Evaluation of the VIDAS toxo competition assay for the detection of antibodies specific to Toxoplasma gondii in human sera

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Serodiagn.

Immunother.

Infect.

Disease

1994; 8: 41-45

Evaluation of the WDAS toxo competition assay for the detection antibodies specific to Toxoplasma gondii in human sera J Dave, A H Balfour, Toxoplasma

Reference

of

J Perkin

Laboratory,

Regional

Public

Health

Laboratory,

Leeds,

UK

Summary The Vitek lmmuno-Diagnostic Assay System (VIDAS) toxo competition (TXC) automated enzyme immunoassay (EIA) for the detection of antibodies in human sera specific for Toxoplasma gondii was evaluated in several groups of sera by comparison with the dye test (DT). The TXC showed good agreement with the DT using a modified procedure in which 75 pl of serum was added directly to the diluent well, which is useful in conserving sera. This automated assay is of potential value in determining the immune status to T. gondii. It is simple to perform, rapid and produces an objective result evaluated against a defined standard. Key words: Toxoplasma gondii, VIDAS toxo competition negative predictive values

assay, sensitivity,

Introduction Toxoplasma gondii is an obligate intracellular protozoan parasite. It can give rise to a wide range of clinical symptoms but the infection is usually self-limiting in the immunocompetent patient. A primary infection during pregnancy can give rise to stillbirths, foetal abnormalities and perinatal infectionsI. Reactivation of toxoplasma infections in immunocompromised patients may lead to encephalitis, myocarditis or pneumonitis*. Serological assays are the mainstay of diagnosis and the dye test (DT) remains the ‘gold standard’3. The need for live parasites, maintained by animal passage, has restricted the use of the DT to reference laboratories. With the increasing demand for testing, particularly in relation to pregnancy and immunocompromised patients, automated assays which are rapid and simple to perform are of potential value in the less specialized diagnostic laboratory.

Received: 14 December 1993 Accepfed: 16 December 1993 Correspondence und reprint requests to: J Reference Laboratory, Regional Public Health Path. York Road, Lekds Lsl5 7TR, UK 0 1994 Butterworth-Heinemann Ltd 0888-0786/94/O I (X)4 1-0s

specificity, positive and

The Vitek Immuno-Diagnostic Assay System (VIDAS) is a novel automated enzyme immunoassay system. It is rapid and simple to perform and developed for the measurement of specific antibodies against a wide range of infectious disease agent@. The assays use only minimal volumes of reagents and wash fluids, supplied as a sealed strip of preloaded wells. One strip is used for each serum tested. VIDAS kits for the detection of IgG6 and IgM specific for T. gondii are available. More recently, a competitive assay measuring the combined activity of specific IgG and IgM, VIDAS toxo competition assay (TXC), has been introduced for screening. In the TXC 125 ~1 of serum is loaded in a dry well from which the system transfers about 75 ~1 into a second diluent well. To conserve sera we modified the protocol by adding 75 ~1 of test serum directly to the diluent well and evaluated the TXC by comparison with the dye test (DT) for the detection of antibodies specific for T. gondii in selected groups of human sera. Materials and methods

Dave. Toxoplasma Laboratorv. Bridle

Sera tested

A total of 587 sera routinely sent to the Toxoplasma Reference Laboratory for detection of antibodies to T.

42

Serodiagn.

Immunother.

Infect. Disease

1994; 6: No 1

gondii were used for this study. These included 151 refrigerated sera processed within 48 h of receipt and 150 sera stored at -70” C after routine testing. Representative samples from selected subgroups included 99 stored sera with high IgM titres from patients with acute infection and 82 sera from a group consisting of neutropenic patients, patients with HIV and heart transplant recipients. Possible false positive reactions were monitored in: (a) 75 DT negative haemagglutination assay (HA) positive sera of which 27 were submitted as latex agglutination (LA) positive; (b) sera with autoantibodies (10 antinuclear factor (ANF) and 10 rheumatoid factor (RF) positive); and (c) 10 sera with IgM specific for Epstein-Barr virus (EBV). All sera were heat inactivated at 56” C for 30 min before initial testing. VIDA S The VIDAS is a novel automated enzyme immunoassay that uses a fluorescence detection systemh. The central processor unit can run up to five assays simultaneously, in batches of up to 30 strips. The TXC assay involves competition between specific antibodies to T. gondii in the patient’s serum and the anti-p30 monoclonal antibody present in a reagent well. The system uses a solid phase receptacle (SPR) which also acts as a pipettor. The pipette interior is precoated with toxoplasma antigen, a sonicate of the RH strain of T. gondii grown in mice. Strips containing the reagents have 10 wells with a foil seal, bar coded and colour coded to match the SPR. The test or control serum (125 ~1) is normally introduced into the initial dry well. during the run the SPR is lowered into the well and samples approximately 75 ~1. The strip is moved to present the reagent wells sequentially to the SPR as the assay proceeds. The final well is a cuvette in which fluorescence is detected with an optical scanner. Wells 2-5 contain the wash and dilution fluids with the anti-P30 monoclonal antibodyenzyme conjugate in well 6. Following further wash steps, the enzyme conjugate and antigen complex on the SPR is exposed to the substrate 4-methyIumbellifery1 phosphate in well 10. In the presence of the enzyme conjugate this is converted to a fluorescent product 4methylumbelliferone, detected by the scanner. The background reading of the cuvette and substrate is subtracted from the final reading to give a relative fluorescence value (RFV). The ratio of the sample and the standard RFV is calculated as the test value. A test value of >1.6 is given in the absence of toxoplasma antibodies in the sample, while in the presence of specific antibody there is competition and the value drops, A standard for calibrating the TXC assay was run fortnightly, while positive and negative controls were run in each batch of up to 28 tests. The minimum level of detection of toxoplasma antibodies by the TXC assay was stated as 2 IU ml-‘. To conserve sera the procedure was modified by loading 75 pl of the test serum directly into the diluent

in the second well, rather than 125 ul into the first well for autosampling by the system. Reproducibility

studies

The reproducibility of the modified TXC assay was measured using three positive sera with a range of DT values (216 000, 256 and 64 IU ml-i) and a negative serum (DT <2 IU ml-‘). Intra-assay reproducibility was measured by running the four samples five times in one run. Inter-assay reproducibility was measured by running the four sera once on each of five runs. Other serological

nssays

All sera were routinely tested by the DT7 and the indirect HAX. Sera with a false positive HA were also tested by a LA assay* (Eiken, Mast Diagnostics). IgM was measured by the p-capture enzyme-linked immunosorbent assay (ELISA)9 in sera with a dye test titre 216 IU ml-’ or if specifically requested on clinical grounds. Sera were regarded as positive in the DT at 22 IU ml-i (titre 28) a titre of 232 in the HA and a titre of 232 in the LA. Values of 235 enzyme immunoassay units (EIU) were regarded as positive in the IgM ELISA”. Sera showing discrepancies between the TXC and DT were retested in both assays with a third run performed if required to obtain a consensus result. Results A total of 587 sera were tested in the study. The number of positive and negative results for both assays, as well as the consensus results after retesting in the TXC are shown in Table 1. Data on sensitivity, specificity, positive and negative predictive values, as well as agreement with the DT are shown in Table 2. Initially 12 gave discrepant results compared to the DT, with four false positives and eight false negatives in the TXC (Table 3). After running the 11 sera available for retesting this fell to nine, two false positives and seven false negatives. Of the 301 routinely submitted sera screened by the TXC assay 114 (38%) were DT positive. In the group of 151 sera tested shortly after receipt, without freezing, there were 65 DT positive sera (43%). Three sera were classed as falsely negative in the TXC, confirmed on retesting, giving a sensitivity of 95.3% and a specificity of 100% with the TXC (Table 2). In the second group of 150 sera stored at -20” C before testing, 49 were DT positive (33%). Initially, there were four false positives in the TXC, giving a specificity of 96% and a sensitivity of 100%. On retesting this dropped to two false positives, increasing the specificity to 98%. For the combined group of 301 sera the TXC assay showed, on initial testing, a specificity of 97.9% and a sensitivity of 97.4% (Table 2). The predictive values of the positive and negative tests were 96.5% and 98.4% respectively. After retesting of discrepant sera the

Dave et al.: VIDAS

Table

1. Comparison

Serum

group

of the TXC with

the DT run with

toxo competition

selected

No. of sera

groups VIDAS

assay for detection

Sequential sera On receipt From storage t-20” C) Combined IgM positive immunocompromised False positive HA (DT-I* ANF+ RF+ EBV IgM+

151 150 301 99 82 75 10 10 10

TXC

Dye Tesi

I = initial agreement. R = consensus result after retesting. “27 of these sera also gave false positive

results

Table

2. Performance

of the TXC assay,

Serum

group

characteristics

in the

62 51 113 95 24 -

Positive

Negative

I

R

No.

%

No.

%

89 97 186 6 58 75 7 7 10

89 99 188 4 58 -

65 49 114 95 27 0 3 3 0

(43) (33)

86 101 187 4 55 75 7 7 10

(57) (67) (62) (4)

(38) (96) (33) (0) (30) (30) (0)

(67) (100) (70) (70) (100)

LA assay.

Sensitivity

Sequential sera On receipt From storage k-20” C) Combined IgM positive lmmunocompromised

Negative R

62 53 115 93 24 0 3 3 0

43

of sera

Positive I

of T. gondii

measured

against

Specificity

the DT on selected

PPV

groups

of sera

NPV

I

R

I

R

I

R

I

95.3 100 97.4 88.9

95.3 100 97.4 88.9

100 96 97.9 100

100 98 99 100

100 92.5 96.5 100

100 96.1 98.2 100

96.6 100 98.4 94.8

Agreement with DT R

96.6 100 98.4 94.8

I

R

98.0 97.3 97.7 98 96.3

98.0 98.7 98.3 100 96.3

I = initial agreement. R = consensus result after retesting. PPV = Positive predictive value. NPV = Negative predictive value. All figures are shown as percentages.

Table

3. Comparison

Serum no.

Serum

of the TXC with

group

the DT - discrepant DT (IlJ mt--‘I

sera HA

TXC 751-11

1 2 3 4 5 6 7 8 9 10

Tested on receipt Tested on receipt Tested on receipt From storage k-20” From storage (-20” From storage t-20” From storage t-20” IgM positive IgM positive lmmunocompromised

12 11

lmmunocompromised

C) C) C) C)

Class shows the consensus classification. A = results agree with the DT. F = results do not agree. + Positive, - Negative, / Not done.

1

2

4 4 2 <2 <2 <2 <2 32 8 4

4 <2 2 <2 <2 <2 <2 64 4 4

2

2I

~32 ~32 128 <32 ~32 ~32 64 I ~32 128

125 pi

7

2

3

+ + + + -

+ + + +

7 I

7

T 7

Class

1

Class

FFFAF+ AF+ A+

-

FFFI F+ I F+ A+ A+ A+

;,

7 ; + + +

;I 7I

FF-

;/

:

44 Table

Serodiagn.

Immunother.

4. Intra- and inter-assay

Infect. Disease reproducibility

Dye Test IU ml-’

in the TXC assay

Intra-assay Mean

>I 6 000 High 256 Mid range 64 Low positive <8 Negative

1994; 6: No 1

Inter-assay

Range

CV%

Mean

Range

CV%

8-9

5.45

9

8-10

11.11

145.6

142-153

2.94

163*

145-179

8.81

240.0

180-409

39.97

223

196-267

11.97

2436.6

2427-2477

1.79

2484.8

2427-2503

2.48

a.2

Relative fluorescence values are shown. CV% = coefficient of variation. Mean of four runs only, five runs were

available

for all other

mean

specificity increased to 99% and the sensitivity remained at 97.4%, with predictive values of 98.2% and 98.4% respectively for the positive and negative tests. A total of 99 IgM positive sera included paired sera from 22 patients. On initial testing the TXC gave 97 positive and two negative results. This changed to 98 positives on retesting, with one false negative result. These two sera were both positive when tested using a loading of 125 ul in the dry well. Of the 75 sera identified as HA positive but DT negative, 27 had been submitted as being LA positive. The LA results were confirmed by retesting, while the remaining 48 sera were LA negative. These sera were all negative in the TXC, as were the ANF, RF and EBV-IgM positive sera (10 of each). No false positive reactions were given in the TXC assay by these selected groups of sera. In the group of 82 sera from immunocompromised patients there were 27 DT positive (22 IU ml-‘) and 55 DT negative sera (Table l), with 79 (96.3%) (Table 2) showing agreement between the TXC assay and DT, giving a sensitivity of 88.9% and a specificity of 100% in the TXC. The three discrepant sera had low DT levels but were negative in the TXC, the two sera available for retesting remained negative. Reproducibility studies

The results for the four sera run in the reproducibility studies are shown in Table 4. These are expressed as relative fluorescence values, the mean and range are shown, as well as the coefficient of variation. The intraassay variation was low for three of the sera l&5.5% but the low positive sample (DT 64 IU ml-i) gave a high value of 40%. The inter-assay values ranged from 2.5 to <12%. Retesting of discrepant sera with 125 /.~l

Of the 12 discrepant sera listed in Table 3, eight were available and retested with 125 ul loaded into the initial dry well. The discrepancy was resolved for two of the sera. For one serum the test value was very near

values.

the cut-off value, with both loading volumes. The other serum was positive with both volumes but had given a negative result twice when diluted into the second well. Discussion Sera submitted for toxoplasma serology were tested in the TXC on receipt or after storage at -20” C. The sensitivity and specificity for the two groups were similar, suggesting that storage at -20” C does not interfere with the TXC assay. For the total 301 samples the overall sensitivity and specificity after resolution of discrepant results was high at 97.4% and 99% respectively. When the assay is used as a screening test followup samples should be requested at 10-20 days to monitor for evidence of a seroconversion, particularly if there is a strong clinical suspicion of toxoplasmosis. The TXC detected specific antibody in 97 of the 99 IgM positive sera. On retesting the two discrepant sera the low levels of specific antibody in the DT were confirmed. The TXC remained negative for one serum but became positive for the second, confirmed as positive by a further test. Both of these sera were positive when 125 ul was loaded into the dry well of the assay strip. Thus, the TXC gave one false negative with the 75 ul loading by comparison with the DT, although agreement was obtained using the recommended loading of 125 ~1. With one serum from the immunocompromised group the TXC had given a negative result for two of three tests at a 75 ul loading taken as negative by consensus, while the 125 l.tl loading gave a positive result. These discrepancies occurred at low levels of specific antibody as measured by the DT (~10 IU ml-‘). For the other six sera tested at both levels of serum loading the TXC results showed no change. There were four sera in the IgM group with DT <2 IU ml-i, as part of a series from patients who had seroconverted. All of the selected sera giving false positive HA or LA results relative to the DT were negative by the TXC, as were the sera from the ANF and RF patients, as well as the sera with IgM specific for EBV. This suggests that false positive results are less likely to occur in the TXC when these types of sera are tested. Some of the sera regularly detected as giving false

Dave et al.: VIDAS

positives by the LA and HA would be eliminated if the TXC was used as a screening test. In the group of 82 sera from immunocompromised patients. three sera (3.7%) gave false negative results in the TXC, however. the DT titres for these sera were low (4 II7 mll”. Many commercial assaysare designed to detect specific antibody only as low as 10 IU mll’. Of the 12 sera in this study which did not show initial agreement between the TXC and DT only one had an antibody level >10 IU ml-’ (serum 8, Table 3). After two retests, the consensus TXC result for this serum was positive, suggesting that the initial negative result can be attributed to a technical error. This sample was also positive when tested by loading 125 ul in the initial dry sampling well. Eleven of the sera in the discrepant group were available for retesting and two of these were resolved on further testing. The remaining nine sera consisted of seven false negatives and two false positives in the TXC. In the false negative group three sera were from immunocompromised patients, one was badly haemolyzed. while the other two samples had TXC values very near the assay cut-off (data not shown). Serum 2 had a low DT value and negative HA, on retesting the DT was negative but there was no serum available to resolve the DT in a further run. On balance. this appears to be a negative serum and was negative in the TXC on three runs. Sera 1 and 3 had low DT titres and were not detected by the TXC with either a 75 yl or I25 yl serum loading. Serum 9, with a DT of 8 IU mll was negative by the TXC assay at 75 ul but gave a positive result when tested with a 125 yl loading in the initial well. The two sera giving false positive reactions in the TXC had test values well below the cut-off value. Serum 7 (Table 3) was from a heart-lung transplant recipient who had received hyperimmune serum immunoglobulin, as well as blood transfusions. These may have interfered with the assay, antibody was also detected in the HA but the DT was negative. The other false positive serum (5) was from a donor screened in an IVF clinic, although the DT was negative antibody was detected by the HA. The problem of non-specific reactions in ELISA systems has been described previously’“, uniform antigen coating is an important factor”. In the TXC assay neat serum is briefly in contact with the antigen coated on the inside of the SPR during the initial sampling stage. In bypassing this initial step and adding the serum directly to the diluent in the second well. some loss of sensitivity might be expected. This is of more significance if antibody levels are very near to the cut-off value. The sera classed as false negatives in this study all had antibody levels of
toxo competition

assay for detection

of T. gondii

45

In the present study we modified the TXC assay procedure by loading 75 ul of serum directly to the dilution well and demonstrated good agreement with the DT. Although this is not recommended by the manufacturers, it is a useful way to conserve the 50 ul of sera which would otherwise be lost in the sample well. This can be important when working with serum from a neonate. The TXC is an automated, 40 min assay,simple to set up and run on the VIDAS workstation. All the reagents, diluents and wash fluids are contained within a sealed strip; this and the sensitized solid phase SPR are the only materials requiring disposal. The assay should be of particular value in laboratories with a VIDAS workstation wishing to include toxoplasma screening in their range of assays. The TXC can be run on a varying number of samples. at short notice. However, it is recommended that positive sera should also be investigated in a quantitative assayfor both IgG and IgM. Further work may also be required at a Toxoplasma Reference Laboratory. Acknowledgements We are grateful to Mrs Glyn Yeadon for her help in the preparation of this manuscript. References Hall SM. Congenital toxoplasmosis.Rr Men J 1992;305: 291-7 Ruskin J, Remington JS. Toxoplasmosisin the compromisedhost. Ann Intern Med 1976; 84: 193-9 Sabin AB, Feldman HA. Dyes asmicrochemicalindicators of a new immunity phenomenonaffecting a protozoan parasite(Toxoplasma).Science 1948; 108:660-3 4 JamesK. Immunoserologyof infectious diseases.Clin Microbial

Rev 1990: 3: 132-52

5 Hopson DK, Niles AC, Murray PR. Comparisonof the Vitek ImmunodiagnosticAssay System with three immunoassaysystemsfor detection of Cytomegalovirus- Specific Immunoglobulin G. J Clirz Microbial

1992: 30: 2983-95

6 Sandin RL, Knapp CC, Hall GS et al. Comparisonof the Vitek ImmunodiagnosticAssay. System with an Indirect Immunoassay(Toxostat Test Kit) for detection of Immunoglobulin G antibodiesto Toxoplasma gondii in clinical specimens.J Clin Microbial 1991; 29: 2763-7 7 Hunter D, Chadwick P, Balfour AH, BridgesJR. Examination of ovine foetal fluids for antibodiesto Toxoplasma gondii by the dye test and an indirect immunofluorescencetest specific for IgM. Br Vet .I 1982; 138: 29-34

8 Balfour AH, Fleck DG, Hughes HPA, Sharp D. Comparative study of three tests(dye test. indirect haemagglutinationtest, latex agglutination test) for the detection of antibodies to Toxoplusma gondii in human sera.J Clin Path 1982;35: 228-32 9 Payne RA, Joynson DHM, Balfour AH, Harford JP, Fleck DG, Mythen M, SaundersRJ. Public Health Laboratory Service enzyme linked immunosorbentassay for the detection of toxoplasmaspecific IgM antibody. J Clin Path 1987; 40: 276-81 10 Wood HC, Wreghitt TG. Techniques.In: Wreghitt TG, Morgan-Capner P. eds. ELISA in the clinical microbiology luborutory. London: PHLS, 1190:6-21 11 ChcssumBS. Denmark JR. Inconsistent ELISA. Latwt 1978:i: 161