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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.

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.

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.

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.

Fasciola hepatica, the causative agent of fasciolosis, is a global threat to public health, animal welfare, agricultural productivity, and food security. In the ongoing absence of a commercial vaccine, independent emergences of anthelmintic-resistant parasite populations worldwide are threatening the sustainability of the few flukicides presently available, and particularly triclabendazole (TCBZ) as the drug of choice. Consequently, prognoses for future fasciolosis control and sustained TCBZ application necessitate improvements in diagnostic tools to identify anthelmintic efficacy. Previously, we have shown that proteomic fingerprinting of F. hepatica excretory/secretory (ES) products offered new biomarkers associated with in vitro TCBZ-sulfoxide (SO) recovery or death. In the current paper, two of these biomarkers (calreticulin (CRT) and triose phosphate isomerase (TPI)) were recombinantly expressed and evaluated to measure TCBZ efficacy via a novel approach to decipher fluke molecular phenotypes independently of molecular parasite resistance mechanism(s), which are still not fully characterised or understood. Our findings confirmed the immunoreactivity and diagnostic potential of the present target antigens by sera from TCBZ-susceptible (TCBZ-S) and TCBZ-resistant (TCBZ-R) F. hepatica experimentally infected sheep.

Background Metabolic reprogramming is a hallmark of cancer cells, enabling them to meet the heightened energetic and biosynthetic demands required for rapid growth and proliferation. Recently, non-canonical functions of metabolic enzymes have garnered significant attention in cancer research. Pyruvate kinase 2 (PKM2) has been identified as a key player in transcriptional regulation within the nucleus, presenting new opportunities for therapeutic interventions in cancer. Methods In this study, the cells (A549 and H1299) were treated with indicator concentration of triclabendazole. The effects of triclabendazole on proliferation was detected by CCK8 assay, colony formation assay, EdU staining, and cell count assay. A tumorigenesis study in nude mice was performed to demonstrate the inhibitory effect of triclabendazole on tumor growth. PKM2 nuclear translocation, HDAC6-mediated deacetylation, glycolytic flux downregulation, and activation of AMPK/mTOR signaling pathway were used to elucidate the mechanistic role of triclabendazole in lung cancer progression. Results This study discovered that triclabendazole, a novel benzimidazole derivative, commonly used against Fasciola hepatolithiasis , effectively inhibited the nuclear translocation of PKM2. This inhibition resulted in the downregulation of glycolytic flux, ultimately suppressing lung cancer cell proliferation. Notably, triclabendazole reduced PKM2 acetylation by promoting the interaction between PKM2 and histone deacetylase 6 (HDAC6), thus blocking PKM2 nuclear localization. Moreover, we also demonstrated that triclabendazole-mediated inhibition of cell proliferation is driven by the downregulation of glycolysis, which enhanced AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling. Consistently, triclabendazole administration significantly inhibited tumor growth in vivo, correlating with the blockade of PKM2 nuclear translocation and lactate production decreased. Conclusion Our findings revealed that triclabendazole inhibits PKM2 nuclear localization and glycolysis through an HDAC6-dependent mechanism, leading to the activation of AMPK/mTOR signaling and suppression of lung cancer cell proliferation. These results suggested that triclabendazole holds promise as a potential therapeutic agent, with the HDAC6-PKM2 axis representing a novel target for lung cancer treatment.

, a globally distributed helminth, causes fasciolosis, a disease with significant health and economic impacts. Variability in triclabendazole (TCBZ) efficacy and emerging resistance are remaining challenges. Evidence suggests that the gut microbiome influences host-helminth interactions and is associated with anthelmintic effects, but its association with human infection and TCBZ efficacy is not well understood. In this study, we investigated the relationship between infection and the gut microbiome through metagenomic shotgun sequencing of 30 infected and 60 age- and sex-matched uninfected individuals from Peru. Additionally, we performed a longitudinal analysis to evaluate microbiome dynamics in relation to TCBZ treatment response. Infection was associated with specific microbial taxonomic and functional features, including higher abundance of sp900547015, A sp000285855, and sp002299635 species, and enrichment of microbial pathways linked to survival under stress and depletion of pathways for microbial growth. Unexpectedly, we identified that responders to TCBZ treatment (who cleared infection) harbored many microbiome features significantly different relative to non-responders, both before and after treatment. Specifically, the microbiomes of responders had a higher abundance Firmicutes A and species as well as phospholipid synthesis and glucuronidation pathways, while non-responders had higher abundance of Actinobacteria species including several from the and genera, and shunt and amino acid biosynthesis pathways. Our findings underscore the impact of helminth infection on gut microbiome and suggest a potential role of gut microbiota in modulating TCBZ efficacy, offering novel insights into -microbiome interactions and paving the way for microbiome-informed treatment approaches.

Triclabendazole is an effective and well‐tolerated treatment for human fascioliasis. A placebo‐ and positive‐controlled, four‐sequence by four‐period crossover study was conducted in 45 healthy participants to assess the effect of therapeutic (10 mg/kg twice daily [b.i.d.] for 1 day) and supratherapeutic (10 mg/kg b.i.d. for 3 days) oral doses of triclabendazole on corrected QT (QTc) interval prolongation. Moxifloxacin (400 mg, oral) was used as a positive control. The highest mean placebo‐corrected change from baseline in QTcF (ΔΔQTcF) on day 4 with triclabendazole was 9.2 at therapeutic dose ms and 21.7 ms at supratherapeutic dose, at 4 h postdose. The upper limit of the two‐sided 90% confidence interval exceeded 10 ms across all timepoints, except at predose timepoint on day 4 in the therapeutic group indicating that an effect of triclabendazole on cardiac repolarization could not be excluded. However, triclabendazole had no clinically significant effects on heart rate and cardiac conduction at the studied doses. In the moxifloxacin group, the mean ΔΔQTcF peak value was 13.7 ms at 3 h on day 4. The assay sensitivity was confirmed. Maximum plasma concentration of triclabendazole, sulfoxide metabolite, and sulfone metabolite occurred at ~3‐, 4‐, and 6‐h postdose, respectively. No deaths, serious adverse events, study discontinuations due to treatment‐emergent adverse events, or clinically relevant abnormalities in laboratory evaluations and vital sign values were observed. This study showed that triclabendazole had no clinically relevant effects on heart rate and cardiac conduction; however, an effect on cardiac repolarization (ΔΔQTcF >10 ms) could not be excluded.

Fasciolosis an economically important global disease of ruminants in the temperate and tropical regions, caused by Fasciola hepatica and F. gigantica, respectively, also poses a potential zoonotic threat. In India alone it causes huge losses to stakeholders. Anthelmintics including triclabendazole have been used to control this menace but the emerging resistance against the available compounds necessitates identification of novel and alternative therapeutic measures involving plant derived natural compounds for their anthelmintic potential. Thymoquinone (T) and curcumin (C), the active ingredients of Nigella sativa and Curcuma longa respectively have been used as antiparasitic agents but the information on their flukicidal effect is very limited. Adult flukes of F. gigantica were in vitro exposed to different concentrations of thymoquinone and curcumin separately for 3h at 37+ 1°C. A significant (p<0.05) reduction in the worm motility at 60 μM concentration of both T and C was observed though all the worms remained alive after 3h exposure, whereas the effect on egg shedding was statistically insignificant. Pronounced tegumental disruptions and erosion of spines in the posterior region and around the acetabulum was evident. A significant (p<0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed, while protein carbonylation increased differentially. A significant inhibition of CathepsinL (CatL) gene expression in thymoquinone treated worms was also evident. Further, in silico molecular docking of T and C with CatL revealed a stronger interaction of curcumin with the involvement of higher number of amino acids as compared to thymoquinone that could be more effective in inhibiting the antioxidant enzymes of F. gigantica. It is concluded that both the compounds understudy will decrease the detoxification ability of F. gigantica, while inhibition of CatL will significantly affect their virulence potential. Thus, both thymoquinone and curcumin appeared to be promising anthelmintic compounds for further investigations.

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.