INTRODUCTION

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The diagnosis of infectious mononucleosis is usually based on typical clinical and hematologic findings and confirmed with a positive test for heterophile antibodies. However, in some cases there is a need for analysis of Epstein-Barr virus (EBV)-specific antibodies, especially when there are atypical symptoms or in the absence of heterophile antibodies. This is often observed with specimens from children, who also may have an unusual EBV antibody pattern. Tests used for the virological diagnosis of mononucleosis should have both high sensitivity and high specificity.

In Norway, tests for detection of both EBV-specific and heterophile antibodies are performed at the majority of the microbiological laboratories. Our regular quality assessment system has revealed the need for better control with the use of commercial tests. Also, over the past few years, some newly developed tests have been introduced. Together with nine other microbiological laboratories, the Department of Virology at the National Institute of Public Health (NIPH) in Oslo, Norway, conducted an evaluation of a total of 12 commercial tests for detection of EBV-specific and heterophile antibodies.

	MATERIALS AND METHODS

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Participants in the study. The microbiological departments in the following counties in Norway participated in the study: Aust- and Vest-Agder, Akershus, Bergen, Nordland, Oslo (NIPH, the National Hospital, and Ullevål Hospital), Rogaland, Sogn and Fjordane, and Vestfold.

Tests evaluated. Table 1 gives information on the EBV serological tests evaluated, including manufacturers, antigens, test principle, and mode of detection. All enzyme-linked immunosorbent assay (ELISA) tests were based on microwell enzyme immunoassay (EIA). Table 2 gives similar information on tests for heterophile antibodies.

Test panel. A test panel consisting of 248 and 241 serum specimens for the EBV antibody and heterophile antibody evaluations, respectively, was selected on the basis of both clinical diagnosis and results of laboratory investigations. Before distribution to the sites, the sera received code numbers. Specimens were assigned to the following groups.

(i) Group A. Group A consisted of specimens from patients with recent primary EBV infection. The diagnosis was confirmed by a positive test for heterophile antibodies and an EBV antibody pattern compatible with recent primary infection. A total of 139 and 140 serum specimens were tested for EBV-specific and heterophile antibodies, respectively.

(ii) Group B. Group B consisted of eight serial dilutions (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1,280) of serum from a patient with recent primary EBV infection. The purpose was to register eventual differences in the level of sensitivity between different tests, in particular for the various immunoglobulin M (IgM) tests for EBV-specific antibodies. The dilutions were prepared at the Department of Virology, NIPH, before distribution to the laboratories. Undiluted serum was not investigated.

(iii) Group C. Group C included 23 serum specimens from immunocompromised patients with current cytomegalovirus (CMV) infection. In this group, detection of CMV pp65 antigen in leukocytes was the criterion for selection.

(iv) Group D. Group D included serum specimens from 40 healthy persons (blood donors) age 18 to 67 years.

Group E. Group E consisted of 38 sera from patients with no previous EBV infection. These specimens were mainly from small children for whom an EBV viral capsid antigen (VCA) IgG immunofluorescence test (EBV VCA IgG immunofluorescence assay [IFA] Kit II; Organon Teknika) was performed to determine past EBV infection.

Performance of the analysis. A plan for the performance of the analysis was designed before the evaluation was started. Each site was to perform one test set for EBV-specific antibodies and one test for heterophile antibodies. In this way the sera were investigated only once in each test. The tests chosen for the individual sites were those in routine use or with which the site had some experience.

	RESULTS

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For evaluation of the results, generally accepted criteria for antibody patterns, also referred to in the manufacturers' instructions, were used. These criteria are stated in Table 3. In addition, weak positive results (+) and borderline results (+/) for EBV IgM and borderline results (+/) for heterophile antibodies were considered positive for group A but were considered negative for groups C, D, and E. 

Group A: patients with recent primary EBV infection. A total of 127 serum samples (91%) showed antibody patterns compatible with recent primary EBV infection for all tests. Results for 12 sera with discordant results are presented in Table 4. Virion and Biotest showed antibody patterns compatible with recent primary EBV infection for all sera in group A. 

Group B: serial dilutions of serum from a patient with recent primary EBV infection. Group B included serial dilutions, in phosphate-buffered saline, of serum from a patient with infectious mononucleosis. The results are shown in Table 6. There was a great variation in the ability to detect antibodies in serial serum dilutions. With Vironostika, only VCA IgG could be detected. Virion had a low sensitivity for EBNA IgG antibodies, whereas Biotest was negative for early antigen (EA) IgM and EA IgG from the dilutions 1:20 and 1:40, respectively, onwards. Test results for heterophile antibodies were not validated for sera belonging to this group.

Group C: 23 serum specimens from immunocompromised patients with current CMV infection. Group C comprised sera from 23 immunocompromised patients with current CMV infection. Table 7 gives a summary of the results. With the exception of serum from one patient who was seronegative, all sera met the criteria for earlier EBV infection. Vironostika and Virion gave several negative EBNA IgG results, leading to an antibody pattern which could be interpreted as a primary infection. One specimen was EBNA negative in all tests, and VCA IgM was positive in Captia and Vironostika. Since it cannot be ruled out that the patient was in a convalescent phase of mononucleosis, this specimen was considered inconclusive and was excluded from the specificity calculations. A combination of VCA IgG and EBNA antibodies were detected by Enzygnost EBV IgG, which therefore gave many positive results. An EBV antibody pattern consistent with reactivation was also detected in two cases. Tests for heterophile antibodies were negative for all sera in group C. 

Group D: serum specimens from 40 healthy persons. Group D included test sera from 40 healthy blood donors. Two of them were EBV seronegative, and 17 sera gave conforming results. Table 8 gives a summary of the results for this group.

Group E: 38 sera from patients with no previous EBV infection. Group E consisted of 38 patient sera. A total of 11 sera were negative in all tests. The other sera gave one or more positive results. A summary of the results is given in Table 9.

Calculation of sensitivity and specificity. (i) Tests for EBV-specific antibodies. The basis for calculation of sensitivity and specificity was as follows. A total of 139 sera belonging to group A gave antibody patterns consistent with current infection. In groups C, D, and E a total of 93 sera gave antibody patterns compatible with previous or no EBV infection, while 8 sera were excluded because of inconclusive results. The calculated sensitivities for the different tests are as follows: Captia, 97% (135/139); Enzygnost, 99% (138/139); Vironostika, 95% (132/139); Virion, 100% (139/139); Biotest, 100% (139/139); and Gull, 96% (134/139). The calculated specificities are as follows: Captia, 90% (84/93); Enzygnost, 100% (93/93); Vironostika, 89% (83/93); Virion, 87% (81/93); Biotest, 86% (80/93); and Gull, 90% (84/93).

(ii) Tests for heterophile antibodies. The basis for calculation of sensitivity and specificity was as follows. A total of 140 sera belonging to group A should give positive results compatible with current infection. A total of 101 sera in groups C, D, and E should give negative results. The calculated sensitivities for the various tests were consequently as follows: Paul-Bunnell-Davidsohn, 81% (113/140); Monosticon, 87% (122/140); Avitex, 95% (133/140); Alexon, 91% (128/140); Clearview, 95% (133/140); and Cards±Mono, 92% (129/140). The calculated specificities are as follows: Paul-Bunnell-Davidsohn, 100% (101/101); Monosticon, 100% (101/101); Avitex, 100% (101/101); Alexon, 98% (99/101); Clearview, 100% (101/101); and Cards±Mono 98% (99/101).

	DISCUSSION

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The ideal test kit for detection of EBV-specific antibodies should have both a high sensitivity for analysis of specimens from patients with a current primary infection and a high specificity for analysis of cases with atypical symptoms that could be due to an EBV infection. A perfect test kit does not exist, but many of the tests that were subjected to our evaluation can be recommended for virological diagnostics when their limitations are known.

EA IgM and IgG detected by Biotest are typical for virus activity associated with primary infection or reactivation. As the results for group E show, it is important to consider that the EA IgM results may be nonspecific, especially with a low optical density compared with the cutoff value (8). If the group E panel had been analyzed only for EBNA IgG, Biotest would have achieved a much higher specificity. In previous evaluations the specificity for this test has been closer to 100% for EBNA IgG (3, 8). Our results indicate that the tests for VCA IgG and EBNA IgG from Gull Laboratories may have problems with specificity. Previous evaluations have shown that the Gull ELISAs for VCA IgM and IgG have both high sensitivity and specificity but that the EBNA IgG test has a low sensitivity and needs improvement (8). It has also been proposed to use a combination of the VCA IgM and IgG tests from Gull and the EBNA IgG test from Biotest (3, 8). Such a combination could perhaps be considered close to an optimal test kit, but for use in practical virological diagnostics there is a need for further investigations.

At present, serological EBV reactivation is not associated with a clearly defined clinical syndrome, although reactivations often are observed in immunocompromised patients (6). It has been reported that symptomatic CMV infection frequently occurs in association with simultaneous EBV reactivation (5). However, most of the patients showing a serological reactivation appeared to have a normal immune system (6). It is also important to remember that VCA IgM can be detected at reactivation, as clearly indicated by our evaluation (6, 7). Investigations have shown that high-avidity VCA IgG antibodies indicating previous EBV infection are often found in EBV IgM-positive sera (1). Assays for VCA IgM are often used as the single test, but it is important to consider that a reactive result may be a false positive. It is therefore recommended that tests for EBNA IgG and for VCA or EA IgG be used in addition to VCA IgM, as our evaluation also demonstrates (8). In this way it is possible to avoid incorrectly diagnosing EBV reactivations as primary infections. Since the patients in group C were immunosuppressed, EBNA IgG antibodies probably exist in relatively low titer, and thus a highly sensitive assay is required. When using a sensitive EBNA IgG test as a screening test, antibodies against the other EBV antigens have to be analyzed in cases of EBNA IgG-negative sera (8).

Enzygnost, which apparently has a higher specificity than the other tests, has no means of distinguishing between VCA IgG and EBNA antibodies. With positive results for IgM and IgG, it is not possible to differentiate between primary infection and reactivation. Other investigations have shown that the IgM test may give many equivocal results (8).

The Virion EBNA IgG test seems to be somewhat less sensitive than some of the other tests. In addition, the interpretation of results requires experience.

Captia and Vironostika are recently developed tests, and both are based on a synthetic peptide antigen. Vironostika EBNA IgG seemed to be somewhat less sensitive than the other tests. Captia VCA IgG and EBNA IgG gave some unspecific reactions in group E.

For the purpose of detecting heterophile antibodies, Avitex and Clearview yielded the best results even though Clearview gave some borderline results for patients with recent primary EBV infection. Cards±Mono and Alexon can also be recommended. Since rapid tests are extensively used in general practice, a high specificity is important because a false-positive result can lead to an erroneous diagnosis (4). The Paul-Bunnell-Davidsohn and Monosticon tests had lower sensitivities than the other tests and cannot be recommended. A previous evaluation has also shown that tests with purified antigens have increased sensitivity and specificity compared with methods based on sheep, horse, or bovine erythrocyte agglutination (2).