Am. J. Trop. Med. Hyg., 60(2), 1999, pp. 188–192
Copyright q 1999 by The American Society of Tropical Medicine and Hygiene
DIFFERENTIAL SERODIAGNOSIS FOR CYSTIC AND ALVEOLAR ECHINOCOCCOSIS
USING FRACTIONS OF ECHINOCOCCUS GRANULOSUS CYST FLUID (ANTIGEN B)
AND E. MULTILOCULARIS PROTOSCOLEX (EM18)
AKIRA ITO, LIANG MA, PETER M. SCHANTZ, BRUNO GOTTSTEIN, YUE-HAN LIU, JUN-JIE CHAI,
SAMI K. ABDEL-HAFEZ, NAZMIYE ALTINTAS, DURGA D. JOSHI,
MARSHALL W. LIGHTOWLERS, AND ZBIGNIEW S. PAWLOWSKI
Department of Parasitology, Gifu University School of Medicine, Gifu, Japan; Epidemiology Branch, Division of Parasitic
Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Institute of Parasitology, Faculty of Veterinary Medicine
and Faculty of Medicine, University of Berne, Berne, Switzerland; Institute of Infectious and Parasitic Diseases, Chongqing
University of Medical Sciences, Chongqing, China; National Hydatid Disease Centre of China, Urumqi, China; Department of
Biological Sciences, Faculty of Science, Yarmouk University, Irbid, Jordan; Department of Parasitology, Medical Faculty of Ege
University, Izmir, Turkey; National Zoonoses and Food Hygiene Research Centre, Kathmandu, Nepal; Molecular Parasitology,
Veterinary Clinical Centre, University of Melbourne, Melbourne, Victoria, Australia; Clinic of Parasitic and Tropical Diseases,
Karol Marcinkowski University of Medical Sciences, Poznan, Poland
Abstract. Echinococcus granulosus cyst fluid and E. multilocularis protoscolex extract were fractionated by a
single step of preparative isoelectric focusing, resulting in an antigen B-rich fraction (8-kD) and an Em18-rich fraction,
respectively. The usefulness of both fractions for differential serodiagnosis of cystic (CE) and alveolar (AE) echinococcosis was evaluated by a large-scale immunoblot analysis on a battery of 354 serum samples. These included
66 from AE patients originating from four different endemic areas, 173 from CE patients originating from seven
different endemic areas, 71 from patients with other parasitic diseases, 15 from patients with hepatomas, and 29 from
healthy individuals. In an immunoblot with the antigen B-rich fraction, 92% (158 of 173) of the CE sera as well as
79% (52 of 66) of the AE sera reacted with the 8-kD subunit. No cross-reactivity occurred with any sera from patients
with cysticercosis, other parasitic diseases, or with hepatomas, or from healthy controls. In an immunoblot with the
Em18-rich fraction, all but two sera from AE patients (64 of 66, 97%) recognized Em18, and only nine of 34 CE
sera from China reacted with it. All other (139) CE sera from six other countries were negative as were all (115)
other non-echinococcosis sera. These findings indicate that antigen B (8-kD) is not species-specific for E. granulosus
but is genus-specific for Echinococcus, and that the Em18 antigen is a reliable serologic marker for species-specific
differentiation of AE from CE.
particularly its smallest subunit of 8/12 kD, offers greater
specificity than detection of antigen 5, but neither antigen B
nor antigen 5 are species-specific for E. granulosus.8–11 For
the serodiagnosis of AE, an ELISA using an affinity-purified
Em2 antigen from E. multilocularis metacestodes has been
shown to be a reliable serologic test.12 Its diagnostic value
has been improved by the combination use of the native Em2
antigen and a recombinant antigen (II/3-10) in an ELISA
known as the Em2plus-ELISA, which is now commercially
available.7,13 Recently, an 18-kD antigen from the E. multilocularis protoscoleces (Em18) was reported by our group
as a highly species-specific and sensitive antigen with potential not only for differentiation of AE from either CE or
other helminth infections, but also for differentiation of active from inactive AE.14—18 In general, there was a good correlation between Em18-immunoblots and the Em2plus-ELISA, although the specificity was somewhat higher in the former.16, 18 More recently, we confirmed that both Em18-immunoblots, particularly the IgG subclass Em18-immunoblot
and the Em2plus-ELISA, were reasonably reliable for longterm follow-up study of patients with AE after treatment
with albendazole.19
This paper reports the isolation of antigen B and Em18
by preparative isoelectric focusing (IEF) and the evaluation
of their usefulness by immunoblot for serologic verification
and differentiation between CE and AE from some different
geographic foci.
Echinococcosis, one of the most lethal parasitic zoonoses,
remains a public health problem of worldwide importance
and has recently gained recognition in some regions as an
emerging or re-emerging disease.1–3 In humans, this disease,
including cystic (CE) and alveolar echinococcosis (AE), is
caused by the larval stage of two species of tapeworms
(Echinococcus granulosus and E. multilocularis, respectively). Differential diagnosis between the two species has significant implications for epidemiologic studies and treatment
of these diseases, especially in the regions such as North
America, central Europe, and China, where both species occur sympatrically. Imaging methods, including ultrasonography, computerized tomography (CT), and magnetic resonance imaging (MRI), can provide a species-specific differential diagnosis in many cases, but diagnosis may be problematic in others.4–6 Serodiagnostic tests based on antibody
detection exhibit high sensitivity and reasonable specificity,
and have played an important role in confirming clinical diagnosis and in epidemiologic studies. However, the use of
crude somatic antigens from E. granulosus hydatid cyst fluid
(HCF) or E. multilocularis metacestodes in serologic tests
frequently results in problems of cross-reactions within and/
or across Echinococcus. Thus, this limits their usefulness in
differential serodiagnosis of CE and AE.7 As a result, increasing effort has been expended in identifying and purifying species-specific echinococcal antigens for application
in highly sensitive assays such as ELISAs and immunoblots.
The two major antigens of E. granulosus HCF (antigen 5
and antigen B) have been extensively characterized for their
diagnostic importance by a diversity of methods. Currently,
it has been generally accepted that detection of antigen B,
MATERIALS AND METHODS
Parasite material. The HCF was collected from fertile
lung and liver cysts obtained from E. granulosus-infected
188
189
SERODIAGNOSIS FOR ECHINOCOCCOSIS
sheep in Urumqi, China.20 The fluid was clarified by centrifugation at 3,000 3 g for 15 min at 48C, dialyzed against
5 mM Tris-HCl (pH 7.4) for 48 hr at 48C, and stored at
2208C. Echinococcus multilocularis metacestodes were obtained following passage in experimentally infected Wistar
rats. Crude E. multilocularis protoscolex (EmPS) antigen extract was prepared as described previously.15
Serum samples. A total of 40 and 37 serum samples collected in Switzerland and the United States, respectively,
were examined in blind tests for preliminary study. Sixtysix individual serum samples from surgically and/or parasitologically confirmed cases of AE were obtained from China
(25), Japan (18), the United States (17), and Poland (6). One
hundred seventy-three serum samples from surgically and
parasitologically confirmed cases of CE were obtained from
China (34), Jordan (36), Australia (39), Turkey (25), the
United States (8), Nepal (10), and Poland (21). All of these
AE and CE cases had liver lesion(s), and 234 of 239 were
obtained before treatment.
Serum samples from patients with other proven parasitic
diseases included cysticercosis (23), schistosomiasis (10),
paragonimiasis (10), clonorchiasis (10), sparganosis (10), fascioliasis (4), and filariasis (4). All of these sera had high
antibody responses to their homologous antigens as determined previously by immunoprecipitation, ELISA, or immunoblot. Twenty-nine serum samples from healthy Japanese students with no evidence of these diseases were used
as controls.
This international collaborative project on echinococcosis
and cysticercosis was approved by the Human Research
Committee of Gifu University. Informed consent was obtained from all serum donors prior to collection of any blood
specimens at all medical institutes involved in the study. All
sera were shipped to Gifu University under dry ice storage.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Antigen preparations were resolved by SDS-PAGE using precast 4–20%
gradient gels (#01-022 for two dimensions with a 6-cm
width; Tefco, Tokyo, Japan) under reducing conditions. Prestained low and broad molecular markers (161-0305 and 1610318, respectively; Bio-Rad, Hercules, CA) were used for
monitoring the electrophoresis of EmPS and HCF, either
crude or fractionated, respectively. Approximately 400 mg of
crude antigens and 20 mg of fractionated antigens were loaded into one large sample well of a 6-cm width gel. Electrophoresis was carried out at a constant current of 25 mA for
approximately 90 min and 70 min for EmPS and HCF, respectively. Transfer to ImmobilonTM polyvinylidene difluoride membranes (Millipore, Bedford, MA) was carried out at
150 mA for 4 hr. Immunoblotting was carried out using human sera diluted 1:100 and a monoclonal antibody against
Em1614 diluted 1:200. Horseradish peroxidase (HRP)–labeled polyclonal antibodies against human IgG and mouse
IgG diluted 1:1,000 dilution were used as previously described.14–18
Preparative isoelectric focusing. The HCF or EmPS
crude antigen extracts were fractionated by preparative IEF
using the Rotofort cell (Bio-Rad) according to the manufacturer’s instructions as described previously.19 Briefly, 40
ml of HCF or EmPS crude extracts were mixed with 2 ml
of ampholyte (40%, pH 3–10; Bio-Rad) and loaded into the
FIGURE 1. Representative immunoblots of the antigen B-rich
fraction from Echinococcus granulosus hydatid cyst fluid (HCF)
probed with individual sera from patients with cystic echinococcosis
(CE), alveolar echinococcosis (AE), cysticercosis (CC), and other
diseases (OD). Lane A, crude HCF; B, antigen B-rich fraction; a–j:
10 SE sera; k–t, 10 AE sera; u and the next nine lanes, 6 CC sera
and 4 OD sera. Arrowheads indicate the three subunits of antigen B
(8, 16 and 24 kD). The 16-kD and 24-kD bands are very faint in
this fraction. Values on the left are in kilodaltons (kD).
Rotofort cell. Samples were subjected to IEF at a constant
power of 12 W. After running for 4 hr, the resulting fractions
were harvested and analyzed by SDS-PAGE and immunoblot as described above.
RESULTS
Preliminary blind tests for differentiation of AE from other diseases were carried out by Em18-immunoblot analysis
using crude antigens of protoscoleces of E. multilocularis.
Approximately 83% (35 of 42) of the sera from patients with
AE and 6% (1 of 17) of the sera from patients with CE were
serologically diagnosed as AE. Four of the seven negative
AE sera were from inactive cases with calcified lesions. All
other sera, including neurocysticercosis (0 of 8), trematodiases (0 of 5), or nematodiases (0 of 5), were negative.
Antigen B. Immunoblot analysis of crude HCF revealed
a very complex profile of immunoreactive components ranging from 8 kD to . 213 kD (lane A in Figure 1). The most
dominant bands included the three regularly spaced subunits
of antigen B at 8, 16, and 24 kD, and the large subunit of
antigen 5 at 38 kD. When crude HCF was applied to the
immunoblot to detect antibodies in individual human sera,
there was little difference between the recognition patterns
of AE sera compared with those of CE sera, and cross-reactions were observed with sera from patients with cysticercosis or fascioliasis. Figure 1 shows an antigen B-rich fraction obtained by preparative IEF of HCF. The fraction had
a pH range of 1.4–3.2 and contained the three subunits of
antigen B with a predominance of 8-kD antigen and traces
of 16-kD and 24 kD antigens, and no apparent contamination
was observed. The 8-kD subunit was highly immunogenic
and strongly recognized by sera from patients with CE, as
well as sera from patients with AE. Probing with sera from
patients with cysticercosis or with other parasitic diseases
failed to detect any of the three subunits.
190
ITO AND OTHERS
FIGURE 2. Representative immunoblots of Echinococcus multilocularis protoscolex crude antigens (A) and the Em18-rich fraction (B)
probed with monoclonal antibody against Em16 (M) and by individual sera from patients with cystic echinococcosis (CE) (10), alveolar
echinococcosis (AE) (10), nine other diseases (OD) including cysticercosis, schistosomiasis, paragonimiasis, sparganosis, clonorchiasis, fascioliasis, filariasis, hepatomas, sarcoidosis, and one healthy control. Arrowheads indicate Em18. Lane C shows CE showing the antibody response
against Em18. This was an exceptional case of CE from China that recognized Em18 (see Results and Table 1). Other CE sera were from
Poland, the United States, Turkey, Jordan, Australia, China, and Nepal. Values on the left are in kilodaltons (kD).
Antigen Em18. Immunoblotting with Em crude antigen
and AE/CE sera revealed numerous bands of a wide molecular range (Figure 2A). A cluster of sharply focused bands
with low molecular weights of approximately 14–20 kD was
commonly recognized by sera from patients with AE. The
designated Em18 (arrowheads in Figure 2) is the band just
above the 16-kD band detected by a monoclonal antibody
against Em16.15
As shown in Figure 2B, preparative IEF of EmPS crude
antigen resulted in an Em18-rich fraction with a pH of 7.6
that contained some contaminants with much higher molecular weights. The lack of reactivity with monoclonal antiTABLE 1
Differential serodiagnosis of cystic (CE) and alveolar echinococcosis
(AE)
Serum samples
No. of
sera
Antigen B
fraction
Em18
fraction
CE
China
Australia
Jordan
Turkey
Nepal
United States
Poland
AE
China
Japan
United States
Poland
Others
Cysticercosis
Schistosomiasis
Paragonimiasis
Clonorchiasis
Sparganosis
Fascioliasis
Filariasis
Hepatoma
Normal
173
34
39
36
25
10
8
21
66
25
18
17
6
115
23
10
10
10
10
4
4
15
29
158 (92%)
32
36
34
23
9
8
16
52 (79%)
20
14
13
5
0 (0%)
0
0
0
0
0
0
0
0
0
9 (5%)
9*
0
0
0
0
0
0
64 (97%)
25
18
16
5
0 (0%)
0
0
0
0
0
0
0
0
0
* See Results.
body against Em16 confirmed the absence of contamination
with the Em16 antigen. The Em18 band could be clearly
identified by immunoblotting using the Em18-rich fraction
because only the Em18 band was located in the low molecular weight zones.
Relative sensitivity and specificity of immunoblots with
Em18- and antigen B-rich fractions. The data on serologic
differentiation of CE and AE originating from different endemic areas of the world are summarized in Table 1. In the
immunoblots with the antigen B-rich fraction, 92% (158 of
173) of CE sera and 79% (52 of 66) of AE sera were positive, and no cross-reactivity occurred with any sera from
patients with other parasitic diseases, patients with hepatomas, or healthy controls. There was no significant difference
in sensitivity when serum samples from different countries
were compared. In immunoblots with the Em18-rich fraction, 97% (64 of 66) of the AE sera recognized Em18, as
did nine of 34 Chinese CE sera (lane C in Figure 2). All
other 139 CE sera collected from six other countries were
negative. No cross-reactivity was observed with any sera
from non-echinococcosis patients or from healthy controls.
DISCUSSION
Currently, isolation and purification of specific Echinococcus antigen components have been achieved largely
through the application of a variety of methods, including
anion exchange chromatography,21,22 affinity with protein A
or monoclonal antibodies,12,22–24 hydrophobic interaction,21
and high-performance liquid chromatography.8,21,22,24 Analytical IEF has been used to identify the Em2a antigen12 and
the antigen 5 subunits.24 We used preparative IEF to isolate
Echinococcus antigens. This method is gentle, non-denaturing, and shows high resolution of proteins that differ in pI
by fractions of a pH unit. The proteins separated in the Rotofort cell can be easily recovered once they are focused.
The benefits of this technique have been demonstrated in
our earlier attempts to purify Em18/16 antigen18 and Taenia
solium glycoprotein antigens.25
SERODIAGNOSIS FOR ECHINOCOCCOSIS
In the present study, a single step of preparative IEF performed with E. granulosus HCF and E. multilocularis protoscolex extract resulted in the isolation of an antigen B-rich
fraction and an Em18-rich fraction, respectively. Both fractions were evaluated for their applicability in differential serodiagnosis of CE and AE by a large-scale immunoblot analysis on a battery of 354 serum samples, including 66 from
AE patients originating from four different endemic areas,
173 from CE patients originating from seven different endemic areas, 71 from patients with other parasitic diseases,
15 from patients with hepatomas, and 29 from healthy individuals.
The immunoblot with the Em18-rich fraction demonstrated that all but two sera from AE patients recognized Em18,
and nine of 34 CE sera from China reacted with it. All other
CE sera from other six countries (139) were seronegative for
Em18 antibody as were all other non-echinococcosis sera
(115). The sensitivity and overall specificity were calculated
to be at least 96% and 97%, respectively (including samples
with equivocal results discussed herein). These results further support our previous observations that Em18 is highly
sensitive and species-specific for E. multilocularis.14–18 The
two cases of AE, one from the United States (Alaska)16 and
the other from Poland, that were serologically negative by
Em18 and Em2plus-ELISA, were shown to have calcified inactive lesions only.26 The nine Chinese CE cases seropositive
for anti-Em18 antibody originated from Sichuan, China,
where both species of Echinococcus exist15 and mixed human AE and CE infections have been documented.27 All nine
cases (four with exceptionally high and five with relatively
lower titers) were also seropositive by the Em2plus-ELISA.18
In contrast, no CE cases from Jordan and Australia, where
only CE has been reported, and from Turkey and Poland,
where only sporadic cases have been reported, showed positive reactions with Em18. Similarly, Wen and others28 reported that the 17.5-kD band in EmPS (corresponding to the
Em18) reacted with the sera of three of 18 Chinese CE cases,
but did not react with any of the 23 sera from CE cases from
Uruguay or Libya, where human AE has not been documented. These findings appeared to support our earlier assumption that at least four of the nine Chinese CE cases
showing exceptionally high optical density values in the
Em2plus-ELISA had also been exposed to E. multilocularis.18
This assumption deserves clarification by further investigation.
Our findings are in contrast to those of Nirmalan and
Craig,29 who recently reported that Em18 was cross-reactive
with approximately 28% of the sera from CE patients from
Uruguay and the United Kingdom, two countries that are
non-endemic for AE. The discrepancy may be attributed to
the differences in antigen preparation or electrophoresis conditions. This problem has highlighted the need to purify the
Em18 antigen. In the present study, we obtained a highly
purified Em18-rich fraction by preparative IEF and found
that the Em18 band could be clearly recognized in immunoblots since only the Em18 band was present in the low
molecular weight regions.
Because Em18 has a relatively low molecular weight, it
is essential to perform SDS-PAGE under the optimal conditions, particularly when using a crude antigen preparation.
Otherwise, Em18 may not be well separated from other an-
191
tigen components with similar molecular weights. In the
study of Nirmalan and Craig,29 they used a somewhat low
(12%) nongradient gel, and this may account for their different results. Furthermore, it is noteworthy that Em18 appears as a sharp band on the blots. This is an important
characteristic that should be considered when interpreting
the immunoblot results. If there is a diffuse band located
near or at the position of the 18-kD band, it is probably a
band that is not from the protoscolex, but from the microvesicle, since when crude antigens were prepared from microvesicles purified by density gradient centrifugation,30 they
showed different patterns from those of the protoscolex, with
additional bands near the positions of Em18 and Em16 (Ito
A and others, unpublished data). Further studies are needed
to confirm if Em18 that appeared as a rather diffuse band as
in the study of Nirmalan and Craig29 is indeed identical to
the Em18 identified in our studies.
The immunoblot with the antigen B-rich fraction revealed
that 92% (158 of 173) of the CE sera and 79% (52 of 66)
of the AE sera were reactive with the 8-kD subunit. No
cross-reactivity occurred with any sera from patients with
cysticercosis or other parasitic diseases, patients with hepatoma, or from healthy controls.
The sensitivity (92%) for the 8-kD subunit obtained in our
study appears to be similar to that (94%) for the 8-kD subunit reported by Shambesh and others,11 as well as that
(90%) for the 12-kD subunit described by Leggatt and others,10 but higher than that (80%) for the 12-kD subunit reported by Ioppolo and others.31 The cross-reactions for the
8-kD subunit with AE sera (80%) demonstrated in our study
is higher than those reported in other two studies (both
40%).10, 11 Based on these data, it seems reasonable to conclude that the smallest subunit of antigen B (8/12-kD) is not
species-specific for E. granulosus but is shared with E. multilocularis. This will limit its usefulness for serologic differentiation of CE from AE.
Interestingly, in our present study, no cross-reactivity to
the 8-kD subunit was observed with sera from patients with
human cysticercosis. This is in contrast to the results of previous studies10,11 in which cross-reactions occurred with approximately 5% of the sera from patients with human cysticercosis. Possible explanations for this difference are that
we used a highly purified antigen preparation or that we
examined an insufficient number of serum samples.
The strategy we are recommending for serodiagnosis of
echinococcosis based on the present results is to perform
immunoblotting or an ELISA using antigen B subunits for
suspected cases of CE, and Em18-immunoblotting or the
Em2plus-ELISA for suspected cases of AE based on image
analysis.
Financial support: This work was supported in part by a Grant-inAid for International Scientific Research (Joint Research, 06044089,
07044243, 09044279) and a Grant-in-Aid for Scientific Research (B)
(10480235, 10557029) from the Ministry of Education, Science,
Sports and Culture of Japan, and by a Grant-in-Aid for the Control
of Emerging and Re-emerging Diseases in Japan from the Ministry
of Health and Welfare of Japan to Akira Ito.
Authors’ addresses: Akira Ito, Department of Parasitology, Asahikawa Medical College, Asahikawa 078, Japan. Liang Ma, Critical
Care Medicine Department, Clinical Center, National Institutes of
Health, Bethesda, MD 20892. Peter M. Schantz, Epidemiologic
192
ITO AND OTHERS
Branch, Division of Parasitic Diseases, Centers for Disease Control
and Prevention, Atlanta, GA 30341-3724. Bruno Gottstein, Institute
of Parasitology, Faculty of Veterinary Medicine and Faculty of Medicine, University of Berne, Berne, Switzerland. Yue-Han Liu, Institute of Infectious and Parasitic Diseases, Chongqing University of
Medical Sciences, Chongqing, China. Jun-Jie Chai, National Hydatid Disease Centre of China, Urumqi, China. Sami K. Abdel-Hafez, Department of Biological Sciences, Faculty of Science, Yarmouk
University, Irbid, Jordan. Nazmiye Altintas, Department of Parasitology, Medical Faculty of Ege University, Izmir, Turkey. Durga D.
Joshi, National Zoonoses and Food Hygiene Research Centre, Kathmandu, Nepal. Marshall W. Lightowlers, Molecular Parasitology,
Veterinary Clinical Centre, University of Melbourne, Melbourne,
Victoria, Australia. Zbigniew S. Pawlowski, Clinic of Parasitic and
Tropical Diseases, Karol Marcinkowski University of Medical Sciences, Poznan, Poland.
Reprint requests: Akira Ito, Department of Parasitology, Asahikawa
Medical College, Asahikawa 078, Japan.
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