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(χ^(2))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.

Opisthorchis viverrini:The carcinogenic human liver fluke

Opisthorchiasis caused by Opisthorchis viverrini remains a major public health problem in many parts of Southeast Asia, including Thailand, Lao PDR, Vietnam and Cambodia. The infection is associated with a number of hepatobiliary diseases, including cholangitis, obstructive jaundice, hepatomegaly, cholecystitis and cholelithiasis. Multi-factorial etiology of cholangiocarcinoma, mechanical damage, parasite secretions, and immunopathology may enhance cholangiocarcinogenesis. Moreover, both experimental and epidemiological evidences strongly implicate liver fluke infection as the major risk factor in cholangiocarcinoma, cancer of the bile ducts. The liver fluke infection is induced by eating raw or uncooked fish products that is the tradition and popular in the northeastern and northern region, particularly in rural areas, of Thailand. The health education programs to prevent and control opisthorchiasis are still required in the high-risk areas.

Knowledge, attitude and practice related to liver fluke infection in northeast Thailand

AIM： To investigate the knowledge, attitude and practice （KAP） in prevention and control of liver fluke infection in northeast Thailand. METHODS： A descriptive KAP survey pertaining to liver fluke infection was carried out in June 2005 to October 2006 using structured questionnaires. Data were collected by questionnaires consisting of general parameters, knowledge, attitude, practice, and a history of participation in the prevention and control of liver fluke infection. RESULTS： A total of 1077 persons who were interviewed and completed the questionnaires were enrolled in the study. The majority were females （69.5%） and many of them were 15-20 years of age （37.26%）. The questionnaires revealed that information resources on liver fluke infection included local public health volunteers （31.37%）, public health officers （18.72%）, televisions （14.38%）, local heads of sub-districts （12.31%）, doctors and nurses （9.18%）, newspaper （5.72）, intemets （5.37%）, and others （12.95%）. Fiftyfive point eleven percent of the population had a good level of liver fluke knowledge concerning the mode of disease transmission and 79.72% of the population had a good level of prevention and control knowledge with regards to defecation and consumption. The attitude and practice in liver fluke prevention and control were also at a good level with a positive awareness, participation, and satisfaction of 72.1% and 60.83% of the persons studied. However, good health behavior was found in 39.26% and 41.42% of the persons studied who had unhygienic defecation and ate raw cyprinoid＇s fish. The result also showed that 41.25% of the persons studied previously joined prevention and control campaigns. CONCLUSION： The persons studied have a high level of liver fluke knowledge and positive attitude. However, improvement is required regarding personal hygiene specifically with hygienic defecation and consumption of undercooked fish.

Amplification of chromosome 21q22.3 harboring trefoil factor family genes in liver fluke related cholangiocarcinoma is associated with poor prognosis

AIM： To determine allelic imbalance on chromosomal region 21q22-qter including trefoil factor family genes （TFF） in cholangiocarcinoma （CCA） patients and analyze the correlation between allelic imbalances and clinicopathological parameters. METHODS： Quantitative PCR amplification was performed on four microsatellite markers and trefoil factor family genes （TFF1, TFF2, and TFF3） using a standard curve and SYBR Green I dye method. The relative copy number was determined by DNA copy number of tested locus to reference locus. The relative copy number was interpreted as deletion or amplification by comparison with normal reference range. Associations between allelic imbalance and clinicopathological parameters of CCA patients were evaluated by χ^2-tests. Kaplan-Meier method was used to analyze survival. RESULTS： The frequencies of amplification at D21S1890, D21S1893, and TFF3 were 32.5%, 30.0%, and 28.7%, respectively. Patients who had amplification at regions covering D21S1893, D21S1890, and TFF showed poor prognosis, whereas patients who had deletion showed favorable prognosis （mean： 51.7 wk vs 124.82 wk, P = 0.012）. Multivariate Cox regression analysis revealed that amplification of D21S1893, D21S1890 and TFF, blood vessel invasion, and staging were associated with poor prognosis. CONCLUSION： D21S1893-D21S1890 region may harbor candidate genes especially TFF and serine protease family, which might be involved in tumor invasion and metastasis contributing to poor survival. The amplification in this region may be used as a prognostic marker in the treatment of CCA patients.

Amplification of D22S283 as a favorable prognostic indicator in liver fluke related cholangiocarcinoma

AIM： To analyze the DNA copy number of target genes NF2, TIMP3, ST13, TOB2, BIK, and TP and the reference microsatellite markers D22S283, D22S423, and D22S274 mapped on 22q12-qter in liver fluke related cholangiocarcinoma （CCA） and define its correlation with clinical parameters. METHODS： Quantitative real time PCR （qPCR） was used for determining allelic imbalances in 65 liver fluke related CCA tissues. Statistical correlations between allelic imbalances and clinicopathological parameters, i.e. age, sex, tumor stage, histological type, blood vessel invasion, nerve invasion and lymphatic invasion were evaluated by means of the X^2 test. Cox regression analysis was used for determining patient＇s survival. RESULTS： Amplifications of the TP （22q13.33）, TOB2 （22q13.2-13.31）, D22S283 （22q12.3）, TIMP3 （22q12.3） and NF2 （22q12.2） were found in 35 （53.8%）, 28 （43.1%）, 27 （41.5%）, 24 （36.9%）, and 24 （36.9%）, respectively. Losses at the D22S423 （22q13.1-13.2）and BIK （22q13.31） were detected in 26 （40%） and 23 （35.4%）, respectively. Significant correlations were observed between lymphatic invasion and allelic losses of BIK （P = 0.025） and D22S283 （P = 0.041）. Univariate and multivariate Cox regression analysis revealed D22S283 amplification as an independent predictor of good prognosis （P = 0.006, death hazard ratio = 0.411, 95% CI = 0.217-0.779） and blood vessel invasion as an independent poor prognostic factor （P = 0.042, death hazard ratio = 1.911, 95% CI = 1.022-3.571） in CCA patients. CONCLUSION： This study provides evidence for the involvement of gene amplification and deletion on chromosome 22q in liver fluke related CCA, This is the first report of D22S283 amplification as an independent indicator of favorable prognosis in liver fluke related CCA.

Immunohistochemical expression of mismatch repair genes:A screening tool for predicting mutator phenotype in liver fluke infection-associated intrahepatic cholangiocarcinoma

AIM： To clarify possible contributions of DNA mismatch repair （MMR） system in carcinogenesis of liver fluke infection-associated intrahepatic cholangiocarcinoma （ICC） by using immunohistochemical assay. METHODS： A total of 29 ICC samples, which had been assessed for genomic instability by a PCR-based method, were used for study. They were examined immunohistochemically to demonstrate protein expression of two MMR genes, hMSH2 and hMLH1. Results obtained were compared with their mutator phenotype assessed previously. RESULTS： Either hMSH2 or hMLH1 protein was obviously expressed in 28 of 29 （96.6%） ICC samples. Positive nuclear localization of hMSH2 or hMLH1 protein was observed in 86.2% （25/29） or 93.1% （27/29） ICC cases, respectively, while their negative nuclear reactivity was only detected in 13.8% （4/29） or 6.9% （2/29） ICC cases analyzed, respectively. CONCLUSION： Our study, probably for the first time, showed through immunohistochemical detection of hMSH2 and hMLH1 gene that DNA MMR system does not play a prominent role in liver fluke infection-associatedcholangiocarcinogenesis. These results confirm previous findings on mutational status of these genes assessed through a PCR-based method. The immunohistochemical analysis has proven to be an effective and sensitive approach for screening MMR deficiency regardless of somatic inactivation or promoter hypermethylation of hMSH2 and/or hMLH1 gene. Furthermore, immunohistochemistry is more advantageous compared to mutator phenotyping assay in terms of simplicity, less time consuming and cost effectiveness for screening possible involvements of target MMR genes in tumorigenesis.

Risk factors for hilar cholangiocarcinoma:A case-control study in China

AIM:To study the association between hilar cholangiocarcinoma(HC) and pre-existing medical conditions. METHODS:Three hundred and thirteen HC patients admitted to the Eastern Hepatobiliary Surgery Hospital(Shanghai,China) in 2000-2005 and 608 healthy controls were enrolled in this study.Association between HC and pre-existing medical conditions was studied with their adjusted odds ratio(OR) calculated by logistic regression analysis. RESULTS:The prevalence of choledocholithiasis(adjusted OR=2.704,P=0.039) ,hepatolithiasis(adjusted OR=3.278,P=0.018) ,cholecystolithiasis(adjusted OR =4.499,P<0.0001) ,cholecystectomy(adjusted OR =7.012,P=0.004) ,biliary ascariasis(adjusted OR= 7.188,P=0.001) ,liver fluke(adjusted OR=10.088,P =0.042) and liver schistosomiasis(adjusted OR=9.913,P=0.001) was higher in HC patients than in healthy controls. CONCLUSION:Biliary tract stone disease(choledocho-lithiasis,hepatolithiasis,cholecystolithiasis) and parasitic liver disease(biliary ascariasis,liver fluke,liver schistosomiasis) are the risk factors for HC in Chinese population.

Trefoil factors:Tumor progression markers and mitogens via EGFR/MAPK activation in cholangiocarcinoma

AIM:To investigate trefoil factor(TFF) gene copy number,mRNA and protein expression as potential biomarkers in cholangiocarcinoma(CCA).METHODS:TFF mRNA levels,gene copy number and protein expression were determined respectively by quantitative reverse transcription polymerase chain reaction(PCR),quantitative PCR and immunohistochemistry in bile duct epithelium biopsies collected from individuals with CCA,precancerous bile duct dysplasia and from disease-free controls.The functional impact of recombinant human(rh) TFF2 peptide treatment on proliferation and epidermal growth factor receptor(EGFR) /mitogenactivated protein kinase(MAPK) signaling was assessed in the CCA cell line,KMBC,by viable cell counting and immunoblotting,respectively.RESULTS:TFF1,TFF2 and TFF3 mRNA expression was significantly increased in CCA tissue compared to disease-free controls,and was unrelated to gene copy number.TFF1 immunoreactivity was strongly increased in both dysplasia and CCA,whereas TFF2 immunoreactivity was increased only in CCA compared to diseasefree controls.By contrast,TFF3 immunoreactivity was moderately decreased in dysplasia and further decreased in CCA.Kaplan-Meier analysis found no association of TFF mRNA,protein and copy number with age,gender,histological subtype,and patient survival time.Treatment of KMBC cells with rhTFF2 stimulated proliferation,triggered phosphorylation of EGFR and downstream extracellular signal related kinase(ERK),whereas co-incubation with the EGFR tyrosine kinase inhibitor,PD153035,blocked rhTFF2-dependent proliferation and EGFR/ERK responses.CONCLUSION:TFF mRNA/protein expression is indicative of CCA tumor progression,but not predictive for histological sub-type or survival time.TFF2 is mitogenic in CCA via EGFR/MAPK activation.

Prognostic significance of microsatellite alterations at 1p36 in cholangiocarcinoma

AIM： To investigate loss of heterozygosity （LOH） and microsatellite instability （MSI） on the chromosomal region 1p36-pter in cholangiocarcinoma （CCA） patients and determine the association between microsatellite alterations and clinicopathological parameters. METHODS： Ten polymorphic microsatellite markers were determined for LOH and MSI using GS-3000 gel scan fragment autoanalyzer. RESULTS： Sixty-eight out of 90 cases （75.6%） showed LOH in one or more loci. LOH was found most frequently at DIS199 （40.0%）, DIS507 （34.6%）, DIS2845 （30.5%）, and DIS2734 （30.1%）. MSI was found in 34 of 90 cases （37.8%） at one or more loci. Fine mapping at lp36 showed two distinctive regions of common loss, which were D1S2845 and the 25.5-cM region between D1S507 and D1S2734, indicating the existence of putative tumor suppressor genes that is likely to play important roles in the development of CCA. Patients with LOH at D1S234 showed less lymphatic invasion （P = 0.017）, whereas patients with LOH at D1S2676 exhibited more lymphatic invasion than those without （P = 0.031）. LOH at D1S2845 showed a significant correlation with nerve invasion （P = 0.029）. Moreover, patients who demonstrated MSI at D1S228 showed a poor prognosis （P = 0.0026）. CONCLUSION： Allelic loss plays a major role in microsatellite alterations at chromosome lp36, which may contribute to carcinogenesis and pathogenesis of liver fluke related CCA and these alterations can be used as molecular prognostic indicators for CCA patients.

Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.

Characteristics and outcomes of cholangiocarcinoma by region in Thailand: A nationwide study

AIM To identify the potential risk factors of cholangiocarcinoma, we determined the characteristics of cholangiocarcinoma patients among 5 different regions of Thailand. METHODS All patients diagnosed with cholangiocarcinoma between 2008 and 2013 were identified using the Nationwide Hospital Admission Data registry(n = 39421). Baseline characteristics, comorbidities and survival were abstracted. RESULTS The annual incidence during the study period was stable in all regions. Most patients lived in the Northeast(62.8%), followed by the North(16.9%), Central(12.3%), Bangkok(5.4%), and South(n = 2.6%) regions(P < 0.0001). Significantly more cholangiocarcinoma patients had diabetes, cirrhosis, and chronic viral hepatitis B/C infection than noncholangiocarcinoma participants(diabetes: 11.42% vs 5.28%; cirrhosis: 4.81% vs 0.92%; hepatitis B: 0.74% vs 0.12%; and hepatitis C: 0.50% vs 0.10%, P < 0.0001 for all, respectively). The overall 1-year mortality rate was 81.7%, with a stable trend over time. CONCLUSION Diabetes and chronic liver diseases may be associated with cholangiocarcinoma in the Thai population.

Examining ecosystem(dis-)services on liver fluke infection in rural Northeast Thailand

Background The direct reliance of humans on and their interactions with freshwater ecosystems in the Lower Mekong Basin have given rise to parasitic infections,which is particularly prevalent in Northeast Thailand where raw fish consumption is practiced.This study examined the interactions between environments,ecosystem(dis-)services,human raw fish consumption habits,and raw fish dish sharing on liver fluke infection risk.Method Water fecal contents and the first intermediate snail host were sampled between June and September of 2019.One hundred twenty questionnaires were surveyed in two villages of different environmental surroundings,one next to a river and the other located inland,in Northeast Thailand.Multivariate regression analyses using linear mixed effect models assessed the influence of social,behavioral and perceptual factors on raw fish consumption frequency,willingness to avoid consumption and liver fluke infection status.Social network analysis compared the degree of raw fish dish sharing between the villages and assessed the probable influence of connections to fish procurement locations and sharing activities on liver fluke infection risk.Results High abundance of the first intermediate snail host and presence of fecal contamination in water could endanger both villages to ecosystem disservices of parasitic transmission.The river-side village relied more on provisioning ecosystem services than the inland village(29.7%vs.16.1%of villages)to consume raw fish as their main source of protein.Males in both villages(64.5 and 40.4 days/year for the respective villages)are also likely to consume koi pla and pla som,higher risk fish dishes,more frequently than females(4.1 and 4.3 days/year for the respective villages).The consumption habits of both villages were driven mostly by deriving cultural ecosystem services.Participation in raw fish dish sharing activities significantly reduced the odds of an individual being willing to avoid the consumption(Odds ratio=0.19).Network analysis suggested that river-side villagers had a more direct raw fish dish sharing interaction and they procured fish from multiple locations;these characteristics might potentially account for more liver fluke infected households in the village.Conclusion Villagers’raw fish consumption is driven by deriving cultural ecosystem services,and the geographic settings of the villages potentially affect villagers’fish procurement locations and infection risk.The findings underscore the linkages between villagers and their surrounding ecosystem environments as pertinent determinants for foodborne parasitic disease risk.

Children education for sustainable control of liverfluke infections

Significant disease burden is caused by infections with human liver flukes, includingClonorchis sinensis,Opisthorchis viverrini andO. felineus. Epidemiology is characterized by high burden in the male than the female, and in the elder than the younger. Chemotherapy is the mainstream for morbidity control, but it could not prevent re-infection. Mean-while, behavioral change on the dietary habit of ingesting raw freshwater fish is challenging. In this opinion, we argue why it is important to educate children for sustainable control of liver fluke infections. Then, the design, development and key messages of an educational cartoon for preventing liver fluke infections is introduced. Technical focuses are emphasized based on a pilot using the education cartoon for preventing clonorchiasis in China. Finally, how to further develop and verify this education strategy is discussed.

Accuracy of a new rapid diagnostic test for urinary antigen detection and assessment of drug treatment in opisthorchiasis

Background Screening for opisthorchiasis,a parasitic worm infection affecting many millions of people in Southeast Asia,has traditionally relied on faecal egg examination such as the formalin-ethyl acetate concentration technique(FECT)and Kato-Katz method.Although the urinary enzyme-linked immunosorbent assay(ELISA)has been used more recently,we developed a urinary antigen-based rapid diagnostic test(RDT)to simplify diagnosis and as a point-of-care testing(POCT)and field applications for surveillance and control of opisthorchiasis.Methods A urinaryOpisthorchis viverrini(OV)-RDT was developed using immunochromatographic methodology with a specific monoclonal antibody against OV.The diagnostic performance of the urinary OV-RDT was compared to that of quantitative faecal FECT and urinary antigen ELISA(n=493).Cross-reactivities of urinary OV-RDT with other helminthiases coexisted withO.viverrini were determined(n=96).A field trial in the application of urinary OV-RDT was compared with urinary antigen ELISA at baseline screening and assessment of drug treatment outcomes in opisthorchiasis(n=1629).The McNemar chi-square,Kruskal-Wallis and Cohen’s kappa coefficient(κ-value)tests were used for statistical analyses.Results Urinary OV-RDT had sensitivity of 94.2%and specificity of 93.2%,compared to faecal FECT.Urinary OV-RDT had high diagnostic agreement(Kappa=0.842-0.874,P<0.001)and quantitative correlation with urinary antigen ELISA(Kruskal-Wallis tests=316.2,P<0.0001)and faecal FECT(Kruskal-Wallis tests=362.3,P<0.0001).The positive rates by OV-RDT,ELISA and FECT were 48.9%,52.5%and 49.3%,respectively.Cross-reactions of urinary OV-RDT with other helminthiases were few(2%).Field trials of urinary OV-RDT yielded comparable prevalence ofO.viverrini between urinary OV-RDT(53.2%)and urinary antigen ELISA(54.0%).OV screening showed high diagnostic agreement(kappa>0.8,P<0.0001)between urinary OV-RDT and urinary antigen ELISA.The cure rates of opisthorchiasis at 1 month post-praziquantel treatment determined by urinary OV-RDT(86.6%)and urinary antigen ELISA(80.5%)were similar(P>0.05).Conclusions The urinary OV-RDT test has high potential as a new tool for screening and evaluating treatment outcomes in opisthorchiasis.The ease of sample collection and simplicity of urinary OV-RDT may facilitate mass screening,control and elimination of opisthorchiasis,thereby contributing to a reduction in the disease burden in Southeast Asia.