1 Institute of Parasitology, Faculties of Veterinary Medicine and Medicine, University of Berne, Länggass-Strasse 122, CH-3012 Berne; 3 Institute of Parasitology, University of Zürich, Winterthurerstrasse 266a, CH-8057 Zürich, Switzerland; 2 The Romark Institute for Medical Research, Tampa, FL 33607, USA
Received 20 April 2004; returned 19 May 2004; revised 26 May 2004; accepted 29 June 2004
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Abstract |
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Methods: Freshly isolated E. granulosus protoscoleces were subjected to nitazoxanide treatment (1, 5 and 10 µg/mL), and the effects on parasite viability were monitored by Trypan Blue staining and scanning electron microscopy. Protoscolex cultures were maintained further, until metacestode development took place. Metacestodes were then subjected to nitazoxanide treatment (10 µg/mL), and corresponding effects were visualized by scanning and transmission electron microscopy.
Results: Dose-dependent protoscolex death within a few days of nitazoxanide treatment was observed. Subsequent in vitro culture of drug-treated protoscoleces confirmed the non-viability of parasites, while further cultivation of non-treated protoscoleces for a period of at least 3 months resulted in stage conversion and the formation of small metacestodes 34 mm in diameter. Nitazoxanide had a deleterious effect on these metacestodes, which was comparable to that of albendazole.
Conclusions: Our study indicates a potential for nitazoxanide as an alternative treatment option against CE.
Keywords: E. granulosus , cystic echinococcosis , in vitro drug treatment , albendazole
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Introduction |
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After an undefined incubation period, E. granulosus metacestodes are formed. These are single, fluid-filled cysts, which are surrounded by a thick, host-derived layer of connective tissue (adventitia). The parasite tissue is delineated by the laminated layer, an acellular, carbohydrate-rich outer surface structure of considerable thickness, which separates the living parasite tissue at the inside from the outer host environment. Most human patients harbour a solitary cyst within a single organ. In 70% of patients, infection occurs in the liver, the lung being affected in 20% of patients. Other sites of infection include kidney, spleen, muscle, skin, abdominal and pelvic cavity, mediastinum, heart, brain, spinal cord and others.3 Within fertile metacestodes, brood capsules are formed out of distinct cells originating from the germinal layer, which themselves contain protoscoleces. Infertile metacestodes only contain the germinal layer. Often the metacestode contains one or more daughter cysts, which give rise to the septate appearance in diagnostic imaging.4 The cysts slowly expand over several years, often without being noticed. Spread of infection into other sites, such as the peritoneal cavity, has been described, and this can be caused by cyst rupture and spillage of protoscoleces. These have the potential to develop into metacestodes in the intermediate host, and into adult parasites when ingested by the final host.2
Treatment of human CE by chemotherapy has been developed following a series of animal studies, which demonstrated the efficacy of benzimidazole carbamates against E. granulosus and Echinococcus multilocularis.510 Albendazole and mebendazole are considered to be equally effective. When evaluated up to 12 months after initiation of benzimidazole treatment, 10%30% of patients exhibit cyst disappearance, 50%70% show degeneration of cysts and 20%30% show no morphological changes in cyst appearance.11,12 Continuous or intermittent treatment with albendazole is recommended for a period of 6 months. Praziquantel, shown to exhibit protoscolicidal activity, has been reported to enhance the effect of benzimidazoles, especially when cyst spillage was considered to pose a problem. In about 75% of patients, metacestodes exhibit clear signs of degenerative changes.11 However, adverse side effects have been observed with both benzimidazoles, including abnormalities in liver function, leucopenia and alopecia, and albendazole has been shown to be teratogenic in rats and rabbits. It should therefore be avoided during pregnancy and lactation.3,11,12 As a consequence, the development of alternative means of treatment of CE is necessary.
Nitazoxanide [2-acetolyloxy-N-(5-nitro-2-thiazolyl) benzamide], originally developed as a veterinary antihelminthic, was first described in 1984 as a human cestocidal drug.13 To date, nitazoxanide is known as a broad-spectrum drug against a wide variety of intestinal parasites and enteric bacteria infecting animals and humans. Specifically for the treatment of chronic diarrhoea in immunocompromised patients (e.g. AIDS), nitazoxanide has become widely used.14 More recently, we have reported on the efficacy of nitazoxanide against E. multilocularis metacestodes in vitro,15 and upon egg infection in the murine model.16 We have also shown that combined nitazoxanide/albendazole treatment of mice infected intraperitoneally with E. multilocularis metacestodes results in a treatment efficacy that is superior to albendazole alone. This effect was partially attributed to the fact that combined application of the two drugs leads to increased albendazole sulphoxide serum levels for extended periods of time.16 In this study, we report on the in vitro efficacy of nitazoxanide against E. granulosus protoscoleces and metacestodes.
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Materials and methods |
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If not otherwise stated, all tissue culture media were purchased from Gibco-BRL (Zürich, Switzerland), and biochemical reagents were from Sigma (St Louis, MO, USA). Nitazoxanide used in this study was obtained from Romark Laboratories (Tampa, FL, USA). Albendazole was purchased from GlaxoSmithKline (Germany).
In vitro culture of E. granulosus protoscoleces and metacestodes
E. granulosus hydatid cysts containing protoscoleces were removed under aseptic conditions from infected sheep presented for routine slaughter in abattoirs in Almaty, Kazakhstan. The largely intact cysts were stored for no longer then 1 week at 4°C, during which time they were transported to Switzerland. In vitro cultivation of E. granulosus protoscoleces and metacestodes was carried out as previously described for E. multilocularis metacestodes.17 Briefly, the hydatid cysts (25 cm in diameter) were cut open and vesicle fluid (containing protoscoleces) was separated from the metacestode tissue and host adventitia. Protoscoleces were allowed to settle in a 50 mL Falcon tube, were washed twice in Hanks balanced salt solution and placed into culture medium (RPMI 1640 containing 12 mM HEPES, 2 mM glutamine, 200 U/mL of penicillin, 200 µg/mL of streptomycin and 0.50 µg/mL of amphotericin B) supplemented with 10% fetal calf serum and phenol red. Cultures were kept in culture flasks (200 mL) placed in an upright position in an incubator at 37°C, 5% CO2, with medium changes every 48 days.
Drug treatment of E. granulosus protoscoleces
Treatment of protoscoleces was initiated within 10 days of in vitro culture. Three T25 tissue culture flasks containing at least 500 protoscoleces in 20 mL culture medium were supplemented with 1, 5 and 10 µg/mL nitazoxanide [stock solution in 10 mg/mL in dimethyl sulphoxide (DMSO)], and one control culture was supplemented with 20 µL DMSO alone. The vitality/viability of protoscoleces was assessed by observation of motile behaviour and a Trypan Blue exclusion test, respectively. The corresponding numbers of viable/non-viable protoscoleces were determined in 10 randomly chosen fields by phase contrast microscopy using a Leitz DMIL inverted microscope at 10x magnification. At day 5 following initiation of drug treatment, the medium±nitazoxanide was removed, parasites were washed twice in 20 mL Hanks balanced salt solution and culture was continued in fresh culture medium without nitazoxanide. Of each culture, a small sample was processed for scanning electron microscopy (SEM) (see below).
Drug treatment of E. granulosus metacestodes
E. granulosus metacestode formation was achieved by continuing the culture of protoscoleces for a period of 34 months. Parasites that had undergone stage conversion into small (14 mm) hydatid cysts were collected, split into groups of 40, and resuspended in 5 mL of culture medium supplemented with nitazoxanide (10 µg/mL) and albendazole (10 µg/mL). Two control cultures were set up in culture medium and medium supplemented with the corresponding amount of DMSO, respectively. Samples for SEM and transmission electron microscopy (TEM) inspection were taken on days 0, 4 and 7 following initiation of drug treatment.
SEM and TEM
Protoscoleces and metacestodes were processed as described by Hemphill & Croft.18 Briefly, parasites were washed once in 100 mM sodium cacodylate buffer pH 7.2, and placed into the primary fixative (2.5% glutaraldehyde in 100 mM sodium cacodylate buffer) for 2 h at room temperature. After addition of the fixative, vesicles were gently broken up with a pipette. After several washes in cacodylate buffer, samples were post-fixed in 2% OsO4 in cacodylate buffer. The specimens were then extensively washed in distilled water.
For SEM analysis, the specimens were dehydrated by sequential incubations in increasing concentrations of ethanol (50%70%), and were finally immersed in hexamethyl-disilazane and air-dried under a fume hood. They were then sputter-coated with gold, and inspected on a JEOL 840 scanning electron microscope operating at 25 kV.
For TEM, fixed metacestode vesicles were pre-stained in 1% uranyl acetate for 30 min at 4°C, followed by several washes in water. They were dehydrated in a graded series of ethanol and subsequently embedded in Epon 812. Polymerization of the resin was carried out at 65°C overnight. Sections 12 µm thick were cut on a Reichert and Jung ultramicrotome, loaded onto poly-L-lysine-coated glass coverslips, and stained with Methylene Blue-Fuchsin. Eighty nanometre sections were loaded onto 300-mesh copper grids (Plano GmbH, Wetzlar, Germany), and contrasting with uranyl acetate and lead citrate was performed, as described previously.18
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Results |
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Following the isolation of protoscoleces from hydatid cysts, the Trypan Blue exclusion test revealed that >85% of the parasites were still viable, and >60% of protoscoleces exhibited distinct movements. Of those, the majority was invaginated (Figure 1A and B), but 10%15% were evaginated; rostellum and suckers were clearly visible (see Figure 1C), and these regions of the parasites were the most motile compartments (data not shown).
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In vitro effects of nitazoxanide treatment on E. granulosus metacestodes
E. granulosus metacestodes were obtained by continuous in vitro maintenance of the cultures initiated with protoscoleces for a period of 34 months. This resulted in the formation of vesiculated parasites of 14 mm in diameter. Inspection of these vesicles by SEM revealed that they exhibited features typical of E. granulosus metacestodes, with a distinct acellular outer laminated layer and an intact germinal layer comprised of a multitude of different, morphologically intact, cell types (Figure 3A and B). Upon treatment with nitazoxanide and albendazole for 3 days, macroscopic morphological changes became evident in >50% of metacestodes, most evident through the loss of turgidity. No damage could be seen in the control vesicles. At days 57, all vesicles were extensively distorted upon drug treatment, but control vesicles showed no alteration. Corresponding effects are illustrated by SEM in Figure 3(C and D). Clearly, at day 7 of treatment with both nitazoxanide and albendazole, in many areas, only cellular debris of the former germinal layer could be seen.
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Discussion |
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Nitazoxanide is a 5-nitrothiazole analogue, with structural similarities to the benzimidazole antihelminthics such as albendazole and its metabolic derivatives albendazole sulphoxide and albendazole sulphone, with a 5-nitrothiazole ring substituting the benzimidazole ring. However, the mode of action of the two drugs appears to be different. Benzimidazole derivatives bind to beta-tubulin, and prevent the uptake of glucose by disrupting cellular microtubular structures. This leads to a reduction in glucose uptake, depletion of glycogen storage, degenerative alterations in the germinal layer and cellular autolysis.19 In bacteria and in the protozoan parasite Cryptosporidium, nitazoxanide has been postulated to be reduced to a toxic radical when the 5-nitro-group on the nitroheterocyclic ring reacts with pyruvate:ferredoxin oxidoreductase.20 Nothing is known about a possible mode of action of nitazoxanide in helminths. However, the enzymes of anaerobic electron transport also could be considered as potential targets.
E. granulosus protoscoleces had been shown earlier to be susceptible to treatment with the albendazole metabolite albendazole sulphoxide.21 Another drug, which is widely used for inactivation of protoscoleces, is praziquantel. Praziquantel is a pyrazinoisoquinoline derivative with a wide antihelminthic spectrum including schistosomes, hermaphroditic flukes and cestodes Whereas it is very effective against adult tapeworms, as well as against E. granulosus protoscoleces in vitro, in vivo studies on E. granulosus metacestode infections have been inconclusive.22,23 However, more recently it was suggested that praziquantel exerts a substantial effect against metacestodes and protoscoleces when applied in combination with albendazole.24,25
In addition to its efficacy against protoscoleces, nitazoxanide also exhibits a considerable damaging effect on the metacestode stage. The high parasiticidal efficacy of nitazoxanide is not surprising, as this compound was shown to exhibit a uniquely wide spectrum of antiparasitic and antimicrobial activities,14 including in vitro and in vivo activity against E. multilocularis metacestodes.15,16 In vivo pharmacokinetic studies in humans and mice have shown that nitazoxanide is substantially absorbed following oral administration, and that it is rapidly hydrolysed to tizoxanide, which is extensively bound to plasma albumin, and later to tizoxanide glucuronate.16,26,27
Previous studies on E. multilocularis metacestodes treated with nitazoxanide and albendazole, respectively, had been able to differentiate between the effects of the two drugs during in vitro and in vivo treatment.15,16 For instance, the progressive destruction of E. multilocularis metacestode tissue through albendazole treatment was characterized primarily by shortening and distortion of microtriches, followed by other alterations (vacuolization of the germinal layer, aberrant mitochondria, lipid droplets, inclusion bodies and loss of cellcell contacts within a time frame of 610 days).28 In contrast, nitazoxanide treatment leads first to a marked release of small vesicles from the tegument into the laminated layer, and the formation of large cytoplasmic vacuoles in undifferentiated cells, accompanied by progressive changes within the germinal layer.15 This indicated that in E. multilocularis metacestodes the two drugs exhibited two different modes of action. In contrast, in this study on E. granulosus metacestodes, we were not able readily to distinguish between the alterations imposed upon the parasites through albendazole and nitazoxanide treatment, respectively. However, a more detailed analysis, involving shorter time frames, will be necessary to provide respective information.
In conclusion, we here demonstrate the in vitro efficacy and parasiticidal activity of nitazoxanide against E. granulosus protoscoleces and metacestodes. In a next step, we will investigate the efficacy of nitazoxanide and its derivatives in an animal model. There, the active compound has to reach the infected organ and the parasite tissue in a sufficiently high concentration.
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Acknowledgements |
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Footnotes |
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References |
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20
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28
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