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In vitro efficacy of triclabendazole and clorsulon against the larval stage of

Echinocccus multilocularis

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Parasitol Res (2013) 112:1655–1660
DOI 10.1007/s00436-013-3321-7
ORIGINAL PAPER
In vitro efficacy of triclabendazole and clorsulon
against the larval stage of Echinococcus multilocularis
David Richter & Joachim Richter & Beate Grüner &
Kathrin Kranz & Juliane Franz & Peter Kern
Received: 13 June 2012 / Accepted: 24 January 2013 / Published online: 28 February 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Alveolar echinococcosis (AE) caused by the ces-
tode Echinococcus multilocularis (E. multilocularis) is en-
demic in wide areas of the Northern hemisphere. Untreated
AE progresses and leads to death in more than 90 % of
cases. Until the advent of benzimidazoles, no antihelminthic
drugs were available to cure AE. Benzimidazoles have
greatly improved the prognosis of patients with AE.
However, benzimidazoles have only a parasitostatic effect
on E. multilocularis. Albendazole (ABZ) must sometimes
be withdrawn because of adverse events. Alternative drugs
are urgently needed. The antihelminthic triclabendazole
(TCZ) and clorsulon (CLS) are more effective than ABZ
to cure infections by the liver flukes Fasciola spp. The
efficacy of TCZ and CLS was investigated on an in vitro
culture of E. multilocularis larval tissue. E. multilocularis
vesicles were evaluated for their morphology before and
after adding TCZ, TCZ sulfoxide (TCZSX) and CLS to
the larval tissue culture. TCZ at the concentrations of
20 μg/ml culture solution led to maximum vesicle damage
within 12 days and of 25 μg/ml within 13 days, and TCZSX
at the concentrations of 20 μg/ml within 20 days and of
25 μg/ml within 14 days. Contrary, CLS added at 5, 10 and
15 μg/ml to culture solution did not lead to any vesicle
damage. TCZ is a promising further candidate drug for the
treatment of AE.
D. Richter : B. Grüner : J. Franz : P. Kern
Department of Internal Medicine III, Comprehensive Infectious
Diseases Center; Section Infectiology and Clinical Immunology,
University Hospitals of Ulm, Ulm, Germany
J. Richter (*) : K. Kranz
Tropical Medicine Unit, University Hospital for Gastroenterology,
Hepatology and Infectious Diseases, Heinrich-Heine-University
Düsseldorf, Moorenstr 5,
40225 Düsseldorf, Alemanya, Germany
e-mail: Joachim.Richter@med.uni-duesseldorf.de
Introduction
Alveolar echinococcosis (AE) is caused by the cestode
Echinococcus multilocularis (E. multilocularis) commonly
known as the small fox tapeworm. The primary life cycle
involves predominantly red, arctic foxes as definitive hosts
and rodents as intermediate hosts. Adult worms harboured
by the carnivore definitive host produce ova which are
excreted with faeces or may be spread on its fur by licking
it with its contaminated tongue. Rodents become infected
when ingesting the ova which after the passage through the
stomach become oncospheres which via the portal vein
reach the liver and evolve to the metacestode.
Not only wild carnivores but also domestic dogs may
become infected and constitute the source of accidental
human infection. AE is endemic in wide areas of the
Northern hemisphere. In the human intermediate host, the
larva (metacestode) most frequently develops in the liver but
may spread to the lungs, brain and other organs (Eckert et al.
2011). The metacestode grows by exogenous budding and
proliferation of parasite tissue infiltrating the host tissue. It
appears macroscopically as a dispersed mass of fibrous
tissue with a conglomerate of scattered cavities each ranging
from a few millimetres to centimetres in size containing a
viscous fluid. Histologically, the hepatic lesion is character-
ized by a conglomerate of small cysts demarcated by a thin
laminated layer with or without an inner germinal layer. Due
to the slow growth of the metacestode and nonspecific
symptoms infection is usually diagnosed 5–15 years after
infection (Ammann et al. 1999; Eckert et al. 2011). If left
untreated, the natural course depends on the immune re-
sponse of the host: infections may either not develop, heal
spontaneously or progress. Progredient lesions lead to death
in more than 90 % of cases (Eckert et al. 2011; Torgerson et
al. 2008; Brunetti et al. 2010). Only if the infection is
diagnosed at an early stage, a cure may be obtained by
1656
surgical removal of the lesion under antihelminthic coverage
(World Health Organization 1996; Brunetti et al. 2010). In
most cases, radical surgery is no more possible. AE at this
stage was not curable until the advent of benzimidazoles,
namely mebendazole (MBZ) (Heath et al. 1975; Torgerson
et al. 2008). Albendazole (ABZ) was later preferred to MBZ
because its active metabolite albendazole sulfoxide
(ABZSX) penetrates better into metacestode tissues (Abdi
et al. 1995; Reuter et al. 1998; Ingold et al. 1999; Reuter et
al. 2000).
Although benzimidazoles have increased the lifespan and
life quality of patients with AE, current therapy options are
not satisfactory. In AE, benzimidazoles in vivo have only a
parasitostatic but not a parasitocidal effect (Ingold et al.
1999; Reuter et al. 2010). Therefore, these compounds must
not seldomly be taken for an indefinite time period.
Unfortunately, therapy with ABZ may cause toxic hepatitis,
alopecia and bone marrow suppressions, reasons for that
therapy must be interrupted or even stopped (Reuter et al.
1998, 2000). Although many substances have been tested,
yet no definitive parasitocidal treatment is available to cure
this infection. Therefore, the development of alternative
drugs is mandatory.
So far, benzimidazoles are still the most effective com-
pounds. Triclabendazole (TCZ) is a particularly liposoluble
benzimidazole derivative which has been successfully ap-
plied to treat plathelminthes such as Fasciola and
Paragonimus spp. (Boray et al. 1983; Millán et al. 2000;
Richter et al. 2002; Keiser et al. 2005). TCZ is more effec-
tive than ABZ in treating Fasciola spp. infections.
Therefore, TCZ might also be effective in the treatment of
AE. In mammals, ABZ and TCZ undergo an efficient first
pass sulfoxidation in the liver to their active metabolites
ABZSX and TCZ sulfoxide (TCZSX). Later, both com-
pounds are than metabolized to their sulfones (Lacey 1990).
All benzimidazoles interfere with the metabolism of the
parasites and inhibit the polymerisation of tubuline to mi-
crotubule. Thereby, the absorption of glucose is impeded.
After having exhausted their reserve, parasites die. The
precise mechanism of TCZ and its metabolites on
Echinococcus spp. is not known. In vitro studies on fascio-
liasis show tegument disruption and inhibition of the mitotic
division of spermatogenic cells of the worm (Fairweather
and Boray 1999a, b; Fairweather 2005).
Tissue resorption is lowest for MBZ as compared to
ABZ, TCZ and their active metabolites (Alvarez et al.
2004, Mottier et al. 2004). Resorption of both ABZ and
TCZ is up to fivefold increased by their ingestion together
with a fatty meal (Lecaillon et al. 1998).
Clorsulon (CLS) is an aminobenzoldisulfonamide fascio-
licide used in veterinary medicine (Elitok et al. 2006). CLS
has been successfully applied in TCZ-resistant fascioliasis
of sheep (Coles and Stafford 2001; Hutchinson et al. 2009).
Parasitol Res (2013) 112:1655–1660
The trematocidal effect of CLS relies on the inhibition of
glycolytic enzymes 3-phosphate-glyceratekinase and phos-
phoglyceratmutase required by the trematodes for glycolysis
(Schulman et al. 1982). CLS penetrates well into tissues and
is mainly confiltrated by renal glomeruli and eliminated via
the urinary tract without being metabolized (Sundlof and
Whitlock 1992).
Materials and methods
Test system
An in vitro culture was applied which has been established
by Hemphill et al. (Hemphill and Gottstein 1995; Hemphill
et al. 2003) and Jura et al. (1998) and was modified as
described by Reuter et al. (2003a): Briefly, metacestode
infected liver tissue obtained from an E. multilocularis-
infected patient is fragmented, passed through a mesh and
suspended in an adequate aliquot of phosphate-buffered
saline (PBS) used as source of infection of gerbils. Gerbils
are infected with an intraperitoneal injection of 0.5 ml sus-
pension. Six to 11 weeks after intraperitoneal infections,
lesions grow containing viable protoscolices. Lesions are
resected and washed in PBS divided into blocks of 1 cm3
in size and cultured in bottles containing culture Dulbecco’s
modified Eagles medium and 10 % fetal calve serum. For
prevention of bacterial superinfection, 1 % penicillin/strep-
tomycin and 5 % moxifloxacin-hydrochloride are added. In
culture, these “tissue blocks” produce vesicles. Parts of the
tissue blocks are used for further passages. Growth of
vesicles is optimized by addition of human Hep G2 liver
cells stimulating vesicle growth by producing cytokines. In
vitro cultures of vesicles and Hep G2 cells were incubated in
culture flasks at 37 °C, with a CO2 concentration of 5 % and
a humidity of 91 %. Vesicle growth was accelerated by
exchange of the medium three times weekly. Vesicles con-
tain the internal germinal layer with muscular, connective
and glycogen storage cells and are covered by the tegument.
Externally, the tegument produces the laminar acellular lay-
er consisting in a glycocalix which protects the parasite.
In vitro efficacy assessment of drug treatments
of metacestodes
Vesicles were evaluated macroscopically and microscopical-
ly. Three criteria for the viability of the in vitro system were
assessed: (1) number of vesicles, (2) vesicle size and (3)
vesicle morphology. Vesicle morphology and damage were
assessed according to a standardised grading (Richter 2010;
Table 1). Assessment was done by two independent readers
who were blinded towards the results of each other (JF and
DR).
Parasitol Res (2013) 112:1655–1660 1657

Table 1 Morphology grading of E. multilocularis larval vesicles (Richter 2010)

1. Viable metacestode. Vesicles are slightly turbid. Vesicle is turgid and surface is smooth. No passive staining by culture medium. Membranes are
intact. Hep G2 liver cells are well attached
2. Turgidity of vesicles slightly decreased. Vesicles acquire red staining by culture medium. Membranes are preserved not fissured and not
transparent. Vesicles do not contain particles
3. Turgidity of vesicles decreased. Membranes exhibit first fissures and irregularities
4. Turgidity of vesicles reduced. Vesicle membranes are very thin and are hardly able to bear the weight of the vesicle. Segmentation occurs with an
apparent budding of an internal vesicle. Vesicle surface is strongly irregular and fissured
5. Turgidity of vesicle is strongly reduced. Membrane is perforated in numerous spots. Vesicle walls have become tiny and partially translucent.
Laminar layer is transparent. Germinal layer may be detached, collapses and may appear as a mobile precipitation

Within 3 weeks of culturing, the number of vesicles
increased to approximately five vesicles per culture bottle
(mean of 30 culture bottles). Vesicle sizes were measurable
from day 30 on. It increased on average to 2 mm after
40 days to 4 mm until after 80 days when measurement
was terminated.
ABZ was provided by Sigma-Aldrich Co. LCC., St. Louis,
USA; TCZ, TCZSX and TCZ sulfone by Witega Laboratories
GmbH, Berlin-Adlershof, Germany and CLS by Merial Inc.,
Duluth, GA, USA. Therapeutic activity of compounds was
assessed by adding each ABZ, ABZSX, TCZ, TCZSX or CLS
to the larval tissue culture. Stock solution was prepared adding
DMSO at 0.1 % (1 μg/ml) according to OECD standards. At
this dosage, the bias due to DMSO (molecular weight 78.13)
on concentrations was minimized (molecular weight of ABZ
—265.34, of ABZSX—281.34, of TCZ—359.66, of TCZSX
—375.66, of CLS—380.66). Every experiment result was
controlled by comparison with a parallel simultaneous exper-
iment where only DMSO was added (Da Violante et al. 2002).
All experiments were performed in duplicates.
The test dosage for the reference standard compounds
ABZ and ABZSX was 1 μg/ml, a dosage which had been
previously found to yield result comparable to the standard
dose of 10 μg/ml (Ingold et al. 1999; Reuter et al. 2003a,
2003b). Test dosages for TCZ and TCZSX were 10, 15, 20
and 25 μg/ml with the intent to find an optimal dosage.
These dosages correspond to the efficacious dosages of
TCZSX observed in plasma of patients treated for fascioli-
asis reached if two doses of 10 mg/kg are taken 12 h apart
each after a fatty meal (Lecaillon et al. 1998).
Bioassay
The observation period lasted 80 days including 35 days
where test substance was added followed by 45 days after
removal of test substances. After the end of the assay, culture
medium was exchanged, and tissue blocks with disrupted
vesicle material were observed for further 7 weeks. The tissue
extracted from the tissue blocks and the vesicle material was
re-injected intraperitoneally into gerbils. Ten weeks later, ger-
bils were sacrified, and peritoneum and liver were re-
examined for larval structures. The validity of all experiments
was assessed by parallel identic procedures with untreated
tissues. A parasitostatic effect was defined as a vesicle damage
in culture followed by a successful re-infection of gerbils. A
parasitocidal effect was defined as a vesicle damage in culture
followed by a failure to re-infect gerbils. Since TCZ is li-
censed for use in humans, and its toxicity has been investigat-
ed extensively, cytotoxicity experiments on mammalian cell
lines were not performed (WHO 1993).

Fig. 1 Vesicle damage after 1

adding different doses of
triclabendazole (TCZ)
2

5
0 5 10 15 20 25 30 35 40
Time in d

Control
1658
Fig 2 Vesicle damage after 1
adding different doses of
triclabendazole sulfoxide
(TCZSX) 2
5
0 5
Control
Results
Effect of ABZ versus control
ABZ led to vesicle degeneration more rapidly as compared
to vesicle degeneration in DMSO alone (control). The fact
that vesicles in two of three assays deteriorated also without
adding ABZ is most probably due to deficient feeding by
Hep G2 cells. As reflected by the morphology grading,
curve membranes became translucent, tension decreased,
and vesicles became increasingly deformed.
Effect of TCZ versus control
TCZ and TCZSX assays showed strictly dosage-dependent
vesicle damage (Figs. 1 and 2), contrary to control vesicles
where these compounds had not been added. The best
results were obtained with TCZSX where a complete dis-
ruption of the vesicles occurred after 2 weeks when the
dosage was 20 or 25 μg/ml and after 28 days at a dosage
of 15 μg/ml. After further 2 weeks, the culture medium
started to stain the cultures. After the end of the assay,
culture medium was exchanged, and tissue blocks were
observed for 7 weeks. Recurrence of vesicle growth did
not occur. Whereas intraperitoneal injection of untreated
culture material into gerbils resulted in re-infection of these,
this did not occur in six gerbils which had been injected with
the TCZSX-treated tissue block material. As expected, treat-
ment of cultures with the inactive metabolite TCZ sulfone
did not result in vesicle damage during a 5-week observa-
tion period.
Effect of CLS versus control
CLS assays showed effects comparable to the controls with
no vesicle damage irrespective of the dosage chosen at 5, 10
Parasitol Res (2013) 112:1655–1660
10 15 20 25 30 35 40
Time in d
15mg/ml
and 15 μg/ml. The lack of any antihelminthic activity was
confirmed by all experiments on tissue blocks.
Discussion
AE is one of the last non-curable helminthic infections of
humans. The urgency of developing new therapy options is
obvious considering that this widespread disease usually leads
to death if left untreated (Torgerson et al. 2008; Brunetti et al.
2010). Most of the compounds investigated so far did not
progress into clinical development (Hemphill et al. 2007).
These include antihelminthics such as ivermectin, nitazoxa-
nide, flubendazole and artemether; antibiotics such as rifam-
picin, clarithromycin and thrimetoprim-sulfamethaxole;
antimycotics such as caspofungin and itraconazole, and many
other substances such as miltefosine, isoprinosine and deriv-
atives, alpha-difluoromethyl-ornithine, genistein and deriva-
tives, pyridinyl imidazoles, thioureides, 2-methoxyestradiol,
cyclosporine and doxorubicine (Liance et al. 1992, 1993;
Miyaji et al. 1993; Lawton et al. 2001; Stettler et al. 2003,
2004; Reuter et al. 2003a; Mathis et al. 2005; Kern et al. 2006;
Naguleswaran et al. 2006; Reuter et al. 2006; Hemphill et al.
2007; Gelmedin et al. 2008; Spicher et al. 2008a, b; Müller et
al. 2009; Hemphill and Müller 2009). Promising experimental
drugs include mefloquine and dicationic compounds (Küster
et al. 2011; Stadelmann et al. 2011). Although praziquantel is
active on the adult Echinococcus parasites in the definitive
host and on E. granulosus scolices in vitro, it has no effect on
the metacestode of E. multilocularis (Bygott and Chiodini
2009). The role of nitazoxanide in combination with albenda-
zole has recently been explored in vivo in disseminated E.
granulosus infection (Peréz-Molina et al. 2011).
Amphotericine B has been shown to be active against the
larval AE in vitro and in vivo and has been given alone or in
combination with benzimidazoles in a salvage context.
Parasitol Res (2013) 112:1655–1660
However, amphotericin B frequently cannot be given for
long because its nephrotoxicity (Reuter et al., 2003a, b;
Kern et al. 2006; Reuter et al. 2006, 2010).
Among benzimidazoles, TCZ and its active sulfoxide
metabolite are particularly interesting to study because of
their higher activity on other plathelminthes, e.g. Fasciola
spp., possibly due to their liposolubility, their proven safety
in human and animals and their approval for human use.
Animal studies have proven that toxicity of TCZ is low even
when given for many months or even years (World Health
Organization 1993). TCZ is licensed for use in humans for
other infections due to plathelminthes such as fascioliasis
(Millán et al. 2000; Richter et al. 2002; Keiser et al. 2005;
World Health Organization 2007). It is also effective in para-
gonimiasis and possibly in other trematode infections such as
Clonorchis sinensis, Opistorchis spp. and Fasciolopsis buskii
infections (Keiser et al. 2005). The most important adverse
event observed in the treatment of fascioliasis with TCZ is
biliary colics with increase in liver enzymes. This effect is not
caused by a conceivable toxicity of TCZ but due to the
mechanical bile duct obstruction resulting from the expulsion
of dying or dead Fasciola worms ensuing effective TCZ
therapy (Richter et al. 1999; Millàn et al. 2000).
Conclusion
TCZ and TCZSX proved efficacious against AE metacestodes
in vitro. Moreover, TCZ and TCZSX are possibly not only
parasitostatic but also parasitocidal, a result to be confirmed in
vivo.
Acknowledgments This work is part of a diploma thesis in Pharma-
cy by David Richter. We are indebted to Dr. S. Wacker, Dr. F. Oswald,
Dr. K. Wahlers, Dr. M. Willmann, Dr. G. Härter, U. Knoll, I. Katz, A.
Helfrich and M. Holl for their cooperation.
References
Abdi YA, Gustafsson LL, Ericsson O, Hellgren U (1995) Handbook of
drugs for tropical parasitic diseases. 2nd ed.; Taylor and Francis
London. pp.12-17 and pp. 78–82
Alvarez LI, Mottier ML, Lanusse CE (2004) Comparative assessment of
albendazol, febendazol and triclabendazol to Fasciola hepatica: ef-
fect of bile in the incubation medium. Parasitology 128(1):73–81
Ammann RW, Fleiner Hoffmann A, Eckert J, und Schweizerische
Echinokokkose-Studiengruppe (SESG) (1999) Schweizerische
Studie für Chemotherapie der alveolären Echinokokkose–Rück-
blick auf ein 20jähriges klinisches Forschungsprojekt. Swiss che-
motherapy trial for alveolar echinococcosis: review of a 20-year
clinical research project. Schweiz Med Wochenschr 129:323–332
Boray JC, Crowfoot PD, Strong MB, Allison JR, Schellenbaum M,
Von Orelli M, Sarasin G (1983) Treatment of immature and
mature Fasciola hepatica infections in sheep with triclabenda-
zole. Vet Rec 113:315–317
1659
Brunetti E, Kern P, Vuitton DE (2010) Writing panel for the WHO-
International Working Group on Echinococcosis. Expert consen-
sus for the diagnosis and treatment of cystic and alveolar echino-
coccosis in humans. Acta Trop 114:1–16
Bygott JM, Chiodini PL (2009) Praziquantel: neglected drug? ineffec-
tive treatment? or therapeutic choice in cystic hydatid disease.
Acta Trop 111:95–101
Coles GC, Stafford KA (2001) Activity of oxyclozanide, nitroxynil,
clorsulon and albendazole against adult triclabendazole-resistant
Fasciola hepatica. Vet Rec 148:723–724
Da Violante G, Zerrouk N, Richard I, Provot G, Chaumeil JC, Arnaud
P (2002) Evaluation of the cytotoxicity effect of dimethyl sulfox-
ide (DMSO) on Caco2/TC7 colon tumor cell cultures. Biol Pharm
Bull 12:1600–1603
Eckert J, Deplazes D, Kern P (2011) Alveolar echinococcosis (Echi-
nococcus multilocularis) and neotropical forms of echinococcosis
(Echinococcus vogeli and Echinococcus oligarthrus). In SR
Palmer Lord Soulsby, PR Torgerson, DWW Brown edts.: Oxford
Textbook of Zoonoses. Oxford University Press, pp. 669–699
Elitok B, Elitok OM, Kabu M (2006) Field trial on comparative
efficacy of four fasciolicides against natural liver fluke infection
in cattle. Vet Parasitol 135:279–285
Fairweather I (2005) Triclabendazole: new skills to unravel an old(ish)
enigma. J Helminthol 79:227–234
Fairweather I, Boray JC (1999a) Fasciolicides: efficacy, actions, resis-
tance and its management. Vet J 158:81–112
Fairweather I, Boray JC (1999b) Mechanism of fasciolicide action and
drug resistance in Fasciola hepatica. In: Dalton JP (ed) Fascioli-
asis. CABI publishing, Dublin, Ireland, pp 225–276
Gelmedin V, Caballero-Gamiz R, Brehm K (2008) Characterization and
inhibition of a p38-like mitogen and mitogen-activated protein kinase
(MAPK) from Echinococcus multilocularis: antiparasitic activities of
p38 APK inhibitors. Biochem Pharmacol 76:1068–1081
Heath DD, Christie MJ, Chevis RAF (1975) The lethal effect of
mebendazole on secondary Echinococcus granulosus cysticerci
and Taenia pisiformis and tetrahydria Mesocestoides corti. Para-
sitology 70:273–285
Hemphill A, Gottstein B (1995) Immunological and morphological
studies on the proliferation of in vitro cultivated Echinococcus
multilocularis metacestode. Parasitol Res 851:605–614
Hemphill A, Müller J (2009) Alveolar and cystic echinococcosis: towards
novel chemotherapeutical treatment options. J Helminthol 83:99–111
Hemphill A, Stettler M, Walker M, Siles-Lucas M, Fink R, Gottstein B
(2003) In vitro culture of Echinococcus multilocularis and Echi-
nococcus vogeli metacestodes: studies on the host-parasite inter-
face. Acta Trop 85:145–155
Hemphill A, Spicher S, Stadelmann B, Mueller J, Naguleswaran A,
Gottstein B (2007) Innovative chemotherapeutical treatment options
for alveolar and cystic echinococcosis. Parasitology 134:1657–1670
Hutchinson GW, Dawson K, Fitzgibbon CC, Martin PJ (2009) Efficacy
of an injectable combination anthelmintic (nitroxynil + clorsulon
+ ivermectin) against early immature Fasciola hepatica compared
to triclabendazole combination flukicides given orally or topically
to cattle. Vet Parasitol 162:278–284
Ingold K, Bigler P, Thormann W, Cavaliero T, Gottstein B, Hemphill A
(1999) Efficacies of albendazole sulfoxide and alebendazole sul-
fone against in vitro cultivated Echinococcus multilocularis meta-
cestodes. Antimicrob Agents Chemother 43:1052–1061
Jura H, Bader A, Frosch M (1998) In vitro activities of benimidazoles
against Echinococcus multilocularis metacestodes. Antimicrob
Agents Chemother 42:1052–1056
Keiser J, Engels D, Büscher G, Utzinger J (2005) Triclabendazole for
the treatment of fascioliasis and paragonimiasis. Expert Opinion
Invest Drugs 14:1513–1526
Kern P, Reuter S, Manfras B, Merkle M, Härter G (2006) In vitro
activities of itraconazol, methiazol and nitazoxanide versus
1660
Echinococcus multilocularis larvae. Antimicrobial Agents Che-
mother 50:2966–2970
Küster T, Stadelmann B, Scholl S, Barna F, Kropf C, Keiser J, Boykin
DW, Stephens CE, Hemphill A (2011) In vitro and in vivo
efficacies of mefloquine-based treatment against alveolar echino-
coccosis. Antimicrob Agents Chemother 55:713–721
Lacey E (1990) Mode of action of benzimidazoles. Parasitol Today
6:112–115
Lawton PN, Walchshofer N, Sarciron M (2001) In vitro and in vivo
effects of Isoprinosine and a dipeptide methyl ester on Echino-
coccus multilocularis protoscolices. J Helminthol 75:251–257
Lecaillon JB, Godbillon J, Campestrini J, Naquira C, Miranda L,
Pacheco R, Mull R, Poltera AA (1998) Effect of food on the
bioavailability of triclabendazole in patients with fascioliasis. Br J
Clin Pharmacol 45:601–604
Liance M, Nemati C, Bories C, Couvreur P (1992) Effects of cyclo-
sprin A on the course of murine alveolar echinococcosis and on
specific cellular and humoral immune responses against Echino-
coccus multilocularis. Int J Parasitol 22:23–28
Liance M, Nemati C, Bories C, Couvreur P (1993) Experience with
doxorubicine-bound polyisohexylcyanoacrylate nanoparticles on mu-
rine alveolar echinococcosis of the liver. Int J Parasitol 23:427–429
Mathis A, Wild P, Boettger C, Kapel CM, Deplazes P (2005) Mito-
chondrial ribosome as the target for the macrolide antibiotic
clarithromycin in the helminth Echinococcus multilocularis. Anti-
microb Agents Chemother 49:3251–3255
Millán JC, Mull R, Freise S, Richter J, The Triclabendazole Study
Group (2000) Efficacy and tolerability of triclabendazole for the
treatment of latent and chronic fascioliasis. Am J Trop Med and
Hyg 63:264–269
Miyaji S, Katakura K, Matsufuji S, Murakami Y, Hayashi S, Oku Y,
Okamoto M, Kamiya M (1993) Failure of treatment with alpha-
difluoromethylornithine against secondary multilocular echino-
coccosis in mice. Parasitol Res 79:75–76
Mottier L, Virkel G, Solana H, Alvarez L, Salles J, Lanusse C (2004)
Triclabendazole biotransformation and comparative diffusion of
the parent drug and its oxidized metabolites into Fasciola hepat-
ica. Xenobiotica 34:1043–1057
Müller J, Limban C, Stadelmann B, Missir AV, Chirita IC, Chifiriuc
MC, Nitulescu GM, Hemphill A (2009) Thioureides of 2-(phe-
noxymethyl)benzoic acid 4-R substituted. A novel class of anti-
parasitic compounds. Parasitol Int 58:128–135
Naguleswaran A, Spicher M, Vonlaufen N, Ortega-Mora LM, Torger-
son P, Gottstein B, Hemphill A (2006) In vitro metacestocidal
activities of genistein and other isoflavones against Echinococcus
multilocularis and Echinococcus granulosus. Antimicrob Agents
Chemother 50:3770–3778
Peréz-Molina JA, Diaz-Menendez M, Gallego JI, Norman F, Monge-
Maillo B, Peréz-Ayala A, López-Vélez R (2011) Evaluation of
nitazoxanide for the treatment of disseminated cystic echinococ-
cosis. Report of five cases and literature review. Am J Trop Med
H 84:351–356
Reuter S, Kratzer W, Kurz S, Wellinghausen N, Kern P (1998) Che-
motherapy of alveolar echinococcosis with benzimidazoles. A
prospective long-term study. Med Klin 93:463–467
Reuter S, Jensen B, Buttenschoen K, Kratzer W, Kern P (2000)
Benzimidazoles in the treatment of alveolar echinococcosis: a
comparative study and review of the literature. J Antimicrob
Chemother 46:451–456
Reuter S, Merkle M, Brehm K, Kern P, Manfras B (2003a) Effect of
amphotericin b on larval growth of Echinococcus multilocularis.
Antimicrob Agents Chemother 47:620–625
Reuter S, Buck A, Grebe O, Nüssle-Kügele K, Kern P, Manfras B
(2003b) Salvage treatment with amphotericin B in progressive
View publication stats
Parasitol Res (2013) 112:1655–1660
human alveolar echinococcosis. Antimicrob Agents Chemother
47:3586–3591
Reuter S, Manfras B, Merkle M, Härter G, Kern P (2006) In vitro activities
of itraconazole, methiazole, and nitazoxanide versus Echinococcus
multilocularis. Antimicrob Agents Chemother 50:2866–2970
Reuter S, Beisler T, Kern P (2010) Combined albendazole and ampho-
tericin B against Echinococcus multilocularis in vitro. Acta Trop
115:270–274
Richter D (2010) In vitro Testung ausgewählter Antihelminthica auf das
Larvenstadium von Echinocccus multilocularis. [In vitro testing of
selected antihelminthica on the larval stage of Echinocccus multi-
locularis]. Diploma thesis in Pharmacy. Institute for Pharmacy and
Medical Faculty, Ernst-Moritz-Arndt-Universität Greifswald and
Department of Internal Medicine III, Section Infectiology and Clin-
ical Immunology, University Hospitals Ulm, Germany
Richter J, Freise S, Mull R, Millán JC, The Triclabendazole Clinical
Study Group (1999) Fascioliasis: sonographic abnormalities of
the biliary tract and evolution after treatment with triclabendazole.
Trop Med Intern Health 4:774–781
Richter J, Knipper M, Göbels K, Häussinger D (2002) Fascioliasis. In
Current Treatment Options in Infectious Diseases 4:313–317
Schulman MD, Ostlind DA, Valentino D (1982) Mechanism of action
of MK-401 against Fasciola hepatica: inhibition of phosphoglyc-
erate kinase. Mol Biochem Parasitol 5:133–145
Spicher M, Roethlisberger C, Lany C, Stadelmann B, Keiser J, Ortega-
Mora LM, Gottstein B, Hemphill A (2008a) In vitro and in vivo
treatments of echinococcus protoscoleces and metacestodes with
artemisinin and artemisinin derivatives. Antimicrob Agents Che-
mother 52:3447–3450
Spicher M, Naguleswaran A, Ortega-Mora LM, Müller J, Gottstein B,
Hemphill A (2008b) In vitro and in vivo effects of 2-
methoxyestradiol, either alone or combined with albendazole,
against Echinococcus metacestodes. Exp Parasitol 119:475–482
Stadelmann B, Küster T, Scholl S, Barna F, Kropf C, Keiser J, Boykin DW,
Stephens CE, Hemphill A (2011) Efficacy of dicatonic compounds
and mefloquine enantiomers against Echinococcus multilocularis
metacestodes. Antimicrob Agents Chemother 55:4866–4872
Stettler M, Siles-Lucas M, Sarciron E, Lawton P, Gottstein B, Hemp-
hill A (2003) In vitro parasitocidal effect of nitazoxanide against
Echinococcus multilocularis metacestodes. Antimicrob Agents
Chemother 47:467–474
Stettler M, Siles-Lucas M, Sarciron E, Lawton P, Gottstein B, Hemp-
hill A (2004) Secondary and primary alveolar echinococcosis:
combined albendazole/nitazoxanide chemotherapy exhibits pro-
found anti-parasitic activity. Int J Parasitol 34:615–624
Sundlof SF, Whitlock TW (1992) Clorsulon pharmacokinetics in sheep
and goats following oral and intravenous administration. J Vet
Pharmacol Ther 5:282–291
Torgerson PR, Schweiger A, Deplazes P, Pohar M, Reichen J,
Ammann RW, Tarr PE, Halkik N, Mullhaupt B (2008) Alveolar
echinococcosis: from a deadly disease to a well-controlled infec-
tion. Relative survival and economic analysis in Switzerland over
the last 35 years. J Hepatol 49:72–77
World Health Organization (1993) Evaluation of certain veterinary
drug residues in food. WHO Tech Rep Ser 832:1–62
World Health Organization (1996) Guidelines for treatment of cystic
and alveolar echinococcosis in humans. WHO Informal Working
Group on Echinococcosis. Bull WHO 74:231–242
World Health Organization (2007) Report of the WHO Informal Meeting
on Use of Triclabendazole in Fascioliasis Control. WHO Headquar-
ters, Geneva, Switzerland: October 17–18, 2006. Available at:
www.who.int/neglected_diseases/preventive_chemotherapy/WHO_
CDS_NTD_PCT_2007.1.pdf. Accessed June 12, 2012.World Health
Organization