Secondary and primary murine alveolar echinococcosis: combined albendazole/nitazoxanide chemotherapy exhibits profound anti-parasitic activity.

In this study, the efficacies of chemotherapy employing nitazoxanide (NTZ), albendazole (ABZ), and a NTZ/ABZ-combination against alveolar echinococcosis (AE) were investigated in an experimental murine model. Following secondary infection, meaning i.p. injection of 20 Echinococcus multilocularis metacestodes, the drugs were administered by intragastric inoculation on a daily bases for a period of 5 weeks. Treatment was started either immediately on the day of infection, or at 2 months p.i., respectively. Application of the NTZ/ABZ-combination starting at 2 months p.i. was proven to be most effective in terms of reducing parasite weight (from 4.42+/-1.03 to 1+/-0.05 g; P=0.01). Inspection of treated parasites by transmission electron microscopy showed that ABZ- and NTZ-treated metacestode tissues, respectively, were heterogeneous in that both largely intact parasites as well as severely altered metacestodes could be observed. NTZ/ABZ-combination treatment induced the most severe ultrastructural alterations, including massive reduction in length and number of microtriches, severely damaged tegumental architecture, and progressive loss of viability of the germinal layer, associated with encapsulation by host connective tissue. A comparative pharmacokinetic study in mice revealed that the application of ABZ and NTZ in combination resulted in a two- to four-fold increase of albendazole sulfoxide serum levels for the period of 4-8 h following drug uptake compared to application of ABZ alone. In a third experiment, mice were orally infected with E. multilocularis eggs, and treated with NTZ starting at 2 months p.i. This resulted in a significantly lower lesion number in treated versus untreated mice (P=0.01). This investigation indicates the potential value for NTZ and/or a combined ABZ/NTZ chemotherapy against AE.

In vitro culture of Echinococcus multilocularis and Echinococcus vogeli metacestodes: studies on the host-parasite interface.

The larval stage of Echinococcus multilocularis causes alveolar echinococcosis (AE) in various mammalians including humans, while Echinococcus vogeli larvae cause a related disease which is also occasionally found in man. Traditionally, Echinococcus metacestodes have been maintained in the laboratory by serial transplantation passages into susceptible animals such as mice or gerbils, enabling the parasite to proliferate asexually. These experimental animal models have been used extensively to investigate host-parasite interactions and to study immunological events occurring at the host-parasite interface. However, with the use of laboratory animals it has always been difficult to investigate in more detail those factors modulating metacestode differentiation, and investigations on gene expression and respective regulation have been hampered by the complexity of the host-parasite interplay. There has been a need for an in vitro culture model which would enable researchers to dissect specific parasite compartments involved in the host-parasite relationship in more detail. This review summarises the studies leading to the development and application of a suitable in vitro culture model for the maintenance and proliferation of E. multilocularis and E. vogeli metacestodes, including the formation of protoscoleces, in a chemically defined medium devoid of host influence. These culture models have been used to study the basic parameters of metacestode in vitro proliferation and differentiation, and for the dissection of the ultrastructure and composition of the acellular laminated layer, the structure of which is predominantly involved in the physical interaction between the parasite and host immune and non-immune cells and tissues. For E. multilocularis, in vitro cultured parasites have been more extensively employed to study the localisation of several antigens, and to generate defined antigens for immunological studies. Although in vitro culture will not completely eliminate the need of animal experimentation, a wider application of this technique could significantly reduce the use of animals, and thus the costs and time required for respective experimental investigations.

In vitro parasiticidal effect of Nitazoxanide against Echinococcus multilocularis metacestodes.

When humans serve as inadvertent intermediate hosts for Echinococcus multilocularis, disease (alveolar echinococcosis [AE]) may result from the expanding parasite metacestode in visceral organs, mostly in the liver. Benzimidazole carbamate derivatives such as mebendazole and albendazole are used for chemotherapeutic treatment of AE. However, these treatments are, in most cases, parasitistatic rather than parasiticidal. As treatment is discontinued, a recurrence of parasite growth has been observed in many AE patients with nonradical resections. The only curative treatment for AE is radical surgical resection of the parasite tissue and support by chemotherapy. As there is a need for new treatment options for AE, the in vitro efficacy of nitazoxanide (NTZ), a broad-spectrum drug used against intestinal parasites and bacteria, was investigated. We showed that in vitro treatment of E. multilocularis metacestodes with NTZ induced high levels of alkaline phosphatase activity in the medium. Concurrently, distinct morphological and ultrastructural alterations were detected. Most significantly, two distinct types of alterations were observed as soon as after 3 h of NTZ treatment. At first, the drug induced a peripheral output of membranous vesicles from the tegumental membrane into the laminated layer. Simultaneously, germinal layer-associated undifferentiated cells produced large vacuoles filled with lipid-like and often electron-dense membranous segments. Other alterations were observed at later time points, including vacuolization of the germinal layer, accumulation of lipid droplets, and lastly, loss of microtriches and separation of the laminated and germinal layers. The pattern of damage induced by NTZ was different from the alterations earlier observed in albendazole sulfoxide-treated vesicles. The nonviability of NTZ-treated metacestodes was confirmed through bioassay, i.e., inoculation of treated and untreated parasites into mice. These experiments demonstrate the in vitro parasiticidal effect of NTZ on E. multilocularis metacestodes.

Culture of Echinococcus multilocularis metacestodes: an alternative to animal use.

This article reviews the use of an in vitro culture model for the maintenance and proliferation of Echinococcus multilocularis metacestodes and the formation of protoscoleces. This model has been used to identify and characterize parasite molecules involved in host-parasite interactions, and is a suitable tool to perform in vitro drug-screening assays. The development of a simple and easy-to-handle assay to determine the effects of drugs on parasite viability, without the need for time-consuming animal experimentation, has opened the way for larger-scale in vitro drug screening.