Epidemiology and Geographical Distribution of Human Trematode Infections.

Digenetic trematodes infecting humans are more than 109 species that belong to 49 genera all over the world. According to their habitat in the definitive hosts, they are classified as 6 blood flukes (Schistosoma japonicum. S. mekongi, S. malayensis, S. mansoni, S. intercalatum, and S. haematobium), 15 liver flukes (Fasciola hepatica, F. gigantica, Clonorchis sinensis, Opisthorchis viverrini, O. felineus, Dicrocoelium dendriticum, D. hospes, Metorchis bilis, M. conjunctus, M. orientalis, Amphimerus sp., A. noverca, A. pseudofelineus, Pseudamphistomum truncatum, and P. aethiopicum), nine lung flukes (Paragonimus westermani, P. heterotremus, P. skrjabini, P. skrjabini miyazakii, P. kellicotti, P. mexicanus, P. africanus, P. uterobilateralis, and P. gondwanensis), 30 heterophyid intestinal flukes (Metagonimus yokogawai, M. takahashii, M. miyatai, M. suifunensis, M. katsuradai, M. pusillus, M. minutus, Heterophyes heterophyes, H. nocens, H. dispar, Haplorchis taichui, H. pumilio, H. yokogawai, H. vanissinus, Centrocestus formosanus, C. armatus, C. cuspidatus, C. kurokawai, Procerovum calderoni, P. varium, Pygidiopsis genata, P. summa, Stictodora fuscata, S. lari, Stellantchasmus falcatus, Heterophyopsis continua, Acanthotrema felis, Apophallus donicus, Ascocotyle longa, and Cryptocotyle lingua), 24 echinostome intestinal flukes (Echinostoma revolutum, E. cinetorchis, E. mekongi, E. paraensei, E. ilocanum, E. lindoense, E. macrorchis, E. angustitestis, E. aegyptica, Isthmiophora hortensis, I. melis, Echinochasmus japonicus, E. perfoliatus, E. lilliputanus, E. caninus, E. jiufoensis, E. fujianensis, Artyfechinostomum malayanum, A. sufrartyfex, A. oraoni, Acanthoparyphium tyosenense, Echinoparymphium recurvatum, Himasthla muehlensi, and Hypoderaeum conoideum), 23 miscellaneous intestinal flukes (Brachylaima cribbi, Caprimolgorchis molenkampi, Phaneropsolus bonnei, P. spinicirrus, Cotylurus japonicus, Fasciolopsis buski, Gastrodiscoides hominis, Fischoederius elongatus, Watsonius watsoni, Gymnophalloides seoi, Gynaecotyla squatarolae, Microphallus brevicaeca, Isoparorchis hypselobagri, Nanophyetus salmincola, N. schikobalowi, Neodiplostomum seoulense, Fibricola cratera, Plagiorchis muris, P. vespertilionis, P. harinasutai, P. javensis, P. philippinensis, and Prohemistomum vivax), one throat fluke (Clinostomum complanatum), and one pancreatic fluke (Eurytrema pancreaticum). The mode of transmission to humans includes contact with cercariae contaminated in water (schistosomes) or ingestion of raw or improperly cooked food, including fish (liver flukes, heterophyid flukes, echinostomes, and throat flukes), snails (echinostomes, brachylaimids, and gymnophallid flukes), amphibia, reptiles (neodiplostomes), aquatic vegetables (fasciolids and amphistomes), and insect larvae or adults (lecithodendriids, plagiorchiids, and pancreatic flukes). Praziquantel has been proven to be highly effective against almost all kinds of trematode infections except Fasciola spp. Epidemiological surveys and detection of human infections are required for a better understanding of the prevalence, intensity of infection, and geographical distribution of each trematode species.

Liver Histopathological and Immunohistochemical Evaluation from Fasciola hepatica Experimentally Infected and Reinfected Sheep.

Fasciolosis is an important economic disease of livestock. There is a global interest in the development of protective vaccines since the current anthelmintic therapy is no longer sustainable. A better knowledge of the host-parasite interaction is needed to design effective vaccines. To date, few studies have evaluated host-parasite interaction by comparing infected and reinfected animals. The present study evaluates the microscopical hepatic lesions in sheep infected and reinfected with Fasciola hepatica during the acute and chronic stages of infection. The histopathological study revealed the presence of necrotizing foci (NF1) associated with larvae migration during the early stages of infection in the primoinfected (PI) and reinfected (RI) groups. In the late stages of infection of the PI group and at the early and late stages of infection in the RI groups, extensive necrotizing/hemorrhagic foci (NF2) were found in the vicinity of enlarged bile ducts, some containing adult flukes, suggesting parasites may have caused NF2 while feeding. The immunohistochemical study revealed an increase in Foxp3+ T cells in both PI and RI groups with respect to the UC group and in the infiltrates adjacent to NF1 in the RI groups with respect to the PI group, suggesting the F. hepatica induce Foxp3 T cell expansion to facilitate parasite survival. In addition, in both the PI and RI groups, and during acute and chronic stages of the infection, a poor expression of iNOS was found accompanied by a strong expression of CD163, suggesting a marked M2 activation of macrophages in the hepatic lesions, which may be related with healing processes, and it also may facilitate parasite survival. The main differences between PI and RI animals were the more severe infiltration of eosinophils and Foxp3+ T cells, whereas RI did not modify M2 activation of macrophages which occurs since the early stages of primoinfection.

Prevalence and associated economic losses of bovine fasciolosis from postmortem inspection in municipal abattoirs in Ethiopia: A systematic review and meta-analysis.

Fasciolosis is a prevalent disease that significantly affects the health and productivity of cattle and causes significant economic loss. Beyond individually available studies with varying prevalence rates, there are no pooled national prevalence studies on bovine fasciolosis. Therefore, the aim of the current study was to determine the combined magnitude and economic significance of fasciolosis among cattle on postmortem examination. Inverse variance (I2), sensitivity analysis, funnel plots, Begg's test, and Egger's regression test were used to assess heterogeneity and publication bias. A random-effects model was used to calculate the pooled burden of fasciolosis among cattle. The pooled prevalence of fasciolosis among cattle on postmortem examination was 31.77 % (95 % CI=27.82-35.71). Among a total of 14,965 livers of slaughtered cattle examined in municipal abattoirs, Fasciola hepatica (54.4 %) was the predominant fluke identified compared to F. gigantica (24.6 %). Mixed infections of both species and unidentified immature flukes were detected in 12.4 % and 7.6 %, respectively, of affected livers. Regarding the severity of the pathological lesions observed, 30.5 %, 44.3 %, and 25.2 % of the livers were lightly, moderately, and seriously infested, respectively. The pooled annual economic loss attributed to fasciolosis-associated liver condemnation among cattle in 40 reported studies was approximately 40,833,983.15 ETB (6,417, 847.73 USD). Therefore, bovine fasciolosis requires integrated control methods to address its influence on animal health and economic impact.

Zoonotic human liver flukes, a type 1 biocarcinogen, in freshwater fishes: genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh.

BACKGROUND: Opisthorchiid flukes, particularly Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, and Metorchis spp. are the most common fish-borne zoonotic human liver flukes (hLFs). Liver fluke infections are more prevalent in resource-deprived and underprivileged areas. We herein estimated the prevalence of the metacercariae (MC) of major hLFs in common large freshwater fishes (lFWF) marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs. METHODS: The current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018-June 2022 using conventional and multiple molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (RFLP), sequencing, and bioinformatic analyses. The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared (χ2) test. RESULTS: The MC of C. sinensis, Opisthorchis spp., and Metorchis spp. were detected in 11 species of common and popular lFWF. In lFWF, the estimated prevalence was 18.7% and the mean load was 137.4 ± 149.8 MC per 100 g of fish. The prevalence was the highest (P < 0.05) in spotted snakehead fishes (Channa punctata, 63.6%). The highest rate of infection (P < 0.05) was observed with the MC of C. sinensis (11.8%). Metacercariae were almost equally (P > 0.05) distributed between the head and body of fishes. The infection rate was slightly higher in cultured (19.6%) fishes. The MC of C. sinensis, O. felineus, O. viverrini, and Metorchis orientalis in fishes were confirmed using PCR, PCR-RFLP and bioinformatics. The cercariae of opisthorchiid (Pleurolophocercus cercariae) flukes were only recovered from Bithynia spp. (3.9%, 42 out of 1089). The ova of hLFs from dogs (4.3%, 5 out of 116) and cats (6.0%, 6 out of 100), and adult flukes (M. orientalis) from ducks (41.1% 113 out of 275) were detected. CONCLUSIONS: The MC of hLFs are highly prevalent in fresh water fishes in Bangladesh. Reservoir hosts, such as street dogs, cats, and ducks carried the patent infection, and residents of Bangladesh are at risk.

A Case of Clonorchiasis in a Non-endemic Region of China.

Clonorchiasis, an important foodborne parasitic disease, is prevalent in several Asian countries. In China, the three provinces with the highest incidence are Guangdong, Guangxi, and Heilongjiang, with no reported cases in Qingdao for nearly a decade. In this study, a 29-year-old male patient was diagnosed with fatty liver due to abnormal liver function during physical examination and was admitted to the hospital multiple times for examination and treatment within 3 years, but his liver function did not improve. Eventually, clonorchis eggs were found in the stool, confirming the diagnosis of clonorchiasis. The purpose of this report is to enhance the understanding of clonorchiasis among clinicians in no-prevalence areas, to familiarize laboratory technicians with egg identification, to strengthen parasite-knowledge training, and to reduce missed and misdiagnosed cases.

Misclassification of Opisthorchis viverrini and Minute Intestinal Fluke Eggs by Routine Laboratory Staff Using Images from the Kato-Katz Method.

BACKGROUND: The Kato-Katz method is a commonly used diagnostic tool for helminth infections, particularly in field studies. This method can yield inaccurate results when samples contain eggs that are similar in appearance, such as Minute Intestinal Fluke (MIF) and Opisthorchis viverrini (OV) eggs. The close resemblance of eggs can be problematic and raises the possibility of false diagnoses. The objectives were to compare the diagnostic performance of the Kato-Katz method for accurately identifying MIF and OV and to provide evidence of possible misclassification.  Methods: Based on questionnaire responses from 15 (young parasitologists and public health staff), the test comprised 50 MIF egg images and 50 OV egg images, for a total of 100 Google Form questionnaires. RESULTS: The morphology of MIF and OV eggs found size and shape similarity and found that the shoulder rims were small, while the OV egg found the knobs had disappeared. The opercular conjunction was apparent, the shoulder rims and miricidium were prominent. The average percentage of correctly classified infections was 61.6 ± 12.1%. The accuracy percentages for both public health staff and young parasitologists in identifying were found to be 59.0 ± 14.8 and 66.8 ± 2.8, respectively. There was no significant difference observed in both groups. CONCLUSION: These findings highlight the need for improving the accuracy of parasite identification. Preserving stool samples before the Kato-Katz method can help mitigate the potential degradation or distortion of parasite eggs. The incorrect classification of both eggs had an impact on treatment plans and the policy of parasite control programs.

Infection with Clonorchis sinensis (Cobbold, 1875) Metacercariae in Fish from the East Lake of Wuhan: Freshwater Fish in Urban Lakes May Act as Infection Sources of Liver Fluke.

The liver fluke disease caused by Clonorchis sinensis is one of the most serious food-borne parasitic diseases in China. Many freshwater fish and shrimps can be infected with C. sinensis metacercariae as the second intermediate hosts in endemic regions. Owing to the lack of infected humans and the good administration of pet dogs and cats in cities of non-endemic regions, few fish are expected to be infected with C. sinensis metacercariae in urban lakes. To determine the infection of C. sinensis metacercariae in freshwater fish and shrimps in urban lakes, a total of 18 fish species and one shrimp species were investigated in the East Lake of Wuhan City. Metacercariae were isolated by artificial digestive juice and identified using morphology and rDNA-ITS2 sequences. Five species of fish, Pseudorasbora parva, Ctenogobius giurinus, Squalidus argentatus, Hemiculter leuciclus, and Rhodeus spp., were infected with C. sinensis metacercariae. The overall prevalence of C. sinensis was 32.5%. The highest prevalence was found in P. parva with 57.9%, while S. argentatus exhibited the highest mean abundance (13.9). Apart from the C. sinensis metacercariae, four species of other trematode metacercariae were also identified across twelve fish species in total. Owing to the consumption of undercooked fish and feeding cats with small fish caught by anglers, there is a potential risk that the small fish infected with C. sinensis metacercariae may act as an infection source to spread liver fluke. Given the complete life cycle of C. sinensis, stray cats and rats were inferred to act as the important final hosts of C. sinensis in urban lakes in non-endemic areas.

Estimation of diagnostic sensitivity and specificity of abattoir registrations and bulk tank milk ELISA as herd-level tests for Fasciola hepatica using Bayesian latent class modelling.

The common liver fluke, Fasciola hepatica, is a trematode parasite found worldwide, typically with a focal distribution due to its requirement for suitable climatic and environmental conditions to complete its lifecycle. Bovine fasciolosis causes suboptimal production and economic losses, including liver condemnation at slaughter. The lack of reliable diagnostic methods is a disadvantage to the increasing demand for surveillance and control. The aim of this study was to evaluate the diagnostic accuracy of bulk tank milk (BTM) antibody testing and aggregated abattoir registrations (AAR) of liver fluke as herd-level tests for F. hepatica infection using Bayesian latent class models. Data from the abattoirs in 2019-2021 and BTM, sampled in the winter of 2020/2021, were collected from 437 herds on the southwest coast of Norway. The BTM samples were analysed with the SVANOVIR® F. hepatica-Ab ELISA test, with results given as an optical density ratio (ODR), and later dichotomized using the recommended cut-off value from the test manufacturer (ODR ≥0.3). Based on the BTM ELISA test, 47.8% of the herds tested positive. The AAR test was defined as the herd-level proportion of female slaughtered animals registered with liver fluke infection during the study period. For this test, three cut-offs were used (a proportion of 0.05, 0.1 and 0.2). The herds were split into two subpopulations ("Coastal" and "Inland"), which were expected to differ in true prevalence of F. hepatica infection based on climate-related and geographical factors. The diagnostic accuracies of both tests were estimated using Bayesian latent class models with minimally informative priors. Post-hoc analysis revealed that the maximum sum of sensitivity (Se) and specificity (Sp) of the tests was achieved with a herd-level proportion of ≥0.1 registered with liver fluke as the AAR test. Using this cut-off, the median estimate for the diagnostic accuracy of the BTM ELISA was 90.4% (84.0-96.2 95% Posterior Credible Interval (PCI)) for Se and 95.3% (90.6-100% PCI) for Sp, while the median estimate of Se for AAR was 87.5% (81.4-93.1% PCI) and the median estimate of Sp for AAR was 91.0% (85.2-96.5% PCI). The cut-off evaluation of the SVANOVIR® F. hepatica-Ab ELISA test for BTM confirmed the manufacturer's recommended cut-off of ODR ≥0.3 to denote positive and negative herds. This study suggests that AAR and BTM ELISA test can be used as herd-level tools to monitor liver fluke infection, so that appropriate interventions against infection can be implemented as necessary.

Identification and functional analysis of mitogenic miRNA of the carcinogenic liver fluke Opisthorchis viverrini.

Opisthorchis viverrini is a pathogenic liver fluke that is known to cause cholangiocarcinoma in chronic infections. The underlying mechanism for this carcinogenesis is believed to be multifactorial, with parasite-derived excretory-secretory (ES) products potentially playing major roles. A recent study on these ES products has identified microRNAs (miRNA) that originate from O. viverrini but their influence on carcinogenesis remains understudied. Hence, we aimed to investigate the role of these miRNAs in the carcinogenesis of O. viverrini-associated cholangiocarcinoma. The mature miRNA sequences were retrieved from published data. Bioinformatics analysis was employed to identify miRNA targets and to identify potentially mitogenic miRNAs. An in vitro study was conducted to test the effects of miRNA on the bile duct epithelial cell lines. The miRNA target prediction analysis revealed that Ov_miRNA_EV_36/ovi-miR-3479a targets cancer-associated pathways. Hence, it was selected and used to assess its effect on the cell proliferation rate of H69 and MMNK-1 cholangiocyte cell lines. The results showed that Ov_miRNA_EV_36/ovi-miR-3479a induced significant cell proliferation in both cell lines when compared to negative controls. These results indicate that Ov_miRNA_EV_36/ovi-miR-3479a may play an essential role in the carcinogenesis of O. viverrini and therefore warrant further investigations.

Calicophoron daubneyi (Paramphistomidae) in deer of the Sumava National Park, Czech Republic - Consequence of prevalent rumen fluke infection in cattle.

A substantial parallel increase in prevalence and geographical spread of the rumen fluke, Calicophoron daubneyi, in livestock in western and central Europe has been recognized in the recent past. In the course of the examination of rectum feces of 471 red deer (Cervus elaphus) and one sika deer (Cervus nippon) from the Fascioloides magna endemic Sumava National Park in the years 2021 and 2022, rumen fluke eggs were detected in four red deer (0.8%) and the sika deer and identified as eggs of C. daubneyi by molecular analysis. Subsequent examination of rectal fecal samples of 247 beef cattle from 22 herds of 14 farms located in or nearby the national park revealed rumen fluke eggs in 53 samples (21.5%) originating from 16 herds of 11 farms, molecularly identified as C. daubneyi eggs as well. One C. daubneyi egg positive red deer and three C. daubneyi egg positive cattle samples also contained fasciolid eggs, respectively, which were detected in 9.5% or 3.6% of the total samples from red deer or cattle, respectively. Results of this investigation reveal the first finding of C. daubneyi in sika deer worldwide and in red deer in mainland Europe and add to the growing number of reports on C. daubneyi in livestock in Europe. Considering that the ratio of cattle excreting rumen fluke eggs exceeded that of deer substantially, it can reasonably be assumed that the C. daubneyi infections in deer are a consequence of the prevalent infection in cattle, illustrating a pathogen spillover event from livestock into wildlife.

The riboflavin (vitamin B2) transporter protein (SmaRT) of the human intravascular parasitic trematode Schistosoma mansoni.

Schistosomes are intravascular parasitic worms infecting >200 million people globally. Here we examine how the worms acquire an essential nutrient - vitamin B2 (riboflavin). We demonstrate that all intravascular life stages (schistosomula, adult males and females) take up radiolabeled riboflavin. This process is impeded in the presence of excess unlabeled riboflavin and at 4 °C. We have identified a transporter homolog in worms designated SmaRT (Schistosoma mansoni riboflavin transporter) that localizes to the tegument and internal tissues of adults. CHO-S cells transfected with plasmid encoding SmaRT import significantly more radiolabeled riboflavin compared to controls. Uptake of radiolabel is impeded when SmaRT-expressing cells are incubated in an excess of unlabeled riboflavin but not by an excess of an irrelevant metabolite. Uptake is mediated in a sodium-independent manner and over a wide range of pH values (pH 5.5-9). This is the first identification of a bone fide riboflavin transporter in any platyhelminth.

Occurrence and Risk Factors Associated with Platynosomum illiciens Infection in Cats with Elevated Liver Enzymes.

Platynosomum spp., a hepatic trematode, causes fatal hepatobiliary disease in cats. Feline platynosomiasis is often underestimated due to a lack of awareness and diagnostic challenges. This study aimed to investigate the occurrence, factors, and clinicopathological abnormalities associated with Platynosomum spp. infection in cats with elevated serum ALT levels. Platynosomum infection was determined using zinc sulfate flotation and formalin-ether sedimentation. DNA sequence analysis of PCR products from the Platynosomum internal transcribed spacer 2 (ITS2) region and cox1 gene was used to identify Platynosomum species. Of a total of 43 cat fecal samples, the proportion of Platynosomum spp. infection by microscopic examination was 11.63% (5/43). All PCR-positive samples were molecularly identified as Platynosomum illiciens. From the logistic regression analysis, the odds of Platynosomum infection in cats without a deworming program were 16 times higher than those of regularly dewormed cats. Demographic data, housing conditions, and predatory behavior were not significantly associated with the infection. Regarding blood profiles, infected cats had higher eosinophil counts (p = 0.014), with no significant differences in ALT (p = 0.791) or ALP (p = 0.970) levels compared to non-infected cats. Our findings demonstrate that eosinophilia in cats with increased serum ALT may suggest P. illiciens infection in endemic areas. We strongly recommend a regular deworming program to mitigate the risk of P. illiciens infection.

Intraductal magnetic resonance imaging of cholangiocarcinoma - a practical possibility.

Intraductal T2 mapping based on a catheter receiver is proposed as a method of visualizing the extent of intraductal and periductal cholangiocarcinoma (CCA). Compared to external receivers, internal receivers provide locally enhanced signal-to-noise ratios by virtue of their lower field-of-view for body noise, allowing smaller voxels and higher resolution. However, inherent radial sensitivity variation and segmentation for patient safety both distort image brightness. We discuss simulated T2 weighted images and T2 maps, and in vitro images obtained using a thin film catheter receiver of a freshly resected liver specimen containing a polypoid intraductal tumor from a patient with CCA. T2 mapping provides a simple method of compensating non-uniform signal reception patterns of catheter receivers, allowing the visualization of tumor extent without contrast enhancement and potentially quantitative tissue characterization. Potential advantages and disadvantages of in vivo intraductal imaging are considered.

Epidemiology of rumen fluke infection in selected buffalo farms in perak, malaysia: prevalence, molecular species identification, and associated risk factors.

Rumen flukes cause heavy economic losses in the ruminant industry worldwide, especially in tropical and subtropical countries. This study estimated the prevalence of rumen flukes in buffaloes, identified the species diversity, and determined risk factors associated with rumen fluke prevalence in Perak, Peninsular Malaysia. A cross-sectional study was conducted, and 321 faecal samples were collected from six buffalo farms. A structured questionnaire was developed, and farmers were interviewed to obtain information regarding risk factors associated with rumen fluke infection. The faecal samples were examined using sedimentation and Flukefinder® techniques. Genomic DNA was extracted from the fluke eggs recovered using the Flukefinder® method, and the internal transcribed spacer 2 (ITS2) fragment was amplified and sequenced to facilitate species identification. The results showed that the overall prevalence of rumen fluke across the sampled farms was 40.2% (129/321). Three rumen fluke species were identified, namely, Fischoederius elongatus, F. cobboldi, and Orthocoelium streptocoelium. Several management factors had a significant association (P < 0.05) with rumen fluke prevalence, including production type, cleaning of the stable, drinking water system, flooding around the farm, grazing system, pasture sharing with other livestock, and deworming program. This work constitutes the first attempt to understand the epidemiology of rumen fluke infection in the region and suggests that good farm management, pasture management, choosing appropriate drugs, and proper husbandry practices may improve buffalo health and production in areas where rumen flukes are prevalent.

Natural variables separate the endemic areas of Clonorchis sinensis and Opisthorchis viverrini along a continuous, straight zone in Southeast Asia.

BACKGROUND: Clonorchiasis and opisthorchiasis, caused by the liver flukes Clonorchis sinensis and Opisthorchis viverrini respectively, represent significant neglected tropical diseases (NTDs) in Asia. The co-existence of these pathogens in overlapping regions complicates effective disease control strategies. This study aimed to clarify the distribution and interaction of these diseases within Southeast Asia. METHODS: We systematically collated occurrence records of human clonorchiasis (n = 1809) and opisthorchiasis (n = 731) across the Southeast Asia countries. Utilizing species distribution models incorporating environmental and climatic data, coupled machine learning algorithms with boosted regression trees, we predicted and distinguished endemic areas for each fluke species. Machine learning techniques, including geospatial analysis, were employed to delineate the boundaries between these flukes. RESULTS: Our analysis revealed that the endemic range of C. sinensis and O. viverrini in Southeast Asia primarily spans across part of China, Vietnam, Thailand, Laos, and Cambodia. During the period from 2000 to 2018, we identified C. sinensis infections in 84 distinct locations, predominantly in southern China (Guangxi Zhuang Autonomous Region) and northern Vietnam. In a stark contrast, O. viverrini was more widely distributed, with infections documented in 721 locations across Thailand, Laos, Cambodia, and Vietnam. Critical environmental determinants were quantitatively analyzed, revealing annual mean temperatures ranging between 14 and 20 °C in clonorchiasis-endemic areas and 24-30 °C in opisthorchiasis regions (P < 0.05). The machine learning model effectively mapped a distinct demarcation zone, demonstrating a clear separation between the endemic areas of these two liver flukes with AUC from 0.9 to1. The study in Vietnam delineates the coexistence and geographical boundaries of C. sinensis and O. viverrini, revealing distinct endemic zones and a transitional area where both liver fluke species overlap. CONCLUSIONS: Our findings highlight the critical role of specific climatic and environmental factors in influencing the geographical distribution of C. sinensis and O. viverrini. This spatial delineation offers valuable insights for integrated surveillance and control strategies, particularly in regions with sympatric transmission. The results underscore the need for tailored interventions, considering regional epidemiological variations. Future collaborations integrating eco-epidemiology, molecular epidemiology, and parasitology are essential to further elucidate the complex interplay of liver fluke distributions in Asia.

FASN-mediated fatty acid biosynthesis remodels immune environment in Clonorchis sinensis infection-related intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer and is highly lethal. Clonorchis sinensis (C. sinensis) infection is an important risk factor for iCCA. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis infection-related iCCA. METHODS: We performed single-cell RNA sequencing, whole-exome sequencing, RNA sequencing, metabolomics and spatial transcriptomics in 251 patients with iCCA from three medical centers. Alterations in metabolism and the immune microenvironment of C. sinensis-related iCCAs were validated through an in vitro co-culture system and in a mouse model of iCCA. RESULTS: We revealed that C. sinensis infection was significantly associated with iCCA patients' overall survival and response to immunotherapy. Fatty acid biosynthesis and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis-related iCCAs. iCCA cell lines treated with excretory/secretory products of C. sinensis displayed elevated FASN and free fatty acids. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage (TAM)-like macrophages and the impaired function of T cells, which led to formation of an immunosuppressive microenvironment and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis-related iCCAs than non-C. sinensis-related iCCAs. Importantly, treatment with a FASN inhibitor significantly reversed the immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in iCCA mouse models treated with excretory/secretory products from C. sinensis. CONCLUSIONS: We provide novel insights into metabolic alterations and the immune microenvironment in C. sinensis infection-related iCCAs. We also demonstrate that the combination of a FASN inhibitor with immunotherapy could be a promising strategy for the treatment of C. sinensis-related iCCAs. IMPACT AND IMPLICATIONS: Clonorchis sinensis (C. sinensis)-infected patients with intrahepatic cholangiocarcinoma (iCCA) have a worse prognosis and response to immunotherapy than non-C. sinensis-infected patients with iCCA. The underlying molecular characteristics of C. sinensis infection-related iCCAs remain unclear. Herein, we demonstrate that upregulation of FASN (fatty acid synthase) and free fatty acids in C. sinensis-related iCCAs leads to formation of an immunosuppressive microenvironment and tumor progression. Thus, administration of FASN inhibitors could significantly reverse the immunosuppressive microenvironment and further enhance the efficacy of anti-PD-1 against C. sinensis-related iCCAs.

RNAscope: a novel method for the detection of Heterobilharzia americana ova in canine liver.

Canine schistosomiasis caused by Heterobilharzia americana can lead to severe morbidity and eventual mortality, in part due to the deposition of fluke ova in the liver and gastrointestinal tract, which promotes an influx of peri-ova inflammatory cells. Although fluke eggs can be identified in H&E-stained histologic sections, cases exist in which only fragments of the ova persist, or the egg is obscured by inflammatory infiltrates, which can confound definitive histologic diagnosis. Unfortunately, antibodies specific to Heterobilharzia are not commercially available for immunohistochemical labeling. Therefore, we aimed to use an RNA in situ hybridization strategy to fluorescently label Heterobilharzia ova. Using the H. americana 18S rRNA sequence, we developed an RNA probe and validated its performance on archival formalin-fixed, paraffin-embedded canine tissue. A positive signal was observed for all identifiable ova, fragmented and whole. Use of this methodology could aid understanding of the pathogenesis of H. americana infection in dogs. This technique augments standard diagnostic methodology, enabling spatial colocalization of fluke ova and inflammatory infiltrates when using fluorescent techniques.

Plastoquinone-Derivative SkQ1 Improved the Biliary Intraepithelial Neoplasia during Liver Fluke Infection.

Carcinogenic food-borne liver fluke infections are a serious epidemiological threat worldwide. The major complications of Opisthorchis felineus infection are chronic inflammation and biliary intraepithelial neoplasia. Although evidence has accumulated that increased reactive oxygen species production is observed in liver fluke infection, a direct relationship between the oxidative stress and biliary intraepithelial neoplasia has not been shown. Quinones and SkQ1, a derivative of plastoquinone, have been demonstrated to be cytoprotective in numerous liver injuries due to their potent antioxidant properties. This study is aimed to assess the level of biliary intraepithelial neoplasia in O. felineus-infected hamsters after treatment with mitochondria-targeted SkQ1. SkQ1 significantly reduced the biliary intraepithelial neoplasia, which was accompanied by a decrease in lipid and DNA oxidation byproducts, mRNA expression and level of proteins associated with inflammation (TNF-α, CD68) and fibrogenesis (CK7, αSMA), and was also associated with an activation of the Keap1-Nrf2 pathway. Thus, a direct relationship was found between oxidative stress and the severity of biliary intraepithelial neoplasia in O. felineus-infected hamsters. The hepatoprotective effect of plastoquinone-derivative SkQ1 was established; therefore, this compound is a promising agent in complex therapy in the treatment of opisthorchiasis.

Visualization and development of colorimetric loop-mediated isothermal amplification for the rapid detection and diagnosis of paramphistome infection: colorimetric PAR-LAMP.

Colorimetric detection can be applied to differentiate between positive and negative conditions. It can be coupled with loop-mediated isothermal amplification to diagnose rumen fluke or paramphistome infection, also called colorimetric PAR-LAMP. This study conducted LAMP using three candidate indicator dyes, namely malachite green (MLG), methyl green (MTG), and neutral red (NTR), and the results were observed by the naked eye. The dye concentration was optimized to obtain the most pronounced positive-negative result discrimination. Subsequently, we conducted target sensitivity tests using the DNA of Fischoederius elongatus at different concentrations. To validate the detection accuracy, the result was confirmed by gel electrophoresis. The sensitivity test presented the lowest detectable DNA concentration or limit of detection (LOD), with 1 pg for MLG, 0.5 ng for MTG, and 50 pg for NTR. Different LODs revealed inhibition of LAMP reaction and reduced efficiency of result presentation for colorimetric-based detection, particularly NTR and MTG. For MLG-LAMP, we observed no cross-reaction of non-target DNA and improved reaction with the DNA of Fischoederius cobboldi and Calicophoron sp., with multi-detection. In addition, naked eye observation and agarose gel electrophoresis (AGE) evaluation of the MLG-LAMP results showed a moderate and strong agreement with LAMP-AGE and microscopic examinations. Based on our results, colorimetric PAR-LAMP is a rapid, comfortable, and point-of-care procedure for the diagnosis of paramphistome infection.

Environmental influence on abundance and infection patterns of snail intermediate hosts of liver and intestinal flukes in North and Central Vietnam.

Liver and intestinal flukes (LIF) are important groups of foodborne zoonotic trematodes (FZTs) in Southeast Asia, including Vietnam. Their complex life cycles require specific freshwater snail species as the obligatory first intermediate hosts. In 2019, we conducted a longitudinal study in Yen Bai and Thanh Hoa provinces in North and Central Vietnam, respectively, to investigate the diversity of LIF and their infection prevalence in relation to snail host abundance and environmental factors. Using a combination of morphological and molecular identification techniques, we identified 10 LIF species infecting 11 snail host species. We observed significant seasonal variation in the mean abundance of several snail host species, with the majority of snails collected during the spring. We also detected seasonal changes in LIF species composition, with the highest species richness reported in the spring. Clonorchis sinensis and Fasciola gigantica, two medically important human liver flukes in Asia, were found only in the spring in Yen Bai. Our study revealed that not all snail host species have the same probability of becoming infected, and we recorded seasonal variations in the prevalence of LIF infection in different snail species in relation to water parameters.