Behaviour and eating habits as determinants for human opisthorchiasis in the Bolsena Lake area, Italy.

In Italy, food-borne outbreaks of opisthorchiasis have occurred since 2003, all of them originating from some lakes in Central Italy where the only European liver fluke, Opisthorchis felineus (Rivolta, 1884) is endemic. The aim of the present study is to investigate the factors that may facilitate the life cycle of O. felineus in the area of Bolsena Lake, the knowledge of the local population about opisthorchiasis, and preventative healthcare measures. We performed a descriptive observational study through the submission of questionnaires to three target populations: fishermen who fish exclusively in Bolsena Lake, restaurateurs whose businesses are close to the lake and general population living in the province of Viterbo. Results showed hazardous behaviours and eating habits in the three target populations, as well as a poor knowledge of opisthorchiasis and preventative healthcare measures.

ABC transporters in the liver fluke Opisthorchis felineus.

ATP-binding cassette transporters (ABC transporters) are essential components of normal cellular physiological machinery in all eukaryotic and prokaryotic species, including parasites. Some ABC transporters, e.g., P-glycoproteins, are involved in the efflux of toxins and xenobiotics from the cell. At present, nothing is known about ABC transporter genes in epidemiologically important liver flukes from the Opisthorchiidae family, including European liver fluke Opisthorchis felineus. Opisthorchiasis caused by O. felineus is a serious public health problem on the territory of Russia and other Eastern European countries. ABC drug transporters are attractive objects of research on molecular markers of resistance and on ways to potentiate sensitivity to anthelmintics through suppression of the transporters themselves with specific inhibitors. Here we aimed at the identification of ABC transporters in the O. felineus transcriptome and identification of P-glycoproteins. In addition, our aim was to assess ABC transcript abundance in the RNA-seq data, to study the mRNA expression of P-glycoprotein genes by Droplet Digital PCR throughout the life cycle of O. felineus, and to test the gene induction in response to xenobiotics or anthelminthic agents. We found 23 nucleotide sequences encoding ABC transporters belonging to different subfamilies, including four sequences of P-glycoproteins. According to the transcript abundance in the RNA-seq data, one of P-glycoproteins (P4) has the highest expression among all ABC genes in the adult worm. P-glycoproteins showed substantially differential mRNA expression throughout the fluke life cycle, with high expression in the adult worms. Putative activity of P-glycoproteins as xenobiotic efflux pumps was found to be linked to the excretory system of O. felineus and to be inhibited by verapamil or tariquidar. Thus, ABC drug transporters in the liver fluke O. felineus are functionally active, indicating that ABC drug transporters are likely to be molecular targets for a combination therapy aimed at prevention of a xenobiotic removal from helminth tissues and at increasing the drug concentration in the tissues.

Development and validation of species-specific molecular diagnostic tool for Opisthorchis felineus (Digenea, Opisthorchiidae) metacercariae.

Opisthorchis felineus (family Opisthorchiidae) is a parasitic flatworm representing a serious threat to humans in some countries. Opisthorchiasis occurs after consumption of raw or undercooked cyprinid fish infected by the metacercarial stage of the parasite. Due to its small size, detection of the parasite in fish fillet is time-consuming and difficult. Furthermore, isolated metacercariae can be identified to genus but not to species level using morphological features and molecular techniques are necessary. In this work, we describe the development of primers for a diagnostic PCR amplification of a 254-bp fragment of the cytochrome c oxidase I in the mitochondrion of Opisthorchis felineus metacercariae isolated from fish fillet, together with a validation protocol for this method.

Modeling impacts of climate change on the potential distribution of the carcinogenic liver fluke, Opisthorchis viverrini, in Thailand.

Global climate change is now regarded as imposing a significant threat of enhancing transmission of parasitic diseases. Maximum entropy species distribution modeling (MaxEnt) was used to explore how projected climate change could affect the potential distribution of the carcinogenic liver fluke, Opisthorchis viverrini, in Thailand. A range of climate variables was used: the Hadley Global Environment Model 2-Earth System (HadGEM2-ES) climate change model and also the IPCC scenarios A2a for 2050 and 2070. Occurrence data from surveys conducted in 2009 and 2014 were obtained from the Department of Disease Control, Ministry of Public Health, Thailand. The MaxEnt model performed better than random for O. viverrini with training AUC values greater than 0.8 under current and future climatic conditions. The current distribution of O. viverrini is significantly affected by precipitation and minimum temperature. According to current conditions, parts of Thailand climatically suitable for O. viverrini are mostly in the northeast and north, but the parasite is largely absent from southern Thailand. Under future climate change scenarios, the distribution of O. viverrini in 2050 should be significantly affected by precipitation, maximum temperature, and mean temperature of the wettest quarter, whereas in 2070, significant factors are likely to be precipitation during the coldest quarter, maximum, and minimum temperatures. Maps of predicted future distribution revealed a drastic decrease in presence of O. viverrini in the northeast region. The information gained from this study should be a useful reference for implementing long-term prevention and control strategies for O. viverrini in Thailand.

Immunization and challenge shown by hamsters infected with Opisthorchis viverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke.

Here we report findings to optimize and standardize conditions to attenuate metacercariae of Opisthorchis viverrini by ionizing radiation to elicit protective immune responses to challenge infection. Metacercariae were gamma-irradiated and the ability of irradiated metacercariae to prevent patent infection of challenge metacercariae in hamsters was determined, as well as their ability to induce a host antibody response. Metacercariae irradiated in a dose-dependent manner, with 3, 5, 10, 12, 20, 25 and 50 Gray, were used to infect Syrian golden hamsters by stomach gavage to ascertain the effect of irradiation on ability of the worms to establish infection. In addition, other hamsters were infected with metacercariae irradiated with 20-50 Gray, followed by challenge with intact/wild-type (non-irradiated) metacercariae to determine the protective effect as established by the numbers of adult flukes, eggs of O. viverrini in hamster faeces and anti-O. viverrini antibody titres. Significantly fewer worms were recovered from hamsters immunized with metacercariae irradiated at 20, 25 and 50 Gray than from control hamsters infected with intact metacercariae or 0 Gray, and the worms showed damaged reproductive organs. Faecal egg numbers were decreased significantly in hamsters immunized with 25 and 50 Gray metacercariae of O. viverrini. Moreover, hamsters administered metacercariae that were protected elicited a robust, specific anti-fluke immunoglobulin G response compared to control hamsters, suggesting a role for antibody in protection elicited by radiation-attenuated metacercariae.

Whole transcriptome profiling of adult and infective stages of the trematode Opisthorchis felineus.

Opisthorchis felineus, the trematode belonging to the family Opisthorchiidae, is a causative agent of the infection called opisthorchiasis or liver fluke infection. Being a close relative of Opisthorchis viverrini and Clonorchis sinensis (oriental liver flukes) it is encountered in northern Eurasia, especially in Russia, Kazakhstan, Belarus, Ukraine, and Baltic countries. Whole genome data for oriental liver flukes revealed their adaptations for life in the bile duct but our knowledge of O. felineus is scarce. To address this knowledge gap and uncover evolutionary aspect of the adaptations on the transcriptomic level, we used RNA-sequencing approach to investigate two stages of the parasite residing in different hosts. Bioinformatic analysis revealed specific features affecting various biochemical pathways and gene networks. Namely, we observed the loss of genes involved in polyamine synthesis, methionine salvage and peroxisome biogenesis. Some of the gene families, like MD-2 lipid binding proteins, calmodulins and cathepsins on the contrary have expanded compared to free living eukaryotes. We identified significant differences between the stages in homeodomain-containing genes, G-protein coupled receptors, and neuroactive signaling systems. Granulin-like growth factors specific for O. felineus were also identified. In this work, we provide the first whole transcriptome investigation of this parasite. We also hope that these results will create a background for further molecular research of helminth infections and opisthorchiasis in particular.

Effects of fermentation time and low temperature during the production process of Thai pickled fish (pla-som) on the viability and infectivity of Opisthorchis viverrini metacercariae.

Contamination of a popular fermented fish dish, pla-som, by Opisthorchis viverrini metacercariae (OVMC) is a possible cause of carcinogenic liver fluke infection in Thailand. Affected individuals are at risk of bile duct cancer, which is a major health problem for people in the Greater Mekong Subregion. In order to investigate concerns about food safety, we studied the effects of fermentation time and low temperature on the viability and infectivity of OVMC during the pla-som production process. Pla-som was prepared at room temperature for up to 1 week in duplicate experiments using cyprinid freshwater fish obtained from an O. viverrini-endemic area. OVMC were then isolated and identified under a stereomicroscope. Complete and viable OVMC were found on days 1-4 of fermentation, while their morphology was degenerated thereafter. After OVMC were fed to hamsters, the percentage of the worm recovery after 1 to 2 months of infection was 52%, 44.7%, 11.3% and 1% for days 1, 2, 3 and 4, respectively. In order to measure the effect of low temperature on OVMC, fish were kept in a refrigerator (4 °C) for up to five days and then subsequently fermented for three days. In fish stored in a refrigerator for 1 and 2 days, viable OVMC were clearly observed and were able to infect hamsters, a worm-recovery percentage of 3.3% and 12.7%, respectively. By contrast, in pla-som prepared from fish stored for 3 to 5 days, OVMC were degenerated and could not infect the host. In conclusion, pla-som fermentation for more than four days and refrigerating fish for three days before pla-som processing can prevent O. viverrini infection. This study may increase awareness of fermented-fish dish preparation to prevent liver fluke infection.

Review and Current Status of Opisthorchis viverrini Infection at the Community Level in Thailand.

Opisthorchis viverrini is remains a public health problem in Thailand, particularly in the northeast and north regions which have the highest incidences of chonalgiocarcinoma (CCA). O. viverrini causes the disease opithorchiasis, and its has been classified as a group 1 biological carcinogen. Humans, dogs, and cats become infected with O. viverrini by ingesting raw or undercooked fish containing infective metacercariae. The first human cases of O. viverrini infection were reported in Thailand 100 years ago, and it's still a problem at the community level. Based on data for the year 2009, more than 6 million people were infected with O. viverrini. Associated medical care and loss of wages in Thailand costs about $120 million annually. This review highlights the current status of O. viverrini infection in communities of Thailand through active surveillance for the five years period from 2010 and 2015. A total of 17 community-based surveys were conducted, most in the northeast region. Some 7 surveys demonstrated a high prevalence over 20%, and the highest was 45.7%. Most commonly infection was found in age group of 35 years and older, males, and agricultural workers. Although, the national prevalence may be decreasing but the results show that the O. viverrini infection is still high in communities of the northeast region. Therefore, the focus in populations living in northeast Thailand should be screening of infection and changing their eating behavior.

Hemozoin "knobs" in Opisthorchis felineus infected liver.

BACKGROUND: Hemozoin is the pigment produced by some blood-feeding parasites. It demonstrates high diagnostic and therapeutic potential. In this work the formation of co-called hemozoin "knobs" - the bile duct ectasia filled up by hemozoin pigment - in Opisthorhis felineus infected hamster liver has been observed. METHODS: The O. felineus infected liver was examined by histological analysis and magnetic resonance imaging (MRI). The pigment hemozoin was identified by Fourier transform infrared spectroscopy and high resolution electrospray ionization mass spectrometry analysis. Hemozoin crystals were characterised by high resolution transmission electron microscopy. RESULTS: Hemozoin crystals produced by O. felineus have average length 403 nm and the length-to-width ratio equals 2.0. The regurgitation of hemozoin from parasitic fluke during infection leads to formation of bile duct ectasia. The active release of hemozoin from O. felineus during in vitro incubation has also been evidenced. It has been shown that the hemozoin knobs can be detected by magnetic resonance imaging. CONCLUSIONS: In the paper for the first time the characterisation of hemozoin pigment extracted from liver fluke O. felineus has been conducted. The role of hemozoin in the modification of immune response by opisthorchiasis is assumed.

Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthorchiasis.

Administration of praziquantel for treatment of liver fluke infection may affect the host, with mild and severe effects after treatment caused by host immune response. Therefore, we focused on the antioxidant property, inflammatory and anthelmintic effects of the traditional folk medicine, G. mangostana pericarp extract, in hamster opisthorchiasis. Syrian hamsters were divided into four groups: normal (control) (N); administered G. mangostana alone (GM); infected with Opisthorchis viverrini alone (OV); and infected with O. viverrini and administered G. mangostana extract for 1.5 months (OVGM). Hamster livers were collected 45 days after infection to determine histopathological changes, i.e. aggregation of inflammatory cells. The morphology of adult O. viverrini (body size and sizes of reproductive organs) was analyzed, as well as worm burden, eggs per worm and eggs per gram of feces. Toxicity was tested by kidney function (blood urea nitrogen and creatinine); the results demonstrated that G. mangostana had no renal toxic effect. ABTS radical-scavenging assay indicated that the extract had antioxidant property. Reduction in aggregation of inflammatory cells surrounding the hepatic bile duct, especially at the hilar region, was found in the OVGM group. Worm burden was similar in both infected groups (treated or untreated with G. mangostana), but the average size of adults in the OV group was larger than in the OVGM group; moreover, eggs per worm and eggs per gram of feces were also comparatively higher. The present study suggests that G. mangostana extract possesses anti-inflammatory and antioxidant properties and can interfere with parasite development by affecting adult size and egg production. This may be useful for controlling the spread of OV infection and other parasites in endemic areas.

Bi-functionality of Opisthorchis viverrini aquaporins.

Aquaporins (AQP) are essential mediators of water regulation in all living organisms and members of the major intrinsic protein (MIP) superfamily of integral membrane proteins. They are potential vehicles or targets for chemotherapy, e.g. in Trypanosoma brucei melarsoprol and pentamidine uptake is facilitated by TbAQP-2. Transcriptome data suggests that there are at least three active aquaporins in the human liver fluke, Opisthorchis viverrini, OvAQP-1, 2 and 3, and crude RNA silencing of OvAQP-1 and 2 has recently been shown to affect parasite swelling in destilled water. In the present work we demonstrate that OvAQP-3 is a major water-conducting channel of the parasite, that it can be detected from the newly excysted juvenile to the adult stage and that it is present in major tissues of the parasite. Furthermore, a comparative functional characterization of the three parasite AQPs was performed by using Xenopus oocyte swelling and yeast phenotypic assays. OvAQP-1, OvAQP-2, and OvAQP-3 were found to conduct water and glycerol while only the latter two were also able to conduct urea. In addition, all OvAQPs were found to transport ammonia and methylamine. Our findings demonstrate that the sequence-based classification into orthodox aquaporins and glycerol-conducting aquaglyceroporins is not functionally conserved in the parasite and implicate a broder range of functions for these channels.

[A man in his thirties with icterus and itching]. / En mann i 30-årene med icterus og kløe.

BACKGROUND: We present a patient from South-East Asia who has been living in Norway for five years. He was referred to our department with jaundice caused by opisthorchiasis/clonorchiasis. This reason for jaundice is highly unusual in Europe. Worldwide, however about 35,000,000 people are infected. CASE PRESENTATION: A male in his thirties, originally from South-East Asia, experienced diffuse itching for five weeks and painless jaundice for two weeks. Blood samples showed increasing cholestasis. Abdominal ultrasound, MRCP and computed tomography showed no sign of bile duct obstruction or liver tumour. Serological tests and liver biopsy revealed no infectious or autoimmune liver disease. ERCP showed normal bile ducts, but large quantities of typical hookworms in the duodenum. Stool samples showed at least one egg typical of Opisthorchis/Clonorchis and a large quantity of hookworm eggs. We interpreted these findings as parasite-induced obstruction of some of the small bile ducts. The patient recovered completely after treatment with Praziquantel. INTERPRETATION: Jaundice due to bile duct obstruction by opisthorchiasis/clonorchiasis is a well-known problem in South-East Asia. It may become more common in Europe as well as a result of increasing migration. Treatment with Praziquantel is simple and effective.

RNA-Seq reveals infection-induced gene expression changes in the snail intermediate host of the carcinogenic liver fluke, Opisthorchis viverrini.

BACKGROUND: Bithynia siamensis goniomphalos is the snail intermediate host of the liver fluke, Opisthorchis viverrini, the leading cause of cholangiocarcinoma (CCA) in the Greater Mekong sub-region of Thailand. Despite the severe public health impact of Opisthorchis-induced CCA, knowledge of the molecular interactions occurring between the parasite and its snail intermediate host is scant. The examination of differences in gene expression profiling between uninfected and O. viverrini-infected B. siamensis goniomphalos could provide clues on fundamental pathways involved in the regulation of snail-parasite interplay. METHODOLOGY/PRINCIPAL FINDINGS: Using high-throughput (Illumina) sequencing and extensive bioinformatic analyses, we characterized the transcriptomes of uninfected and O. viverrini-infected B. siamensis goniomphalos. Comparative analyses of gene expression profiling allowed the identification of 7,655 differentially expressed genes (DEGs), associated to 43 distinct biological pathways, including pathways associated with immune defense mechanisms against parasites. Amongst the DEGs with immune functions, transcripts encoding distinct proteases displayed the highest down-regulation in Bithynia specimens infected by O. viverrini; conversely, transcription of genes encoding heat-shock proteins and actins was significantly up-regulated in parasite-infected snails when compared to the uninfected counterparts. CONCLUSIONS/SIGNIFICANCE: The present study lays the foundation for functional studies of genes and gene products potentially involved in immune-molecular mechanisms implicated in the ability of the parasite to successfully colonize its snail intermediate host. The annotated dataset provided herein represents a ready-to-use molecular resource for the discovery of molecular pathways underlying susceptibility and resistance mechanisms of B. siamensis goniomphalos to O. viverrini and for comparative analyses with pulmonate snail intermediate hosts of other platyhelminths including schistosomes.

Alcohol and alkalosis enhance excystation of Opisthorchis viverrini metacercariae.

The northeastern region of Thailand has long been known as an endemic area of the human liver fluke infection which is caused by Opisthorchis viverrini. Humans are infected by ingestion of uncooked cyprinoid fish in traditional dishes such as "koi-pla," "pla-som," "pla-jom," and "pla-ra." To date, the prevalence of this parasite infection remains high because of cultural behavior and local beliefs. The popular misunderstanding among people in this area is that alcohol, lemon juice, and fish sauce can kill the parasites. Thus, they believe that they can eat raw fish without the risk of infection. This study attempts to clarify the effects of ethyl alcohol and acidosis-alkalosis on O. viverrini metacercariae excystation. Metacercariae of O. viverrini were obtained from infected cyprinoid fish in a natural reservoir. Most metacercariae were obtained from small cyprinoid fish. Metacercariae were divided into three experimental groups and were treated with solutions containing four different concentrations of ethyl alcohol, four different concentrations of salt, and a range of acidic/basic pH. Metacercariae excystation was observed at the assigned times, and the data were then analyzed. Salt had no effect on excystation. Interestingly, the optimal conditions for O. viverrini excystation were pH 9 and 25 % ethyl alcohol. The present study suggests that raw fish should not be eaten while drinking alcohol or when consuming other ingredients with pH 9, because both alcohol and pH 9 could induce O. viverrini metacercariae excystation, leading to the early development of parasites in the hepatobiliary system.

Curcumin prevents bile canalicular alterations in the liver of hamsters infected with Opisthorchis viverrini.

Opisthorchis viverrini infection causes inflammation and liver injury leading to periductal fibrosis. Little is known about the pathological alterations in bile canaliculi in opisthorchiasis. This study aimed to investigate bile canalicular alterations in O. viverrini-infected hamsters and to examine the chemopreventive effects of curcumin on such changes. Hamsters were infected with O. viverrini and one group of animals was fed with 1% dietary curcumin supplement. Animals were examined during the acute infection phase, days 21 and 30 post-infection (PI) and chronic infection phase (day 90 PI). Scanning electron microscopy revealed that in the infected group fed with a normal diet, bile canaliculi became slightly tortuous by 30 day PI and more tortuous at day 90 PI. Transmission electron microscopy showed a reduction in microvilli density of canaliculi starting at day 30 PI, with a marked loss of microvilli at day 90 PI. These ultrastructral changes were slightly seen at day 21 PI, which was similar to that found in infected animals fed with 1% curcumin-supplemented diet. Notably, curcumin treatment prevented the reduction of microvilli density, reduced the dilation of bile canaliculi, and decreased the tortuosity of the bile canaliculi relative to non-infected animals on a normal diet at days 30 and 90 PI. These results suggest that curcumin reduces alteration of bile canaliculi and may be a promising agent to prevent the onset of bile duct abnormalities induced by O. viverrini infection.

Molecular changes in Opisthorchis viverrini (Southeast Asian liver fluke) during the transition from the juvenile to the adult stage.

BACKGROUND: The Southeast Asian liver fluke (Opisthorchis viverrini) chronically infects and affects tens of millions of people in regions of Asia, leading to chronic illness and, importantly, inducing malignant cancer (= cholangiocarcinoma). In spite of this, little is known, at the molecular level, about the parasite itself, its interplay with its hosts or the mechanisms of disease and/or carcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we generated extensive RNA-Seq data (Illumina) representing adult and juvenile stages of O. viverrini, and combined these sequences with previously published transcriptomic data (454 technology) for this species, yielding a combined assembly of significantly increased quality and allowing quantitative assessment of transcription in the juvenile and adult stage. CONCLUSIONS: This enhanced assembly reveals that, despite the substantial biological similarities between the human liver flukes, O. viverinni and Clonorchis sinensis, there are previously unrecognized differences in major aspects of their molecular biology. Most notable are differences among the C13 and cathepsin L-like cysteine peptidases, which play key roles in tissue migration, immune evasion and feeding, and, thus, represent potential drug and/or vaccine targets. Furthermore, these data indicate that major lineages of cysteine peptidases of socioeconomically important trematodes have evolved through a process of gene loss rather than independent radiation, contrasting previous proposals.

[The strategy of a parasite is to preserve and continue its species; some trends for achieving the goal].

The authors show that the strategy of parasites, which is to preserve and continue its species is accomplished mainly by providing of a trophic substrate. Opisthorichiasis is used as an example to show that the nutriceutic biomass of cholangiocytes may be increased due to gene mutations, induced proliferation of liver stem cells, their differentiation to committed ones and cholangiocellular differon elements; moreover, the proliferative processes of mesenchymal components become active in other organs of a host. During their ontogenesis in the intermediate and final hosts, the parasites work out the mechanisms for prolonging their life span to complete a full development cycle (Margaritifera margaritifera glochidia); however, predominantly the parasite-host symbiosis is attended by the latter's lameness. Predation is one of the types of symbiotic relations.

Co-infection with Opisthorchis viverrini and Haplorchis taichui detected by human fecal examination in Chomtong district, Chiang Mai Province, Thailand.

Diseases caused by the liver fluke, Opisthorchis viverrini and the minute intestinal fluke, Haplorchis taichui, are clinically important, especially in the Northeast and North regions of Thailand. It is often difficult to distinguish between these trematode species using morphological methods due to the similarity of their eggs and larval stages both in mixed and co-infections. A sensitive, accurate, and specific detection method of these flukes is required for an effective epidemiological control program. This study aimed to determine the prevalence of O. viverrini and H. taichui infections in human feces by using formalin-ether sedimentation and high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR methods. Fecal specimens of people living along the Mae Ping River, Chomtong district were examined seasonally for trematode eggs using a compound microscope. Positive cases were analyzed in HAT-RAPD, DNA profiles were compared with adult stages to determine the actual species infected, and specific DNA markers of each fluke were also screened. Our results showed that out of 316 specimens, 62 were positive for fluke eggs which were pre-identified as O. viverrini and H. taichui. In addition, co-infection among these two fluke species was observed from only two specimens. The prevalence of H. taichui infections peaked in the hot-dry (19.62%), gradually decreased in the rainy (18.18%), and cool-dry seasons (14.54%), respectively. O. viverrini was found only in the hot-dry season (6.54%). For molecular studies, 5 arbitrary primers (Operon Technologies, USA) were individually performed in HAT-RAPD-PCR for the generation of polymorphic DNA profiles. The DNA profiles in all 62 positives cases were the same as those of the adult stage which confirmed our identifications. This study demonstrates the mixed infection of O. viverrini and H. taichui and confirms the extended distribution of O. viverrini in Northern Thailand.

The current status of opisthorchiasis and clonorchiasis in the Mekong Basin.

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.