Evaluation of a Commercially Available Enzyme-Linked Immunosorbent Assay for Giardia lamblia Antigen in Stool

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, June 1991, p /91/ $02.00/0 Copyright C 1991, American Society for Microbiology Vol. 29, No. 6 Evaluation of a Commercially Available Enzyme-Linked Immunosorbent Assay for Giardia lamblia Antigen in Stool DAVID G. ADDISS,1* HENRY M. MATHEWS,1 JAMES M. STEWART,1 SUSANNE P. WAHLQUIST,1 RUBY M. WILLIAMS,1 ROBERT J. FINTON,2 HARRISON C. SPENCER,' AND DENNIS D. JURANEK1 Parasitic Diseases Branch, Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Office of Epidemiology, Fulton County Health Department, Atlanta, Georgia Received 9 October 1990/Accepted 26 February 1991 The lack of a quick, simple, and inexpensive diagnostic test has limited the ability of public health officials to rapidly assess and control outbreaks of Giardia lamblia in child day-care centers. We evaluated the performance of a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of a G. lamblia-associated antigen in stool. Stool specimens were collected from the diapers of 426 children attending 20 day-care centers, fixed in 10% Formalin and polyvinyl alcohol, and examined by microscopy by Formalin concentration and trichrome staining techniques. Specimens were also tested visually and spectrophotometrically by ELISA. Of 99 tests positive by microscopy, 93 were visually positive by ELISA (sensitivity, 93.9%). Of 534 tests negative for G. lamblia by microscopy, 32 (6.0%) were ELISA positive. However, on the basis of examination of multiple specimens from the same child, none of these could be considered false-positive ELISAs; the specfficity of the ELISA was therefore 100%. The sensitivity of both microscopy and ELISA improved as the number of specimens per child increased. An optical density value of >0.040 was 98.0% sensitive and 100% specific for G. lamblia. This ELISA, which appeared to be more sensitive for G. lamblia than did microscopic examination of stool, should be useful as an epidemiologic tool, particularly in day-care settings, and may also have a role in confirming clinical diagnoses of giardiasis. Giardia lamblia is endemic in child day-care centers (DCCs) in the United States (2, 12), with estimates of 5 to 15% of diapered children attending DCCs being infected. G. lamblia is also a cause of epidemic diarrhea in DCCs; conventional measures to control these outbreaks, including the diagnosis, treatment, and follow-up of infected children and their household contacts, may be costly for local public health agencies to implement and of questionable effectiveness (13). Diagnosis of Giardia infection by microscopic examination of stool for ova and parasites can be time-consuming and expensive; furthermore, examination of a single stool specimen may miss 10 to 50% of Giardia infections because of intermittent or low-level shedding of cysts in stool (3). The need for rapid, inexpensive methods to diagnose Giardia infection has led to the recent development of enzyme-linked immunosorbent assays (ELISAs) for the detection of G. lamblia-associated antigens in stool. As part of an epidemiologic study of endemic giardiasis in DCCs in Fulton County, Ga., we evaluated the first commercially available ELISA kit for G. lamblia in stool (ProSpecT/ Giardia, Alexon, Inc., Mountain View, Calif.). This ELISA detects a G. lamblia-specific antigen (GSA-65) associated with the Giardia cyst wall and, to a lesser extent, with the trophozoite (11). In a previous study, this ELISA appeared to be both sensitive and specific for Giardia infection in hospitalized adults and refugees attending a screening clinic (10), but neither its use in children nor its correlation with standard microscopic examination of several stools from the same person had been evaluated. Similarly, most studies of other ELISAs for Giardia antigen in stool (4, 6, 7, 14, 15) have * Corresponding author evaluated only single specimens from each study participant. Because microscopic examination of a single stool specimen for G. lamblia lacks sensitivity, the use of a single specimen as a "gold standard" in these studies may have resulted in artificially low estimates of test specificity. MATERIALS AND METHODS After informed consent was obtained, stool specimens were collected from the soiled diapers of children attending 17 randomly selected DCCs and three DCCs with outbreaks of diarrheal illness in Fulton County, Ga., from February 1989 to March When diapers were changed, stool specimens were collected by the investigators or by caregivers at the centers and placed in separate vials of 10% Formalin and polyvinyl alcohol preservative (Para-Paks, Meridian Diagnostics). At the Centers for Disease Control, specimen containers were labeled numerically so that laboratory investigators were unaware of the source of the specimen. Whenever possible, three or more specimens were collected on different days from each child. Each specimen was examined microscopically after being processed by three different methods. Stools fixed in 10% Formalin were concentrated by the Formalin-ethyl acetate sedimentation technique (8). The concentrate was placed on a slide and covered with a 22-mm coverslip; the entire slide was then examined microscopically for the presence of ova and parasites. Using the Kinyoun carbol-fuchsin (modified acid-fast) procedure (1), a stained slide was also prepared from the concentrate and examined microscopically for the presence of Cryptosporidium species. A permanent slide of stool fixed in polyvinyl alcohol was stained with trichrome (8) and examined microscopically for the presence of G. lamblia and other protozoa. All slides were read for at least

2 1138 ADDISS ET AL. J. CLIN. MICROBIOL. TABLE 1. Results of 633 tests for G. lamblia by both ELISA and microscopic examination for ova and parasites, with classification of discordant specimens Test result category No. Total Positive by microscopic examination Positive by ELISA Negative by ELISA... 6a Negative by microscopic examination Negative by ELISA Positive by ELISA False-positive ELISA... 0 False-negative microscopy Repeat microscopic examination of same specimen was positive... 8 Children with intermittent cyst shedding; other stools from these children were positive on microscopic examination... 7 Posttreatment: specimens were obtained a few days after children were treated for giardiasis; specimens obtained before treatment were positive for G. lamblia on microscopic examination Probable prepatent or recent infection; specimens obtained a few days later were positive for G. lamblia on microscopic examination... 2 Indeterminate Children were treated for giardiasis after these initial specimens were obtained; all subsequent stools were negative for G. lamblia by microscopic examination and ELISA... 4 Other specimens from these children were positive for G. lamblia on microscopic examination, but were obtained more than 1 week later; therefore, the children could have become infected after these specimens were obtained... 8 " See Table min by one of two microscopists (S.P.W. or R.M.W.), who noted the presence and number of organisms per 10 fields at x40, as follows: none; few, <3 organisms; moderate, 3 to 9 organisms; or many,.10 organisms. An independent reviewer confirmed all positive or questionable specimens and a 10% subset of negative specimens that were randomly selected by computer. The first Formalin-preserved stool specimen from each child and all additional specimens from children with evidence of Giardia infection were tested by ELISA, according to the instructions of the manufacturer, by one of the authors (H.M.M.), who was blinded to the source of the stool and the results of microscopic examination. Two readers (H.M.M. and another reader) independently scored their visual interpretation of the colormetric reactions using the indicator card provided by the manufacturer (from 0 to 4+; a faint color lighter than the 1+ standard on the indicator card was coded as 0.5). If two readers disagreed regarding positive or negative results, a third reader scored all the tests in that run. Aliquots of 200,ul each were then transferred to a microtitration tray for determination of the optical density (OD) at 490 nm with a microplate spectrophotometer (Molecular Devices, Palo Alto, Calif.). Lyophilized antigen (GSA-65) from a single lot was reconstituted and stored at -20 C for use as a positive control; immediately before each run, the antigen was warmed to room temperature. In addition, aliquots of stools from two people with giardiasis and from an uninfected asymptomatic person were fixed in 10% Formalin in February 1989, stored at room temperature, and tested with each run to evaluate antigen stability after fixation in Formalin. If microscopic examination and ELISA results were discordant, the specimen was relabeled with a new number and evaluated again for G. lamblia by both methods. Some specimens were therefore tested more than once; however, laboratory investigators did not know whether a specimen had previously been tested. To evaluate the specificity of the ELISA, 131 stool specimens from a Bolivian village where G. lamblia and other intestinal parasites are endemic were also examined by microscopy and tested by ELISA as described above. Definitions. A child was defined as infected if Giardia cysts or trophozoites were observed by microscopic examination of one or more stool specimens; the specificity of microscopic examination for G. lamblia was therefore considered to be 100%. A child was defined as uninfected if no Giardia cysts or trophozoites were visualized on microscopic examination of three or more stool specimens. Except where otherwise noted, a specimen was considered to have a positive visual ELISA result if two readers indicated a visual colorimetric result of 0.5 or more. The Spearman rank test was used to test for correlations between variables. RESULTS A total of 1,148 stool specimens were obtained from 426 diapered children; 609 specimens were examined by both microscopy and ELISA. Twenty-four specimens with discordant or questionable results were repeated by microscopy and ELISA, for a total of 633 tests examined by both methods. Of the 633 tests, 99 (15.6%) were positive for G. lamblia by microscopy; 93 (93.9%) of these were visually positive by ELISA (Table 1). Of the six tests that were visually negative by ELISA, five tests had fewer than five cysts per slide on microscopic examination and ELISA OD readings of to 0.093; the sixth had only a few trophozoites on microscopic examination (Table 2). The remaining 534 tests were negative for G. lamblia by microscopy; of these, 32 (6.0%) were visually positive by ELISA (Table 1). These discordant tests were classified as falsely positive by ELISA, falsely negative by microscopy, or indeterminate on the basis of subsequent stool specimens from the same child or repeat examinations of the same specimen. Twenty discordant tests were classified as falsely negative by microscopy, and the remaining 12 tests were considered to be indeterminate for the reasons listed in Table 1; the specificity of the ELISA for G. lamblia was therefore 100%. Of 119 true-positive tests (99 specimens positive by microscopy and an additional 20 positive only by ELISA), 99 (83.2%) were identified as positive by microscopy and 113 (95.0%) were positive by ELISA (Table 3).

3 VOL. 29, 1991 EVALUATION OF AN ELISA FOR GIARDIA ANTIGEN IN STOOL 1139 TABLE 2. Results for six discordant tests that were positive for G. lamblia on microscopic examination for ova and parasites and negative by visual ELISA reading Child no. Specimen no. OD Microscopic finding OD Results of repeat ELISA testing Visual reading Four cysts seen Positive; two of two readers Few trophozoites Negative; two of two readers Two cysts seen Positive; one of three readers 8893 (1103) Five cysts seen Four cysts seen Positive; three of three readers 8888 (8595) One cyst seen Positive; two of three readers Initial specimens. In the diagnostic laboratory, only one test on an individual stool specimen. Because the average stool specimen might be received from each patient, and prepatent or incubation period for giardiasis is approxireading of the ELISA by multiple technicians may not be mately 9 days (16), we reasoned that children were already practical. To estimate the sensitivity and specificity of the infected during the 4 days before they produced a stool that ELISA in a routine diagnostic laboratory, we compared was microscopically positive for G. lamblia. One would visual ELISA results for a single reader (H.M.M.) with therefore expect a test with 100% sensitivity for Giardia microscopic results of initial stool specimens from all 426 infection to be positive on any specimen obtained during children. Twenty-nine (6.8%) children were positive for G. these preceding 4 days. To compare the ability of microslamblia by microscopy on the first stool (Table 3). Two of copy and ELISA to detect G. lamblia in individuals known these specimens (specimens 1103 and 8595, Table 2) were to be infected, we identified 31 specimens obtained from 15 visually negative by ELISA; no follow-up specimens were G. lamblia-positive children during the 4 days before a available from either child. Eight children had microscopi- microscopically positive specimen was obtained. The sensically negative specimens that were ELISA positive. Seven tivities of microscopy and ELISA were 70.0 and 83.9%, of these specimens were from children shown to have G. respectively (Table 3). lamblia on subsequent specimens or on repeat microscopic One or more specimens. Collection of more than one stool examination of the same stool. The eighth child, who was specimen from the same child permitted us to assess the excluded from the calculations of test sensitivity presented degree to which multiple specimens may improve the sensiin Table 3, was treated for symptomatic giardiasis after tivity of both microscopic examination and ELISA. We submission of the first stool specimen; the subsequent spec- reviewed data for all 38 children who had a stool specimen imen was negative. Of 36 children with initial true-positive positive by microscopy within 10 days after submitting their specimens (29 by microscopy and 7 more positive only by first specimen. Specimens submitted more than 10 days after ELISA), 29 (80.6%) were positive by microscopy and 34 the initial specimen were excluded because some children, (94.4%) were positive by ELISA. particularly in outbreak centers, were initially negative for Known infected children. Clinicians may be more inter- G. lamblia and were found to be positive 2 to 3 weeks later. ested in the degree to which the ELISA correctly identifies These children were considered likely to have had newly people infected with G. lamblia than in the sensitivity of the acquired infections rather than false-negative initial test TABLE 3. Results and sensitivity of ELISA and microscopic (ova and parasite) examination for G. lamblia in stool Result (no.) of microscopic examination Sensitivity of both methods vs and ELISA Sensitivity of ELISA all true positivesb Micro +, Micro +, Micro -, Micro -, vs microscopya ELISA Microscopic ELISA + ELISA - ELISA + ELISA - exam All specimens (n = 633 tests) 93 6c /99 (93.9) 113/119 (95.0) 99/119 (83.2) Initial specimens (n = 426 tests) 27 2c /29 (93.1) 34/36 (94.4) 29/36 (80.6) All specimens obtained during the /31 (83.9) 22/31 (71.0) days before a microscopically positive specimen Specimens submitted within 10 days after first specimen from each child First specimen /38 (81.6) 29/38 (76.3) Two specimens /38 (86.8) 33/38 (86.8) Three specimens /38 (94.7) 36/38 (94.7)d a Values are number of ELISA-positive, microscopically positive tests/number of microscopically positive tests (%). b Values are number of tests positive by ELISA or microscopy/number of "true-positive" tests (t). "True positives" include specimens positive for G. lamblia by microscopic examination as well as ELISA-positive/microscopically negative specimens from children who were microscopically positive for G. lamblia on a subsequent stool specimen or repeat examination of the same specimen. Specimens considered "indeterminate" (Table 1) are excluded. I See Table 2. d The sensitivity of microscopy did not reach 100%o because two children had specimens that were ELISA positive but microscopically negative on initial examination; on repeat examination of the same stool, a few Giardia cysts were found.

4 1140 ADDISS ET AL. 10 0* 2.oo M.... :::::::: Uninfected Infected FIG. 1. ELISA OD results for microscopically negative specimens from uninfected children (those who had three or more specimens negative for G. lamblia by microscopic examination) and microscopically positive specimens from infected children. results. The sensitivities of both microscopy and ELISA improved as the number of specimens increased; for a single specimen, the sensitivities of microscopy and ELISA were 76.3 and 81.6%, respectively. For three specimens, both methods were 94.7% sensitive (Table 3). These values probably underestimate test sensitivity since the four children who remained negative for G. lamblia by either method were treated for giardiasis after the first specimen was obtained. Correlation of ELISA OD with visual readings and microscopy. To determine the OD that would best distinguish between G. lamblia-positive and G. Iamblia-negative specimens, OD values of 99 microscopically positive specimens were compared with OD values of specimens from 261 uninfected people with three or more microscopically negative specimens (Fig. 1). All negative specimens had ODs of c.0.040; two positive specimens (specimens 821 and 1103, Table 2) had ODs of c A positive OD value of >0.040 would therefore be 98.0% sensitive and 100% specific. The OD was highly correlated with the mean visual score (Table 4) (r2 = 0.92, P = ). To a lesser extent, both the J. CLIN. MICROBIOL. OD and the mean visual ELISA reading were also correlated with the number of organisms seen on microscopy (r2 = 0.66 and 0.70, respectively; P = 0.001). The lower degree of correlation appeared to be due primarily to the fact that many of the discordant (microscopically negative/visual ELISA positive) specimens considered to be true positives had a relatively high OD. Correlation between readers. ELISA readers agreed on positive or negative visual results for all but 10 (1.6%) tests, which had ODs of between and None of the nine people who read ELISAs during the study were significantly more likely than the others to score these tests as negative; therefore, the discrepancies did not appear to be due to differences in the ability to perceive slight colorimetric changes. Actual visual scores (0 to 4+) were highly correlated (r2 = 0.90 to 1.00, P ) for eight of the nine readers. Scores for the remaining reader were less highly correlated with those of one other reader (r2 = 0.58, P = 0.001). Lack of evidence for ELISA cross-reactivity. No evidence of cross-reactivity with Cryptosporidium species or other organisms was found. Sixty-nine DCC specimens were microscopically positive for Cryptosporidium species; the ELISA was positive only for the seven DCC specimens that were also microscopically positive for G. lamblia. Of 131 single stool specimens from Bolivia, 115 were negative for G. lamblia by both microscopy and ELISA; the specificity of the ELISA was therefore 100% in this group. Forty-four of these specimens were positive for other organisms, including 20 (17%) for Ascaris lumbricoides, 20 (17%) for Trichuris trichiura, 11 (10%) for Entamoeba histolytica, and 5 (5%) for Taenia species. Decay in antigen reactivity with time. The OD of the lyophilized positive controls provided by the manufacturer ranged from to (mean + standard deviation, ) and did not significantly change with time (Fig. 2). In contrast, the ELISA reactivity of the Formalinpreserved G. lamblia-positive stool prepared in our laboratory dropped sharply in the first week, followed by a gradual decay over a period of 9 months. Overall, the OD of the Formalin-preserved stools decreased by 78.2% (an average of 8.7% per month), from a mean of during the first month to a mean of during the ninth month. Intermittent detection of antigen and cysts. Two children in the same DCC who were considered infected with G. lamblia had stool specimens that were negative by both microscopic examination and ELISA. Both children had stools that were microscopically positive for G. lamblia within 26 days before and 25 days after these negative specimens; thus, the negative stools were "bracketed" by G. lambliapositive stools. Neither child was treated for giardiasis; TABLE 4. Correlation of ELISA OD with ELISA visual readings and number of organisms seen on microscopya OD range Visual reading (04+)b No. of organisms seen on microscopyc No. of Mean ± SD Range None Few Moderate Many tests ± ± ± ± ± a There were 633 tests performed on stools from 426 children. b A trace of color (less than 1+ on indicator card) was scored as 0.5. c Few, c2 organisms per 10 (x40) fields; moderate, 3 to 9 organisms per 10 fields; many,.10 organisms per 10 fields.

5 VOL. 29, 1991 EVALUATION OF AN ELISA FOR GIARDIA ANTIGEN IN STOOL STOOL I STOOL2-- GSA a 0 U4 (I) w ,'.1,,',V. ^ y,", _ Days After Preparation of Formalin Specimen FIG. 2. ELISA OD over time for Formalin-preserved, G. lamblia-positive stools and the lyophilized positive test kit control (GSA-65) provided by the manufacturer. however, because of the length of time between specimens, self-cure and reinfection cannot be entirely excluded as a possibility. OD values for these specimens were and DISCUSSION.I % % 0 % I I.I The lack of a quick, simple, and inexpensive diagnostic test has limited the ability of public health officials to rapidly assess and control outbreaks of G. lamblia in DCCs. Although ELISAs for Giardia antigen in stool have recently been developed in several laboratories and appear to be promising (4-7, 9, 14, 15), these tests have not been rigorously assessed in the DCC setting. We evaluated the performance of a commercially available ELISA using stool specimens from diapered children attending DCCs in Fulton County, Ga. Compared with microscopic examination of stool specimens for ova and parasites, the ELISA was more sensitive and virtually 100% specific for G. lamblia. Because many specimens can be read by a single technician in a short period of time, the ELISA is also potentially less expensive than microscopic examination of stools. The ELISA is particularly useful for epidemiologic investigations when rapid results are required for large numbers of specimens and the etiologic agent is known to be G. lamblia. An OD cutoff of >0.040 was 98% sensitive and 100% specific for Giardia infection. Rosoff et al. (10) recommended the same positive cutoff value for this ELISA in a study of hospitalized patients and people attending a refugee screening clinic. However, the assay is designed to be read visually, and for routine use in the diagnostic laboratory, OD determination does not appear to be necessary. In our laboratory, visual ELISA results and OD values were highly correlated, and reader-to-reader variation was minimal. Our data suggest that if even a faint colorimetric change is observed, the test should be interpreted as positive for G. lamblia. The sensitivities of this and other ELISAs have compared favorably with microscopic examination in previous studies (4-7, 9, 14, 15). Nash et al. (9) examined serial stool specimens from 18 infected adult volunteers; antigen-positive but cyst-negative stools occurred primarily during treatment and in the prepatent period. We also observed ELISApositive but cyst-negative stools in children who had recently begun antimicrobial therapy and in those from outbreak centers who were apparently infected after the initial specimens had been obtained. Investigators have expressed concern about the potential lack of specificity of other Giardia antigen assays (17), but we were unable to document a single false-positive ELISA result. Although the ELISA appeared to be more sensitive for G. lamblia than microscopic examination was, on a single stool specimen the sensitivity was <100% in children known to be infected. As with microscopy, the sensitivity of ELISA improved as the number of specimens increased. Two different types of false-negative ELISA results (discordant and concordant) were observed. Discordant results (those that were microscopically positive on the same test) had borderline OD readings and were likely to be ELISA positive on repeat testing of the same specimen. These false-negative results may have been caused by test-to-test variation in the laboratory. By contrast, two specimens with negative ELISAs and negative microscopic results were obtained from children who were microscopically positive both on previous and subsequent specimens. These specimens had lower OD readings and did not become ELISA positive on repeat testing. Although self-cure and reinfection could not be excluded as a possibility, these findings suggest that shedding of both cysts and Giardia antigen into stool may occur intermittently. The presence of GSA-65 in the stool

6 1142 ADDISS ET AL. could therefore be expected to be irregular in infected people who shed cysts intermittently. The functional nature of the antigen detected by the ELISA remains obscure, but the antigen has been shown to be associated primarily with the Giardia cyst wall (11). An ideal stool test for G. lamblia would be 100% sensitive for the presence of trophozoites in the small intestine. We found only one stool specimen that contained trophozoites (in low numbers) and no Giardia cysts; this specimen was negative by ELISA. It is unknown whether GSA-65 is associated with all human Giardia strains. However, our results in children attending DCCs in Georgia and people in a Bolivian village were comparable to those of Rosoff et al. (10), who studied this ELISA with specimens from refugees and symptomatic hospital patients in California. OD readings of Formalinfixed positive control specimens decreased gradually during the study, suggesting that the antigen is not stable over long periods of time in 10% Formalin at room temperature. For maximum ELISA sensitivity, preserved specimens should be tested within a few months after being submitted. To limit costs, only the first stool specimen from each child was tested by ELISA, unless that specimen was ELISA positive or any of the child's specimens were positive for G. lamblia by microscopy. A few more ELISApositive, cyst-negative discordant specimens may have been identified if all specimens had been tested by both ELISA and microscopy. However, because the prevalence of Giardia infection in this population was low, virtually all of the untested specimens would have been negative by both methods, i.e., concordantly negative. Thus, routine testing of only the first stool specimen by ELISA may have led us to overestimate the proportion of discordant test results and underestimate the performance of the ELISA. In summary, when compared with microscopic examination of stool, the ELISA was more sensitive and 100% specific for Giardia infection. The assay appears to be useful as an epidemiologic tool, especially in day-care settings, and in the investigation and control of outbreaks of giardiasis. The ELISA may also have a role in confirming the clinical diagnosis of giardiasis by physicians. It is unlikely to replace microscopic examination of stool for ova and parasites as a routine diagnostic test, however, because other potential pathogens would escape detection. Although more sensitive than microscopic examination, intermittent antigen shedding and laboratory variation may result in an occasional falsenegative ELISA, particularly on a single stool specimen. Additional studies are needed to further characterize the nature of the antigen and evaluate the diagnostic potential of the ELISA, especially in people with chronic giardiasis who are negative for G. lamblia on microscopic examination of stool. ACKNOWLEDGMENTS We thank the directors, staff, children, and parents of the DCCs participating in this study; Barry Whitset and William Elsea, Fulton County Health Department; Chandra Jones, Morehouse University School of Medicine; and Henry Bishop and James Sullivan at the J. CLIN. MICROBIOL. Centers for Disease Control. We also thank Phyllis Moir for editorial assistance, Ralph Bryan for providing stool specimens from Bolivia, Meridian Diagnostics for providing specimen containers, and Alexon, Inc., for providing reagents for the ELISA. REFERENCES 1. Ash, L. R., and T. C. Orihel A guide to laboratory procedures and identification, p Society of Clinical Pathologists Press, Chicago. 2. Black, R. E., A. C. Dykes, S. P. Sinclair, and J. G. Wells Giardiasis in day-care centers: evidence of person-to-person transmission. Pediatrics 60: Burke, J. A The clinical and laboratory diagnosis of giardiasis. Crit. Rev. Lab. Sci. 7: Carlson, J. R., P. S. Sullivan, D. J. Harry, M. A. Stork, S. A. Thorton, and H. L. DuPont Enzyme immunoassay for the detection of Giardia lamblia. Eur. J. Clin. Microbiol. Infect. Dis. 7: Goldin, A. J., W. Apt, X. Aguilera, I. Zulantay, D. C. Warhurst, and M. A. Miles 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: Green, E. L., M. A. Miles, and D. C. Warhurst Immunodiagnostic detection of Giardia antigen in faeces by a rapid visual enzyme-linked immunosorbent assay. Lancet ii: Janoff, E. N., J. C. Craft, L. K. Pickering, T. Novotny, M. J. Blaser, C. V. Knisley, and L. B. Reller Diagnosis of Giardia lamblia infection by detection of parasite-specific antigens. J. Clin. Microbiol. 27: Melvin, D. M., and M. M. Brooke Laboratory procedures for the diagnosis of intestinal parasites, 3rd ed., p Centers for Disease Control laboratory manual. U.S. Department of Health and Human Services publication no. (CDC) Centers for Disease Control, Atlanta. 9. Nash, T. E., D. A. Herrington, and M. M. Levine Usefulness of an enzyme-linked immunosorbent assay for detection of Giardia antigen in feces. J. Clin. Microbiol. 25: Rosoff, J. D., C. A. Sanders, S. S. Sonnad, P. R. DeLay, W. K. Hadley, F. F. Vincenzi, D. M. Yajko, and P. D. O'Hanley Stool diagnosis of giardiasis using a commercially available enzyme immunoassay to detect Giardia-specific antigen 65 (GSA 65). J. Clin. Microbiol. 27: Rosoff, J. D., and H. H. Stibbs Isolation and identification of a Giardia lamblia-specific stool antigen (GSA 65) useful in coprodiagnosis of giardiasis. J. Clin. Microbiol. 23: Sealy, D. P., and S. H. Schuman Endemic giardiasis and day care. Pediatrics 72: Steketee, R. W., S. Reid, T. Cheng, J. S. Stoebig, R. G. Harrington, and J. P. Davis Recurrent outbreaks of giardiasis in a child day care center, Wisconsin. Am. J. Public Health 79: Ungar, B. L. P., R. H. Yolken, T. E. Nash, and T. C. Quinn Enzyme-linked immunosorbent assay for the detection of Giardia lamblia in fecal specimens. J. Infect. Dis. 149: Wienecka, J., E. Olding-Stenkvist, H. Schroder, and G. Huldt Detection of Giardia antigen in stool samples by a semiquantitative enzyme immunoassay (EIA) test. Scand. J. Infect. Dis. 21: Wolfe, M. S Giardiasis. Pediatr. Clin. North Am. 26: Yolken, R. H., and B. Ungar ELISA detection of Giardia lamblia. Lancet ii:1120. (Letter.)