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Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F . hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F . hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F . hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

This study has been proposed new, sensitive, straightforward, and validated spectrophotometric and spectrofluorimetric methods for estimating of amprolium (AMP) and triclabendazole (TCB) in laboratory prepared pharmaceutical wastewater. Both methods were based on the reaction of AMP and TCB with Eosin-Y in acidic medium, targeting the tertiary amino groups of the concerned analytes, generating an orange-red ion pair complexes. The resulting products were measured by spectrophotometric (Method I) and spectrofluorimetric (Method II) tools. Regarding method I, the absorbance was measured at 547 nm with a lower detection limit of 45 ng/mL and quantitation limit of 136 ng/mL for AMP with corresponding values of 191 ng/mL and 579 ng/mL for TCB. The proposed method exhibited a linear pattern over the concentration range of 100–5000 and 1500–10,000 ng/mL for amprolium and triclabendazole, respectively. Meanwhile in method II, the produced complex was measured at λem 552 nm for AMP or 555 nm for TCB after excitation at λex 470 nm with a lower detection limit of 142 ng/mL and quantitation limit of 429 ng/mL for AMP with corresponding values of 67 ng/mL and 202 ng/mL for TCB. The proposed method exhibited a linear pattern over the concentration range with linearity ranges of 500–2000 and 100–1500 ng/mL for amprolium and triclabendazole, respectively. The International Conference on Harmonization (ICH) requirements for linearity, accuracy, precision, specificity, and robustness were followed in the comprehensive validation of the proposed methods, allowing its application successfully to determine the studied analytes in wastewater samples.

Background Fasciola hepatica is of worldwide significance, impacting on the health, welfare and productivity of livestock and regarded by WHO as a re-emerging zoonosis. Triclabendazole (TCBZ), the drug of choice for controlling acute fasciolosis in livestock, is also the drug used to treat human infections. However TCBZ-resistance is now considered a major threat to the effective control of F. hepatica . It has yet to be demonstrated whether F. hepatica undergoes a genetic clonal expansion in the snail intermediate host, Galba truncatula , and to what extent amplification of genotypes within the snail facilitates accumulation of drug resistant parasites. Little is known about genotypic and phenotypic variation within and between F. hepatica isolates. Results Six clonal isolates of F. hepatica (3× triclabendazole-resistant, TCBZ-R and 3× triclabendazole-susceptible, TCBZ-S) were generated. Snails infected with one miracidium started to shed cercariae 42–56 days post-infection and shed repeatedly up to a maximum of 11 times. A maximum of 884 cercariae were shed by one clonally-infected snail ( Fh LivS1) at a single time point, with > 3000 clonal metacercariae shed over its lifetime. Following experimental infection all 12 sheep were FEC positive at the time of TCBZ treatment. Sheep infected with one of three putative TCBZ-S clones and treated with TCBZ had no parasites in the liver at post-mortem , whilst sheep each infected with putative TCBZ-R isolates had 35–165 adult fluke at post-mortem , despite TCBZ treatment. All six untreated control animals had between 15–127 parasites. A single multi-locus genotype was reported for every fluke from each of the six clonal isolates. Adult F. hepatica showed considerable variation in weight, ranging from 20–280 mg, with variation in weight evident within and amongst clonal isolates. Conclusions A genetic clonal expansion occurs within G. truncatula , highlighting the potential for amplification of drug resistant genotypes of F. hepatica . Variation in the weight of parasites within and between clonal isolates and when comparing isolates that are either susceptible or resistant to TCBZ represent inherent variation in liver fluke and cannot be attributed to their resistance or susceptibility traits.

Recent estimates suggest that liver condemnation due to liver fluke in Ethiopia causes an annual economic loss of about 48.4 million Ethiopian birr. For approximately ten decades, the treatment of fasciolosis has relied solely on triclabendazole (TCBZ). In many clinical practices, combined tetramisole with Oxyclozanide and albendazole drugs have been used as treatment options for ovine fasciolosis. Therefore, this study aimed to compare the effectiveness of triclabendazole (TCBZ), combined tetramisole with Oxyclozanide (TETRA), and albendazole (ALBE) for the treatment of ovine fasciolosis and their effects on biochemical parameters. In this study, a field experimental trial was carried out in 75 purposively selected forty-five naturally Fasciola-infected sheep from May to November 2023 in the West Dembiya district, Ethiopia. The infected animals were randomly divided into three Groups: Group I received triclabendazole, Group II received combined tetramisole with Oxyclozanide, Group III received albendazole, and the triclabendazole treatment Group was considered the positive control Group. Fecal and serum samples were collected on days 0 (before treatment) and 7, 14, and 21 after treatment. A fecal egg count reduction test (FECRT) and biochemical analysis were carried out. Descriptive statistics and repeated-measures ANOVA were used to analyze the data. The current study revealed that TCBZ was the most effective drug, with a FECRT of 97.8%, followed by TETRA (96.6%) and ALBE (84% reduction in the mean fecal egg count). The results also indicated that after treatment, the concentrations of the biochemical parameters significantly changed ( P  < 0.05), particularly after 21 days of treatment. In all the Groups, treatment and time had significant effects on the concentrations of the biochemical parameters and eggs per gram count (EPG). In terms of the EPG, albendazole substantially differed from combined tetramisole with Oxyclozanidee and triclabendazole ( p  < 0.05), whereas combined tetramisole with Oxyclozanide and triclabendazole did not ( p  > 0.05). The study also revealed that the observed clinical presentation improved after treatment. In conclusion, triclabendazole and combined tetramisole with Oxyclozanide strongly affected ovine fasciolosis. If the accessibility of triclabendazole is limited on the market, the use of the 2nd promising drug (combined tetramisole with Oxyclozanide) is mandatory. Biochemical parameters are the most promising biomarkers for liver damage and are used to select the most effective anthelmintic medicines for the treatment of ovine fasciolosis.

Fasciola gigantica, the causative agent of tropical fasciolosis, is a food-borne zoonotic trematode that affects around 80% livestock of Bangladesh. Triclabendazole (TCBZ), nitroxynil (NTON) and oxyclozanide (OCZN) are frequently used against fascioliasis; however, the current status of potency of these flukicides was unknown. In this study, in vitro efficacy of TCBZ, NTON and OCZN at various concentrations on F. gigantica has been evaluated by relative motility (RM), morphological distortions of apical cone through an inverted microscope, architectural and ultra-structural changes through histopathological and scanning electron microscopy (SEM). It is observed that TCBZ, NTON and OCZN at higher concentrations significantly (P < 0.05) reduced RM of the flukes compared to untreated control. NTON at 150 μg mL−1 was the most potent to reduce the motility within 4 h whereas TCBZ and OCZN were much delayed. Histopathological changes showed swollen, extensive cracking, numerous vacuoles and splitting of the tegument surrounding the spines; spine dislodged from its socket in treated flukes compared to untreated worms. Histopathological changes were more conspicuous at higher doses of TCBZ, NTON and OCZN. SEM has shown the disruption of the apical cone, apart from swelling of the tegument on the ventral surface corrugation and disruption of the ventral apical cone. All these changes indicate that NTON is the most potent in killing flukes in vitro among the tested flukicides and suggest the presence of TCBZ-resistant fluke populations in Bangladesh. It is imperative to explore the in vivo effects of these flukicides and subsequently their molecular mechanisms.

Purpose Fasciola hepatica is a globally distributed trematode that causes significant economic losses. Triclabendazole is the primary pharmacological treatment for this parasite. However, the increasing resistance to triclabendazole limits its efficacy. Previous pharmacodynamics studies suggested that triclabendazole acts by interacting mainly with the β monomer of tubulin. Methods We used a high-quality method to model the six isotypes of F. hepatica β-tubulin in the absence of three-dimensional structures. Molecular dockings were conducted to evaluate the destabilization regions in the molecule against the ligands triclabendazole, triclabendazole sulphoxide and triclabendazole sulphone. Results The nucleotide binding site demonstrates higher affinity than the binding sites of colchicine, albendazole, the T7 loop and pβVII ( p  < 0.05). We suggest that the binding of the ligands to the polymerization site of β-tubulin can lead a microtubule disruption. Furthermore, we found that triclabendazole sulphone exhibited significantly higher binding affinity than other ligands ( p  < 0.05) across all isotypes of β-tubulin. Conclusions Our investigation has yielded new insight on the mechanism of action of triclabendazole and its sulphometabolites on F. hepatica β-tubulin through computational tools. These findings have significant implications for ongoing scientific research ongoing towards the discovery of novel therapeutics to treat F. hepatica infections.

Fascioliasis is a neglected zoonotic disease that infects humans and ruminant species worldwide. In the absence of vaccines, control of fascioliasis is primarily via anthelminthic treatment with triclabendazole (TCBZ). Parasitic flatworms, including Fasciola hepatica, are active secretors of extracellular vesicles (EVs), but research has not been undertaken investigating EV anthelmintic sequestration. Adult F. hepatica were cultured in lethal and sub-lethal doses of TCBZ and its active metabolites, in order to collect EVs and evaluate their morphological characteristics, production and anthelmintic metabolite content. Transmission electron microscopy demonstrated that F. hepatica exposed to TCBZ and its metabolites produced EVs of similar morphology, compared to non-TCBZ exposed controls, even though TCBZ dose and/or TCBZ metabolite led to measurable structural changes in the treated F. hepatica tegument. qNano particle analysis revealed that F. hepatica exposed to TCBZ and its metabolites produced at least five times greater EV concentrations than non-TCBZ controls. A combined mass spectrometry and qNano particle analysis confirmed the presence of TCBZ and the TCBZ–sulphoxide metabolite in anthelmintic exposed EVs, but limited TCBZ sulphone was detectable. This data suggests that EVs released from adult F. hepatica have a biological role in the sequestration of TCBZ and additional toxic xenobiotic metabolites.

Leishmaniasis is a neglected tropical disease, with approximately 1 million new cases and 30,000 deaths reported every year worldwide. Given the lack of adequate medication for treating leishmaniasis, drug repositioning is essential to save time and money when searching for new therapeutic approaches. This is particularly important given leishmaniasis's status as a neglected disease. Available treatments are still far from being fully effective for treating the different clinical forms of the disease. They are also administered parenterally, making it challenging to ensure complete treatment, and they are extremely toxic, in some cases, causing death. Triclabendazole (TCBZ) is a benzimidazole used to treat fasciolosis in adults and children. It presents a lower toxicity profile than amphotericin B (AmpB) and is administered orally, making it an attractive candidate for treating other parasitoses. The mechanism of action for TCBZ is not yet well understood, although microtubules or polyamines could potentially act as a pharmacological target. TCBZ has already shown antiproliferative activity against , and . However, further investigations are still necessary to elucidate the mechanisms of action of TCBZ. Cytotoxicity assay was performed by MTT assay. Cell inhibition (CI) values were obtained according to the equation CI = (O.D treatment x 100/O.D. negative control). For Infection evaluation, fixated cells were stained with Hoechst and read at Operetta High Content Imaging System (Perkin Elmer). For growth curves, cell culture absorbance was measured daily at 600 nm. For the synergism effect, Fractional Inhibitory Concentrations (FICs) were calculated for the IC50 of the drugs alone or combined. Mitochondrial membrane potential (DYm), cell cycle, and cell death analysis were evaluated by flow cytometry. Reactive oxygen species (ROS) and lipid quantification were also determined by fluorimetry. Treated parasites morphology and ultrastructure were analyzed by electron microscopy. The selectivity index (SI = CC50/IC50) of TCBZ was comparable with AmpB in promastigotes and amastigotes of . Evaluation of the cell cycle showed an increase of up to 13% of cells concentrated in S and G2, and morphological analysis with scanning electron microscopy showed a high frequency of dividing cells. The ultrastructural analysis demonstrated large cytoplasmic lipid accumulation, which could suggest alterations in lipid metabolism. Combined administration of TCBZ and AmpB demonstrated a synergistic effect against intracellular amastigote forms with cSFICs of 0.25. Considering that TCBZ has the advantage of being inexpensive and administrated orally, our results suggest that TCBZ, combined with AmpB, is a promising candidate for treating leishmaniasis with reduced toxicity.

We conducted a retrospective cohort study of children who had chronic fascioliasis in the highlands of Peru to determine triclabendazole treatment efficacy. Children passing Fasciola eggs in stool were offered directly observed triclabendazole treatment (>1 doses of 10 mg/kg). Parasitologic cure was evaluated by using microscopy of stool 1–4 months after each treatment. A total of 146 children who had chronic fascioliasis participated in the study; 53% were female, and the mean ± SD age was 10.4 ± 3.1 years. After the first treatment, 55% of the children achieved parasitologic cure. Cure rates decreased after the second (38%), third (30%), and fourth (23%) treatments; 17 children (11.6%) did not achieve cure after 4 treatments. Higher baseline egg counts and lower socioeconomic status were associated with triclabendazole treatment failure. Decreased triclabendazole efficacy in disease-endemic communities threatens control efforts. Further research on triclabendazole resistance and new drugs to overcome it are urgently needed.

Triclabendazole is a poorly-water soluble (0.24 μg/mL) compound classified into the Class II/IV of the Biopharmaceutical Classification System. It is the drug of choice to treat fascioliasis, a neglected parasitic disease worldwide disseminated. Triclabendazole is registered as veterinary medicine and it is only available for human treatment as 250 mg tablets. Thus, the aim of this work was to develop novel drug delivery systems based on nanotechnology approaches. The chitosan-based nanocapsules and nanoemulsions of triclabendazole were fully characterized regarding their particle size distribution, polydispersity index and zeta potential, in-vitro release and stability in biological media. Cytotoxicity evaluation and cellular uptake studies using CaCo-2 cell line were also investigated. The results indicated an average hydrodynamic size around ~160 nm were found for unloaded nanoemulsions which were slightly increased up to ~190 nm for loaded one. In contrast, the average hydrodynamic size of the nanocapsules increased from ~160 nm up to ~400 nm when loaded with triclabendazole. The stability studies upon 30 days storage at 4, 25 and 37°C showed that average size of nanoemulsions was not modified with varying amounts of loaded TCBZ while an opposite result was seen in case of loaded nanocapsules. In addition, a slight reduction of zeta potential values over time was observed in both triclabendazole nanosystems. Release of TCBZ from nanoformulations over 6 h in simulated gastric fluid was 9 to 16-fold higher than with untreated TCBZ dispersion. In phosphate buffer saline solution there was no drug release for neither nanocapsules nor nanoemulsions. Cell viabilities studies indicated that at certain concentrations, drug encapsulation can lower its cytotoxic effects when compared to untreated drug. Confocal laser scanning microscopy study has shown that nanocapsules strongly interacted with Caco-2 cells in vitro which could increase the passage time of triclabendazole after oral administration. The results of this study constitute the first step towards the development of nanoformulations intended for the oral delivery of anti-parasitic drugs of enhanced bioavailability.