Monoclonal antibody detection of Giardia lamblia cysts in human stool by direct immunofluorescence

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1 Journal of Wilderness Medicine 1, (1990) Monoclonal antibody detection of Giardia lamblia cysts in human stool by direct immunofluorescence S.C. ZELL*, M. BUDHRAJA, J.L. RIGGS and S.K. SORENSON Department ofinternal Medicine, University ofnevada School ofmedicine and Veteran's Administration Medical Center, Reno, Nevada, USA United States Geological Survey Water Resources Division, Sacramento, California, USA Viral and Rickettsial Disease Laboratory, Division oflaboratories, California State Department ofhealth Services, 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 fluorescent monoclonal antibody to Giardia lamblia was evaluated for its applicability in the detection of cysts in human stool. Using positive and control stool specimens, the monoclonal antibody incubation technique identified correctly all specimens prepared from the positive donor pool with 100% sensitivity. Monoclonal antibody detection of Giardia lamblia may prove to be an excellent screening test for cysts in human stool. Key words: antibody; monoclonal; Giardia lamblia Introduction Traditionally, definitive diagnosis of giardiasis depends upon finding the trophozoite or cyst by microscopic examination of stool or duodenal aspirate. Ova and parasite analysis of stool involves multiple steps, including visualization of saline preparations and iodinestained emulsions of fresh stool samples, centrifugation to concentrate cysts, and trichrome staining [1]. Parasite identification requires recognition of specific internal morphologic protozoal characteristics by an experienced technician [2]. Monoclonal antibodies conjugated to fluorescent compounds offer the ability to more rapidly screen samples for the presence of infectious organisms and may eliminate the need for highly skilled technicians to analyze samples [3]. A fluorescent monoclonal antibody to G. lamblia cysts has been developed for the purposes of detecting and analyzing cyst contamination in freshwater drinking supplies [4]. We tested the applicability of this monoclonal 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 Administration Medical 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 /90 $ Chapman and Hall Ltd.

2 204 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 developed by immunizing mice with cysts obtained from infected human stool specimens. Giardia cysts from pooled stool specimens were crudely purified according to the procedure of Sheffield and Bjorvatn [5] and were used to immunize BalblC mice for hybridoma production. Splenocytes from the immunized mice were fused with SP 2/0 myeloma cells and grown in 96-well plates. Monoclonal antibody production was determined by immunofluorescence using G. Lamblia cysts affixed to glass microscope slides as the antigen. Producer colonies were expanded and clones were obtained by three successive soft agar clonings. Antibody titers from such clones were measured. The antibody subclass was determined by immunoprecipitation to be mouse IgG 2a. After conjugation with fluorescein isothiocyanate [6], labelled monoclonal antibody was diluted 1: 10 and yielded a 4+ positive reaction when incubated with G. LambLia cysts from pooled human stool specimens. A positive donor pool was made by pooling stool specimens from symptomatic patients having G. Lamblia cysts noted on prior ova and parasite analysis. An asymptomatic, healthy 26-year-old male consented to treatment with metronidazole 750 mg orally T.LD. for 10 days; two weeks later, he provided stool for the control group that tested negative for ova and parasites. All stool was stored in 2% formalin. An aliquot from 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 a microscopic slide. A thick and thin smear was made and then heat fixed. 10 slides were made from each dilution for a total of 50 slides per group. The slides were coded to conceal their origins. A technician, experienced in analyzing filtered water samples for Giardia cysts with a fluorescent antibody [7], performed the monoclonal antibody incubation 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 to each slide and incubated at 37 C for 30 min, rinsed with PBS and distilled water, and allowed to air dry. One drop of elvanol was then placed on the slide, which was mounted with a cover slip and scanned under epifluorescent illumination at 200 power. Positive identification was reported when an oval, uniform pattern of immunofluorescence of correct size (10-20 f-lm in length, 5-15 f-lm in width) was seen encircling the cyst wall as a bright green halo (Fig. 1). Results All 50 slides prepared from the aggregate donor pool tested positive, even at the 1: 10 dilution. This suggests that the monoclonal antibody for G. Lamblia is extremely sensitive. Specificity can not be commented upon, since it requires investigating the false positivity rate in large populations parasitized with other organisms. However, our technician did not report any slide prepared from the negative control stool specimen as being positive.

3 Monoclonal antibody detection of Giardia lamblia cysts 205 Fig. 1. Appearance of Giardia lamblia cysts after staining with fluorescent monoclonal antibody (magnification, X 200). Cysts appear oval shaped, have an outer uniform green halo about their wall, and are of [!m in length.

4 206 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 laboratory technician of specific morphologic features. Direct microscopy has been the mainstay of diagnosing giardiasis, although attention has recently been given to immunologic techniques [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 fluorescence pattern was obtained. However, serum antibody levels against G. lamblia were sought from symptomatic subjects only. The clinical utility of serologic testing is unclear in asymptomatic carriers of the parasite and persons with hypogammaglobulinemia unable to manifest a systemic antibody response [10]. Thus, efforts at documenting infection with G. lamblia are probably best done by direct demonstration ofthe parasite in human stool. Riggs et al. [11] immunized guinea pigs with human G. lamblia cysts, conjugated the guinea pig sera to fluorescein isothiocyanate and incubated the mixture with stool smears prepared from humans symptomatic with giardiasis. Upon incubation, G. lamblia cysts fluoresced brightly against a clear background; however, the serum antibody obtained was most likely polyclonal, as it cross-reacted with cysts of Chilomastix mesnili. Sauch immunized New Zealand white rabbits with G. lamblia cysts and obtained rabbit sera that was later incubated with Giardia cysts attached to a special membrane filter. Utilizing an indirect immunofluorescence method, goat anti-rabbit IgG was added to the filter, yielding a bright green halo pattern around cysts that were identified as G. lamblia based upon strict morphologic criteria [12]. Our current work advances prior knowledge by utilizing a fluorescent monoclonal antibody to G. lamblia cysts. To investigate its performance as a diagnostic test, we presented stool specimen slides from both a positive and control group to a technician having no knowledge of each slide's source. The high degree of sensitivity for direct fluorescent monoclonal antibody detection is evident. In addition, the threshold for cyst detection by this technique is noteworthy, since a 1: 10 dilution of a centrifuged stool concentrate from which direct staining is done routinely during ova and parasite analysis yielded a positive identification. Utilization of monoclonal antibody to screen freshlyprepared stool specimens, without requiring cyst concentration by centrifugation, would be a valuable method. The fluorescent monoclonal antibody technique for detection of G. lamblia cysts in 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 not require recognition of internal organism morphology, thereby eliminating the need for a highly skilled taxonomist of protozoa to identify cysts. We currently plan to utilize this method in a longitudinal study to determine Giardia cyst acquisition rates in travelers to wilderness 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.

5 Monoclonal antibody detection of Giardia lamblia cysts 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 Conference XII. Denver: American Water Works Association, 1984, Dick, H.M. Monoclonal antibodies in clinical medicine. Br Med J, 1985; 291, 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). 5. Sheffield, H.G. and Bjorvatn, B. Ultrastructure of the cyst of Giardia lamblia. Am J Trop Hyg, 1977; 26, Riggs, J.L. Immunofluorescent staining. In: Lennette, E.H., Schmidt, N.J. eds. Diagnostic procedures for viral, rickettsial and chlamydiai infections. 5th ed. Washington, DC: American Public Health Association, 1979; Sorenson, S.K., Riggs, J.L., Dileanis, P.D. and Suk, T.J. Isolation and detection of Giardia cysts from water using direct immunofluorescence. Water Resources Bulletin, 1986; 22, Ridley, M.J. and Ridley, D.S. Serum antibodies and jejunal histology in giardiasis associated with malabsorption. J Clin Path, 1976; 29, Visvesvara, G.S., Smith, P.D., Healy, G.R. and Brown, W.R. An immunofluorescence test to detect serum antibodies to Giardia lamblia. Ann Intern Med, 1980; 93, Hughes, W.S., Gerda, J.J., Hotzapple, P. and Brocks, F.P. Primary hypogammaglobulinemia and malabsorption. Ann Intern Med, 1971; 74, Riggs, J.L., Dupuis, K.W., Nakamura, K. and Spath, D.P. Detection of Giardia lamblia by immunofluorescence. Applied and Environmental Microbiology, 1983; 45, Sauch, J. Use of immunofluorescence and phase-contrast microscopy for detection and identification of Giardia cysts in water samples. Applied and Environmental Microbiology, 1985; 50,