Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis (family Opisthorchiidae) are parasitic flatworms that pose serious threats to humans in certain countries and cause opisthorchiasis/clonorchiasis. Opisthorchiid flukes parasitize the biliary tract of the host, causing cholangitis, cholecystitis, cholelithiasis and cholangiocarcinoma. In this review, we primarily focus on recent microRNAs (miRNAs) studies of opisthorchiid flukes and their definitive hosts. Many miRNAs are conserved and expressed in a developmentally stage specific manner in the three opisthorchiid flukes, which play important roles in the growth and development of Opisthorchiidae spp., as well as host-pathogen interactions. Some miRNAs might be potential biomarkers related to carcinogenesis of cholangiocarcinoma. Therefore, this review provides the basis for further investigating the roles of miRNAs in opisthorchiid flukes and their definitive hosts, as well as promoting the development of novel approaches to prevent and treat opisthorchiasis/clonorchiasis.

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.
CONCLUSIONS: 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.

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.

Cyprinid fishes are commonly acted as second intermediate hosts of Opisthorchis viverrini and Haplorchis spp. This research investigated the prevalence and intensity of both parasites in cyprinid species. This process is regularly used as a guideline for disease surveillance and as a preventive measure in the Upper Northeastern region of Thailand. Fish samples were collected from both lentic and lotic water sources between November 2020 and October 2021, and classified them by their species. Acid pepsin-HCL solution was used to digest the individual samples and create a build-up of precipitate in order to view metacercariae using a stereomicroscope. A total of 3583 fish were caught and classified into 23 species. Out of these fish, 5.49% of them were found O. viverrini, 26.54% Haplorchis taichui, 10.04% Haplorchis pumilio, and 9.29% Haplorchoides sp. Ten species of fish were observed to have been infected with O. viverrini. Amongst the collected fish, M. obtusirostris had the highest prevalence of infection at 52.94%, while B. gonionotus had only 1.71%. The prevalence of O. viverrini infection was highest at 27.41% in Udon Thani Province. No prevalence of O. viverrini infection was observed in Bueng Kan Province. High prevalence of O. viverrini infection in cyprinid fishes was found in both lentic and lotic water sources in almost all provinces in the region. The prevalence of infection and intensity depended on the fish species and fish habitats.

Infection by the zoonotic fish-borne trematode, Opisthorchis viverrini, remains a crucial health issue in Thailand and neighboring countries. Recently, molecular analysis revealed two populations of putative O. viverrini: one found primarily in human hosts (\"human-specific\" population) and the other primarily in cats (\"cat-specific\" population). It is unclear how the infective stages (metacercariae) of these different populations circulate among definitive and reservoir hosts in nature. To gain an insight into this, mitochondrial cox1 and nad1 gene sequences of metacercariae from fish intermediate hosts were examined. None of 192 metacercariae from cyprinid fish in Lao PDR and Thailand had sequences typical of \"cat-specific\" O. viverrini, suggesting that cyprinid fish are not the main second intermediate hosts of this population. Interestingly, all 20 O. viverrini-like metacercariae from snakehead fish (Channa striata) shared 99.51-100% sequence identity with eggs from cats naturally infected in a previous study. Hence, we propose a modification of the known transmission dynamics of O. viverrini: consumption of metacercariae within snakehead fish provides another pathway for cats and (occasionally) humans to acquire infection. We also performed morphological comparisons of eggs, metacercariae, and adult flukes (raised in hamsters) of both Opisthorchis populations. The \"cat-specific\" population has eggs that are narrower and adults that are shorter and wider than in the human-specific population. The metacercaria of the \"cat-specific\" population is elliptical, while that of the \"human-specific\" population is oval, occasionally rounded. Our results confirmed that O. viverrini-like metacercariae from snakehead fish are the infective stages of the \"cat-specific\" fluke. This provides a new insight into the dissemination and transmission of each population in the second intermediate host. The identity of the cat-specific population is discussed.

UNASSIGNED: Inflammation caused by Opisthorchis viverrini infection increases the risk of cholangitis, cholecystitis, and leads to bile duct cancer (cholangiocarcinoma or CCA). However, only certain infected individuals are susceptible to CCA, suggesting the involvement of host factors in cancer development. In addition, there are reports indicating differences in the locations of CCA.
UNASSIGNED: This study aims to investigate cellular inflammatory responses in the common bile duct (CB), intrahepatic bile duct (IHB), and gallbladder (GB) in susceptible and non-susceptible hosts following O. viverrini infection.
UNASSIGNED: Thirty Syrian golden hamsters (a susceptible host) and 30 BALB/c mice (a non-susceptible host) infected with O. viverrini were studied at six time points (five animals per group). Histopathological evaluations were conducted on samples from the IHB, CB, and GB. Inflammatory cell infiltration was quantitatively assessed and compared between groups and time points. Statistical analysis was performed using one-way ANOVA, with a significance level of p < 0.05.
UNASSIGNED: Inflammation was significantly more pronounced in the IHB compared to the other two biliary locations. In comparison between susceptible and non-susceptible hosts, the intensity of inflammation was higher in the OV+H group than in the OV+M group (p < 0.05).
UNASSIGNED: This study highlights the association between host response to inflammation, tissue location, and host susceptibility, with the IHB showing particular susceptibility to inflammation and pathological changes. These findings contribute to our understanding of the increased risk of CCA in susceptible hosts.

OBJECTIVE: This study aimed to assess the diagnostic potential of cell-free DNA (cfDNA) and cell-free miRNA (cf-miRNA) for distinguishing between Healthy, asymptomatic opisthorchiasis viverrini and cholangiocarcinoma in a preliminary manner.
METHODS: In this study, 36 participants were enrolled into three health status groups: a healthy control group (HC), Opisthorchis viverrini-infected group (OV), and a cholangiocarcinoma group (CCA), each comprising 12 participants. Concentration measurements of cfDNA and cf-miRNA from plasma were conducted. Additionally, ultra-low-pass whole-genome sequencing (ULP-WGS) was employed to investigate DNA alterations.
RESULTS: The study revealed a significant elevation in plasma cfDNA concentration in the cholangiocarcinoma (CCA) group compared to healthy controls (HC) and Opisthorchis viverrini-infected (OV) groups (P < 0.001). The cfDNA concentration demonstrated a sensitivity of 75.00% and specificity of 95.83% for differentiating cholangiocarcinoma, with a cut-off of > 30.50 ng/ml plasma. Likewise, the concentration of cf-miRNA in the CCA group significantly differed from that in the HC and OV groups, demonstrating a sensitivity of 83.33% and specificity of 95.83% with a cut-off set at > 70.50 ng/ml plasma. Furthermore, a positive correlation between plasma concentrations of cfDNA and cf-miRNA suggests a potential relationship between these two biomarkers. These findings indicated the diagnostic potential of cfDNA and cf-miRNA in distinguishing cholangiocarcinoma, emphasizing their role as promising biomarkers for further investigation and clinical applications.
CONCLUSIONS: Elevated plasma concentrations of cfDNA and cf-miRNA could serve as potential diagnostic tools for distinguishing cholangiocarcinoma from other conditions. cf-miRNA was superior to cfDNA in terms of sensitivity.

UNASSIGNED: Steatotic liver disease (SLD) prevalence is rising worldwide, linked to insulin resistance and obesity. SLD prevalence can surpass 10% even among those with normal weight. In Lao People\'s Democratic Republic (Lao PDR), where Opisthorchis viverrini (OV) trematode infection and type 2 diabetes mellitus (T2DM) are common, infection related liver morbidity such as cholangiocarcinoma (CCA) is high, but data on SLD prevalence is lacking. The objective of this study was to estimate the prevalence and explore determinants of SLD in rural southern Lao PDR for lean and non-lean populations.
UNASSIGNED: A cross-sectional community-based study assessed SLD prevalence using abdominal ultrasonography (US). Factors investigated for association with SLD were identified by interview, serological tests (Hepatitis B surface antigen (HBsAg); lipids and HbA1c), anthropometrical measurements, and parasitological assessments (OV infection). Uni- and multivariable logistic regression analyses with SLD as endpoint were conducted separately for lean (body mass index (BMI) <23.0 kg/m2) and non-lean (BMI ≥ 23.0 kg/m2) participants.
UNASSIGNED: 2,826 participants were included. SLD prevalence was 27.1% (95% confidence interval (95% CI) 24.0%-30.4%), higher among non-lean (39.8%) than lean individuals (17.4%). Lean individuals with OV infection had a statistically significant association with lower odds of SLD (adjusted odds ratio (aOR) 0.49, 95% CI 0.33 - 0.73). T2DM showed a significant positive association with SLD in both lean (aOR 3.58, 95% CI 2.28 - 5.63) and non-lean individuals (aOR 3.31, 95% CI 2.31 - 4.74) while dyslipidemia was significantly associated only in the non-lean group (aOR 1.83, 95% CI 1.09 - 3.07). Females participants exhibited elevated odds of SLD in both lean (aOR 1.43, 95% CI 1.02 - 2.01) and non-lean SLD (aOR 1.50, 95% CI 1.12 - 2.01).
UNASSIGNED: SLD prevalence is notably high among Laotian adults in rural areas, particularly in females and in non-lean individuals. Lean individuals with OV infection exhibited lower SLD prevalence. SLD was more prevalent in individuals with T2DM, independent of BMI. SLD adds to the burden of infection-related liver morbidity in Lao PDR.

Cats are recognized as significant reservoir hosts for human opisthorchiasis, particularly in areas with a high prevalence of infection. Despite this, the precise role of cats in the transmission of Opisthorchis viverrini between humans and felines remains unclear. This study investigates the association between these two hosts through both spatial and non-spatial analyses in the endemic Thanya sub-district of Thailand. A total of 105 owned cats were randomly sampled from 15 villages within the sub-district for stool examination. A questionnaire was administered to 66 cat owners to explore the human-pet relationship. Household locations were collected using GPS devices. Non-spatial analyses revealed a positive association between the two hosts (P= 0.011; OR 7, 95% CI: 1.6-30.9), highlighting two independent significant risk factors: cat owners consuming raw fish (P = 0.028; OR = 4.52, 95% CI: 1.25-19.45) and feeding cats raw fish (P = 0.011; OR = 16.41, 95% CI: 2.78-317.04) according to multivariate analysis. Spatial analysis provided further support to the non-spatial findings (p = 0.0123; OR = 3.45, 95% CI = 0.88-13.61). Multiple autologistic regression confirmed two significant risk factors: cat owners consuming raw fish (p = 0.054; OR = 3.37, 95% CI: 0.98-11.59) and feeding cats raw fish (p = 0.014; OR = 7.43, 95% CI: 1.49-37.05). Risk mapping identified the western part of the study site as a hotspot for O. viverrini infection. Hyper-endemic focusing revealed a union of human and cat buffers at 0.46 km², with an overlapping area of 0.22 km² (47.83%). This study underscores the impact of owners\' behaviors, specifically consuming and feeding raw fish to cats, on the increased probability of infection in cats. It emphasizes the need for effective opisthorchiasis control through health education targeting cat owners in endemic areas.

BACKGROUND: Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O. viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O. viverrini infection in field-collected human feces.
METHODS: To detect O. viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O. viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods.
RESULTS: The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O. viverrini DNA. On the basis of the detection limit, the assay could detect O. viverrini DNA at concentrations as low as 10-1 ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1 ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O. viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections.
CONCLUSIONS: The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O. viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O. viverrini infection, marking an initial step in the development of point-of-care diagnosis.