The use of semi-automated EBV IgG avidity determination for the diagnosis of infectious mononucleosis

The diagnosis of acute Epstein-Barr virus (EBV) infection is based frequently on the combination of positive viral capsid antigen (VCA) IgM antibodies and negative EB viral nuclear antigen 1 (EBNA-1) IgG antibodies. However, both VCA IgM and EBNA-1 IgG can provide false positive and false negative results. Therefore, situations in which the EBV serology remains unclear are not uncommon. Determination of EBV IgG avidity can clarify the EBV status in these patients. So far, mainly immunofluorescence assays have been used for this purpose. These tests are laborious, their evaluation is subjective, and automation is difficult. Therefore, two commercially available microtiter plate enzyme immunoassays (EIA) were compared for their usefulness for semi-automated EBV IgG avidity determination. One assay is based on a mixture of EBV antigens, the other assay uses a synthetic peptide of the VCA-complex. Patient sera of confirmed acute and past EBV infections were tested for avidity by both assays. The results with the antigen mixture assay proved to be highly sensitive (100%) and specific (100%). Avidity index calculations on the basis of one-point-quantification titers gave better results than calculations using OD values. Determination of EBV IgG avidity by the peptide assay was complicated by the fact that it was less sensitive than the antigen mixture assay for IgG detection in acute EBV infections. On the other hand, about 30% of the samples had to be retested with the peptide assay in a higher dilution because the IgG units in initial testing fell outside the range covered by the standard curve. Using OD values of the peptide EIA, the sensitivity was 99% but the specificity of detection of acute EBV infections was only 86%. Thus, while the peptide EBV avidity assay is unsuitable as a confirmatory assay, avidity testing with the antigen mixture assay is a useful tool to resolve equivocal EBV serologies. Avidity assays on the basis of EIA can be automated which should lead to wider use of this methodology. J. Med. Virol. 54:145–153, 1998. © 1998 Wiley-Liss,Inc.     

Evaluation of the Architect Epstein-Barr Virus (EBV) Viral Capsid Antigen (VCA) IgG,VCA IgM,and EBV Nuclear Antigen 1 IgG Chemiluminescent Immunoassays for Detection of EBV Antibodies and Categorization of EBV Infection Status Using Immunofluorescence Assays as the Reference Method

Commercial immunoassays for detecting IgG and IgM antibodies against Epstein-Barr virus (EBV), viral capsid antigens (VCA), and IgGs toward EBV nuclear antigen-1 (EBNA-1) are routinely used in combination to categorize EBV infection status. In this study, we evaluated the performances of the Architect EBV VCA IgG, VCA IgM, and EBNA-1 IgG chemiluminescent microparticle assays (CMIAs) in EBV serological analyses using indirect immunofluorescence assays and anticomplement immunofluorescence assays as the reference methods for VCA IgG, VCA IgM, and EBNA-1 IgG antibody detection, respectively. A total of 365 serum samples representing different EBV serological profiles were included in this study. The κ values (concordances between the results) obtained in the Architect CMIA and those in the reference assays were 0.905 (P < 0.0001) for VCA IgM, 0.889 (P < 0.0001) for VCA IgG, and 0.961 (P < 0.0001) for EBNA-1 IgG. The sensitivities and specificities were, respectively, 91.08% and 99.48% for VCA IgM, 99.23% and 86.27% for VCA IgG, and 96.77% and 99.16% for EBNA-1 IgG. The sensitivities and specificities of the Architect CMIA panel were, respectively, 99.15% and 98.6% for diagnosing a primary infection, 97.62% and 93.39% for diagnosing a past EBV infection, and 92.42% and 97.82% for diagnosing the absence of an EBV infection. In summary, we demonstrated that the Architect EBV antibody panel performs very well for EBV antibody detection and correctly categorizes clinically relevant EBV infection states.     

Performance of the Architect EBV Antibody Panel for Determination of Epstein-Barr Virus Infection Stage in Immunocompetent Adolescents and Young Adults with Clinical Suspicion of Infectious Mononucleosis

The Architect EBV antibody panel is a new chemiluminescence immunoassay system used to determine the stage of Epstein-Barr virus (EBV) infection based on the detection of IgM and IgG antibodies to viral capsid antigen (VCA) and IgG antibodies against Epstein-Barr nuclear antigen 1 (EBNA-1). We evaluated its diagnostic accuracy in immunocompetent adolescents and young adults with clinical suspicion of infectious mononucleosis (IM) using the RecomLine EBV IgM and IgG immunoblots as the reference standard. In addition, the use of the antibody panel in a sequential testing algorithm based on initial EBNA-1 IgG analysis was assessed for cost-effectiveness. Finally, we investigated the degree of cross-reactivity of the VCA IgM marker during other primary viral infections that may present with an EBV IM-like picture. High sensitivity (98.3% [95% confidence interval {CI}, 90.7 to 99.7%]) and specificity (94.2% [95% CI, 87.9 to 97.8%]) were found after testing 162 precharacterized archived serum samples. There was perfect agreement between the use of the antibody panel in sequential and parallel testing algorithms, but substantial cost savings (23%) were obtained with the sequential strategy. A high rate of reactive VCA IgM results was found in primary cytomegalovirus (CMV) infections (60.7%). In summary, the Architect EBV antibody panel performs satisfactorily in the investigation of EBV IM in immunocompetent adolescents and young adults, and the application of an EBNA-1 IgG-based sequential testing algorithm is cost-effective in this diagnostic setting. Concomitant testing for CMV is strongly recommended to aid in the interpretation of EBV serological patterns.     

Evaluation of a multiplex flow immunoassay for detection of epstein-barr virus-specific antibodies

Conventional methods for the detection of Epstein-Barr virus (EBV)-specific antibodies include the immunofluorescence assay (IFA) and enzyme immunoassay (EIA). While sensitive and specific, these methods are labor-intensive and require separate assays for each analyte. This study evaluated the performance of a multiplex bead assay (BioPlex 2200; Bio-Rad Laboratories, Hercules, CA) for the simultaneous detection of immunoglobulin G (IgG) and IgM class antibodies to the EBV viral capsid antigen (VCA) and IgG class antibodies to Epstein-Barr virus nuclear antigen-1 (EBNA-1). Serum specimens (n = 1,315) submitted for routine EBV-specific antibody testing by EIA (Grifols-Quest, Inc., Miami, FL) were also tested by the multiplex bead assay using the BioPlex 2200 automated analyzer. Specimens showing discordant results were tested by IFA. Following IFA resolution, the BioPlex VCA IgM, VCA IgG, and EBNA-1 IgG assays demonstrated 97.9%, 91.4%, and 96.9% agreement, respectively, with the results obtained by EIA. Furthermore, the BioPlex assays showed an overall agreement of 94.1% with the EIA when the specimens were categorized by disease state (susceptible, acute, or past infection) based on the EBV-specific antibody profiles. These findings indicate that the BioPlex EBV assays demonstrate a performance comparable to that of the conventional EIA, while allowing for a more rapid (2.3 h for 100 samples versus 4.5 h by the EIA) and higher-throughput ( approximately 400 samples per 9 h versus 200 samples by the EIA) analysis of the EBV-specific antibody response.     

Prevalence of primary versus reactivated Epstein-Barr virus infection in patients with VCA IgG-, VCA IgM- and EBNA-1-antibodies and suspected infectious mononucleosis.

BACKGROUND: In Epstein-Barr virus (EBV) infection, IgG- and IgM-antibodies to viral capsid antigen (VCA) and IgG-antibodies to Epstein-Barr nuclear antigen 1 (EBNA-1) can occur simultaneously both in late primary infection and during subclinical viral reactivation in immunocompetent persons, and the differential diagnosis is of importance. OBJECTIVES: To study the prevalence of primary infection and serological reactivation in patients with suspected primary EBV infection and with all three parameters present. STUDY DESIGN: Fifty serum samples from 43 consecutive patients referred for suspected infectious mononucleosis and positive for VCA IgG-, VCA IgM- and EBNA-1-antibodies by EIA, were tested for IgG-antibody avidity with an EBV IgG immunoblot. Sera were also tested for heterophile antibodies (HA). To verify the presence of IgM-antibodies an EBV IgM immunoblot was performed when high-avidity IgG-antibodies were found. RESULTS AND CONCLUSIONS: Of 43 patients with suspected primary EBV infection and VCA IgG-, VCA IgM- and EBNA-1-antibodies present, only 18 patients (42%) had a late primary infection. Twenty-one patients (49%) had high-avidity IgG-antibodies, indicating an IgM response due to reactivation, thus suggesting other causes for their symptoms. In 10 of these 21 patients the presence of IgM-antibodies was confirmed by immunoblot, indicating reactivation as a cause of IgM-antibodies in at least 23% of the 43 patients studied. Of 18 patients with primary infection, HA were detected in 16 (94%) of 17 patients tested. Only one (5%) of the patients with high-avidity antibodies had HA. Absence of HA in patients with this serological pattern is therefore a good indicator of reactivation, and conversely, the presence of HA is a good indicator of primary infection. In HA negative patients, avidity testing could be used for differential diagnosis.     

Assessment of rapid ELISA test for detection of Epstein-Barr virus infection.

A rapid test for the detection of IgM and IgG Epstein-Barr nuclear antigen (EBNA-1) has been extensively marketed. If IgM to Epstein-Barr viral capsid antigen (EBV VCA) is taken as evidence of current EBV infection, one observer detected 17 of 38 such samples and the other 22 of 38 as acute. The positive predictive value of the test was 63%, and the greatest difficulty was posed by the detection of IgM EBV VCA positive, heterophile antibody negative samples. Significant false positive results were obtained in sera with evidence of current Toxoplasma gondii, cytomegalovirus, and adenovirus infection. Rheumatoid factor was not a problem. Modification of the test protocol improved its performance: the positive predictive value rose to 87% and the negative predictive value to 81%. Although our modifications did not increase the speed of the test, there was reliable information on the EBNA-1 state. The test is best used as an adjunct to other EBV serology, and laboratories should be aware of the limitations of the rapid test.     

Seroepidemiology of EBV and interpretation of the “isolated VCA IgG” pattern

The presence of VCA IgG in the absence of VCA IgM and EBNA‐1 IgG antibodies makes classifying EBV infection more difficult as this serological picture can be seen in the case of past infection with EBNA‐1 IgG loss or non‐appearance, or acute infections with the early disappearance or delayed onset of VCA IgM. The aim of this study was to assess the prevalence of this pattern in 2,422 outpatients with suspected EBV infection examined in 2005–2006, and to interpret its significance by means of immunoblotting. One hundred and seventy‐seven (7.3%) of the patients were VCA IgG‐positive, VCA IgM‐negative and EBNA‐1 IgG‐negative, 15 of whom (8.5%) presented with heterophile antibodies. Analysis by age class showed that the prevalence of isolated VCA IgG ranged from 4.5% in the subjects aged 1–10 years to 9% in those aged >60 years. Immunoblotting allowed 18.9% of the cases to be classified as acute and 81.1% as past infections, the latter being observed in about 37% of the patients aged less than 10 years and in 100% of those aged >30 years. Therefore, in our case series, the presence of isolated VCA IgG was associated usually with past infection, particularly among adults. In children aged less than 10 years, it was associated mainly with acute infection but as past infection may be present in about one‐third of such children, this possibility should not be overlooked. J. Med. Virol. 81:325–331, 2009. © 2008 Wiley‐Liss, Inc.     

Measurement of EBV-IgG anti-VCA avidity aids the early and reliable diagnosis of primary EBV infection

Current serological methods for the diagnosis of Epstein-Barr virus (EBV) infection still differentiate poorly between primary infection and reactivation. This is particularly true when IgG and IgM antibodies are present simultaneously and only a single serum sample is provided for analysis. The demonstration of the IgG avidity state has the potential to distinguish recent from past or reactivated infection. An analysis of the kinetics of avidity maturation of anti-VCA antibodies in primary EBV infection was undertaken with longitudinally collected sets of sera from 28 well-characterised EBV cases and in sera from 35 cases with previous EBV infection and recent primary infection due to HIV, CMV, or hepatitis A. Antibodies directed against the viral capsid antigen (VCA) and Epstein-Barr nuclear antigen (EBNA-1) were sought, using a commercial enzyme immunoassay (EIA). In parallel with standard IgG anti-VCA detection, serum was incubated with 8 M urea to disrupt low-avidity complexes to allow calculation of the percentage avidity. In cases with primary EBV infection, the mean avidity rose from 54% at 6 weeks to 82% by 28 weeks after the onset of symptoms, but remained lower than that of the control sera (96%). The addition of the avidity measurement improved the sensitivity of IgG and IgM anti-VCA testing in diagnosis of primary EBV infection from 93% to 100%. The specificity of IgM anti-VCA testing alone was poor, with 14 of 35 cases (49%) demonstrating false-positive results, but it improved to 97% by the demonstration of high-avidity IgG anti-VCA. The combination of negative IgG anti-EBNA and low-avidity IgG anti-VCA had a sensitivity and specificity of 100%. The routine addition of IgG anti-VCA avidity estimation to diagnostic EBV serology is recommended.     

The seroepidemiology of infection due to Epstein-Barr virus in southern India

We investigated the seroepidemiology of infection due to Epstein-Barr virus (EBV) in 181 south Indian subjects aged 0-25 years using the indirect immunofluorescence method to titrate antibodies to viral capsid antigen (VCA), nuclear antigen (EBNA), and early antigen (EA). The age-specific prevalence of IgG antibodies to VCA rose rapidly to 90% by the age of 5 years. The prevalence of VCA-specific IgM and the geometric mean titre of VCA-specific IgG antibodies were highest between the ages of 6 months and 2 years, the median age of primary infection being 1.4 years. Thus primary EBV infection occurs early in life. EA antibody prevalence was highest (55%) in the third year of life and remained between 30% and 40% thereafter. This pattern of EA antibody prevalence suggests that the latent EBV infection that persists lifelong after primary infection may be reactivated in many individuals. EBNA antibody prevalence was low until the age of 2 years but rose to 80% in the fourth year. Geometric mean titres of antibodies to EA and EBNA were low and stable at all ages. These results are similar to data from areas where EBV-associated Burkitt's lymphoma is endemic and indicate a high EBV infection load early in life.     

Epstein-Barr virus antibodies in Kawasaki disease

The prevalent ages at onset for Kawasaki Disease (KD) and Epstein-Barr virus (EBV) infection are known to be similar in Korea and Japan. We evaluated the correlation between EBV infection and KD. The antibodies to EBV such as anti-viral capsid antigen (VCA) IgG and IgM, anti-diffuse and restricted early antigen IgG (anti-EADR IgG), and the anti-EBV determined nuclear antigen IgG (anti-EBNA IgG) were examined in 29 KD patients at five separate times sequentially during a period of one year, and also in 14 other children with a past history of KD. The results of each group were compared with those of age-matched controls. The positive rates of anti-VCA IgG and IgM at presentation in the KD patients were 41.4% (12/29) and 0% (0/29), respectively. Only one patient was found to be anti-VCA IgM-positive within two months. There were no cases of anti-VCA IgG except one, anti-EADR IgG and anti-EBNA IgG positive to negative seroconversion during the year. The children with a past history of KD showed higher anti-EBNA IgG-positive rates than the controls (p=0.04). There was no difference in the seropositive rates of the antibodies to EBV, cytomegalovirus, herpes simplex virus and herpes zoster virus. In conclusion, children with KD were noted to have normal immune responses to EBV infection. Children with a past history of KD seemed to be infected with EBV at a later age than children with no history of KD.     

Evaluation of 12 Commercial Tests for Detection of Epstein-Barr Virus-Specific and Heterophile Antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc.), Clearview IM (Unipath Ltd.), and Cards±OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards±OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

Evaluation of 12 commercial tests for detection of Epstein-Barr virus-specific and heterophile antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc. ), Clearview IM (Unipath Ltd.), and Cards+/-OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards+/-OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

Evaluation of four commercial systems for the diagnosis of Epstein-Barr virus primary infections

To compare the performance of four diagnostic commercial systems for Epstein-Barr virus (EBV) serology (for IgM and IgG virus capsid antigen [VCA] and EBV nuclear antigen [EBNA] antibodies), a collection of 125 samples from clinically suspected infectious mononucleosis cases was studied. Indirect immunofluorescence (IIF) for VCA IgM and IgG antibodies and anticomplement immunofluorescence for EBNA antibodies (Meridian Bioscience Inc.) were used as reference methods. By these methods, the cases were classified EBV primary infection (presence of IgM to VCA or IgG to VCA in the absence of EBNA antibodies; n = 82), EBV past infection (presence of VCA IgG and EBNA antibodies in the absence of VCA IgM; n = 26), or no infection (negative for the three markers; n = 17). The following systems were tested: two chemiluminescent immunoassays (CLIAs; the Liason [CLIA-L; DiaSorin] and the Immulite 2000 [CLIA-I; Siemens]), immunofiltration (IF; All.Diag), and an enzyme-linked immunosorbent assay (ELISA; DiaSorin). In the IgM assays, sensitivities ranged from 67.1% (ELISA) to 92.2% (CLIA-L) and specificities ranged from 93.8% (CLIA-L) to 100% (IF). In the VCA IgG assays, sensitivities varied from 79.4% (IF) to 94.4% (CLIA-I) and specificities varied from 94.4% (IF and CLIA-L) to 100% (CLIA-I and ELISA). In EBNA assays, sensitivities ranged from 78.1% (IF) to 93.8% (CLIA-I) and specificities ranged from 32.3% (CLIA-L) to 91.4% (IF). In relation to EBV profiles, the corresponding figures for sensitivity (in detecting primary infection) for IF, CLIA-L, CLIA-I, and ELISA were 92.7%, 93.8%, 89%, and 89.6%, respectively, and those for specificity (to exclude primary recent infection) were 90.7%, 94.6%, 97.7%, and 95.2%, respectively. Although there were limitations in some individual markers, especially CLIA-L for EBNA IgG, the systems evaluated appear to be useful for diagnosis of EBV infection.     

High incidence of elevated antibody titers to Epstein-Barr virus in patients with uveitis

Summary. We assayed Epstein-Barr virus (EBV) antibody titers in patients’ sera using indirect immunofluorescence and tested for the presence of antibody to EBV immediate-early BZLF1 protein ZEBRA by Western blotting to explore the association of EBV infection with uveitis. IgG and IgA antibodies to viral capsid antigen (VCA), IgG antibodies to early antigen (EA), and antibodies to EBV nuclear antigen were detected at higher titers in sera of patients with uveitis than in the sera of healthy controls. Neither IgM antibody to VCA nor EA was detected in the patients’ sera. Anti-ZEBRA-IgG antibodies were detected in most patients’ sera, but not in those of healthy controls. These results suggest that uveitis might be a disease accompanied by EBV reactivation.     

Evaluation of an Immunofiltration Assay That Detects Immunoglobulin M Antibodies against the ZEBRA Protein for the Diagnosis of Epstein-Barr Virus Infectious Mononucleosis in Immunocompetent Patients

The performance of an immunofiltration assay (IMFA) that detects immunoglobulin M (IgM) antibodies to the Epstein-Barr virus (EBV) ZEBRA (BamHI Z EBV replication activator) protein was evaluated for the diagnosis of EBV infectious mononucleosis (IM) in immunocompetent patients. The test panel consisted of 47 sera displaying an EBV-specific antibody profile compatible with an acute primary EBV infection from patients with clinical and biological features of EBV IM, 20 sera from healthy individuals either with a past EBV infection or who were EBV seronegative, 20 sera displaying an equivocal EBV antibody pattern (viral capsid antigen IgG positive [VCA IgG⁺], VCA IgM⁺, and EBV nuclear antigen-1 IgG⁺), and 15 sera obtained from patients with a mononucleosis-like syndrome owing to cytomegalovirus, human herpesvirus 6, or parvovirus B19. Overall, the sensitivity and the specificity of the assay were found to be 92.5%, and 97.3%, respectively. The sensitivity of the assay for the diagnosis of heterophile antibody-negative EBV IM was 86.2%. The IMFA is rapid, easy to perform, and, thus, suitable for point-of-care testing, and it may be used as a first-line test for the diagnosis of acute EBV IM in immunocompetent patients.Diagnosis of Epstein-Barr virus (EBV) infectious mononucleosis (IM) is commonly made on the basis of characteristic clinical manifestations and the detection of heterophile antibodies (HA). Nevertheless, HA may be absent, particularly in young children (14) but also in as many as 20% of adults with EBV IM (7). In these cases, demonstration of the presence of EBV viral capsid antigen (VCA) immunoglobulin G (IgG) and/or IgM antibodies, along with the absence of IgG antibodies to EBV nuclear antigen-1 (EBNA-1), allows the diagnosis of EBV primary infection (9). Detection of EBV-specific antibodies is accomplished by the use of commercial enzyme immunoassays, indirect immunofluorescence assays, line blot immunoassays (9), or, as established more recently, a multiplexed bead assay (3). These methods have long turnaround times, are labor-intensive, or require specific instruments or skilled technologists for their performance. In addition, interpretation of EBV VCA IgG/IgM and EBNA-1 IgG reactivity profiles is not always straightforward (9).The ZEBRA (BamHI Z EBV replication activator) protein is encoded by the immediate early BZLF1 gene. ZEBRA is expressed during the lytic cycle in EBV-permissive cells and plays a critical role in transactivating several immediate early, early, and late EBV genes (5). Antibodies against ZEBRA are produced during primary EBV infection (11, 15, 18), and thus, the detection of ZEBRA-specific IgMs may allow an early diagnosis of EBV IM. In the present study, we evaluated a rapid and easy-to-perform immunofiltration assay (IMFA) detecting IgMs to the EBV ZEBRA protein for the biological diagnosis of IM in immunocompetent patients.     

Serological diagnosis of Epstein-Barr virus infection by novel ELISAs based on recombinant capsid antigens p23 and p18

A new pair of Epstein-Barr virus ELISAs (Biotest Anti-EBV VCA IgG and VCA IgM ELISA) was evaluated for usefulness for routine diagnosis of acute EBV infections. The ELISAs are based on two viral capsid antigens (VCA), p23 (BLRF2, full-length) and p18 (BFRF3, carboxy-half), that are combined by autologous gene fusion. In total, 179 sera were tested in direct comparison with classical VCA immunofluorescence assays (IFA). With the help of clinical data and additional reference serology, i.e., heterophile antibodies, anti-EA IgG (IFA) and anti-EBNA-1 IgG (ELISA), the patients were divided into the following categories: seronegatives (46), acute primary infections (67), previous infections (39), suspected reactivations (20) and constellations with intermediate serological patterns (7). The VCA IgG and VCA IgM ELISAs showed overall agreement to IFA of 95.0% and 94.4%, respectively. The calculated analytical performance (sensitivity; specificity) of VCA IgG and VCA IgM was 94.0%; 97.8% and 97.1%; 96.5%, respectively. A certain delay in seroconversion of anti-p23-p18 IgG may account for a significant difference in sensitivity of the VCA IgG ELISA between primary (88.4%) and previous infections (100%). In summary, the new recombinant VCA ELISAs yielded good correlation to VCA IFA and in combination with EBNA-1 IgG allow rapid, sensitive, and specific diagnosis of infectious mononucleosis or EBV immune status in general.     

Antibody response to Epstein-Barr virus antigens in patients with chronic viral infection

We tested antibody titres against Epstein-Barr virus (EBV) antigens in patients suffering from chronic viral disease and compared them with those determined in sex- and age-matched healthy controls. Patient sera showed signs of active EBV infection [antibodies against early antigen (EA) and/or viral capsid antigen (VCA) in the IgM or IgA classes] significantly more frequently than the control group. Correspondingly, geometric mean titres (GMT) of antibodies against all viral antigens were elevated in the patients. The strongest association with EBV was observed in patients whose clinical symptoms closely resembled infectious mononucleosis: 92% of the subjects in this subgroup possessed anti-EA and 41 and 25% had IgM and IgA anti-VCA antibody, respectively. In patients with signs of lymphoproliferation only and in those suffering from frequent respiratory infections the association with EBV was less marked but still significant. Patients with transient defects in humoral and cellular immunity mounted higher titres against VCA in the IgG class than those without immune defects.     

Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, tonsillar carcinoma and control groups

In the sera of 17 patients with nasopharyngeal carcinoma (NPC) and of 19 patients with tonsillar carcinoma (TC) the titres of IgA, IgG and IgM antibodies to EBV VCA (viral capsid antigen) and of IgG antibodies to EBV EA (early antigen) were determined by the indirect immunofluorescence (IF) method. Significant difference was observed in the frequency of IgA antibodies to EBV VCA and IgG antibodies to EBV EA between NPC patients and controls. There was also a significant difference between the frequency of IgM antibody to EBV VCA and EBV EA antibody titres in TC patients and controls. The geometric mean titre (GMT) of IgG antibodies to EBV VCA was significantly higher in the NPC and TC patients as compared to controls.     

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

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 four commercially available Epstein-Barr virus enzyme immunoassays with an immunofluorescence assay as the reference method

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.