JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2763-27670095-1137/91/122763-05$02.00/0Copyright © 1991, American Society for Microbiology

Vol. 29, No. 12

Comparison of the Vitek Immunodiagnostic Assay Systemwith an Indirect Immunoassay (Toxostat Test Kit) for

Detection of Immunoglobulin G Antibodies to Toxoplasma gondiiin Clinical Specimens

R. L. SANDIN,t C. C. KNAPP, G. S. HALL,* J. A. WASHINGTON, AND I. RUTHERFORD

Department of Microbiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received 4 June 1991/Accepted 10 September 1991

The value and convenience of testing for specific anti-Toxoplasma gondii antibodies have led to thedevelopment of various antibody detection methods, such as the enzyme immunoassay. Two enzyme

immunoassays, the Vitek Immuno-Diagnostic Assay System (VIDAS; Vitek Systems, Hazelwood, Mo.) and theToxostat Test Kit (TST; Whittaker Bioproducts, Walkersville, Md.), were compared for their ability to detectT. gondii immunoglobulin G antibodies in fresh human sera. Specimens were tested according to theinstructions of each manufacturer. Of 304 serum specimens tested, the results for 282 (93%) agreed in bothassays (77 were positive and 205 were negative). The results for the remaining 22 (7%) specimens were

discrepant; 20 of the 22 specimens tested low positive or equivocal by Toxostat and negative by VIDAS. Uponretesting, 8 of these 22 specimens were in concordance, increasing the level of agreement to 95.3%. Intra-assayreproducibility was tested with four to nine replicates of each of seven samples (four positive, one negative, andtwo equivocal) by both tests and six replicates of one standard by VIDAS. Coefficients of variation (CVs) forVIDAS were 6, 8, 10, 15, 18, 19, and 23% for the samples and 14% for the standard. For Toxostat the CVswere 2, 8, 10, 10, 13, and 16%. In general, CVs for VIDAS were slightly larger than the CVs for Toxostat.Day-to-day variability over 3 days was tested by VIDAS for only six samples (three positive, two negative, andone equivocal) and four standards; CVs ranged from 2 to 15% for the samples and from 6 to 13% for thestandards. In conclusion, VIDAS is a rapid, convenient, non-labor-intensive, and easily performed test for thedetection of immunoglobulin G antibodies to T. gondii in serum specimens.

Serological tests constitute the cornerstone in the diagno-sis of infection with Toxoplasma gondii. This is because theparasite is very difficult to culture (4), the clinical diagnosisis often difficult to make, and the number of organisms frombiopsy specimens of involved lymphoid tissues and othersites present on histopathological slides may be very small(1). Three of the earliest and best-characterized serologicaltests for the diagnosis of toxoplasmosis have been theSabin-Feldman dye test (11), the indirect hemagglutinationassay (6), and the indirect immunofluorescence test (14).Various enzyme immunoassays (EIAs) have also been eval-uated for their ability to detect anti-T. gondii antibodies inserum (8, 9, 12, 13, 17, 18).The Vitek Immuno-Diagnostic Assay System (VIDAS;

Vitek Systems, Hazelwood, Mo.) is a novel, versatile, highlyautomated instrument which uses EIA technology for thedetection of antibodies in patient sera as well as antigensfrom different organisms in other body fluids. Its perfor-mance in the detection of antibodies to the human immuno-deficiency virus type 1 was very favorable when it wascompared with standard procedures, according to a prelim-inary report (7). No published reports exist, however, com-paring VIDAS with other EIAs for their ability to detect T.gondii immunoglobulin G (IgG) antibodies in human sera.Thus, we report here the results of a comparative study ofthe VIDAS T. gondii IgG Assay (TXG) with the ToxostatTest Kit (TST; Whittaker Bioproducts, Walkersville, Md.)

* Corresponding author.t Present address: Pathology Service, H. Lee Moffitt Cancer

Center and Research Institute, Tampa, FL 33612-9497.

(16) for the detection of IgG anti-T. gondii antibodies in 304fresh, unfrozen serum samples as well as the results of intra-and interassay reproducibility studies with VIDAS.

(This work was presented in poster form during the 91stGeneral Meeting of the American Society for Microbiology,Dallas, Tex., 5 to 9 May 1991.)

MATERIALS AND METHODSSerum samples. A total of 304 serum samples were ob-

tained from specimens submitted to the Department ofMicrobiology at the Cleveland Clinic Foundation for evalu-ation of IgG antibodies to T. gondii. Each serum sample wasstored at 4 to 8°C for 1 to 3 days and was tested by theVIDAS T. gondii IgG Assay (TXG) and the TST. Only onesample from each patient was accepted for the study.Samples that had the same results by both methods were

not retested; samples with discrepant results during theinitial run were retested one or more times by each method.Seven serum samples (four positive, one negative, and twoequivocal by VIDAS) were selected for testing intra-assayreproducibility by both methods. One standard was testedfor intra-assay reproducibility by TXG alone. This consistedof retesting of samples during one run between four and ninetimes, depending on the amount of serum available. Day-to-day reproducibility studies were performed on the VIDASby test repetitions of six samples (three positive, two nega-tive, and one equivocal) on 3 consecutive days and of fourstandards on 4 to 15 different days. Samples used in theintra-assay and day-to-day variability studies were stored at-70°C for up to 3 weeks prior to use.TXG. The Vidas TXG (Vitek), an enzyme-linked fluores-

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2764 SANDIN ET AL.

.-

.......

.o~.>.40;,g:ib

s

0 e. XC

il iii 1111 11e ,.d'l I.. '.. s

il il.1 1. Il. 1 1 fl. l:l il 1 Il

FIG. 1. The SRSs used in the assay contain all the requiredpipette-tip-like disposable devices called SPRs.

cent immunoassay, was performed by using the VIDASinstrument according to the directions of the manufacturer,as follows. Solid-phase receptacles (SPRs), pipette tip-likedisposable devices, serve both as the solid phase as well asthe pipettor for the assay. The interior surface of each SPRis coated with sonicated, inactivated tachyzoites of the RHstrain of T. gondii from peritoneal extracts of infected mice.SPRs are used in conjunction with sealed reagent strips(SRSs), which are 10-well polypropylene strips that containthe required assay reagents, including dilution, wash, andconjugate fluids, in separate wells (Fig. 1). A maximum of 30SRSs can be processed by the VIDAS TXG in a single run.A 200-pul serum sample was introduced into well 1 of theSRS. Each strip was placed into the tracks of the instrument,and all subsequent steps were performed automatically bythe VIDAS TXG module in less than 60 min. Each sampleundergoes preliminary dilution and wash steps within wells 2to 5, during which samples are cycled in and out of the SPRfor specified lengths of time. Anti-T. gondii IgG antibodiesthat may be present in the test serum bind to the T. gondiiantigens that coat the SPR, while unbound sample compo-nents are washed away. The detector antibody conjugatepresent in well 6 (goat anti-human IgG conjugated to alkalinephosphatase) is then cycled in and out of the SPR to attachto any human IgG bound to the antigen on the SPR wall.Final wash steps in the subsequent three wells removeunbound anti-human antibody conjugate.The final well, well 10, is an optically clear cuvette

containing a fluorescent substrate, 4-methylumbelliferylphosphate. Upon being introduced into the SPR, any re-maining enzyme catalyzes the conversion of this substrate toa fluorescent product, 4-methylumbelliferone. The intensityof fluorescence is measured by the VIDAS optical scanner.Results are subsequently analyzed automatically by thecomputer interface. A standard, representing a T. gondiiantibody titer of approximately 1:16 by the indirect immu-nofluorescence test, is run, and results for the sample arecompared with results for the standard. Test values aregenerated for each sample from the ratio of the relativesample fluorescence reading to that of the standard. A

reagents within separate wells. They are used in conjunction with

negative and a positive control are also run with each set ofSRSs. Test values are compared with predetermined thresh-old values to yield an interpretation which is then printed bythe instrument. Test values of <0.27 were considered nega-tive results. Test values of between 0.27 and 0.33 wereconsidered equivocal results and the test was repeated witha fresh specimen. If it is not practical to obtain a freshspecimen, the original specimen should be evaluated onceagain. Test values greater than or equal to 0.34 were consid-ered positive results and, thus, provide evidence of previousexposure to the parasite.TST. The TST (Whittaker Bioproducts) is an enzyme-

linked immunoassay for the detection of T. gondii IgGantibodies in human sera and was performed according tothe directions of the manufacturer, as follows. Briefly,96-well microplates precoated with purified, whole, unsoni-cated Toxoplasma organisms were used in this assay. Wellswere prewashed three times with phosphate-buffered saline-Tween solution, and 100 ,ul of each patient serum samplediluted 1:20 was transferred to the appropriate wells. Plateswere placed on a plate shaker for 15 min at room tempera-ture. Wells were then washed three times with phosphate-buffered saline-Tween to remove unbound antibody. Then,100 pul of lx alkaline phosphatase-conjugated anti-humanIgG was added to each well and the plates were placed on aplate shaker for 15 min at room temperature. Wells werewashed three times with phosphate-buffered saline-Tweento remove unbound conjugate. To each well, 100 pil ofsubstrate, phenolphthalein monophosphate, was then added.Plates were incubated at room temperature for 15 min. Tostop the reaction, 200 pul of stop reagent solution, tribasicsodium phosphate, was added to each well, the plates weremixed in a plate shaker for 2 min, and the A550 was thendetermined on an EIA reader (model EL 308; Biotek).Positive and negative controls were tested in each run.Absorbance values were interpreted by comparison with alow-range-positive calibrator. The ratio of patient sampleabsorbance to that of a low-range-positive calibrator is thepredictive index value. A predictive index value of <0.80was considered negative and indicates no evidence of previ-

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T. GONDII IgG DETECTION BY VIDAS 2765

TABLE 1. Results with 304 serum samples tested by TXG andTST on an initial runa

No. of samples with the following TST result:TXG result

Negative Equivocal Positive

Negative 205 10 9Equivocal 0 0 1Positive 1 1 77

a There was 93% agreement between the two tests.

ous exposure to the parasite. Values ranging from 0.80 to0.99 were considered equivocal. Samples were run in dupli-cate and, if the results were equivocal, the result was

reported as equivocal and a new patient serum sample was

requested. Values greater than or equal to 1.00 were positiveand indicate prior exposure. Semiquantitation was evaluatedas follows: predictive index values of between 1.00 and 2.49were considered low-range positives, those between 2.50and 4.59 were considered mid-range positives, and thoseequal to or above 4.60 were considered high-range positives.

Statistical evaluation. The level of agreement betweenTXG and TST was calculated as a function of the percentageof concordant samples during the initial sample run andfollowing a rerun of the discrepant samples. Coefficients ofvariation (CVs) were calculated for each method duringintra-assay and day-to-day reproducibility studies.

RESULTS

Initial run by both assays. Of 304 serum samples tested byTXG and TST, 282 provided concordant results by bothmethods during an initial run; 77 were positive and 205 werenegative (Table 1). The agreement between the assays was

93%. Of 22 samples with discrepant results during the initialrun, 9 were positive by TST and negative by TXG, 10 wereequivocal by TST and negative by TXG, and 1 was positiveby TST and equivocal by TXG (Table 1). Nine additionalsamples from three patients whose original samples showeddiscrepancies were received in the laboratory. Testing ofthese nine samples by both assays revealed the same patternof discrepancies as those in the original samples: positive orequivocal by TST and negative by TXG. The reported 93%agreement between both assays does not reflect these nineadditional samples.

Retesting of discrepant samples. The 22 discrepant sampleswere retested one or more times by both assays (Table 2). Ofthe 22 samples, 8 showed concordant results between TXGand TST during the repeat run, and the agreement increasedto 95.3%. By TST, 12 of 22 samples (55%) showed the same

interpretive results when the original and repeat runs were

compared; by TXG, 16 of 22 samples (73%) were identical.Intra-assay reproducibility studies. Seven samples were

selected for testing of intra-assay reproducibility by bothmethods; four low-range positive, one negative, and twoequivocal samples were tested by TXG during the initial run.

One TXG standard (lot 910415-1) was also tested. Descrip-tive statistics and CVs calculated for each sample by bothmethods are given in Table 3. In general, CVs by TXGranged from 6.1 to 23.5% and were slightly greater than theCVs by TST, which ranged from 2 to 16.5%.

Day-to-day reproducibility studies. Table 4 provides thedescriptive statistics and CVs for a day-to-day reproducibil-ity study performed with the VIDAS TXG on six samplesrun on 3 different days (three positives, two negatives, and

TABLE 2. Results for all serum samples after retesting 21 of 22serum samples that showed discrepancies on initial testing

No. of samples with the following TST result:TXG result

Negative Equivocal Positive

Negative 211 4 4Equivocal 0 2 3Positive 0 1 78

a There was no serum remaining for 1 of the 22 specimens which showeddiscrepant results on initial testing. Thus, only 21 of the 22 discrepant serumsamples were retested. There was 95.3% agreement between the two tests.

one equivocal during the initial run) and on four standardsrun on 4 to 15 different days. The CVs ranged from 2 to 15%.Since the specimens used for the intra-assay reproducibilitystudies by TXG were different from those used during theday-to-day reproducibility studies, the CVs from the twostudies could not be compared.

DISCUSSION

Because the Sabin-Feldman Dye test is considered the"gold standard" for the laboratory identification of infectionwith T. gondii and VIDAS TXG and TST are both EIAs,agreement between immunoassays rather than sensitivityand specificity were calculated. Of 304 serum samplestested, results for 93% of them agreed by both tests duringthe initial sample rn. Most discrepant samples followed aconsistent pattern: positive or equivocal by TST and nega-

TABLE 3. Descriptive statistics and CVs calculated for samplesselected for intra-assay reproducibility study

performed by TST and TXG

Relative fluorescence units orpredictive inner value

Test and No boriginal value Min- Max- CV

Mean Median SD imum imumvalue value

TXGSamples

0.39 (+) 5 0.422 0.42 0.033 0.39 0.46 7.80.35(+) 7 0.397 0.40 0.024 0.35 0.42 6.10.34(+) 10 0.383 0.37 0.072 0.26 0.51 18.80.34(+) 10 0.356 0.36 0.068 0.25 0.47 19.10.32 (equiv) 10 0.309 0.31 0.030 0.26 0.36 9.70.30 (equiv) 10 0.329 0.31 0.077 0.23 0.50 23.50.14(-) 10 0.171 0.17 0.026 0.14 0.21 15.0

Standard 7 1,156.8 1,124 160.0 912.0 1,387 13.8

TST1.40 (+) 7 1.582 1.62 0.125 1.40 1.70 7.91.24(±) 9 1.453 1.48 0.189 1.23 1.77 13.01.15 (+) 9 1.408 1.48 0.232 1.05 1.69 16.51.14 (+) 8 0.934 0.91 0.092 0.86 1.14 9.80.97 (equiv) 8 0.966 0.97 0.019 0.94 1.00 2.00.82 (equiv) 7 0.691 0.71 0.070 0.62 0.82 10.1

a +, positive; equiv, equivocal; -, negative.b Number indicates total number of repetitions plus the original run.

'Values for TXG are reported as relative fluorescence units and values forTST are reported as the predictive inner value.

d Values for the samples are test values, or ratios of the relative fluores-cence reading of the sample to that of the standard.

e The standard was lot 910415-1.

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TABLE 4. Descriptive statistics and CV of a day-to-dayreproducibility study performed by TXG on six

samples and CV for four standards

Sample anda % CV % Matched' ~ Fractionoriginal value matched

TXG sample0.60 (+) 9.6 100 7/70.59 (+) 7.8 100 7/70.34(+) 2.4 100 3/30.30 (equiv) 4.8 75 3/40.24 (-) 14.1 100 3/30.20 (-) 15.1 100 7/7

Standards1,223.63 10.51,201.33 12.61,109.75 6.3810.25 11.8

a Values for samples are test values. All samples were run on threeconsecutive days. +, positive; equiv, equivocal; -, negative.

b Percentage of times rerun sample matched the original category.C Fraction of rerun samples that matched original category.d Values for standards are mean relative fluorescence values. The four

standards were run on 15, 8, 4, and 4 different days for the values from top tobottom, respectively.

tive by VIDAS TXG. Several possible explanations exist tointerpret this finding. It has been reported that there arepotential inconsistencies in test kits used for EIAs (2, 10).The uneven attachment of antigen to the polystyrene in platewells or solid-phase receptacles could lead to variations inthe amount of antigen that coats the solid phase (2). Theantigenic determinants of T. gondii detected by the twosystems may well be different, as may the enzymes used, thedegree of enzyme labeling, the site of cross-linkage betweenenzyme and antibody, the test configuration, and the detec-tion system (10). The above differences can also help toexplain some of the variations in reproducibility obtainedbetween TXG and TST in the intra-assay and day-to-dayvariability studies. Lot-to-lot variability could also influenceresults, as could the learning process inherent in workingwith a new instrument.Plasma constituents in serum samples may also preferen-

tially affect the activity of one enzyme over another (10).This was observed when nine additional samples from threepatients whose original samples showed discrepancies be-tween TXG and TST were tested. All nine samples wereonce again discrepant and followed the same pattern ob-served with the first set of samples. One of the three patientshad recently undergone a heart transplant and had receivedseveral doses of hyperimmune serum globulin to decreasethe chance of infection and rejection. The serum fromanother one of these patients produced nonspecific reactionswhen it was tested for the presence of anti-T. gondii IgMantibodies by an immunofluorescence assay.The TXG in combination with the VIDAS instrument

offers the user several advantages over other EIAs whichuse the more traditional microtiter plate system. The self-contained nature of the disposable SRSs and SPRs can helpto alleviate the technologist's concerns about handling spec-imens potentially infected with hepatitis B virus or humanimmunodeficiency virus type 1. A high degree of automationmakes the test procedure relatively non-labor-intensive andhelps to free up the technologist's time. The instrumentlends itself to the processing of as few or as many samples ata time (maximum, 30 SRSs per run) as might be necessary

without having to resort to a batching process. The instru-ment is versatile; when it is combined with different softwarepackages, the same hardware can be used for antigen as wellas antibody detection. Finally, the assay is quick. The totalprocessing time for the TXG was less than 1 h.On the other hand, the TXG requires a relatively large

volume of serum for processing (i.e., 200 ,ul). Efforts by thecompany are in progress to decrease the required volume to100 ,ul. The TST requires only 10 to 20 ,ul of serum. Atpresent, the TXG is qualitative in nature and is not used forthe detection of changes in antibody levels, such as thosethat may occur with activation, but it is used to screen forprevious exposure to T. gondii. The TXG also does notdistinguish between IgG antibodies acquired recently andthose present in chronic (latent) infections. Several novel,still experimental approaches have been described recentlythat can distinguish between these two categories of IgGs ina single test (i.e., without concurrent detection of IgMantibodies). Suzuki et al. (15) reported that the use ofpurified acetone-treated, tachyzoite-specific antigens candistinguish between antibodies to acute-stage-specific anti-gens and antibodies developed during chronic infections insingle serum samples. Hedman et al. (5) used urea, ahydrogen bond-destroying agent, to selectively elute anti-bodies with low antigen-binding avidity from immobilizedantigen. Antibodies with low antigen-binding avidity werefound to be more characteristic of recent primary infectionswith T. gondii than they were of chronic infections. Thiscould have great diagnostic value during pregnancy, becausereactivation of latent T. gondii infection does not result incongenital toxoplasmosis, while acute primary infectionduring pregnancy may (3). Both stages could be totallyasymptomatic. In the immunosuppressed host, on the otherhand, reactivation of latent infection could be life-threaten-ing.

Highly versatile, rapid, accurate and automated assays,such as the TXG performed on VIDAS hardware, couldincrease laboratory productivity by increasing the numberand frequency of in-house tests that would otherwise be sentout to reference laboratories that can be offered by hospitallaboratories.

REFERENCES1. Brooks, R. G., R. E. McCabe, and J. S. Remington. 1987. Role

of serology in the diagnosis of toxoplasmic lymphadenopathy.Rev. Infect. Dis. 9:1055-1062.

2. Chessum, B. S., and J. R. Denmnark. 1978. Inconstant ELISA.Lancet i:161.

3. Desmonts, G., and J. Couvreur. 1974. Congenital toxoplasmo-sis: a prospective study of 378 pregnancies. N. Engl. J. Med.290:1110-1116.

4. Fleck, D. G. 1989. Laboratory techniques. Annotation: diagno-sis of toxoplasmosis. J. Clin. Pathol. 42:191-193.

5. Hedman, K., M. Lappalainen, I. Seppaia, and O. Makela. 1989.Recent primary toxoplasmosis infection indicated by a lowavidity of specific IgG. J. Infect. Dis. 159:736-740.

6. Jacobs, L., and M. N. Lunde. 1957. A hemagglutinin test fortoxoplasmosis. J. Parasitol. 43:308-314.

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13. Smith, S. B., and C. F. Repetti. 1987. Evaluation of a rapidscreening immunoassay for antibodies to Toxoplasma gondii. J.Clin. Microbiol. 25:2207-2208.

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15. Suzuki, Y., P. Thulliez, and J. S. Remington. 1990. Use ofacute-stage-specific antigens of Toxoplasma gondii for serodi-agnosis of acute toxoplasmosis. J. Clin. Microbiol. 28:1734-1738.

16. Van Enk, R. A., K. K. James, and K. D. Thompson. 1991.Evaluation of three commercial enzyme immunoassays forToxoplasma and cytomegalovirus antibodies. Am. J. Clin.Pathol. 95:428-434.

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18. Wilson, M., D. A. Ware, and K. W. Walls. 1987. Evaluation ofcommercial serodiagnostic kits for toxoplasmosis. J. Clin. Mi-crobiol. 25:2262-2265.

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