Johannes Eckert

Biological, Epidemiological, and Clinical Aspects of Echinococcosis, a Zoonosis of Increasing Concern

SUMMARY Echinococcosis in humans is a zoonotic infection caused by larval stages (metacestodes) of cestode species of the genus Echinococcus. Cystic echinococcosis (CE) is caused by Echinococcus granulosus, alveolar echinococcosis (AE) is caused by E. multilocularis, and polycystic forms are caused by either E. vogeli or E. oligarthrus. In untreated cases, AE has a high mortality rate. Although control is essentially feasible, CE remains a considerable health problem in many regions of the northern and southern hemispheres. AE is restricted to the northern hemisphere regions of North America and Eurasia. Recent studies have shown that E. multilocularis, the causative agent of AE, is more widely distributed than previously thought. There are also some hints of an increasing significance of polycystic forms of the disease, which are restricted to Central and South America. Various aspects of human echinococcosis are discussed in this review, including data on the infectivity of genetic variants of E. granulosus to humans, the increasing invasion of cities in Europe and Japan by red foxes, the main definitive hosts of E. multilocularis, and the first demonstration of urban cycles of the parasite. Examples of emergence or reemergence of CE are presented, and the question of potential spreading of E. multilocularis is critically assessed. Furthermore, information is presented on new and improved tools for diagnosing the infection in final hosts (dogs, foxes, and cats) by coproantigen or DNA detection and the application of molecular techniques to epidemiological studies. In the clinical field, the available methods for diagnosing human CE and AE are described and the treatment options are summarized. The development of new chemotherapeutic options for all forms of human echinococcosis remains an urgent requirement. A new option for the control of E. granulosus in the intermediate host population (mainly sheep and cattle) is vaccination. Attempts are made to reduce the prevalence of E. multilocualaris in fox populations by regular baiting with an anthelmintic (praziquantel). Recent data have shown that this control option may be used in restricted areas, for example in cities, with the aim of reducing the infection risk for humans.

Light and electron microscopic studies on the effect of praziquantel on Schistosoma mansoni, Dicrocoelium dendriticum, and Fasciola hepatica (Trematoda) in vitro.

The fine structure of the tegument of three trematode species,Schistosoma mansoni, Dicrocoelium dendriticum, andFasciola hepatica, was studied by means of light scanning (SEM) and transmission electron microscopy (TEM) after in vitro exposure to 0, 1,10, and 100 μg/ml of the anthelmintic praziquantel for 5, 15, 30, and 60 min. InS. mansoni andD. dendriticum the resulting vacuolization of the tegument was confined to numerous small areas scattered all over the surface of the parasites and this finally led to the disruption of the apical tegumental layer. No changes were found in the tegument ofF. hepatica after treatment with praziquantel.

Veterinary aspects of alveolar echinococcosis - a zoonosis of public health significance.

Human alveolar echinococcosis (AE), caused by the metacestode stage of Echinococcus multilocularis, is a serious zoonosis which caused up to 100% lethality in untreated patients before the 1970s, when modern methods of treatment were not yet established. AE occurs in large areas of the northern hemisphere mostly with low country-wide prevalences, but high prevalences of up to 4% have been reported from small population groups in highly endemic foci, e.g. from China. AE includes many veterinary aspects which are the topic of this review. Recent studies have shown that E. multilocularis has a wider geographic range than previously anticipated. There is evidence for growing populations of red foxes (Vulpes vulpes) in some areas, for increasing invasion of cities by foxes and also for establishment of the parasite cycle in urban areas. These and other factors may lead to an increased infection risk for humans. Significant progress has been made in the development of sensitive and specific new techniques for the intra vitam and post mortem diagnosis of intestinal E. multilocularis infection in definitive hosts, notably the detection of coproantigen by enzyme-linked immunosorbent assay and of copro-DNA by PCR. Both tests can also be used for the identification of E. multilocularis in faecal samples collected in the environment. Recommendations are given for chemotherapy and chemoprophylaxis of the intestinal infection in definitive hosts. In recent years, infections with the metacestode stage of E. multilocularis have not only been diagnosed in humans in several regions, including at least eight countries in central Europe, but also in animal species which do not play a role in the transmission cycle (wild and domestic pigs, dogs etc.). From 1987 to 2000 our group in Zurich has diagnosed 10 cases of AE in dogs and 15 in captive monkeys. In 2 dogs, concurrent infections of the intestine and of the liver with adult and larval stages of E. multilocularis, respectively, were observed for the first time. Clinical data are presented, and methods of diagnosis and treatment (surgery, chemotherapy) are described. Furthermore, small liver lesions caused by E. multilocularis were diagnosed in 10% of 90 slaughter pigs, and 2.9% of 522 breeding sows had specific serum antibodies against parasite antigens. In view of the unpredictable epidemiological situation, all possible measures for preventing E. multilocularis infections in humans and in domestic animals should be initiated by the veterinary and health authorities.

Human Alveolar Echinococcosis after Fox Population Increase, Switzerland

An increase in fox population has led to an increase in incidence of human alveolar echinococcosis.

Specific lgG1 and lgG2 antibody responses of dogs to Leishmania infantum and other parasites

Sera from dogs naturally infected with Leishmania infantum were analysed for the IgG subclass specificity of their antibody response by ELISA. Dogs infected with L. infantum produced both IgGl and IgG2 antibodies with IgG2 being associated with asymptomatic infections and IgGl being associated with disease (symptomatic dogs, non‐ or low‐responsive to chemotherapy). The differential responses of IgG] and IgGl serum antibodies in asymptomatic and symptomatic dogs may indicate a dichotomous immune response to infection with L. infantum. To confirm this, on a broader scale, sera from dogs naturally exposed to an asymptomatic protozoan infection, Toxoplasma gondii, were also analysed as were sera from dogs exposed to the helminths, Dirofilaria immitis and Toxocara canis. Antibodies specific for T. gondii antigen detected in sera from 17 dogs were of the IgG2 subclass only. Both IgGl and IgG2 antibodies to D. immitis andl. canis were present in the sera of naturally infected dogs but IgGl appeared to be the predominant subclass. Furthermore, in dogs experimentally infected with T. canis, selective regulation ofIgG2 and IgGl responses was apparent since production of the two subclasses occurred at different times following infection, with IgGl levels declining as IgGl levels rose. Thus, the analysis of IgG subsets in parasitized dogs provides evidence of a dichotomous response to infection: IgGl is associated with asymptomatic protozoan infections and IgGl is associated with helminth infections and disease caused by protozoan infection.

An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs

For the sensitive detection of eggs of Echinococcus multilocularis in fox faeces by PCR we have evaluated a method based on the previous concentration of helminth eggs by a combination of sequential sieving of faecal samples and flotation of the eggs in zinc chloride solution. The eggs were microscopically detected in the fractions retained in 40 and 20 microns mesh sieves. DNA of the taeniid eggs retained in the 20 microns sieve was obtained after alkaline lysis and PCR was performed using E. multilocularis species-specific primers. Compared to the parasitological findings after examination of the small intestines of the foxes, the specificity of the PCR was 100% (no false-positive result with 20 foxes free of E. multilocularis) and the sensitivity was 94% (33 positive results from total 35 foxes proven to be infected with E. multilocularis). Both false-negative results were obtained with faeces from foxes harbouring immature worms. Using faecal volumes between 2 and 20 ml, no inhibition of PCR was observed as was demonstrated by the amplification of size-modified target in parallel reactions. The tests were undertaken with fresh faeces stored in 70% ethanol, but egg detection by PCR was also possible after inactivation of eggs by freezing the faeces at -80 degrees C for one week or by incubation at +70 degrees C for 2 h.

Alveolar Echinococcosis in Humans: The Current Situation in Central Europe and the Need for Countermeasures

Alveolar echinococcosis (AE) in humans is caused by a larval stage (metacestode) of Echinococcus multilocularis, which exhibits a tumor-like growth, initially in the liver, with the potential to induce serious disease. At the end of the 1980s, E. multilocularis was known to occur in four countries of Central Europe, but has now been identified in ten countries. Red foxes are the principal definitive hosts of E. multilocularis and sources of human infection, but dogs and cats can also be infected. Growing populations of foxes and their increasing immigration to urban areas are new risk factors. Human AE is rare but its potential high fatality rate, considerable costs of treatment and the persisting infection risk should be reasons for health authorities in European countries to establish coordinated systems of surveillance and risk assessment in combination with measures to reduce morbidity and mortality of AE in the human population. Here, J. Eckert and P. Deplazes outline the current epidemiological situation in Central Europe, and discuss options for surveillance, prevention and control.

Chemotherapy for larval echinococcosis in animals and humans: Report of a workshop

Mebendazole, its fluorine analogue flubendazole, and other benzimidazole derivatives are active against many gastrointestinal and tissue-stage helminths. This article reviews the published literature and proceedings of a workshop on the use of benzimidazoles against larval echinococcosis (hydatid disease). Orally administered high doses (30–50 mg/kg body weight) of mebendazole given daily for 20–90 days to rodents or sheep infected with larvalEchinococcus granulosus cause damage or destruction of the cyst wall, loss of cyst fluid, and death of protoscolices. Similar treatment of rodents infected withE. multilocularis with mebendazole, flubendazole, fenbendazole, and albendazole for 60–300 days leads to reduction of weight, inhibition of growth and of metastases formation ofE. multilocularis tissue, and to prolonged host survival time although the metacestodes are not killed. Mebendazole or flubendazole treatment of human patients infected withE. granulosus is followed by subjective improvement in most, and evidence of regression of cysts in some; in other patients, cysts continue to grow or have been proven viable even after several months of high-dose mebendazole therapy. In patients infected withE. multilocularis, the progressive course of the disease appeared to be arrested, but treatment apparently did not kill the parasite. Side effects in some patients have included allergic reactions, alopecia, and reversible neutropenia. Some possible reasons for differnet responses to treatment include inadequate plasma drug absorption from the gut and age, condition, and location of cysts. Many remaining questions concerning the risk versus benefits of mebendazole therapy can be answered only through controlled clinical trials.

Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection ofEchinococcus coproantigens in fecal samples from dogs, dingoes or foxes infected with eitherE. granulosus orE. multilocularis. The ELISA was based on protein-A-purified polyclonal antibodies [anti-E. granulosus excretory/secretory (E/S) antigens]. The specificity of the assay as determined in 155 samples derived from carnivores that were free of helminth infection (n=37) or infected with non-Echinococcus cestodes (n=76) or with various nematodes (n=42) was found to be 98% overall. The diagnostic sensitivity was strongly dependent on the homologous worm burden. All 13 samples from foxes harboring >1,000E. multilocularis worms and 13 of 15 (87%) samples from dogs or dingoes containing >200E. granulosus worms were ELISA-positive, whereas 34 of 46 samples from foxes harboring <1,000E. multilocularis and 9 of 10 samples from dogs or dingoes bearing <200E. granulosus tested negative. Experimental prepatent infections of dogs withE. granulosus revealed positive ELISA reactions within the prepatent period (10–20 days post-infection) for six animals bearing >1,000E. granulosus each; a low worm burden (<1,000 tapeworms/animal) resulted in ELISA positivity in only 2 of 3 animals at 30 days post-infection at the earliest. All five dogs that had been experimentally infected withE. multilocularis tested positive in the coproantigen ELISA as early as on day 5 post-infection.

Intraspecific variation of Echinococcus granulosus and related species with emphasis on their infectivity to humans

Four species are presently recognised within the genus Echinococcus, namely Echinococcus granulosus, E. multilocularis, E. oligarthrus and E. vogeli, which are infective to humans. Evidence for strain diversity within the species E. granulosus, previously mainly based on morphological, biological and biochemical features, has been principally confirmed by recent genetic studies. Several molecular techniques are now available which allow the identification of E. granulosus strains. Epidemiological evidence and molecular studies indicate that the so-called sheep, cattle and cervid strains of E. granulosus are infective to humans, while the horse, camel and pig strains may be less or not infective, but this question warrants further studies. A recent study indicates that E. granulosus infecting patients in Poland shares close molecular affinity with a genotype of pig origin (G7) but exhibits some clear differences. Therefore, it may represent a previously undescribed genotype of E. granulosus, designated as G9. Phylogenetic analysis of molecular data has demonstrated the need to reappraise the taxonomic status of currently recognised strains. Clear evidence for strain variation in the other species of Echinococcus does not exist at present.