Evaluation of Four Commercially Available Epstein-Barr Virus Enzyme Immunoassays with an Immunofluorescence Assay as the Reference Method

Abstract

Four commercially available enzyme immunoassays (EIAs) (Novitec, Biotest, Virotech, and DiaSorin) were evaluated, with an indirect immunofluorescence assay as the reference method, for Epstein-Barr virus (EBV) VCA (viral capsid antigen) immunoglobulin G (IgG), VCA IgM, or EBNA (EBV nuclear antigen) IgG at three different locations (Homburg, Stuttgart, and Dresden). Serum samples from 66 immunocompetent patients with infectious mononucleosis, 73 patients without prior EBV infection, and 96 patients with past EBV infections and 29 serum samples with possible cross-reactions to other herpesviruses were included. In addition, 25 samples from an extensively pretested panel that is commercially available (Boston Biomedica) were tested. Each sample was tested at only one location. The four EIAs varied considerably in performance. When analyzing for EBV diagnosis, the Novitec assay performed the best, with 4.9% discrepant diagnoses, followed by the Biotest, Virotech, and DiaSorin assays, with 6.8, 11.7, and 14.0% discrepant diagnoses, respectively. On the basis of single-parameter analysis, the Novitec assay also showed the lowest number of discrepant results, with 3.5%, compared with the Virotech, Biotest, and DiaSorin assays, which produced 5.4, 6.4, and 8.6% discrepant results, respectively. VCA assays using affinity-purified native antigens performed better than assays with recombinant or synthetic antigens. The synthetic EBNA-1s showed the lowest concordance with the reference compared to recombinant p72. Commercially available EBV EIAs differed considerably in performance; however, some proved to be reliable and convenient alternatives to the indirect immunofluorescence assay for routine diagnostics. Native antigens, rather than synthetic peptides, are favored for EBV serology testing.

Evaluation of 4 methods for the serological diagnosis of Epstein–Barr virus infection using an immunofluorescence assay as the reference method

“…IFA displayed a considerably reduced sensitivity compared to the 2 other methods for the detection of anti-EBNA antibodies. Gartner et al evaluated 4 commercially available EIAs using IFA as the reference method (18). They reported that 2 of the 4 EIAs agreed well with the reference IFA results for the distinction of a primary infection from seronegativity and a past infection, possibly constituting reasonable alternatives to the standard IFA method.…”

Serological diagnosis of Epstein-Barr virus infection: Problems and solutions

“…the P3HR-1 or Raj cell lines) [2,23,45,[50][51][52] , whereas EIAs use purified native or recombinant proteins, synthetic peptides or fusion proteins (complete proteins or fragments of the proteins encoded by the EBV genes) [42,48] . The type and preparation of the antigens used are probably responsible for the differences in the sensitivity and specificity of the various assays [42,53,54] . IFA has been used as the reference method, although its sensitivity is the same as or less than that of EIAs [43] , automated versions of which allow a large number of samples to be tested and are commonly used in laboratories with a large routine workload.…”

“…IFAs usually use EBV-transformed B cell lines from patients with Burkitt's lymphoma (e.g. the P3HR-1 or Raj cell lines) [2,23,45,[50][51][52] , whereas EIAs use purified native or recombinant proteins, synthetic peptides or fusion proteins (complete proteins or fragments of the proteins encoded by the EBV genes) [42,48] . The type and preparation of the antigens used are probably responsible for the differences in the sensitivity and specificity of the various assays [42,53,54] .…”

“…[2,41] . Using only three parameters (VCA IgG, VCA IgM and EBNA-1 IgG), it is generally easy to distinguish acute and past infections in immunocompetent patients [42,43] . The presence of VCA IgG and VCA IgM in the absence of EBNA-1 IgG indicates acute infection, and the presence of VCA IgG and EBNA-1 IgG in the absence of VCA IgM is typical of past infection (Table 2).…”

“…Consequently, EBNA-1 IgG, and VCA IgG and IgM may be simultaneously present in patients with primary EBV infection if VCA IgM persist and EBNA-1 IgG have already been produced (a phase that has been variously defined as "recent infection", "primary infection, transient phase or convalescence", "past infection, IgM persisting"), or in those with reactivation and the simultaneous presence of VCA IgM and EBNA-1 IgG [42] . Reactivation is still relatively rare and often short in immunocompetent subjects, and is generally considered of no clinical relevance [113,114] ; however, it can cause serious complications in immunocompromised patients.…”