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Pierangeli - Usefulness and validation of a coproantigenos en NEuquen

Parasitology International 59 (2010) 394–399
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Parasitology International
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Usefulness and validation of a coproantigen test for dog echinococcosis screening in
the consolidation phase of hydatid control in Neuquén, Argentina☆
N.B. Pierangeli a,⁎, S.V. Soriano a, I. Roccia b, H.F.J. Bergagna c, L.E. Lazzarini a, A. Celescinco c, A.V. Kossman a,
M.S. Saiz a, J.A. Basualdo d
Cátedra de Microbiología y Parasitología, Escuela de Medicina, Universidad Nacional del Comahue, Buenos Aires 1400, (8300) Neuquén, Argentina
Departamento de Zoonosis, Subsecretaria de Salud, Provincia de Neuquén, Fotheringham 121, (8300) Neuquén, Argentina
Dirección de Zoonosis y Vectores, Municipalidad de Neuquén, Av. Argentina 307, (8300) Neuquén, Argentina
Cátedra de Microbiología y Parasitología, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, Calle 60 y 120, (1900) La Plata, Buenos Aires, Argentina
a r t i c l e
i n f o
Article history:
Received 10 November 2009
Received in revised form 14 May 2010
Accepted 14 May 2010
Available online 28 May 2010
Canine echinococcosis
Arecoline purgation
a b s t r a c t
Hydatidosis is endemic in Neuquén, Patagonia, Argentina, even though sanitary authorities have been
performing a control programme since 1970. At present, the programme is in consolidation phase, and dogs
have being evaluated by arecoline purgation. The aims of this study were to evaluate diagnostic performance
of a coproantigen (CAg) ELISA test developed “in house” and to assess CAg detection in prepatent period. We
examined 8 dogs experimentally infected with Echinococcus granulosus and 403 rural dogs in an endemic
area in Neuquén using CAg ELISA test and arecoline purgation. Within the experimental dog group,
sensitivity and specificity of the test were 93.6% and 88.5% respectively. In rural dogs group, the overall
prevalence of canine echinococcosis was 3.7% using arecoline purgation and 12.4% by the CAg test; sensitivity
and specificity of the test using arecoline purge as standard were 73.3% and 89.9% respectively. Possible cross
reactions in CAg test were evaluated in rural dogs: CAg was undetectable in 96.4% of the dogs infected only
with taeniids non-E. granulosus, and in 90.1% of dogs infected with non-taeniid helminths. The CAg test could
detect infections within prepatent period and produced negative results after worm expulsion. Our test
showed adequate diagnostic performance with experimentally and naturally infected dogs, in the
epidemiological situation of Neuquén. Employment of this sensitive and practical method for surveillance
in the control programme in Neuquén would improve screening of canine echinococcosis by detecting
infected dogs even with low burdens or within prepatent period.
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Cystic echinococcosis, caused by Echinococcus granulosus, is an
endemic zoonosis in many countries all over the world, with an
important impact in animal and human health. The infection in
definitive hosts has a key role in the transmission dynamics of the
disease. Detection of adult E. granulosus in dogs is very important in
order to evaluate its prevalence, in epidemiological studies, and to
develop surveillance and control programmes of hydatidosis [1].
Diagnosis of the infection with E. granulosus in definitive hosts is
difficult because eggs are morphologically indistinguishable from
those of other taeniid cestodes, and because emission of eggs is
variable. The gold standard method to evaluate Echinococcus infection
in definitive hosts is the recovery of adult worms in the intestine after
necropsy. Even though this technique is close to 100% sensitive and
Abbreviations: CAg, coproantigen.
☆ Location: The study was carried out in (a), (b) and (c).
⁎ Corresponding author. Tel./fax: + 54 299 443 0820.
E-mail address: [email protected] (N.B. Pierangeli).
1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
specific, it is ethically questionable and not suitable for large trials [2].
Conventional ante mortem method is based on purging dogs with
arecoline hydrobromide followed by examination of faecal material
discharged. This technique is very specific but has a highly variable
sensitivity, is biohazardous, time-consuming, and some dogs suffer
undesired side effects [3].
Immunological methods are used to detect serum antibodies
against oncosphere or protoscolex antigens in dogs [4,5]. These
methods do not differentiate present from past infection and show
variable sensitivity and specificity [6].
Detection of parasitic specific antigens in faecal samples (coproantigens) has become useful for the diagnosis of intestinal infection with
several pathogens [7,8]. Coproantigen (CAg) ELISA assay has been
developed for diagnosis of canine echinococcosis, using polyclonal
antibodies either against somatic or excretory/secretory antigens,
from adult E. granulosus [9,10] or from protoscoleces [11]. This
method has the advantage of detecting infection in the prepatent
period, it showed 88% sensitivity and a good specificity (77–88%),
and ELISA values decreased to negative 2 or 3 days after elimination
of E. granulosus [12–16]. Results of CAg used to evaluate canine
N.B. Pierangeli et al. / Parasitology International 59 (2010) 394–399
echinococcosis in different geographical regions confirm the usefulness
of this assay in epidemiological surveys [13,14,17–20]. However, several
studies have demonstrated that sensitivity of CAg tests is dependent on
the worm burden, and that the diagnostic parameters of sensitivity and
specificity vary in different epidemiological situations [12,18].
In Neuquén, where hydatidosis is endemic, provincial sanitary
authorities have been carrying out a control programme of cystic
echinococcosis since 1970. This programme is based on anthelmintic
treatment of dogs, sanitary education, slaughter control and notification of confirmed human cases. At present, the control programme is
in consolidation phase, and infection in dogs is still evaluated by purge
with arecoline hydrobromide. In 1970, at the beginning of the
programme echinococcosis prevalence in dogs was 28.2% and it
decreased to 2.9% in 1990. According to official data, prevalence of dog
echinococcosis in Neuquén has being remained in a steady state at
about 1.0% since 1996 [21]. However, the overall mean annual
incidence of human hydatid disease in the province of Neuquén for
the period 1995–2004 was 24.4 per 100,000 inhabitants within the
total population and 9.7/100,000 in the 0–14 year group [22].
Considering this particular epidemiological situation in Neuquén,
the purge with arecoline seems to be inadequate to evaluate canine
echinococcosis [12,18]. It would be necessary to change the arecoline
purge used for the surveillance of dog echinococcosis for a more
sensitive and practical method as the CAg test. Commercial kits for
CAg assay are not available in Argentina, so it was necessary to
develop a test “in house” and therefore to evaluate its efficacy before
using it in the Province Hydatid Control Programme.
In order to improve screening of canine echinococcosis in a longterm control area, the aims of this study were to evaluate the
diagnostic performance (sensitivity, specificity, predictive value and
precision) of the coproantigen ELISA test developed “in house”, with
both experimentally and naturally infected dogs, as well as to assess
coproantigen detection in the prepatent period.
2. Materials and methods
Fig. 1. Geographic location. A: Argentina
Argentina. B: Sanitary Area IV in Neuquén.
in South America and Neuquén
Group A: 8 dogs were used for validation of the test with
experimental infection and in time-course experiments. Dogs from
urban areas of Neuquén of mixed breed aged between 6 month and
5 years old were kept in individual kennels and treated orally with
praziquantel (5 mg/kg) and albendazole (50 mg/kg) one week before
infection. They were maintained on commercial dried food and water
ad libitum according to biosafety and ethical regulations. Two dogs
from this group (Group A1) were kept as uninfected controls and 6
were infected with E. granulosus (Group A2).
Group B: 403 rural dogs under Province Control Programme
belonging to Sanitary Zone IV of Neuquén were included in the field
trial. To assess possible cross reactions, 367 dogs of Group B that
showed infection with other parasites but were negative for E.
granulosus, were classified in 3 sub groups, as follows: Group B1: 268
helminths-free dogs; Group B2: 28 dogs with natural infection with
taeniid different from E granulosus and Group B3: 71 dogs with
natural infection with non-taeniid helminths. Presence or absence of
helminths in these dogs was assessed by both arecoline purgation
and/or coprological exam.
2.1. Experimental design and study area
2.3. Parasite material and experimental infections
This study included two parts. The first part was carried out with
experimentally infected dogs and comprises validation of the CAg
ELISA test as well as detection of CAg in time-course experiments. For
these purposes a prospective experimental design was carried out
between March and September 2005. In the second one, we carried
out a field trial to validate the CAg test against arecoline purgation
with natural infected dogs in a rural area of the province of Neuquén,
Patagonia Argentina. We also assessed the influence of the possible
low burden of E. granulosus and the presence of other helminths in
naturally infected dogs on the sensitivity and on the specificity of
coproantigen detection. In this case, we applied a descriptive
observational cross sectional design between June 2005 and October
2008. The Province of Neuquén is located in the north of the
Argentinean Patagonia and its public health system is organized in 6
Sanitary Zones (Fig. 1). The study area corresponds to small rural
communities of the Sanitary Zone IV, where human hydatid disease
showed one of highest incidence rates in the province [22]. The main
economic activity is raising livestock for subsistence; goats are the
most common livestock, but sheep and cattle are also raised. The
canine population in the studied area composed by shepherd and
guarding dogs is included in the Province Hydatid Control
Programme, in which dogs' owners carried out dosing of rural dogs
with praziquantel every 6 weeks [21].
2.2. Animals
The dogs evaluated in this study were classified in 2 groups, A and
B, as follows.
For experimental infections, fertile pulmonary hydatid cysts were
obtained from goats slaughtered in abattoirs of Chos Malal, in the
north of the Province of Neuquén. Protoscoleces viability was assessed
by observation of flame cell activity with 100× with an optic
microscope and vital stain with 0.4% Tripan Blue. Dogs from Group
A2 were orally infected with a dose of 20.000–50.000 protoscoleces
with a viability of 80–95%.
2.4. Collection of samples
To validate the CAg test with Group A dogs, we randomly selected
35 fresh faecal samples from uninfected dogs (Group A1) and 47
samples of confirmed infected dogs (Group A2). Infection was
confirmed by arecoline purgation at 35 days post infection (dpi). All
faecal samples were stored at − 20 °C for further CAg assay.
For Group B, trained staff of the Control Programme obtained
faecal samples during periodic visit to farms. Dogs were brought
voluntarily by their owners to the testing area and were dosed with
4 mg/kg of arecoline hydrobromide according to standard procedures
[23]. After purge, the first portion of faeces was collected separately in
a plastic container and the rest of the purge was examined in a black
tray. Helminths found in purge were identified as previously
described [24]. Only dogs from which both samples could be obtained
were included in this study. Once in our laboratory, the first portion of
faeces was separated in 2 fractions: one was stored at − 20 °C for CAg
detection, and the other was kept with 4% formaldehyde for
coprological exam.
N.B. Pierangeli et al. / Parasitology International 59 (2010) 394–399
2.5. Time-course experiments
2.9. Data analysis
Time course experiments were performed with 6 dogs of Group
A2. Fresh faecal samples from these dogs were collected 2 days before
infection and daily post infection from days 2 dpi to 35 dpi. At day 35
dpi arecoline purgation was performed [19]. To evaluate the effect of
purgation on CAg excretion, dog faecal samples were collected daily
until 7 days post arecoline treatment, and were frozen until use.
Afterwards, they were treated orally with 5 mg/kg of Praziquantel to
eliminate parasite infection. All faecal samples were processed for
CAg. Detection of CAg in pre patent period was evaluated in this
In the experimental assay cut off value for CAg test was calculated
in 2 ways. First, experimental cut off value was defined as the mean of
optical density (OD) of 35 negative control samples plus 3 standard
deviations (SD). In the second case, the receiver operating characteristic (ROC) analysis was applied to determine the optimal cut off value
and the area under the curve (AUC) was used to evaluate the global
diagnostic performance of the CAg test.
Diagnostic parameters were calculated as follows: sensitivity
(SENS) = TP × 100/(TP + FN); specificity (SPEC) = TN × 100/(TN +
FP); positive predictive value (PPV) = TP × 100/(TP + FP); negative
predictive value (NPV) = TN × 100/(TN + FN); diagnostic efficiency
(DE) = (TP + TN) × 100/(TP + FP + TN + FN).
Data from dogs in the field trial were analyzed using SPSS
statistical software (SPSS 17.0, Chicago, IL). Diagnostic performance
of the test in natural infected dogs was evaluated using arecoline
purge as gold standard because it was not possible to do necropsy to
dogs for legal and ethical reasons. SENS, SPEC, PPV, NPP, DE were
calculated as above mentioned. Bayesian's formula was also used to
estimate PPV and NPV with 95% confidence interval (CI) taking into
account prevalence (PREV) of dog echinococcosis in the region where
the CAg is going to be applied [29].
Inter-assay precision of the CAg test was assessed with 4 positive
controls (2 with high and 2 with low concentrations of EgSA) in 30
different runs.
2.6. Coprological exam and arecoline purgation
Parasitological exam of faecal samples was processed by standard
sedimentation method and was observed at light microscope with
100× and 400×. Genera and species of helminths were identified as
previously described [24].
Arecoline purgation was performed according to standard procedures [23]. Dogs were given an oral dose (4 mg/kg) of arecoline
hydrobromide. Purgation usually took approximately 30 min, although some dogs that did not purge successfully required a second
arecoline dose. The complete purge samples were collected from the
ground, mixed with 5% formal saline, and initially examined in blackbacked trays. All purges were conserved in separated vessels and then
examined under a dissecting microscope in our laboratory. All
heminths found were identified as previously described [24] and
the total number of Echinococcus worms was counted.
2.7. E. granulosus somatic antigen, hyperimmune rabbit antisera and
Extract of E. granulosus somatic antigen (EgSA) was prepared as
described by Allan et al. [9]. For this purpose, about 800 adult E.
granulosus worms were obtained on 35 dpi by purging one of the
infected dogs from Group A2 with arecoline hydrobromide. Hyperimmune rabbit anti EgSA antiserum was prepared as described previously
[25]. Purification of the IgG fraction from hyperimmune rabbit antisera
was obtained by precipitation with ammonium sulphate 40% and
dialysis [26]. Conjugation of antisera with horse radish peroxidase type
VI enzyme (Sigma) was performed by the protocol of Wilson and
Nakane [27]. Both IgG fractions and conjugate were sterilized by passing
through a 0.22 μm filter and stored in 200 μl aliquots at −20 °C until use.
2.8. Coproantigen ELISA procedure
Canine faecal supernatant were prepared at a ratio of 1:1 with buffer
PBS 0.15 M with 0.3% Tween 20, were shaken vigorously with a vortex,
and were centrifuged at 3500 rpm for 30 min at room temperature.
The CAg test was performed using a sandwich ELISA protocol
described by Allan et al. [9] with the following modifications: wells of
microtiter plates (Nunc Maxisorp) were coated with 100 μl of rabbit IgG
anti EgSA at a dilution of 1:300 (14 μg/ml) in 0.05 M carbonate buffer pH
9.6 at 4 °C overnight; 100 μl of anti EgSA conjugate diluted 1:200 was
added to each well; ABTS (2,2′-Azino-bis-3-Ethylbenzthiazoline-6Sulfonic Acid) (Sigma) diluted in 0.05 M citric acid and 2% hydrogen
peroxide was used as substrate solution. The reaction was stopped by
adding 100 μl 0.1 M fluorhydric acid. The plates were read at 410 nm in a
StatFax ELISA reader [28]. The cut off value was calculated as described
below. In all plates, blank (PBS buffer instead of faecal sample), positive
controls (faecal sample with 5 μg/ml of EgSA), and negative controls
(faecal sample from helminths-free dogs) were tested. All fecal samples
and controls were assayed in duplicate.
3. Results
3.1. Coproantigen detection in time-course experiments
All the 6 dogs of Group A2 were successfully infected with E.
granulosus from goat origin. The pattern of CAg excretion was similar
in all dogs: CAg was undetectable from day 3 until 6–7 dpi, followed
by a peak in OD values over the cut off point, remaining positive until
arecoline purging at 35 dpi (Fig. 2). Worms were recovered from all
the infected dogs; burdens ranged from 300 to more than 1000. After
purge, there was a rapid reduction on OD values below the cut off
3.2. Validation of coproantigen test with experimental infected dogs
The experimental cut off calculated as mean plus 3 SD of OD values
of 35 samples from uninfected control dogs (Group A1) was 0.235. On
this basis, diagnostic parameters of the test with the experimental
infected dogs (Group A2) were: SENS = 93.6%; SPEC = 88.5%;
PPV = 91.7%; NPV = 91.2% and DE = 91.5% (Table 1). In concordance,
optimal cut off value estimated by ROC curve was 0.235 and it
corresponds to the better combination of SENS and SPEC with the data
analyzed. For this cut off point in the ROC curve, SENS was 93.6%, SPEC
88.6% and the AUC was calculated as 0.934 (Fig. 3).
Evaluation of inter-assay precision of the CAg test in 30 different
runs for 2 levels of positive controls showed that CV% varied from 6.2
to 8.8% (Table 2).
3.3. Arecoline purgation and coprological exam in rural dogs
E. granulosus was present in arecoline purgation of 15/403 (3.7%)
rural dogs. In 13 of these dogs E granulosus worm burden varied from
5 to 100 worms per dog, and only 2 dogs harboured 500 and more
than 1000 respectively. Other adult helminths found in the purge
were: Taenia hydatigena 59/403 (14.6%) and Toxocara canis 43/403
(10.7%) either in single or associated infections.
Coprological exam was positive for intestinal worms in 148/403
(36.7%) rural dogs. The most frequent helminths were: T. canis 67/403
N.B. Pierangeli et al. / Parasitology International 59 (2010) 394–399
Fig. 2. Detection of coproantigen by ELISA during the experimental infection of dogs with Echinococcus granulosus. Mean value plus standard deviation of optical density (OD) from 6
dogs between 2 and 42 days post infection (dpi) were considered.
(16.6%), taeniids 29/403 (7.2%) and Toxascaris leonina, Trichuris vulpis,
and Ancylostoma caninum in minor percentages.
3.4. Validation of coproantigen test in rural dogs with E. granulosus
using arecoline purge as gold standard
In the field trial we included 403 rural dogs under Control
Programme from Neuquén. The overall prevalence of canine
echinococcosis was 3.7% (15/403) using arecoline purgation and
12.4% (50/403) by CAg ELISA test. CAg test was positive in 11/15 dogs
with positive arecoline purgation, and it was negative in 349/388 dogs
with negative arecoline purgation. SENS was calculated as 73.3%;
SPEC = 89.9%; PPV = 22.0%; NPV 98.9%; DE= 89.3%. When PPV and NPV
were estimated by Bayesian's formula prevalence of canine echinococcosis in Neuquén was considered to be 12%. In this case, PPV was 49.7%
(95% CI 44.8–54.6) and NPV was 96.1% (95% CI 94.2–98.0).
3.5. Evaluation of the effect of non-E. granulosus helminths' infection on
coproantigen detection in rural dogs
CAg values in field dogs were also analyzed taking into account the
presence or absence of infection with other helminths (Table 3). In
Group B1 (helminths-free dogs) CAg was negative in 245/268 (91.4%).
Group B2 included 28 dogs with taeniid different from E. granulosus
infection confirmed by purge and only one of them was positive for CAg.
CAg was positive in 7/71 (9.9%) dogs from Group B3 harbouring nontaeniid helminthes. In this group the most common helminths were T.
canis (66/71), T. vulpis (2/71), T. leonina (2/71) and A. caninum (2/71).
4. Discussion
In the last years CAg test has been increasingly adopted in order to
detect E. granulosus infection in dogs as an important tool for
surveillance in endemic areas showing diverse diagnostic performances
Table 1
Diagnostic characteristic of the coproantigen ELISA test evaluated with dogs
experimentally infected with Echinococcus granulosus.
Samples from infected dogs
Samples from uninfected dogs
44 (TP)a
3 (FN)c
4 (FP)b
31 (TN)d
TP: true positive.
FP: false positive.
FN: false negative.
TN: true negative.
[12]. However, only few studies demonstrated its usefulness in the
consolidation phase of control programmes for cystic echinococcosis.
Moreover, considering that commercial kits for CAg test are not
available in Argentina, we had to develop our own test “in house”, so
its efficacy must be established before using it in local control
programme. This is the first validation trial of CAg test against arecoline
purgation in Neuquén both with experimental and rural dogs.
Although sanitary authorities have been carrying out a hydatid
control programme since 1970 in Neuquén province, prevalence of dog
echinococcosis evaluated by arecoline purgation has remained at a low
and stable level of 1.0% since 1996, but E. granulosus has not been
eradicated [21,30–33]. Data of human hydatid disease in Neuquén
Province in a recent period (1995–2004) showed high mean annual
incidences both for the total population and for the 0–14 year group.
This situation suggests that standard control measures, despite longterm implementation, are not able to produce a sustained improvement
of the epidemiological status of the disease in Neuquén, and so it is
necessary to enhance surveillance of the control programme [22]. After
more than 30 years of periodic treatment of dogs with praziquantel
every 6 weeks, probably such dogs should harbour a low number of
worms in their intestines. In these circumstances sensitivity of
purgation diminishes as the percentage of infected dogs decreases
[23]. This fact could explain the low values of positive arecoline
purgation found in Neuquén in the last ten years. Nevertheless, in dog
populations harbouring low burdens of E. granulosus CAg showed to be
more sensitive than arecoline purgation [12]. In the status of
consolidation phase in Neuquén, purge with arecoline seems to be
inappropriate to evaluate canine echinococcosis, so it is necessary to
replace it with a more sensitive and practical method that can be used at
large scale.
In contrast with other authors, in the present study we used somatic
antigens from adult E. granulosus from goat origin to immunize rabbits
in order to obtain polyclonal hyperimmune antisera, because these
antigens are easy to produce and they do not need in vitro culture as
excretory–secretory products do. IgG purified fraction of rabbit
polyclonal antibodies raised against somatic crude extract proved to
capture antigens from faecal supernatants properly.
In time course experimental infections, 6 dogs were successfully
infected with E. granulosus cyst from goat origin. CAg was detected in
the pre patent period after 7 dpi and test results remained positive
until the end of the experiences, in line with other authors who used
either antisera against somatic [9,13] or excretory–secretory antigens
[10,17]. In our experimentally infected dogs, we observed fluctuation
in OD values in daily detection of CAg in agreement with other studies
that showed that elimination of antigens in faeces is uneven, probably
due to physiological conditions of dogs [9,10,13,17]. After arecoline
purgation, elimination of the worm burden was followed by a rapid
N.B. Pierangeli et al. / Parasitology International 59 (2010) 394–399
Fig. 3. Receiver operating characteristic (ROC) curve representing the plot of sensitivity versus 1-specificity for ELISA test for the detection of Echinococcus granulosus coproantigen in
experimental infected dogs. The ROC curve was constructed with 35 CAg data from dogs of Group A1 (uninfected dogs) and 47 from Group A2 (experimental infected dogs) using the
SPSS statistical software (SPSS 17.0, Chicago, IL).The best cut off point (arrow) corresponds to optical density (OD) = 0.235 (sensitivity 0.936; specificity 0.886).
disappearance of detectable CAg values within 2–3 days after
treatment. This fact is advantageous compared to the detection of
serum antibodies and seems to be useful for surveillance in areas with
periodic anthelmintic treatment.
Considering that we couldn't validate our CAg test against
necropsy as the gold standard for ethical reasons, first of all we had
necessarily to assess its diagnostic performance with experimentally
infected dogs, in order to differentiate dogs certainly infected with E.
granulosus as unique specie from those without infection. We could
use only 8 dogs in the experimental infection trial for biohazard and
legal regulations on experimental trials in our country. Despite the
fact that the sample size of experimental dogs infected seems to be
small, we selected randomly several samples from each dog in order
to process a greater number of fecal samples of truly infected and
uninfected dogs. CAg detection in this group of dogs showed high
values of sensitivity and specificity when diagnostic evaluation was
established empirically and also when ROC curve analysis was
applied. These data indicate that this test developed “in house”
could differentiate accurately between true positive and true negative
samples and has an adequate global diagnostic performance.
Evaluation of inter-assay precision showed tolerable CV% and
demonstrated the reproducibility of the test.
Although the CAg test developed “in house” showed to be suitable
for echinococcosis detection in experimental dogs, it was necessary to
proof its diagnostic performance in a field trial in an area under
control programme, in order to evaluate real conditions, as low worm
burden and the presence of other helminths. Moreover, rural dogs
could present different physiological conditions related to diet and
digestive activity that could influence CAg detection, when compared
to dogs fed regularly with commercial food, like dogs from group A.
Table 2
Inter-assay precision of the coproantigen ELISA test for Echinococcus granulosus detection
evaluated with positive controls.
Mean O.D.b
Low positive controls
High positive controls
Number of runs.
Mean of optical density at 410 nm.
Standard deviation.
Percentage of coefficient of variation.
Field trial was performed in Sanitary Zone IV of Neuquén, where
mean annual incidence of human hydatidosis in the period 1995–2004
was reported as one of the highest in the province (51.5 cases per
100,000 inhabitants for the total population and 27.9 per 100,000 for the
0–14 years group) suggesting that active transmission of the disease
continues to occur [22]. However, previous arecoline purgation official
data showed that prevalence of dog echinococcosis in Sanitary Zone IV
was 0.96% in 2002 and 1.18% in 2003 [21]. Our results indicated that the
overall prevalence of dog echinococcosis detected by arecolina
purgation was 3.7% and CAg test showed to be more sensitive as it
was positive in 12.4% of rural dogs. It must be taken into account that
purge is not 100% sensitive and only 15 dogs were certainly positive for
E. granulosus by this method, and that in 13/15 of these dogs worm
burden was under 100 worms. On this basis, the sensitivity of the CAg
test was 73.3% and specificity was 89.9% in the field trial. These values
were found to be similar to those reported by other authors in field trials
that used purge or necropsy as confirmation of the infection [12,19].
Predictive value of a diagnostic test is the probability that a result will
indicate the true state of infection. Prevalence of infection influences on
the predictive value. Consequently a decrease in infection prevalence
reduces the PPV and increases the NPV [1]. Negative predictive value
was high both with experimentally infected dogs (91.2%) and in the field
trial (98.8%), and therefore, a negative result of CAg test confirms that
such dog is free of infection. In contrast, PPV was high in experimentally
infected dogs (91.7%) but low (49.7%) in rural dogs, in accordance with
other studies [19,34]. This data could be the consequence of the low
number of true positive infected dogs (15/403) detected by purge due to
the low worm burden and to the low prevalence of echinococcosis in the
studied population of rural dogs. This could be explained in part by the
fact that arecoline purge is not the gold standard method. Even though
false positive results can be accepted more readily than false negative in
surveillance of a hydatid control programme [17], confirmation of CAg
positive results by detecting E. granulosus DNA in faeces using PCR
(Polymerase chain reaction) is highly desirable [35–37].
Table 3
Evaluation of the effect of non-E. granulosus helminths' infection on coproantigen
detection in rural dogs (Sanitary Zone IV, Neuquén, Argentina).
B1: Helminths-free dogs
B2: Taeniid non-E. granulosus infected dogs
B3: Helminths non-taeniid infected dogs
CAg + N (%)
CAg − N (%)
23 (8.6)
1 (3.6)
7 (9.9)
245 (91.4)
27 (96.4)
64 (90.1)
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To evaluate possible cross reactions in CAg test rural dogs were
classified in 3 groups on the basis of the presence or absence of
confirmed infection with other helminths. In the helminths-free dog
group, CAg was negative in 91.4% of dogs, showing the high specificity
of the test. Taenia genus is considered the main source of antigenic
cross reactions in the Echinococcus immunodiagnosis. In the present
field trial only 1/28 dogs infected with Taenia hydatigena was CAg
positive, indicating low cross reaction with other taeniids antigens. In
group B3, 9.9% of nematode-infected dogs were CAg positive. Other
authors found undetectable or lower percentages of cross reaction in
dogs infected with nematodes [9,10]. In rural dogs infected only with
nematodes, CAg was positive in 3/82 (3.6%) in Spain [11] and in 2/37
(5.4%) in Uruguay and Brazil [17]. Our results observed in Group B3
could be explained by the fact that some of these dogs would
correspond to dogs certainly infected with E. granulosus detected by
CAg test, but they did not shed worms after arecoline purgation.
In the study area prevalence of canine echinococcosis assessed by
CAg test was three times higher than by arecoline purgation.
Although the first value could be influenced by a percentage of
false positive results, it seems to reflect more accurately the real
epidemiological situation in Neuquén, where important active
transmission of the disease has been demonstrated. Our results are
in accordance with the findings of Benito et al. [34] who refer that in
an area under control programme where dogs are treated regularly
with praziquantel, recent infections could be undetectable by
purgation, but demonstrable by CAg test.
In conclusion, the CAg test developed “in house” showed an
adequate diagnostic performance with experimentally or naturally
infected dogs, in Neuquén. The CAg test has the potential to replace
arecoline purgation for the diagnosis of dog echinococcosis in
Neuquén. Our experience in developing and validation of a CAg test
could be useful to other groups that are considering improving
echinococcosis diagnosis in endemic areas. Employment of this
sensitive and practical method for large scale surveillance in the
control programme in Neuquén would improve screening of canine
echinococcosis in this endemic area by detecting infected dogs with
low burdens or within prepatent period.
The authors are grateful to Hermosina Ruth Astete for efficient
technical assistance and to Prof. Luciana Di Pascuale for language
revision. We are also thankful to Vet. Med. Marcelo Infante, Vet. Med.
Claudio Brusoni, Juan Carlos Arias and technicians of the Environmental and Zoonosis Department of the Province of Neuquén for
collection of samples from rural areas and Chos Malal. This study was
supported by grant 04/N005 from the Research Department of the
Universidad Nacional del Comahue.
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