Impact of Wortmannilactone F and G31P on Clonorchis Sinensis-infected mice.

Clonorchis sinensis could induce inflammation, epithelial hyperplasia and fibrosis in the intrahepatic bile duct as a food-borne parasite, which was associated with the development of cholangiocarcinoma (CCA). Praziquantel was the most effective drug on treatment of this kind of parasite. However, new drugs with minimal toxicity to the host were urgently needed due to the side effects of Praziquantel and its CCA risk. In this study, helminth mitochondria respiratory chain blocker Wortmannilatone F (WF) and IL-8 analogue CXCL8 (3-72) K11R/G31P were used to treat BALB/C mice infected by Clonorchis sinensis. We investigated the gross and histopathological morphology of the liver, inflammation-associated cytokine IL-6, lipid peroxidation-related proteins cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), collagen fiber accumulation and fibroblast-specific protein 1 (FSP1), malignant markers proliferating cell nuclear antigen (PCNA) and cytokeratin 19 (CK19), as well as the disinfection effect on these parasites in vitro. WF inhibited and killed the worms dramatically, and the combination of WF with G31P improved the condition of the hepatobiliary duct tissue greatly. These outcomes indicated that the combination of WF and G31P was a potential therapeutic method to treat the Clonorchis sinensis infection.

Dopaminergic antagonists inhibit bile chemotaxis of adult Clonorchis sinensis and its egg production.

Human clonorchiasis, caused by Clonorchis sinensis, is endemic in East Asian countries. C. sinensis metacercariae excyst in the duodenum of mammalian hosts, migrate to the intrahepatic bile duct, and mature into adults in the milieu of bile. We have previously shown that newly excysted juvenile C. sinensis move chemotactically toward bile and bile acids. Here, the chemotactic behavior of adult C. sinensis (CsAd) toward bile and bile acids was investigated. CsAds moved toward 0.05-5% bile and were most attracted to 0.5% bile but moved away from 10% bile. Upon exposure to 1-10% bile, CsAds eventually stopped moving and then died quickly. Among bile acids, CsAds showed strong chemotaxis toward cholic acid (CA) and deoxycholic acid. On the contrary, CsAds repelled from lithocholic acid (LCA). Moreover, at higher than 10 mM LCA, CsAds became sluggish and eventually died. Dopamine D1 receptor antagonists (LE-300 and SKF-83566), D2/3 receptor antagonists (raclopride and its derivative CS-49612), and a dopamine re-uptake inhibitor inhibited CA-induced chemotaxis of CsAds almost completely. Clinically used antipsychotic drugs, namely chlorpromazine, haloperidol, and clozapine, are dopaminergic antagonists and are secreted into bile. They completely inhibited chemotaxis of CsAds toward CA. At the maximum doses used to treat patients, the three tested medicines only expelled 2-12% of CsAds from the experimentally infected rabbits, but reduced egg production by 64-79%. Thus, antipsychotic medicines with dopaminergic antagonism could be considered as new anthelmintic candidates for human C. sinensis infections.

Development and application of anthelminthic drugs in China.

China was once a country plagued by parasitic diseases. At the beginning of the founding of the People's Republic of China, nearly 80% of the population suffered from parasitic diseases because of poverty and poor sanitary conditions. After nearly 70 years of development, China has made remarkable achievements in the prevention and control of parasitic diseases, and the prevalence of parasitic diseases has been greatly reduced. In addition to organizational leadership from the government and various preventive measures, drug treatment and drug research & development are important and irreplaceable links in prevention and control work. Since the 1950s, China has begun to introduce, produce and imitate antiparasitic drugs from abroad, such as santonin, benzimidazole, and praziquantel. Chinese scientists have also contributed to the optimization of production techniques, improvements in drug formulation, the application in the clinic and the mechanisms of actions of generic drugs. At the same time, China has independently developed tribendimidine (TrBD, a broad spectrum anthelminthic), and its anthelminthic spectrum has been comprehensively studied. It is active against almost 20 parasites, is especially superior to benzimidazoles against Necator americanus, and surpasses the effectiveness of praziquantel against Clonorchis sinensis. In the treatment of tapeworm disease, the traditional Chinese medicines pumpkin seeds and betel nuts have good curative effects for taeniasis. Chinese scientists have explored the action modes and clinical administration methods of pumpkin seeds and betel nuts, which is still the main clinical regimen for the disease. This paper reviews the history and progress of the study of anthelmintics in intestinal helminth infections since the founding of the People's Republic of China and aiming to support clinicians and drug researchers in China and other countries.

Chemotactic migration of newly excysted juvenile Clonorchis sinensis is suppressed by neuro-antagonists.

The metacercariae of the Clonorchis sinensis liver fluke excyst in the duodenum of mammalian hosts, and the newly excysted juveniles (CsNEJs) migrate along the bile duct via bile chemotaxis. Cholic acid is a major component of bile that induces this migration. We investigated the neuronal control of chemotactic behavior of CsNEJs toward cholic acid. The migration of CsNEJs was strongly inhibited at sub-micromolar concentration by dopamine D1 (LE-300 and SKF-83566), D2 (spiramide, nemonapride, and sulpiride), and D3 (GR-103691 and NGB-2904) receptor antagonists, as well as a dopamine reuptake inhibitor (BTCP). Neuropeptides, FMRFamide, peptide YY, and neuropeptide Y were also potent inhibitors of chemotaxis. Meanwhile, serotonergic, glutamatergic, and cholinergic inhibitors did not affect chemotaxis, with the exception of fluoxetine and CNQX. Confocal immunofluorescence analysis indicated that dopaminergic and cholinergic neurons were colocalized in the somatic muscle tissues of adult C. sinensis. Our findings suggest that dopaminergic neurons and neuropeptides play a major role in the chemotactic migration of CsNEJs to bile, and their inhibitors or modulators could be utilized to prevent their migration from the bile duct.

A Clonorchiasis Case of a Leopard Cat, Prionailurus bengalensis euptilurus, Diagnosed by Ultrasonography and Egg Detection in Republic of Korea.

A clonorchiasis case in a captive leopard cat, Prionailurus bengalensis euptilurus, was confirmed by ultrasonographic findings and egg morphologies found in the bile juice sample in the Korea. The leopard cat was introduced from the wild habitat of Gyeongsangnam-do, to Cheongju Zoo in Cheongju-si, Chungcheongbuk-do, Korea in August 2014. Physical examinations were basically performed for quarantine and check-up health. The cat was comparatively good in health except anorexia. The cyst-like bile duct dilation and the increased echogenicity of gall bladder wall and hepatic parenchyma were observed by ultrasonography. Ultrasound-guided needle biopsy was conducted for collecting bile juice and the specimens were observed under light microscope. The numerous small trematode eggs were detected in the bile juice sample of the light microscopy. The eggs were 25-33 (28±3) µm by 18-22 (20±1) µm in size and showed typical characteristics of Clonorchis sinensis egg, i.e., a dominantly developed operculum, shoulder rim and dust-like wrinkles in surface. To treat the liver fluke infection, 20 mg/kg of praziquantel was orally administered only once to the case. Follow-up studies including fecal examinations were conducted during 2 years after treatment. But no more eggs were detected from the case. In the present study, we described the first clonorchiasis case of leopard cat, which was confirmed by ultrasonographic findings and egg morphologies from the bile juice sample in Korea.

Bile acids drive chemotaxis of Clonorchis sinensis juveniles to the bile duct.

Clonorchiasis is a neglected tropical disease caused by Chinese liver fluke, Clonorchis sinensis infection. C. sinensis is a biological carcinogen causing cholangiocarcinoma in humans. In the mammalian host, C. sinensis newly excysted juveniles (CsNEJs) migrate from the duodenum into the bile duct. Bile drives the chemotactic behavior of CsNEJs. Little is known about which components of bile induce the chemotaxis. We designed a chemotaxis assay panel and measured the chemotactic behavior of CsNEJs in response to bile or bile acids. The CsNEJs migrated toward 0.1-1% bile but away from 5-10% bile. The CsNEJs showed strong chemoattraction to cholic acid ≥25 mM, but chemorepulsion to lithocholic acid ≥0.25 mM. To the CsNEJs, mixture of cholic acid and lithocholic acid was chemoattractive at a ratio greater than 25:1 but chemorepulsive at one smaller than that. Regarding migration in the mammalian hosts, high concentration of lithocholic acid in the gallbladder bile may repel CsNEJs from entering it. However, bile in the hepatic bile duct has a chemoattractive strength of cholic acid but a trace amount of lithocholic acid. Collectively, our results explain why the CsNEJs migrate principally to the hepatic bile ducts, bypassing the gallbladder.

The role of evolutionary biology in research and control of liver flukes in Southeast Asia.

Stimulated largely by the availability of new technology, biomedical research at the molecular-level and chemical-based control approaches arguably dominate the field of infectious diseases. Along with this, the proximate view of disease etiology predominates to the exclusion of the ultimate, evolutionary biology-based, causation perspective. Yet, historically and up to today, research in evolutionary biology has provided much of the foundation for understanding the mechanisms underlying disease transmission dynamics, virulence, and the design of effective integrated control strategies. Here we review the state of knowledge regarding the biology of Asian liver Fluke-host relationship, parasitology, phylodynamics, drug-based interventions and liver Fluke-related cancer etiology from an evolutionary biology perspective. We consider how evolutionary principles, mechanisms and research methods could help refine our understanding of clinical disease associated with infection by Liver Flukes as well as their transmission dynamics. We identify a series of questions for an evolutionary biology research agenda for the liver Fluke that should contribute to an increased understanding of liver Fluke-associated diseases. Finally, we describe an integrative evolutionary medicine approach to liver Fluke prevention and control highlighting the need to better contextualize interventions within a broader human health and sustainable development framework.

Phylogenetic characterization of Clonorchis sinensis proteins homologous to the sigma-class glutathione transferase and their differential expression profiles.

Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system.

[Research on chemotherapy measures in different Clonorchis sinensis infectiosity endemic areas].

OBJECTIVE: To explore the suitable chemotherapy measures in different Clonorchis sinensis infectiosity endemic areas. METHODS: The interventional groups and control groups were set up in the heavy, moderate and light C. sinensis infectiosity areas in Guangdong Province, respectively. In the intervention groups, the chemotherapy was administered among all the residents aged above 3 years in the heavy endemic areas, the chemotherapy was given among the focus populations in the moderate endemic areas, and the chemotherapy was administered among the residents infected with C. sinensis confirmed by stool examinations in the light endemic areas. No measures were carried out in the control groups. RESULTS: One year after the interventions, the C. sinensis infection rates of the interventional groups decreased by 47.90%, 86.52% and 100%, the abnormal rates of liver B ultrasonic examinations decreased by 26.50%, 31.00% and 100%, the ALT abnormal rates decreased by 48.70%, 62.30% and 100%, and the AST abnormal rates decreased by 55.50%, 39.90% and 100% in the heavy, moderate and light endemic areas, respectively. There was no decreasing trend of above-mentioned indexes in the control groups. CONCLUSION: In the different C. sinensis infectiosity endemic areas, the different chemotherapy measures should be carried out.

[Current treatments for clonorchiasis].

The paper briefly reviews the current techniques for treatment for human clonorchiasis, which are both widely used in medical practice and developed at experimental research laboratories. It describes specific examples of chemotherapy, including combined therapy, clonorchiasis vaccines and drug resistance. Particular emphasis is placed on the prospects of use of minor interfering RNA as a source of new-generation diagnostic and remedial agents.

RNAi-mediated silencing of enolase confirms its biological importance in Clonorchis sinensis.

Clonorchis sinensis (C. sinensis) infection is still a common public health problem in freshwater fish consumption areas in Asian countries. More molecular evidence are required to speed up the prevention strategies to control this kind of infectious disease. In the present study, to confirm the biological importance of Csenolase followed by our previous observations of the key metabolic enzyme, we explored the RNA silence effect of the Csenolase-derived RNA interference (RNAi) in C. sinensis. The extramembranous region aa105-226 was selected as the target sequence of RNA silence. Csenolase-derived double strand RNA (dsRNA-Csenolase, 366 bp) was synthetized and delivered into C. sinensis by soaking approach. The penetration of dsRNA into adult worms and metacercariae was tracked using fluorescently labeled RNA. Western blotting and qRT-PCR experiments were performed to determine dsRNA-Csenolase-silencing effect. Our results showed that, after incubating for 120 h, dsRNA-Csenolase could effectively target and downregulate the expression of Csenolase in both adult worms (P < 0.001) and metacercariae (P < 0.01), resulting in a remarkable killing effect on C. sinensis adult worms (P < 0.01). Fluorescent Cy3-labeled dsRNA was mostly deposited in the uterus and vitellarium of adult worm and in the cyst wall of metacercaria. The present study is the first report of RNAi trials in C. sinensis, allowing further applications in identifying functional genes in C. sinensis.

Differential activation of diverse glutathione transferases of Clonorchis sinensis in response to the host bile and oxidative stressors.

BACKGROUND: Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. METHODOLOGY/PRINCIPAL FINDINGS: We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa µ-class GST (Cs26µGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses. CONCLUSIONS/SIGNIFICANCE: Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26µGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions.

Efficacy and safety of tribendimidine against Clonorchis sinensis.

BACKGROUND: Clonorchiasis is of considerable public health importance, particularly in the People's Republic of China (PR China), where most of the 15 million individuals infected with Clonorchis sinensis are currently concentrated. Praziquantel is the drug of choice, but tribendimidine might be an alternative. METHODS: We performed a randomized open-label trial in Guangxi, PR China, to assess the efficacy and safety of 400 mg tribendimidine once, 400 mg tribendimidine daily for 3 days, and 75 mg/kg praziquantel in 1 day divided in 3 doses against parasitological-confirmed C. sinensis infections. Cure and egg reduction rates were determined 3 weeks posttreatment using available case analysis. Clinical symptoms were documented at baseline, and adverse events were recorded and graded 3 and 24 hours after each dose. RESULTS: A total of 74 patients were included in the final analysis. Single-dose tribendimidine achieved a cure rate of 44%, whereas cure rates of 58% and 56% were obtained for tribendimidine administered for 3 days and praziquantel, respectively. High egg reduction rates (97.6%-98.8%) were observed for all treatment regimens. Single-dose tribendimidine was the best-tolerated treatment scheme. Patients treated with praziquantel experienced significantly more adverse events than did tribendimidine recipients (P < .05). CONCLUSIONS: Tribendimidine has an efficacy comparable to praziquantel in the treatment of C. sinensis infection and resulted in fewer adverse events compared to praziquantel. Larger clinical trials are warranted among C. sinensis-infected patients to determine the potential of tribendimidine against clonorchiasis and other helminthiases. Clinical Trials Registration.Controlled-Trials.com, ISRCTN80829842.

[In vitro effect of seven anthelmintic agents against adult Clonorchis sinensis].

OBJECTIVE: To observe the in vitro effect of praziquantel, tribendimidine, levamisole, artemether, artesunate, albendazole and mebendazole against adult Clonorchis sinensis. METHODS: Seventy rats infected with 50-100 C. sinensis metacercariae for 5-7 weeks were euthanized, and adult C. sinensis were collected from the common bile duct Three to four worms were placed in each well of a 24-well falcon plate, and treated by Hanks' balanced salt solution-20% calf serum containing aforementioned drugs at various concentrations. The motor activity and morphology change of the worms were observed under an inverted microscope at 4, 24, 48 and 72 h post treatment. RESULTS: Praziquantel could reduce the motor activity of the worms rapidly which resulted in detachment of oral sucker from the well wall, curl of the worm body and emergence of vacuoles from the tegument. The minimal concentration of praziquantel to kill adult C. sinensis was 0.1 g/ml. After adult C. sinensis exposed to tribendimidine at concentrations of 0.5, 1 and 10 g/ml, they revealed in paralysis, looseness and stretch of the worm body rapidly or immediately. The minimal concentration of tribendimidine to kill adult worms was 0.05 g/ml. When worms exposed to levamisole at 10 and 20 g/ml, there was a gradual decrease in the worm's motor activity accompanied by looseness of the worm body. But 48 h post exposure, most worms showed apparently recovery of motor activity. In a higher levamisole concentration of 50 g/ml, all worms revealed in stretch and paralysis which was similar to that induced by tribendimidine. When adult C. sinensis were exposed to artemether or artesunate 10 and 50 g/ml, the motor activity of worm body and oral sucker reduced which accompanied by worm contraction, then followed by looseness of the worm body and emergence of vacuoles along the tegument. At 72h post exposure, the worm mortalities induced by the two concentrations of the two drugs were about half, respectively. In adult C. sinensis exposed to albendazole and mebendazole at concentrations of 10 and 50 g/ml, only stimulation of motor activity of oral sucker was seen which revealed in vigorous contraction within 24 h post exposure. During 72 h observation period, no any other changes in worm activity and morphology were seen. CONCLUSION: Praziquantel and tribendimidine exhibit strong in vitro killing effect on adult C. sinensis. The minimal concentration of levamisole used to kill adult worm is 50 times higher than that of tribendimidine. The higher concentrations of artemether and artesunate show slower action to reduce the worm activity and kill part of the worms. Higher concentrations of albendazole and mebendazole exhibit no killing effect on C. sinensis, besides stimulating the motor activity of worm oral sucker.

Obstructive jaundice caused by Clonorchiasis-associated duodenal papillitis: a case report.

We describe an unusual presentation of Clonorchis sinensis infection with obstructive jaundice due to duodenal papillitis which was relieved dramatically by endoscopic sphincterotomy. A 26-yr-old male presented with complaints of fatigue, weight loss and painless jaundice. The history was significant for frequent ingestion of raw freshwater fish. The patient underwent endoscopic retrograde cholangiopancreatography for evaluation of obstructive jaundice. The duodenal papilla was markedly edematous with a bulging configuration and hyperemic changes at the orifice. Cholangiography revealed mild bile duct dilatation and irregular wall changes with multiple indentations. However, there were no biliary stricture or stones noted as the cause of obstructive jaundice. We performed an endoscopic sphincterotomy for effective bile drainage through the duodenal papilla. After the sphincterotomy, the patient's jaundice was dramatically improved. Pathology of the duodenal papilla showed eosinophilic infiltration of the mucosa. Parasitic eggs, consistent with the diagnosis of C. sinensis, were found in the bile sample.

Comparative effect of mebendazole, albendazole, tribendimidine, and praziquantel in treatment of rats infected with Clonorchis sinensis.

The aim of the study is to understand the anti-Clonorchis sinensis properties of mebendazole and albendazole, and compare to praziquantel and tribendimidine. Two hundred and thirty rats were divided into five batches for experimental treatment. In four batches, each rat was infected orally with 50 or 100 C. sinensis metacercariae. Twenty-eight to 46 days post-infection, groups of rats were treated orally with single doses of mebendazole, albendazole, praziquantel, tribendimidine, or multiple daily doses of albendazole. While in the remaining batch, mebendazole or praziquantel was administered to groups of rats infected each with 50 metacercariae for 7 or 14 days. In each batch of test, untreated but infected rats served as control. All rats were euthanized 2-4 weeks post-drug administration for assessment of efficacy. In the first batch of test, rats treated with mebendazole or tribendimidine at single doses of 150, 75, and 37.5 mg/kg resulted in worm burden reductions of 99.0%, 94.0%, and 73.1%, or 98.0%, 80.6%, and 60.4%, respectively. When rats were treated with albendazole at the same dose levels, no or poor effect was seen. In the second batch of test, promising effect against adult C. sinensis in rats treated with mebendazole or tribendimidine at single doses of 100 and 50 mg/kg were also observed, but under the single dose of 25 mg/kg, only tribendimidine still remained the effect. In the third batch of test, the aforementioned three single dose levels of mebendazole, albendazole and praziquantel were applied. Again, mebendazole exhibited higher effect and albendazole exhibited no or poor effect. While praziquantel, administered at a higher dose of 300 mg/kg, also showed promising effect. In the fourth batch of test, oral administration of albendazole at a daily dose of 150 or 100 mg/kg for 2 or 3 days resulted in moderate or higher efficacy with worm burden reductions of 79.2% and 91.9%, respectively. In the fifth batch of test, single mebendazole doses of 150 or 75 mg/kg exhibited promising effect against 14-day-old C. sinensis in rats with worm burden reductions of 95.3% and 86.4%, respectively, but mebendazole was short of the effect against 7-day-old worms. Under the same dose level, praziquantel possessed an effect against both 7- and 14-day-old juvenile C. sinensis. The results confirm that in infected rats, mebendazole administered orally at a single dose of 150 mg/kg exhibits potential effect against juvenile (14-day-old) and adult C. sinensis. No or less effect is obtained from albendazole under the same dose levels, but extension of treatment course may enhance the effect of albendazole against this species of fluke. The single effective dose ranges of mebendazole and tribendimidine against C. sinensis in rats are similar with a broad window, while the window for praziquantel is narrow.

Effectiveness of mefloquine against Clonorchis sinensis in rats and Paragonimus westermani in dogs.

The aim of the study is to explore the effect of mefloquine against Clonorchis sinensis and Paragonimus westermani. For anti-C. sinensis study, a total of 71 rats were divided into four batches for oral infection of each rat with 50 C. sinensis metacercariae. Five to 7 weeks post-infection, groups of rats were treated orally with mefloquine at single doses or multiple daily doses while infected, but untreated rats served as control. All treated rats were euthanized 2 weeks post-treatment for assessment of efficacy. For anti-P. westermani study, two batches of eight and ten dogs were each infected intraperitoneally with 100 P. westermani metacercariae. Eighty-five to 96 days post-infection, groups of two or three dogs were treated orally with mefloquine and groups of two dogs were treated with praziquantel at a single dose or multiple doses. In each batch of test, three untreated but infected dogs served as control. All treated dogs were euthanized 26-30 days post-treatment for evaluation of efficacy. In rats infected with C. sinensis and treated orally with mefloquine at a single dose of 75 and 150 mg/kg, no effect against C. sinensis was observed. When the dose of mefloquine was increased to 250 mg/kg, one third (five out of 15) rats died 3-5 days post-treatment. Although the mean worm burden was lower than that of the control, the difference between the treated and control groups was not statistically significant (P>0.05) with worm burden reduction of 22.4%. Whereas, the group of infected rats received mefloquine at a daily dose of 100 mg/kg for 3 days, one out of five rats died after the last administration. The mean worm burden was significantly lower than that of the control with worm burden reduction of 67.6% (P<0.01). In the first test of mefloquine against P. westermani, three infected dogs received two oral doses of the drug, 50 mg/kg, given at a 4-h interval, the mean worm burden were similar to that of the control. While other two dogs were treated with praziquantel at the same dose schedule, the worm burden reduction of 78% was observed. In the second test, three and two dogs were treated with mefloquine 50 mg/kg daily for 5 days or 100 mg/kg daily for 2 days; the mean worm burdens of the two groups were lower than that of the control with worm burden reduction of 65.6% and 51.9%, respectively. However, only the difference of mean worm burdens between mefloquine 50 mg/kg given daily for 5 days and the control was statistically significant (P<0.05). Other two dogs treated with praziquantel at a single dose of 100 mg/kg were cured. The results indicate that under the appropriate dose schedule mefloquine exhibits less effect against C. sinensis in rats and P. westermani in dogs.

Effect of artemether, artesunate, OZ78, praziquantel, and tribendimidine alone or in combination chemotherapy on the tegument of Clonorchis sinensis.

We investigated the morphological effects of half-strength treatments with praziquantel, artemether, artesunate, OZ78 and tribendimidine as well as combinations of praziquantel with artemether, artesunate, OZ78 and tribendimidine and an artesunate-tribendimidine combination in rats harboring adult Clonorchis sinensis. Rats were infected with C. sinensis, dosed orally with single agents or combination treatments and flukes recovered at 3 or 5 days post-treatment. The number of flukes was counted, the viability recorded and surface changes monitored by scanning electron microscopy. Drug effects induced by the individual drugs at sub-curative doses 3 days post-treatment were minor with the exception of flukes recovered from rats treated with artemether and tribendimidine. Treatment with the praziquantel combinations of artesunate, OZ78 and tribendimidine did not produce a greater disruption of the tegument than the individual drugs 3 days post-treatment. On the other hand, at this time point many worms treated with artemether-praziquantel had died and eruptions, roughening or blebbing were observed on all worms examined. Five days post-treatment flukes exposed to any of the praziquantel combinations in rats had died. Rats treated with an artesunate-tribendimidine combination resulted in a rapid death of flukes, 3 days post-treatment all worms had been expelled. In conclusion, we have confirmed the promising clonorchicidal properties of different drug combinations in rats. Differences in the extent and time-scale of tegumental disruption have been observed. The effect of drug combinations against C. sinensis requires further scientific inquiry, e.g. in transmission electron microscopy studies and in the C. sinensis-rabbit model.

[Therapeutic effect of tribendimidine, artesunate and praziquantel administered to hamsters infected with Clonorchis sinensis].

OBJECTIVE: To observe the effect of tribendimidine, artesunate and praziquantel in treatment of hamsters (Mesocricetus auratus) infected with Clonorchis sinensis. METHODS: A total of 93 hamsters, each infected with 30 C. sinensis metacercariae, were treated intragastrically with above-mentioned drugs at a single dose. (1) In order to observe the effect of the drugs against juvenile C. sinensis, 20 out of 31 infected hamsters were randomly divided into 4 groups (5 hamsters per group) 14 d post-infection: artesunate 300 mg/kg, tribendimidine 100 mg/kg or 200 mg/kg, and praziquantel 200 mg/kg. Other 6 hamsters were divided equally into 2 groups 24 d post-infection and treated with tribendimidine 200 mg/kg and artesunate 300 mg/kg, respectively. The remained 5 untreated hamsters served as control. (2) Twenty-two hamsters were randomly divided into 5 groups (4-5 hamsters per group) 28 d post-infection and treated with tribendimidine 25 mg/kg or 50 mg/kg, artesunate 25 mg/kg and praziquantel 50 mg/kg, respectively. Other untreated hamsters served as control. (3) Forty hamsters 28 d after infection were randomly divided into 8 groups (4-6 hamsters per group) and treated with tribendimidine 50 mg/kg, 100 mg/kg or 200 mg/kg, artesunate 100 mg/kg or 200 mg/kg, praziquantel 100mg/kg or 200mg/kg, respectively. The remained hamsters served as control. All hamsters were sacrificed 14 d post-treatment and worms were recovered from the bile duct and liver tissue. The mean worm burden and its reduction were calculated. The differences of mean worm burden between each treated group and the corresponding control were analyzed statistically. RESULTS: In hamsters infected with 14-d-old C. sinensis and treated orally with tribendimidine at a single dose of 100 or 200 mg/kg, the mean worm burdens were significantly lower than that of the control (P<0.01) with a worm reduction of 90.6% and 85.9% respectively. The mean worm burden obtained from the infected hamsters treated with praziquantel at a single dose of 200 mg/kg was also significantly lower than that of the control (P<0.05) with a worm reduction of 71.9%. However, the difference of mean worm burden between artesunate and control groups was not statistically significant. The juvenile parasites developed into adult worms 24 d after infection. By administering tribendimidine 200 mg/kg to the adult C. sinensis-infected hamsters, the mean worm burden was significantly lower than that of the control (P<0.01) with a worm reduction of 89.8%. Whilst the administration of artesunate at a higher dose of 300 mg/kg, all hamsters were cured. Further tests indicated that tribendimidine in a lower dose of 25 mg/kg to the hamsters 28 d after infection resulted in a significantly lower mean worm burden compared to the control (P<0.05) with a worm reduction of 71.8%. With an increased dose of tribendimidine 100 mg/kg, all hamsters were cured. The worm reduction was only 20.0% and 56.4% when 25 mg/kg and 100 mg/kg of artesunate were administered. With 200 mg/kg artesunate, the worm reduction reached as high as 98.5% and the mean worm burden was significantly lower than that of the control (P<0.01). Furthermore, administration of praziquantel at a dose of 100 mg/kg or 200 mg/kg at 28 d post-infection resulted in a significantly lower mean worm burden than that of the control (P<0.05) with a worm reduction of 78.9% and 83.5% respectively. CONCLUSION: In hamster model, tribendimidine and praziquantel exhibit promising effect against both juvenile and adult C. sinensis, while artesunate is only efficacious against adult worms.

Efficacy of artesunate and artemether against Clonorchis sinensis in rabbits.

Artesunate and artemether display promising clonorchicidal properties in rats. In this study, we assessed the efficacy of artesunate and artemether against Clonorchis sinensis in rabbits. Rabbits were each fed with 300 C. sinensis metacercariae. At day 28 postinfection, the rabbits were administered oral artesunate at doses of 7.5-120 mg/kg and oral artemether of 15-120 mg/kg. Two groups of rabbits were treated with single oral praziquantel at 75 and 150 mg/kg. Untreated rabbits served as controls. Fourteen days after treatment, all rabbits were sacrificed, and C. sinensis adults were collected from the bile ducts and counted. At the highest doses tested (120 mg/kg) artesunate and artemether achieved statistical significant worm burden reductions of 88.8% and 67.2%, respectively. These rates were lower than worm burden reductions observed with praziquantel (88% and 100%, respectively). It is suggested that artesunate and artemether have moderate anthelminthic efficacy against C. sinensis in rabbits.