Immunoglobulin M Toxoplasma Antibodies

CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, JUlY 1994, p. 401-405 Vol. 1, No. 4 1071-412X/94/$04.00+0 Copyright C) 1994, American Society for Microbiology The Vitek Immunodiagnostic Assay for Detection of Immunoglobulin M Toxoplasma Antibodies ERMANNO CANDOLFI,1'2 RAYMUND RAMIREZ,1 MARIE-PAULE HADJU,1 CAROL SHUBERT,3 AND JACK S. REMINGTON' 2* Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto,' and Division of Infectious Disease and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford,2 Califomia, and Vitek Systems, Inc., Hazelwood, Missouri3 Received 14 January 1994/Returned for modification 15 February 1994/Accepted 10 March 1994 We compared the Vitek immunodiagnostic assay for detection of immunoglobulin M (IgM) toxoplasma antibodies (VIDAS TXM) with an IgM enzyme-linked immunosorbent assay (ELISA) which we developed. A total of 407 serum samples from 391 adults were used for this study. We also examined 17 serial serum samples from four women who had seroconverted during gestation. We observed a sensitivity of 99.5%, a specificity of 93%, a positive predictive value of 93.6%, a negative predictive value of 99.5%, and a global agreement of 96.3%. In each case of seroconversion the VIDAS TXM became positive at the same time as did the IgM ELISA. The diagnosis of acute acquired Toxoplasma infection depends primarily on results of serological tests. A variety of methods which vary considerably in their sensitivity and specificity are being used to detect immunoglobulin M (IgM) Toxoplasma antibodies (12). Because IgM antibodies may persist for months or even years following the acute infection (2, 6), their greatest value is that a negative result virtually rules out a recently acquired infection, unless sera are tested so early after the acute infection that an antibody response has not yet occurred. Demonstration of their presence can lead to appropriate additional testing by a reference laboratory. Commercial kits for the detection of toxoplasma IgM antibodies are increasingly being used in laboratories throughout the United States and Europe. A potential problem with many of these kits is the unacceptably high numbers of false-positive and false-negative results (1, 14). In addition, interpretation of the results is frequently flawed and can result in unnecessary abortion or undue concern on the part of a pregnant woman and her family. Thus, the use of a commercially available kit as a reference with which to compare a newly developed method is not necessarily justified. We have evaluated commercial kits for demonstration of IgM toxoplasma antibodies for years, and many lacked sufficient sensitivity and/or specificity (unpublished observations). We compared the Vitek immunodiagnostic assay for detection of IgM toxoplasma antibodies (VIDAS TXM) with an IgM enzyme-linked immunosorbent assay (ELISA) which we developed (11) and which we and other reference laboratories have used for many years. The excellent results recently obtained with the VIDAS TXM prompted this report. MATERIALS AND METHODS Serum samples. A total of 407 serum samples from 391 adults were used for this study. The sera were submitted from outside sources to our Toxoplasma serology laboratory. They were chosen so that approximately 50% had been found to be IgM positive and 50% had been found to be IgM negative by our IgM ELISA. A total of 333 (84.3%) were from women, of whom 208 (62%) were pregnant. Of the 391 patients, 39 * Corresponding author. Mailing address: Research Institute, Palo Alto Medical Foundation, 860 Bryant St., Palo Alto, CA 94301. 401 (9.5%) had lymphadenopathy, 12 (2.9%) had received a heart transplant, and 5 (1.2%) had AIDS. The remaining 24 patients presented with a variety of clinical features including pneumonia, renal transplantation, Hodgkin's disease, and hepatitis. There was no clinical information provided with 122 (29.7%) of the samples. Tests were performed with sera that had been refrigerated for up to 48 h or frozen for longer periods. In addition to the sera for overall evaluation of the VIDAS system, we examined 17 serial serum samples from four women who had seroconverted during gestation. These sera were supplied by Phillip Thulliez, Paris, France. VIDAS TXM. The VIDAS TXM system (biomerieux Vitek Inc., Hazelwood, Mo.) uses an automated enzyme-linked fluorescent immunoassay for the semiquantitative detection of IgM toxoplasma antibodies. The assay was performed according to the directions of the manufacturer. The instrument features a disposable pipette-like tip device, which is a solidphase receptacle that serves as the solid phase as well as the pipettor for the assay. The interior of the solid-phase receptacle was coated at the time of production with a monoclonal antibody directed to human IgM which served to capture IgM. Reagents for the assay are contained in a foil-sealed 10-well polypropylene reagent strip. The first well was for the sample, and the remaining wells contained the various reagents, including Toxoplasma gondii antigen bound to a mouse monoclonal antibody directed to the P30 antigen of T gondii and labeled with alkaline phosphatase. The T. gondii antigen bound to the mouse monoclonal antibody is cycled in and out; this allows it to attach to the patient's IgM antibody to T. gondii that has bound to the solid-phase receptacle wall. Unbound material was removed by washing. The last well of the strip is an optically clear cuvette in which the fluorometric measurement is made by the VIDAS optical scanner. One reagent strip and one solid-phase receptacle were used for each sample for the control or standard to be tested. A minimum of 100,ul of serum, control, or standard was pipetted into the sample well of each reagent strip which was introduced into the instrument and processed automatically. The total time for the assay is 40 min, and a maximum of 30 VIDAS TXM tests could be processed in a single VIDAS module. The intensity of the fluorescence is proportional to the quantity of IgM antibodies present in the sample. Final semiquantitative results are expressed as a test value, which is the ratio of relative sample

402 CANDOLFI ET AL. fluorescence to that of the standard. Interpretation was based on the following thresholds: <0.55 = absence of IgM antibodies; between 0.55 and 0.65 = equivocal result; >0.65 = presence of IgM antibodies. The manufacturer recommends that all specimens with equivocal results be retested. We did so, and the retest value was used as the final result. Samples which repeatedly tested as equivocal in the present study were reported as such. Negative and positive controls were tested with each run. When a discrepancy was found between the VIDAS TXM and IgM ELISA, confirmatory tests, including the IgA ELISA and the differential agglutination test (AC/HS) described below, were run. VIDAS TXM precision was determined by intra-assay and interassay testing of negative, positive, and precision controls. Intra-assay precision testing was completed before clinical testing was begun. Intra-assay precision was determined by running three consecutive runs on the VIDAS of each negative, positive, and calibrator (standard) control. Interassay precision was determined by testing negative, positive, and calibrator (standard) controls each in triplicate, for 10 days, concurrent with testing of the sera of the patients. VIDAS TXM reproducibility was determined by testing a panel of three samples coded in a blind manner, each in triplicate, on three consecutive days. Additional serological tests. The Sabin-Feldmann dye test (7), double-sandwich IgM ELISA (11), AC/HS (5), and IgA ELISA (13) were performed as previously described (15). A titer of -1:16 was considered positive in the dye test. For samples from those who seroconverted, the dye tests were also performed in the laboratory of Phillip Thulliez. The positivities and negativities of sera from the laboratory in Paris and those in the United States agreed perfectly. For the IgM ELISA, a value of 0.0 to 1.6 was considered negative, 1.7 to 1.9 was considered equivocal,.2.0 was considered positive, and.6.0 was considered to reflect more recently acquired infection. In the IgA ELISA, a positive result was.1.4 and a negative result was <1.4. When IgA ELISA is used as a confirmatory test, a positive result adds support to a diagnosis of recently acquired infection but does not prove it (13). The AC/HS test was used to help distinguish recently acquired infection from a latent infection as previously described (5). Briefly, the method is a differential agglutination which compares the titers obtained with formalin-fixed tachyzoites (HS antigen) with those obtained with acetone-fixed tachyzoites (AC antigen). The AC antigen preparation contains stage-specific antigens which are recognized by IgG antibodies formed against toxoplasma tachyzoites early in infection. These antibodies have specificities different from those of antibodies formed later in infection. We attempted to resolve discrepancies by using results of the IgA ELISA and AC/HS tests. Technical performance comparison. Sensitivity was defined as the percentage of specimens positive in the IgM ELISA that were identified by the VIDAS TXM; specificity was defined as the percentage of specimens negative in the IgM ELISA that were identified as negative in the VIDAS TXM. The positive predictive value was defined as the probability that a positive VIDAS TXM result would be positive in the IgM ELISA. The negative predictive value was defined as the probability that a negative VIDAS TXM result would be negative in the IgM ELISA. Overall agreement was defined as the percentage of specimens that were positive or negative in the IgM ELISA and that gave the same positivity or negativity in the VIDAS TXM (9). Statistical test. An analysis of variance test was used for the comparison of the distribution of the data in the VIDAS TXM and in the IgM ELISA. TABLE 1. Discrepant results between VIDAS TXM and IgM ELISA Value (result) for: Sample Dye test titere VIDAS IgM IgA AC/HS tese TXM ELISA ELISA ACHte" 159 256 0.74(+) 1.4(-) 2.6(+) 400/800 (A) 187 4,096 0.69 (+) 1.5 (-) 2.0 (+) 1,600/3,200 (A) 188 4,096 0.68 (+) 1.3 (-) 3.4 (+) 1,600/1,600 (A) 196 2,048 0.77 (+) 1.3 (-) 1.6 (+) 800/3,200 (A) 3 128 0.66 (+) 0.8 (-) 0.6 (-) 100/800 (NA) 59 128 0.74 (+) 1.5 (-) 0.5 (-) 50/800 (NA) 77 1,024 0.84 (+) 1.6 (-) 0.7 (-) 50/1,600 (NA) 79 256 0.67 (+) 0.9 (-) 0.8 (-) 50/3,200 (NA) 118 128 0.81(+) 0.9(-) 0.6(-) 50/800 (NA) 125 32 1.04 (+) 1.1 (-) 0.8 (-) 50/400 (NA) 133 128 1.17(+) 1.1(-) 0.7(-) 50/800 (NA) 170 64 0.86 (+) 1.1 (-) 0.6 (-) 50/200 (NA) 189 2,048 0.77 (+) 1.4 (-) 0.4 (-) 50/>3,200 (NA) 249 32 0.96 (+) 0.1 (-) 0.0 (-) <501<100 (NA) 5 64 0.25 (-) 2.0 (+) 0.5 (-) 50/400 (NA) a Values are reciprocals of titers. b NA, not an acute-phase pattern; A, acute-phase pattern. RESULTS CLIN. DIAGN. LAB. IMMUNOL. Results of the comparison of the VIDAS TXM and IgM ELISA are as follows. Of the 407 samples tested, 205 (50.7%) were positive in the IgM ELISA. Of the 205, 204 (99.5%) were positive and 1 was negative in the VIDAS TXM. Of the 201 (49.3%) serum samples that were negative in the IgM ELISA, 187 (93%) were negative and 14 (7%) were positive in the VIDAS TXM. Three serum samples gave equivocal results in the first run in the VIDAS TXM. Only 1 (0.2%) of the 407 samples that was negative in the IgM ELISA gave repeated equivocal results in the VIDAS TXM. This sample was not used in the remainder of the analysis, and the results are not included in the tables. The VIDAS TXM, compared with the IgM ELISA, had a 99.5% sensitivity, a 93% specificity, a 93.6% positive predictive value, a 99.5% negative predictive value, and an overall agreement of 96.3%. Discrepant results were noted for 15 serum samples. The data are presented in Table 1. Each was positive in the dye test; 14 were positive in the VIDAS TXM and negative in the IgM ELISA, and 1 was negative in the VIDAS TXM and positive in the IgM ELISA. Four of the serum samples (samples 159, 187, 188, and 196) positive in the VIDAS TXM and negative in the IgM ELISA were considered likely to have come from patients with relatively recent infections, since both the IgA ELISA and the AC/HS test were positive. The 10 remaining serum samples (samples 3, 59, 77, 79, 118, 125, 133, 170, 189, and 249) were positive in the VIDAS TXM and negative in the IgM ELISA and were likely from patients infected in the distant past, since the IgA ELISA and AC/HS tests were negative (Table 1). Only one serum sample was negative by VIDAS TXM and positive by IgM ELISA and was likely from a patient in the chronic stage of the infection, since the IgA ELISA and the AC/HS test were negative. The test values for quantification of the IgM antibodies by the VIDAS TXM and the IgM ELISA were compared. They revealed a good correlation between the distributions of the data (P = 0.0001; correlation coefficient = 0.837) (Fig. 1). Reproducibility and precision. Overall, with all samples tested, the results of precision tests (inter- and intra-assay) and reproducibility studies (Tables 2 and 3) for the VIDAS TXM

1 VOL. 1, 1994 VIDAS FOR THE DIAGNOSIS OF TOXOPLASMOSIS 403 8.0. 7.8 + 7.4 + A 7.2. A 7.0. AA 6.8 4 A A 6.6 * A 6. 4. A A A 6.2 + A A A 6.0 + A A 6.8 + A AA 5.6 * A A 5.4 + A A A 6.2. A 6.0 * A A AA B = 4.8 A A AA w 4.6 * A B A A > 4.4 + - 4.2 + A A A A 4.0 + A AA AA A B 3.8 + A A A A > 3.6 + A A A A A CZ cn 3.4 * A A A C 3.2. A A A A A 3.0 + A B A A A % 2.8. A AA A A B A Q 2.6 + A A A A A 2.4. B CBAA A AA A 2.2 * A A A AABA BAB 2.0. BB A A AAB C AA A 1.8 * B BABABB A 1.6 + A D A BAA A A A A 1.4 4 C BAAABABFC A 1.2 4 B AA AAAA B 1.0 4 B C A AA A AA 0.8 + A AADBDAAA 0.6 + C AAB CB A 0.4 + C AHAD 0.2 + ZZH11A A 0.0 * ZII8AA + + + 1 + + + 1 4 + 4 1+ 4 't 4 1,.,4 4,1 4,4 -, O-,4 4 4+, s1 4 4 1 + 4 4 f 4 4 4,. 4 4 + + 4 4 4 + 4 0 1 2 3 4 f; 6 7 8 9 10 11 12 13 IgM-ELISA Value FIG. 1. Scatter plot of results in the VIDAS TXM plotted against results obtained in the IgM ELISA. Letters represent the number of observations (times of occurrence) at any given point, where A = one observation, B = two observations, C = three observations, and so on, through the alphabet. In instances in which there were more than 26 observations at one point, the observations in excess of 26 are hidden (i.e., not seen), since Z corresponds to 26 observations. In this plot, there are 55 hidden observations. had coefficients of variation of less than 7.46 and 3.2%, cases (cases 2 and 3), the IgA ELISA did not become positive respectively. until some time after the tests for IgM had become positive. Seroconverters. The results for the four women who sero- We had previously noted that the level of IgA antibody in converted during gestation are shown in Table 4. In each of patients with acute acquired toxoplasma infection rises later these cases, the VIDAS TXM became positive at the same than does the level of IgM antibody. time as did the IgM ELISA. It is of interest that in two of the DISCUSSION TABLE 2. Intra- and interassay precision of the VIDAS TXM The data presented above reveal that there is excellent agreement between the results obtained with the VIDAS TXM Result Type of Intra-assay Interassay and those obtained with a comparison method, the doublevaluea Mean CV (%) Mean CV (%) Negative RFV 81.68 4.68 81.44 3.59 TV 0.08 6.12 0.07 6.43 Positive RFV 2,017.91 4.81 1,999.08 2.36 TV 1.93 4.81 1.96 3.66 Calibrator RFV 1,013.14 6.73 1,051.33 5.97 TV 1.10 6.66 1.03 7.46 a RFV, relative fluorescence value; TV, test value. b CV, coefficient of variation. TABLE 3. Reproducibility of the VIDAS TXM Serum sample RFV (mean)' CV (%)b Negative 61.8 3.2 Low positive 978 2.8 High positive 2,172 0.65 a RFV, relative fluorescence value. b CV, coefficient of variation.

404 CANDOLFI ET AL. CLIN. DIAGN. LAB. IMMUNOL. TABLE 4. Serologic test results for women who seroconverted during gestation Value (result) for": Patient Specimen Dye test result date' (lu/ml) VIDAS IgM IgA AC/HS test TXM ELISA ELISA A/Sts 1 9/8/89 <2 0.03 (-) 0.0 (-) 0.0 (-) <50/<100 (NA) 9/29/89 80 5.46(+) 10.0(+) 17.1(+) 200/100 (A) 10/12/89 800 6.01(+) 9.5(+) 15.4(±) 800/800 (A) 2 4/18/90 <2 0.07 (-) 0.4 (-) 0.0 (-) Not done 5/14/90 <2 0.07 (-) 0.0 (-) 0.0 (-) <50/<100 (NA) 6/28/90 80 0.92 (+) 2.1 (+) 0.0 (-) <50/<100 (NA) 7/30/90 80 0.90 (+) 2.0 (±) 0.1 (-) <50/<100 (NA) 9/13/90 1,600 1.55(+) 4.2(+) 1.0(-) 800/800 (A) 9/21/90 1,600 1.52(+) 3.6(+) 1.4(±) 1,600/1,600 (A) 3 3/13/90 <2 0.08(-) 0.0(-) 0.0(-) <50/<100 (NA) 4/17/90 20 0.53 (-) 1.8 (E) 0.0 (-) <50/<100 (NA) 5/14/90 200 1.83(+) 7.1(+) 0.3(-) 100/100 (A) 6/5/90 1,600 1.94 (+) 6.4 (±) 1.6 (±) 400/400 (A) 4 12/28/89 20 0.11 (-) 0.6 (-) 0.5 (-) <50/<100 (NA) 1/26/90 400 1.97(+) 4.0(+) 7.6(+) 200/<100 (A) 2/8/90 800 2.3 (+) 6.0 (+) 8.4 (+) 400/400 (A) 6/8/90 400 0.51 (-) 1.3 (-) 2.5 (±) 400/400 (A) a Month/day/year. NA, not an acute-phase pattern; A, acute-phase pattern; E, equivocal (or low-positive) result. sandwich IgM ELISA developed in our laboratories (11). This was true for the sera chosen for this study specifically because they were previously shown to be either positive or negative in our IgM ELISA. In addition, we studied sera from pregnant women who seroconverted during gestation. The latter group is critical to such a study, since early detection of IgM antibodies in them is very important (10, 12). Moreover, a positive result in an IgM test will not infrequently lead to abortion; in recent years, this has occurred in approximately 20% of women diagnosed in our serology laboratory as possibly having acquired their acute infection during or just prior to gestation (lla). Such abortions, which are often performed unnecessarily and at the request of the concerned pregnant woman, are all the more tragic when they occur in women who have false-positive IgM tests (12). In the United States, a single serum sample is usually all that is available on which to base a decision of whether a pregnant patient has recently acquired infection with T. gondii. This amplifies the need for dependable, reproducible accuracy of the method used for demonstration of the presence or absence of IgM antibodies. For this reason, the relative performances of commercially available assays require critical assessment, with the results being made available to all practicing laboratories. At present there is no evidence that in the United States there is satisfactory (if any) monitoring of either the performance or the quality control of commercial kits (for IgM toxoplasma antibodies). The fact that many of these kits are faulty has been clearly demonstrated on numerous occasions in our laboratory (unpublished data) as well as by other investigators (1, 14). How, then, is a physician able to make the proper interpretation of the test result? Among the discrepant results observed in our study, the presence of a positive VIDAS TXM and negative IgM ELISA was the most frequent (3.4% among all the sera studied). In four of these cases, the VIDAS TXM appeared to have greater sensitivity than the IgM ELISA for samples from patients in the acute stage of infection. This greater sensitivity was also noted with 10 additional serum samples which were from patients who appeared to have been infected in the more distant past (chronic pattern in the AC/HS test and negative IgA ELISA). In one case the VIDAS TXM was less sensitive. This serum sample was classified as non-acute phase by the AC/HS test and was negative by IgA ELISA. The presence of IgM antibodies in the chronic stage of the infection is not rare (2, 6), and detection of these IgM antibodies induces considerable variations in sensitivity and specificity among the assays (4, 8). These discrepancies may be due to differences in the antigen preparations used in the two methods; the IgM ELISA uses as the antigen preparation a lysate of the tachyzoite form of the parasite that contains both cell membrane and cytoplasmic antigens. In contrast, the VIDAS TXM depends solely on detection of the major membrane antigen of T. gondii. It should be recognized that the lack of an accepted standard for a reference test for detection of IgM antibodies in an infection that is most often asymptomatic makes the interpretation of serological test comparison difficult and suggests that at least certain of the discrepant results we observed in the present study cannot be classified definitely as being false-positive or false-negative results. In conclusion, the VIDAS TXM was user friendly and had excellent reproducibility, sensitivity, and specificity. Further investigation is necessary to define its ability to detect IgM toxoplasma antibodies in the infected immunocompromised patient, the fetus, and the newborn (3). ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service grants A104717 and A130320 from the National Institutes of Health; E. Candolfi is a recipient of a special NATO grant. REFERENCES 1. Ashburn, D., R. Evans, L. J. Skinner, M. W. Chatterton, A. W. L. Joss, and D. 0. Ho-Yen. 1992. Comparison of relative uses of commercial assays for Toxoplasma gondii IgM antibodies. J. Clin. Pathol. 45:483-486. 2. Bobic, B., D. Sibalic, and 0. Djurkovic-Djakovic. 1991. High levels

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