Journal of Wilderness Medicine 1,203-207 (1990)

Monoclonal antibody detection of Giardia lambliacysts in human stool by direct immunofluorescence

S.C. ZELL*, M. BUDHRAJA, J.L. RIGGS and S.K. SORENSON

Department of Internal Medicine, University ofNevada School of Medicine and Veteran's AdministrationMedical Center, Reno, Nevada, USAUnited States Geological Survey Water Resources Division, Sacramento, California, USAViral and Rickettsial Disease Laboratory, Division ofLaboratories, California State Department ofHealthServices, Berkeley, California, USA

Diagnosing giardiasis traditionally involves ova and parasite analysis, a time consuming method.Recently developed for purposes of analyzing freshwater drinking supplies, a fluorescentmonoclonal antibody to Giardia lamblia was evaluated for its applicability in the detection of cystsin human stool. Using positive and control stool specimens, the monoclonal antibody incubationtechnique identified correctly all specimens prepared from the positive donor pool with 100%sensitivity. Monoclonal antibody detection of Giardia lamblia may prove to be an excellentscreening test for cysts in human stool.

Key words: antibody; monoclonal; Giardia lamblia

Introduction

Traditionally, definitive diagnosis of giardiasis depends upon finding the trophozoite orcyst by microscopic examination of stool or duodenal aspirate. Ova and parasite analysisof stool involves multiple steps, including visualization of saline preparations and iodine­stained emulsions of fresh stool samples, centrifugation to concentrate cysts, andtrichrome staining [1]. Parasite identification requires recognition of specific internalmorphologic protozoal characteristics by an experienced technician [2]. Monoclonalantibodies conjugated to fluorescent compounds offer the ability to more rapidly screensamples for the presence of infectious organisms and may eliminate the need for highlyskilled technicians to analyze samples [3]. A fluorescent monoclonal antibody to G.lamblia cysts has been developed for the purposes of detecting and analyzing cystcontamination in freshwater drinking supplies [4]. We tested the applicability of thismonoclonal antibody technique as a clinical diagnostic tool in the detection of G.lamblia cysts in human stool.

Materials and methods

The evaluation was conducted cooperatively between the Veterans AdministrationMedical Center in Reno, Nevado, the United States Geological Survey Office in

*To whom correspondence should be addressed at: Department of Medicine, VA Medical Center,1000 Locust Street, Reno, NV 89520, USA

0953-9859/90 $03.00 + .12 © 1990 Chapman and Hall Ltd.

204 Zellet al.

Sacramento, California and the Viral and Rickettsial Disease Laboratory of the Califor­nia State Department of Health Services, Berkeley, California.

The hybridoma producing the anti- Giardia cyst monoclonal antibody was developedby immunizing mice with cysts obtained from infected human stool specimens. Giardiacysts from pooled stool specimens were crudely purified according to the procedure ofSheffield and Bjorvatn [5] and were used to immunize BalblC mice for hybridomaproduction. Splenocytes from the immunized mice were fused with SP 2/0 myeloma cellsand grown in 96-well plates. Monoclonal antibody production was determined byimmunofluorescence using G. Lamblia cysts affixed to glass microscope slides as theantigen. Producer colonies were expanded and clones were obtained by three successivesoft agar clonings. Antibody titers from such clones were measured. The antibodysubclass was determined by immunoprecipitation to be mouse IgG 2a. After conjugationwith fluorescein isothiocyanate [6], labelled monoclonal antibody was diluted 1: 10 andyielded a 4+ positive reaction when incubated with G. LambLia cysts from pooled humanstool specimens.

A positive donor pool was made by pooling stool specimens from symptomaticpatients having G. Lamblia cysts noted on prior ova and parasite analysis. An asymp­tomatic, healthy 26-year-old male consented to treatment with metronidazole 750 mgorally T.LD. for 10 days; two weeks later, he provided stool for the control group thattested negative for ova and parasites. All stool was stored in 2% formalin. An aliquotfrom both groups was washed by centrifugation twice in phosphate buffered saline(PBS). Each pellet was resuspended in PBS to provide slurries of varying dilutions (111,1/2, 114, 1/5, 1110). After resuspension, 45 f-ll from each dilution were applied to amicroscopic slide. A thick and thin smear was made and then heat fixed. 10 slides weremade from each dilution for a total of 50 slides per group. The slides were coded toconceal their origins. A technician, experienced in analyzing filtered water samples forGiardia cysts with a fluorescent antibody [7], performed the monoclonal antibodyincubation on the slides, and reported the results as positive or negative for Giardia cysts.50 f-ll of monoclonal antibody conjugated to fluorescein isothiocyanate were added toeach slide and incubated at 37°C for 30 min, rinsed with PBS and distilled water, andallowed to air dry. One drop of elvanol was then placed on the slide, which was mountedwith a cover slip and scanned under epifluorescent illumination at 200 power. Positiveidentification was reported when an oval, uniform pattern of immunofluorescence ofcorrect size (10-20 f-lm in length, 5-15 f-lm in width) was seen encircling the cyst wall as abright green halo (Fig. 1).

Results

All 50 slides prepared from the aggregate donor pool tested positive, even at the 1: 10dilution. This suggests that the monoclonal antibody for G. Lamblia is extremelysensitive. Specificity can not be commented upon, since it requires investigating the falsepositivity rate in large populations parasitized with other organisms. However, ourtechnician did not report any slide prepared from the negative control stool specimenas being positive.

Monoclonal antibody detection of Giardia lamblia cysts 205

Fig. 1. Appearance of Giardia lamblia cysts after staining with fluorescent monoclonalantibody (magnification, X 200). Cysts appear oval shaped, have an outer uniform greenhalo about their wall, and are of 10-20 [!m in length.

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Discussion

Zellet al.

G. lamblia is one of the most frequently identified human intestinal parasites.Identification of the trophozoite or cyst requires recognition by a skilled laboratorytechnician of specific morphologic features. Direct microscopy has been the mainstay ofdiagnosing giardiasis, although attention has recently been given to immunologictechniques [8]. Visvesvara et al. [9], utilizing a suspension of G. lamblia trophozoites,incubated sera from symptomatic patients experiencing giardiasis. Upon addition of anti­human gamma globulin conjugated with fluorescein isothiocyanate, a bright fluorescencepattern was obtained. However, serum antibody levels against G. lamblia were soughtfrom symptomatic subjects only. The clinical utility of serologic testing is unclear inasymptomatic carriers of the parasite and persons with hypogammaglobulinemia unableto manifest a systemic antibody response [10].

Thus, efforts at documenting infection with G. lamblia are probably best done bydirect demonstration of the parasite in human stool. Riggs et al. [11] immunized guineapigs with human G. lamblia cysts, conjugated the guinea pig sera to fluoresceinisothiocyanate and incubated the mixture with stool smears prepared from humanssymptomatic with giardiasis. Upon incubation, G. lamblia cysts fluoresced brightlyagainst a clear background; however, the serum antibody obtained was most likelypolyclonal, as it cross-reacted with cysts of Chilomastix mesnili. Sauch immunized NewZealand white rabbits with G. lamblia cysts and obtained rabbit sera that was laterincubated with Giardia cysts attached to a special membrane filter. Utilizing an indirectimmunofluorescence method, goat anti-rabbit IgG was added to the filter, yielding abright green halo pattern around cysts that were identified as G. lamblia basedupon strict morphologic criteria [12].

Our current work advances prior knowledge by utilizing a fluorescent monoclonalantibody to G. lamblia cysts. To investigate its performance as a diagnostic test, wepresented stool specimen slides from both a positive and control group to a technicianhaving no knowledge of each slide's source. The high degree of sensitivity for directfluorescent monoclonal antibody detection is evident. In addition, the threshold for cystdetection by this technique is noteworthy, since a 1: 10 dilution of a centrifuged stoolconcentrate from which direct staining is done routinely during ova and parasite analysisyielded a positive identification. Utilization of monoclonal antibody to screen freshly­prepared stool specimens, without requiring cyst concentration by centrifugation, wouldbe a valuable method.

The fluorescent monoclonal antibody technique for detection of G. lamblia cystsin human stool may prove to be an excellent screening test due to its superb sensitivity,reproducibility, ease and rapidity to perform. In addition, positive identification does notrequire recognition of internal organism morphology, thereby eliminating the need for ahighly skilled taxonomist of protozoa to identify cysts. We currently plan to utilize thismethod in a longitudinal study to determine Giardia cyst acquisition rates in travelers towilderness areas in the Lake Tahoe Basin.

References

1. Garcia, L.S., and Ash, L.R. Diagnostic parasitology: clinical laboratory manual. 2nd ed. St.Louis: C.V. Mosby Co., 1979.

Monoclonal antibody detection of Giardia lamblia cysts 207

2. Schaefer, F.W., Hibler, c.P., Meyer, E.A., et al. Giardia methods workshop. In: Jakubowski,W., ed. Proceedings of the American Water Works Association Water Technology ConferenceXII. Denver: American Water Works Association, 1984,487-98.

3. Dick, H.M. Monoclonal antibodies in clinical medicine. Br Med J, 1985; 291, 762-4.4. Riggs, J.L., Nakamura, K. and Crook, J. Identifying Giardia lamblia by immunofluorescence in

controlling waterborne giardiasis. G.S. Logsdon, ed., American Society of Civil Engineers, NY,N.Y. 1988; (in press).

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11. Riggs, J.L., Dupuis, K.W., Nakamura, K. and Spath, D.P. Detection of Giardia lamblia byimmunofluorescence. Applied and Environmental Microbiology, 1983; 45, 698-700.

12. Sauch, J. Use of immunofluorescence and phase-contrast microscopy for detection andidentification of Giardia cysts in water samples. Applied and Environmental Microbiology,1985; 50, 1434-8.