An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Histopathological assessment of the viability of hepatic alveolar echinococcosis.

AIMS: Infections by the larval stage of the tape worms Echinococcus multilocularis and Echinococcus granulosus s.l. are potentially fatal zoonoses affecting humans as dead-end hosts. Histopathological evaluation of hepatic echinococcosis is an integral part of patient management, including the distinction between alveolar (AE) and cystic echinococcosis (CE), which are associated with different disease courses and treatments. To improve histopathological assessment of Echinococcus lesions, we aimed to develop robust criteria to evaluate their viability and decay. METHODS AND RESULTS: Histomorphological criteria for determining parasitic viability based on the morphology of parasite structures and different stages of their decay were defined based on a clinically and molecularly defined cohort comprising 138 specimens from 112 patients (59 AE and 53 CE); 618 AE lesions were assessed for histopathological viability comparing haematoxylin and eosin (H&E) staining with mAbEm18 and mAbEm2G11 immunostaining. Moreover, parasite viability was systematically mapped in cross-sections of five additional AE lesions. Protoscoleces in CE and AE displayed variable states of degeneration. Albendazole had no significant effect on the morphology of parasite structures. Viability assessment revealed high agreement between H&E and mAbEm18, but not mAbEm2G11 staining, suggesting mAbEm18 staining as reliable for parasite viability assessment. H&E and mAbEm18 staining displayed a central-peripheral gradient of parasite viability and decay across parasitic lesions, with decayed cystic lesions located more towards the lesion centre while the most viable cystic lesions were located more peripherally. CONCLUSIONS: Histopathological criteria corroborated by mAbEm18 staining provide a simple and reliable tool to assess the viability of AE lesions, knowledge of which is a valuable decision-making tool for further treatment.

Environmental distribution of Echinococcus- and Taenia spp.-contaminated dog feces in Kyrgyzstan.

Recently, there have been epidemics of human cystic echinococcosis (CE) and alveolar echinococcosis (AE) in Kyrgyzstan. This study investigated 2 districts for the presence of Echinococcus granulosus s.l. and Echinococcus multilocularis eggs; species identity was confirmed by polymerase chain reaction in dog feces and the level of environmental contamination with parasite eggs in 2017­2018 was also investigated. In the Alay district 5 villages with a high reported annual incidence of AE of 162 cases per 100 000 and 5 villages in the Kochkor district which had a much lower incidence of 21 cases per 100 000 were investigated. However, the proportion of dog feces containing E. granulosus s.l. eggs was ~4.2 and ~3.5% in Alay and Kochkor respectively. For E. multilocularis, the corresponding proportions were 2.8 and 3.2%. Environmental contamination of Echinococcus spp. eggs was estimated using the McMaster technique for fecal egg counts, weight and density of canine feces. The level of environmental contamination with E. multilocularis eggs was similar at 4.4 and 5.0 eggs per m2 in Alay and Kochkor respectively. The corresponding values for E. granulosus s.l. were 8.3 and 7.5 eggs per m2. There was no association between village or district level incidence of human AE or CE and the proportion of dog feces containing eggs of Echinococcus spp. or the level of environmental contamination. Increased contamination of taeniid eggs occured in the autumn, after the return of farmers with dogs from summer mountain pastures.

First isolation of Echinococcus granulosus sensu lato genotype 7 in the archipelago of Cape Verde.

There are no scientific data available on the occurrence of the Echinococcus granulosus sensu lato (s.l.) cluster in definitive hosts (domestic dogs), intermediate hosts (domestic livestock) nor humans in Cape Verde. In this pilot study, environmental dog fecal samples (n = 369) were collected around food markets, official slaughterhouses, as well as home and small business slaughter spots in 8 of the 9 inhabited islands from the Cape Verde archipelago, between June 2021 and March 2022. Additionally, during the same period, 40 cysts and tissue lesions were opportunistically collected from 5 islands, from locally slaughtered cattle (n = 7), goats (n = 2), sheep (n = 1) and pigs (n = 26). Genetic characterization by a multiplex polymerase chain reaction assay targeting the 12S rRNA gene confirmed the presence of E. granulosus s.l. in fecal and tissue material. In total, 17 cyst samples from Santiago (n = 9), Sal (n = 7) and São Vicente (n = 1) and 8 G6/G7-positive dog fecal samples from Santiago (n = 4) and Sal (n = 4) were identified as E. granulosus s.l. G7 by sequence analysis (nad2, nad5 and nad1 genes). This study discloses the transmission of E. granulosus s.l. G7, in pig, cattle and dog in Cape Verde.

Differences in clinical aspects of human cystic echinococcosis caused by Echinococcus granulosus sensu stricto and the G6 genotype in Neuquén, Argentina.

Most human cystic echinococcosis (CE) cases worldwide are attributed to Echinococcus granulosus sensu stricto (s.s), followed by the G6 and G7 genotypes. While E. granulosus s.s. has a cosmopolitan distribution, the G6 genotype is restricted to areas where camels and goats are present. Goats are the primary livestock in the Neuquén province in Argentina where the G6 genotype has been reported to be responsible for a significant percentage of CE human cysts genotyped. In the present study, we genotyped 124 Echinococcus cysts infecting 90 CE-confirmed patients. Echinococcus granulosus s.s. was identified in 51 patients (56.7%) with 81 cysts and the G6 genotype in 39 patients (43.3%) harbouring 43 cysts. Most CE cases ≤18 years were male suggesting pastoral work could be a risk factor for the infection. Echinococcus granulosus s.s. was significantly found more frequently in the liver (32/51 patients) and the G6 genotype in the lungs and extrahepatic localizations (27/39). The patients infected with E. granulosus s.s., presented up to 6 cysts while patients infected with G6 presented a maximum of 2. The diameter of lung cysts attributed to E. granulosus s.s. was significantly larger compared to lung cysts from G6. Following the WHO ultrasound classification of liver cysts, we observed inactive cysts in 55.6% of G6 cysts and only 15.3% of E. granulosus s.s cysts. In conclusion, we provide evidence of differences in clinical aspects of CE caused by E. granulosus s.s. and the G6 genotype of E. granulosus s.l. complex infecting humans.

Estimated specific antibody-based true sero-prevalences of canine filariosis in dogs in Central Europe and the UK.

Dirofilariosis is a vector-borne disease mainly caused by Dirofilaria immitis and Dirofilaria repens. In contrast to the known endemicity of dirofilariosis in southern and south-eastern Europe, information on the distribution of D. repens in Central-Europe is fragmentary. We tested 8877 serum samples from dogs from Austria, Denmark, Germany, Italy, Lithuania, Poland, Switzerland and the UK using an ELISA detecting filarial-specific antibodies, hypothesising higher occurrence of D. repens. Based on two overlapping frequency distributions, presumed negative samples had a mean optical density (OD) value of 0.097, representing 97.45% of all samples. Presumed positive samples, representing 2.55% of all sera, had a mean OD value of 0.287. Test prevalence based on the calculated cut-off was 3.51% for all sera (4.36% for Austria, 1.94% for Denmark, 1.39% for Germany, 3.37% for Italy, 6.90% for Lithuania, 6.99% for Poland, 0.77% for Switzerland and 0.0% for the UK, respectively). The bimodal distribution, representing overlapping distributions of OD values from positive and negative dogs, enabled the assignment of a probability of true infection status to each dog. Mean probabilities of true infection status across groups, based on the postal codes of origin, allowed us to estimate and map true prevalences. For all countries, except the UK, the true prevalence was lower than the test prevalence. The large number of serum samples and the use of a non-gold standard analytical method allowed us to create a more realistic picture of the distribution of D. repens in Central Europe and the UK.

Alveolar echinococcosis: what triggers emergence in North America, Central Europe and Asia?

PURPOSE OF REVIEW: Infection with the larval (metacestode) stage of Echinococcus multilocularis causes alveolar echinococcosis (AE), a serious hepatic disorder. The parasite has increased its infection extensity in wildlife and domestic dogs, mainly due to urbanization and spatial extension of wildlife hosts in Europe, Asia as well as North America, resulting in emerging infection risk for humans. RECENT FINDINGS: In hyperendemic areas such as Kyrgyzstan and China, ecological and socioeconomic changes have been associated with the unpredictable increase of AE cases. In North America, the appearance of the European-like genotype is of concern. In Europe, the annual increase of human case numbers reached a plateau even in hyperendemic situations. Therefore, we conclude that most of the exposed individuals are resistant to parasite invasion and/or to disease development. Thus, AE develops in a few healthy individuals, but preferentially in immunosuppressed patients. SUMMARY: In the future, improved diagnostic strategies will allow more precise estimations of transmission routes including the role of food, water and direct dog contact, which should yield improved public health recommendations. Finally, understanding protective innate and acquired immune mechanisms as well as parasite-driven immune-evasion processes will be essential to develop curative therapies in nonoperable patients and, futuristically, appropriate vaccines.

Agranulocytosis leads to intestinal Echinococcus multilocularis oncosphere invasion and hepatic metacestode development in naturally resistant Wistar rats.

Susceptibility to Echinococcus multilocularis infection considerably varies among intermediate (mostly rodents) and dead-end host species (e.g. humans and pig), in particular regarding intestinal oncosphere invasion and subsequent hepatic metacestode development. Wistar rats are highly resistant to infection and subsequent diseases upon oral inoculation with E. multilocularis eggs, however, after immunosuppressive treatment with dexamethasone, rats become susceptible. To address the role of the cellular innate immunity, Wistar rats were individually or combined depleted of natural killer (NK) cells, macrophages (MΦ) and granulocytes (polymorphonuclear cells, PMN) prior to E. multilocularis egg inoculation. Although NK cell and MΦ depletion did not alter the resistance status of rats, the majority of PMN-depleted animals developed liver metacestodes within 10 weeks, indicating that PMN are key players in preventing oncosphere migration and/or development in Wistar rats. In vitro studies indicated that resistance is not caused by neutrophil reactive oxygen species or NETosis. Also, light microscopical examinations of the small intestine showed that oral inoculation of E. multilocularis eggs does not elicit a mucosal neutrophil response, suggesting that the interaction of oncospheres and neutrophils may occur after the former have entered the peripheral blood. We suggest to consider granulocytes as mediators of resistance in more resistant species, such as humans.

Amplification of cestode DNA from the peri-anal region of naturally infected foxes by PCR and LAMP: proof of concept for a potential sampling strategy for diagnosing human taeniosis.

The diagnosis of human taeniosis can be achieved through coproscopy, ELISA or PCR. An important limitation of these methods is the high turnaround time for stool sample collection and preparation, indicating the need for a straightforward sampling strategy. Due to the high metabolic activity and reproductive potential of Taenia spp., we hypothesise that parasite DNA (cells and eggs) present in the peri-anal region of the host can be exploited as a target for molecular diagnosis. We evaluated the feasibility of recovering parasite DNA from the peri-anal area of foxes naturally infected with Taenia spp. Before necropsy, cotton swabs were rubbed at the peri-anal region of foxes. DNA was extracted using alkaline lysis coupled with a commercial DNA isolation kit (method A) or alkaline lysis alone (method B). DNA was used in the multiplex-PCR assay (previously described and called here swab-PCR) and a novel LAMP assay detecting Taenia spp. commonly found in foxes (swab-LAMP). The results of these assays from 105 foxes were compared with the presence of intestinal helminths determined at necropsy and by the sedimentation and counting technique (SCT). The sensitivity of swab-PCR for detecting Taenia (n = 68) was 89.8% (95% CI, 77.7-96.6) and 89.5% (66.9-98.7) using methods A and B, respectively. The sensitivity of the swab-LAMP assay was 83.7% (70.3-92.7) using method A and 89.5% (66.9-98.7) with method B. We postulate that peri-anal swab sampling followed by simplified DNA extraction and LAMP might be a suitable strategy for surveillance of human taeniosis in resource-limited settings in the future.

Atopic dermatitis in West Highland white terriers - Part III: early life peripheral blood regulatory T cells are reduced in atopic dermatitis.

BACKGROUND: Regulatory T (Treg) cells are involved in homeostasis of immune regulation and suppression of inflammation and T-cell polarisation. Current knowledge regarding the role of Treg cells in the initiation of allergic disease is limited for both people and dogs. OBJECTIVES: To explore the role of circulating Treg cells and their possible influencing factors, on the development of atopic dermatitis (AD). METHODS AND MATERIALS: This study followed part of a birth cohort of West Highland white terrier dogs and classified them according to eventual clinical signs of AD (i.e. allergic versus healthy). The Treg phenotypes were assessed longitudinally by flow cytometry at 3, 3-12 and 12-36 months of age, and associated with development of AD. Different early life antigenic factors [endotoxins and allergens in house dust, Toxocara canis-specific immunoglobulin (Ig)E/IgG, allergen-specific and total IgE, skin microbiota] were measured at three months of age, and a possible association with Treg cell levels was assessed. RESULTS: The percentages of CD4+ CD25+ Foxp3+ Treg cells in healthy dogs were significantly higher at in 3-month-old (mean 4.5% healthy versus 3.3% allergic; P = 0.021) and <1-year-old (4.0% healthy versus 2.9% allergic; P = 0.028) dogs when compared to percentages of Treg cells in dogs that developed AD. There was a significantly positive correlation between the relative abundance of Lachnospiraceae on the skin and CD4+ CD25+ Foxp3+ Treg cells in puppies that became allergic (r = 0.568, P = 0.017). CONCLUSION AND CLINICAL IMPORTANCE: Further large-scale studies are needed to identify the practical value of these findings in AD diagnosis, treatment and prevention.

Human Alveolar Echinococcosis, Croatia.

Alveolar echinococcosis is a parasitic disease caused by the tapeworm larval stage of Echinococcus multilocularis. This zoonotic disease has not been known to occur in Croatia. We report a confirmed case of human alveolar echinococcosis in a patient in Croatia who had never visited a known E. multilocularis-endemic area.

Conquering Switzerland: the emergence of Angiostrongylus vasorum in foxes over three decades and its rapid regional increase in prevalence contrast with the stable occurrence of lungworms.

Angiostrongylus vasorum, Crenosoma vulpis and Capillaria aerophila are the most common lungworms of domestic and wild canids. We investigated the short- and long-term lungworm prevalence changes in the Swiss fox population with a focus on A. vasorum. Between 2012 and 2017, lungs and hearts of 533 foxes from north-eastern Switzerland were necropsied and blood samples tested for circulating A. vasorum antigen. Angiostrongylus vasorum prevalence increased steadily from 21.5% in 2012 to 81.8% in 2017. In contrast, C. aerophila and C. vulpis prevalences fluctuated between 41.8 and 74.7%, and 3.6 and 14.9%, respectively. Based on 3955 blood samples collected between 1986 and 2017 from three geographic areas and during four time periods, antigen seropositivity increased from 2.4 to 62.0%. In north-eastern Switzerland, seropositivity was initially low (1.9 and 1.7% in the first two time periods) but increased in the following two decades to 22.2 and 62.0%, respectively. Our findings depict the spectacular expansion of A. vasorum in the past three decades. Regionally, the prevalence in foxes increased 4-fold within 6 years in some regions. This underpins the important role of foxes as reservoir hosts, likely explaining the increasing number of cases of canine angiostrongylosis in Switzerland. Our findings are representative of central Europe and may help anticipating future developments in areas where A. vasorum is present but (still) infrequent.

Long-term (35 years) cryopreservation of Echinococcus multilocularis metacestodes.

The metacestode of Echinococcus multilocularis is the etiological agent of alveolar echinococcosis. The metacestode stage used for research is maintained in rodents by serial passages. In order to determine whether cryopreservation of E. multilocularis metacestodes would be suitable for long-term maintenance and replace serial passages, isolates of different geographic origin were cryopreserved in 1984-1986. The aim of the current study was to test the viability of cryopreserved isolates following long-term cryopreservation (up to 35 years) and to determine the phylogenetic clades these isolates belonged to. Cryopreserved isolates were tested for viability in vitro and in vivo in gerbils. In vitro results of 5 isolates indicated protoscolex survival in 13 of 17 experiments (76%) and metacestode survival in 5 of 12 (42%) in vivo experiments. In vivo results showed 'abortive lesions' in 13 of the 36 animals, 15 were negative and 8 harboured proliferating metacestode tissue containing protoscoleces. Genetic analysis confirmed the isolates belonged to European, Asian and North-American clades. In conclusion, the results of the current study indicate that metacestodes of E. multilocularis are able to survive long-term cryopreservation. Therefore, cryopreservation is a suitable method for long-term storage of E. multilocularis metacestode isolates and reduces the number of experimental animals.

Unravelling Spirocerca vulpis from red foxes from Switzerland: a 20-year-old record.

Spirocerca vulpis is a parasitic nematode of red foxes associated with gastric nodule formation. Since its description in 2018, this species has been reported in red foxes from Spain, Bosnia and Herzegovina, Italy, and Portugal. We present here the analysis of uncharacterized nematodes obtained from gastric nodules of a red fox from Switzerland in 1999. The specimens were identified as S. vulpis based on the observation of teeth-like structures in the buccal capsule and a 99.4% sequence identity to S. vulpis DNA from Spain. Phylogenetic analysis demonstrated the clustering of the Swiss sequences in a different group from specimens of other geographical locations. Altogether, this study constitutes the first report of S. vulpis in Switzerland and a report of the oldest specimen of this species in the world. Our findings highlight the widespread distribution of S. vulpis in Europe which may be facilitated by the free-roaming nature of red foxes.

The genomes of four tapeworm species reveal adaptations to parasitism.

Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.

ALVEOLAR ECHINOCOCCOSIS IN WESTERN LOWLAND GORILLAS (GORILLA GORILLA GORILLA): ALBENDAZOLE WAS NOT ABLE TO STOP PROGRESSION OF THE DISEASE.

Echinococcus multilocularis is the etiologic agent of alveolar echinococcosis (AE), a severe and potentially fatal larval cestode infection primarily affecting the liver. AE is known to occur in dead-end intermediate hosts, including humans and nonhuman primates. Between 1999 and 2016, AE was diagnosed in seven western lowland gorillas (Gorilla gorilla gorilla), all from a Swiss zoo. Six gorillas died of the disease. One individual is still alive, receives continuous albendazole medication, and shows no clinical signs. Most infected animals remained asymptomatic for years. Only one young gorilla showed early signs of acute discomfort and abdominal pain. In the final stage of the disease, affected animals died suddenly, or showed a short course of nonspecific but severe clinical signs, including lethargy, recumbency, abdominal enlargement, and anorexia. Postmortem examination confirmed hepatic AE complicated by peritonitis in most cases. Echinococcus multilocularis infection may remain undetected because of a very long incubation period. Hematological and biochemical parameters rarely showed abnormalities in this phase. Thus, inclusion of abdominal hepatic ultrasound examination and serology is recommended for early AE detection in routine examinations of gorillas in endemic areas or where food is potentially contaminated with E. multilocularis eggs. Ultrasound or computed tomography was useful to monitor progression and to estimate the volumetric extension of the hepatic lesions. Current medication with albendazole, which proved to be effective for human patients, was not able to stop progression of hepatic lesions in gorillas. Therefore, its therapeutic value remains questionable in gorillas. However, long-term oral albendazole treatment proved to be safe, and therapeutic plasma levels published for humans were achieved. Preventive measures such as thermo-treatment of food or vaccination of gorillas and other nonhuman primates should be considered in areas where E. multilocularis is present.

Oral Application of Recombinant Bacillus subtilis Spores to Dogs Results in a Humoral Response against Specific Echinococcus granulosus Paramyosin and Tropomyosin Antigens.

Bacillus subtilis is known as an endospore- and biofilm-forming bacterium with probiotic properties. We have recently developed a method for displaying heterologous proteins on the surface of B. subtilis biofilms by introducing the coding sequences of the protein of interest into the bacterial genome to generate a fusion protein linked to the C terminus of the biofilm matrix protein TasA. Although B. subtilis is a regular component of the gut microflora, we constructed a series of recombinant B. subtilis strains that were tested for their ability to be used to immunize dogs following oral application of the spores. Specifically, we tested recombinant spores of B. subtilis carrying either the fluorescent protein mCherry or else selected antigenic peptides (tropomyosin and paramyosin) from Echinococcus granulosus, a zoonotic intestinal tapeworm of dogs and other carnivores. The application of the recombinant B. subtilis spores led to the colonization of the gut with recombinant B. subtilis but did not cause any adverse effect on the health of the animals. As measured by enzyme-linked immunosorbent assay and immunoblotting, the dogs were able to develop a humoral immune response against mCherry as well as against E. granulosus antigenic peptides. Interestingly, the sera of dogs obtained after immunization with recombinant spores of E. granulosus peptides were able to recognize E. granulosus protoscoleces, which represent the infective form of the head of the tapeworms. These results represent an essential step toward the establishment of B. subtilis as an enteric vaccine agent.

Intense Focus of Alveolar Echinococcosis, South Kyrgyzstan.

Human alveolar echinococcosis (AE) is a highly pathogenic zoonotic parasitic disease caused by Echinococcus multilocularis. An ultrasound study in southern Kyrgyzstan during 2012 revealed a prevalence of 4.2% probable or confirmed AE and an additional 2.2% possible AE, representing an emerging situation. The risk for probable or confirmed AE was significantly higher in dog owners.

Evaluation of kinase-inhibitors nilotinib and everolimus against alveolar echinococcosis in vitro and in a mouse model.

Infection with the larval stage (metacestode) of the fox tapeworm Echinococcus multilocularis leads to a primary hepatic disease referred to as alveolar echinococcosis (AE). The progressive disease can be lethal if untreated. In cases where complete parasite resection by surgery is not feasible, the current treatment regimens of AE consist of chemotherapy with the parasitostatic benzimidazoles albendazole or mebendazole over decades. Kinase-inhibitors currently administered in various cancer treatments are of increasing interest also as anti-parasitic drugs due to previous promising in vitro results. In order to search for novel drug targets and treatment regimens, nilotinib (AMN107; Tasigna®), an Abl-tyrosine kinase inhibitor and everolimus (RAD001; Afinitor®), a serine/threonine-kinase inhibitor, were tested for their treatment efficacy against metacestode vesicles of E. multilocularis in vitro and in BALB/c mice. In vitro treatment with 200 µM nilotinib caused drug-induced alterations after 12 days, and everolimus exerted parasite damage at concentrations dosing from 40 to 100 µM after 5 and 12 days of in vitro exposure. Nilotinib (100 mg/kg) + erythromycin (to increase nilotinib plasma levels: 10 mg/kg intraperitoneal) or everolimus (5 mg/kg) were formulated in honey and administered daily for three weeks and subsequently twice a week for an additional three weeks in experimentally infected mice. Treatments did not result in any reduction of parasite growth compared to untreated control groups, whereas oral treatment with albendazole (200 mg/kg) was highly effective. Combined application of the kinase-inhibitors with albendazole did not lead to a synergistic or additive treatment efficacy compared to albendazole treatment alone. These results show that neither nilotinib nor everolimus represent valuable alternatives to the current treatment regimens against AE.

First detection of Echinococcus multilocularis in Croatia.

Echinococcus multilocularis has been spreading through Europe but has not yet been reported in Croatia. We report the results of a surveillance programme to detect E. multilocularis in red foxes (Vulpes vulpes) in different parts of Croatia. PCR-based screening of faecal samples from 238 red foxes in 2015 and 150 in 2016 indicate prevalences of 7.5% in 2015 and 6.6% in 2016 (overall 7.2%, CI 4.9 to 10.3). Positive samples were confirmed by sequencing parts of the nad1 gene and the gene encoding mitochondrial 12S rRNA. Geographic locations of all examined and positive cases were mapped to provide data on the distribution of E. multilocularis. Our results provide the first detection of E. multilocularis in Croatia and extend the southern boundary of this parasite's endemic area.