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Background: Alveolar echinococcosis (AE) is a chronic zoonosis caused by the larval form of Echinococcus multilocularis (E. multilocularis). Current chemotherapy against AE has relied on albendazole and mebendazole, which only exhibit parasitostatic and not parasiticidal efficacy. Therefore, novel compounds for the treatment of this disease are needed., Methods: Phosphoglucose isomerase (PGI) assays were used for compound screening of seven neonicotinoids. The anti-parasitic effects of thiacloprid were then evaluated on E. multilocularis metacestode vesicles, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFF) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. Glucose consumption in E. multilocularis protoscoleces and germinal cells was assessed by measuring uptake of 2-deoxyglucose (2-DG). Molecular docking was used to evaluate the potential binding sites of thiacloprid to acetylcholine receptors. In vivo efficacy of thiacloprid was evaluated in mice by secondary infection with E. multilocularis. In addition, ELISA and flow cytometry were used to evaluate the effects of cytokines and T lymphocyte subsets after thiacloprid treatment. Furthermore, collagen deposition and degradation in the host lesion microenvironment were evaluated., Results: We found that thiacloprid is the most promising compound, with an IC 50 of 4.54 ± 1.10 μM and 2.89 ± 0.34 μM, respectively, against in vitro-cultured E. multilocularis metacestodes and germinal cells. Thiacloprid was less toxic for HFF and RH mammalian cell lines than for metacestodes. In addition, thiacloprid inhibited the acetylcholinesterase activity in protoscoleces, metacestodes and germinal cells. Thiacloprid inhibited glucose consumption by protoscoleces and germinal cells. Subsequently, transmission electron microscopy revealed that treatment with thiacloprid damaged the germinal layer. In vivo, metacestode weight was significantly reduced following oral administration of thiacloprid at 15 and 30 mg/kg. The level of CD4 + T lymphocytes in metacestodes and spleen increased after thiacloprid treatment. Anti-echinococcosis-related cytokines (IL-2, IL-4, IL-10) were significantly increased. Furthermore, thiacloprid inhibited the expression of matrix metalloproteinases (MMPs 1, 3, 9, 13) and promoted collagen deposition in the host lesion microenvironment., Conclusions: The results demonstrated that thiacloprid had parasiticidal activity against E. multilocularis in vitro and in vivo, and could be used as a novel lead compound for the treatment of AE., (© 2021. The Author(s).)

Background: Alveolar echinococcosis (AE) is a fatal zoonosis caused by the larvae of Echinococcus multilocularis. However, current chemotherapy treatment options are based on benzimidazoles [albendazole (ABZ) and mebendazole], which have limited efficacy. Therefore, novel drugs are necessary for the treatment of this disease., Methods: The anthelmintic effects of crocin were tested on E. multilocularis metacestodes, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFFs) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. The in vivo efficacy of crocin was investigated in mice following secondary infection with E. multilocularis. Furthermore, collagen deposition and degradation in host tissues around the metacestodes were evaluated., Results: In vitro, crocin had a median effective concentration of 11.36 μM against cultured E. multilocularis metacestodes, while it reduced germinal cell viability at a median inhibitory concentration of 10.05 μM. Crocin was less toxic to HFFs and RH mammalian cell lines than to metacestodes. Transmission electron microscopy revealed that crocin treatment resulted in structural damage in the germinal layer. In addition, 60.33 ± 3.06% of protoscoleces were killed by treatment with 10 μM crocin for 7 days, indicating that crocin has a parasiticidal effect. In vivo, the metacestode weight was significantly reduced after the administration of crocin at 50 mg/kg and 100 mg/kg (55.1 and 68.1%, respectively). Metacestode pathology showed structural disruption of the germinal and laminated layers after crocin treatment. The crocin- and ABZ-treated groups presented significant increases in the levels of interleukin (IL)-2 and IL-4. Furthermore, crocin inhibited the expression of matrix metalloproteinases (MMPs) (MMP2 and MMP9) and promoted collagen deposition in the metacestode., Conclusions: Crocin was demonstrated to exert parasiticidal activity against E. multilocularis in vitro and in vivo, and can be developed as a novel drug for the treatment of AE., (© 2021. The Author(s).)

Background: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca 2+ /CaM-CaMKII) in infected mice., Methods: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction., Results: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm., Conclusions: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca 2+ /CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca 2+ /CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Alveolar echinococcosis is one of the most dangerous parasitic zoonoses. This disease, widely distributed in the northern hemisphere, is caused by the metacestode stage of the tapeworm Echinococcus multilocularis. All surgical and non-surgical patients should perform chemotherapy with benzimidazoles, mainly with albendazole. However, the efficacy of albendazole is variable due to its deficient pharmacokinetic properties. Therefore, the need to find new therapeutic alternatives for the treatment of alveolar echinococcosis is evident. Menthol is a natural compound of low toxicity, used in industries such as cosmetics and gastronomy and generally recognized as safe by the Food and Drug Administration. In addition, menthol has important pharmacological effects and is effective against a wide variety of organisms. The development of prodrugs allows improving the pharmacokinetic properties of the parental drug. To improve lipophilicity and therefore the bioavailability of menthol, a novel prodrug called menthol-pentanol was developed by masking the functional polar group of menthol by linking n-pentanol by a carbonate bond. The aim of the current work was to evaluate the in vitro and in vivo efficacy of menthol and menthol-pentanol against E. multilocularis. Menthol-pentanol had a greater protoscolicidal effect than menthol. In addition, the prodrug demonstrated a similar clinical efficacy to albendazole. The increase in lipophilicity of the prodrug with respect to menthol was reflected in an increase in its antiparasitic activity against E. multilocularis. Thus, menthol-pentanol appears as a promising candidate for further evaluation as a potential alternative for the treatment of alveolar echinococcosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report the case of a 65-year-old female patient with hepatic alveolar echinococcosis (AE) caused by Echinococcus multilocularis. This infrequent zoonosis has a considerable morbidity and mortality. The malignant appearing hepatic mass was initially misdiagnosed as cholangiocarcinoma of the right hepatic lobe (segments VII, VIII, and IVa, sized 10.9 cm × 7.6 cm) involving the right and middle hepatic vein and extending close to the left hepatic vein. During exploratory laparotomy, the frozen-section biopsy was indicative of AE (World Health Organization [WHO] classification: stage P 3 N 0 M 0 ). Due to the high operative risk, it was decided to pretreat the patient with albendazole as inductive therapy in order to remove the AE secondarily in accordance with the patient's request. After year-long treatment with albendazole (under strict control of the maximum blood levels), a right hemihepatectomy was successfully performed. Postoperative treatment with albendazole had to be stopped prematurely after 11 months due to considerable subjective intolerance and a more-than-tenfold elevation of transaminases despite normal therapeutic albendazole blood levels. A 18 F-FDG-PET/CT scan revealed no evidence of AE residues. Conducting follow-up examinations by 18 F-FDG-PET/CT scans every 2 years is planned in order to recognize possible recurrence at an early stage., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

The essential oil (EO) of Thymus capitatus, seven fractions (F1-F7) obtained from silica gel chromatography, and several pure EO components were evaluated with respect to in vitro activities against Echinococcus multilocularis metacestodes and germinal layer (GL) cells. Attempts to evaluate physical damage in metacestodes by phosphoglucose isomerase (PGI) assay failed because EO and F1-F7 interfered with the PGI-activity measurements. A metacestode viability assay based on Alamar Blue, as well as transmission electron microscopy, demonstrated that exposure to EO, F2 and F4 impaired metacestode viability. F2 and F4 exhibited higher toxicity against metacestodes than against mammalian cells, whereas EO was as toxic to mammalian cells as to the parasite. However, none of these fractions exhibited notable activity against isolated E. multilocularis GL cells. Analysis by gas chromatography-mass spectrometry showed that carvacrol was the major component of the EO (82.4%), as well as of the fractions F3 (94.4%), F4 (98.1%) and F5 (90.7%). Other major components of EO were β-caryophyllene, limonene, thymol and eugenol. However, exposure of metacestodes to these components was ineffective. Thus, fractions F2 and F4 of T. capitatus EO contain potent anti-echinococcal compounds, but the activities of these two fractions are most likely based on synergistic effects between several major and minor constituents.

To evaluate the effect of mitogen-activated protein kinase (MAPK) signal transduction pathway inhibitors against alveolar echinococcosis in vitro and in vivo, Echinococcus multilocularis metacestode cysts and protoscolices were obtained from infected mice. Protein chip technology was utilized to screen for key highly expressed target proteins in the MAPK pathway in this parasite and their corresponding inhibitors. Four-week-old Balb/c female mice used for the in vivo experiment underwent inoculation of E. multilocularis by intraperitoneal injection, as well as intragastric administration of MAPK inhibitors for 6 wk. We included 6 groups of mice: a phosphate-buffered saline (PBS) group (negative control); an albendazole-treated group (positive group); and 4 experimental groups treated with TRx0237 mesylate, GDC-0994, pifithrin-β hydrobromide, or Selonsertib. Echinococcus multilocularis protoscolices were collected and cultured in 1066 medium with penicillin/streptomycin and 10% fetal bovine serum. The in vitro experiment included a PBS group (negative control), a dimethyl sulfoxide-treated group (solvent group), and 4 inhibitor-treated groups as in the in vivo experiment (experimental groups). Each inhibitor group received 4 drug concentrations (5, 30, 55, and 80 μM), and the experiment was performed in triplicate per sample. Fluorescence microscopy was used to evaluate the survival rate of the protoscolices every 48 hr beginning from the first 24 hr. The same grouping was used to evaluate cytotoxicity on E. multilocularis germinal cells and L02 cells. The average weights of E. multilocularis metacestode cyst tissue from each group of the in vivo experiment were 873 mg (PBS), 335 mg (albendazole), 323 mg (TRx0237 mesylate), 420 mg (GDC-0994), 340 mg (pifithrin-β hydrobromide), and 642 mg (Selonsertib). Results showed albendazole, TRx0237 mesylate, and pifithrin-β hydrobromide had significant inhibitory effects on inhibition of E. multilocularis. We found a positive correlation between drug concentrations and the inhibitory effects seen in the in vitro experiment, with the differences in contrast with the control group becoming statistically significant after 72 hr of treatment ( P < 0.05). The inhibition rates of TRx0237 mesylate to germinal cells by drug concentration were 23.73, 46.59, 74.71, and 77.44%. Other drugs had no effect on germinal cells. All the inhibitors had low toxicity on L02 cells. Inhibitors of the MAPK signal transduction pathway showed significant inhibitory effects on E. multilocularis, suggesting these may be potential candidates for the treatment of alveolar echinococcosis.

Human cystic and alveolar echinococcosis are helmintic zoonotic diseases caused by infections with the larval stages of the cestode parasites Echinococcus granulosus and E. multilocularis, respectively. Both diseases are progressive and chronic, and often fatal if left unattended for E. multilocularis. As a treatment approach, chemotherapy against these orphan and neglected diseases has been available for more than 40 years. However, drug options were limited to the benzimidazoles albendazole and mebendazole, the only chemical compounds currently licensed for treatment in humans. To compensate this therapeutic shortfall, new treatment alternatives are urgently needed, including the identification, development, and assessment of novel compound classes and drug targets. Here is presented a thorough overview of the range of compounds that have been tested against E. granulosus and E. multilocularis in recent years, including in vitro and in vivo data on their mode of action, dosage, administration regimen, therapeutic outcomes, and associated clinical symptoms. Drugs covered included albendazole, mebendazole, and other members of the benzimidazole family and their derivatives, including improved formulations and combined therapies with other biocidal agents. Chemically synthetized molecules previously known to be effective against other infectious and non-infectious conditions such as anti-virals, antibiotics, anti-parasites, anti-mycotics, and anti-neoplastics are addressed. In view of their increasing relevance, natural occurring compounds derived from plant and fungal extracts are also discussed. Special attention has been paid to the recent application of genomic science on drug discovery and clinical medicine, particularly through the identification of small inhibitor molecules tackling key metabolic enzymes or signalling pathways.

Intestinal cestodes are infecting millions of people and livestock worldwide, but treatment is mainly based on one drug: praziquantel. The identification of new anti-cestodal compounds is hampered by the lack of suitable screening assays. It is difficult, or even impossible, to evaluate drugs against adult cestodes in vitro due to the fact that these parasites cannot be cultured in microwell plates, and adult and larval stages in most cases represent different organisms in terms of size, morphology, and metabolic requirements. We here present an in vitro-drug screening assay based on Echinococcus multilocularis protoscoleces, which represent precursors of the scolex (hence the anterior part) of the adult tapeworm. This movement-based assay can serve as a model for an adult cestode screen. Protoscoleces are produced in large numbers in Mongolian gerbils and mice, their movement is measured and quantified by image analysis, and active compounds are directly assessed in terms of morphological effects. The use of the 384-well format minimizes the amount of parasites and compounds needed and allows rapid screening of a large number of chemicals. Standard drugs showed the expected dose-dependent effect on movement and morphology of the protoscoleces. Interestingly, praziquantel inhibited movement only partially within 12 h of treatment (at concentrations as high as 100 ppm) and did thus not act parasiticidal, which was also confirmed by trypan blue staining. Enantiomers of praziquantel showed a clear difference in their minimal inhibitory concentration in the motility assay and (R)-(-)-praziquantel was 185 times more active than (S)-(-)-praziquantel. One compound named MMV665807, which was obtained from the open access MMV (Medicines for Malaria Venture) Malaria box, strongly impaired motility and viability of protoscoleces. Corresponding morphological alterations were visualized by scanning electron microscopy, and demonstrated that this compound exhibits a mode of action clearly distinct from praziquantel. Thus, MMV665807 represents an interesting lead for further evaluation.

The parasitic tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a serious zoonotic infection present in Europe that can be fatal. The United Kingdom currently has E. multilocularis free status but the possibility of introduction exists, most likely via an imported or returning dog or other deliberately introduced animal that has not had anthelmintic treatment. We have developed a model to predict the probability of successfully eliminating a focal outbreak of E. multilocularis using a programme of anthelmintic bait distribution. We investigated three different potential control programmes, each with 36 monthly campaigns commencing five, ten or 15 years after disease introduction over an area of 2827km 2 . We assumed equilibrium disease prevalence of 30%, 40% and 55% based on the range of values reported across Europe. However, for all of these scenarios, equilibrium had not been reached at five to 15 years after introduction and simulated local prevalence values were between 0.5% and 28%. We found that it is possible to eliminate the disease with a 38%-86% success rate if control is started five years after introduction, dropping to 0% to 56% if control is delayed until 15 years after introduction, depending upon the prevalence equilibrium. We have also estimated the costs involved in these programmes to be from €7 to €12 million (2013 prices)., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes.

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.

This study investigated the effects of oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine (MEF) and identified proteins that bind to MEF in parasite extracts and human cells by affinity chromatography. In a pilot experiment, MEF treatment was applied 5 days per week and was intensified by increasing the dosage stepwise from 12.5 mg/kg to 200 mg/kg during 4 weeks followed by treatments of 100 mg/kg during the last 7 weeks. This resulted in a highly significant reduction of parasite weight in MEF-treated mice compared with mock-treated mice, but the reduction was significantly less efficacious compared with the standard treatment regimen of albendazole (ABZ). In a second experiment, MEF was applied orally in three different treatment groups at dosages of 25, 50 or 100 mg/kg, but only twice a week, for a period of 12 weeks. Treatment at 100 mg/kg had a profound impact on the parasite, similar to ABZ treatment at 200 mg/kg/day (5 days/week for 12 weeks). No adverse side effects were noted. To identify proteins in E. multilocularis metacestodes that physically interact with MEF, affinity chromatography of metacestode extracts was performed on MEF coupled to epoxy-activated Sepharose(®), followed by SDS-PAGE and in-gel digestion LC-MS/MS. This resulted in the identification of E. multilocularis ferritin and cystatin as MEF-binding proteins. In contrast, when human cells were exposed to MEF affinity chromatography, nicotinamide phosphoribosyltransferase was identified as a MEF-binding protein. This indicates that MEF could potentially interact with different proteins in parasites and human cells., (Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

This study aimed to investigate the pharmacology and anti-parasitic efficacy of albendazole-chitosan microspheres (ABZ-CS-MPs) for established intraperitoneal infections of Echinococcus multilocularis metacestodes in an experimental murine model. Male outbred Kunming mice infected with E. multilocularis Metacestodes were administered with three ABZ formulations, namely, ABZ-CS-MPs, Liposome-Albendazole (L-ABZ), and albendazole tablet (ABZ-T). Each of the ABZ formulations was given orally at three different doses of 37.5, 75, and 150 mg/kg, three times a week for 12 weeks postinfection. After administering the drugs, we monitored the pharmacological performance and anti-parasitic efficacy of ABZ-CS-MPs compared with L-ABZ, and ABZ-T treated mice. ABZ-CS-MPs reduced the weight of tissues containing E. multilocularis metacestodes most effectively compared with the ABZ-T group and untreated controls. Metacestode grown was Highly suppressed during treatment with ABZ-CS-MPs. Significantly higher plasma levels of ABZ metabolites were measured in mice treated with ABZ-CS-MPs or L-ABZ compared with ABZ-T. In particular, enhanced ABZ-sulfoxide concentration profiles were observed in the mice given 150 mg/kg of ABZ-CS-MPs, but not in the mice treated with L-ABZ. Histological examination showed that damages caused disorganization of both the germinal and laminated layers of liver hyatid cysts, demolishing their characteristic structures after treatment with ABZ-CS-MPs or L-ABZ. Over time, ABZ-CS-MPs treatment induced a shift from Th2-dominant to Th1-dominant immune response. CS-MPs As a new carrier exhibited improved absorption and increased bioavailability of ABZ in the treatment of E. multilocularis infections in mice.

The larval (metacestode) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a mainly hepatic disease characterized by continuous asexual proliferation of metacestodes by exogenous budding, resulting in the tumor-like, infiltrative growth of the parasite lesion. Current chemotherapeutical treatment of AE relies on the use of benzimidazoles, albendazole (ABZ) and mebendazole, but these drugs act parasitostatic rather than parasitocidal, and due to their low success rate they imply a lifelong application causing severe side effects. Thymol is one of the major components of the essential oils of Thymus and is a widely known anti-microbial agent. The aim of the present work was to compare the efficacy of albendazole (ABZ) and thymol separately or combined on E. multilocularis protoscoleces and metacestodes. For this purpose, microscopical examinations at different time points were carried out. Moreover the tegumentary enzyme gamma glutamyl transferase (GGT) was measured to quantify the damage in metacestodes. Even though treatments of in vitro cultured E. multilocularis protoscoleces or metacestodes with ABZ or/and thymol showed that the drugs have an adverse effect on parasite viability, the combination of the two compounds at the concentration of 10μg/ml showed the maximum anti-parasitic effect. Three days postincubation the first effects of the treatment were detected on protoscoleces and a marked reduction in viability (33%) was registered at day 18. Incubation of E. multilocularis metacestodes in the presence of ABZ 10μg/ml+thymol 10μg/ml during 10 days resulted in dramatic alterations such as strongly irregular and fissured surface and markedly disrupted vesicles. Scanning electron microscopy showed that protoscoleces as well as the germinal layer of E. multilocularis metacestodes were dramatically damaged following ABZ or/and thymol treatment. Also an important increase of tegumentary enzyme GGT was registered after 72h postincubation with both drugs. The data reported in this article demonstrate a clear in vitro effect of ABZ+thymol against E. multilocularis protoscoleces and metacestodes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Deworming wild foxes by baiting with the anthelmintic praziquantel is being established as a preventive technique against environmental contamination with Echinococcus multilocularis eggs. Improvement of the cost-benefit performance of baiting treatment is required urgently to raise and maintain the efficacy of deworming. We established a spatial model of den site selection by urban red foxes, the definitive host, to specify the optimal micro-habitats for delivering baits in a new modeling approach modified for urban fox populations., Methods: The model was established for two cities (Obihiro and Sapporo) in Hokkaido, Japan, in which a sylvatic cycle of E. multilocularis is maintained. The two cities have different degrees of urbanization. The modeling process was designed to detect the best combination of key environmental factors and spatial scale that foxes pay attention to most (here named 'heeding range') when they select den sites. All possible models were generated using logistic regression analysis, with "presence" or "absence" of fox den as the objective variable, and nine landscape categories customized for urban environments as predictor variables to detect the best subset of predictors. This procedure was conducted for each of ten sizes of concentric circles from dens and control points to detect the best circle size. Out of all models generated, the most parsimonious model was selected using Akaike's Information Criterion (AIC) inspection., Results: Our models suggest that fox dens in Obihiro are located at the center of a circle with 500 m radius including low percentages of wide roads, narrow roads, and occupied buildings, but high percentages of green covered areas; the dens in Sapporo within 300 m radius with low percentages of wide roads, occupied buildings, but high percentages of riverbeds and green covered areas. The variation of the models suggests the necessity of accumulating models for various types of cities in order to reveal the patterns of the model., Conclusions: Our denning models indicating suitable sites for delivering baits will improve the cost-benefit performance of the campaign. Our modeling protocol is suitable for the urban landscapes, and for extracting the heeding range when they select the den sites.

The current chemotherapeutic treatment of alveolar echinococcosis (AE) in humans is based on albendazole and/or mebendazole. However, the costs of treatment, life-long consumption of drugs, parasitostatic rather than parasiticidal activity of chemotherapy, and high recurrence rates after treatment interruption warrant more efficient treatment options. Experimental treatment of mice infected with Echinococcus multilocularis metacestodes with fenbendazole revealed similar efficacy to albendazole. Inspection of parasite tissue from infected and benzimidazole-treated mice by transmission electron microscopy (TEM) demonstrated drug-induced alterations within the germinal layer of the parasites, and most notably an almost complete absence of microtriches. On the other hand, upon in vitro exposure of metacestodes to benzimidazoles, no phosphoglucose isomerase activity could be detected in medium supernatants during treatment with any of these drugs, indicating that in vitro treatment did not severely affect the viability of metacestode tissue. Corresponding TEM analysis also revealed a dramatic shortening/retraction of microtriches as a hallmark of benzimidazole action, and as a consequence separation of the acellular laminated layer from the cellular germinal layer. Since TEM did not reveal any microtubule-based structures within Echinococcus microtriches, this effect cannot be explained by the previously described mechanism of action of benzimidazoles targeting β-tubulin, thus benzimidazoles must interact with additional targets that have not been yet identified. In addition, these results indicate the potential usefulness of fenbendazole for the chemotherapy of AE., (Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The search for novel therapeutic options to cure alveolar echinococcosis (AE), due to the metacestode of Echinococcus multilocularis, is ongoing, and these developments could also have a profound impact on the treatment of cystic echinococcosis (CE), caused by the closely related Echinococcus granulosus s.l. Several options are being explored. A viable strategy for the identification of novel chemotherapeutically valuable compounds includes whole-organism drug screening, employing large-scale in vitro metacestode cultures and, upon identification of promising compounds, verification of drug efficacy in small laboratory animals. Clearly, the current focus is targeted towards broad-spectrum anti-parasitic or anti-cancer drugs and compound classes that are already marketed, or that are in development for other applications. The availability of comprehensive Echinococcus genome information and gene expression data, as well as significant progress on the molecular level, has now opened the door for a more targeted drug discovery approach, which allows exploitation of defined pathways and enzymes that are essential for the parasite. In addition, current in vitro and in vivo models that are used to assess drug efficacy should be optimized and complemented by methods that give more detailed information on the host-parasite interactions that occur during drug treatments. The key to success is to identify, target and exploit those parasite molecules that orchestrate activities essential to parasite survival., (© A. Hemphill et al., published by EDP Sciences, 2014.)

Artemisinin is an antimalarial sesquiterpene lactone that contains a 1,2,4-trioxane heterocycle. Dihydroartemisinin and artesunate demonstrated activity against Echinococcus multilocularis metacestodes in vitro but were not effective in a mouse model. In this study, the in vitro effects of a small library of synthetic ozonides (1,2,4-trioxolanes) were investigated. Initial compound screening against E. multilocularis metacestodes was performed at 20μM, and selected ozonides were further assessed in dose-response studies in metacestode cultures and mammalian cells. Transmission electron microscopy (TEM) was employed to characterise compound-induced structural alterations. At 20μM, the most potent ozonides (OZ401, OZ455, OZ491 and OZ494) led to death of ca. 60-100% of the parasites. Subsequent dose-response experiments demonstrated that OZ401, OZ455 and OZ491, which contain an aminopropylether substructure, were the most potent, with 50% inhibitory concentrations ranging from 11μM to 14μM. Cytotoxicity for these three ozonides, assessed in human foreskin fibroblasts, rat hepatoma cells and green monkey epithelial kidney (Vero) cells, was evident only at high concentrations. TEM demonstrated that OZ401 and OZ491 treatment induced considerable metabolic impairment in metacestodes at 1 day post exposure. At Day 3 post exposure, the germinal layer was severely distorted, although some intact cells were still visible, demonstrating that not all cell types in the parasite tissue were equally affected. Complete destruction of the germinal layer was noted at 5 days post exposure. Synthetic ozonides could represent interesting leads that will be further investigated in a suitable in vivo model of E. multilocularis infection., (Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemotherapy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating proliferation of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite's stem cell population., (© K. Brehm and U. Koziol, published by EDP Sciences, 2014.)

Alveolar echinococcosis (AE) caused by the cestode Echinococcus multilocularis (E. multilocularis) is endemic in wide areas of the Northern hemisphere. Untreated AE progresses and leads to death in more than 90% of cases. Until the advent of benzimidazoles, no antihelminthic drugs were available to cure AE. Benzimidazoles have greatly improved the prognosis of patients with AE. However, benzimidazoles have only a parasitostatic effect on E. multilocularis. Albendazole (ABZ) must sometimes be withdrawn because of adverse events. Alternative drugs are urgently needed. The antihelminthic triclabendazole (TCZ) and clorsulon (CLS) are more effective than ABZ to cure infections by the liver flukes Fasciola spp. The efficacy of TCZ and CLS was investigated on an in vitro culture of E. multilocularis larval tissue. E. multilocularis vesicles were evaluated for their morphology before and after adding TCZ, TCZ sulfoxide (TCZSX) and CLS to the larval tissue culture. TCZ at the concentrations of 20 μg/ml culture solution led to maximum vesicle damage within 12 days and of 25 μg/ml within 13 days, and TCZSX at the concentrations of 20 μg/ml within 20 days and of 25 μg/ml within 14 days. Contrary, CLS added at 5, 10 and 15 μg/ml to culture solution did not lead to any vesicle damage. TCZ is a promising further candidate drug for the treatment of AE.

The metacestode stage of Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a lethal zoonosis with very limited treatment options. Chemotherapy of AE currently employs benzimidazoles (BZs); however, these exert only a parasitostatic action in vivo and have to be given life-long. In the search for novel drug targets, we have concentrated on parasite signalling pathways. Here we report significant antiparasitic effects of imatinib, an ABL kinase inhibitor that is in clinical use to treat certain cancers. At concentrations of 25 μM, imatinib was highly effective in killing Echinococcus stem cells, metacestode vesicles and protoscoleces in vitro. Moreover, already at concentrations as low as 10 μM, imatinib significantly inhibited the formation of metacestode vesicles from parasite stem cells, inactivated 50% of vesicles after 7 days, and induced morphological alterations in the metacestode upon short-term treatment. We also demonstrate that E. multilocularis larvae express enzymes with high homology to previously identified ABL-like kinases that act as imatinib targets in Schistosoma mansoni. In particular, amino acids known to mediate the binding of imatinib to target kinases are well conserved between human and Echinococcus ABL kinases. Taken together, these data demonstrate effective inactivation of Echinococcus larvae using imatinib concentrations that do not significantly affect cultivated human cells, indicating that imatinib might be a promising alternative to BZs in anti-AE chemotherapy. Furthermore, imatinib can also act as a lead substance for the identification of related compounds with higher antiparasitic activity, the identification of which will be facilitated by the Echinococcus ABL kinase sequences determined in this study., (Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Two series of η(6)-areneruthenium(II) phosphite complexes were prepared, characterized, and evaluated in vitro for their toxic potential against Echinococcus multilocularis metacestodes. Neutral complexes of general formula [(η(6)-p-cymene)RuCl(2){P(OR)(3)}] (R = Et, (i)Pr, Ph) with two easily exchangable chloride ligands showed only minor toxicity, whereas the substitution of these moieties against a β-diketonate (2,2,6,6-tetramethylheptanedionate) ligand led to hydrolytically stable complex salts of type [(η(6)-p-cymene)Ru(β-diketonate){P(OR)(3)}][BF(4)] (R = Et, (i)Pr, Ph) with comparable in vitro toxicity (50% PGI release at c = 1.4 - 4.7 μM) to the reference drug nitazoxanide (50% PGI release at c = 1.2 μM). In addition, the latter complexes were highly toxic against rat hepatoma cells (IC(50) = 0.40-2.0 μM) and less toxic against human foreskin fibroblasts (IC(50) = 1.1-2.9 μM) and Vero cells (IC(50) = 1.2-8.9 μM). The measured cytotoxicities against mammalian cells are, to the best of our knowledge, among the highest ever observed for ruthenium-based complexes. In conclusion, complex salts of type [(η(6)-p-cymene)Ru(β-diketonate){P(OR)(3)}][BF(4)] might be interesting candidates for further development toward anthelmintic drugs and/or highly cytotoxic metal compounds.

Benzimidazoles, namely albendazole (ABZ) and mebendazole, are the only drugs licensed for the treatment of inoperable alveolar echinococcosis. In addition, amphotericin B (AMB) has shown effect against Echinococcus multilocularis as salvage treatment in humans. Both benzimidazoles and AMB are only parasitostatic against E. multilocularis and toxicity may limit long-term use. In the present study we examined the effect of combined treatment between ABZ and AMB on E. multilocularis larvae in an in vitro model. Vesicles were grown in a tissue culture model of metacestodes and hepatocytes. Drugs were added to the culture and the destructive effect on the vesicles was visually observed. Sequential application of ABZ and AMB yielded effective destruction of vesicles which was faster than the application of AMB alone. However, simultaneous application of ABZ and AMB had an inhibitory effect on vesicle destruction. After discontinuation of drug application, regrowth of vesicles occurred, hereby proving the parasitostatic effect of combined treatment against E. multilocularis larvae. Due to an inhibitory effect between ABZ and AMB against E. multilocularis larvae, we discourage from the simultaneous application of both drugs. If our in vitro results hold true in vivo, sequential application of ABZ and AMB would be an effective means for long-term suppression of larval growth. Long-term tolerance of both drugs could be improved by a reduction of side effects., (2010 Elsevier B.V. All rights reserved.)

The cytostatic drugs Vincristine (VCR), Navelbine (NAV), and Methotrexate (MTX) were evaluated for their growth inhibitory potential against metacestodes of Echinococcus multilocularis (Em) by in vitro and in vivo assays. In vitro cultures of E. multilocularis were exposed to IC 90, IC 80, and IC 5 concentrations of VCR, NAV, or MTX for 1 week, then parasite tissue cultures were kept for 1 week without drug exposure in vitro, and thereafter, metacestode tissues were injected intra-peritoneally into Meriones unguiculatus. Metacestode growth was monitored for several months post-infection (p.i.) by body weight control, magnetic resonance imaging (MRI), and autopsy at 5 months p.i. Weight monitoring of infected M. unguiculatus did not provide conclusive evidence for Em-metacestode growth, while MRI could detect growing Em-metacestode in the MTX-treated group at 8 weeks (p.i.), whereas metacestodes exposed to VCR and NAV were at 17 weeks (p.i.) detectable. MRI disclosed progressive and massive growth of Em-metacestode in the VCR- and MTX-exposed groups, while the NAV-pretreated Em-metacestodes' volume did not exceed 4 cm(3). At autopsy, Em-metacestodes of less than 4 cm(3) were found in infected M. unguiculatus, which was not detected by MRI. In summary, the cytostatic drugs Methotrexate, Navelbine, and Vincristine--as applied in the present work--did not show parasitocidal or clear parasitostatic effects on metacestodes of E. multilocularis. While parasite growth in vivo was inhibited in NAV- and VCR-pretreated Em-metacestodes, MTX pretreatment seemed to enhance parasite proliferation. Magnetic resonance imaging appears suitable to monitor in vivo the effects of drugs on growth progression and regression only of larger Em-metacestode tissues.

Objectives: The disease alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is fatal if treatment is unsuccessful. Current treatment options are, at best, parasitostatic, and involve taking benzimidazoles (albendazole, mebendazole) for the whole of a patient's life. In conjunction with the recent development of optimized procedures for E. multilocularis metacestode cultivation, we aimed to develop a rapid and reliable drug screening test, which enables efficient screening of a large number of compounds in a relatively short time frame., Methods: Metacestodes were treated in vitro with albendazole, the nitro-thiazole nitazoxanide and 29 nitazoxanide derivatives. The resulting leakage of phosphoglucose isomerase (PGI) activity into the medium supernatant was measured and provided an indication of compound efficacy., Results: We show that upon in vitro culture of E. multilocularis metacestodes in the presence of active drugs such as albendazole, the nitro-thiazole nitazoxanide and 30 different nitazoxanide derivatives, the activity of PGI in culture supernatants increased. The increase in PGI activity correlated with the progressive degeneration and destruction of metacestode tissue in a time- and concentration-dependent manner, which allowed us to perform a structure-activity relationship analysis on the thiazolide compounds used in this study., Conclusions: The assay presented here is inexpensive, rapid, can be used in 24- and 96-well formats and will serve as an ideal tool for first-round in vitro tests on the efficacy of large numbers of antiparasitic compounds.

The effect of dialysable leucocyte extract (transfer factor TF) on immune response of mice infected with Echinococcus multilocularis and treated with albendazole (ABZ) was observed. TF administration increased the parasite-suppressed proliferative response of T and B lymphocytes of infected mice from weeks 8 to 12 or 14 post infection (p.i.), respectively, with the most stimulative effect after TF+ABZ therapy. The CD4 T cell presence in the spleen of infected mice with TF or TF+ABZ therapy was increased from weeks 6 to 12 or 14 p.i., respectively. The production of IFN-gamma (Th1 cytokine) after TF or TF+ABZ therapy was significantly higher from weeks 6 to 12 p.i., and during this time, the significantly inhibited IL-5 synthesis (Th2 cytokine) was detected, particularly after TF+ABZ therapy. The superoxide anion (O2-) production in peritoneal macrophages of infected mice treated with TF or TF+ABZ was stimulated from weeks 8 to 18 p.i. The immunomodulative effect of TF reduced the growth of larval cysts till week 14 p.i. with a comparable intensity to the anthelmintic drug ABZ. Combined therapy TF+ABZ resulted in the greatest parasite restriction and reduced the cyst development till the end of the experiment.

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a mainly hepatic disease characterized by continuous asexual proliferation of metacestodes by exogenous budding, resulting in the tumor-like, infiltrative growth of the parasite lesion. Current chemotherapeutical treatment of AE relies on the use of benzimidazoles (albendazole, mebendazole), but these drugs act parasitostatic rather than parasitocidal, and in case of side effects such as liver toxicity, patients are left without valuable alternatives. 2-ME2 is a natural metabolite of estradiol, with a documented anti-angiogenic and broad spectrum anti-tumour activity. Treatments of in vitro cultured E. multilocularis metacestodes with 2-ME2 (2-10 microM) showed that the drug has an adverse effect on parasite viability. First, 2-ME in vitro treatment downscaled the transcription of the 14-3-3-pro-tumorogenic zeta-isoform in E. multilocularis metacestodes. Second, scanning and transmission electron microscopy showed that the germinal layer of E. multilocularis metacestodes was dramatically damaged following 2-ME2-treatment, and the effect was dose-dependent. Similar results were obtained with E. granulosus metacestodes. Bioassays were performed in mice injected with 2-ME2-treated and albendazole-treated metacestodes, or parasites-treated with both 2-ME and albendazole in combination. These assays indicated that, despite inducing considerable damage in vitro, neither of the drugs was capable of exerting a true parasiticidal effect, but best results were achieved with a combination of both compounds. In vivo treatment in E. multilocularis-infected mice for a period of 6 weeks showed that a combined 2-ME2/albendazole based treatment lead to a reduction in parasite weight, but the results did not show statistical difference from the application of albendazole alone.

Background: Long-term benzimidazole therapy benefits patients with non-resectable alveolar echinococcosis (AE). Methods to assess early therapeutic efficacy are lacking. Recently, AE liver lesions were reported to exhibit increased F-18-fluorodeoxyglucose (FDG) uptake in positron emission tomography (PET). To assess the value of FDG-PET for diagnosis and follow-up of AE patients., Patients/methods: Twenty-six consecutive patients with newly diagnosed AE were enrolled. Baseline evaluation included CT and FDG-PET. Thirteen patients (11 women; median age 50 years, range 40-76) were resected, the remaining 13 (8 women; median age 60 years, range 39-72) had non-resectable disease, were started on benzimidazoles, and CT and FDG-PET were repeated at 6, 12 and 24 months of therapy. Twelve consecutive patients with newly diagnosed cystic echinococcosis (CE) of the liver were also subjected to baseline FDG-PET., Results: In 21/26 AE patients, baseline PET scans showed multifocally increased FDG uptake in the hepatic lesions' periphery, while liver lesions were FDG negative in 11/12 CE patients. Thus, sensitivity and specificity of FDG-PET for AE vs. CE were 81% and 92%, respectively. In 5 of 10 non-resectable patients with increased baseline FDG uptake, the intensity of uptake decreased (or disappeared) during benzimidazole therapy, in 3 by >or=2 grades within the initial 6 months., Conclusions: FDG-PET is a sensitive and specific adjunct in the diagnosis of suspected AE and can help in differentiating AE from CE. The rapid improvement of positive PET scans with benzimidazole therapy in some patients indicates that absent FDG uptake does not necessarily reflect parasite viability.

Selected immunological parameters in healthy mice and mice infected with Echinococcus multilocularis and the effect of free and liposomized albendazole (lip.ABZ) upon these parameters in relation to the reduction of parasite growth were investigated over 26 weeks. Proliferative response of splenic T and B lymphocytes, number of CD4+ and CD8+ spleen T cell subpopulations, serum concentration of IFN-gamma and IL-5, and generation of superoxide anion (O2-) by peritoneal macrophages were the chosen parameters. Both drug forms were given to mice at a dose of 10 mg kg(-1) twice a week from week 4 to week 10 post infection (p.i.) (6 weeks in total). The reduction of cyst growth after treatment with ABZ and lip.ABZ was similar up to week 4 after last dose, but the parasitostatic effect of lip.ABZ lasted 4 weeks longer than the effect of free drug. After administration of both drug forms, the proliferative responses of T and B cells were restored, and also the number of CD4+ and CD8+ increased markedly. In lip.ABZ-treated mice, stimulation of mentioned lymphocyte parameters, except that of CD8+ numbers, persisted for longer period than after ABZ therapy, where values peaked at week 12 p.i., then declined more rapidly. A very strong stimulatory effect was seen on B lymphocytes during the period of lip.ABZ administration, although interestingly, numbers of CD8+ cells were higher in free ABZ-treated group. Low concentrations of IFN-gamma (Th1 response) were present in infected, untreated mouse serum. Only moderate IFN-gamma elevation was observed after treatment with free ABZ. A profound increase of its concentration was seen shortly after administration of lip.ABZ, and persisted until the experiment ended. In infected untreated mice, concentration of IL-5 (Th2 response) was highest on week 2 p.i. Significantly more IL-5 was recorded in serum of mice treated with free ABZ treatment than with lip.ABZ from week 12 to 18 p.i. (weeks 2-8 after the last dose). After the initial increase of superoxide anions (weeks 4-11 p.i.), generation of O2- by peritoneal macrophages was gradually inhibited by E. multilcoularis infection. In general, treatment abolished this suppression and macrophages from lip.ABZ-treated mice produced elevated amounts of O2- over a longer period than macrophages from ABZ-treated mice. Our data indicate that anthelmintic potency of ABZ could be increased after incorporation into liposomes, not only because of improved pharmacokinetics and consequent bioavailability, but also because of significant stimulation of Th1-type cytokine IFN-gamma response and effector macrophage functions.