Diagnostic Microbiology and Infectious Disease

48 (2004) 117123

www.elsevier.com/locate/diagmicrobio

Parasitology

Echinococcus granulosus in Jordan: assessment of various antigenic

preparations for use in the serodiagnosis of surgically confirmed cases
using enzyme immuno assays and the indirect haemagglutination test
Manal A. Nasrieh, Sami K. Abdel-Hafez*
Department of Biological Sciences, Yarmouk University, Irbid, Jordan
Received 10 June 2003; received in revised form 19 September 2003

Abstract
The Enzyme linked immunosorbent Assay (ELISA), indirect haemagglutination (IHA), and immunoblot techniques (IB) were used for
the serodiagnosis of surgically confirmed cystic echinococcosis (CE) caused by the tapeworm Echinococcus granulosus. Antigens used for
the detection of IgG or total antibodies included crude sheep hydatid fluid (CSHF), autoclaved antigen B (AAB), boiled antigen B (BAB),
and homogenate protoscoleces antigen (HPA). The overall sensitivity of the ELISA and IHA tests used for the serodiagnosis of 57 surgically
confirmed human cases was 91.2% and 68.4%, respectively. The sensitivity of both tests was comparable in groups whose sera were
collected one week before surgery and up to one year after surgery at 95.8% and 87.5%, respectively. In contrast, the sensitivity of the
ELISA was significantly higher than that of IHA for sera of patients collected after one year of surgery. There was a positive correlation
(r 0.61) between the titers of antibodies detected by the ELISA and IHA. Using the IB technique, antigen B fractions (8/12, 16, and 24
KDa) were detectable by sera of 68.4% using either CSHF or AAB, 49.1% using BAB and 22.8% using HPA as detecting antigens. The
overall sensitivity of the three AgB fractions was identical or similar to that of the 8/12 KDa fraction alone, indicating that the detection
of the latter fraction is sufficient for the serodiagnosis of CE infection in humans. In conclusion, the ELISA is the test of choice for the
serodiagnosis of CE and the follow up of cases following surgery using CSHF as an antigen. The IB test is a confirmatory test when antigen
B fractions of CSHF or AAB are detected. 2004 Elsevier Inc. All rights reserved.
Keywords: Cystic echinococcosis; Echinococcus granulosus; Hydatidosis; Jordan; Serodiagnosis

1. Introduction
Cystic echinococcosis (CE) or unilocular hydatidosis because of the infection with the metacestode stage of the tiny
tainiid tapeworm Echinococcus granulosus is a serious global
zoonotic disease of human and various herbivores acting as
intermediate hosts and the domestic dog as the definitive host
(Schantz et al., 1995). The disease is prevalent in sheep raising
countries in various part of the world including the Middle East
(Andersen et al., 1993, 1997; Schantz et al., 1995). In Jordan,
human CE is one of the most important endemic infectious
diseases (Abdel-Hafez & Kamhawi, 1997; Al-Qaoud et al.,
2003). The enzyme linked immunosorbent assay (ELISA),
indirect haemagglutination (IHA) test and immunoblot (IB)
technique are the most used serodiagnostic tests for CE. However, there is a great deal of variability in specificity and

sensitivity of these tests among different laboratories. This

variability is influenced by the type and procedures of the
antigen preparation (Gottstein et al., 1986; Njeruh and
Gathuma, 1989; Craig, 1993, 1997; Lightowlers and Gottstein,
1995; Shambesh et al., 1995; Ioppolo et al., 1996; Mc Vie et
al., 1997; Ersfed et al., 1997; Poretti et al., 1999; Irabuena et
al., 2000; Gonzalez-Sapienza et al., 2000; Ortona et al., 2000).
The present investigation evaluates the IHA, ELISA, and IB
techniques as diagnostic tests using a battery of different hydatid fluid and protoscolex antigenic preparations and sera of
surgically confirmed cases from Jordan.

2. Materials and methods

2.1. Collection of sera of surgically confirmed patients

* Corresponding author.
E-mail address: skhafez@yu.edu.jo (S.K. Abdel-Hafez).
0732-8893/04/$ see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.diagmicrobio.2003.09.018

Blood samples of 57 surgically confirmed Jordanian CE

patients were collected by venipuncture. The blood was

118

M.A. Nasrieh, S.K. Abdel-Hafez / Diagnostic Microbiology and Infectious Disease 48 (2004) 117123

collected at different periods ranging from 7 days before

surgery and up to 7 years post-surgery. Surgeries were
performed in major public and private hospitals in 8 governorates including Karak (20), Tafieleh (19), Zarqa (10),
Amman (4), Irbid (2), Jarash and Ajloun (1 each). Data on
sex, age, and cyst location of each case were collected from
hospital records and the patients themselves. The location of
cysts in these patients included the liver or the lungs, and in
some patients multiple cysts were found in the liver and
either one of the following organs: lungs, spleen, or kidneys.
In 3 patients the locations of extracted cysts were not recorded in hospital registry and were identified as unknowns.
The blood sample of each patient was allowed to clot and
separate overnight at 4C, and the serum was collected and
stored at 20C until used.
2.2. Preparation of antigens
The Bradford method (Bradford, 1976) was used to determine the protein content of all antigens prepared as described below and preparations were stored at 20C until
further use.
2.2.1. Crude sheep hydatid fluid (CSHF) antigen
This antigen was prepared as described by Moosa and
Abdel Hafez (1994). Briefly, hydatid fluid from sheep liver
and lung cysts was collected and centrifuged at 1000 g for
30 min at 4C to settle down the PSCs, brood capsules, and
membranes. The supernatant was aspirated and lyophilized.
For the preparation of soluble antigen, 200 mg of the lyophilized antigen were reconstituted in 1 ml deionized water, dialyzed overnight against 0.1 M phosphate buffer solution (pH 7.2) at 4C, and used as CSHF antigen.
2.2.2. Homogenate protoscoleses antigen (HPA)
Homogenate protoscoleses antigen (HPA) was prepared
as described by Allan and Craig (1989). The PSCs were
collected from sheep liver and lung hydatid cysts. After
washing twice with 0.15 M phosphate buffer saline (PBS,
pH 7.2), the PSCs were frozen and thawn three times and
homogenized using a glass homogenizer (23 ml capacity)
for 20 30 strokes. The homogenate was centrifuged at
15,000 g at 4C for 20 min and the supernatant was used
as HPA.
2.2.3. Preparation of antigen B (AgB)
The thermostable antigen B component of CSHF was
prepared either by autoclaving or boiling. Autoclaved AgB
(AAB) was prepared as described by Njeruh and Gathuma
(1989). Briefly, concentrated CSHF was heated in a universal bottle in autoclave at a pressure of 1.66 atmospheres and
at temperature of 110C for 10 min. The heated antigen was
centrifuged at 15,000 g for 20 min at 4C. The supernatant
was used as AAB.
Boiled antigen B (BAB) was prepared as described by
Rogan et al. (1991). Concentrated CSHF was boiled in a

water bath maintained at 100C for 15 min, centrifuged at

15,000 g for 20 min at 4C and the supernatant used as
BAB.
2.3. Enzyme linked immunosorbent assay (ELISA)
This technique was performed as described by Moosa
and Abdel Hafez (1994) with modification. Briefly, flatbottomed microtiter plates were coated with 10 ug/ml CSHF
antigen prepared in 0.05 M carbonate-bicarbonate buffer
(pH 9.6) and kept overnight at 4C. After washing with a
buffer made of 0.05% Tween 20 prepared in PBS, wells
were blocked with 5% skimmed milk protein (Regilait,
France) in PBS for 1 h at room temperature. Tested sera
were diluted to 1:200 using 1% skimmed milk protein in
PBS. After washing, the plates containing tested sera were
incubated for 1 h at room temperature. After further washings, wells were loaded with horse-raddish peroxidase conjugated rabbit IgG fraction against human IgG (Cappel,
Durham NC) (1:5000 dilution) and incubated for 1 h at
room temperature. After washing, a substrate solution containing O-phenyl diamine (OPD) (Sigma, St. Louis) and
H2O2 in citrate buffer (0.1 M, pH 4.5) was added to each
well, and the plates were read at 490 nm wavelength. Positive and negative serum controls were included in each
plate. The cut off point was calculated as the average of OD
of negative controls 3SD.
2.4. Indirect haemagglutination test (IHA)
This technique was performed as described by Moosa
and Abdel-Hafez (1994) with slight modification. Sheep
blood was collected in 0.11 M sodium citrate and washed
with PBS to remove the plasma and the buffy coat. A 7%
suspension of blood cells in PBS and an equal volume of
7% formaldehyde suspension in PBS were prepared. Onequarter of 7% formaldehyde suspension was added to the
red blood cell PBS suspension. The mixture was incubated
in a water bath for 1 h at 37C with frequent gentle shaking.
The rest of 7% formaldehyde/PBS solution was added to the
red blood cells and incubated in water bath for 24 h at 37C
with gentle shaking. The formalinized sheep red blood cells
(SRBCs) were washed 8 10 times and resuspended as 2.5%
suspension in PBS containing 0.02% sodium azide and
stored at 4C. A 1:40,000 tannic acid/PBS solution was
mixed 1:1 v/v with 2.5 formalinized SRBC and incubated in
water bath at 37C for 15 min before being centrifuged at
100 g for 15 min. The tanned formalinized SRBCs were
washed twice, resuspended to 2.5% concentration in PBS,
and divided into 2 lots: one was mixed 1:1 v/v with CSHF/
PBS antigen preparation (150 ug/ml). The second lot was
diluted with 1:1 v/v with PBS and used as a negative
control. The two lots were incubated in water bath for 30
min before centrifugation at 100 g for 10 min. After
further washing with PBS, the cells were resuspended to a

M.A. Nasrieh, S.K. Abdel-Hafez / Diagnostic Microbiology and Infectious Disease 48 (2004) 117123

final volume of 2.5% in PBS containing 0.02% sodium

azide and stored at 4C until use.
Tested sera including positive and negative controls were
diluted in twofold dilution series in PBS (1:16, 1:32, 1:64,
1:128, and 1:200, 1:400 up to 1:25,600). Of each diluted
serum samples, 100 ul were added to U-shaped microtiter
plate wells. Then, 100 ul of antigen coupled SRBC was
added to the tested sera in each well. Plates were incubated
for 1224 h at 4C. An IHA titer 32 was considered
negative as determined by several pooled negative serum
samples.
2.5. Immunoblot (IB) technique
The immunoblot technique was performed as described
by Towbin et al. (1979) and Boto et al. (1984). Immunoblotting was carried out following separation of antigenic
fractions by 12.5% SDS-PAGE using a transblot cell (BioRad, Italy). The gel was immersed in a 20% methanol Tris
glycine transfer buffer (pH 8.3) for 30 min. All other IB cell
components including the nitrocellulose transfer blot sheets
(Bio-Rad, Richmond, CA) were saturated with transfer
buffer for 30 min. The IB components were arranged properly in the Transblot cell with the gel and nitrocellulose
sheet sandwiched between the two sets of spongy and filter
paper pads. The sandwich was closed and held to the Transblot cell with the gel towards the cathode () and the
nitrocellulose sheet towards the anode (). The protein
transfer was done at 100 V for 2.5 h. The gel was removed,
fixed, stained, and destained as for SDS-PAGE to ensure
transfer of fractions to nitrocellulose paper. Nitrocellulose
paper strips (35 mm wide) were blocked with 5% skimmed
milk protein in tris buffer saline (TBS, PH 7.2) for 1 h then
washed three times with 0.05% Tween 20 in TBS (TBS-T).
The strips were immersed in diluted serum samples (1:50 in
1% skimmed milk/TBS) and incubated for 1 h at room
temperature. After washing in TBS-T, the strips were immersed in diluted solutions of horse-raddish peroxidase conjugated to rabbit IgG fraction against human IgG (Cappel)
(1:500 in 1% skimmed milk/TBS) and incubated for 1 h.
After further washing, the strips were incubated in a substrate solution composed of 60 l H2O2 (C.B.H. UK) added
to 0.06g 4-chloro-1- naphthol (Sigma) prepared in 20%
methanol/TBS solution. Immunoreactive fractions appeared
as dark bands.

3. Results
Of 57 surgically confirmed CE cases from Jordan, 45
(78.9%) were females, 13 (22.8%) were 20 years of age,
42 (73.7%) had liver cysts only, 3 (5.3%) had lung cysts
only, and 9 (15.8%) had multiple cysts in more than one
organ including the liver and lungs (6 cases), the liver and
spleen (2 cases), and the liver and kidneys (1 case). The
highest number of cases (86%) originated from the rural

119

governorates of Karak, Tafieleh, and Zarqa. Serologically,

91.2% and 68.4% of the 57 surgically confirmed cases were
positive as determined by the ELISA and IHA, respectively
(Table 1). Of the liver cases, 92.9% and 71.4% were ELISA
and IHA seropositive, respectively. The sera of 42.9% of
patients with liver cysts yielded very strong reactions
() in the ELISA, while 47.6% had high titers of
1:800 or more using the IHA. All three lung cases were
weakly ELISA positive (), but only 1 case was IHA
positive. The sera of 7 and 5 of the 9 patients with multiple
cysts were ELISA and IHA positive, respectively (Table 1).
None of the ELISA negative samples were IHA positive.
The correlation coefficient between the optical density representing IgG antibody levels as determined by ELISA and
the antibody titers as determined by IHA was calculated at
r 0.61.
An attempt to correlate between seropositivity rates and
length of time of serum sample collection post surgery was
made (Table 2). The sensitivity of both the ELISA and IHA
was high (95.8% and 87.5%, respectively) for serum samples collected during the first year postsurgery. However,
the IHA sensitivity dropped sharply to 58.3% and 42.9%
after 1 and over 5 years of surgery, respectively. In contrast,
the ELISA sensitivity was still relatively high in patients
even after 5 years postsurgery in which 78.6% of the cases
were still detectable serologically (Table 2).
Table 3 shows the immunoreactivity of serum samples of
surgically confirmed cases with antigen B fractions using
different antigenic sources and preparation methods. The
percent detectability rate of at least one of antigen B fractions (8 24 KDa) was significantly higher in all patients
when CSHF or AAB were used as a detecting antigen. The
least number of cases were detectable by HPA antigenic
preparation where only 22.8% of the cases showed reactivity with 8/12 KDa fraction. There was no statistically significant difference (p 0.05) in the percentages of samples
that showed immunoreactivity with 8/12 KDa fractions and
the overall immunoreactivity irrespective of which antigenic source was used (Table 3).

4. Discussion
The distribution of the new series of CE cases reinforced
earlier finding pertaining to the predominance of liver involvement and the high endemicity of the disease throughout Jordan with higher incidence in females and rural communities (see review by Abdel-Hafez and Kamhawi, 1997).
The higher surgical incidence among females compared to
males (3.75:1) have been attributed to occupational roles
that different sexes play in the community (Abdel-Hafez
and Kamhawi, 1997). However, other factors such as hormonal effects on the immunology of the host and subsequently on the progression of parasitic diseases are worth
investigation (see review by Morales-Montor, 2002). Recently, Al-Qaoud et al., (2003) observed higher female to

120

M.A. Nasrieh, S.K. Abdel-Hafez / Diagnostic Microbiology and Infectious Disease 48 (2004) 117123

Table 1
Grades and percentages of seropositivity of the sera of 57 surgically confirmed CE patients from various Governorates of Jordan as determined by
ELISA and IHA. Detecting antigen used was crude sheep hydatid fluid
Titer grade

Cyst locality (number of cases)

Liver (42)

Multiple (9)

Lung* (3)
%

Total (57)
No

17
12
8
15

29.8
21.1
14.0
26.3

52

91.2

4
14
12
9

7.0
24.6
21.1
15.8

39

68.4

No

Positivity grade

11
10
6
12

26.2
23.8
14.3
28.6

2
1
0
0

Total

39

92.9

Reciprocal titer
64128
200400
8001600
1600

3
7
11
9

7.1
16.7
26.2
21.4

0
1
0
0

0.0
33.3
0
0.0

0
4
1
0

0.0
44.4
11.1
0.0

1
2
0
0

Total

30

71.4

33.3

55.6

100

No

66.7
33.3
0.0
0.0

No

Unknown* (3)

No

3
0
1
3

ELISA
33.3
0.0
11.1
33.3

1
1
1
0

77.8

%
33.3
33.3
33.3
0
100

IHA
33.3
66.6
0.0
0.0
100

* Number of lung and unknown cases (cyst locations were not recorded in hospital registry) were too small to draw conclusions.

Cases with multiple cyst localities include those with liver and lung cysts (6 cases), liver and spleen (2 cases), and liver and kidneys (1 case).

Grades of seropositivity were determined on the basis of optical density (O.D) of sample tested and the cut off point (COP). COP was determined as the
O.D. 3SD of 6 8 wells containing negative control sample. Thus, O.D. of sample between COP- 2x COP was graded as (), 2x COP -3x COP
as (), 3x COP-4xCOP as () and 4x COP as ().

male ratio in surgical incidence of CE in Jordan at ages

above 15 years old while the reverse was observed in
younger age groups.
The sensitivity of ELISA was significantly higher than
that of IHA and the ELISA technique detected all IHA
negative cases. This is consistent with recent studies (Kaur
et al., 1999; Ortona et al., 2000) but contrasts some other
earlier finding that showed that both techniques have comparable sensitivity and are complementary to each other

(Al-Yaman et al., 1988; Craig, 1993; Moosa and AbdelHafez, 1994). Furthermore, the sensitivity of the ELISA in
the present series of cases was comparable to or higher than
that reported earlier in Jordan and elsewhere (Al-Yaman et
al., 1988; Moosa and Abdel-Hafez, 1994; Craig, 1993; Kaur
et al., 1999; Zarzosa et al., 1999). In contrast, Hira et al.,
(1990), and Sbihi et al (2001) found a higher sensitivity of
98.1% and 96.5%, respectively. This variability in sensitivity may reflect the time lapse between surgical operation

Table 2
ELISA and IHA seropositivity levels of sera of 50 CE patients from Jordan as tested at various periods of surgical intervention
Titer grade

Time lapse between surgery and serum collection

7 days1 year (24)*

5 years (14)*

15 years (12)*

No.

No.

Positivity grade

6
6
11

25.0
25.0
45.8

3
2
6

25.0
16.7
50.0

6
2
3

42.9
14.3
21.4

Total

23

95.8

11

91.7

11

78.6

Reciprocal titer
64128
200400
400

2
9
10

8.3
37.5
41.7

0
0
7

0.0
0.0
58.3

2
2
2

14.3
14.3
14.3

Total

21

87.5

58.3

42.9

No.

ELISA

IHA

* Numbers in parenthesis indicate number of cases examined.

Seropositivity grades were determined as shown under Table 1.

M.A. Nasrieh, S.K. Abdel-Hafez / Diagnostic Microbiology and Infectious Disease 48 (2004) 117123

121

Table 3
Detection of antigen B immunoreactive bands using various antigenic preparations. Serum samples of 57 surgically confirmed cases were reacted with
PAGE fractions of crude sheep hydatid fluid (CSHF), autoclaved antigen B (AAB), boiled antigen B (BAB) or homogenated protoscoleces antigen
(HPA) preparations
Detecting

Number and % of detected cases in following organs (number of cases)

Ag fraction

Liver (42)

Multiple (9)

Lung (3)*

No.

No.

CSHF
8/12
16
24

29
15
17

69.0
35.7
40.5

0
0
0

Total positive

31

73.8

AAB
8/12
16
24

30
16
17

Total positive

Unknown (3)*

Total (57)

No.

No.

No.

0.0
0.0
0.0

5
4
4

55.6
44.4
44.4

2
1
0

66.7
33.3
0.0

36
20
21

63.2
35.1
36.8

0.0

66.7

66.7

39

68.4

71.4
38.1
40.5

2
1
2

66.6
33.3
66.6

5
3
5

55.6
33.3
55.6

2
0
0

66.7
0.0
0.0

39
20
24

68.4
35.1
42.1

29

69.0

66.6

66.7

66.7

39

68.4

BAB
8/12
16
24

21
15
21

50.0
35.7
50.0

0
1
1

0.0
33.3
33.3

2
1
3

22.2
11.1
33.3

1
0
0

33.3
0.0
0.0

24
17
25

42.1
29.8
43.9

Total positive

23

54.8

33.3

33.3

33.3

28

49.1

HPA
8/12
16
24

11
0
0

26.2
0.0
0.0

0
0
0

0.0
0.0
0.0

1
0
0

11.1
0.0
0.0

1
0
0

33.3
0.0
0.0

13
0
0

22.8
0.0
0.0

Total positive

11

26.2

0.0

11.1

33.3

13

22.8

* Number of lung and unknown cases (cyst locations were not recorded in hospital registry) were too small to draw conclusions.
Cases with multiple cyst localities include those with liver and lung cysts (6 cases), liver and spleen (2 cases), and liver and kidneys (1 case).

Calculated as reactive with any of the three AgB fractions.

and serum collection. Despite the fact that antibodies to CE

can remain detectable over 1 year after surgery (Craig,
1993; Rickards and Lightowlers, 1986; Moosa and AbdelHafez, 1994; Zarzosa et al., 1999, present study), high titers
of detectable antibodies in patients after surgery have be
explained as indicative of nonsuccessful surgeries and/or
recurrence of the disease; a condition that may apply to the
present series of patients also.
The sera of CE patients with liver cysts showed the
highest seropositivity in both ELISA and IHA (92.9% and
71.4%, respectively, Table 1) which agrees with that obtained by others (Craig et al., 1986; Schantz and Gottstein,
1986). These authors indicated that the degree of antibody
responses may be related to the location of cyst where
hydatid cysts in human lungs, spleen, and kidney tend to be
associated with lower serum antibody levels. In particular,
lung cysts are the least likely to be serologically diagnosed
(Moosa and Abdel-Hafez, 1994).
Comparison between the sensitivity of the ELISA and
IHA tests using serum samples of old and recent cases

showed that the IHA test was less sensitive in detecting

older cases (Table 2). Thus, for follow up of cases after
surgery, the ELISA is far more superior to the IHA as a
diagnostic test. Indeed, only 42.9% of cases tested after five
years of surgery were IHA seropositive (Table 2).
The present investigation compared the usefulness of
various sources and methods of antigen B preparations to
specific antibodies against CE in surgically confirmed patients by the IB method. The use of heat stable antigen B
fractions (8/12, 16, and 24 KDa) as specific detecting antigens for E. granulosus has been confirmed by previous
studies (Shepherd and McManus, 1987; Al-Yaman and
Knobloch, 1989; Maddison et al., 1989; Siracusano et al.,
1991; Verastegui et al., 1992; Kharebov et al., 1997). Cross
reaction in Western blot (IB) to antigen B fractions have
been observed only with other Echinococcus species (E.
multilocularis and E. vogeli) and patients with cysticercosis
(Maddison et al., 1989; Legatt et al., 1992, Verastegui et al.,
1992; Craig, 1997; Ito et al., 1999), all of which do not exist
in Jordan.

122

M.A. Nasrieh, S.K. Abdel-Hafez / Diagnostic Microbiology and Infectious Disease 48 (2004) 117123

The similarity between seropositivity rates using CSHF

and AAB as detecting antigens is expected. CSHF contains
many host and parasite antigens including AgB fractions
while AAB contains the heat stable components of AgB
only. The sensitivity of antigen B components whether in
CSHF or AAB for detection by sera of CE patients was
within previous ranges reported earlier (56.7%96.5%)
(Maddison et al., 1989; Njeruh and Gathuma, 1989; Siracusano et al., 1991; Verastegui et al., 1992; Moosa and
Abdel-Hafez, 1994; Craig, 1997; Kharebov et al., 1997; Ito
et al., 1999; Sbihi et al., 2001). The significantly lower
sensitivity obtained by using BAB as a detecting antigen
may be a result of incomplete purification and loss of some
of the component of antigen B by boiling of CSHF for 15
minutes. Indeed, fraction 8/12 was the most affected component in BAB compared to CSHF or AAB preparation
(Table 3). HPA which is prepared by homogenization appeared to yield little detectable AgB compared to what is
present in CSHF. In all of the four antigenic preparations the
smallest fraction 8/12 KDa provided sensitivity identical or
very close to that provided by overall seropositivity of all
fractions. This confirms with observation made by Maddison et al. (1989) and Leggatt et al. (1992) who indicated that
fraction 8/12 is the most species specific fraction to E.
granulosus.
In conclusion, the ELISA is the test of choice for the
diagnosis of CE in humans and particularly in the follow up
of surgical cases using CSHF as a detecting antigen. The IB
can be used as a confirmatory test when antigen B fractions
in CSHF are detected. The use of purified Antigen B fractions does not add any advantage over CSHF in terms of the
sensitivity of the test. Only the AAB preparation was comparable in sensitivity to CSHF. However, the use of AAB in
IB diagnostic testing has the advantage of combining both
specificity and sensitivity.

Acknowledgments
This work received financial support from NIAID-NIH
(Grant No. AI-45194), European commission (EC Contract
IC18-CT98-0354), and Yarmouk University Research
Council.

References
Abdel-Hafez, S. K. & Kamhawi, S. A. (1997). Cystic echinococcosis in the
Levant Countries (Jordan, Palestinian Autonomy, Israel, Syria and
Lebanon). In F.L. Andersen, H. Ouhelli & M. Kachani. (Eds.) Compendium on Cystic Echinococcosis in Africa and Middle Eastern Countries with Special Reference to Morocco. Provo, UT: Brigham Young
University, pp. 292316.
Allan, J. C., & Craig, P. S. (1989). Coproantigens in gut tape worm
infection. Hymenolopis diminuta in rats. Parasitol Res 76, 68 73.
Al-Qaoud, K. M., Craig, P. S., & Abdel-Hafez, S. K. (2003). Retrospective
surgical incidence and case distribution of cystic echinococcosis (CE)
in Jordan between 1994 2000. Acta Trop 87, 207214.

AL-Yaman, F. M., Abdel-Hafez, S. K., Assaf, L. M., & Malkawi, F. K.

(1988). Evaluation of various serodiagnostic tests for human hydatidosis. Jap J Parasitol 37, 133138.
AL-Yaman, F. M., & Knobloch, J. (1989). Isolation and partial characterization of species-specific and cross reactive antigens of E. granulosus
cyst fluid. Mol Biochem Parasitol 37, 101108.
Anderson F. L., Chai J., & Liu F. (1993). Compendium on Cystic Echinococcosis with Special References to Xinjiang Uygur Autonomous Region. The Peoples Republic of China. Provo, Utah: Brigham Young
University 235pp.
Andersen F. L., Ouhelli H., Kachani M (Eds.) (1997). Compendium on
Cystic Echinococcosis in Africa and Middle Eastern Countries with
Special Reference to Morocco. Provo, Utah: Brigham Young University, 345pp.
Boto, W. M., Owers, K. G., & Levy, D. A. (1984). Antigens of Dirofilaria
immitis which are immunogenic in canine host: detection by immunostaining of protein blots with antibodies of occult dogs. J Immunol 133,
975980.
Bradford, M. M. (1976). A rapid and sensitive method of quantification of
microgram quantities of protein utilizing the principle of dye binding.
Anal Biochem 72, 248 254.
Craig, P. S. (1993). Immunodiagnosis of Echinococcus granulosus. In: FL
Andersen, J Chai, & F Liu (eds.). Compendium on Cystic Echinococcosis with Special Reference to Xinjiang Uygur Autonomous Region.
The Peoples Republic of China. Provo, Utah: Brigham Young University, pp, 85118.
Craig, P. S. (1997). Immunodiagnosis of Echinococcus granulosus and a
comparison of techniques for diagnosis of canine echinococcosis. In:
FL Andersen, H Ouhelli, & M Kachani (eds.). Compendium on Cystic
Echinococcosis in Africa and Middle Eastern Countries with Special
Reference to Morocco. Provo, Utah: Brigham Young University, pp,
85118.
Craig, P. S., Zeyhl, E., & Romig, T. (1986). Hydatid disease research and
control in Turkana II. The role of the immunological techniques for the
diagnosis of hydatid disease. Trans Roy Soc Trop Med Hyg 80, 183
192.
Ersfeld, K., Gasser, R. B., & Craig, P. S. (1997). The immunodiagnostic
potential of Echinococcus granulosus adult worm antigens in human
cystic echinococcosis. Parasitol Res 83, 90 92.
Gonzalez-Sapienza, G., Lorenzo, C., & Nieto, A. (2000). Improved immunodiagnosis of cystic hydatid disease by using a synthetic peptide with
higher diagnostic value than that of its parent protein, Echinococcus
granulosus antigen B. J Clin Microbiol 38, 3979 3983.
Gottstein, B., Schantz, P. M., Todorov, T., Saimot, A. G., & Jacquier, P.
(1986). An international study of the serological differential diagnosis
of human cystic and alveolar echinococcosis. Bull World Health Organ
64, 101105.
Hira, P. R., Bahr, G. M., Shweiki, H. M., & Behbihani, K. (1990). An
enzyme linked immunosorbent assay using an are 5 antigen for the
diagnosis of cystic hydatid disease. Ann Trop Med Parasitol 84, 157
162.
Ioppolo, S., Notargiacomo, S., Profumo, E., Franchi, C., Ortona, E.,
Rigano, R., & Siracusano, A. (1996). Immunological responses to
antigen B from Echinococcus granulosus cyst fluid in hydatid patients.
Parasite Immunol 18, 571578.
Irabuena, O., Nieto, A., Ferreira, A. M., Battistoni, J., & Ferragut, G.
(2000). Characterization and optimization of bovine Echinococcus
granulosus cyst fluid to be used in immunodiagnosis of hydatid disease
by ELISA. Rev Inst Med Trop Sao Paulo 42, 255262.
Ito, A., Ma, L., Schantz, P. M., et al. (1999). Differential serodiagnosis for
cystic and alveolar echinococcosis using fractions of Echinococcus
granulosus cyst fluid (antigen B) and E. multilocularis protoscolex
(Em18). Am J Trop Med Hyg. 60, 188 192.
Kaur, M., Mahajan, R. C., & Malla, N. (1999). Diagnostic accuracy of
rapid enzyme linked immunosorbent assay for diagnosis of human
hydatidosis. Indian J Med Res 110, 18 21.

M.A. Nasrieh, S.K. Abdel-Hafez / Diagnostic Microbiology and Infectious Disease 48 (2004) 117123
Kharebov, A., Nahmlas, J., & Elon, J. (1997). Cellular and humoral
immune-responses of hydatidosis patients to Echinococcus granulosus
purified antigen. Am J Trop Med Hyg 57, 619 625.
Leggatt, G. R., Yang, W., & McManus, D. P. (1992). Serological evaluation of the 12Kda subunit of antigen B in E. granulosus cyst fluid by
immunoblot analysis. Trans Roy Soc Trop Med Hyg 86, 189 192.
Lightowlers, M. W., & Gottstein, B. (1995). Echinococcosis hydatidosis:
antigens immunological and molecular diagnosis. In: RCA Thompson,
& AJ Lymbry (eds.). Echinococcus and Hydatid Disease. Wallingford,
UK: CAB International, pp 355410.
Maddison, S. E., Slemenda, S. B., Schantz, P. M., Freid, J. A., Wilson, M.,
& Tsang, V. C. W. (1989). A specific diagostic antigen of E. granulosus with an apparent molecular weight of 8KDa. Am J Trop Med Hyg
40, 377383.
McVie, A., Ersfeld, K., Rogan, M. T., & Craig, P. S. (1997). Expression
and immunological characterization of Echinococcus granulosus recombinant antigen B for IgG4 subclass detection in human cystic
echinococcosis. Acta Trop 67, 19 35.
Moosa, R. A., & Abdel-Hafez, S. K. (1994). Serodiagnosis and seroepidemiology of human unilocular hydatidosis in Jordan. Parasitol Res
80, 664 671.
Morales-Montor, J. (2002). Does host neuroendocrine system regulate the
immune response during parasitic infections? Mod Aspects Immunol 2,
110 116.
Njeruh, F. M., & Gathuma, J. M. (1989). Diagnosis of human hydatid
disease in surgically confirmed cases by the use of indirect haemagglutination test based on thermostable lipoprotein and on unfractionated hydatid cyst fluid. Ann Trop Med Parasitol 83, 229 303.
Ortona, E., Rigano, R., Margutti, P., et al. (2000). Native and recombinant
antigens in the immunodiagnosis of human cystic echinococcosis.
Parasite Immunol 22, 553559.
Poretti, D., Felleisen, E., Grimm, F., et al. (1999). Differential immunodiagnosis between cystic hydatid disease and other cross-reactive pathologies. Am J Trop Med Hyg 60, 193198.
Rickard, M. D., & Lightowlers, M. W. (1986). Immunodiagnosis of hydatid disease. In: RCA Thompson (eds.). The Biology of Echinococcus
and Hydatid disease. London: George, Allen and Unwin, pp. 217249.
Rogan, M. T., Craig, P. S., Zeyhle, E., Romig, T., Lubano, G. M., &
Liudeshan, L. (1991). Evaluation of a rapid dot-ELISA as a field test

123

for the diagnosis of cystic hydatid disease. Trans Roy Soc Trop Med
Hyg 85, 773777.
Sbihi, Y., Rmiqui, A., Rodriguez-Cabezas, M. N., Orduna, A., RodriguezTorres, A., & Osuna, A. (2001). Comparative sensitivity of six serological tests and diagnostic value of ELISA using purified antigen in
hydatidosis. J Clin Lab Anal 15, 14 18.
Schantz, P. M., Chai, J., Craig, P. S., et al. (1995). Epidemiology and
control of hydatid disease. In: R. C. A. Thompson, & A. J. Lymbry
(eds.). Echinococcus and Hydatid Disease. Wallingford, U.K.: CAB
International, pp. 233331.
Schantz, P. M., & Gottestein, B. (1986). Echinococcus (hydatidosis). In: K
F Walls, & P M Schantz (eds.). Immunoserology of parasitic diseases.
Vol I: Helminthic diseases. New York: Academic Press, pp. 69 107.
Shambesh, M. K., Craig, P. S., Gusbi, A. M., Echtuish, E. F., & Wen, H.
(1995). Immunoblot evaluation of the 100 and 130 KDa antigens in
camel hydatid cyst fluid for the serodiagnosis of human cystic echinococcosis in Libya. Trans Roy Soc Trop Med Hyg 89, 276 279.
Shepherd, J. C., & McManus, D. P. (1987). Specific and cross reactive
antigens of Echinococcus granulosus cyst fluid. Mol Biochem Parasitol
25, 143154.
Siracusano, A., Loppolo, S., Notargiacomo, S., Ortona, R., Teggi, A.,
DeRosa, F., & Vicari, G. (1991). Detection of antibodies against
Echinococcus granulosus major antigens and their subunits by immunoblotting. Trans Roy Soc Trop Med Hyg 85, 239 243.
Siracusano, A., Loppolo, S., Notargiacomo, S., et al. (1991). Detection of
antibodies against Echinococcus granulosus major antigens and their
subunits by immunoblotting. Trans Roy Soc Trop Med Hyg 85, 239
243.
Towbin, H., Staophelin, T., & Gordon, J. (1979). Electrophoretic transfer
of protein from polyacrylamide gel to nitrocellulose sheets: procedures
and some applications. Proc Natl Acad Sci USA 76, 4350 4354.
Verastegui, M., Moro, P., Guerara, A., Rodriguez, T., Miranda, E., &
Gilmon, R.H. (1992). Enzyme linked immunoelectrotransfer blot test
for diagnosis of human hydatid disease. J Clin Microbiol 30, 1557
1561.
Zarzosa, M. P., Orduna Domingo, A., Gutierrez, P., et al. (1999). Evaluation of six serological tests in diagnosis and postoperative control of
pulmonary hydatid disease patients. Diag Microbiol Infect Dis 35,
255262.