Echinococcus multilocularis alkaline phosphatase as a marker for metacestode damage induced by in vitro drug treatment with albendazole sulfoxide and albendazole sulfone

Abstract

Alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis. The disease affects the human liver and occasionally other organs and is fatal if treatment is unsuccessful. The present chemotherapy of AE is based on the administration of benzimidazole carbamate derivatives, such as mebendazole and albendazole. Albendazole treatment has been found to be ineffective in some cases, parasitostatic rather than parasiticidal, and the recurrence rate is rather high. Therefore, chemotherapy usually involves the lifelong uptake of massive doses of albendazole and new treatment options are urgently needed. In order to avoid costly and time-consuming animal experimentation, a first step in searching for novel parasiticidal compounds could be the in vitro drug screening of novel compounds by employing metacestode cultivation. However, presently used techniques (e.g., transmission electron microscopy) for determination of parasite viability involve costly equipment and time-consuming preparation of rather large amounts of parasite material. We therefore searched for a parasite marker which can be easily traced and the presence or absence of which is indicative of parasite viability. In this study we show that the increase of E. multilocularis alkaline phosphatase activity in culture supernatants during in vitro drug treatment with albendazole derivatives correlates with the progressive degeneration and destruction of the metacestode tissue. The inexpensive and rapid assay presented here will serve as an ideal tool for performing first-round in vitro tests on the efficacy of a large number of antiparasitic compounds.

FIG. 1

Demonstration of EmAP activity as revealed by dot blotting in E. multilocularis vesicle fluid, insoluble metacestode walls, soluble metacestode tissue components, and medium supernatants after different time points of in vitro culture in the presence and absence of 10 μg/ml of ABZSO or ABZSN. Number of days of in vitro treatment is indicated.

FIG. 2

Time course experiment demonstrating elevated EmAP activity in culture supernatants of E. multilocularis metacestodes treated with 10 μg of ABZSO or ABZSN per ml. Controls include RPMI 1640 medium alone (without parasites; designated medium), RPMI 1640 medium plus metacestodes (control), or RPMI 1640 medium plus metacestodes plus the corresponding amount of DMSO (DMSO). Note the increase of EmAP levels starting at day 4 in drug-treated cultures.

FIG. 3

SEM nontreated (A and B) or ABZSO-treated (C to F) E. multilocularis metacestodes. GL, germinal layer; LL, laminated layer. (A and B) Control metacestodes cultured in vitro in the presence of DMSO (1:1,000) but in the absence of any drugs. Note that most cells exhibit an intact morphology. Bar = 1.2 mm (A) or 200 μm (B). (C to F) SEM of metacestodes cultured in vitro in the presence of ABZSO for 10 days (C and D) and of ABZSN for 14 days (E and F). (C) Large portions of the germinal layer have disintegrated after 10 days of drug treatment and are detached from the laminated layer (bar = 900 μm). (D) Higher-magnification view of image in panel A (bar = 200 μm). (E) After 14 days, only metacestode “ghosts,” comprised of the acellular laminated layer, are found (bar = 1.2 mm). (F) Higher-magnification view onto the interior surface of the laminated layer. Note the presence of largely destroyed cells (bar = 180 μm).

FIG. 4

Comparative assessment of ultrastructure (A and B; bars = 4 and 3 μm, respectively) and EmAP immunogold labeling (C and D; bars = 2.3 and 1.9 μm, respectively) in control (A and C) versus ABZSO-treated (B and D) metacestodes 10 days after initiation of drug treatment. Note the lack of the typical microfibrillar pattern in the matrix of control metacestodes (A) compared to the amorphous appearance of the laminated layer (LL) in drug-treated parasites (B). The density of EmAP immunogold labeling within the laminated layer in drug-treated parasites (D) is significantly lower than in control metacestodes (C). GL, germinal layer.