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Triclabendazole (TCBZ) is the only chemical that kills early immature and adult Fasciola hepatica (liver fluke) but widespread resistance to the drug greatly compromises fluke control in livestock and humans. The mode of action of TCBZ and mechanism(s) underlying parasite resistance to the drug are not known. Due to the high prevalence of TCBZ resistance (TCBZ-R), effective management of drug resistance is now critical for sustainable livestock production. Here, we discuss the current status of TCBZ-R in F. hepatica, the global distribution of resistance observed in livestock, the possible mechanism(s) of drug action, the proposed mechanisms and genetic basis of resistance, and the prospects for future control of liver fluke infections using an integrated parasite management (IPM) approach., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Triclabendazole is reported to be highly effective in treatment of human fascioliasis. We present 7 of 19 selected cases of human fascioliasis referred to our center in the Cusco region of Peru that failed to respond to triclabendazole. These were mostly symptomatic adults of both sexes that continued passing Fasciola eggs in the stool despite multiple treatments with 2 doses of triclabendazole at 10 mg/kg per dose. We documented the presence of eggs by rapid sedimentation and Kato Katz tests after each treatment course. We found that repeated triclabendazole courses were not effective against fascioliasis in this group of people. These findings suggest that resistance to triclabendazole may be an emerging problem in the Andes.

Fasciolosis is a zoonosis affecting ruminants, caused by the liver flukes Fasciola (F.) hepatica, and F. gigantica, which infect at least 2.4 million people worldwide. This disease may occur in cluster or family infections or after travel in high-risk areas such as the Nile Delta in Egypt, Iran, Turkey, South-East Asia, Mexico, the Caribbean and the Andean Altiplano. In Europe, fasciolosis occurs more frequently in Portugal, Spain and France, although autochthonous infections have also been reported from Ireland and Germany. Infectious metacercariae are ingested with contaminated water or raw or undercooked vegetables. During their larval stage immature flukes migrate through the liver producing an acute febrile syndrome some weeks after infection, followed by a chronic-latent stage which may last for years or decades. Acute fasciolosis is characterized by fever, high eosinophilia and hepatosplenomegaly. At this stage ova are usually not yet produced. Diagnosis relies on the detection of specific antibodies and/or antigens in serum. Typical imaging features include multiple, ill-defined, fleeting hypodense or hypoechoic areas in the liver. Intraabdominal bleeding due to fluke's penetration of the bowel wall or liver capsule, may occur. In chronic latent fasciolosis the diagnosis is achieved by specific serology tests, detection of eggs in bile and parasitological examinations of multiple enriched stools samples. Ultrasonography may sometimes reveal a dilated and thickened common bile duct or crescent-like parasites in the gallbladder or bile ducts. Some patients exhibit sludge which typically does not sediment. Triclabendazole at a single dose of 10  mg/kg body weight is the treatment of choice., (© Georg Thieme Verlag KG Stuttgart · New York.)

Fasciolosis caused by Fasciola spp. is considered the most important helminth infection of ruminants in tropical countries. Anthelmintic resistance has become a global concern. This study compared the efficacy of the commonly used anthelmintics, determined the toxicity level and any indication of resistance. Thirty two water buffaloes naturally-infected with Fasciola spp. were used to determine the efficacy of triclabendazole (TBZ), albendazole (ABZ), and bromofenofos (BRO) using Fecal Egg Count Reduction Test (FECRT). To test the toxicity of the drugs given, serum glutamic-pyruvic transaminase (SGPT) was evaluated before and within one week after treatment. One dose administration of ABZ registered an efficacy of 79.17%, 73.33% for TBZ and 70.83% for BRO. Efficacy in two dose- treatment group was 83.33% for both BRO and ABZ, and 90.00% for TBZ. Two dose-treatment was effective for TBZ (90%), ineffective for BRO and ABZ. SGPT levels were not significantly different between pre-treatment and post- treatment across all treatments. Giving one or two doses of anthelmintics, at one month interval, does not increase the efficacy of the three drugs tested. The study also implies that anthelmintic resistance may have developed in the animals.

This study describes the synthesis of compound (7), a highly hydrosoluble phosphonooxymethyl prodrug of compound alpha (4). Compound (7) improved the aqueous solubility of its precursor compound (4) by 50,000 times and it is stable at neutral pH. The prodrug showed faciolicidal activity when evaluated in vitro against excysted Fasciola hepatica metacercariae. The in vivo evaluation of (7) was carried out via oral, intramuscular and subcutaneous administration in sheep artificially infected with F. hepatica metacercariae. At an intramuscular dose of 4 mg/kg, the activity of (7) was similar to that of compound alpha (4) at an oral dose of 15 mg/kg., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Triclabendazole (TCBZ) is widely used for control of Fasciola hepatica (liver fluke) in animals and humans and resistance to this drug is now widespread. However, the mechanism of resistance to TCBZ is not known. A T687G single nucleotide polymorphism (SNP) in a P-glycoprotein gene was proposed as a molecular marker for TCBZ resistance in F. hepatica (Wilkinson et al., 2012). We analyzed this Pgp gene from TCBZ-susceptible and TCBZ-resistant populations from Australia to determine if the SNP was a marker for TCBZ resistance. From the 21 parasites studied we observed 27 individual haplotypes in the Pgp sequences which comprised seven haplotypic groups (A-G), with haplotypes A and B representing 81% of the total observed. The T687G SNP was not observed in either of the resistant or susceptible populations. We conclude that the T687G SNP in this Pgp gene is not associated with TCBZ resistance in these Australian F. hepatica populations and therefore unlikely to be a universal molecular marker for TCBZ resistance., (Copyright © 2014. Published by Elsevier B.V.)

Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae each by oral gavage. Four of them were treated with single dose of TCBZ at 10mg/kg at four weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96h post treatment. Juvenile flukes were manually retrieved from the goat livers and processed for scanning electron microscopy. In control flukes, the anterior region was adorned with sharply pointed spines projecting away from the surface, while in the posterior region, spines become shorter and narrower, loosing serration and with the appearance of distinct furrows and papillae. The dorsal surface retained the same pattern of surface architecture similar to that of ventral surface. Flukes obtained from 24h post-treatment did not show any apparent change and were still very active. However, there were limited movements and some blebbing, swelling, deposition of tegumental secretions and some flattening displayed by the flukes of 48h post-treatment. All the worms were found dead 72h post-treatment and showed advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing off the tegument to expose the basal lamina, formation of pores and isolated patches of lesions. By 96h post-treatment, the disruption was extremely severe and the tegument was completely sheared off causing deeper lesions that exposed the underlying musculature. The disruption was more severe at posterior than anterior region and on ventral than dorsal surface. The present study further establishes the time-course of TCBZ action in vivo with 100% efficacy against the juvenile tropical liver fluke., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Fascioliasis is a zoonotic infestation caused by the trematode fasciola hepatica. It presents a wide spectrum of clinical pictures ranging from fever and eosinophilia to ambiguous gastrointestinal symptoms in the acute phase. However, it may often be overlooked, especially in the acute phase, because of uncertain symptoms. Fasciola hepatica can have an initial presentation similar to the presentation of malignancy. Here, we report a case of a hepatic mass caused by fasciola hepatica.

Despite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

Three methods of diagnosing Fasciola hepatica (F. hepatica) infection (a coproantigen ELISA, Bio-X Diagnostics, Belgium, Faecal Egg Count (FEC), and a serum IgG ELISA,Bio-X Diagnostics, Belgium) were evaluated in artificially infected cattle, with and without drug treatment. Specifically, the potential value of the coproantigen ELISA in the quantitation of F. hepatica infection was sought. Twelve steers were each infected with 100, 200 or 500 metacercariae (n=4 cattle/group). On day 84, post infection (PI), 2 animals from each group were treated orally with triclabendazole (TCBZ). Faecal and blood samples were collected weekly after infection from all animals, as well as over 5 consecutive days (days 105-109 PI) for the six animals remaining infected to determine the repeatability of these assays. Cattle were killed 126 days PI and the coproantigen, FEC and IgG levels were compared with the number of fluke recovered. Animals first tested positive for infection with the serum ELISA, with 11/12 animals positive on day 28, and IgG responses increased to day 42 PI. The coproantigen ELISA was first positive on day 42 (3/12 animals), with all animals positive by day 56 PI. The first F. hepatica egg was detected on day 49 from an animal infected with 500 metacercariae; however only on one occasion (day 84) did all animals return positive FEC. Within one week of treatment with TCBZ, all six treated animals had returned to negative status by coproantigen ELISA and FEC whereas IgG levels persisted. Weekly variation in both coproantigen level and FEC was evident throughout the trial. Results from the consecutive daily collections varied greatly between days for both methods, with 2-6-fold differences in coproantigen levels and 2-4-fold variation in FEC. Strong correlations were observed between fluke burdens (day 126) and day 125 coproantigen levels (R(2)=0.8718) and FEC (R(2)=0.8368). The coproantigen ELISA was more sensitive than FEC (FEC displayed false negatives) and detected infection earlier. This ELISA showed good correlation to fluke burdens in these cattle and has promise as a test for detecting low fluke burdens., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Human fascioliasis has the widest latitudinal, longitudinal and altitudinal distribution of any vector-borne disease, yet only 3 cases have been reported from South Africa, the last in 1964. We report 2 cases from the same geographic area associated with local consumption of watercress, suggesting an endemic focus.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by the inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R-VPL]. In the first experiment, flukes were initially incubated for 2 h in R-VPL (100 μM), then incubated for a further 22 h in R-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg/ml, or 0.1327 μM). For controls, flukes were incubated for 24 h in R-VPL and TCBZ.SO on their own. In a second experiment, flukes were removed from the incubation media following cessation of movement. In the third experiment, Sligo flukes were incubated in lower concentrations of R-VPL (10 μM) and TCBZ.SO (15 μg/ml, or 0.0398 μM). Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R-VPL alone had minimal effect on either isolate. After treatment with TCBZ.SO alone, there was greater surface disruption to the Cullompton than Sligo isolate. However, combined treatment of R-VPL+TCBZ.SO led to more severe surface changes to the Sligo isolate than with TCBZ.SO on its own; this potentiation of drug activity was not seen with the Cullompton isolate. The phenomenon was evident at both concentrations of TCBZ.SO. Inclusion of R-VPL in the incubation medium also reduced the time taken for the flukes to become inactive; again, this effect was more distinct with the Sligo isolate. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Fasciolosis caused by Fasciola hepatica, is the most prevalent parasitic disease in dairy cattle from the northern region of Cajamarca, Peru. The control of this parasite is based on the use of Triclabendazole (TCBZ), a drug that has been used for more than fifteen years in this area. Recent studies, however, have reported a lack of clinical efficacy after treating dairy cattle. This research was aimed to determine the efficacy of TCBZ in a clinical trial. Eleven dairy cows all positive to F. hepatica identified by presence of eggs in feces, were treated with TCBZ (Fasinex(®) 10%) at 12 mg/kg body weight. Fourteen and thirty days after treatment, the animals were analyzed for F. hepatica eggs in their feces by the fecal egg count reduction test. The results found show an overall efficacy of 31.05% and 13. 63% (14 and 30 days post treatment, respectively). Furthermore, an in vivo efficacy test was conducted in sheep with metacercariae obtained from eggs isolated from a cow clinically resistant to TCBZ. Eleven sheep divided in two groups, a control group with no treatment (n=5) and a treated group (n=6) were all infected with two hundred metacercariae. One hundred and six days after infection all the animals demonstrated F. hepatica eggs in their feces, confirming the presence of adult parasites in their livers. The animals were then treated with TCBZ (Fasinex(®) 10%) at 10mg/kg body weight. Fifteen days later, the animals were sacrificed and the number of F. hepatica in their livers counted. The results of this experiment showed an efficacy of the flukicide of 25.2% confirming the resistance to TCBZ of the F. hepatica isolated from dairy cattle in Cajamarca, Peru., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The experiments described here were designed to characterize the status of susceptibility/resistance to albendazole (ABZ) and triclabendazole (TCBZ) of a Fasciola hepatica isolate (named CEDIVE isolate) recovered from infected sheep (Gualeguay, Argentina) and maintained under laboratory conditions. Two separate clinical efficacy experiments were performed on sheep artificially infected with the CEDIVE isolate. Experiment 1: sheep were randomly distributed either in an untreated control group or in an ABZ (7.5 mg/kg) treated group (n=4 each). Additionally, the systemic exposure of ABZ metabolites was assessed in those ABZ-treated infected animals. In Experiment 2, an untreated control group and a TCBZ (10 mg/kg) treated group was included (n=4 each). The fluckicidal efficacy of ABZ and TCBZ was assessed by the comparison of the number of flukes recovered from untreated and treated sheep at 15 days post-treatment. The efficacy against the CEDIVE isolate of F. hepatica was 29% (ABZ) and 100% (TCBZ). The plasma drug exposure (expressed as AUC and C(max)) observed in the ABZ treated animals (Experiment 1) was in agreement with data obtained in the previous studies, which indicate that the low ABZ efficacy was not related to the quality of the pharmaceutical product and/or to a low systemic availability of the active drug/metabolite. The results reported here clearly show that the CEDIVE isolate of F. hepatica behaves as resistant to ABZ and susceptible to TCBZ., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Isothermal microcalorimetry (IMC) is an analytical tool that continuously measures the heat flow generated by chemical, physical or biological processes. We have demonstrated that IMC is a useful tool to analyze drug effects on helminths, including adult Fasciola hepatica. Here, we used IMC to examine the activity of triclabendazole and its metabolites triclabendazole sulphone and triclabendazole sulphoxide on juvenile and adult F. hepatica. Worms (one adult or 2-3 juveniles) were placed in 4 or 20 ml glass ampoules containing RPMI 1640 and the test compound (25-100 μg/ml) and the heat flow and motility of worms was examined with TAM48 and TAMIII isothermal microcalorimetry instruments. IMC was found to be precisely document drug effects on juvenile F. hepatica and confirmed the pronounced effect of the benzimidazole derivatives on the motor activity of F. hepatica. Juvenile F. hepatica incubated with 100 μg/ml triclabendazole, triclabendazole sulphone and triclabendazole sulphoxide showed no movements 8.3, 35 and 6h post-incubation (all p<0.001). The metabolic heat of triclabendazole sulphoxide treated worms (100 μg/ml) was reduced by 50% and 76% 24 and 120 h post-incubation, respectively. Limitations of calorimetric measurements were observed using adult F. hepatica as only three worms could be measured simultaneously and also control worms showed a considerable decrease in heat flow. Adult F. hepatica exposed to triclabendazole, triclabendazole sulphone and triclabendazole sulphoxide showed no movements after 31 (p=0.009), 49 (p>0.05) and 88 (p>0.05)h. In conclusion, IMC is useful to document drug effects on juvenile F. hepatica and since rapid technological developments in this field are occurring IMC might also hold promise to study adult F. hepatica in the near future., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Albendazole (ABZ) has been used for control of ovine fasciolosis in Sweden for several decades. However, increasing prevalence of Fasciola hepatica in Sweden requires attention to diagnostic tools and current control strategies. The coproantigen reduction test (CRT) using commercial Bio-X K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) was recently suggested as a novel method for diagnosis of patent F. hepatica infection. The aim of this study was to compare the efficacy of albendazole and triclabendazole (TCBZ) treatment against F. hepatica in naturally infected sheep in south-western Sweden by CRT, and also to evaluate the usefulness of this test as a diagnostic marker for anthelmintic efficacy following treatment with ABZ. Three weeks after housing, 24 serologically positive ewes on a commercial farm in Sweden were randomly allocated into three groups, which were either dewormed with 5mg/kg ABZ, 10mg/ml TCBZ or left untreated. Twenty-six days after initial administration of these anthelmintics, all of the sheep in the ABZ group and untreated control groups were treated with 10mg/kg TCBZ. While TCBZ caused elimination of coproantigen and eggs in faeces 7 days after application, ABZ treatment failed completely. Neither anthelmintic resistance, underdosing, nor lack of efficacy due to the presence of immature flukes can be out ruled as possible causes. Despite some deviations in conformity between coproantigen levels and presence of fluke eggs, the CRT was a useful tool for measuring treatment efficacies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In the present work, we evaluate in vivo the activity of carboxylesterase of Fasciola hepatica exposed to triclabendazole. We observed a statistically significant increase in enzyme activity at 24 and 48 h post treatment (P<0.01 and P<0.001, respectively). The zymogram of cytosolic fractions identified a protein of 170 kDa containing the carboxylesterase activity. The densitograms of the zymograms confirmed the phenomenon of enzyme induction under the experimental conditions of the assay. These results provide not only the understanding of the importance of this metabolic pathway in flukes but carboxylesterase would also be an enzyme that could participate more actively in the development of anthelmintic resistance at TCBZ., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A faecal egg count reduction test, using composite samples, was developed in order to assess the efficacy of the flukicide, triclabendazole (TCBZ) on commercial sheep farms in England and Wales. First, a comparison between individual counts and composite counts was conducted using sheep on two farms with different levels of infection. Faecal samples were collected from 50 sheep on each farm at the time of TCBZ treatment and 21&emsp14;days later. The results showed that a composite fluke egg count (CFEC) was as sensitive as using individual samples, and the test was subsequently validated on an additional 18 sheep farms. The pre- and post-treatment CFECs using five composite samples were subjected to bootstrap analysis. The variance in fluke egg counts of composite samples was high, but analyses indicated that 20 individual samples analysed as two composites was as sensitive as using five composites. The two-composite assay was evaluated on another five farms. On seven out of 25 farms sampled, egg counts either did not decrease significantly or increased following treatment, suggesting TCBZ resistance on these farms. This assay represents a practical field test that can be used in the first instance to evaluate the efficacy of TCBZ on sheep farms where resistance is suspected.

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were treated with compound alpha at a dosage of 15 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24h, 48 h and 72 h post-treatment (pt). They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system: specifically, the uterus, Mehlis' gland, ovary and vitellaria. As judged by the appearance of the uterus, normal egg production ceased within 24h of treatment; this phenomenon preceded significant changes to the other reproductive organs. Over the 3-day pt period, there was a progressive decline in the number of oogonia in the ovary, together with an increase in the number of eosinophilic and apoptotic oocytes and vacuolation and shrinkage of the ovarian tubules. There was a shift in the cell population within the vitelline follicles at 48 h pt, with relatively greater numbers of mature vitelline cells and fewer immature cells. The follicles were vacuolated and the shell globule clusters in the mature cells were disorganised. Greater disruption was seen at 72 h pt, with a reduction in the size of the follicles and rupture of cells, releasing their content into the lumen of the follicles. These histological observations were confirmed and extended at the TEM level. Thus, examination of electron micrographs showed that disruption of the shell globule clusters was evident at 48 h pt, which coincided with the start of the breakdown of the mature cells and of the nurse cell network. These degenerative changes were more conspicuous at 72 h pt. In the Mehlis' gland, shrinkage and vacuolation of the cells and their cytoplasmic extensions became progressively greater from 48 h to 72 h pt, and secretory activity declined. The changes in the reproductive organs and inhibition of egg production are put in context of the overall time-course of drug action., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This report presents two cases of human fascioliasis from different states in India. Although only few cases of human fascioliasis have been reported from India previously, both these cases were encountered within a span of three months at this tertiary care centre. Case 1 had significant symptoms with episodes of fever, abdominal pain and eosiniphilia and underwent multiple diagnostic procedures before the correct diagnosis was reached. Case 2, who had few symptoms, had fascioliasis diagnosed with minimal evaluation. These different presentations seen at two ends of the clinical spectrum of disease along with findings of peripheral eosinophilia, and radiological findings led to a presumptive diagnosis that was then confirmed by microscopic examination of bile. Morphometric analysis of ova from these cases was suggestive of infestation with F. gigantica or a F. gigantica-like hybrid. Both patients were treated with triclabendazole which was imported from Geneva. The need to be aware of the possibility of occurrence of this disease and the inclusion of drugs used for treating the disease, in the Indian drug list, should be emphasized.

Sheep infected with the Cullompton isolate of Fasciola hepatica were treated with triclabendazole at a concentration of 10 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the liver and, where present, from the gall bladder at 48, 72 and 96 h post-treatment (pt). Gross changes to the spermatogenic cells of the testis were examined by histology and ultrastructural alterations were visualised via transmission electron microscopy. Disruption was progressive in nature, with the testis tubules becoming shrunken, vacuolated and gradually more denuded of cellular content over the 96-h time period. From 48 h pt, the number of primary and secondary spermatogonia decreased and multinucleate spermatogonial cells were frequent. Later, developmental stages were uncommon, giving rise to much empty space within the tubules. By 72 h pt, the tubules contained many apoptotic and degraded cells and had an extremely disorganised appearance. At 96 h pt, the tubules were almost completely empty, with the exception of the remains of degraded spermatogenic cells. These results indicate that triclabendazole severely disrupts spermatogenesis in the liver fluke from 48 h pt in vivo.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 μM), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ + NADPH + TCBZ (15 μg/ml), or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO; 15 μg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with KTZ + TCBZ, but more particularly KTZ + TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: in the syncytium, for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

In the winter of 2008, cattle on a farm in the province of Neuquen, Argentina died from subacute and chronic liver fluke disease despite four previous treatments with Triclabendazole (TCBZ). In the spring of 2009, a preliminary efficacy test revealed good performance using nitroxynil, whereas TCBZ efficacy was only 18% by egg counts of Fasciola eggs in the faeces. Resistance to Fasciola hepatica to TCBZ has never been reported in South America, so in January of 2010 a controlled trial was conducted to confirm and to define the degree of resistance in this herd. In a clinical trial, the fluke egg output was monitored on Days 14 and 21 and serum enzymes gamma-glutamyl transpeptidase (GGT) and glutamic-oxaloacetic transaminase (GOT) on Days 0 and 21 in 36 calves treated with TCBZ or with closantel. The results showed a reduction of 100% in fluke egg output at Days 14 and 21 for closantel. The mean epg in the TCBZ-treated groups did not decrease. Because of the fact that in this study TCBZ treatment in cattle had no effect, even at double the recommended dose, it is highly indicative that resistance of F. hepatica against TCBZ is present on this farm. The GGT and GOT levels decreased in the closantel-treated group as a result of the treatment at 21 days after dosing. To evaluate the importance of TCBZ resistance in F. hepatica in Argentina, a study on more farms from endemic areas is needed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An in vivo study in the laboratory rat model was carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from combination treatment of triclabendazole (TCBZ) and the metabolic inhibitor, ketoconazole (KTZ). Rats were infected with the TCBZ-resistant Oberon isolate of F. hepatica and divided into 3 groups at 12 weeks post-infection. The first group was dosed orally with TCBZ at a dosage of 10mg/kg and KTZ at a dosage of 10mg/kg. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.). A second group of rats was treated with TCBZ alone (10mg/kg) and sacrificed at 96 h p.t. The third group acted as untreated controls. Surface changes were monitored by scanning electron microscopy (SEM). In flukes from the TCBZ+KTZ-treated group, the results showed a progressive and time-dependent increase in the level of disruption to the tegumental syncytium. Swelling, furrowing, blebbing and sloughing of the syncytium increased with time p.t. Another feature seen was a thick layer of tegumental shedding in some fluke samples at different times p.t. By comparison, flukes treated with TCBZ alone remained unaffected. The results demonstrated that the Oberon isolate is only sensitive to drug action in the presence of ketoconazole, indicating that combining triclabendazole with a metabolic inhibitor could be used to preserve the effectiveness of the drug against TCBZ-resistant populations of F. hepatica., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Eight indoor-reared cross-bred sheep with no prior exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Twelve weeks after infection, sheep were treated with 10mg/kg triclabendazole. Two sheep were euthanised per time period; at 48 h, 72 h and 96 h post-treatment (pt). Two untreated control sheep were euthanised at 96 h pt. Flukes were recovered from the liver and, if present, from the gall bladder of the sheep. They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system; specifically, the uterus, vitelline follicles, Mehlis' gland and ovary. Over the 4-day post-treatment period, there was a progressive reduction in the number of oogonia and oocytes in the ovary and evidence of apoptosis. Vacuolation and a decrease in the number of Mehlis' gland cells were observed from 48 h pt onwards and disruption of the normal role of the gland in egg formation was evident. The vitelline follicles showed a gradual decrease in size and became vacuolated; the population structure in each follicle changed to be one consisting mainly of mature cells and the production of shell protein material declined. The follicle became disorganised as the cells broke down and released their contents into the lumen of the follicle. While the uterus appeared to contain eggs at 48 h pt in whole-mount specimens, no properly-formed eggs were observed in histological sections. By 96 h pt, the uterus was completely devoid of eggs. Overall, egg production was seen to be severely affected by TCBZ treatment and flukes were incapable of producing normal eggs within 2 days of treatment. The implications of this in terms of the epidemiology of the disease are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The objective of the present study was to analyse the reproductive viability (using histopathologic studies) of Fasciola hepatica from cattle artificially infected and treated subcutaneously with a new experimental formulation of triclabendazole (8mg/kg b.w.). The results of the efficacy controlled test, which only takes into account the presence of live adult flukes, indicated that, whilst in the control group (n=7) 533 live specimens were recovered, in the test groups (doses of 8 and 12mg/kg b.w.) only 195 and 47 adults were recovered, respectively. These numbers indicate efficacies of 69% and 95.6%, respectively. It was observed in that dose of 8mg/kg b.w. some specimens remained viable, but they were infertile, which severely compromises the biological cycle of the trematode. In the testis tubules of flukes treated with the low dose of TCBZ (8mg/kg), very few cells were present and the vitelline follicles were markedly reduced in size and each follicle contained very few cells. This would have direct implications for the pathogenesis of the parasitosis since the remaining parasites would produce little clinical-productive manifestations, would stimulate the immune response and would find it difficult to establish future re-infestations/re-infections. Consequently, these observations will also prompt a review of certain methodological and interpretative aspects related to efficacy tests, where the only discriminative factor is the reduction of the adult parasite load. On one hand, histopathological studies could be complementary to the efficacy controlled test for TCBZ or other BZD formulations., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A sheep trial was performed to standardise a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole (TCBZ) in Fasciola hepatica). The CRT employs the BIO K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) to test for the presence of F. hepatica coproantigens in a faecal sample. If it is coproantigen-positive, the CRT protocol recommends that faecal samples are re-tested for coproantigens at 14 days post-treatment (dpt), with negative testing at this point indicating TCBZ success. Initial work aimed to confirm the sensitivity of the BIO K201 ELISA for Fasciola infection and investigate whether coproantigens represent a robust reduction marker of TCBZ efficacy. Thirty-eight, indoor-reared sheep were artificially infected with F. hepatica isolates known to be susceptible (Cullompton) and resistant (Sligo) to TCBZ action, respectively. Treatment was administered at 12 weeks post-infection (wpi), with 2 sheep groups, infected with each isolate, culled at 2 and 4 weeks post-treatment (wpt), respectively. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period. Additional work focused on the effect of temperature on faecal sample collection and storage. Faecal samples collected from sheep positive for F. hepatica infection were sub-sampled and left at room temperature. Individual sub-samples were tested by ELISA on consecutive days and these readings compared to the original test result on the day of collection. In addition, ELISA values were compared between faecal sub-samples prepared on the day of sampling and post storage at -20°C. Also, an immunocytochemical study was performed to determine the tissue site of origin of the coproantigen protein in the fluke. Results showed that the BIO K201 ELISA was sensitive for Fasciola coproantigens, with coproantigens detectable from 5 wpi onwards. The suitability of coproantigens as a diagnostic marker of TCBZ efficacy was supported by the absence and presence of coproantigens in TCBZ-treated Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) F. hepatica infections at 2 and 4 wpt, respectively. Study results suggest that low to moderate temperature has little, if any, impact on coproantigen stability in faecal samples, but that higher temperatures may have. Immunolabelling for the coproantigen showed that it was specific to the gastrodermal cells of both adult and juvenile flukes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The aim of this study was to compare the efficacy of triclabendazole (TCBZ) and nitroxynil against a TCBZ-resistant Fasciola hepatica strain in a naturally infected sheep flock. The efficacies were measured by the faecal egg count reduction test. The level of F. hepatica antigens was tested in faeces; and haematological indices such as total proteins (TP), albumin, hepatic enzymes and total IgG were also studied. The results confirmed the resistance of F. hepatica against TCBZ in the flock with an efficacy during the first month post-treatment between 59.4% and 73.8%. In the nitroxynil group, the efficacy during the same period ranged between 81.3% and 86%, likely because the efficacy of this drug against 7- to 9-week-old immature stages is only 50-90%. Anemia was showed in all groups and white blood cells were always higher than the reference range. The values of TP and albumin were within normal range in most of the sheep, and an increase in hepatic enzymes confirmed the liver damage. Regarding total IgG, some negative correlations were found with egg excretion, and in relation to the level of antigens in faeces, these ones decreased immediately after treatment. We conclude that nitroxynil could be an alternative in case of TCBZ resistance.

Eight indoor-reared crossbred sheep with no pre-exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. At 12 weeks post-infection, sheep were dosed with 10mg/kg triclabendazole. Two sheep per time period were euthanized at 48h, 72h and 96h post-treatment. Two control sheep were euthanized alongside the 96h triclabendazole-treated sheep. Flukes were recovered from each of the sheeps' liver and, if present, from the gall bladder, and they were processed for scanning electron microscopy (SEM). Flukes recovered 48h post-treatment were active. Disruption to the tegument took the form of swelling, widespread blebbing and some loss of the tegument covering the spines. By 72h post-treatment, all flukes recovered were dead and a number were recovered from the gall bladder. Typically, the posterior end of the flukes was elongated and in this region the tegumental syncytium had sloughed away. Tegumental loss was more widespread on flukes recovered from the gall bladder. At 96h post-treatment, only one fluke was recovered from the liver and three from the gall bladder. All the flukes were dead and they were totally devoid of tegumental syncytium.

Uptake of triclabendazole by the liver fluke, Fasciola hepatica has been studied by experiments designed to block either oral uptake of drug, by use of ligatures, or trans-tegumental diffusion, by allowing the drug to bind to an excess of bovine serum albumin (BSA) in the medium. Changes to the tegumental system, musculature and gut were assessed using transmission electron microscopy. Flukes were incubated in vitro for 24 h in TCBZ.SO (15 microg/ml). Disruption to the tegument and muscle was similar in ligatured and non-ligatured flukes, suggesting that closing the oral route did not affect drug uptake. The ultrastructure of the gastrodermal cells remained unchanged. Non-ligatured flukes were also incubated for 24 h in vitro in TCBZ.SO (15 microg/ml) in the presence of red blood cells (RBCs). Oral uptake of blood was demonstrated, but gut ultrastructure remained normal, whereas the tegument was severely disrupted. In separate experiments, ligatured and non-ligatured flukes were incubated in TCBZ.SO (15 microg/ml) in the presence of BSA (30 mg/ml) for 24 h in vitro. There was a marked decrease in the degree of tegumental disruption observed compared with TCBZ.SO action alone; again, the gut remained normal. The findings support previous morphological and pharmacological studies indicating that trans-tegumental uptake of triclabendazole predominates in the liver fluke.

Twenty-four shed-reared lambs were each infected orally with 250 metacercariae of Fasciola hepatica, using either the triclabendazole (TCBZ)-sensitive Cullompton isolate or the TCBZ-resistant Sligo isolate. Twelve weeks after infection the lambs were treated with TCBZ (10mg/kg) or with the experimental fasciolicide, Compound Alpha (Cpd alpha), a benzimidazole derivative of TCBZ (15mg/kg). The lambs were euthanised 48, 72 and 96h after TCBZ treatment, or 24, 48 and 72h after Cpd alpha treatment, and flukes were collected from the liver and/or gall bladder of each animal. Untreated animals harbouring 12-week infections were euthanized 24h after administration of anthelmintic to the treatment groups, and the untreated flukes provided control material. A semi-quantitative assessment of the degree of histological change induced by the two drugs after different times of exposure was achieved by scoring the intensity of three well-defined lesions that developed in the testes and uteri of a representative sample of flukes from each lamb. In general, it was found that in those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), there was progressive loss of cell content due to apparent failure of cell division to keep pace with expulsion of the mature or effete products. Further, actively dividing cell types tended to become individualised, rounded and condensed, characteristic of apoptotic cell death. Protein synthetic activity was apparently inhibited in the Mehlis' secretory cells. In the uterus, where successful formation of shelled eggs represents the culmination of a complex sequence of cytokinetic, cytological and synthetic activity involving the vitelline follicles, the ovary and the Mehlis' gland, histological evidence indicating failure of ovigenesis was evident from 24h post-treatment onwards. The development of these lesions may be related to the known anti-tubulin activity of the benzimidazole class of anthelmintics, to the induction of apoptosis in cells where mitosis or meiosis has aborted due to failure of spindle formation, and to drug-induced inhibition of protein synthesis. The semi-quantitative findings indicated that Cpd alpha is slightly less efficacious than TCBZ itself in causing histological damage to the reproductive structures of TCBZ-sensitive flukes, and that, like TCBZ, it caused no histological damage in flukes of the TCBZ-resistant isolate. This study illustrates the potential utility of histological techniques for conveniently screening representative samples of flukes in field trials designed to validate instances of drug resistance or to test the efficacy of new products against known drug-resistant and drug-susceptible fluke isolates. It also provides reference criteria for drug-induced histopathological changes in fluke reproductive structures which may aid interpretation of TEM findings.

Seventy indoor-reared sheep were divided into 10 groups to test the efficacy of the experimental fasciolicide, compound alpha (15mg/kg) against triclabendazole (TCBZ)-resistant and TCBZ-susceptible F. hepatica infections. Activity against the Sligo TCBZ-resistant isolate was tested at three time points post-infection (p.i.): 3 days, 4 weeks and 12 weeks (Groups 1-3, respectively). A parallel trial was carried out using TCBZ (10mg/kg) (Groups 5-7): this provided a direct comparison between the efficacies of the two drugs. Group 4 served as an untreated Sligo control. Groups 8 and 9 were setup to test the efficacy of TCBZ and compound alpha against 12-week-old and 4-week-old TCBZ-susceptible, Cullompton infections, respectively. Group 10 served as an untreated Cullompton control. Sheep were sacrificed at 16 weeks p.i. and efficacies were determined. All remaining flukes were collected and measured, before being processed for whole-mount staining to assess the condition of their reproductive structures (testis, vitellaria, ovary and uterus). A second study was carried out to test the activity of compound alpha (15mg/kg) against mature 12-week-old TCBZ-susceptible F. hepatica infections in sheep. Eighteen sheep were divided into two groups, A and B. Group A was treated and Group B served as an untreated control group. Efficacy was determined by reduction in faecal egg counts. The results showed that, whilst compound alpha was very active against adult TCBZ-susceptible flukes, producing a 100% reduction in faecal egg counts, it only caused a 62.5% reduction in fluke burden against juvenile flukes. Moreover, compound alpha was not effective against any stage of infection with TCBZ-resistant F. hepatica in sheep. Data from the trial also revealed biological differences between the two isolates. Thus, Sligo flukes were smaller in size and produced fewer eggs than the Cullompton flukes and their cysts were less infective to sheep. However, they reached the bile ducts more quickly and their eggs appeared in the faeces >2 weeks earlier.

Fascioliasis causes important economic losses in ruminant species all over the world. Its control is largely based on the use of the flukicidal compound triclabendazole (TCBZ). However, its chemically related benzimidazole anthelmintic albendazole (ABZ) is being successfully used to control TCBZ-resistance flukes. This research gains some insights into the comparative molecular behaviour of both anthelmintics within the target fluke. The goals of the current work were: (i) to assess the competitive binding of ABZ and TCBZ to cytosolic proteins of F. hepatica, and (ii) to evaluate the enantioselective biotransformation of ABZ in microsomal fractions obtained from TCBZ-susceptible and TCBZ-resistant strains of the liver fluke. Cytosolic proteins from fluke specimens bound TCBZ with greater affinity (83%) than ABZ (44%) and the fraction of TCBZ bound to cytosolic proteins was not displaced by ABZ. The microsomes from both -susceptible and resistant flukes sulphoxidized ABZ into ABZ sulphoxide (ABZSO). However, this oxidative activity was 49% higher in microsomes from TCBZ-resistant flukes (P < 0.001) with a predominant production of the (+) ABZSO enantiomer. As earlier shown for TCBZ, the results reported here confirm an enhanced ability for ABZ oxidation in TCBZ-resistant flukes. While this enhanced oxidative metabolism of ABZ may cooperate to the resistance phenomenon, other pharmacodynamic-based mechanisms may be involved, which would explain why, although being chemically-related, ABZ remains efficacious against TCBZ resistant flukes under field conditions.

Eight indoor-reared crossbred sheep with no pre-exposure to Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Anthelmintic dosing occurred at 4 weeks post-infection with 10 mg/kg triclabendazole. Two treated sheep were euthanized at 48 h, 72 h and 96 h post-treatment with triclabendazole. Two control sheep were euthanized alongside the 48 h triclabendazole-treated sheep. Juvenile flukes were recovered from each of the sheeps' liver and processed for scanning electron microscopy (SEM). Flukes were still active 48 h post-treatment and displayed limited morphological disruption. There was some blebbing and sloughing of the tegument around the oral sucker. In several of the specimens, an extra layer had been deposited on the fluke surface, giving it a flattened appearance. At 72 h post-treatment, only one fluke remained alive and the disruption varied in degree. In the majority of flukes, there was severe swelling of the tegument, accompanied by isolated areas of flattening along the lateral margins of the flukes and in the tail region. Limited areas of sloughing occurred in the tail region. In more seriously affected specimens, the syncytium had been stripped away to reveal the basal lamina and some deeper lesions were also observed. By 96 h post-treatment, all the flukes were dead and were grossly disrupted. They were totally devoid of tegument and deep lesions exposed the internal tissues of the fluke.

Praziquantel is still very effective for the treatment of schistosomiasis, but there are rising concerns on the potential risk of developing resistances because of the extensive use of this drug. Triclabendazole, a systemic anthelmintic, is very effective against other trematodes such as Paragonimus spp. and Fasciola spp. It has been reported to be effective in vitro and in experimental animals against Schistosoma mansoni. However, its antischistosomal efficacy in humans has not yet been evaluated. The objective of the study was to evaluate the efficacy of triclabendazole at the dosage currently used for the treatment of human fascioliasis (10mg/kg body weight) in subjects co-infected with S. mansoni and Fasciola spp. The study was carried out in Behera, a highly endemic area for both parasites, by personnel of the Egyptian Ministry of Health and Population. Ten subjects (m = 4, f = 6; age, 8-58years), who were infected at the same time by Fasciola spp. and S. mansoni, were enrolled. Six weeks after therapy, seven subjects were still excreting ova of S. mansoni, whereas none was excreting Fasciola spp. ova. At the given dosage, triclabendazole appeared not to be sufficiently effective in the treatment of S. mansoni.

Juvenile triclabendazole-resistant liver flukes, Fasciola hepatica, were incubated in vitro with 10 microg/ml of the sulphoxide metabolite of the experimental fasciolicide, compound alpha [5-chloro-2-methylthio-6-(1-naphthyloxy)-1H-benzimidazole], for 6 and 18 h. Following treatment, the specimens were examined using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and tubulin immunocytochemistry. The SEM results revealed a posterior-directed disruption comprised predominantly of swelling and blebbing of the tegument; these changes were more severe and extensive after the longer 18-h incubation. Along with swelling of the tegument and blebbing, the TEM results also revealed swelling of the mitochondria and basal infolds. A decrease in the number of both T1 and T2 secretory bodies was observed in the syncytium and cytoplasmic connections after the 18-h treatment. The circular muscle bundles were also disrupted, in that the organisation of the muscle fibres was irregular and the total number of muscle fibres was reduced. The immunocytochemical studies revealed no significant disruption to the distribution of tubulin immunoreactivity within the tegumental syncytium, the cytoplasmic connections or the associated tegumental cells. The results indicate that alpha.SO is capable of disrupting the tegument of 4-week-old triclabendazole-resistant liver flukes, though the morphological changes were not associated with any significant differences in tubulin immunostaining.

Fascioliasis and paragonimiasis, which are caused by liver flukes (Fasciola) and lung flukes (Paragonimus), are emerging public health problems. Several hundred millions of people are at risk of the two diseases that cause considerable morbidity and delay socio-economic development. Triclabendazole, a benzimidazole derivative, has been routinely used since 1983 in veterinary medicine to control infections with Fasciola spp. in domestic herbivorous animals. In 1986, a veterinary formulation of triclabendazole was first administered to two human patients with fascioliasis. Clinical data obtained thus far suggest that triclabendazole is the most efficacious and best tolerated drug for the treatment of fascioliasis. Moreover, the drug holds promise as a useful therapeutic alternative to praziquantel in the treatment of paragonimiasis. This review of triclabendazole includes an overview of the pharmacodynamics and pharmacokinetics, toxicology and efficacy against the major food-borne trematodes in laboratory animals. Data from case reports and clinical trials in humans infected with Fasciola spp. and Paragonimus spp. are summarised and the current state of triclabendazole regarding treatment of human fascioliasis and paragonimiasis is described. Efforts to facilitate broader registration of this drug should go hand-in-hand with research and development on novel drugs against food-borne trematodiasis, better access to improved sanitation, sound health education and the development of new technologies for assuring food safety.

Triclabendazole (TCBZ) is a halogenated benzimidazole compound that possesses high activity against immature and adult stages of the liver fluke, Fasciola hepatica. The intensive use of TCBZ in endemic areas of fascioliasis has resulted in the development of liver flukes resistant to this compound. TCBZ sulphoxide (TCBZSO) and TCBZ sulphone (TCBZSO2) are the main molecules recovered in the bloodstream of TCBZ-treated animals. In order to gain some insight into the possible mechanisms of resistance to TCBZ, the goals of the work described here were: to compare the ex vivo transtegumental diffusion of TCBZ parent drug and its sulpho-metabolites (TCBZSO and TCBZSO2) into TCBZ-susceptible and -resistant liver flukes; and to assess the comparative pattern of TCBZ biotransformation by TCBZ-susceptible and -resistant F. hepatica. For the tegumental diffusion studies, TCBZ-susceptible (Cullompton) and -resistant (Sligo) adult flukes collected from untreated infected sheep were incubated (15-180 min) in KRT buffer containing either TCBZ, TCBZSO or TCBZSO2 (5 nmol.ml-1). For the metabolism studies, microsomal fractions obtained from TCBZ-susceptible and -resistant flukes were incubated for 60 min with TCBZ (40 microM), and the amount of the formed metabolic product (TCBZSO) was measured. Drug/metabolite concentrations were quantified by HPLC. All the assayed TCBZ-related molecules penetrated through the tegument of both TCBZ-susceptible and -resistant flukes. However, significantly lower (approximately 50%) concentrations of TCBZ and TCBZSO were recovered within the TCBZ-resistant flukes compared to the TCBZ-susceptible ones over the 180 min incubation period. The rate of TCBZ sulphoxidative metabolism into TCBZSO was significantly higher (39%) in TCBZ-resistant flukes. The flavin-monooxigenase (FMO) enzyme system appears to be the main metabolic pathway involved in the formation of TCBZSO in both TCBZ-susceptible and -resistant flukes. The altered drug influx/efflux and enhanced metabolic capacity identified in TCBZ-resistant liver flukes may account for the development of resistance to TCBZ.

In this study, the susceptibility of two isolates of Fasciola hepatica--the Fairhurst and Oberon isolates--to treatment with triclabendazole was investigated, both in vivo and in vitro. The Fairhurst isolate originated in England, but has since been maintained in Australia; the Oberon isolate originated in Australia. Triclabendazole had a very high efficacy against the Fairhurst isolate. In sheep (dose: 10 mg/kg), the efficacy ranged from 78.4% at 2 weeks post-infection to 98.5% at 6 weeks post-infection. In cattle (dose: 12 mg/kg) efficacy was 89% at 2 weeks post-infection and 100% at 12 weeks. In contrast, against the Oberon isolate, triclabendazole had 0% efficacy against 2-week-old flukes in sheep (dose: 10 mg/kg) and 5% against 4-week-old flukes. Surface changes to flukes of the two isolates were assessed by scanning electron microscopy following treatment in vitro for 24 h in triclabendazole sulphoxide (15 and 50 microg/ml). Disruption took the form of blebbing, swelling and furrowing of the tegument and was greater in the Fairhurst than the Oberon isolate. Surface changes generally were more severe in the anterior than posterior region of the fluke and the dorsal surface was also consistently more severely affected than the ventral surface. Disruption was more severe at the higher drug concentration for both isolates. The morphological data is consistent with the efficacy data, which indicates that the Fairhurst isolate of F. hepatica is susceptible to triclabendazole treatment, whilst the Oberon isolate is refractory.

1. A study performed > 10 years ago and case reports published recently suggest that triclabendazole is effective for the treatment of patients with fascioliasis. 2. To confirm the efficacy of a human formulation of triclabendazole, we enrolled 165 patients into the present study and divided the subjects into two groups: (i) those who had fascioliasis, as evidenced by the presence of ova in their stools; and (ii) patients with clinical and laboratory data suggesting fascioliasis. 3. Patients were randomly allocated to receive 10 mg/kg, p.o., triclabendazole for 1, 2 or 3 days (single-, double- and triple-dose groups, respectively). Medical history and physical and laboratory examinations were performed at baseline and at 7, 14, 30 and 60 days after treatment. Results were based on 152 patients who completed the study. 4. A sharp decrease in the proportion of clinical signs and symptoms was observed in all groups immediately after treatment. Ova disappeared from the stools of all patients in the single- and double-dose groups. Thirty days after treatment, ova were identified in the stools of two patients in the triple-dose group who received a second course of triclabendazole. 5. All cases were cured on day 60. However, the cure rate was lower when the patients with suggestive fascioliasis were included in the analysis. The cure rate was not significantly different (P > 0.05) among the three dose groups. No cases of toxic hepatitis were observed. 6. In conclusion, oral administration of 10 mg/kg of the human pharmaceutical preparation of triclabendazole for 1-3 days is safe and effective in the treatment of human fascioliasis.

The benzimidazole derivative, triclabendazole is the main drug used against Fasciola hepatica, although its precise mode of action remains to be fully determined. Previous studies have suggested that triclabendazole acts as a microtubule inhibitor in the same manner as tubulozole-C. Consequently, flukes from triclabendazole-susceptible and triclabendazole-resistant isolates were treated with tubulozole-C (1x10(-6 )M) in vitro and changes in tegumental morphology and tubulin distribution within the tegument were monitored by scanning and transmission electron microscopy, together with tubulin immunocytochemistry. Tubulozole-C caused severe disruption within the tegument of triclabendazole-susceptible flukes. Tubulin immunoreactivity diminished within the tegumental syncytium of the triclabendazole-susceptible flukes following treatment with tubulozole-C. In contrast, tubulozole-C caused only minor disruption of the tegument in triclabendazole-resistant F. hepatica and did not significantly alter the pattern of tubulin immunostaining within the tegumental syncytium. The results of the present study indicate that tubulozole-C and triclabendazole share the same target molecule and that triclabendazole-resistant flukes are also cross-resistant to tubulozole-C.

Human fascioliasis cases in Japan have been reported infrequently and the most appropriate therapy for this disease remains to be determined. This case report describes a patient with the infection unsuccessfully treated with multiple high doses of praziquantel for over 1 year and eventually cured by the administration of triclabendazole in two single doses of 10 mg/kg and 12.5 mg/kg 5 months apart.

A study was developed to evaluate the influence of triclabendazole (Fasinex) and netobimin (Hapasil) the antigenaemia in sheep naturally infected with Fasciola hepatica during 16 weeks. A sandwich-ELISA (enzyme-linked immunosorbent assay) using a rabbit polyclonal IgG antibody to F. hepatica antigens was employed and the data obtained were compared to those from coprological and indirect-ELISA techniques. Triclabendazole reduced the values of circulating antigens at weeks 2-4 post-treatment and faecal output at weeks 2-8 post-treatment, but antibodies showed positive values until the end of the study. Netobimin did not reduce circulating antigens of the trematode nor egg-excretion; and IgG antibodies did not decrease throughout the study.

Infection with Fasciola hepatica, a liver trematode, is not frequently reported in the United States. We describe 2 patients, both originally from Cape Verde, who illustrate the spectrum of clinical presentations of F. hepatica as well as the means of treating infection with this parasite. Patient 1 had extensive disease and underwent multiple diagnostic procedures before the correct diagnosis was reached. Patient 2, who had few symptoms, had fascioliasis diagnosed by a noninvasive evaluation. Both patients were treated with triclabendazole without experiencing significant side effects. Fascioliasis that has been imported to the United States may elude prompt or accurate diagnosis. Obtaining a detailed travel history and recognizing the clinical presentation early in the course of infection may permit timely and noninvasive identification of infection. Triclabendazole is now the recommended drug for treating for fascioliasis because of its efficacy, safety, and ease of use.

We report a case of temporary biliary obstruction due to fascioliasis. This case report shows that in Central Europe, fascioliasis is one of the differential diagnoses of abdominal pain, especially if it is associated with eosinophilia. Successful medical treatment is possible even with obstruction of the bile duct.