Investigation of Plasma Cell-Free DNA and MiRNA in Cholangiocarcinoma and Opisthorchiasis Viverrini Patients.

OBJECTIVES: 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. CONCLUSION: 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.

Update on the risk factors for opisthorchiasis and cholangiocarcinoma in Thailand.

This study aimed to identify the recent risk factors for Opisthorchis viverrini infection and cholangiocarcinoma (CCA) to improve disease prevention. The participants were divided into the following 3 groups based on their health status: healthy control (nonOV and nonCCA), those with O. viverrini infection (OV), and those with CCA. A questionnaire was used to explore their lifestyle and behaviors. Multivariate logistic regression and backward elimination were used to identify the significant risk factors. The results showed that the significant risk factors for both O. viverrini infection and CCA were age>50 years (odd ratio (OR)=8.44, P<0.001, 95% confidence intervals (CI) 2.98-23.90 and OR=43.47, P=0.001, 95% CI 14.71-128.45, respectively) and raw fish consumption (OR=8.48, P< 0.001, 95% CI 3.18-22.63 and OR=3.15, P=0.048, 95% CI 1.01-9.86, respectively). A history of O. viverrini infection was identified as an additional risk factor for CCA (OR=20.93, P=0.011, 95% CI 2.04-215.10). This study provided an update on the risk factors for O. viverrini infection and CCA. Asymptomatic patients with O. viverrini infection, particularly those>50 years old, should be carefully monitored to prevent CCA.

Differential plasma proteomes of the patients with Opisthorchiasis viverrini and cholangiocarcinoma identify a polymeric immunoglobulin receptor as a potential biomarker.

In Southeast Asian countries, nitrosamine compounds and the liver fluke Opisthorchis viverrini have long been identified as carcinogens for cholangiocarcinoma (CHCA). In order to effectively treat O. viverrini infections and prevent the development of CHCA, methods for disease detection are needed. This study aims to identify biomarkers for O. viverrini infection and CHCA. In the discovery phase, technical triplicates of five pooled plasma pools (10 plasma each) of healthy control subjects (noOVCCA), O. viverrini subjects (OV), and cholangiocarcinoma subjects (CCA), underwent solution-based digestion, with the label-free method, using a Thermo Scientific™ Q Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer and UltiMate 300 LC systems. The noOVCCA, OV, and CCA groups demonstrated different profiles and were clustered, as illustrated by PCA and heat map analysis. The STRING and reactome analysis showed that both OV and CCA groups up-regulated proteins targeting immune system-related proteins. Differential proteomic profiles, S100A9, and polymeric immunoglobulin receptor (PIGR) were specifically expressed in the CCA group. During the validation phase, another 50 plasma samples were validated via the PIGR sandwich ELISA. Using PIGR >1.559 ng/ml as a cut-off point, 78.00% sensitivity, 71.00% specificity, and AUC = 0.8216, were obtained. It is sufficient to differentially diagnose cholangiocarcinoma patients from healthy patients and those with Opisthorchiasis viverrini. Hence, in this study, PIGR was identified and validated as a potential biomarker for CHCA. Plasma PIGR is suggested for screening CHCA, especially in an endemic region of O. viverrini infection.

Susceptibility patterns of Bithynia siamensis siamensis and Bithynia funiculata to Opisthorchis viverrini infection: an indication of the risk of opisthorchiasis transmission in non-endemic areas.

Among the snail species acting as hosts for medically significant trematodes, only three taxa of Bithynia are responsible for transmitting the carcinogenic liver fluke Opisthorchis viverrini to humans in different geographical areas. Although B. siamensis goniomphalos is the primary species responsible for O. viverrini transmission in endemic areas, B. siamensis siamensis and B. funiculata remain potential hosts for transmission. This study objects to determine the susceptibility of B. siamensis siamensis and B. funiculata to O. viverrini to assess the risk of O. viverrini transmission in non-endemic areas. The snails of both species were first introduced to O. viverrini eggs, after which O. viverrini infection was investigated using specific PCR primers after a period of 1, 7, 14, 28, and 56 days post-infection (dpi). Opisthorchis viverrini infection in both B. siamensis siamensis and B. funiculata was high in the early period (1 and 7 dpi) while decreasing over time. It was also shown that the odds of susceptibility to O. viverrini infection in B. siamensis siamensis were 64.5% higher relative to the odds of susceptibility in B. funiculata (P < 0.05). Results of this study provide an early insight into the Bithynia-Opisthorchis relationship and thus have great potential to assess risk and raise awareness of opisthorchiasis in non-endemic regions, especially in regions endemic for B. siamensis siamensis.

Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta as a potential biomarker for Opisthorchis viverrini infection and cholangiocarcinoma.

The human liver fluke Opisthorchis viverrini (Ov), the primary risk factor for cholangiocarcinoma (CHCA), is a parasite endemic to southeast Asian countries. With no effective treatments for CHCA currently available, early diagnosis and treatment of Ov infection remains the only practical method for the prevention of CHCA. In this study, plasma phosphoproteomes of patients in the non-Ov infection, non-cholangiocarcinoma subject group (non-OVCCA), the asymptomatic Ov infected group (OV), and the CHCA group (CCA), were investigated to identify potential biomarkers for Ov infection and CHCA. The AKT signalling pathway was found to be up-regulated. Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (PIK3CB), an upstream signalling molecule, was selected as a potential biomarker and evaluated using indirect enzyme-linked immunosorbent assay (ELISA). Results demonstrated evidence that levels of PIK3CB in both the OV group and CCA group was statistically different compared to the non-OVCCA group (P < 0.01). However, the levels of PIK3CB between the OV group and the CCA group were found not to be statistically different. Sensitivity and specificity for OV using OD450 cut-off at >1.570 was 76 and 72%, respectively. For CCA, sensitivity and specificity using OD450 cut-off at >1.398 was 68 and 76%, respectively. Application of indirect ELISA detecting plasma PIK3CB will be of great benefit for screening of opisthorchiasis and CHCA.

Infectivity and development of Opisthorchis viverrini metacercariae in immunosuppressed Barbonymus gonionotus fingerlings (Cyprinidae).

Opisthorchiasis caused by Opisthorchis viverrini infection, is a serious public health problem in Lao PDR, Cambodia, Vietnam and Thailand. Cyprinid fish are required for the transmission of O. viverrini metacercariae to fish eating-mammal hosts including humans. The transmission of infective metacerariae is an important event, potentially resulting in the development of cholangiocarcinoma in cases of chronic opisthorchiasis. For a better understanding of the development of O. viverrini metacercariae in the second intermediate host, Barbonymus gonionotus (Cyprinidae), the immune response of infected B. gonionotus through fibrous tissue formation and superoxide dismutase (SOD) activity were investigated. The immunosuppressant (prednisolone) was administered to B. gonionotus infected with O. viverrini cercariae to study infectivity and development of the metacercariae. Additionally, the effects of prednisolone were studied by the investigation of fibrous tissue formation around the metacercariae, as well as SOD activity. In our study, prednisolone administered prior to infection in B. gonionotus had higher metacercarial recovery when compared to the non-administered control group. On the other hand, the administration of prednisolone after the O. viverrini infection allowed metacercariae to develop to the infective stage. Energy consumption from encystation to the infective stage required 443.9-463.7degree-days. The results show that immunosuppression decreases fibrous tissue and SOD activity in infected fish, and also affects the ability of O. viverrini metacercariae to develop to the infective stage. The immune response to metacercarial development of O. viverrini in cyprinid fish should be further studied to gain a greater understanding of this process, and hence, a better understanding of the mechanism(s) involved in metacercarial wall formation.

TREMATODE INFECTION OF FRESHWATER SNAIL, FAMILY BITHYNIIDAE IN THAILAND.

Opisthorchis viverrini is restricted to and requires for its aquatic life cycle only Bithynia snail as first intermediate host but many species of cyprinid fish as second intermediate hosts. A survey in Thailand of trematode infection in freshwater snails of the family Bithyniidae carried out during October 2008 - July 2009 found a total of 5,492 snails, classified into ten species distributed in various geographic areas. Bithyniafuniculata and Gabbia pygmaea were localized to the north, B. s. goniomphalos, Wattebledia siamensis and W. crosseana to northeast and B. s. siamensis, Hydrobioides nassa and G. wykoffi to central region. W. baschi and G. erawanensis was found only in the south and Erawan waterfall, Kanchanaburi Province, respectively. Trematode infection rate was 3.15%. Cercariae were identified as belonging to six types, namely, amartae , monostome, mutabile, O. viverrini, virgulate, and unknown. The prevalence of cercarial infection in B. s. goniomphalos of amartae, mutabile, O. viverrini, virgulate, and unknown type cercaria was 0.55%, 0.74%, 1.07%, 2.87%, and 0.37%, respectively, and in B. s. siamensis monostome (1.10%) and virgulate (0.55%). Only virgulate cercariae were shed from W. crosseana (3.85%) and W. siamensis (5.19%). Cercariae of the unknown type were found in G. wykoffi (1.69%). No infection of O. viverrini cercariae was detected in the other species.

Reprint of "Experimental infection of Opisthorchis viverrini cercariae to the cyprinid fish, Barbonymus gonionotus".

Opisthorchis viverrini is an important public health problem, a major cause of cholangiocarcinoma in the Greater Mekong subregion including Lao PDR, Cambodia, Vietnam and Thailand. Humans acquire the infection by consumption of raw, fermented or partially cooked freshwater cyprinid fish containing infective metacercariae. This study examined the effect of cercarial infection dosage (25-200 cercariae), age (1-60 day) and size (1-24 mm) of Barbonymus gonionotus fish on infection success of O. viverrini cercariae. Additionally, the site of cyst formation of O. viverrini cercariae and subsequent development to the metacercariae was examined. The result showed that cercarial infection dose (F4,95=8.52, P<0.001) age (P<0.001, ƛLR(2)=954.72) and size (P<0.001, ƛLR(2)=1204.84) were significantly associated with number of O. viverrini metacercariae recovery with in every additional fish age, the O. viverrini metacercarial recovery rate increased by 5.4% (P<0.001). Most metacercariae were found in the body of the fish (81.4%). We concluded that O. viverrini cercarial infection dosage, and age and size of fish were important determinants for a successful infection to fish.

Temperature dependence of Opisthorchis viverrini infection in first intermediate host snail, Bithynia siamensis goniomphalos.

Determining of the success of a parasite's infectiveness in its snail host clearly depends on environmental conditions. Temperature, one of the most influential factors impinging on metabolism of cold-blooded animals, is believed to be an important factor in parasitic infection in snails. In order to elucidate the influence of temperature, sex and size of snails on infectivity of Opisthorchis viverrini to its first intermediate host, Bithynia siamensis goniomphalos, 960 snails were divided into 2 groups by sex. Each group was subdivided by their size into small and medium sub-groups. Each snail was fed with embryonated uterine-eggs of O. viverrini at different temperatures (16-37°C, 3°C intervals). Dissections were carried out 1, 7, 14, 28 and 56 days thereafter and detection of O. viverrini infection was undertaken by PCR using specific primers. Infection was strongly temperature-dependent, as temperature increases of 1°C resulted in increased odds of infection 5.4% (P<0.01). A temperature of 34°C gave the highest rate of infection of 44.14%. We also found that the odds of infection in small sized snails was 39.8% higher relative to medium sized snails (P<0.05). Relative to day 1, the decrease in the odds of infection was detected when the day post infection was longer (P<0.01). Proportion of infection in female was not different to male significantly.

Localization of the cystogenous glands of Opisthorchis viverrini cercariae.

Opisthorchiasis in northeastern Thailand is an important etiology of cholangiocarcinoma. To form the infectious stage, free swimming cercariae penetrate cyprinid fish, shed their tails, and then secret a cystic substance to cover their larval stage to form metacercariae in the fish body. We determined the location of the cystogenous glands in Opisthorchis viverrini cercariae. The cercariae and metacercariae were obtained from the naturally infected snail host, Bithynia siamensis goniomphalos and from cyprinid fish, respectively. The cyst walls of the metacercariae were separated and used to immunize inbred male BALB/c mice to obtain cyst wall antibodies. The general characteristics of the O. viverrini cercariae and metacercariae were studied by hematoxylin and eosin (H&E) staining of sections. The location and ultrastructure of the cystogenous glands of cercariae were studied by immunoperoxidase, immunofluorescence and transmission electron microscopy. The structures and organelles of cercariae and metacercariae could be identified, but the cystogenous glands could not be detected in H&E sections. The immunoperoxidase and immunofluorescence sections revealed positive reactions for cystogenous glands predominanted in the lateral part of the cercariae and were clearly seen in the cyst wall of the metacercariae. The ultrastructure of the cystogenous glands contained semitranslucent electron dense oval shaped granules. If interference occurs during the formation of the cysts by fish immune response, the metacercariae may not develop to maturity. It may be easily digested or degraded by human stomach acid and pepsin. This may be an efficient method for control of O. viverrini infection which requires further detailed study.

Experimental infection of Opisthorchis viverrini cercariae to the cyprinid fish, Barbonymus gonionotus.

Opisthorchis viverrini is an important public health problem, a major cause of cholangiocarcinoma in the Greater Mekong subregion including Lao PDR, Cambodia, Vietnam and Thailand. Humans acquire the infection by consumption of raw, fermented or partially cooked freshwater cyprinid fish containing infective metacercariae. This study examined the effect of cercarial infection dosage (25-200 cercariae), age (1-60 day) and size (1-24mm) of Barbonymus gonionotus fish on infection success of O. viverrini cercariae. Additionally, the site of cyst formation of O. viverrini cercariae and subsequent development to the metacercariae was examined. The result showed that cercarial infection dose (F4,95=8.52, P<0.001) age (P<0.001, ƛLR(2)=954.72) and size (P<0.001, ƛLR(2)=1204.84) were significantly associated with number of O. viverrini metacercariae recovery with in every additional fish age, the O. viverrini metacercarial recovery rate increased by 5.4% (P<0.001). Most metacercariae were found in the body of the fish (81.4%). We concluded that O. viverrini cercarial infection dosage, and age and size of fish were important determinants for a successful infection to fish.

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

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

DNA barcode identification of freshwater snails in the family Bithyniidae from Thailand.

Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke (Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5' region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy.

Species-specific primers designed from RAPD products for Bithynia funiculata, the first intermediate host of liver fluke, Opisthorchis viverrini, in North Thailand.

Bithynia funiculata is the first intermediate host of the human liver fluke Opisthorchis viverrini in northern Thailand but its identification through morphological analysis is often problematic due to the shortage of gastropod taxonomists. As a consequence, we focused on the development of species-specific primers for use as an identification tool. Our work involved recovery of a 502-base pair (bp) amplicon of unknown function through species-specific primers whose effectiveness was tested by analyzing specimens of B. funiculata from 3 locations in northern Thailand. This primer set did not amplify other species in the Bithyniidae or in other gastropod families. By providing a tool to confirm morphological identifications of B. funiculata, and by enabling the identification of juvenile specimens and those with damaged shells, these primers will improve estimates of the prevalence of parasitic infections in this snail.

Effect of soil surface salt on the density and distribution of the snail Bithynia siamensis goniomphalos in northeast Thailand.

Opisthorchis viverrini infection is associated with human cholangiocarcinoma and northeast Thailand has the highest incidence of this disease in the world. Bithynia siamensis goniomphalos is the major freshwater snail intermediate host of O. viverrini in this area and an analysis based on geographical information systems was used to determine the effect of variation in soil surface salt on the density and distribution of this snail. A malacological survey was carried out in 56 water bodies in the Khorat basin, northeast Thailand at locations with various soil surface salt levels. Mollusk samples were collected from 10 ecologically representative water body sites with 10-20 sampling stations in each. The shoreline of clear, shallow water bodies was found to be the preferred B. s. goniomphalos habitat. The snails were exclusively found in water with salinity levels ranging between 0.05 and 22.11 parts per thousand (ppt), which supports the notion that B. s. goniomphalos prefers water with some saline content over pure, freshwater. The highest snail population densities were in rice fields, ponds, road-side ditches and canals within a water salinity range of 2.5-5.0 ppt. However, the presence of B. s. goniomphalos was negatively correlated with water salinity (P ≤0.05), both with regard to density and distribution. The areas with the highest density of B. s. goniomphalos were those with less than 1% soil surface salt (potential index = 0.314), while the lowest densities were found in areas exceeding 50% soil surface salt (potential index = 0.015).