Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy.

Abstract

Recently, extensions of the range of Echinococcus multilocularis in Europe and North America and drastic increases in fox populations in Europe put an increasing proportion of the human population at risk of alveolar echinococcosis. To obtain data on the local infection pressure, studies of the prevalence of the parasite in the animals that transmit the parasite, foxes, dogs, and cats, are urgently required. Such investigations, however, have been hampered by the need for necropsy of the host animal to specifically diagnose infection with the parasite. In this study, a nested PCR and an improved method for DNA extraction were developed to allow the sensitive and specific diagnosis of E. multilocularis infections directly from diluted fecal samples from foxes. The target sequence for amplification is part of the E. multilocularis mitochondrial 12S rRNA gene. The specificity of the method was 100% when it was tested against 18 isolates (metacestodes and adult worms) of 11 cestode species, including E. granulosus. The sensitivity of the method was evaluated by adding egg suspensions and individual eggs to samples of diluted feces from uninfected foxes. The presence of one egg was sufficient to give a specific signal. To confirm the PCR results, an internal probe which hybridized only with E. multilocularis amplification products but not with the DNA of other cestodes was constructed. In order to investigate the applicability of this method for epidemiological studies, 250 wild foxes from a area in southern Germany where echinococcosis is highly endemic were examined by both necropsy and PCR of rectal contents. The sensitivity correlated with the parasites' number and stage of maturity. It ranged from 100% (>1,000 gravid worms) to 70% (<10 nongravid worms). On the basis of positive PCR results for 165 foxes, the sensitivity of the traditional and widely used necropsy method was found to be not higher than 76%. We therefore present this PCR system as an alternative method for the routine diagnosis of E. multilocularis in carnivores.

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JOURNAL OF CLINICAL MICROBIOLOGY,
0095-1137/98/$04.00?0
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
July 1998, p. 1871–1876Vol. 36, No. 7
Detection of Echinococcus multilocularis in the Definitive Host:
Coprodiagnosis by PCR as an Alternative to Necropsy
ANKE DINKEL,1* MARKUS VON NICKISCH-ROSENEGK,2BIRGIT BILGER,1
MICHAEL MERLI,1RICHARD LUCIUS,2AND THOMAS ROMIG1
Department of Parasitology, University of Hohenheim, 70599 Stuttgart,1and Department of
Molecular Parasitology, Humboldt-University Berlin, 10115 Berlin,2Germany
Received 25 November 1997/Returned for modification 13 February 1998/Accepted 23 March 1998
Recently, extensions of the range of Echinococcus multilocularis in Europe and North America and drastic
increases in fox populations in Europe put an increasing proportion of the human population at risk of alveolar
echinococcosis. To obtain data on the local infection pressure, studies of the prevalence of the parasite in the
animals that transmit the parasite, foxes, dogs, and cats, are urgently required. Such investigations, however,
have been hampered by the need for necropsy of the host animal to specifically diagnose infection with the
parasite. In this study, a nested PCR and an improved method for DNA extraction were developed to allow the
sensitive and specific diagnosis of E. multilocularis infections directly from diluted fecal samples from foxes.
The target sequence for amplification is part of the E. multilocularis mitochondrial 12S rRNA gene. The spec-
ificity of the method was 100% when it was tested against 18 isolates (metacestodes and adult worms) of 11
cestode species, including E. granulosus. The sensitivity of the method was evaluated by adding egg suspensions
and individual eggs to samples of diluted feces from uninfected foxes. The presence of one egg was sufficient
to give a specific signal. To confirm the PCR results, an internal probe which hybridized only with E. multi-
locularis amplification products but not with the DNA of other cestodes was constructed. In order to investigate
the applicability of this method for epidemiological studies, 250 wild foxes from a area in southern Germany
where echinococcosis is highly endemic were examined by both necropsy and PCR of rectal contents. The
sensitivity correlated with the parasites’ number and stage of maturity. It ranged from 100% (>1,000 gravid
worms) to 70% (<10 nongravid worms). On the basis of positive PCR results for 165 foxes, the sensitivity of
the traditional and widely used necropsy method was found to be not higher than 76%. We therefore present
this PCR system as an alternative method for the routine diagnosis of E. multilocularis in carnivores.
Alveolar echinococcosis (AE), caused by the metacestode of
the fox tapeworm Echinococcus multilocularis, is a potentially
lethal human disease. In its natural wildlife hosts (canines and
rodents) the parasite is present across most regions of the
northern hemisphere, whereas transmission to humans seems
to occur predominantly focally. In Alaska and parts of north-
ern China, where AE poses one of the most serious health
problems, the disease seems to be transmitted mainly by do-
mestic dogs. In central Europe and North America, AE in
humans is a comparatively rare disease; wild foxes (Vulpes
vulpes) are thought to be the principal transmitters, although
the role of domestic dogs and cats has not been satisfactorily
evaluated. There is, however, concern due to extensions of the
parasite’s range in Europe and North America, the drastic
increases in wild fox populations in most parts of Europe, the
increasingly close association of wild foxes with human habi-
tations, and in some regions, sharply increasing rates of prev-
alence of E. multiocularis in foxes which locally may exceed
70% (14, 21). The exact route of transmission to humans is
unknown. Ingestion of contaminated berries or herbs from
forests were thought to be potential sources of infection; in
some studies, farming was found to be a risk factor (11, 18).
For several European regions, detailed information on the
prevalence rates in foxes is available; on the contrary, few data
on the role of domestic dogs and cats, which may carry the
parasite as a spillover of the wildlife cycle in areas where
echinococcosis is endemic, exist. The reason for the paucity of
data on these hosts, which may be of prime importance in
carrying the disease to humans, is the difficult diagnostic pro-
cedure, which largely relies on inspection of the dead animal’s
intestine and visual identification of the worms. This tech-
nique, although sensitive, extremely specific, and applicable to
the examination of species of wildlife, is also expensive and
hazardous and largely prevents the examination of owned do-
mestic animals. Detection of eggs in feces is not a method for
specific diagnosis, since the eggs of all taeniid cestodes of foxes
and dogs are morphologically indistinguishable.
In the search for alternative methods for the diagnosis of
infections with Echinococcus spp. in definitive hosts (hosts
containing the adult tapeworms), antibody serology has up to
now proved to be unsatisfactory. With varying levels of success,
several approaches have been used to develop test systems for
coprodiagnosis. The methods include the use of monoclonal
antibodies to label artificially hatched eggs (5) and the immu-
nological detection of coproantigens (1, 6). Recently, PCR was
introduced as a tool for the coprodiagnosis of E. multilocularis
infection (4). In the following sections we present a novel
approach to coprodiagnosis by PCR with unprocessed fecal
samples.
MATERIALS AND METHODS
Collection of samples. (i) E. multilocularis metacestodes. Seventeen E. mul-
tilocularis isolates were used to evaluate the PCR method: five isolates originated
from common voles (Microtus arvalis) and eight originated from water voles
(Arvicola terrestris) trapped in an area of the Swabian Jura in southern Germany
of high endemicity, one isolate was from Clethrionomys rufocanus from eastern
Hokkaido, Japan, and three isolates were from Microtus oeconomus from Alaska.
The diagnosis was based on parasitological examination. The parasite material
* Corresponding author. Mailing address: Department of Parasitol-
ogy, University of Hohenheim, Emil-Wolff-Str. 34, 70599 Stuttgart,
Germany. Phone: 0049-711-4593076. Fax: 0049-711-4592276. E-mail:
dinkelan@uni-hohenheim.de.
1871

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was removed within 24 h after the death of the host, cleaned from the host tissue
as much as possible, and stored in 70% ethanol.
(ii) E. multilocularis adult worms and eggs. Two isolates of adult worms with
mature eggs and two isolates of immature worms originated from the intestines
of wild foxes (V. vulpes) shot by hunters in southern Germany. After removal
from the intestinal mucosa, the worms were rinsed in water, frozen for 5 days at
?80°C for safety reasons, and stored in 70% alcohol. Eggs were isolated as
described by Mu ¨ller (17); in short, a suspension containing gravid proglottids
underwent digestion with proteinase K for 1 h followed by purification on 60%
Percoll. The purified egg suspension was then either stored at 4°C in phosphate-
buffered saline or frozen at ?80°C.
(iii) Fecal samples. A total of 250 fecal samples originated from wild foxes shot
by hunters in an area of southern Germany of high endemicity (Baden-Wu ¨rt-
temberg) and 42 samples from an area of northeastern Germany of low ende-
micity (Brandenburg). In addition, four samples from captive foxes and four
samples from dogs were tested. All fecal samples were removed from the rectum,
avoiding contamination, frozen at ?80°C for 5 days for safety reasons, and sub-
sequently stored at ?20°C.
(iv) Other helminths. For specificity screening, the following samples of ces-
todes other than E. multilocularis were used: Echinococcus granulosus (three
isolates, metacestodes, from Kenya and Germany), Taenia crassiceps (one iso-
late, a metacestode, from Germany), Taenia hydatigena (three isolates, a meta-
cestode and adults, from Kenya and Switzerland), Taenia martis (two isolates,
metacestodes, from Germany), Taenia mustelae (one isolate, a metacestode,
from Germany), Taenia ovis (one isolate, an adult, from Australia), Taenia pisi-
formis (one isolate, an adult, from Australia), Taenia polyacantha (two isolates,
metacestodes, from Germany), Taenia serialis (one isolate, an adult, from Aus-
tralia), Taenia taeniaeformis (two isolates, a metacestode and an adult, from
Germany and Switzerland), and Mesocestoides leptothylacus (one isolate, an
adult, from Germany). In addition, several adult nematodes of fox origin (Tox-
ocara sp. and Uncinaria sp.) were tested.
Examination of fox intestines. Small intestines were opened with gut scissors
and were visually inspected for the presence of E. multilocularis and other
helminths. After removal of coarse intestinal contents, smear samples were taken
from locations at 10-cm intervals by scraping the mucosa with microscopic glass
strips which were pressed on square polystyrene petri dishes (8). The samples
were examined with a stereomicroscope at ?8 to ?50 magnification. All proce-
dures were performed under appropriate safety conditions. The numbers and
developmental stages of the parasites seen were recorded.
Extraction of DNA. (i) Cestode tissue. Samples of parasite tissue were digested
as described elsewhere (3), with the following modifications. Samples (up to
0.3 g) were cut into small pieces and were digested in the presence of 900 ?g of
proteinase K (Boehringer GmbH, Mannheim, Germany) at 56°C for 6 to 12 h in
0.5 ml of 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 50 mM NaCl, 2% sodium
dodecyl sulfate, and 20 mM dithiothreitol. DNA was extracted as described
before (20) with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-
isoamyl alcohol. The DNA was precipitated with 3 M sodium acetate (pH 4.8)
(1:10) and ethanol (2:1) at ?20°C (3). After vacuum drying the precipitate was
suspended in 100 ?l of double-distilled H2O.
(ii) Fecal samples. Fecal samples (minimum amount, 0.5 g) were diluted 1:2
(vol/vol) with distilled water. A total of 1,500 ?l of the resulting suspension was
used to extract the DNA. Since proteinase K proved to be ineffective in digesting
the embryophore of cestode eggs, a DNA extraction method based on alkaline
hydrolysis as described previously (4) was modified as follows. To each 1,500 ?l
of the fecal suspension, 108 ?l of 1 M KOH and 30 ?l of 1 M dithiothreitol were
added. After vortexing, the sample was incubated at 65°C for 30 min and neu-
tralized with 270 ?l of 2 M Tris-HCl (pH 8.3) and 40.5 ?l of 25% HCl. The DNA
was extracted with 1,950 ?l of phenol-chloroform-isoamyl alcohol (25:24:1), and
the aqueous phase was transferred to a 12-ml tube. The DNA was purified and
concentrated with the Prep-A-Gene purification kit (Bio-Rad Laboratories
GmbH, Munich, Germany). A total of 5,400 ?l of binding buffer was added to
1,800 ?l of the aqueous phase and mixed briefly. A total of 30 ?l of the
Prep-A-Gene matrix was added, and the samples were incubated at 37°C for 60
min with frequent agitation. After centrifugation, the pellet was washed once
with 1,000 ?l of binding buffer and three times with 1,000 ?l of washing buffer.
To remove the ethanol the pellet was vacuum dried. The DNA was eluted by
resuspending the matrix in 100 ?l of double-distilled H2O and incubating the
mixture for 15 min at 50°C. After centrifugation, the supernatant containing the
DNA was ready to be used in the PCR.
PCR. The target sequence for amplification is part of the E. multilocularis
mitochondrial 12S rRNA gene, which has been used in phylogenetic studies (24).
The PCR was conducted in two steps. For the first PCR, the primer pair P60.for.
and P375.rev. amplified a 373-bp fragment (Fig. 1). A total of 10 ?l of DNA was
added to a 90-?l reaction mixture containing 20 mM Tris-HCl (pH 8.5), 16 mM
(NH4)2SO40.2 mM MgCl2, 50 mM KCl, each deoxynucleoside triphosphate at a
concentration of 0.2 mM, 40 pmol of each primer, and 2 U of Taq polymerase
(AGS GmbH, Heidelberg, Germany). The sample fluid was covered with 55 ?l
of mineral oil to prevent evaporation. Thermal cycling of the amplification
mixture was performed in a DNA Thermal Cycler 480 (Perkin-Elmer) for 50
cycles. A cycle represents denaturation for 60 s at 93°C, annealing for 90 s at
55°C, and elongation for 120 s at 73°C. In a second step, the primer pair
Pnest.for. and Pnest.rev. (Fig. 1) was used for a nested PCR. It is located
downstream of the first primer pair and amplifies a 250-bp fragment. The reac-
tion mixture consisted of 3 ?l of amplification product, 20 mM Tris-HCl (pH
8.5), 16 mM (NH4)2SO4, 1.5 mM MgCl2, each deoxynucleoside triphosphate at
a concentration of 0.2 mM, 50 pmol of each primer, and 2 U of Taq polymerase
(AGS GmbH). The nested PCR was performed for 40 cycles, with each cycle
consisting of denaturation for 60 s at 93°C, annealing for 60 s at 59°C, and
elongation for 120 s at 73°C. After amplification, 10 ?l of the PCR products was
visualized on a 1.5% agarose gel containing 1 ?g of ethidium bromide per ml.
Control for contamination. To exclude the possibility of contamination with
specific DNA, in each test run (usually done with 16 samples) 2 negative controls
were included and underwent the entire procedure starting with DNA extraction.
Control for inhibition. Due to unknown factors present in some fecal samples,
PCRs may occasionally be inhibited and may therefore give false-negative results
(25). To control for such inhibitions, 100 ng of E. multilocularis DNA was added
to each negative sample and the first PCR was repeated. The test sample was
recorded as negative only if a signal was obtained; if not, the result was consid-
ered inconclusive.
Hybridization of PCR products. To control the specificity of the PCR, an
internal oligonucleotide, E.multi.1. (Fig. 1), was constructed. Agarose gels were
blotted onto nylon membranes (Quiagen GmbH, Hilden, Germany) and, after
prehybridization at 68°C for 1 h, were probed with E.multi.1., which was 5? end
labeled with digoxigenin. Hybridization was performed at 48°C for 2 h. For
detection the DIG Luminescent Detection Kit (Boehringer Mannheim) was
used. The hybridization signal was visualized with E. multilocularis DNA after
amplification with P60.for.-P375.rev. and Pnest.for.-Pnest.rev. but not with
P60.for.-P375.rev. amplification products of other cestode DNAs (Fig. 2).
FIG. 1. Sequence of part of the mitochondrial 12S rRNA gene from E. mul-
tilocularis. Primers and probe are underlined.
FIG. 2. PCR amplification of DNA from 10 different tapeworm species with
P60.for.-P375.rev. A total of 10 ?l of the PCR products was separated on a 1.5%
agarose gel and stained with ethidium bromide (a). PCR products were analyzed
by Southern transfer and hybridized with internal oligonucleotide E.multi.1. la-
beled at the 5? end with digoxigenin (b). Lanes A, E. multilocularis; lanes B,
E. granulosus; lanes C, T. taeniaeformis; lanes D, T. hydatigena; lanes E, T. pisi-
formis; lanes F, T. serialis; lanes G, T. martis; lanes H, T. ovis; lanes I, T. mustelae;
lanes J, T. polyacantha; lanes M, size marker.
1872DINKEL ET AL. J. CLIN. MICROBIOL.

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RESULTS
PCR of metacestode tissue. The primer pair P60.for.-P375.
rev. amplified the target sequences of all 12 cestode species
which were tested (Fig. 3). The second (nested) PCR with
Pnest.for.-Pnest.rev. was found to selectively amplify E. multi-
locularis DNA. All 17 isolates of E. multilocularis metacestodes
yielded the same characteristic band of 250 bp. In contrast, this
band was never visualized after amplification of other cestode
DNAs with Pnest.for.-Pnest.rev. (Fig. 3), including three iso-
lates of E. granulosus.
PCR of fecal samples. (i) Sensitivity. To determine the num-
ber of E. multilocularis eggs necessary to give a positive PCR
result, an egg suspension was diluted to obtain 100-?l batches
with mean egg contents of from 200 eggs to 1 egg. These
batches were added to 1,500 ?l of diluted feces from captive
foxes free of E. multilocularis. To be certain that a signal could
be obtained from suspensions with only one egg, single eggs
were also added to diluted feces. One egg was found to be
sufficient to give a specific signal (Fig. 4). The same result was
obtained by adding 10 pg of E. multilocularis DNA (one egg
contains approximately 8 pg of DNA [19]) to 1,500 ?l of
diluted fox feces free of E. multilocularis.
Even in the case of immature infections prior to the produc-
tion of eggs, whole E. multilocularis worms may be present
in the feces, and therefore, DNA may be detectable. To test
whether the DNA extraction method used for eggs would also
be suitable for somatic cestode tissue, six immature E. multi-
locularis worms (without visible eggs) were subjected to the
DNA extraction by using alkaline lysis. The subsequent PCR
gave positive results in all cases.
(ii) Specificity. Four fecal samples from captive foxes and
four fecal samples from dogs free of E. multilocularis gave
negative PCR results. To exclude the possibility that some
organism other than E. multilocularis present in the intestine or
food of wild foxes may give a positive signal, fecal samples from
42 foxes which were from the area of Brandenburg, which is of
low endemicity, and which were negative by intestinal inspec-
tion (22) were tested by PCR. All gave negative results.
As an additional control for specificity, nested PCR products
from 60 positive fecal samples were randomly selected and
underwent hybridization with the specific probe E.multi.1.
With all samples a hybridization signal was obtained (Fig.
5).
(iii) Comparison of methods. A total of 250 wild foxes from
an area of high endemicity were examined for E. multilocularis
by both necropsy (intestinal inspection) and PCR of fecal sam-
ples (rectal content). Nine of the 250 fecal samples (3.6%)
were found to contain factors inhibiting the PCR and therefore
gave no result; data for these nine foxes (seven positive and
two negative at postmortem examination) were excluded from
the following calculations. The E. multilocularis prevalence
based on necropsy results was 59% (142 of 241), the prevalence
based on PCR results was 68% (165 of 241), and the overall
prevalence (foxes positive by at least one method) was 75%
(181 of 241).
The overall sensitivity of PCR, based on the 142 positive
results at necropsy, was 89%. Sensitivity was influenced by the
worm burden (Table 1); it ranged from 100% (foxes with
?1,000 worms seen at necropsy) to 78% (foxes with ?10
worms). Infections with worms containing mature eggs were
more reliably detected by PCR (97%) than infections with
immature worms (78%) (Table 1).
FIG. 3. PCR amplification with P60.for.-P375.rev. (a) followed by amplifica-
tion with Pnest.for.-Pnest.rev. (b) of DNA from 12 different tapeworm species. A
total of 10 ?l of PCR products was separated on a 1.5% agarose gel and stained
with ethidium bromide. Lanes A, E. multilocularis; lanes B, E. granulosus; lanes
C, T. hydatigena; lanes D, T. martis; lanes E, T. taeniaeformis; lanes F, T. crassi-
ceps; lanes G, T. mustelae; lanes H, T. ovis; lanes I, T. pisiformis; lanes J, T. poly-
acantha; lanes K, T. serialis; lanes L, M. leptothylacus; lanes N, negative control;
lanes M, size marker.
FIG. 4. Nested PCR amplification of DNA from eggs added to 1,500 ?l of
diluted fox feces free of E. multilocularis. A total of 10 ?l of PCR products was
separated on a 1.5% agarose gel and stained with ethidium bromide. Lane A,
positive control; lane B, one egg; lanes C, D, E, F, G, H, and I, egg suspensions;
lane C, 200 eggs; lane D, 100 eggs; lane E, 50 eggs; lane F, 20 eggs; lane G, 10
eggs; lane H, 2 eggs; lane I, 1 egg; lane J, negative control; lane M, size marker.
FIG. 5. Nested PCR amplification of DNA from nine positive fox fecal sam-
ples; ethidium bromide staining of 10 ?l of PCR products after 1.5% agarose gel
electrophoresis showed the specific 250-bp band (a). The reaction products were
analyzed by Southern transfer and hybridized with internal oligonucleotide
E.multi.1. labeled at the 5? end with digoxigenin (b). Lanes A, positive control;
lanes B, C, D, E, F, G, H, I, and J, positive fox fecal samples; lanes K, negative
control.
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By reciprocal calculation, the sensitivity of the necropsy
method, based on 165 PCR-positive specimens, was 76%.
Within the group of foxes negative for E. multilocularis at
necropsy, the presence of other cestode species (Taenia spp.
and Mesocestoides sp.) did not influence the PCR results: of 39
PCR-positive foxes, 54% harbored other cestodes, whereas
58% of 62 PCR-negative foxes harbored other cestodes.
(iv) Cost and processing capacity. The approximate cost for
processing one fecal sample by PCR (consumables only, not
counting equipment and labor) was approximately US$10. The
processing capacity for one person was some 70 samples per
week, approximately equal to the capacity for postmortem
examinations.
DISCUSSION
At present, routine diagnosis of E. multilocularis infections
in definitive hosts largely depends on necropsy and the visual
detection of the worms in the intestines. Serological screening
is generally considered unsuitable for the reliable diagnosis of
infections with Echinococcus spp. in definitive hosts because of
a poor correlation between antibody titers and the presence of
worms in the intestine (7, 12). Recent approaches to the de-
tection of coproantigen by enzyme-linked immunosorbent as-
say showed high sensitivities with heavy infections only (6, 7),
while the diagnostic sensitivity for the detection of E. multi-
locularis in foxes with individual worm burdens of less than 20
worms may be as low as 38% (7). A method based on PCR for
the detection of E. multilocularis DNA directly from fecal
samples, tested with only 29 fecal samples of foxes (4), proved
to be difficult to reproduce because of false-negative results
due to the presence of PCR-inhibitory substances (16). In
order to overcome such problems the investigators (16) im-
proved this technique at the cost of a very time consuming
DNA extraction protocol. Therefore, the applicability of this
method for epidemiological purposes is only limited. As an
alternative approach, Mathis et al. (15) have published a
method of PCR identification of E. multilocularis eggs after
isolation of the eggs from fecal samples. Although sensitive
and specific, this approach is suitable for the diagnosis of
gravid infections only (with eggs present in the feces). Copro-
antigen enzyme-linked immunosorbent assays, although not
suitable for the detection of light infections, are still considered
suitable for epidemiological purposes since they will reliably
detect heavy infections, which are responsible for the bulk of
environmental contamination. However, there are situations in
which the detection of light infections is equally important
(e.g., surveillance of chemotherapy studies and diagnosis of
infections in domestic animals with contact with humans). A
sensitive test is even more important, since in our representa-
tive sample of foxes, 25% were in the category of foxes con-
taining 1 to 10 worms.
For the first time, we developed a method that was evaluated
against the traditional postmortem examination using a large
number of foxes in the routine laboratory and that compared
favorably with the traditional method concerning specificity,
sensitivity, cost, and the processing time needed.
The specificity of our test system was evaluated against a
variety of cestode species including E. granulosus and other
helminths regularly found in the intestines of foxes in the study
area. Nevertheless, 39 (16%) of 241 foxes were negative at
necropsy and reacted positively by the PCR. We therefore had
to exclude the possibility that amplification of non-E. multi-
locularis DNA present in the intestinal contents of wild foxes
may give an amplification product of a similar size. This was
done by testing fecal samples from foxes from areas of very low
endemicity (none of which gave a signal) and by hybridiz-
ing the nested PCR product with an E. multilocularis-specific
probe, which succeeded in all cases. We are therefore certain
that the DNA of E. multilocularis was amplified. The proba-
bility of accidental contamination was minimal since negative
control samples were included in each test run (36 negative
control samples in 18 test runs), none of which ever gave a
signal. Theoretically, positive PCR results can also be obtained
by amplifying DNA from immature E. multilocularis metaces-
todes which have been ingested by the fox together with the
intermediate host (voles). However, calculations that consider
the rate of mature and immature infections in voles and the
prevalence and life span of the adult worm in foxes indicate
that voles with immature metacestodes cannot be present in
the intestines of more than 2% of foxes at a given time. There-
fore, positive PCR results which are not confirmed by necropsy
in most cases cannot be considered false-positive results but
must be attributed to low-intensity infections overlooked dur-
ing the visual examination of the intestines.
PCR test systems for viral, bacterial, and protozoan organ-
isms are known to detect extremely small quantities of DNA
(2, 9, 10, 13). In our system, signals were obtained from single
E. multilocularis eggs, which have a DNA content of approxi-
mately 8 ng (19). However, even in foxes with mature infec-
tions, eggs or gravid proglottids are not shed continuously and
are not homogeneously distributed within the feces. This ex-
plains the moderate decrease in sensitivity from 100% for
animals with heavy infections to 92% for animals with very
light infections (10 or fewer worms seen at necropsy). A sur-
prisingly high PCR sensitivity was found with fecal samples
TABLE 1. PCR results for fecal samples (rectal contents) from wild foxes positive for E. multilocularis by necropsy
No. of worms seen
at necropsy
Mature eggsa
No mature eggsb
Total
No.
examinedc
No.
positived
Sensitivity
(%)e
No.
examined
No.
positive
Sensitivity
(%)
No.
examined
No.
positive
Sensitivity
(%)
?1,000
101–1,000
11–100
1–10
14
30
22
13
14
29
22
12
100
97
100
92
55 100
92
73
70
19
43
44
36
19
41
38
28
100
95
86
78
13
22
23
12
16
16
Total 79 7797 6349 78142126 89
aWorms containing eggs which appeared mature at microscopic examination (the presence of eggs in feces is likely).
bWorms containing no eggs or eggs appearing immature (the presence of eggs in feces is unlikely).
cTotal number of fecal samples (from individual foxes) examined by PCR.
dNumber of fecal samples positive by PCR.
eSensitivity of the PCR method with fecal samples from foxes with different worm counts at necropsy.
1874DINKEL ET AL. J. CLIN. MICROBIOL.

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from foxes with immature infections (100% with heavy infec-
tions; it was still 70% for animals with 10 or fewer worms). This
may be attributable to the detection of tissue fragments or
whole worms shed with the feces, although the presence of
undetected mature, egg-producing worms (in addition to the
immature stages seen at necropsy) can in no case entirely be
excluded. For 3.6% of all samples the PCR result was incon-
clusive due to inhibition. Fecal samples are known to occasion-
ally contain factors, which are as yet unidentified, which in-
terfere with the amplification process (25), rendering these
samples unsuitable for PCR testing. Although the percentage
of such samples in our study was acceptably small, further
efforts are necessary to overcome this obstacle.
To date, routine examination of definitive hosts for the pres-
ence of E. multilocularis is limited to a very few parasitology or
veterinary laboratories. Safety precautions taken to exclude
accidental infection of personnel and contamination of the
environment require laboratories with high levels of safety and
with specialized facilities for heat decontamination, since the
infectious eggs are largely resistant to chemical disinfectants
(23). The high cost required to construct and maintain such
laboratories prevents the routine monitoring of E. multilocu-
laris in wild and domestic animals, as is done with rabies, for
example. Freezing of the carcasses at ?80°C for 4 days is also
a suitable means of destroying cestode eggs before performing
postmortem examination in a routine laboratory. However, the
large freezer capacity needed also confines this approach to a
few institutions. Compared with the facilities necessary for
necropsy, PCR equipment is cheap and is usually present in
every routine laboratory. Fecal samples can easily be rendered
noninfectious by freezing them for some days at ?80°C before
entering the laboratory. Since in our system DNA extraction is
done directly from feces without preprocessing, the workload
for one person (using one set of PCR equipment) would be
some 70 samples per week, which also compares favorably with
the workload for postmortem examination. A total of 3.6% of
fecal samples are unsuitable for PCR due to inhibition factors.
However, not all fox carcasses delivered by hunters or from
other sources are suitable for postmortem examination due to
decomposition of the intestine; in our laboratory, the rate of
occurrence of such specimens ranges from 4 to 10%.
Domestic dogs and cats are suitable hosts for E. multilocu-
laris and may be important transmitters of echinococcosis to
humans. The evaluation of their epidemiological role has until
now been impossible because, for obvious reasons, represen-
tative samples for necropsy could not be obtained. Coprodiag-
nosis by PCR with fresh fecal samples will overcome this prob-
lem in the near future, although the test system will have to be
evaluated separately for each host species.
PCR of fox feces for the detection of E. multilocularis is an
important step in simplifying the routine diagnosis of infection
with the parasite. In our study this technique was evaluated
with fresh samples removed from the rectums of foxes that had
been shot. To exploit the full potential of the technique, it
should in future be evaluated with deposited fox feces from the
environment. However, the influences of various factors (e.g.,
age, temperature, and desiccation) on the reliability of the test
system are unknown, and to the authors’ knowledge, no diag-
nostic PCR system that uses fecal samples from the environ-
ment has been developed. Therefore, the practicality of the
method needs to be determined by testing large numbers of
fecal samples randomly collected from areas where prevalence
rates (and their temporal variations) have previously been es-
tablished by necropsy of adequate numbers of animals.
Our sample of 241 foxes showed a rate of E. multilocularis
prevalence of 59% by necropsy examination which, by adding
PCR as a second method, increased to 75% (that is, samples
positive by at least one method). Since some infections may fail
to be detected by both methods, the real prevalence may be
even slightly higher. With 165 PCR-positive foxes, it was for
the first time possible to determine the sensitivity rate of the
necropsy method, which has been in use (with modifications)
since the 1970s. Our set of data showed a sensitivity of 76%.
Since most of the recently published rates of E. multilocularis
prevalence in definitive hosts were established by using nec-
ropsy with smear samples as the only method of detection (14),
this sensitivity rate is of prime importance for interpretation of
these data.
ACKNOWLEDGMENTS
This work was financially supported by the local government of
Baden-Wu ¨rttemberg (ministries of agriculture, research, and social
affairs) and the German Federal Ministry of Health.
We thank Kirsten Tackmann (Wusterhausen, Germany) for kindly
providing fox fecal samples. We also thank Peter Deplazes (Zu ¨rich,
Switzerland), Marshall Lightowlers (Werribee, Australia), Kenishi Ta-
kahashi (Sapporo, Japan) and Eberhard Zeyhle (Nairobi, Kenya) for
supplying cestode material from various species. We thank Ute Mac-
kenstedt and Brigitte Frank (Hohenheim, Germany) for general sup-
port and advice.
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