infected and healthy groups. Interestingly, compared with healthy subjects, increase of TLR-2, COX-2, and SOD-2 and decreased CAT mRNA expression levels were observed in O. viverrini-infected group. The results indicate that O. viverrini antigen induces upregulation of TLR-2, COX-2, and SOD-2 and downregulation of CAT genes in opisthorchiasis patients, suggesting that imbalance of oxidant/anti-oxidant transcripts during re-infection may be involved in the inflammatory-driven carcinogenesis. These molecules may be used as the chemopreventive target for intervention of opisthorchiasis patients in an endemic area. [ABSTRACT FROM AUTHOR]

Abstract: Together with host and environmental factors, the systematics and population genetic variation of Opisthorchis viverrini may contribute to recorded local and regional differences in epidemiology and host morbidity in opisthorchiasis and cholangiocarcinoma (CCA). In this review, we address recent findings that O. viverrini comprises a species complex with varying degrees of population genetic variation which are associated with specific river wetland systems within Thailand as well as the Lao PDR. Having an accurate understanding of systematics is a prerequisite for a meaningful assessment of the population structure of each species within the O. viverrini complex in nature, as well as a better understanding of the magnitude of genetic variation that occurs within different species of hosts in its life cycle. Whether specific genotypes are related to habitat type(s) and/or specific intermediate host species are discussed based on current available data. Most importantly, we focus on whether there is a correlation between incidence of CCA and genotype(s) of O. viverrini. This will provide a solid basis for further comprehensive investigations of the role of genetic variation within each species of O. viverrini sensu lato in human epidemiology and genotype related morbidity as well as co-evolution of parasites with primary and secondary intermediate species of host. [Copyright &y& Elsevier]

Abstract: This review highlights the current status and control of liver fluke infections in the Mekong Basin countries where Opisthorchis and Clonorchis are highly endemic. Updated data on prevalence and distribution have been summarized from presentations in the “96 Years of Opisthorchiasis. International Congress of Liver Flukes”. It is disturbing that despite treatment and control programs have been in place for decades, all countries of the Lower Mekong Basin are still highly endemic with O. viverrini and/or C. sinensis as well as alarmingly high levels of CCA incidence. A common pattern that is emerging in each country is the difference in transmission of O. viverrini between lowlands which have high prevalence versus highlands which have low prevalence. This seems to be associated with wetlands, flooding patterns and human movement and settlement. A more concerted effort from all community, educational, public health and government sectors is necessary to successfully combat this fatal liver disease of the poor. [Copyright &y& Elsevier]

Opisthorchis viverrini ( O. viverrini; known as human liver fluke) is a major health problem in the northeastern region of Thailand. Infection with O. viverrini is the cause of hepatobiliary disease and cholangiocarcinoma (CCA). Previous studies demonstrated specific antibodies to crude O. viverrini antigens in serum from O. viverrini-infected patients. However, no studies have measured specific antibodies to O. viverrini antigens in saliva from patients with opisthorchiasis and CCA. The objective of the study was to detect specific antibodies to crude O. viverrini antigens in saliva from patients with opisthorchiasis and CCA, and to evaluate their use for diagnosis of O. viverrini infection. Saliva samples from 23 control subjects, 30 opisthorchiasis patients, and 38 CCA patients were collected. ELISA was established for detection of salivary IgA and IgG to crude O. viverrini antigens. ANOVA was used to compare salivary IgA and IgG levels among groups. Salivary IgA to crude O. viverrini antigens in CCA patients was significantly higher than controls ( p = 0.007). Salivary IgG in CCA patients was significantly higher than opisthorchiasis patients and controls ( p = 0.010 and p < 0.001, respectively). The cut-off value from salivary IgG test demonstrated higher accuracy for positivity of O. viverrini infection than salivary IgA. In conclusion, specific antibodies to crude O. viverrini antigens were detected in saliva of patients with opisthorchiasis and CCA. Salivary antibodies reflect serum immune response to O. viverrini infection, and salivary IgG tends to be a good candidate for diagnosis of O. viverrini infection. [ABSTRACT FROM AUTHOR]

Abstract: Snails are the critical amplifying hosts of the liver fluke Opisthorchis viverrini, the causative agent of hepatobiliary disease and cholangiocarcinoma in the Mekong area of Southeast Asia. Bithynia funiculata, B. siamensis goniomphalos and B. s. siamensis are the first intermediate hosts of O. viverrini in Thailand. Morphological similarity between Bithynia species and subspecies creates problems for their taxonomic identification and an understanding of Bithynia systematics. In this study, multilocus enzyme electrophoresis (MEE) was applied to define genetic markers that could prove useful for investigating the systematics and population genetics of this genus in Thailand. Of the 34 enzymes examined, 20 encoding a presumptive 24 loci showed sufficient staining intensity and resolution for genetic interpretation. Of these, three loci were monomorphic and eight loci were diagnostic among the three Bithynia taxa. The remaining 13 loci were diagnostic between combinations of the three taxa. Fixed genetic differences were detected at 67–73% of loci among these taxa which in turn differed from a closely related species, Hydrobioides nassa, at 88% of loci. Seventy three percent fixed genetic differences were detected between B. funiculata and the two sub-species B. s. siamensis and B. s goniomphalos. Our data reveals similarly large genetic divergence, 67% fixed genetic differences, between B. s. siamensis and B. s. goniomphalos, which may well represent different species rather than subspecies as currently defined. The genetic markers detected will form the basis for subsequent comprehensive studies on the systematics and population genetics of Bithynia snails as well as for their role in the transmission of O. viverrini and opisthorchiasis. [Copyright &y& Elsevier]

Abstract: Opisthorchis viverrini is a carcinogenic foodborne trematode endemic in Southeast Asia especially in Thailand and the Lao People''s Democratic Republic. Opisthorchiasis causes hepatobiliary diseases and cholangiocarcinoma (bile duct cancer). Currently there is substantial evidence on genetic variation of O. viverrini but the information on population genetic structure is lacking. Because microsatellite DNA of this parasite is not available, we for the first time isolated and utilized microsatellite DNA as genetic markers to examine genetic diversity and the population structure of O. viverrini. Partial genomic DNA libraries were constructed by conventional and enrichment methods which yielded microsatellite-containing clones of 0.18–0.25% and 16.84%, respectively. Within 41 microsatellite loci isolated 36.59% were perfect, 60.98% were interrupted and 2.44% were compound microsatellites. The CA repetitions were the most frequent, followed by GT and CAT. Primers specific to the flanking regions of 12 microsatellite loci were developed to genotype 150 O. viverrini individuals from geographical localities in Thailand and Lao PDR. Allele numbers per locus ranged from 2 to 15, with the mean expected heterozygosity of 0.03–0.66. Analyses of O. viverrini from 5 localities revealed a high level of genetic diversity and had significant deviation from Hardy–Weinberg equilibrium. Significant heterozygote deficiency as well as heterozygote excess was detected across all localities indicating the possibility of selfing (inbreeding) as a predominant reproductive mode. Significant genetic differentiation (F ST) was also detected between worms from different localities with varying levels of genetic heterogeneity. We discuss our results in terms of what these novel microsatellite markers reveal about the epidemiology and transmission dynamics of this medically important parasite, both in terms of the current study and their potential for future comprehensive population genetic studies O. viverrini sensu lato in Southeast Asia. [Copyright &y& Elsevier]

Previous molecular genetic analyses indicate that Opisthorchis viverrini is a complex of at least 2 cryptic species in Thailand and the Lao People's Democratic Republic (PDR). Opisthorchis viverrini sensu lato (s.l.) populations can be divided into 6 genetic groups, which correlate with 5 different river wetlands systems. The present study examines the infectivity, growth, fecundity, and body size of O. viverrini s.l. isolates from these systems. Worm recovery was lowest in the Songkhram River, which differed significantly from all other wetland systems. In addition, the Chi River and Nam Ngum River populations also differed significantly. A similar pattern was found for fecundity. These results support the genetic/molecular data indicating a subdivision of O. viverrini populations on the basis of watershed and that propose the existence of cryptic species in Thailand and the Lao PDR. [ABSTRACT FROM AUTHOR]

Abstract: The food-borne trematodes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis, have long been recognized as the cause of major human health problems, with an estimated 40 million infected persons. Of the three species of liver fluke, only O. viverrini is classified as a type 1 carcinogen because of its role as an initiator of chronic inflammation and the subsequent development of cholangiocarcinoma. At present, there are no techniques for the early diagnosis of cholangiocarcinoma and it is fatal for most patients. There is considerable variation in parasite prevalence and disease presentation in different geographical areas, the latter of which may be associated with genetic differences among parasites. In the present study, multilocus enzyme electrophoresis was used to provide a comprehensive genetic characterization of O. viverrini from different geographical localities in Thailand and the Peoples’ Democratic Republic of Laos. Parasites from different localities were compared genetically at 32 enzyme loci. The results of the genetic analyses are sufficient to reject the null hypothesis that O. viverrini represents a single species. Therefore, O. viverrini consists of at least two genetically distinct, yet morphologically similar (i.e. cryptic) species. Moreover, there was also separation of the different populations of snails (i.e. the first intermediate hosts) into two distinct genetic groups that corresponded with the delineation of O. viverrini into two species. This suggests that there may be a history of co-evolution in this host–parasite lineage. Additionally, five distinct genetic groups of parasites were detected, each of which occurred within a different and independent river wetland system. Our findings have major implications for the implementation of effective control and surveillance programs targeted to these medically important food-borne parasites. [Copyright &y& Elsevier]

Opisthorchis viverrini is a food borne trematode, important because of the sheer numbers of people infected and its serious morbidities such as hepatobiliary diseases and cholangiocarcinoma (CHCA). Although infections are identified throughout Southeast Asia, the epi-center is northeast Thailand, where high prevalence coexists with a high incidence of CHCA. In this review, we present the basic population features and the factors influencing transmission between the different hosts. [Copyright &y& Elsevier]

Cholangiocarcinoma (CCA) caused by chronic liver fluke infection is a major public health problem in Northeast Thailand. Identification of CCA risk groups is urgently needed for the control of CCA in this region. Periductal fibrosis (PDF) induced by chronic inflammation of bile ducts is known as a pre-neoplastic lesion of CCA. We aimed to identify the serum CCA and PDF biomarkers using mass spectrometry (UPLC-ESI-QqQ) with multiple reaction mode (MRM) analysis. Here, serum levels of serotransferrin glycoforms at the glycopeptide level were measured in the sera of CCA (n = 100), PDF (n = 50), and healthy control (n = 100) subjects. The results indicated that serotransferrin peptide levels were generally the same between the control and PDF groups, whereas CCA patients had reduced levels. Moreover, 56 serotransferrin glycoforms were detected, with nine increased in CCA compared to control subjects. Among them, the serum levels of four glycoforms were increased in PDF and CCA patients compared to control subjects. In particular, highly sialylated multi-branched glycans of serotransferrin serum were significantly correlated with poor prognosis and tumor stage in CCA patients. Taken together, these glycoforms could be used as risk biomarkers and prognosis and diagnosis markers of CCA. [ABSTRACT FROM AUTHOR]

The group 1 carcinogen, the liver fluke Opisthorchis viverrini is the causative agent of opisthorchiasis and subsequent bile duct cancer (cholangiocarcinoma; CCA), which is an important public health problem in Southeast Asia. Bithynia snails are known to be the sole intermediate host of O. viverrini , and distributed throughout endemic areas of opisthorchiasis. Since 2001, the genetic variation investigation of O. viverrini has progressively been investigated. Comprehensive genetic variation studies of O. viverrini and Bithynia snails were undertaken and consecutively published in 2007 by Saijuntha and colleagues. These studies provided genetic evidence that O. viverrini and Bithynia snails are both species complex with evidence of co-evolution. Later, several studies have provided data in support of this finding, and have continuously to date reinforced that both O. viverrini and Bithynia are species complexes. Moreover, studies have shown that genetic variation of O. viverrini is related to geographical, temporal, fish host species including geographical genetic variation of its snail host, Bithynia siamensis sensu lato. This is significant and important in our understanding of the evolution and phylogenetic relationships between species within the O. viverrini and Bithynia species complexes. A comprehensive knowledge of the systematics and population genetics of O. viverrini and Bithynia snails provides a sound basis to instigate and develop effective prevention and control programs targeting opisthorchiasis and CCA in the endemic areas of Southeast Asia. Thus, this review examines the historical series of investigations of the systematics and population genetics of O. viverrini including Bithynia spp. in Southeast Asia since molecular genetic investigations commenced some 20 years ago. • Systematic and population genetic investigations of Opisthorchis viverrini and its Bithynia snail host are reviewed. • Studies of the parasite and snail host by time periods since the past two decades are updated. • In both O. viverrini and Bithynia snails, many cryptic species have been discovered. • The further requirement for comprehensive molecular genetic analyses of O. viverrini and Bithynia snail complexes across their ranges is discussed. [ABSTRACT FROM AUTHOR]

• Social-ecological model reveals links between liver fluke infection risk and places. • Land use and modernization affect raw fish dish choices, thereby infection levels. • Interpersonal-level family tradition reinforces raw fish eating as cultural identity. • Community-level health support contributes to individual-level behavior change. • Water management policies and developments may unintentionally impact infection risks. Human liver fluke infection through the consumption of raw freshwater fish is one of the foodborne parasitic infections of global concern. Despite decades of health campaign efforts, high prevalence of infection remains in different areas of the Lower Mekong Basin. This necessitates the consideration of the infection differences between places and the human-environment complexities of disease transmission. This paper used the socio-ecological model as a framework to unraveled the social science dimensions of liver fluke infection. We conducted questionnaire surveys in Northeast Thailand to gather participants' knowledge on liver fluke infection and reasons for raw fish consumption. We synthesized our findings with prior work to identify factors influencing liver fluke infection at four socio-ecological levels. At the individual level, gender and age differences in food consumption habits and personal hygiene of open defection presented the behavioral risks. At the interpersonal level, family tradition and social gathering affected the disease risk. At the community level, physical-social-economic environments of land use and modernization, community health infrastructure and health volunteer support accounted for the varying degree of infection. At the policy level, impacts of regional and national regulations on disease control, health system organization structure, and government development projects were of concerned. The findings provide insights into how infection risks are shaped by people's behavior, social connectedness, interactions with places, and the interplay of these multi-level socio-ecological influences. The framework therefore allows a more comprehensive understanding of liver fluke infection risks to inform a culturally sensitive and sustainable disease control program. Summary: Through the lens of social science, this study uses the social-ecological framework to illustrate the interplay among multi-level contextual factors on foodborne parasitic infection risk. [Display omitted] [ABSTRACT FROM AUTHOR]

Opisthorchis viverrini (Ov) is one of the most important human parasitic diseases in Southeast Asia. Although the concept of connectivity is widely used to comprehend disease dispersal, knowledge of the influences of landscape connectivity on Ov transmission is still rudimentary. This study aimed to investigate the role of landscape connectivity in Ov transmission between the human and the first intermediate snail hosts. Fieldwork was conducted in three villages respectively in Kamalasai District, Kalasin Province, Phu Wiang District, Khon Kaen Province, and Nong Saeng District, Udon Thani Province. Bithynia snails were collected to examine parasitic infections, water samples were analyzed for fecal contamination, and locations of septic tanks and connections between habitat patches with observable water movement were surveyed. Euclidean distance, topological link and distance, and graph measures were employed to quantify the connectivity between human and snail habitats. The findings showed that snail patches with higher fecal contents were generally located nearer to septic tanks. The statistically significant results for the topological link and distance measures highlighted the importance of water in functionally facilitating Ov transmission. Graph measures revealed differences in landscape connectivity across the sites. The site with the largest landscape component size and the most mutually connected snail patches coincided with the presence of Ov parasite, reinforcing its higher risk for human to snail transmission. The site with the dissected landscape structure potentially limited the transmission. This study underscored the potential effect of landscape connectivity on Ov transmission, contributing to the understanding of the spatial variation of Ov infection risk. [ABSTRACT FROM AUTHOR]

Highlights: [•] The three most important liver flukes infect more than 45 million people worldwide. [•] At least two of these species induce cholangiocarcinoma. [•] All three species have similar life cycles; however the zoonotic component of these life cycles differs between species. [•] Evidence for cryptic species is found within Opisthorchis viverrini. [ABSTRACT FROM AUTHOR]

Abstract: Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and the Lao People''s Democratic Republic (Lao PDR; Laos). Currently, more than 600 million people are at risk of infection with these fish-borne trematodes and/or their close relatives. Opisthorchiasis has been studied extensively in Thailand, where about 8 million people are infected with the liver fluke. Here we review the pathogenesis, control and re-emergence of O. viverrini infection, in particular in Thailand and, to a lesser extent in Lao PDR given the contiguous geographical range of O. viverrini through these two regions. We also review the association of O. viverrini infection and cholangiocarcinoma, bile duct cancer, and highlight new findings on pathogenesis of liver fluke-induced cholangiocarcinogenesis. Last, we comment on national control strategies in Thailand for the control of O. viverrini infection aimed at reduction in the prevalence of O. viverrini-associated liver cancer in the longer term. [Copyright &y& Elsevier]

Abstract: Infection by the liver fluke (Opisthorchis viverrini) causes hepatobiliary disease and bile duct cancer (cholangiocarcinoma, CCA) in endemic areas in Southeast Asia. Measurements of humoral immune response particularly parasite-specific antibodies are useful not only for serodiagnosis but they have been implicated as risk factors of CCA. In this study, we used indirect Enzyme Immunosorbent Assay (ELISA) to measure O. viverrini-specific immunoglobulins in serum, urine and saliva and assessed efficacies in diagnosis of opisthorchiasis and evaluated the relationship of antibodies among clinical specimens in a sample population in endemic areas in Khon Kaen, Thailand. By employing the Receiver Operation Characteristics (ROC) analysis, diagnostic efficacy based upon the area under the curve (AUC) revealed that serum, salivary IgG and IgA performed better than urine for diagnosis of opisthorchiasis. Seropositive cases were found in both parasite egg-negative as well as O. viverrini egg-positive groups. The levels of serum IgG correlated with intensity of O. viverrini infection (P <0.05). Diagnostic sensitivities based on serum and salivary IgG, IgA also positively associated with the intensity of infection. Correlations between serum antibodies and those in saliva were found to be greater in egg-negative than egg-positive individuals for O. viverrini. Our findings indicated a complex interrelation between antibody responses in different clinical specimens triggered by liver fluke infection. More comprehensive examinations are needed to determine the potential utility of salivary antibody detection which, in combination with the conventional fecal examination method, may better assist in the identification of individuals with opisthorchiasis. Furthermore, it may provide a better indicator of the risk of disease, particularly CCA. [Copyright &y& Elsevier]

Liver fluke infection through the consumption of raw or undercooked freshwater fish is a major public health problem in the Mekong Region. Despite the extensive efforts of liver fluke health campaigns, Northeast Thailand still reports high human infection prevalence as consumption of raw fish dishes has diminished but not ceased. This study examines the roles of social-cultural factors, particularly the influences of masculinity and misinformation, on liver fluke infection risk. Participant observation, questionnaire surveys and semi-structured interviews were conducted in four villages in Kalasin Province, Thailand, to scrutinize reasons for raw fish consumption, gender differences in raw fish culture, processes of liver fluke information dissemination, and the extent of information mismatch. Our results show that one of the key reasons (76.9%) underlying continued raw fish consumption are deeply embedded cultural practices associated with ways of rural life. About 30% of the participants indicated that they would not avoid eating raw fish, regardless of knowing the health consequences. Gender difference is evident, with 75.6% of males consuming raw fish salad (koi pla), compared to 42.7% of females. Some male participants associate raw meat consumption with virility and strength. Such beliefs underscore the cultural linkage of koi pla consumption with masculinity. Misconceptions of liver fluke life cycle and risk of infection remain, as only 15.3% of the participants correctly selected raw fish as the food source for liver fluke infection while 84.2% misunderstood that other raw foods could lead to infection. The multi-layered and hierarchical structure of public health information dissemination from medical professionals to health officers and village health volunteers to villagers has contributed to information mismatch between different layers. Our study builds on others which call for multi-pronged scientific and social strategies, as well as culturally attuned approaches to public health messaging. The study raises masculinity and misinformation as relevant considerations in disease prevention. Incorporating grounded research and gendered perspectives are part of appreciating the cultural roots of raw fish consumption. Realizing the significant role of village health volunteers in information dissemination and in supplying coherent public health messages is vital for effective health campaigns. [ABSTRACT FROM AUTHOR]

Summary: Objectives: To isolate and characterize an asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and evaluate its expression profile, biochemical activity, and potential as an immunodiagnostic antigen. Methods: The full length mRNA encoding an asparaginyl endopeptidase (family C13), Ov-aep-1, was isolated by immunoscreening of a cDNA bacteriophage library of adult O. viverrini using sera from patients infected with O. viverrini. Investigation of Ov-aep-1 transcripts in developmental stages of the parasite, and phylogenetic analysis, immunohistochemical localization, and recombinant protein expression and enzymology were employed to characterize the Ov-AEP-1 protein. Immunoblotting was used to assess the potential of this enzyme for immunodiagnosis of human opisthorchiasis. Results: Ov-AEP-1 is characteristic of the C13 cysteine protease family. Ov-aep-1 transcripts were detected in adult and juvenile worms, eggs, and metacercariae. Phylogenetic analysis indicated that Ov-AEP-1 is closely related to homologous proteins in other trematodes. Recombinant Ov-AEP-1 was expressed in bacteria in inclusion bodies and refolded to a soluble form. Excretory–secretory (ES) products derived from adult O. viverrini and refolded recombinant Ov-AEP-1 both displayed catalytic activity against the diagnostic tripeptide substrate, Ala–Ala–Asn-aminomethylcoumarin. Rabbit antiserum raised to recombinant Ov-AEP-1 identified the native AEP-1 protease in both somatic extract and ES products of adult worms. Anti-Ov-AEP-1 IgG immunolocalized the anatomical site of expression to the gut of the fluke, implying a physiological role in digestion of food or activation of other digestive enzymes. Recombinant Ov-AEP-1 was recognized by serum antibodies from patients with opisthorchiasis but not other helminth infections, with a sensitivity and specificity of 85% and 100%, respectively. The positive and negative predictive values are 100% and 67%, respectively. Conclusions: The liver fluke, O. viverrini, has a gut-localized asparaginyl endopeptidase. Refolded recombinant Ov-AEP-1 is catalytically active and has potential for immunodiagnosis of human opisthorchiasis. [Copyright &y& Elsevier]