Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

SAFETY AND PHARMACOKINETICS OF PRAZIQUANTEL IN EUROPEAN POND TURTLES (EMYS ORBICULARIS).

Spirorchiidosis, caused by blood flukes of the genus Spirorchis, is a disease of great concern for the critically endangered European pond turtle (EPT; Emys orbicularis) in Switzerland. The endogenous life cycle of the parasite often leads to systemic inflammatory reactions, thrombosis, and death. Praziquantel (PZQ) is the treatment of choice against adult Spirorchis spp. in green (Chelonia mydas) and in loggerhead (Caretta caretta) sea turtles and is therefore considered for the treatment of EPT. This study aimed to establish a safe, easily applicable PZQ treatment for EPT, based on pharmacokinetics and tolerability. Three application methods were tested in a total of 12 adult EPT. Each turtle received a total of 75 mg/kg PZQ (three doses of 25 mg/kg in 3-h intervals [q3h × 3]) via IM (n = 3 turtles), SC (n = 3 turtles), or PO (n = 6 turtles) administration. Blood was collected 3, 6, 24, and 48 h after the first administration to determine the plasma concentration of PZQ using high-performance liquid chromatography coupled to mass spectrometry. Maximum measured R-PZQ concentrations (Cmax) were reached after 6 h. The mean Cmax of the total PZQ (sum of R- and S-PZQ) in the PO-treated EPT group was 1,929 ng/ml. Significantly higher concentrations were measured after IM and SC injection (mean Cmax of total PZQ = 12,715 ng/ml and 10,114 ng/ml, respectively). Transient side effects were evident after IM administration (local swelling and lameness), whereas no adverse drug effects were observed after PO and SC administration. Based on these results and the ease of administration to EPT, SC injection of PZQ at 25 mg/kg q3h times 3 serves as promising treatment application for the future.

In Vitro Activities of Dithiocarbamate Derivatives against Echinococcus multilocularis Metacestode Vesicles.

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the severe zoonotic disease alveolar echinococcosis. New treatment options are urgently needed. Disulfiram and dithiocarbamates were previously shown to exhibit activity against the trematode Schistosoma mansoni. As both parasites belong to the platyhelminths, here we investigated whether these compounds were also active against E. multilocularis metacestode vesicles in vitro. We used an in vitro drug-screening cascade for the identification of novel compounds against E. multilocularis metacestode vesicles with disulfiram and 51 dithiocarbamates. Five compounds showed activity against E. multilocularis metacestode vesicles after five days of drug incubation in a damage marker release assay. Structure-activity relationship analyses revealed that a S-2-hydroxy-5-nitro benzyl moiety was necessary for anti-echinococcal activity, as derivatives without this group had no effect on E. multilocularis metacestode vesicles. The five active compounds were further tested for potential cytotoxicity in mammalian cells. For two compounds with low toxicity (Schl-32.315 and Schl-33.652), IC50 values in metacestode vesicles and IC50 values in germinal layer cells were calculated. The compounds were not highly active on isolated GL cells with IC50 values of 27.0 ± 4.2 µM for Schl-32.315 and 24.7 ± 11.5 µM for Schl-33.652, respectively. Against metacestode vesicles, Schl-32.315 was not very active either with an IC50 value of 41.6 ± 3.2 µM, while Schl-33.652 showed a low IC50 of 4.3 ± 1 µM and should be further investigated in the future for its activity against alveolar echinococcosis.

The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure-Activity Relationships at TRPMPZQ Orthologs.

The anthelmintic drug praziquantel remains a key clinical therapy for treating various diseases caused by parasitic flatworms. The parasite target of praziquantel has remained undefined despite longstanding usage in the clinic, although a candidate ion channel target, named TRPMPZQ, has recently been identified. Intriguingly, certain praziquantel derivatives show different activities against different parasites: for example, some praziquantel analogs are considerably more active against cestodes than against schistosomes. Here we interrogate whether the different activities of praziquantel analogs against different parasites are also reflected by unique structure-activity relationships at the TRPMPZQ channels found in these different organisms. To do this, several praziquantel analogs were synthesized and functionally profiled against schistosome and cestode TRPMPZQ channels. Data demonstrate that structure-activity relationships are closely mirrored between parasites and their TRPMPZQ orthologs, providing further support for TRPMPZQ as the therapeutically relevant target of praziquantel.

Establishment and application of unbiased in vitro drug screening assays for the identification of compounds against Echinococcus granulosus sensu stricto.

Echinococcus multilocularis and E. granulosus s.l. are the causative agents of alveolar and cystic echinococcosis, respectively. Drug treatment options for these severe and neglected diseases are limited to benzimidazoles, which are not always efficacious, and adverse side effects are reported. Thus, novel and improved treatments are needed. In this study, the previously established platform for E. multilocularis in vitro drug assessment was adapted to E. granulosus s.s. In a first step, in vitro culture protocols for E. granulosus s.s. were established. This resulted in the generation of large amounts of E. granulosus s.s. metacestode vesicles as well as germinal layer (GL) cells. In vitro culture of these cells formed metacestode vesicles displaying structural characteristics of metacestode cysts generated in vivo. Next, drug susceptibilities of E. multilocularis and E. granulosus s.s. protoscoleces, metacestode vesicles and GL cells were comparatively assessed employing established assays including (i) metacestode vesicle damage marker release assay, (ii) metacestode vesicle viability assay, (iii) GL cell viability assay, and (iv) protoscolex motility assay. The standard drugs albendazole, buparvaquone, mefloquine, MMV665807, monepantel, niclosamide and nitazoxanide were included. MMV665807, niclosamide and nitazoxanide were active against the parasite in all four assays against both species. MMV665807 and monepantel were significantly more active against E. multilocularis metacestode vesicles, while albendazole and nitazoxanide were significantly more active against E. multilocularis GL cells. Albendazole displayed activity against E. multilocularis GL cells, but no effects were seen in albendazole-treated E. granulosus s.s. GL cells within five days. Treatment of protoscoleces with albendazole and monepantel had no impact on motility. Similar results were observed for both species with praziquantel and its enantiomers against protoscoleces. In conclusion, in vitro culture techniques and drug screening methods previously established for E. multilocularis were successfully implemented for E. granulosus s.s., allowing comparisons of drug efficacy between the two species. This study provides in vitro culture techniques for the reliable generation of E. granulosus s.s. metacestode vesicles and GL cell cultures and describes the validation of standardized in vitro drug screening methods for E. granulosus s.s.

Targeted and non-targeted proteomics to characterize the parasite proteins of Echinococcus multilocularis metacestodes.

The larval stage of the cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis. To investigate the biology of these stages and to test novel compounds, metacestode cultures represent a suitable in vitro model system. These metacestodes are vesicles surrounded by an envelope formed by the vesicle tissue (VT), which is formed by the laminated and germinal layer, and filled with vesicle fluid (VF). We analyzed the proteome of VF and VT by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified a total of 2,954 parasite proteins. The most abundant protein in VT was the expressed conserved protein encoded by EmuJ_000412500, followed by the antigen B subunit AgB8/3a encoded by EmuJ_000381500 and Endophilin B1 (protein p29). In VF, the pattern was different and dominated by AgB subunits. The most abundant protein was the AgB8/3a subunit followed by three other AgB subunits. In total, the AgB subunits detected in VF represented 62.1% of the parasite proteins. In culture media (CM), 63 E. multilocularis proteins were detected, of which AgB subunits made up 93.7% of the detected parasite proteins. All AgB subunits detected in VF (encoded by EmuJ_000381100-700, corresponding to AgB8/2, AgB8/1, AgB8/4, AgB8/3a, AgB8/3b, and AgB8/3c) were also found in CM, except the subunit encoded by EmuJ_000381800 (AgB8/5) that was very rare in VF and not detected in CM. The relative abundance of the AgB subunits in VF and CM followed the same pattern. In VT, only the subunits EmuJ_000381500 (AgB8/3a) and EmuJ_000381200 (AgB8/1) were detected among the 20 most abundant proteins. To see whether this pattern was specific to VF from in vitro cultured metacestodes, we analyzed the proteome of VF from metacestodes grown in a mouse model. Here, the AgB subunits encoded by EmuJ_000381100-700 constituted the most abundant proteins, namely, 81.9% of total protein, with the same order of abundance as in vitro. Immunofluorescence on metacestodes showed that AgB is co-localized to calcareous corpuscles of E. multilocularis. Using targeted proteomics with HA-tagged EmuJ_000381200 (AgB8/1) and EmuJ_000381100 (AgB8/2), we could show that uptake of AgB subunits from CM into VF occurs within hours.

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis.

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed. Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1'681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.

Immunomodulatory components of Trichinella spiralis excretory-secretory products with lactose-binding specificity.

The immunomodulatory potential of Trichinella spiralis muscle larvae excretory-secretory products (ES L1) has been well documented in vitro on dendritic cells (DCs) and in animal models of autoimmune diseases. ES L1 products possess the potential to induce tolerogenic DCs and consequently trigger regulatory mechanisms that maintain immune homeostasis. The use of ES L1 as a potential treatment for various inflammatory disorders proved to be beneficial in animal models, although the precise immunomodulatory factors have not yet been identified. This study aimed at the isolation and characterization of ES L1 components that possess galectin family member properties. Galectin-1-like proteins (TsGal-1-like) were isolated from ES L1 based on the assumption of the existence of a lactose-specific carbohydrate-recognition domain and were recognized by anti-galectin-1 antibodies in Western blot. This TsGal-1-like isolate, similar to galectin-1, induced DCs with tolerogenic properties and hence, the capacity to polarize T cell response towards a regulatory type. This was reflected by a significantly increased percentage of CD4+CD25+Foxp3+ regulatory T cells and significantly increased expression of IL-10 and TGF-ß within this cell population. Proteomic analysis of TsGal-1-like isolate by mass spectrometry identified nineteen proteins, seven with annotated function after blast analysis against a database for T. spiralis and the UniProt database. To our surprise, none of the identified proteins possesses homology with known galectin family members. Nevertheless, the isolated components of ES L1 possess certain galectin-1 properties, such as specific lactose binding and the potential to elicit a regulatory immune response, so it would be worth further investigating the structure of sugar binding within isolated proteins and its biological significance.

Dual inhibition of the Echinococcus multilocularis energy metabolism.

Alveolar echinococcosis is caused by the metacestode stage of the zoonotic parasite Echinococcus multilocularis. Current chemotherapeutic treatment options rely on benzimidazoles, which have limited curative capabilities and can cause severe side effects. Thus, novel treatment options are urgently needed. In search for novel targetable pathways we focused on the mitochondrial energy metabolism of E. multilocularis. The parasite relies hereby on two pathways: The classical oxidative phosphorylation including the electron transfer chain (ETC), and the anaerobic malate dismutation (MD). We screened 13 endochin-like quinolones (ELQs) in vitro for their activities against two isolates of E. multilocularis metacestodes and isolated germinal layer cells by the phosphoglucose isomerase (PGI) assay and the CellTiter Glo assay. For the five most active ELQs (ELQ-121, ELQ-136, ELQ-271, ELQ-400, and ELQ-437), EC50 values against metacestodes were assessed by PGI assay, and IC50 values against mammalian cells were measured by Alamar Blue assay. Further, the gene sequence of the proposed target, the mitochondrial cytochrome b, was analyzed. This allowed for a limited structure activity relationship study of ELQs against E. multilocularis, including analyses of the inhibition of the two functional sites of the cytochrome b. By applying the Seahorse XFp Extracellular Flux Analyzer, oxygen consumption assays showed that ELQ-400 inhibits the E. multilocularis cytochrome bc 1 complex under normoxic conditions. When tested under anaerobic conditions, ELQ-400 was hardly active against E. multilocularis metacestodes. These results were confirmed by transmission electron microscopy. ELQ-400 treatment increased levels of parasite-released succinate, the final electron acceptor of the MD. This suggests that the parasite switched to MD for energy generation. Therefore, MD was inhibited with quinazoline, which did not induce damage to metacestodes under anaerobic conditions. However, it reduced the production of succinate compared to control treated parasites (i.e., inhibited the MD). The combination treatment with quinazoline strongly improved the activity of the bc 1 inhibitor ELQ-400 against E. multilocularis metacestodes under anaerobic conditions. We conclude that simultaneous targeting of the ETC and the MD of E. multilocularis is a possible novel treatment approach for alveolar echinococcosis, and possibly also other foodborne diseases inflicted by platyhelminths, which cause substantial economic losses in livestock industry.

Albendazole reduces hepatic inflammation and endoplasmic reticulum-stress in a mouse model of chronic Echinococcus multilocularis infection.

BACKGROUND: Echinococcus multilocularis causes alveolar echinococcosis (AE), a rising zoonotic disease in the northern hemisphere. Treatment of this fatal disease is limited to chemotherapy using benzimidazoles and surgical intervention, with frequent disease recurrence in cases without radical surgery. Elucidating the molecular mechanisms underlying E. multilocularis infections and host-parasite interactions ultimately aids developing novel therapeutic options. This study explored an involvement of unfolded protein response (UPR) and endoplasmic reticulum-stress (ERS) during E. multilocularis infection in mice. METHODS: E. multilocularis- and mock-infected C57BL/6 mice were subdivided into vehicle, albendazole (ABZ) and anti-programmed death ligand 1 (αPD-L1) treated groups. To mimic a chronic infection, treatments of mice started six weeks post i.p. infection and continued for another eight weeks. Liver tissue was then collected to examine inflammatory cytokines and the expression of UPR- and ERS-related genes. RESULTS: E. multilocularis infection led to an upregulation of UPR- and ERS-related proteins in the liver, including ATF6, CHOP, GRP78, ERp72, H6PD and calreticulin, whilst PERK and its target eIF2α were not affected, and IRE1α and ATF4 were downregulated. ABZ treatment in E. multilocularis infected mice reversed, or at least tended to reverse, these protein expression changes to levels seen in mock-infected mice. Furthermore, ABZ treatment reversed the elevated levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the liver of infected mice. Similar to ABZ, αPD-L1 immune-treatment tended to reverse the increased CHOP and decreased ATF4 and IRE1α expression levels. CONCLUSIONS AND SIGNIFICANCE: AE caused chronic inflammation, UPR activation and ERS in mice. The E. multilocularis-induced inflammation and consecutive ERS was ameliorated by ABZ and αPD-L1 treatment, indicating their effectiveness to inhibit parasite proliferation and downregulate its activity status. Neither ABZ nor αPD-L1 themselves affected UPR in control mice. Further research is needed to elucidate the link between inflammation, UPR and ERS, and if these pathways offer potential for improved therapies of patients with AE.

Evaluation of the PrioCHECK™ Trichinella AAD kit to detect Trichinella spiralis, T. britovi, and T. pseudospiralis larvae in pork using the automated digestion method Trichomatic-35.

Trichinellosis is a potentially deadly parasitic zoonosis that is contracted by consuming undercooked infected meat. Reliable detection of infectious Trichinella spp. larvae in meat is therefore pivotal to ensure consumer's safety. The recently authorised PrioCHECK™ Trichinella Alternative Artificial Digestion (AAD) test kit appears promising when used with the standard magnetic stirrer method, but evaluation with other apparatus types is lacking. In this study, the performance of the AAD kit in an adapted Trichomatic-35 (TM35) instrument was evaluated, first, at the Swiss National Reference Laboratory for trichinellosis (NRL); second, in a ring trial involving four Swiss official laboratories. Proficiency pork samples spiked with larvae of Trichinella spiralis, T. britovi, or T. pseudospiralis were tested with the AAD kit and with the reference pepsin-HCl digestion method in TM35 instruments. At the NRL, both methods yielded identical qualitative and similar quantitative results independently of the Trichinella species. In the ring trial, satisfactory results were obtained for 47/50 (94.0%) (AAD) and 62/67 (92.5%) (reference method) of the analysed samples. Technical problems impairing analysis were more frequently observed with the AAD kit (n = 22) than with the reference method (n = 5) and were mainly (16/22) reported by one of the external labs. When no technical issues were recorded, the performance of both methods was comparable, in agreement with the observations at the NRL; however, these results suggest a need for further training with the kit and standardisation of the adapted TM35 instruments.

Maca against Echinococcosis?-A Reverse Approach from Patient to In Vitro Testing.

Drug-based treatment of alveolar echinococcosis (AE) with benzimidazoles is in most cases non-curative, thus has to be taken lifelong. Here, we report on a 56-year-old male AE patient who received standard benzimidazole treatment and biliary plastic stents, and additionally self-medicated himself with the Peruvian plant extract Maca (Lepidium meyenii). After 42 months, viable parasite tissue had disappeared. Based on this striking observation, the anti-echinococcal activity of Maca was investigated in vitro and in mice experimentally infected with Echinococcus multilocularis metacestodes. Albendazole (ABZ)-treated mice and mice treated with an ABZ+Maca combination exhibited a significantly reduced parasite burden compared to untreated or Maca-treated mice. As shown by a newly established UHPLC-MS/MS-based measurement of ABZ-metabolites, the presence of Maca during the treatment did not alter ABZ plasma levels. In vitro assays corroborated these findings, as exposure to Maca had no notable effect on E. multilocularis metacestodes, and in cultures of germinal layer cells, possibly unspecific, cytotoxic effects of Maca were observed. However, in the combined treatments, Maca inhibited the activity of ABZ in vitro. While Maca had no direct anti-parasitic activity, it induced in vitro proliferation of murine spleen cells, suggesting that immunomodulatory properties could have contributed to the curative effect seen in the patient.

Impact on Bile Acid Concentrations by Alveolar Echinococcosis and Treatment with Albendazole in Mice.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis is a chronic, progressive liver disease widely distributed in the Northern Hemisphere. The main treatment options include surgical interventions and chemotherapy with benzimidazole albendazole (ABZ). To improve the current diagnosis and therapy of AE, further investigations into parasite-host interactions are needed. This study used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess serum and liver tissue bile acid profiles in the i.p. chronic E. multilocularis-infected mouse model and evaluated the effects of the anthelmintic drug ABZ. Additionally, hepatic mRNA and protein expression of enzymes and transporters regulating bile acid concentrations were analyzed. AE significantly decreased unconjugated bile acids in serum and liver tissue. Taurine-conjugated bile salts were unchanged or increased in the serum and unchanged or decreased in the liver. Ratios of unconjugated to taurine-conjugated metabolites are proposed as useful serum markers of AE. The expression of the bile acid synthesis enzymes cytochrome P450 (CYP) 7A1 and aldo-keto reductase (AKR) 1D1 tended to decrease or were decreased in mice with AE, along with decreased expression of the bile acid transporters Na+/taurocholate cotransporting polypeptide (NTCP) and bile salt efflux pump (BSEP). Importantly, treatment with ABZ partially or completely reversed the effects induced by E. multilocularis infection. ABZ itself had no effect on the bile acid profiles and the expression of relevant enzymes and transporters. Further research is needed to uncover the exact mechanism of the AE-induced changes in bile acid homeostasis and to test whether serum bile acids and ratios thereof can serve as biomarkers of AE and for monitoring therapeutic efficacy.

Short communication: Efficacy of albendazole in Echinococcus multilocularis-infected mice depends on the functional immunity of the host.

Alveolar echinococcosis (AE) is a deadly parasitic disease that requires lifelong treatment with albendazole. Development of host immunity is pivotal with regard to the clinical outcome of AE, but its influence on conventional albendazole treatment is unknown. Using T-cell deficient athymic nude mice, we demonstrated that functional immunity is required for albendazole to be efficacious against murine AE. These results call for attention given the increasing number of immunocompromised patients with AE.

Drug repurposing applied: Activity of the anti-malarial mefloquine against Echinococcus multilocularis.

The current chemotherapeutical treatment against alveolar echinococcosis relies exclusively on benzimidazoles, which are not parasiticidal and can induce severe toxicity. There are no alternative treatment options. To identify novel drugs with activity against Echinococcus multilocularis metacestodes, researchers have studied potentially interesting drug targets (e.g. the parasite's energy metabolism), and/or adopted drug repurposing approaches by undertaking whole organism screenings. We here focus on drug screening approaches, which utilize an in vitro screening cascade that includes assessment of the drug-induced physical damage of metacestodes, the impact on metacestode viability and the viability of isolated parasite stem cells, structure-activity relationship (SAR) analysis of compound derivatives, and the mode of action. Finally, once in vitro data are indicative for a therapeutic window, the efficacy of selected compounds is assessed in experimentally infected mice. Using this screening cascade, we found that the anti-malarial mefloquine was active against E. multilocularis metacestodes in vitro and in vivo. To shed more light into the mode of action of mefloquine, SAR analysis on mefloquine analogues was performed. E. multilocularis ferritin was identified as a mefloquine-binding protein, but its precise role as a drug target remains to be elucidated. In mice that were infected either intraperitoneally with metacestodes or orally with eggs, oral treatment with mefloquine led to a significant reduction of parasite growth compared to the standard treatment with albendazole. However, mefloquine was not acting parasiticidally. Assessment of mefloquine plasma concentrations in treated mice showed that levels were reached which are close to serum concentrations that are achieved in humans during long-term malaria prophylaxis. Mefloquine might be applied in human AE patients as a salvage treatment. Future studies should focus on other repurposed anti-infective compounds (MMV665807, niclosamide, atovaquone), which showed stronger in vitro activity against E. multilocularis than mefloquine.

In vitro metabolomic footprint of the Echinococcus multilocularis metacestode.

Alveolar echinococcosis (AE) is a zoonotic disease that is deadly if left untreated. AE is caused by the larval metacestode stage of the cestode Echinococcus multilocularis. Better knowledge on the host-parasite interface could yield novel targets for improvement of the treatment against AE. We analyzed culture media incubated with in vitro grown E. multilocularis metacestodes by 1H nuclear magnetic resonance spectroscopy to identify the unknown metabolic footprint of the parasite. Moreover, we quantitatively analyzed all amino acids, acetate, glucose, lactate, and succinate in time-course experiments using liquid chromatography and enzymatic assays. The E. multilocularis metacestodes consumed glucose and, surprisingly, threonine and produced succinate, acetate, and alanine as major fermentation products. The metabolic composition of vesicle fluid (VF) from in vitro grown E. multilocularis metacestodes was different from parasite-incubated culture medium with respect to the abundance, but not the spectrum, of metabolites, and some metabolites, in particular amino acids, accumulated in the VF. Overall, this study presents the first characterization of the in vitro metabolic footprint of E. multilocularis metacestodes and VF composition, and it provides the basis for analyses of potentially targetable pathways for future drug development.

Activity of Thymus capitatus essential oil components against in vitro cultured Echinococcus multilocularis metacestodes and germinal layer cells.

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 ß-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.

The importance of being parasiticidal an update on drug development for the treatment of alveolar echinococcosis.

The lethal disease alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis. Current chemotherapeutical treatment of AE relies on albendazole and mebendazole, with the caveat that these compounds are not parasiticidal. Drugs have to be taken for a prolonged period of time, often life-long, which can cause adverse effects and reduces the patients' quality of life. In some individuals, benzimidazoles are inactive or cause toxicity, leading to treatment discontinuation. Alternatives to benzimidazoles are urgently needed. Over the recent years, in vivo and in vitro models for low-to-medium throughput drug discovery against AE have been set in place. In vitro drug tests include the phosphoglucose-isomerase (PGI) assay to measure physical damage induced to metacestodes, and viability assays to assess parasiticidal activity against metacestodes and stem cells. In vitro models are also employed for studies on mechanisms of action. In vivo models are thus far based on rodents, mainly mice, and benefits could be gained in future by comparative approaches in naturally infected dogs or captive monkeys. For the identification of novel drugs against AE, a rare disease with a low expected market return, drug-repurposing is the most promising strategy. A variety of chemically synthesized compounds as well as natural products have been analyzed with respect to in vitro and/or in vivo activities against AE. We here review and discuss the most active of these compounds including anti-infective compounds (benzimidazoles, nitazoxanide, amphotericin B, itraconazole, clarithromycin, DB1127, and buparvaquone), the anti-infective anti-malarials (artemisinin, ozonids, mefloquine, and MMV665807) and anti-cancer drugs (isoflavones, 2-methoxyestradiol, methotrexate, navelbine, vincristine, kinase inhibitors, metallo-organic ruthenium complexes, bortezomib, and taxanes). Taking into account the efficacy as well as the potential availability for patients, the most promising candidates are new formulations of benzimidazoles and mefloquine. Future drug-repurposing approaches should also target the energy metabolism of E. multilocularis, in particular the understudied malate dismutation pathway, as this offers an essential target in the parasite, which is not present in mammals.