Re-evaluation of commercially available enzyme-linked immunosorbent assay for the detection of Giardia lamblia and Cryptosporidium spp from stool specimens.

Abstract

This study aimed to detect Giardia lamblia and Cryptosporidium spp infection from stool specimens. A total of 345 stool specimens were examined by microscopy (both direct smear and formalin concentration) and EIA techniques (ProSpecT Microplate Assay) for G. lamblia and Cryptosporidium spp. Of 73 tests positive for G. lamblia, 41(56.2%) were positive by microscopy, and 71(97.3%) were positive by EIA. Of 16 tests positive for Cryptosporidium spp, 5 (31.3%) were positive by microscopy, and 16(100%) were positive by EIA technique. The results demonstrate that this EIA method is quick, simple, and more sensitive than the microscopy method and should be used for the detection of G. lamblia and Cryptosporidium spp where the prevalence of these protozoan parasites is a public health problem.

Full text

Page 1

26 Vol 36 (suppl 4) 2005
RE-EVALUATION OF COMMERCIALLY AVAILABLE ENZYME-
LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF
GIARDIA LAMBLIA AND CRYPTOSPORIDIUM SPP
FROM STOOL SPECIMENS
Apichai Srijan, Boonchai Wongstitwilairoong, Chittima Pitarangsi, Oralak Serichantalergs, CD Fukuda,
Ladaporn Bodhidatta and CJ Mason
Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), US Army
Medical Component, Bangkok, Thailand
Abstract. This study aimed to detect Giardia lamblia and Cryptosporidium spp infection from stool specimens.
A total of 345 stool specimens were examined by microscopy (both direct smear and formalin concentration) and
EIA techniques (ProSpecT Microplate Assay) for G. lamblia and Cryptosporidium spp. Of 73 tests positive for
G. lamblia, 41(56.2%) were positive by microscopy, and 71(97.3%) were positive by EIA. Of 16 tests positive
for Cryptosporidium spp, 5 (31.3%) were positive by microscopy, and 16(100%) were positive by EIA technique.
The results demonstrate that this EIA method is quick, simple, and more sensitive than the microscopy method
and should be used for the detection of G. lamblia and Cryptosporidium spp where the prevalence of these
protozoan parasites is a public health problem.
often leads to persistent infection or death in
immunocompromized patients. Asymtomatic infection
may also occur (Dubey et al, 1990; McDonald, 1996).
The examination of stool specimens for detection
of intestinal parasites in our laboratory involves six
different techniques of microscopic analysis: (1) direct
saline and (2) iodine wet mounts of fresh stool, a (3)
DMSO (Dimethylsulfoxide)-mAFB stained smear of
fresh stool, an (4) iodine wet mount and (5) DMSO-
mAFB stained smear of a sample concentrated by the
formalin-ethylacetate sedimentation concentration
technique from a 10% buffered formalin preserved
stool, and a (6) trichrome stained smear from a Zinc
sulfate polyvinyl alcohol (Zinc PVA) preserved stool
(Bronsdon et al, 1984; NCCLS, 1997; Kellogg and
Elder, 1999; Garcia, 2001). In patients with either
giardiasis or cryptosporidiosis, the use of such routine
diagnostic methods may be insufficient to detect these
organisms. Additionally, diagnosis of these infections
by microscopic examination can be time-consuming
and expensive. Recent studies have found enzyme-
linked immunosorbent assays (ELISA) to be sensitive,
cost-effective, simple and a rapid method for the
detection of G. lamblia and Cryptosporidium in stool
specimens (Nash et al, 1987; Addiss et al, 1991; Garcia
and Shimizu, 1997; Aldeen et al, 1998). The assays
employed in this evaluation are manufactured by
Alexon-Trend (now Remel) and are currently available
(Remel, Inc, 12076 Santa Fe Drive, Lenexa, KS
66215). ProSpecT Giardia and Cryptosporidium
Microplate assays are solid phase immunoassays
for the detection of Giardia (GSA 65) and Crypto-
sporidium (CSA) Specific Antigens in stool specimens.
Correspondence: Apichai Srijan, Department of Enteric
Diseases, Armed Forces Research Institute of Medical
Sciences (AFRIMS), US Army Medical Component,
315/6 Rajavithi Road, Phyathai, Bangkok 10400,
Thailand.
Tel: 66 (0) 2644-4888 Ext 2580-1; Fax: 66 (0) 2644-4980
E-mail: apichais@afrims.org
INTRODUCTION
Diarrheal disease is a major cause of childhood
illness in rural areas world-wide. Giardia lamblia and
Cryptosporidium are enteric protozoan parasites that
take up residence in the small intestine, causing
giardiasis and cryptosporidiosis, respectively.
Giardiasis occurs world-wide, but is more prevalent
in warmer climates. It is also more common in children
than in adults. After ingestion, the cysts excyst in the
duodenum thereby releasing trophozoites that rapidly
multiply and colonize the microvillous surface of the
jejunum. This process can lead to endothelial
dysfunction, malabsorbtion of essential nutrients,
nausea, diarrhea, abdominal distension, cramps, weight
loss, flatulence, anemia and general weakness lasting
from a few weeks to several months. In the young this
can lead to morbidity and even death. Chronic infection
may also be asymptomatic (Black et al, 1977; Wolfe,
1979; Garcia, 2001). Cryptosporidiosis is also
recognized as a significant human disease in world-
wide distribution. Transmission of Cryptosporidium
is direct, by either the fecal-oral route or ingestion of
water contaminated with infective oocysts. Illness is
characterized by acute, severe diarrheal illness, and

Page 2

Vol 36 (suppl 4) 200527
RE-EVALUATION OF ELISA FOR DETECTION OF G. LAMBLIA AND CRYPTOSPORIDIUM
These antigens are produced by the organisms within
the host intestinal tract and can be detected in stool
specimens with or without the visible presence of cysts,
trophozoites or oocysts. In this study the above assays
were used and re-evaluated to determine the true
prevalence of G. lamblia and Cryptosporidium spp in
the study area.
MATERIALS AND METHODS
After informed consents were obtained, stool
specimens were collected from children 3 months to 5
years of age from the Sangkhla Buri district in the west
of Thailand along the Thai-Myanmar border. All stool
specimens were processed by preparing a 1:3
suspension of stool and preservative, namely 10%
buffered formalin and Zinc PVA (Kellogg and Elder,
1999). Saline and iodine wet mounts of fresh stools
were also prepared. The slides of fresh stool smears
were fixed in absolute methanol for 10 minutes and
air dried before performing DMSO-mAFB staining
(Bronsdon et al, 1984 ) for detecting Cryptosporidium
and Cyclospora oocysts. An iodine wet mount and
DMSO-mAFB smear were prepared from the formalin
preserved specimens after performing a formalin-
ethylacetate sedimentation concentration. Trichrome
staining was performed on the stool specimens
preserved in Zinc PVA (Bronsdon et al, 1984; NCCLS,
1997; Kellogg and Elder, 1999; Garcia, 2001).
Part of each specimen was kept in a cryogenic vial
at -20˚C for weekly shipment to AFRIMS in a
box on dry ice. There it was tested for G. lamblia
and Cryptosporidium spp antigens by enzyme
immunoassay (EIA: ProSpecT Microplate Assay,
Alexon-Trend 14000 Unity St. NW, Ramsey, MN
55303). Positive ProSpecT Giardia/Cryptosporidium
Microplate Assay results were followed-up by
performing individual assays of ProSpecT Giardia
Microplate Assay and ProSpecT Cryptosporidium
Microplate Assay. EIA results were read both visually,
according to the manufacturer’s guidelines (0 to 4+),
and spectrophotometrically via optical density (OD)
readings.
RESULTS
From October 2001 through October 2002, a total
of 345 stool specimens were examined by microscopy
as described above and ProSpecT Giardia/
Cryptosporidium Microplate Assay. Of 84 positive
results by ProSpecT Giardia/Cryptosporidium
Microplate Assay, subsequently, 71 tested positive by
ProSpecT Giardia Microplate Assay and 16 were
positive by ProSpecT Cryptosporidium Microplate
Assay. Upon further analysis of the original 84
positives, 9 stool samples were positive by both
ProSpecT Giardia and Cryptosporidium Microplate
Assays and 6 were negative when tested by the
individual assays (Table 1).
Of 73 tests positive for G. lamblia, 41 (56.2%)
were microscopy positive, and 71 (97.3%)
were positive by EIA. Of 16 tests positive for
Cryptosporidium spp, 5 (31.3%) were microscopy
positive, and 16 (100%) were positive by EIA
technique. The sensitivity of EIA ProSpecT Giardia/
Cryptosporidium, Giardia, and Cryptosporidium
Microplate Assays were 95.5, 94.9, and 100%,
respectively (Table 2).
Visual readings and OD results showed good
correlation when comparing positive results. They
yielded the same results, except one specimen, in which
the EIA Giardia Assay was negative by visual reading
(0), but the OD yielded a positive result (OD = 0.081).
All negative specimens had OD readings equal to or
lower than 0.05 (Table 3). Microscopic examination
also revealed a number of co-infections: 13 (3.8%)
Ascaris lumbricoides, 1 (0.3%) Trichuris trichiura, 2
(0.6%) Blastocystis hominis, 4 (1.2%) Trichomonas
Table 1
Results of EIA ProSpecT Giardia/Cryptosporidium, Giardia, and Cryptosporidium Microplate Assays.
EIA Microplate Assays No. of stool specimens
Giardia/Cryptosporidium positive results
Giardia positive results alone
Cryptosporidium positive results alone
Both Giardia and Cryptosporidium positive results
Both Giardia and Cryptosporidium negative results
84
62
7
9
6

Page 3

SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH
28Vol 36 (suppl 4) 2005
hominis, and 6 (1.7%) Cyclospora spp. These
organisms showed no evidence of cross reactivity with
Giardia lamblia or Cryptosporidium spp by EIA.
DISCUSSION
By virtue of its rural location, Sangkhla Buri is
characterized by a high incidence of both G. lamblia
and Cryptosporidium spp, 18.4 and 3.4%, respectively
(Boonchai et al, 2002, unpublished data). The endemic
nature of these intestinal parasites and their ability to
be transmitted person-to-person (Black et al, 1977;
Garcia, 2001) calls for reliable diagnostic methods.
Accurate and rapid diagnosis can lead to imple-
mentation of preventative measures and - in cases of
giardiasis - treatment to limit the spread of disease.
Choices for diagnostic testing are ever increasing.
Historically, a diagnosis of giardiasis relied on
detection of cysts and/or trophozoites in the stool by a
highly trained microscopist, while a diagnosis of
cryptosporidiosis relied on detection of small oocysts,
again, by microscopic examination. These examina-
tions are labor-intensive, subjective, require a well-
trained microscopist who sees repeated positives,
require consistency in staining, and rely on an infected
person shedding the cysts, trophozoites, or oocysts,
an event which is known to be intermittent.
For G. lamblia detection, advances in laboratory
techniques include DFA, CIE, and ELISA. At least
one study comparing two of these methods against the
“gold standard”-microscopy-found DFA to be no more
sensitive than microscopy (Aldeen et al, 1998).
Detecting Cryptosporidium is being performed by an
array of stains: auramine, DMSO-mAFB (Bronsdon,
1984), DFA, and ELISA. The drawback of DFA is its
high cost. In contrast, EIA techniques to detect these
two pathogens are recognized as rapid, easy to use,
highly sensitive and specific, less labor-intensive and
subjective than microscopic methods, and less
dependent on expensive equipment (Dagan et al, 1995;
Parisi and Tierno, 1995).
Our experience with ProSpecT Microtiter Assays
confirmed earlier reports citing their ease of use and
increased sensitivity when compared to “gold
standard” methods (Dagan et al, 1995; Parisi and
Tierno, 1995). In contrast to microscopy, ELISA
Table 2
Results and sensitivity of EIA and microscopic examination for G.lamblia and Cryptosporidium in stool
specimens.
Result (no.) of microscopic examination and EIASensitivitya
of EIA vs
microscopy
EIA Microplate Assays
Specimens (n= 345)
M+ E+M+ E-
M- E+
M- E-
Giardia/Cryptosporidium
Giardia
Cryptosporidium
42
37
5
2
2
0
42
34
11
259
272
329
42/44 (95.5)
37/39 (94.9)
5/5 (100)
M: microscopic examination, E: EIA Microplate Assays
a Values are number of EIA positive /number of microscopic positive tests (%)
Table 3
Visual reading and OD of positive results by EIA.
Giardia/CryptosporidiumGiardia Cryptosporidium
Visual ODVisual OD VisualOD
No.of positive
Mean
SD
Min
Max
84
3
84 70
3
7116
2.5
1.366
1
4
16
1.306
0.96
0.063
3.926
1.372
0.539
0.067
2.153
1.738
1.772
0.057
3.947
1.168
1
4
0.617
1
4

Page 4

Vol 36 (suppl 4) 200529
RE-EVALUATION OF ELISA FOR DETECTION OF G. LAMBLIA AND CRYPTOSPORIDIUM
detected 30 additional G.lamblia cases and 11
additional Cryptosporidium cases in an infant and child
population. The lack of cross-reactivity with five other
parasites highlighted the test’s specificity. Freezing of
specimens did not adversely affect test results.
Additionally, high correlation between visual and OD
readings made this group of three ELISA tests
independent of equipment expenditures. Thus, these
ELISA tests served as reliable and sensitive adjuvants
to microscopic ova and parasite examinations.
ACKNOWLEDGEMENTS
We would like to express our appreciation to the
Director of the Kwai River Christian Hospital and
hospital staff, and all staff in the AFRIMS Enteric
Diseases Department.
REFERENCES
Addiss DG, Mathews HM, Stewart JM, et al. Evaluation
of a commercially available enzyme-linked
immunosorbent assay for Giardia lamblia antigen
in stool. J Clin Microbiol 1991;29:1137-42.
Aldeen WE, Carroll K, Robison A, Morrison M, Hale
D. Comparision of nine commercially available
enzyme-linked immunosorbent assays for
detection of Giardia lamblia in fecal specimens. J
Clin Microbiol 1998;36:1338-40.
Black RE, Dykes AC, Sinclair SP, Wells JG. Giardiasis
in day-care centers: evidence of person-to-person
transmission. Prediatrics 1977;60:486-91.
Bronsdon MA. Rapid dimethyl sulfoxide-modified
acid-fast stain of Cryptosporidium oocysts in stool
specimens. J Clin Microbiol 1984;19:952-3.
Dagan R, Fraser, El-On J, Kassis I, Deckelbaum R,
Turner S. Evaluation of an enzyme immunoassay
for the detection of Cryptosporidium spp in stool
specimens from infants and young children in field
studies. Am J Trop Med Hyg 1995;52:134-8.
Dubey JP, Speer CA, Fayer R, eds. Cryptosporidiosis
of man and animals. Boston: CRC Press, 1990.
Garcia LS. Diagnostic medical parasitology. 4th ed.
Washington, DC: ASM Press, 2001:36-104,741-
85.
Garcia LS, Shimizu RY. Evaluation of nine
immunoassay kits (enzyme immunoassay and
direct fluorescence) for detection of Giardia
lamblia and Cryptosporidium parvum in human
fecal specimens. J Clin Microbiol 1997;35:1526-
9.
Kellogg JA, Elder CJ. Justification for use of a single
trichrome stain as the sole means for routine
detection of intestinal parasites in concentrated
stool specimens. J Clin Microbiol 1999;37:835-
7.
McDonald V. Cryptosporidiosis. In: Cox FEG, ed. The
Welcome Trust illustrated history of tropical
diseases. London: The Welcome Trust, 1996:256-
63.
Nash TE, Herrington DA, Levine MM. Usefulness of
an enzyme-inked immunosorbent assay for
detection of Giardia antigen in feces. J Clin
Microbiol 1987;25:1169-71.
National Committee for Clinical Laboratory Standards.
Procedures for the recovery and identification of
parasites from the intestinal tract, M28-A. Wayne,
Pa: National Committee for Clinical Laboratory
Standards, 1997;17(23).
Parisi MT, Tierno Jr PM. Evaluation of new rapid
commercial enzyme immunoassay for detection of
Cryptosporidium oocysts in untreated stool
specimens. J Clin Microbiol 1995;33:1963-5.
Wolfe MS. Managing the patient with giardiasis:
clinical diagnostic and therapeutic aspects. In:
Jakubowski W, Hoff JC, eds. Waterborne
transmission of giardiasis. Cincinnati, OH: EPA,
US, 1979:39-52.