Effect of Temperature on the Killing of Opisthorchis viverrini Eggs In Vitro

Contaminated liver fluke egg in the environment has led to the high prevalence of human opisthorchiasis associated with cholangiocarcinoma in Southeast Asia. To find the effective lessening methods of Opisthorchis viverrini eggs in the contaminated environment, we investigated the temperature conditions for killing of these trematode eggs in vitro. Numerous O. viverrini eggs were obtained in the proximal part of uteri of adult worms from experimental hamsters. Mature eggs with miracidium were allocated by experimental groups (2 control: positive and negative and 4 treatment: 50, 60, 70, and 80°C) with 0.85% saline, and treated by the experimental plan. Eggs in each experimental groups were observed under the confocal microscope after stain with Propidium Iodide (PI) to evaluate the effect of temperatures. Eggs in 70 and 80°C groups were all killed after over 10 min heated. Majority of eggs in 60°C (10, 15, and 30 min heated), 70 and 80°C (5 min heated) groups were inactivated. However in 50°C group, below half of eggs were to be killed in all time lapse (10, 15 and 30 min). In order to prevent O. viverrini infection and cholangiocarcinoma, direct treatment of sewage by heating at 70 or 80°C at least 10 min is essential. Therefore, treatment of O. viverrini eggs at a high temperature is a potential method for controlling egg contamination in sewage.

Liver Fluke-Associated Biliary Tract Cancer

Cholangiocarcinoma (CCA) is an aggressive cancer arising from epithelial cells of the bile duct. Most patients with CCA have an unresectable tumor at the time of diagnosis. In Western countries, the risk of CCA increases in patients with primary sclerosing cholangitis, whereas liver fluke infection appears to be the major risk factor for CCA in Asian countries. A diagnosis of liver fluke infection often relies on stool samples, including microscopic examination, polymerase chain reaction-based assays, and fluke antigen detection. Tests of serum, saliva and urine samples are also potentially diagnostic. The presence of liver fluke along with exogenous carcinogens magnifies the risk of CCA in people living in endemic areas. The “liver fluke-cholangiocarcinoma” carcinogenesis pathways consist of mechanical damage to the bile duct epithelium, immunopathologic and cellular reactions to the liver fluke's antigens and excretory/secretory products, liver fluke-induced changes in the biliary tract microbiome and the effects of repeated treatment for liver fluke. A vaccine and novel biomarkers are needed for the primary and secondary prevention of CCA in endemic areas. Importantly, climate change exerts an effect on vector-borne parasitic diseases, and awareness of liver fluke should be enhanced in potentially migrated habitat areas.

Comparative Characterization of Four Calcium-Binding EF Hand Proteins from Opisthorchis viverrini

Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2–42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21–23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca²⁺, Mg²⁺, Zn²⁺, and Cu²+. All OvCaBPs showed mobility shifts with Ca²⁺ and Zn²⁺. OvCaBP1 showed also positive results with Mg²⁺ and Cu²⁺. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.

Opisthorchiasis in infant remains from the medieval Zeleniy Yar burial ground of XII-XIII centuries AD

We present a paleoparasitological analysis of the medieval Zeleniy Yar burial ground of the XII-XII centuries AD located in the northern part of Western Siberia. Parasite eggs, identified as eggs of Opisthorchis felineus, were found in the samples from the pelvic area of a one year old infant buried at the site. Presence of these eggs in the soil samples from the infant’s abdomen suggests that he/she was infected with opisthorchiasis and imply consumption of undercooked fish. Ethnographic records collected among the population of the northern part of Western Siberia reveal numerous cases of feeding raw fish to their children. Zeleniy Yar case of opisthorchiasis suggests that this dietary custom has persisted from at least medieval times.

Chronic Opisthorchis viverrini Infection and Associated Hepatobiliary Disease Is Associated with Iron Loaded M2-like Macrophages

Chronic Opisthorchis viverrini-induced hepatobiliary disease is associated with significant leukocyte infiltration, including activated macrophages; however, the polarization of infiltrating macrophages remains to be fully characterized. In this study, we characterized macrophage polarization and phenotype in chronic O. viverrini-induced hepatobiliary disease in humans and hamsters using gene expression and histochemical analysis. Chronic O. viverrini infection and associated hepatobiliary diseases were associated with iron loaded M2-like macrophages in both humans and hamsters. This study provides suggestive evidence that iron loaded M2-like macrophages promote hepatobiliary disease in chronic O. viverrini infection.

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.

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.

Praziquantel Treatment in Trematode and Cestode Infections: An Update / 감염과화학요법

Status and emerging issues in the use of praziquantel for treatment of human trematode and cestode infections are briefly reviewed. Since praziquantel was first introduced as a broadspectrum anthelmintic in 1975, innumerable articles describing its successful use in the treatment of the majority of human-infecting trematodes and cestodes have been published. The target trematode and cestode diseases include schistosomiasis, clonorchiasis and opisthorchiasis, paragonimiasis, heterophyidiasis, echinostomiasis, fasciolopsiasis, neodiplostomiasis, gymnophalloidiasis, taeniases, diphyllobothriasis, hymenolepiasis, and cysticercosis. However, Fasciola hepatica and Fasciola gigantica infections are refractory to praziquantel, for which triclabendazole, an alternative drug, is necessary. In addition, larval cestode infections, particularly hydatid disease and sparganosis, are not successfully treated by praziquantel. The precise mechanism of action of praziquantel is still poorly understood. There are also emerging problems with praziquantel treatment, which include the appearance of drug resistance in the treatment of Schistosoma mansoni and possibly Schistosoma japonicum, along with allergic or hypersensitivity reactions against praziquantel treatment. To cope with and overcome these problems, combined use of drugs, i.e., praziquantel and other newly introduced compounds such as triclabendazole, artemisinins, and tribendimidine, is being tried.

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). 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.

Liver Flukes: the Malady Neglected

Liver fluke disease is a chronic parasitic inflammatory disease of the bile ducts. Infection occurs through ingestion of fluke-infested, fresh-water raw fish. The most well-known species that cause human infection are Clonorchis sinensis, Opisthorchis viverrini and Opisthorchis felineus. Adult flukes settle in the small intrahepatic bile ducts and then they live there for 20-30 years. The long-lived flukes cause long-lasting chronic inflammation of the bile ducts and this produces epithelial hyperplasia, periductal fibrosis and bile duct dilatation. The vast majority of patients are asymptomatic, but the patients with heavy infection suffer from lassitude and nonspecific abdominal complaints. The complications are stone formation, recurrent pyogenic cholangitis and cholangiocarcinoma. Approximately 35 million people are infected with liver flukes throughout the world and the exceptionally high incidence of cholangiocarcinoma in some endemic areas is closely related with a high prevalence of liver fluke infection. Considering the impact of this food-borne malady on public health and the severe possible clinical consequences, liver fluke infection should not be forgotten or neglected.

Mutations of KRAS and TP53 in a minor proportion of Opisthorchis viverrini-associated cholangiocarcinomas in a hamster model.

BACKGROUND/AIMS: KRAS oncogene and TP53 tumor suppressor gene have been known as common genes involving in many cancers including cholangiocarcinoma (CCC). Activation of these genes could lead to uncontrolled proliferation and cancer ultimately. The aim of this study was to investigate mutation of KRAS exon 1 and TP53 exon 5-8 in Opisthorchis viverrini (OV)-induced cholangiocarcinoma (CCA) in a hamster model. METHODS: Twenty-seven CCAs were obtained from Syrian golden hamsters induced by OV infection and N-nitrosodimethylnitrosamine (N-NDDM) administration. The tumor tissues were processed for histopathology. Genomic DNA extracted from paraffin sections by microdissection was amplified for KRAS exon 1 and TP53 exon 5-8 mutations by PCR-direct sequencing. RESULTS: Histopathologically, the tumors were classified into tubular (81.5%, 22/27), papillary (3.7%, 1/27), mucinous (3.7%, 1/27) and mixed types (11.1%, 3/27). Of the 27 CCAs, PCR-direct sequencing of KRAS showed G[see text]A transition at codon 37 exon 1 in one CCA sample (3.70%). Point mutations of p53 exon 6 (G[see text]C transversion at codon 119 and 218 and A[see text]C transversion at codon 217) were found in 3 CCA samples (11.1%). CONCLUSIONS: The results suggest that mutation of TP53 particularly at exon 6 may be involved in cholangiocarcinogenesis and a novel mutation of KRAS exon 1 was firstly reported in OV-induced hamster CCA.

Local perceptions and practices in regard to opisthorchiasis in two villages in Lao PDR.

The aim of the present study was to assess local perceptions and practices in regard to opisthorchiasis in a village receiving treatment and health education compared to a village where no intervention was offered. The study was conducted two years after a health education campaign had been carried out. Focus group discussions and semi-structured interviews were performed among 68 men and women. The results of the study show there were distinct differences in perceptions about opisthorchiasis when comparing the two villages. It appears that introducing health education alongside treatment does have an effect on knowledge and change in behavior. Eating raw fish was mainly practiced by men, however women were not aware they were exposed to opisthorchiasis while preparing food. Although there is no locally derived term, a medically appointed term for opisthorchiasis was acknowledged. Due to the vague disease symptoms, no treatment seeking behavior was found in relation to the disease. Further studies are needed to shed light on gender differences in regard to eating behavior and to assess infection risk among different Lao dishes containing freshwater fish.

Ultrasound screening for Opisthorchis viverrini-associated cholangiocarcinomas: experience in an endemic area.

In Thailand, liver cancer is the most common malignancy in males and the third most common among females. In the Northeast region, cholangiocarcinoma (CHCA) is the prevalent type, with Opisthorchis viverrini (OV), an endemic liver fluke, being considered the cause. We evaluated the role of ultrasound (U/S) for cholangiocarcinoma screening as part of a larger cohort to characterize the linkage between liver fluke infestation and CHCA in Khon Kaen (Northeast Thailand). Most people (77%) had normal U/S findings while only 0.5% had suspected CHCA; thus, U/S should be used primarily for those with the highest risk, presenting symptoms and/or being OV positive.

Enzyme markers to identify and characterize Opisthorchis viverrini in Thailand and Lao PDR.

We conducted an allozyme electrophoretic study to explore potential enzyme markers to distinguish Opisthorchis viverrini in Thailand and Lao PDR. Twenty-eight enzymes encoding presumptive 32 loci were established. The enzymes glucose-6-phosphate dehydrogenase and pyruvate kinase were diagnostic between two geographically separate isolates from Thailand. Twelve enzymes, ie, aconitate hydratase, aldolase, creatine kinase, enolase, esterases, fumarate hydratase, aspartate aminotransferase, glucose-phosphate isomerase, alanine aminotransferase, isocitrate dehydrogenase, malic enzyme, and pyruvate kinase, also provided diagnostic markers for these two isolates from Thailand and one isolate from Lao PDR.

Methylene blue staining method for identification of Opisthorchis viverrini egg.

Methylene blue staining method was used to distinguish O. viverrini eggs from Haplorchis taichui and Prosthodendrium molenkampi eggs. All eggs were obtained from dissected adult worms, fixed in 10% formalin, and stained with methylene blue prior to light microscopy observation. The distinct musk-melon-like texture of the O. viverini eggshell surface and the thread-like texture of H. taichui eggshell surface were recognized, while P. molenkampi eggs showed a smooth eggshell. We also evaluated the sensitivity and specificity of the method by training investigators to differentiate surface textures. After training, the investigators were randomly tested with 10 slides containing fluke eggs. The sensitivity and specificity were 95% and 95%, respectively.

Detection of opportunistic and non-opportunistic intestinal parasites and liver flukes in HIV-positive and HIV-negative subjects.

We assessed the frequency and distribution of infection with opportunistic and non-opportunistic intestinal parasites and the liver fluke, Opisthorchis viverrini, in HIV-seropositive and HIV-seronegative subjects. Age- and sex-matched HIV-seropositive (n = 78) and HIV-seronegative patients (n = 78) from two hospitals in Khon Kaen Province, Thailand, participated in this study from November 1998 to August 2000. These subjects were divided according to the presence of diarrhea and CD4 counts. A single stool sample was obtained and analyzed by using specific techniques. Opisthorchis viverrini, was the most common parasite (19.2%) in each group. The prevalence rates of Cryptosporidium spp (11.5%) and Strongyloides stercoralis (17.9%) in the HIV-seropositive group were significantly (p < 0.05) higher than those in the HIV-seronegative group (1.0% for Cryptosporidium spp and 7.7% for S. stercoralis infections). The prevalences of these two parasites were 28% for Cryptosporidium spp and 20% for S. stercoralis in HIV-seropositives with diarrhea and CD4 counts lower than 100 cells/mm3, and were higher compared with patients without diarrhea or with high CD4 counts. These results suggest that infection with these parasites increases during HIV infection. The epidemiological distribution of Cryptosporidium and S. stercoralis may have implications for AIDS-related diseases.

Association of parasitic infections and cancers.

Recent advances in the fields of molecular biology, epidemiology and infectious diseases have led to significant revelations to clarify the relationship between cancer and infective agents. This article reviews the relationship between parasitic infections and carcinogenesis and the possible mechanisms involved. Few parasites, e.g., Schistosoma haematobium and Opisthorchis viverrini have been found to be strongly associated with bladder cancer and cholangiocarcinoma respectively. The evidence for the association of several other parasites and cancers has also been postulated.

Epidemiological survey of intestinal parasitic infections of diarrhoeal patients in Nepal and Lao PDR.

Intestinal parasitological survey was conducted to clarify the distribution of intestinal parasites in Nepal and Lao Peoples' Democratic Republic (Lao PDR) from 2001 to 2003. A total of 401 diarrheal fecal specimens were collected from patients aged one to 75 years (250 from Nepal and 151 from Lao PDR). The specimens were examined using the formalin-ether sedimentation (FES) and sucrose centrifugal flotation (SCF) techniques. Nine species (3 Nematoda, 1 Cestoda, and 5 Protozoa) of parasites were recovered from Nepal, whereas seven species (3 Nematoda, 1 Trematoda, and 3 Protozoa) from Lao PDR. Of the parasites detected, (14.4%) was the most common in Nepal, and was Opisthorchis viverrini (29.8%) in Lao PDR. Infection rates were markedly different among age groups in both countries; higher rates were observed in age groups of 10-29 years than in 0-9 years group. Scanning electron microscopic observation revealed that trematoda eggs recovered from Lao PDR were identified as O. viverrini, which could not be done by a light microscope.

Studies on concomitant antigens of Bithynia funiculata for detection of antibody to Opisthorchis viverrini: effect of different centrifugal speeds on antigen preparation.

Antigens derived from somatic extracts of Bithynia funiculata, an intermediate snail host of O. viverrini, have been demonstrated to be highly heterogeneous in molecular weight (MW). These antigens have been suggested to be of potential use for serodiagnosis. In this study, B. funiculata somatic antigens were extracted using five different centrifugal speeds, namely 10,000 (C1); 20,000 (C2); 30,000 (C3); 40,000 (C4) and 50,000 (C5) rpm, with the aim of removing some non-specific antigens and determining the optimal centrifugal speed to obtain the highest efficiency of the test for which they will be used. The enzyme-linked immunosorbent assay technique was used to compare the reactivity of the five different centrifugal speed-prepared antigens. The sensitivity and specificity of all five antigens were compared by testing against sera from 81 opisthorchiasis patients, 30 parasite-free healthy individuals and 50 individuals infected with other helminthic infections, using mean + 4SD of all healthy individuals as the cut-off value. The sensitivity of these antigens was 69.1, 84.0, 80.2, 84.0 and 70.4, respectively; while the specificity was 66.2, 76.2, 82.5, 86.2 and 71.2, respectively. Immunoreactive components of each antigen were analyzed by SDS-PAGE and Western blot technique. The assay showed that three pairs of antigens with MW of 29 and 30, 47 and 50, and 86 and 90 kDa of all five antigens, which have previously been shown to be highly immunogenic, still reacted with a pooled serum from patients with opisthorchiasis. However, the C4 antigens gave more distinct components. Our results showed that 40,000 rpm is the optimal speed for antigen preparation for use in the serological diagnosis of opisthorchiasis, as demonstrated by the most satisfactory results of both sensitivity and specificity in the indirect ELISA and Western blot technique.