High prevalence of Opisthorchis viverrini infection in a riparian population in Takeo Province, Cambodia

Opisthorchis viverrini infection was found to be highly prevalent in 3 riverside villages (Ang Svay Chek A, B, and C) of the Prey Kabas District, Takeo Province. This area is located in the southern part of Cambodia, where the recovery of adult O. viverrini worms was recently reported. From May 2006 until May 2010, fecal examinations were performed on a total of 1,799 villagers using the Kato-Katz thick smear technique. In the 3 villages, the overall positive rate for helminth eggs ranged from 51.7 to 59.0% (av. 57.4%), and the percentage positive for O. viverrini was 46.4-50.6% (47.5%). Other helminths detected included hookworms (13.2%), echinostomes (2.9%), Trichuris trichiura (1.3%), Ascaris lumbricoides (0.6%), and Taenia spp. (0.06%). The prevalence of O. viverrini eggs appeared to reflect a lower infection in younger individuals (<20 years) than in the adult population (>20 years). Men (50.4%) revealed a significantly higher (P=0.02) prevalence than women (44.3%). The Ang Svay Chek villages of the Prey Kabas District, Takeo Province, Cambodia have been confirmed to be a highly endemic area for human O. viverrini infection.     

Unusual protein pattern of Opisthorchis viverrini

The proteins of homogenized adult Opisthorchis viverrini were separated by polyacrylamide gel electrophoresis and isoelectric focusing in the presence of detergents. By far the most abundant component(s) had a Mr of 18,000 to 19,000, represented about 47% of the total parasite protein, and did not separate in isotypes.     

Characterization of the Opisthorchis viverrini genome

The methylations of trematode genomic DNA were analyzed using restriction enzymes and Southern blot hybridization. Restriction enzymes MspI, HpaII, HhaI were used to probe CpG methylation while MboI, Sau3A, DpnI were used for A methylation. The results revealed that Opisthorchis viverrini, Fasciola gigantica and Gigantocotyle siamensis had neither CpG nor A methylations. The presence of highly repeated DNA elements was also demonstrated in O. viverrini genomic DNA.     

Opisthorchis viverrini and cholangiocarcinoma in Northeast Thailand

Population-based research is beginning to show clearly the devastating impact that Opisthorchis viverrini infection has on the Laos-descendent population of Northeast Thailand who love to eat uncooked meat and fish, one of which being the parasite's intermediate host: cyprinoid fish. Here, Melissa Haswell-Elkins, Paiboon Sithithaworn and David Elkins discuss the parasite's life cycle and epidemiology, highlighting the close relationship between this infection and bile duct carcinoma, which is a major cause of death among adults in rural Northeast Thailand.     

Examining Landscape Determinants of Opisthorchis viverrini Transmission

Liver fluke (Opisthorchis viverrini, O.v.) infection, along with its associated cholangiocarcinoma, is a major public health problem in Southeast Asia. Despite the vast amount of epidemiological research, human O.v. prevalence remains high and varies greatly across the region. This paper examines the landscape determinants that influence O.v. transmission in relation to the three hosts of its life cycle and identifies areas that require further research so as to advance the understanding of the spatial variation in disease risk. A critical agent functionally connects all sequential life cycle stages of O.v. is water. Seasonality and water quality appear to affect the habitats and population dynamics of the two intermediate hosts, Bithynia snails and cyprinid fish. Land use practice through the construction of irrigation ditches increases the connections between the hosts, thereby functionally facilitating the disease transmission. Multi-season sampling data of host infections and habitat characteristics are needed for integration with analyses of landscape connectivity and human behavior to allow better understanding of the interactions among the landscape determinants on the spatial-temporal dynamics of disease transmission.     

Immunodepression in hamsters experimentally infected with Opisthorchis viverrini

Hamsters experimentally infected with Opisthorchis viverrini had significant depression of the antibody response to sheep red blood cells. The depression was more severe with prolonged infections. Lymphoproliferative response to phytohaemagglutinin stimulation was also depressed when compared with uninfected controls. The immunodepression in these animals was abolished by anthelmintic treatment, suggesting that the immunodepressive effect is reversible and is associated with active infection.     

Opisthorchis viverrini: an underestimated parasite in world health

Infection with Opisthorchis viverrini and its associated cholangiocarcinoma (CCA) is an underestimated problem in the Mekong region of Southeast Asia, despite the widespread use of praziquantel and health education measures for parasite control. Although data from Cambodia, Laos and Vietnam are rare, data from Thailand often show wide-ranging variability in epidemiological parameters, including human morbidity and the prevalence and incidence of CCA. The recent discovery of high levels of population genetic variability in O. viverrini in different wetlands in Thailand and Laos, which indicates the presence of sibling species, suggests that we have underestimated the complexity of this epidemiological situation. Future research should determine the relationship between the genetic variability of O. viverrini and patterns of opisthorchiasis-related disease.     

Prevalence and Intensity of Opisthorchis viverrini Metacercarial Infection in Fish from Phnom Penh,Takeo, and Kandal Provinces,Cambodia

The prevalence and intensity of Opisthorchis viverrini metacercariae (OvMc) were investigated in fish from 3 southern administrative regions along the Mekong River in Cambodia, i.e., Phnom Penh, Takeo, and Kandal Provinces from 2017 to 2020. A total of 295 freshwater fish (24 species) were transported to our laboratory with ice and examined using the artificial digestion method. In Phnom Penh, among 4 fish species positive for OvMc, 9 (23.7%) of 38 specimens examined were infected, and their intensity of infection averaged 4.3 metacercariae per infected fish. In Takeo Province, among 10 fish species positive for OvMc, 24 (38.1%) out of 63 fish examined were infected, and their intensity of infection was av. 14.4 metacercariae per infected fish. In particular, all of 3 Osteochilus schlegelii fish examined were infected, and their infection intensity was high, 34.7 metacercariae per fish. In Kandal Province, among 6 fish species positive for OvMc, 46 (90.2%) out of 51 specimens examined were infected, and their infection intensity was 24.0 metacercaraie per infected fish. All fish of Systomus orphoides (n=17), Barbonymus altus (n=14), and Rasbora aurotaenia (n=2) were infected, and their intensity of infection averaged 37.7, 21.6, and 18.5 metacercariae per fish, respectively. Metacercariae of Haplochis yokogawai, Haplorchis taichui, and Centrocestus formosanus were detected in fish from Takeo and Kandal Provinces. From these results, it has been confirmed that a variety of fish species from Phnom Penh, Takeo, and Kandal Provinces are commonly infected with OvMc, and preventive measures to avoid human O. viverrini infection should be performed in Cambodia.     

Ultrasonography assessment of hepatobiliary abnormalities in 3359 subjects with Opisthorchis viverrini infection in endemic areas of Thailand

A cross sectional study on hepatobiliary abnormalities in opisthorchiasis was performed in 8936 males and females aged from 20 to 60 years from 90 villages of Khon Kaen province, Northeast Thailand. All were stool-examined for Opisthorchis viverrini infection by standard quantitative formalin/ethyl acetate concentration technique. Of these, 3359 participants with stool egg positive underwent ultrasonography of the upper abdomen. The hepatobiliary abnormalities detected by ultrasound are described here. This study found a significantly higher frequency of advanced periductal fibrosis in persons with chronic opisthorchiasis (23.6%), particularly in males. Risks of the fibrosis included intensity of infection, and age younger than 30 years. Height of left lobe of the liver, cross-section of the gallbladder dimensions post fatty meal, sludge, and, interestingly, intrahepatic duct stones were significantly associated with the advanced periductal fibrosis. Eleven suspected cholangiocarcinoma (CCA) cases were observed. This study emphasizes the current status of high O. viverrini infection rate and the existence of hepatobiliary abnormalities including suspected CCA in opisthorchiasis endemic areas of Thailand.     

Spatial distribution of, and risk factors for, Opisthorchis viverrini infection in southern Lao PDR

Background

Opisthorchis viverrini is a food-borne trematode species that might give rise to biliary diseases and the fatal cholangiocarcinoma. In Lao PDR, an estimated 2.5 million individuals are infected with O. viverrini, but epidemiological studies are scarce and the spatial distribution of infection remains to be determined. Our aim was to map the distribution of O. viverrini in southern Lao PDR, identify underlying risk factors, and predict the prevalence of O. viverrini at non-surveyed locations.

Methodology

A cross-sectional parasitological and questionnaire survey was carried out in 51 villages in Champasack province in the first half of 2007. Data on demography, socioeconomic status, water supply, sanitation, and behavior were combined with remotely sensed environmental data and fed into a geographical information system. Bayesian geostatistical models were employed to identify risk factors and to investigate the spatial pattern of O. viverrini infection. Bayesian kriging was utilized to predict infection risk at non-surveyed locations.

Principal Findings

The prevalence of O. viverrini among 3,371 study participants with complete data records was 61.1%. Geostatistical models identified age, Lao Loum ethnic group, educational attainment, occupation (i.e., rice farmer, fisherman, and animal breeder), and unsafe drinking water source as risk factors for infection. History of praziquantel treatment, access to sanitation, and distance to freshwater bodies were found to be protective factors. Spatial patterns of O. viverrini were mainly governed by environmental factors with predictive modeling identifying two different risk profiles: low risk of O. viverrini in the mountains and high risk in the Mekong corridor.

Conclusions/Significance

We present the first risk map of O. viverrini infection in Champasack province, which is important for spatial targeting of control efforts. Infection with O. viverrini appears to be strongly associated with exposure to the second intermediate host fish, human behavior and culture, whereas high transmission is sustained by the lack of sanitation.     

Opisthorchis viverrini: influence of maternal infection in hamsters on offspring infected with homologous parasite and their IgG antibody response

We investigated the influence in hamsters of a maternal Opisthorchis viverrini infection on their offspring infected with homologous parasites and the kinetics of the O. viverrini-specific IgG antibody responses. No significant difference (P > 0.05) was found in the specific IgG antibody response and the number of O. viverrini eggs per gram feces (EPG) between infected offspring from infected mothers and infected offspring from uninfected mothers. A significant difference (P < 0.05) of EPG per worm was found between infected offspring from infected mothers and infected offspring from uninfected mothers only when the offspring were infected with O. viverrini after weaning at 5 weeks of age. The worm loads in infected offspring from infected mothers were significantly less than that in infected offspring from uninfected mothers. This study demonstrated that maternal infection effects worm fecundity and the worm load in an infected offspring.     

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.     

8-nitroguanine formation in the liver of hamsters infected with Opisthorchis viverrini

Nucleic acid damage by reactive nitrogen and oxygen species may contribute to the carcinogenesis associated with chronic infection and inflammation. We examined 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation and nitric oxide (NO) production in hamsters infected with Opisthorchis viverrini (OV). Formation of 8-nitroguanine was assessed immunohistochemically with an antibody specific for 8-nitroguanine. 8-nitroguanine formation was found mainly in the cytoplasm and slightly in the nucleus of inflammatory cells and epithelial lining of bile duct at inflammatory areas in the liver. 8-nitroguanine immunoreactivity reached the highest intensity on day 30. A time profile of 8-nitroguanine formation was closely associated with that of plasma nitrate/nitrite. HPLC with an electrochemical detector revealed that the amount of 8-oxodG in the liver reached the maximal level on day 21. The mechanisms of 8-oxodG and 8-nitroguanine formation via O2*- and NO production triggered by OV infection were discussed in relation to cholangiocarcinoma development.     

The ecology of the Bithynia first intermediate hosts of Opisthorchis viverrini

Opisthorchiasis, together with its associated cholangiocarcinoma, is one of the most important human parasitic diseases on continental Southeast Asia. A great deal of epidemiological data from humans is available on this disease, particularly from the northeast of Thailand, however, only limited information is available on those aspects of the life cycle relating to its Bithynia (Gastropoda) and cyprinid fish intermediate hosts. Here we review the information which is available on the Bithynia hosts of Opisthorchis viverrini. Only one major ecological study has been carried out at one site on a single species of Bithynia. We show not only that detailed ecological studies are required to clarify the epidemiology of opisthorchiasis, but also that the taxonomic status of the Bithynia species transmitting O. viverrini requires clarification.     

Distribution and Abundance of Opisthorchis viverrini Metacercariae in Cyprinid Fish in Northeastern Thailand

To increase public health awareness for prevention of opisthorchiasis caused by eating raw freshwater fish, the distribution and abundance of Opisthorchis viverrini metacercariae (OV MC) was investigated in freshwater fish obtained from 20 provinces in northeastern Thailand between April 2011 and February 2012. A cross-sectional survey was conducted on 12,890 fish consisting of 13 species randomly caught from 26 rivers, 10 dams, and 38 ponds/lakes. Fish, were collected in each of the rainy and winter seasons from each province. Fish were identified, counted, weighed, and digested using pepsin-HCl. Samples were examined for OV MC by a sedimentation method, and metacercariae were identified under a stereomicroscope. OV MC were found in 6 species of fish; i.e., Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti, and Puntioplites proctozysron from localities in 13 provinces. Among the sites where OV MC-infected fish were found, 70.0% were dams, 23.7% were ponds/lakes, and 7.7% were rivers. The mean intensity of OV MC ranged from 0.01 to 6.5 cysts per fish (or 1.3-287.5 cysts per kg of fish). A high mean intensity of OV MC per fish (>3 cysts) was found in 5 provinces: Amnat Charoen (6.5 cysts), Nakhon Phanom (4.3), Mukdahan (4.1), Khon Kaen, (3.5) and Si Sa Ket (3.4). In conclusion, OV MC are prevalent in natural cyprinid fish, with the infection rate varying according to fish species and habitats.     

Fecal bacterial contamination in natural water reservoirs as an indicator of seasonal infection by Opisthorchis viverrini in snail intermediate hosts

Opisthorchis viverrini, a carcinogenic liver fluke, requires Bithynia snails as the first intermediate host, which release cercariae after ingesting fluke eggs from contaminated water. Fecal bacterial contamination and O. viverrini-infected Bithynia snails were investigated in samples collected from natural water reservoirs in Ban Phai, Chonnabot and Muang Districts (Ban Lerngpeuy) in Khon Kaen Province, northeast Thailand, where there is a high incidence of cholangiocarcinoma. Water was sampled and examined six times (February, April, June, August, October and December 2006). The most probable number (MPN) index and coliform counts were utilized to evaluate fecal contamination; the cercarial shedding method was conducted for detecting infected snails. The data revealed that all water samples had a high MPN index number, and fecal coliform levels above the WHO standard. This indicated that water in these reservoirs was contaminated with feces or manure constituents. Water sampling from Ban Lerngpeuy showed full-scale bacterial contamination (> 1609 MPN index) throughout the year. This finding was correlated with the highest prevalence of O. viverrini-infected snails, which were found nearly all year round in this area. Slightly lower fecal contamination levels were detected in water samples from Chonnabot and Ban Phai, with high MPN index numbers and coliform counts from April to October. This corresponded with the higher recovery of infected snails in June and August, but with relatively lower prevalence than those found in Ban Lerngpeuy. Among the sampling sites, the people in Ban Lerngpeuy live nearer to the reservoir than do those in Ban Phai and Chonnabot. These results indicate that fecal bacterial contamination in natural water reservoirs is an important indicator of seasonal transmission of O. viverrini eggs to snail intermediate hosts. Sanitation improvement is essential and future investigations on the sources of contamination are needed.     

Early stage biliary and intrahepatic migration of Opisthorchis viverrini in the golden hamster

In Thailand, infection with the liver fluke Opisthorchis viverrini is a serious health problem, with over 8.6 million human infections each year. Early stage biliary intrahepatic migration and infection of O. viverrini in the Syrian golden hamster were used to study the growth and development of this fluke. Fifty metacercariae of O. viverrini were introduced into each hamster by gastric intubation. Worms were found to migrate rapidly from the stomach to the gall bladder and hepatic duct, where they remained in relatively constant numbers until the end of week 8. Sexual development of worms was rapid, with full development of the uterus and testes by one and one half weeks and the appearance of eggs in the uterus by the beginning of the third week of infection. Worm growth as indicated by body length had ceased by week 8. Hamsters demonstrated development of a full reproductive cycle with in three to four weeks, and this generation continued until 8 weeks.