Jpn. J. Infect. Dis., 54, 72‑74, 2001

MethodDetection of Giardia lamblia Cysts in Non‑Fixed Long‑Term
Stored Human Feces by Direct Immunofluorescence Assay
Norihito Morimoto. *, Chizu Komatsu. , Masaaki Nishidal and Tetsuro SugiuraI･2
1Department of Clinical Laboratory, Kochi Medical School Hospital and2clinical Laboratory Medicine, Kochi Medical School,Kohasu, Oko‑cho, Nankoku, Kochi 783‑0043, Japan(Received November 8, 2000. Accepted April 18, 2001)

SUMMARY: Giaydia lamblia cysts in fecal specimens are detected by conventional morphologlCal methods.
The direct immunofluorescence monoclonal antibody assay (DFA) is also applied to detect G. lamblia cysts infeces, but little isknOwn about the usefulness of DFA in fecal specimens stored under various conditions. The
aim of the present study was to evaluate the DFA for detection of G. lamblia cysts and to compare these resultswith the direct smear method in long‑term storage ofnon‑fixed fecal specimens. Fecal specimens with G. lamblia
cysts were stored in a refrigerator at 5oC (14 samples), afreezer at ‑20oC (9 samples), or in 3.9% formalin‑saline
solution at room temperature (28 samples). G. lamblia cysts were detected by DFA in all stored specimens, while

they were detected in only 56% of refrigerated and 93% offrOzen specimens by the direct smear method. The
storage period of all samples testlng negative by direct smear was more than 24 months. Many degenerated cysts
were recognized by DFAIWhen cysts were negative by the direct smear method. Our results indicate that DFA isa useful method for detectlng G. lamblia cysts in fecal samples that have undergone long‑termstorage.

samples) for 30‑48, 1 0‑29, and 4‑49 months, respectively.

INTRODUCTION

Direct smearmethod: Stored fecal samples were mixed

GiaTdia lamblia is the most common human intestinalin a血op of saline on a clean slide and examined microscopl‑

parasite distributed worldwide, causlng diarrhea, abdominal

cally a鮎r placlng then under a 18 × 18 mm cover slip. Speci一ailments, and weight loss in infected people ( 1 ). Identification

mens were examined for cysts of G. lamblia under a BX60of G. lamblia cysts is made based on morphological charac‑Upright Research Microscope (OLYMPUS, Inc., Tokyo)

teristics in fresh fecal specimens by conventional methods

uslng 40X objectives.

such as direct smear, concentration teclmiquesuslng formalin‑

I?FA: DFA (Cyst‑a‑GloTM kit; Waterbome, Inc., New

ethyl acetate, and cyst preparations stained with chlorazol

Orleans, La., USA) was applied to detect G. lamblia cysts inblack E or hematoxylin. Recently, nuorescent‑labeled mono‑each of the stored fTecal specimens. Specimens were examinedclonal antibody to the cyst‑wall proteins became available

under a BX60 Upright Research Microscope uslng 40×for detec.tion of G･ lamblia cysts in feces (2)･ A recent large

Objectives. Afluorescence microscope with a 470‑ to 490‑

prospectlVe Study clearly showed an increased detection rate

nm exciter filter, a 505‑nm dichromatic beam splitter, and afor G. lamblia cysts using the direct immunonuorescence

5 15‑ to 550‑nm barrier filter was also used. The frozen andmo.noclonal antibody assay (DFA) Compared with conventional

refrigerated direct‑smear fecal samples were classified into

stalning methods (3). Although subsequent reports generally

two grワups: G･ lamblia cyst‑positive (n = 18 samples) ands叩pOrt血e improved sensitivity of DFA staining (4,5), clinical‑negatlVe (〟 ‑ 5 samples). Degeneration rates with DFA

experience with DFA diagnosis of GiaydiainfeCtionsis limited
thus far. Moreover, the detection of G. lamblia cysts in longterm‑stored feces by DFA has yet to be defined. The purposeof this study, therefore, was to evaluate theusefulness ofDFAin detectlng G. lamblia cysts and to compare results with DFAresults of these two groups were compared.

Morphological degeneration of cysts: A degenerated cyst
was defined as a nonellipsoid‑shaped cyst by DFA. The
degeneration rate of G. lamblia cysts was determined as:Degeneration rate ‑ Number or degenerated cysts/

Number oftotal cysts X 100 (%)

to those of conventional morphologlCal tests of various stored

Number of degenerated cysts and total cysts were countedby DFA.

human fecal specimens.

StatisticaJ analysis: Statistical analysis Was Performed byMTERIALS AND METHODS

the unpaired i‑test for degeneration rate. A value of less thanFecal samples: A total of 5 I G. lamblia cyst‑positive fecal

0.05 was considered significant.

specimens from 1 8 patients were collected at Kochi Medical
School Hospital from April 1996 to January 2000. These fecal
specimens were diagnosed positive for G. lamblia cysts by

RESUIXS

the direct smearmethod. Fecal samples were stored randomly

Using the smear specimens, G. lamblia cysts were detected

in a refrigerator at 5oC (9 samples), in afreezer at 120oC (14samples), or in 3.9% formalin‑saline at room temperature (28in lOO% of the formalin‑fixed specimens by direct method,but only in 56 % of the refrigerated feces and 93 % of thefrozen feces. DFA detected 100 % of the G. lamblia cysts in*Corresponding author: Tel: +8 1‑88‑866158 I 1 , Fax: +81188‑880‑

all three stored specimens. Cysts were detected by the direct

2462, E‑mail: morimoto@med.kochi‑ms.acJp

smear method in fecal samples stored at various conditions

72

Table 1. Detection of G. lamblia cysts by direct smear* and DFA**Re舟igerated(n‑9)

Frozen (n‑ 14)

Formaline(n‑28)

ConseⅣation period (Months)

Direct smear
DFA<2424<24く

3/3

2/6

1 3/1 4

3/3

6/6

14/1

く24

424<1 4/1 4

1 4/1 4

1

14/1

4/1

44

* : Direct smear: Smear was examined under regularmicroscope.
** : DFA: Direct immunonuoresccnce monoclonal antibody assay.

for less than 24 months, but the storage period ofall negative

under bright microscopy (6). However, they did not examine

samples was greater than 24 months by the direct smear

the usefulness of immunonuorescence in samples stored formethod. 1n particular, the detection rate of refrigerated feces

a long period. Our study has clarified the difference in thedetection of G. lamblia cysts between the direct smear method†as 33% (Table l)･ Deg?nerated cysts were observed by DFA

and DFA in long term‑stored feces kept inthe refdgerator orln Samples testing negatlVe by the direct smear method (Fig.

1). These cysts were not detected by bright field microscopy

freezer. Interestingly, formalin solution was found to bewhen the fluorescence optical path was closed (Fig. 2A, B).

adequate for long‑term storage of fecal samples. Formalinsolution results in protein degeneration but does not have any

influence on DFA reactivlty.

DISCUSSION

Humans may excrete 1 50 to 20000 cysts/g into feces daily

Because Giardiasis breaks out as a result ofwaterbome or(7), but even under such conditions stool examinations may

foodbome infection, storing and detection of G. lamblia cysts
is necessary not only in fecal samples, but also in food orwater samples. Moreover, when fresh fTeces cannot be obtainedsuch as in cases where samples are transported from a distantarea, feces are stored in the freezer or rehgerator for a
relatively long period･ In the present study, G. lamblia cysts

were detected by DFA in all of the fecal samples stored underthe various conditions, even when cysts were not identified

by the direct smear method. DFA results wereunaffected by
the storage conditions fわr the samples or cyst degeneration,

indicating that DFA has a high reactivityto G. lamblia cysts.
Erlandsen et al. has reported that cysts in fecal samples
can be detected after cycles of freezlng and thawlng byimmunofluorescence, even when the cyst wall cannot be seenーl即 0 0 伽 40 20
Fig. 2A. Bright丘eldmicroscopy. Cysts were not detected by dhect smear

method (arrowhead). Scale bar ‑ 10 Elm.

t[o!一t!J3t[3 PJO3一q

B

Positiye Negative(〟‑21)(〟‑5)

Direct smear
Fig. 2B. Giardia lamblia cysts detected by DFA in the refrigerated
Fig. 1. Rate of degeneration of G. lamblL'a cysts. Results of the direct

feces. Despite degeneration of many cysts a洗er storage of morethansmear method were used to classify feces as G.lamblia cyst‑positive

24 months,fluorescence was not different between typical‑formor ‑negative, andthese results were compared to those obtained by(arrowhead) cysts and untypical‑formcysts (*) in the same field as

the identification of degenerated cysts by DFA.

Fig. 2A. Scalebar‑ 10 〝m.

73

be negative by the direct smear method in some patients with

3. Farthing, M. I. G. (1999): Giafldiay由. p.562‑584. In Herbert,

M. G. (eds), Protozoal Diseases. Section 10. Amold Co.,chronic diarrhea, desplte the presence of G. lamblia tropho‑
degenerated cysts or very few intact cysts even in fresh feces

New York.
4. Garcia, L S. and Shimizu, R. Y. (1997): Evaluation of

from individuals with G. lamblia infection. Degenerated cysts

nine immunoassay kits (en.zyme immunoassay andmay not be detected by the direct smear method in these feces.

direct nuorescence) for detectlOn Of Giaydia lamblia andzoites in the duodenum (1). Moreover, We fわund either

Considering that the storage period and degeneration rate in

CJ二岬tOSPOridium parvum in human fecal specimens. J.

fresh feces had no slgnificant effect on cyst detection by DFA

Clin. Microbio1., 35, 1526‑1529.
5. Alles, A. J., Waldron, M. A., Sierra, L. S. and Mattia, A.

in various stored samples, it appears that G. lamblia cysts
can be detected by DFA in cyst‑negative samples by theR･ (1995): Prospective coヮparison of direct immun0‑

conventional method when the immunologlCal cyst wall isfluorescence and conventional stainlng methods forpreserved.

detection of Giaydia and CtyPtOSPOridium spp. in human

fecal specimens. J. Clin. Microbio1., 33, 1632‑1634.

In conclusion, DFA is a useful method for detectlng G.

6. Erlandsen, S. L., Sherlock, LA. andBemick, W. J. (1990):

lamblia cysts in long term‑stored feces.

The detection of Giaydia muris and Giardia lamblia cysts

by immunofluorescence in animal tissues and fecal

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7. Porter, A. (1916): An enumerative study of the cysts ofbiol. Rev., 55, 706‑732.

2. Marshall, M. M., Naumovitz, D., Ortega, Y and Sterling,Gialldia (lamblia) intestinalis in human dysenteric faeces.

Lancet, 1, 1166‑1169.

C. 良. (1997): Waterborne protozoan pathogens. Clin･Microbiol. Rev., 10, 67‑85.

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