World Journal of Medical Sciences 9 (2): 113-122, 2013

ISSN 1817-3055
IDOSI Publications, 2013
DOI: 10.5829/idosi.wjms.2013.9.2.8114

Diagnostic Potential of Target Giardia lamblia Specific Antigen for

Detection of Human Giardiasis Using Coproantigen Sandwich ELISA
D. Kamel, 1A. Farid, 2E. Ali,
I. Rabia, 2M. Hendawy and 1A.M. El Amir
2

Zoology Department, Faculty of Science, Cairo University

2
Theodore Bilharz Research Institute, Giza, Egypt

Abstract: Giardiasis is endemic in all regions of the world. Giardia lamblia (G. lamblia) cysts are spread in
Egypt via the fecal-oral route, through ingestion of the cyst with contaminated food or water. The symptoms
of giardiasis vary from the asymptomatic passage of cysts to chronic diarrhea, malabsorption and weight loss.
Although microscopy has the advantage of low cost and ability to simultaneously detect other gastrointestinal
parasite, the limitation of this method is that G. lamblia cysts are small and similar to many pseudoparasites.
This study aimed to verify the advantages and disadvantages of enzyme-linked immunoassaying versus
microscopy for diagnosing G. lamblia in human. The 65 kDa glycoprotein Giardia specific antigen (GSA-65
kDa) is considered to be an antigen of interest. It is shared by cysts and trophozoites of G. Lamblia as
determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and
immunoblotting and very specific to this parasite. Sandwich ELISA was more sensitive (95.12%) than ordinary
parasitological diagnosis (78.05% for direct smear or 85.36% for MIFC method). Also, the NPV of sandwich
ELISA (95.12%) is higher than that of parasitlogical procedure (82.35% for direct smear or 87.5% for
MIFC method). On the other hand, the specificity (92.85%) and PPV (92.85%) of sandwich ELISA is lesser
than that of parasitological procedure (100% and 100%, respectively). Sandwich ELISA, using purified
anti-GSA-65 kDa IgG pAb, is recommended as a diagnostic test for Giardia spp. diagnosis more than ordinary
microscopy methods.
Key words: Giardia lamblia

Diagnosis

Coproantigen

INTRODUCTION

Sandwich ELISA

to many pseudoparasites such as yeast. Also, the

trophozoites break up rapidly in the stool, so cannot be
used to measure the severity of infection. Other limitation
is that its sensitivity is quite low because Giardia has
periodic expulsion in alternative days or various hours
of day and its quantity will be decreased when the disease
becomes chronic [4].
Direct immunofluorescence microscopy using
monoclonal antibody to detect giardia antigen offer
greater sensitivity compared to light microscopy, but they
are not available in all parasitology laboratories due to the
high cost and limited access to the required equipments.
Recently, enzyme-linked immunosorbent assay (ELISA)
has been considered as cost effective diagnostic method
which can detect small quantities of coproantigens of
parasite even in mild infections and if the live parasite
itself is absent in the fecal samples [4-6]. It can detect

The World Health Organization (WHO) ranks

diarrheal diseases as the second most common cause of
morbidity and mortality in children in the developing
world [1]. Giardiasis is a diarrheal illness infects the small
intestine and caused by a small flagellate protozoan
called G. lamblia. It is the most common nonviral
nonbacterial cause of diarrhea worldwide with a
prevalence range from 2-7% in developed countries and
increased to 20-30% in most developing countries [2].
The most trusted diagnostic test is direct examination of
feces or intestinal tissue samples for cysts or trophozoites
of parasite [3]. Although microscopy has the advantage
of low cost and ability to simultaneously detect other
gastrointestinal parasite, the limitation of this method is
that G. lamblia cysts are small and similar in appearance

Corresponding Author: I. Rabia, Theodore Bilharz Research Institute, Giza, Egypt.

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World J. Med. Sci., 9 (2): 113-122, 2013

different soluble antigens dispersed in fecal matter rather

than detecting cysts, trophozoites, or antigens on the
surfaces of these morphologic forms.
Most studies have approached the antigenic
characterization of trophozoites of Giardia strains.
Studies conducted in order to characterize the cysts have
expanded our knowledge of the antigenic composition of
the parasite and have permitted the identification of
specific antigens for the immunologic diagnosis of
giardiasis. Specific antigens present in the cysts such as
proteins with a molecular weight 66-103 kDa are
recognized by antibodies produced in rabbits [7-9]. The
65 kDa glycoprotein (GSA 65) is considered the most
efficient and important antigen for diagnosis of G.
lamblia. It is shared by cysts and trophozoites of G.
duodenalis as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis
and immunoblotting and very specific to this parasite
[9,10]. GSA 65 possesses the qualities of a diagnostically
ideal stool antigen. (i) It is specific to G. lamblia. (ii) It is
stable in the host gastrointestinal tract. (iii) It is stable in
standard laboratory fixatives. (iv) It is present in
immunologically
detectable
quantities
[11,12].
Furthermore, GSA 65 specifically occurs in Giardia and is
the major antigen in the feces of individuals with
giardiasis. The antigen is employed in the
immunodiagnosis of giardiasis and show both increase in
the sensitivity and specificity of immunoenzymatic
assays.
This Study aimed to verify the advantages and
disadvantage of ELISA versus microscopy for
diagnosing G. lamblia.

after the void volume (110 ml) had been passed. The
column elute is monitored at 280 nm. Four distinct
fractions were obtained. Giardia specific antigen (GSA-65
kDa) was collected from fraction IV, concentrated,
dialyzed and its protein content was determined by using
protein assay kit (Bio-Rad, Richmond, CA, USA) [15].
Sodium
dodecyl-sulfate
Polyacrylamide
Gel
Electrophoresis (SDS PAGE): Molecular weight was
determined by SDS PAGE according to Harlow and Lane
[16]; Myers [17] and Thaumaturgo et al. [18].
Immunization of Rabbits for Production of Polyclonal
Antibodies (pAb) According to Yinghai et al., [19]: Rabbit
anti-G. lamblia antibodies were obtained by immunization
of New Zealand white rabbits with GSA-65 kDa. The
rabbit received 1 mg of GSA-65 kDa, as the priming dose
by intramuscular (i.m) injection at four sites. Two week
later, two booster doses (1 mg / rabbit) were given at
weekly intervals.
Purification of Rabbit anti-G. lamblia Serum (IgGpAb):
Purification steps of rabbit anti-G. lamblia serum
(IgGpAb) were based on ammonium sulphate precipitation
method [20] and caprylic acid purification method [21].
At the end of the two purification procedures, protein
content was measured according to Bradford [15].
Labeling of Rabbit anti-G. lamblia Serum IgGpAb with
Horseradish Peroxidase (HRP) (Periodate Method):
According to Nakane and Kawaoi [22] and Tijssen and
Kurstak [23], 5 mg HRP (Sigma) was resuspended in 1.2 ml
distilled water; followed by the addition of 0.3 ml freshly
prepared sodium periodate and incubation at room
temperature for 20 min. HRP solution was dialyzed against
1 mM sodium acetate buffer (pH 4) at 4C with several
changes overnight. IgGpAb solution (5 mg/ml in 0.02M
carbonate buffer, pH 9.6) was prepared. The HRP was
removed from dialysis tubing and was added to 0.5 ml of
antibody solution. The mixture was incubated at room
temperature for 2 hr. 100 l sodium borohydride was
added and the solution was incubated at 4C for 2 hr. The
HRP conjugate pAb was dialyzed with several changes
against 0.01 M PBS (pH 7.2).

MATERIALS AND METHODS

Animals: 4 month old New Zealand white rabbits (~3 Kg)
from Theodor Bilharz Research Institute (TBRI). Animals
were free from G. lamblia and other parasitic infections
after examination. Animals were kept at TBRI for 5 weeks.
Preparation of Parasite Antigens: Fecal samples were
collected from heavy infected patient from the laboratory
in TBRI. G. lamblia cysts were isolated from fecal samples
according to OHandley et al. [13]. The (GSA-65 kDa) was
purified according to Sheehan and Gerald [14], in brief
Giardia lamblia cysts isolated from fecal samples were
concentrated using an Amicon 8400 ultrafiltration unit
with membrane (3000 Da cut-off). The concentrated
giardia lamblia cysts were applied to a 120 ml DEAESephadex G-25-ion exchange chromatography equilibrated
in 0.1 M Tris-HCl, pH 7. Fractions of 5 ml were collected,

Application of Rabbit anti-G. lamblia Serum IgGpAb

Study Population: This study was conducted on 41 G.
lamblia infected out patients clinic and 22 patients
infected with other parasites (E. histolytica and
Blastocyst). In addition, 20 individuals were served as
parasite free-healthy negative control.
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World J. Med. Sci., 9 (2): 113-122, 2013

Parasitological Examination: Faecal samples were

collected in clean, wide-mouthed covered containersand
examined by direct smear and merthiolate iodine
formaldehyde concentration methods (MIF).

RESULTS
Purification of G. lamblia GSA-65 kDa by DEAESephadex G-25-ion Exchange Chromatography: The
eluted antigen was represented by a single peak with
maximum OD value equal to 0.695 at fraction number
(Fig.1). The eluted protein fractions resulted from the
purification method was analyzed by 12.5% SDS-PAGE
under reducing condition and showed only one band at
65 kDa which representing GSA-65 kDa (Fig. 2).

Direct Smear: A piece of stool was taken and emulsified

in normal saline, then examined using the x10 and x40
magnification of the ordinary light microscope.
Merthiolate-iodine-formaldhyde
Concentration
Technique (MIFC): 1 gm of faecal specimen was added to
5ml MIF solution, mixed well and filtrated in other cup.
This was followed by the addition of 7ml ether. The
prepared specimen was centrifuge for 5 min at 3000 g. A
drop of mixed sediment was placed on a slide, covered
and examined under light microscope [24] (MIF solution
is a mixture of 2 solutions with ratio 4:1. Solution A
composed of 0.1% merthiolate, 36-40% formaldehyde,
glycerin and dist. H2O. Solution B composed of potassium
iodide, iodine and dist. H2O).

Estimation of Total Protein Content of G. lamblia

Antigens: The crude antigen obtained from positive G.
lamblia stool samples contains 7mg/ml of total protein as
measured by Bio-Rad Protein assay while it was 3.5mg/ml
after purified by DEAE-sephadex G-25 ion-exchange
chromatography.
Reactivity of Target Antigen by Indirect ELISA: The
antigenicity of the purified antigen was characterized by
indirect ELISA. Stool samples from G. lamblia infected
patients gave a strong reaction against GSA-65 kDa with
mean OD reading equal to 1.309 and no cross reactions
were recorded with sample of animals or patients infected
with other parasites e.g., E. histolytica and blastocyste.

Detection of G. lamblia Antigens in Patient's Stool by

Sandwich ELISA: positive stool samples with giardia or
other parasites (E. histolytica and Blastocyst) and free
non-infected samples were individually diluted 1:3 with
PBS [25,26].
The microtitration plates were coated with 100 l/well
of purified anti-G. lamblia IgGpAb, (1/100 for IgG in
carbonate buffer 0.06 M pH 9.6) and incubated overnight
at room temperature. Plates were washed 3 times with 0.1
M PBS/T (pH 7.4). The remaining sites in the wells were
blocked by 200 l/well of 0.1% BSA/PBS/T and incubated
for 2 hr at 37C and were washed trice with PBS/T. 100l
of serum samples was pipetted into each well in duplicate
and incubated for 2 hr at 37C and then wells were washed
3 times. 100l/well of peroxidase-conjugated anti-G.
lamblia IgGpAb diluted 1/50 was then added and
incubated for 1 hr at room temperature. The assay was
completed according to De Jonge et al. [25] and Qiu et al.
[26].

Reactivity and Specificity of anti-G. lamblia Serum:

IgGpAb against GSA-65 kDa rabbit blood samples were
withdrawn before each immunizing dose and tested for the
presence of specific anti-G. lamblia antibodies by indirect
ELISA. An increasing antibody level started 1 wk after the
1st booster dose (Fig. 3). Three days after the 2nd booster
dose, immune sera gave a high titer against GSA-65 kDa
with the highest optical density at 1/100 dilution.
Specificity of Anti-G. lamblia Serum IgGpAb against
GSA-65 kDa: Reactivity of anti-G. lamblia pAb against
GSA-65 kDa and other parasite antigens (E. histolytica
and Blastocyste) was determined by indirect ELISA. AntiG. lamblia IgGpAb diluted 1/100 gave a strong reactivity
to 65KDa GSA. The OD readings at 492 nm, for G.
lamblia, was 2.84 compared to 0.462 and 0.281 for E.
histolytica and Blastocyste, respectively.

Statistical Analysis: The data are presented as mean

standard deviation of mean (X SD). Correlation
coefficient (r) was used to find the relation between the
ELISA O.D and parasitological data according to
Snedecor and Cochran (1981) [27]. The data were
considered significant if P values were equal to or
less than 0.05. Statistical analysis was performed
with the aid of the SPSS computer program (version
6.0 windows).

Purification of Rabbit Anti-G. lamblia Serum IgGpAb:

The total protein content of anti-G. lamblia pAb was
measured before (crude) and after different purification
steps including ammonium sulfate precipitation method
followed by 7% caprylic acid precipitation method. It was
12.5 mg/ml, 5.9 mg/ml and dropped to 3.1 mg/ml,
respectively.
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World J. Med. Sci., 9 (2): 113-122, 2013

Fig. 1: Elute profile of 65kDa GSA on DEAE sephadex G-25

Fig. 4: 12.5% SDS-PAGE of anti-G. lamblia IgG pAb

before and after purification (stained with
coomassie blue).
Table 1: Reactivity of purified target antigen by indirect ELISA
Parasitic antigen

OD readings at 492 nm SD

G. lambila
E. histolytica
Blastocyste

(1.309 0.342)
(0.264 0.201)
(0.182 0.082)

* OD= optical density, SD= standard deviation.

Table 2: Reactivity of rabbit anti-Giardia antibodies against many parasitic
antigens by indirect ELISA (OD reading= 492 nm)
Parasitic antigen
G. lambila
E. histolytica
Blastocyste

Fig 2: SDS-PAGE of target antigen eluted from ion

exchange chromatography column

OD readings at 492 nm
2.84
0.462
0.281

* OD= optical density

Fig. 3: Reactivity of raised rabbit anti-Giardia antibodies

(diluted 1/100) against 65kDa GSA by indirect
ELISA.

Fig. 5: Shows the results of titration, from which the 1/50

g/ml concentration of the conjugate gave the
highest OD reading against 65kDa GSA

The purity of IgG after each steps of purification was

assayed by 12.5% SDS-PAGE under reducing condition.
The purified IgGpAb was represented by H-and L-chain
band at 53 and 31 kDa, respectively (Fig. 4). The pAb
appears free from other proteins.

Conjugation of Purified Rabbit anti-G. lamblia IgGpAb:

Seven mg of rabbit anti-G. lamblia IgGpAb was
conjugated with HRP. IgG antibody was assessed against
65 kDa GSA in ELISA assay. 1/50 g/ml of the conjugate
gave the highest OD reading against GSA-65 kDa (Fig. 5).
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World J. Med. Sci., 9 (2): 113-122, 2013

Table 3: Detection of G. lamblia cyst in stool samples of infected human
Positive cases

Negative cases

-------------------------------------------------------------

------------------------------------------------------

Direct smear

MIFC method

Direct smear

Healthy control (n= 20)

--

--

20

20

Giardia (n= 41)

32

35

Entameaba Histolytica (n= 17)

--

--

17

17

Blastocyst (n= 5)

--

--

Groups

MIFC method

Table 4: Key features of parasitological tests

Detection method

Sensitivity

Specificity

PPV

NPV

Direct smear

78.05 %

100 %

100 %

82.35%

MIFC method

85.36 %

100 %

100 %

87.5 %

PPV: Positive Predictive Value, NPV: Negative Predictive Value.

Table 5: Detection of GSA-65 kDa in stool samples of infected human
Positive cases

Negative cases

--------------------------------------------------

------------------------------------------------------------

Groups

No.

Healthy control (n= 20)

--

Giardia (n= 41)

39

Entameaba Histolytica (n= 17)

Blastocyst (n= 5)

X SD

No.

X SD

20

0.29 0.07

1.98 0.35

0.36 0.032

0.51 0.14

14

0.31 0.10

--

--

0.27 0.16

Study Population
Parasitological Examination: According to stool analysis
by direct smear, 32 patients from 41 patients were positive
with G. lamblia cyst while, by MIFC method 35 patients
were positive. 20 normal and 22 patients infected with
other parasites (17 E. histolytica and 5 Blastocyst) all are
negative by both methods (Table 3).
Both direct smear and MIFC methods gave 100%
specificity and positive predictive value (PPV), while the
sensitivity of MIFC method (85.36%) was higher than that
of direct smear (78.05%). Again, MIFC method recorded
high negative predictive value (NPV) (87.5%) than direct
smear (82.35%) (Table 4).

Fig. 6: Shows the results of titration, from which the

1/100 concentration of the coating gave the
highest OD reading against 65kDa GSA

Standardizationof Sandwich ELISA Used for Detection of

G. lamblia Antigen: The optimum concentration of
coating anti-G. lamblia IgGpAb displays in the highest
OD reading. It was found to be 1/100 (Fig.6).

was significantly higher than both the healthy control

group (0.29 0.07) and other parasite groups (0.31 0.10
and 0.27 0.16).
Two out of 41 G. lamblia infected samples showed
false negative results and the sensitivity of the assay was
95.12%.
All the 20 negative controls were below the cut off
value while 3 out of 22 of other parasites groups were at
the border line of the cut off value giving 92.85 %
specificity. The PPV and NPV were 92.85 % and 95.12 %,
respectively (Table 6).

Application of sandwich ELISA for detection of

G. lamblia Antigen (GSA-65 kDa) in Stool of Infected
Human: In order to measure the incidence of positivity for
G. lamblia in stool samples. Table (5) shows the results
of 65 kDa GSA detection among different studied groups.
Cut off value for positivity was 0.428 as mean2 SD.
The OD values of G. lamblia infected group (1.98 0.35)
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World J. Med. Sci., 9 (2): 113-122, 2013

Table 6: Key features of ELISA test
Detection method

Sensitivity

Specificity

PPV

NPV

ELISA

95.12 %

92.85 %

92.85 %

95.12 %

Table 7: Summarizes the sensitivity, specificity, PPV and NPV of parasitological and sandwich ELISA results used for detection of GSA-65 kDa in stool
samples of human
Detection method
Parasitological

Sensitivity

Specificity

PPV

NPV

Direct smear

78.05 %

100 %

100 %

82.35%

MIFC method

85.36 %

100 %

100 %

87.5 %

ELISA

95.12 %

92.85 %

92.85 %

95.12 %

DISCUSSION
One of the most common intestinal protozoan
parasites is G. lamblia; about 200 million people in Asia,
Africa and Latin America have symptomatic infections
[28], with 280 million infections per year [29]. Since it has
a fecal-oral transmission cycle and is contracted by
ingestion of contaminated water or food or by person-toperson contact, the highest disease burden is found in
areas where sanitary conditions are poor. The highest
rates of infection are therefore encountered in developing
countries 10-30% in young children [30]. Because of the
impact on socioeconomic development, as well as on
domestic animals such as cattle and sheep especially in
developing countries, Giardia is included in the
Neglected Disease Initiative of the World Health
Organization [29-31].
The diagnosis of giardiasis is frequently based
on microscopic examination of stool samples by
visualizing the organism, either the trophozoites or the
cysts [30]. Diagnosis via microscopical examination of a
single stool specimen has a low sensitivity and may
therefore miss up to 50% of Giardia infections [30,33,34],
particularly if there is low parasite density, insufficient
microscopic quality, intermittent excretion of cysts or the
probability of parasite hiding by bile pigments [29,35].
Microscopic examination of three consecutive stool
specimens is required to reach a sensitivity of over 90%
[30, 34].
Given these difficulties the development of
sensitive, cost-effective and rapid diagnostic methods
is of the most importance [30]. ELISA for detection
of the specific antigens in stools is a rapid, sensitive
and economic method to confirm infection and
coproantigens of that parasite which could be traced
and diagnosed even if the live parasite is absent in the
fecal samples [4,6].
In the present study, stool samples were collected in
order to purify and analyze GSA-65 kDa. It was purified
118

from stool samples by DEAE sephadex G-25 ion exchange

chromatography in which 65 kDa GSA appeared as a
single band at 65 kDa by reducing SDS-PAGE. Then, the
total protein content of crude G. lamblia in positive
giardia stool sample was 7 mg/ml, while it was 3.5 mg/ml
after purification with DEAE-sephadex G-25 ionexchange
chromatography.
Recently, Barazesh et al. [36] introduced a rapid,
simple and economical method to purify G. lamblia cysts
from fecal samples of the patients suffering from
giardiasis. They introduced and run a modified method
that in fact is a mixture of various purification methods like
one-and two-phase sucrose gradient isolation, percollsucrose gradient isolation and a modified two-phase
method run by 0.85 and 1.5 M sucrose with some
changes.
In our study, anti-G. lamblia IgGpAb was prepared
by immunization schedule of rabbits with GSA-65 kDa
according to Tendler et al. [37]. Two purification steps
were processed, ammonium sulfate and 7% caprylic acid
precipitation methods. The purity of IgGpAb was assayed
by 12% SDS-PAGE under reducing condition. The
purified anti-G. lamblia IgGpAb was represented by Hand L-chain bands at 53 and 31 kDa, respectively;
indicating that the pAb is free from other proteins. The
protein content of pAb was 3.1 mg/ml IgG refering to the
crude one (12.5 mg/ml). These yields were reasonable in
comparison with the other purified immunoglobulin from
any biological fluid following similar purification
procedures [38,39].
Then, the reactivity of the purified anti-G. lamblia
IgGpAb against GSA-65 kDa and other parasite antigens
(E. histolytica and Blastocyste) was determined. The
purified HRP-labeled IgGpAb was used for the detection
of 65 kDa GSA in stool (coproantigen) of infected patients
by sandwich ELISA. The optimization of various reagents
used in sandwish ELISA was done. The optimum dilution
of purified IgGpAb as a coating layer was 1/100 where as
a peroxidase conjugated layer was 1/50.

World J. Med. Sci., 9 (2): 113-122, 2013

This was in agreement with Duque-Beltrn et al.

[40] who purified Giardia cysts from human fecal
samples by sucrose and percoll gradients. Gerbils
(Merionesunguiculatus) were infected to obtain
trophozoites. Rabbits were inoculated with either cyst or
trophozoite antigens of 14 Colombian Giardia isolate to
develop antibodies against the respective stages. The
anti-G. lamblia IgGpAb was purified by sequential
caprylic acid and ammonium sulfate precipitation. A
portion of these pAb was linked to alkaline phosphatase
(conjugate). The optimal concentration of pAb for antigen
capture was 40 g/ml and the optimal conjugate dilution
was 1:100.
Later on, Barazesh et al. [4] purified giardia parasite
from faeces of patients and injected into rabbit to extract
serum pAb. Then the produced rabbit anti-G. lamblia
IgGpAb was purified by ion-exchange chromatography
and its identity was confirmed by SDS-PAGE. The purified
pAb was conjugated by periodate method. This pAb was
used to design direct and indirect ELISA kits to measure
conjugation titer. In both ELISA methods, ODs were 1 by
using of 1/4000 dil. of conjugation.
We compared between the ordinary parasitological
examination methods and sandwish ELISA in the
diagnosis of giardiasis. The study was conducted on 41
G. lamblia infected patients, 22 other parasites infected
patients and 20 healthy controls. According to
parasitological examination by MIFC method, 35 from 41
patients were positive with G. lamblia cyst with a
sensitivity of 85.36%, a specificity of 100%, a PPV of 100%
and a NPV of 87.5 %. While, according to sandwich
ELISA, 39 patients were positive giving higher sensitivity
than parasitological examination. It had a sensitivity of
95.12%, a specificity of 92.85%, a PPV of 92.85% and a
NPV of 95.12%.
Do ruman et al. [41] evaluate the value of the direct
fluorescent antibody (DFA) techniques reported to have
high sensitivity and specificity and the ELISA test used
to determine antigens in stool samples in the routine
diagnosis of G. intestinalis. When 44 stool samples in
which G. intestinalis cysts and/or trophozoites had been
seen during native Lugol examination were investigated,
positivity detected with the trichrome staining,
monoclonal ELISA and DFA methods were found to be 37
(84.0%), 39 (88.6%) and 35 (79.5%), respectively. DFA
detected Crytosporidiumparvum cysts in addition to G.
intestinalis in one sample. Twenty-seven (61.4%) of the
samples were positive with all three methods. When
compared with the DFA method, the ELISA method had
a sensitivity of 88.6%, a specificity of 88.8%, a PPV of
79.5% and a NPV of 20.0% while the trichrome staining

method had a sensitivity of 85.7%, a specificity of 77.8%,

a PPV of 81.1% and a NPV of 22.2%. There was no
statistically significant difference between the DFA and
ELISA tests and between the DFA test and the trichrome
staining method for diagnosing G. intestinalis.
Akav et al. [42] compared the results of a EIA test (to
detect 65 kDa GSA) for the diagnosis of giardiasis with
the microscopic examination. 280 patients with diarrhea
and 60 controls were included in the study. 78 of 280
samples were positive by microscopy, whereas only 74 of
280 samples gave positive reaction by the EIA assay. EIA
sensitivity exceeded 92% and its specificity was 99%.
Al-Saeed and Issa [43] used ELISA in the detection
of G. lamblia antigen in stool specimens. Of 84 stool
samples, 42 were positive and 42 were negative for
Giardia spp. stages by using microscopic stool
examination. Samples that were positive by microscopy
were positive by ELISA test and 13 samples that were
negative by microscopy were positive by ELISA test.
The sensitivity of the ELISA test was therefore 76.4% and
the specifcity was 100%.
Gutirrez-Cisneros et al. [44] assessed and compared
the performance of two immunochromatographic tests for
the simultaneous detection of G. duodenalis and
Cryptosporidium spp. in faeces. 254 faeces samples were
tested using the two immunochromatography strips
Cryto-Giardia (CerTestBiotec) and Stick Crypto-Giardia
(Operon). In the diagnosis of G. duodenalis, the
sensitivity and specificity of the kits were 97% and 100%,
respectively for the CerTest; and 97% and 95% for
Operon. In the diagnosis of Cryptosporidium spp.CerTest
strip rendering a sensitivity of 100%, compared to with a
sensitivity of 92% using Operon.
Jelinek and Neifer [30] evaluated two EIAs assays to
detect antigens in stool specimens, Ridascreen Giardia
and G. lamblia Serazym Giardia. Every specimen was
examined by a conventional microscopic examination
(CME), PCR and tested by both EIA kits. When
microscopy was used as the reference standard, the
Ridascreen Giardia showed a sensitivity of 72.9% and a
specificity of 100%. Serazym Giardia had a sensitivity of
93.8% and a specificity of 100%.
In conclusion, sandwich ELISA was more sensitive
(95.12%) than ordinary parasitological diagnosis
procedure. Also, the NPV of sandwich ELISA (95.12%) is
higher than that of parasitlogical procedure. On the other
hand, the specificity and PPV of sandwich ELISA is lesser
than that of parasitological procedure. Moreover
sandwich ELISA, using purified anti-65 kDa GSA IgGpAb,
is recommended as a diagnostic test for Giardia spp.
diagnosis more than ordinary microscopy methods.
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World J. Med. Sci., 9 (2): 113-122, 2013

REFERENCES
1.

2.

3.

4.

5.

6.

7.

8.

9.

10. Vidal,
A.M.B. and W.R. Catapani, 2005.
Enzyme-linked immunosorbent assay (ELISA)
immunoassaying versus microscopy: advantages
and drawbacks for diagnosing giardiasis. Sao Paulo
Med. J., 123: 282-285.DOI:10.1590/S151631802005000600006. PMID:16444388.
11. Rosoff, J.D. and H.H. Stibbs, 1986. Physical
and chemical characterization of a Giardia
lamblia-specific
antigen
useful
in
the
coprodiagnosis of giardiasis. J. Clin. Microbiol.,
24: 1079-1083.PMID:3536998.
12. Rosoff, J.D., C.A. Sanders, S.S. Sonnad, P.R. De lay,
W.K. Hadley, F.F. Vincenzi, D.M. Yajko and
P.D. O'Hanley', 1989. Stool Diagnosis of Giardiasis
Using a Commercially Available Enzyme
Immunoassay To Detect Giardia-Specific Antigen
65(GSA 65). J. Clin. Microbiol., 27: 1997-2002.PMID:
2674196. PMCID: PMC267726.
13. OHandley, R.M., C. Cockwill, T.A. McAllister,
M. Jelinski, D.W. Morck and M.E. Olson, 1999.
Duration of naturally acquired giardiosis and
cryptosporidiosis in dairy calves and their
association with diarrhea. J. Am. Vet. Med. Assoc.,
214: 391-396. PMID:10023403.
14. Sheehan, D. and R.F. Gerald, 1996. Ion-exchange
chromatography. Meth. Mol. Biol., 59: 145-150.
15. Bradford, M.M., 1976. A rapid and sensitive
method for the quantitation of microgram
quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem., 72: 245-254.
PMID: 942051.
16. Harlow,
E.
and
D.
Lane,
1988.
Antibodies: A laboratory manual. 1st Edn. New York:
Cold Spring Harbor Laboratory Press, ISBN-10:
0879693142 | ISBN-13: 978-0879693145.
17. Myers, R.L., 1995. Animal and human
immunoglobulins. In: Immunology a Laboratory
manual. 2ndedn. Academic Press, New York,
McGraw-Hill Science/Engineering/Math. ISBN-10:
0697113132. ISBN-13: 978-0697113139.
18. Thaumaturgo, N., M.M. Vilar, R. Edelenyi and
M. Tendler, 2002. Characterization of Sm14 related
components in different helminthes by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis
and western blotting analysis. Mem Inst Oswaldo
Cruz, Rio de Janeiro, 97: 115-116.DOI:
org/10.1590/S0074-02762002000900024.
PMID:12426606.

W.H.O., 2005. Making every mother and child

count, World Health Report,World Health
Organization, Geneva, Switzerland 2005.
Yoder,
J.S.
and
M.J.
Beach, 2007.
Giardiasis surveillance-United States, 2003-2005.
MMWR Surveill Summ.,56: 11-18. Available
from:http://www.cdc.gov/mmwr/preview/mmwrhtml
/ss5607a2.htm. [Accessed 23 sep 2013].
Wilson, J.M. and F.C. Hankenson, 2010.
Evaluation of an Inhouse Rapid ELISA Test for
Detection of Giardia in Domestic Sheep (Ovis aries).
J. of the American Association for Laboratory
AnimalScience, 49:809-913.PMID: 21205445. PMCID:
PMC2994047.
Barazesh, A., J. Majidi, E. Fallah, R. Jamali,
J. Abdolalizade and R. Gholikhani, 2010.
Designing of enzyme linked immunosorbent
assay (ELISA) kit for diagnosis copro-antigens of
Giardia Lamblia. Afr. J. Biotechnol., 9: 5025-5027.DOI:
10.5897/AJB10.024.
Goldin, A.J., A.R.T. Wener, X.
Aguilera,
I. Zulantay, D.C. Warhurst and M.A. Miles,
1990. Efficient diagnosis of giardiasis among
nursery and primary school children in Santiago,
Chile by capture ELISA for the detection of
fecal Giardia antigens. Am. J. Trop. Med. Hyg.
42: 538-545.PMID:2372087.
Aldeen, W.E., D. Hale, A.J. Robinson and K. Carroll,
1995. Evaluation of a commercially available ELISA
assay for detection of Giardia lamblia in fecal
specimens. Diagn. Microbiol. Infect. Dis.,
21: 77-79.PMID:7628196.
Gillin, F.D., D.S. Reiner, M.J. Gault, H. Douglas,
S. Das, A. Wunderlich and J.F. Sauch, 1987.
Encystation and expression of cyst antigens
by
Giardia
lamblia
in vitro. Science,
235: 1040-1043.PMID: 3547646.
Reiner, D.S., M. Mccaffery and F.D. Gillin, 1990.
Sorting of cyst wall proteins to a regulated secretory
pathway during differentiation of the primitive
eukaryote, Giardia lamblia. Europ. J. Cell Biol.,
53: 142-153. PMID:2076701.
Guimares, S., M.I.L. Sogayar and M.F. Franco, 1999.
Giardia duodenalis: inter-strain variability of proteins,
antigens, proteases, isoenzymes and nucleic acids.
Rev. Inst. Med. Trop. So Paulo., 41: 45-58.
PMID:10436670.
120

World J. Med. Sci., 9 (2): 113-122, 2013

19. Yuzhi, H., X. Yazhong and F. Wei, 2007.

Preparation and Application of Polyclonal
Antibody against a Recombinant Laccase.
Cellular
and
Molecular
Immunology,
4(4): 315-317.
20. Nowotny, A., 1979. Basic exercises in
immunochemistry.
2nd
rev. Springer-verlag,
Berlin, New York, pp: 232-234.ISBN: 978-3-54009453-1.
21. Mckinney, M.M. and A. Parkinson, 1987. A simple,
non-chromatographic
procedure
to
purify
immunoglobulins from ascites fluid. J. Immunol.
Meth., 96: 271-278.PMID:3805742.
22. Nakane, P.K. and A. Kawaoi, 1974. peroxidaselabeled antibody. A new method of conjugation. J.
Histochem. Cytochem., 22: 1084-1091.DOI:
10.1177/22.12.1084. PMID:4443551.
23. Tijssen, P. and P. Kurstak, 1984. Highly efficient and
simple methods for the preparation of peroxidase and
active peroxidase-antibody conjugate for enzyme
immunoassays. Anal. Biochem., 136: 451-457.
PMID: 6372541.
24. Blagg, W., N.S. Mansour and G.L. Khalaf, 1955.
A new concentration technique for the
demonstration of protozoa and helminthic eggs in
feces. Am. J. Trop. Med. Hyg., 4: 23-28.
25. De Jonge, N., P.G. Kremsner, F.W. Krijger,
G. Schommer, Y.E. Fffliel, D. Kornelis,
R.J.M. Van Zeg, G.J. Van Dam, H. Feldmeier
and A.M. Deelder, 1990. Detection of the
schistosome circulating cathodic antigen by enzyme
immunoassay using biotinylated monoclonal
antibodies. Trans. R. Sco. Trop. Med. Hyg., 84: 815818.PMID:2128984.
26. Qiu, L.S.,
Z.
Feng,
Y.H.
Zhang and
H. Li, 2000. Circulating antigen detection in
schistosomiasis japonica patients by sandwich
ELISA using polyclonal and monoclonal
antibodies. Zhongguo Ji Sheng Chong Xue Yu
Ji Sheng Chong Bing Za Zhi, 18: 92-93.PMID:
12567723.
27. Snedecor, G.W. and W.G. Cochran, 1981.
Statistical methods (8th edn), Lowa State University
Press, Lowa, USA, pp: 83.
28. Feng, Y. and L. Xiao, 2011. Zoonotic Potential and
Molecular Epidemiology of Giardia Species and
Giardiasis. Clinical
Microbiology
Reviews,
24: 110-140.DOI: 10.1128/CMR.00033-10.

29. El-Nahas, H.A., D.A. Salem, A.A. El-Henawy,

H.I. El-Nimr,
H.A.
Abdel-Ghaffar
and
A.M. El-Meadawy, 2013. Giardia Diagnostic
Methods in Human Fecal Samples: A Comparative
Study Cytometry Part B (Clinical Cytometry), 84: 4449. Published on line 18 October 2012 in Wiley
Online
Library
(wileyonlinelibrary.com).
DOI:10.1002/cyto. b.21048.
30. Jelinek, T. and S. Neifer, 2013. Detection of
stool samples: a comparison of two Giardia lamblia
enzyme-linked immunosorbent assays. DOI:
10.12688/ f1000research.2-39.v1.
31. Roxstrom-Lindquist, K., D. Palm, D. Reiner,
E.
Ringqvist
and
S.G. Svard, 2006.
Giardia immunity-an update. Trends in Parasitology,
22: 26-30.PMID:16303332.
32. Geurden, T., J. Vercruysse and E. Claerebout,
2010. Is Giardia a significant pathogen in
production animals? Exp. Parasitol., 124:98-106.DOI:
10.1016/j.exppara.2009.03.001. PMID:19285075.
33. Schunk, M., T. Jelinek, K. Wetzel and
H. Nothdurft, 2001. Detection of Giardia lamblia
and Entamoeba histolytica in stool samples by
two enzyme immunoassays. Eur J. Clin. Microbiol.
Infect. Dis., 20: 389-391.DOI:10.1007/PL00011279.
PMID:11476438
34. Weitzel, T., S. Dittrich, I. Mhl, E. Adusu and
T. Jelinek, 2006. Evaluation of seven commercial
antigen detection tests for Giardia and
Cryptosporidium in stool samples. Clin. Microbiol.
Infect., 12: 656-659.PMID: 16774562.
35. Gharavi, M.J., S.h. Fallahi and B. Qara-gozlou, 2005.
Evaluation of Giardia detection by routine parasitical
assays and antigen
detection
techniques.
th
In: 5 National Iranian Congress of Parasitology,
Tehran, Iran, pp: 346-349.
36. Barazesh, A., J. Majidi, E.
Fallah and
R. Gholikhani, 2011. Introducing a simple and
economical method to purify Giardia lamblia
cysts. Afr. J. Biotechnol., 10: 8498-8501.DOI:
10.5897/AJB11.391.
37. Tendler, M., M.S.S. Almeida, R.M. Pinto, D. Noronha
and N. Katz, 1991. Schistosoma mansoni-New
Zealand rabbit model: Resistance induced by
infection followed by active immunization with
protective antigen. J. Parasitol., 77: 138141.DOI:10.1590/S0074-02761987000800043.
PMID:1899450.
121

World J. Med. Sci., 9 (2): 113-122, 2013

38. Bride, P., P. Jones, D. Allen, W. Donachie, J. Huntley,

I. Mcconnell and J. Hopkins, 1995. Analysis of
the expression and secretion of isotypes of
sheep B cell immunoglobulins with a panel of
isotype-specific monoclonal antibodies. Res. Vet.
Sci., 59: 189-194.PMID:8588089.
39. Yang, Y.B. and K. Harrison, 1996. Influence of
column type and chromatographic conditions
on
the ion-exchange chromatography of
immunoglobulins. Chromatography, 743: 171-180.
PMID: 8817880.
40. Duque-Beltrn,
S.,
R.S.
Nicholls-Orejuela,
A.
Arvalo-Jamaica,
R.
Guerrero-Lozano,
S. Montenegro and M.A. James, 2002. Detection of
Giardia duodenalis Antigen in Human Fecal Eluates
by Enzyme-linked Immunosorbent Assay Using
Polyclonal Antibodies. Mem. Inst. Oswaldo. Cruz.,
Rio de Janeiro, 97: 1165-1168.Available from:
http://dx.doi.org/10.1590/S0074-02762002000800018.
[Accessed 23 sep 2013].

122

41. Do ruman Al. F., S. Ku timur, T. Ozekinci, N.

Balaban and M.N. Ilhan, 2006. The use of enzyme
linked immunosorbent assay (ELISA) and direct
fluorescent antibody (DFA) methods for diagnosis
of Giardia
intestinalis.
Turkiye
Parazitol
Derg., 30: 275-278.PMID:17309026.
42. Akav, H., S. Kustimur and U. Beyazova, 2009.
Diagnosis of Giardia infection by detection of
specific antigen. Haseki Tip Bulteni, 47: 13-16.
43. Al-Saeed, A.T. and S.H. Issa, 2010. Detection of
Giardia lamblia antigen in stool specimens using
enzyme-linked immunosorbent assay. Journal, Health
Mediterranean Eastern, 16: 362-364.PMID:20795416.
44. Gutirrez-Cisneros, M.J.,
R.
Martnez-Ruiz,
M. Subirats, F.J. Merino, R. Milln and I. Fuentes,
2011. Assessment of two commercially available
immunochromatographic assays for a rapid
diagnosis
of
Giardia
duodenalis
and
Cryptosporidium spp. in human fecal specimens.
Enferm. Infec. Microbiol. Clin., 3: 201-203.DOI:
10.1016/j.eimc.2010.09.005. PMID: 21342732.