Diagnostic Microbiology and Infectious Disease 79 (2014) 310–316

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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio

Virology

Comparative evaluation of the new ARCHITECT EBV assays considering different testing algorithms Eva Sickinger a,⁎, Mario Berth b, Angela Vockel a, Hans-Bertram Braun a, Michael Oer a, Carsten Buenning a a b

Abbott GmbH & Co. KG, Max-Planck-Ring 2, 65205 Wiesbaden-Delkenheim, Germany Algemeen Medisch Laboratorium, Immunology Department, Emiel Vloorsstraat 9, 2020 Antwerp, Belgium

a r t i c l e

i n f o

Article history: Received 10 December 2013 Received in revised form 7 March 2014 Accepted 28 March 2014 Available online 13 April 2014 Keywords: Epstein-Barr virus (EBV) Testing algorithms Infection stage determination VCA IgM VCA IgG and EBNA-1 IgG

a b s t r a c t In the current evaluation, Epstein-Barr virus (EBV) serology was performed on 1113 routine serum samples. Although the initial request for all samples from the general practitioner was EBV IgM testing, 80.9% were classified as past infections. The ARCHITECT® viral capsid antigen (VCA) IgM, VCA IgG, and EBV nuclear antigen (EBNA) 1 IgG assays showed good results for sensitivity and specificity, being 100.0%, 98.3%, and 100.0% and 99.9%, 95.4%, and 99.6%, respectively. Using an algorithm based on initial EBNA-1 IgG testing, followed by VCA IgG and IgM for samples that were not EBNA-1 IgG reactive, the number of tests per sample could be reduced to nearly 50% compared to parallel testing. The high sensitivity and specificity of the ARCHITECT® EBNA-1 IgG assay in combination with a low number of grayzone results are a precondition for the chosen test algorithm. Thus, the newly developed ARCHITECT® EBV panel is suitable for accurate and costefficient EBV serology in a routine clinical laboratory. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Epstein-Barr virus (EBV), also called human herpes virus 4, is one of the most common viruses in humans. In adults above 25 years, the seroprevalence is N95% (Ooka et al., 1991; Rickinson and Kieff, 2001). The virus is mainly transmitted by saliva. Transmission by transplantation or blood products containing lymphocytes has been shown, and sexual transmission may be possible (Crawford et al., 2002; Schooley, 1995). EBV is the causative agent of infectious mononucleosis (IM) but is also associated with other non-malignant and malignant diseases such as Burkitt's lymphoma and nasopharyngeal carcinoma (Williams and Crawford, 2006). Primary EBV infections in childhood are often asymptomatic, while leading to IM in 35–50% of adolescents and up to 75% in young adults (Balfour et al., 2013; Lennette et al., 1995; Steven, 1996; Williams and Crawford, 2006). In rare cases, complications during acute EBV infections are seen such as rupture of the spleen, upper airway obstruction, bacterial super infection, and central nervous system complications. The main objective of EBV serology testing in immune competent patients with suspicion of infectious mononucleosis is to confirm or rule out an acute EBV infection and to differentiate it from other infections or illnesses with similar symptoms, e.g., cytomegalovirus, HIV, and toxoplasmosis (Berth and Bosmans, 2010). Immune status determination of transplant donors and recipients to assess the risk of possible post-transplant lymphoproliferative disorder is another important application for EBV serology (Gulley and Tang, 2010). ⁎ Corresponding author. Tel.: +49-(0)6122-58-1381; fax: +49-(0)6122-58-1473. E-mail address: [email protected] (E. Sickinger). http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.022 0732-8893/© 2014 Elsevier Inc. All rights reserved.

Immunoassays to determine the presence of EBV specific antibodies are highly sensitive and specific (Hess, 2004; Kreuzer et al., 2013) and offer the advantage of automation. In order to reduce costs and labor, the ideal EBV panel should be able to detect the serological status of acute, past, or absence of infection from a single blood specimen. The exact determination of the stage of an EBV infection is typically achieved by a combination of several assays for detection of EBV specific markers, namely, IgG and IgM antibodies against the EBV viral capsid antigen (VCA) and IgG antibodies against the EBV nuclear antigen (EBNA) (de Ory et al., 2011; Hess, 2004; Odumade et al., 2011). VCA IgG and VCA IgM antibodies in the absence of EBNA-1 IgG antibodies are typically found in patients with acute primary infections. In contrast, past infections are characterized by the presence of VCA IgG and EBNA-1 IgG antibodies in the absence of VCA IgM antibodies. VCA IgM antibodies normally disappear within a few weeks (Lamy et al., 1982; Schillinger et al., 1993) but may sometimes persist longer up to the period when EBNA-1 IgG antibodies are already produced. Serology may be further complicated by the fact that some individuals, especially children, might not produce VCA IgM antibodies during primary infection (Schillinger et al., 1993; Sumaya and Ench, 1985), and others lack EBNA-1 IgG antibodies—either the individuals are EBNA-1 non-responders or the individuals may have lost the EBNA-1 IgG antibodies under circumstances such as immunosuppression (Hess, 2004). Unresolved serological patterns, e.g., isolated VCA IgM, isolated VCA IgG, isolated EBNA-1 IgG, or simultaneous reactivity of all 3 markers, are observed in up to 10% of patients (De Paschale et al., 2009; Klutts et al., 2009). As yet, no consensus exists on a general testing algorithm. Several factors influence the testing mode such as assay performance,

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reimbursement schemes, laboratory workflow, requested clinical diagnosis, and population. Not surprisingly, algorithms in practice vary from initial EBNA determination, parallel determination of 2 markers such as VCA IgM and VCA IgG, or running all 3 assays of the EBV panel together (Health Protection Agency, 2012). The aim of the current study was to first evaluate the newly commercially available ARCHITECT ® EBV panel consisting of 3 assays, VCA IgG and IgM and EBNA-1 IgG in comparison to the corresponding DiaSorin LIAISON® EBV assays in terms of sensitivity and specificity for each individual marker (VCA IgM, VCA IgG, EBNA-1 IgG) as well as their combined use as an aid to determine the stage of a potential EBV infection. In a second step, we analyzed whether a testing approach using ARCHITECT ® EBNA-1 IgG alone as initial test may be a suitable and efficient measure for EBV serology in a routine clinical laboratory. 2. Materials and methods Testing was performed at the Algemeen Medisch Laboratorium, Antwerpen, Belgium, and at Abbott GmbH & Co.KG, Germany. 2.1. ARCHITECT ® EBV VCA IgM, EBV VCA IgG, and EBV EBNA-1 IgG assays The 3 ARCHITECT® EBV assays (ABBOTT, Wiesbaden, Germany) are 2-step immunoassays for the qualitative detection of IgG or IgM antibodies to VCA or IgG antibodies to EBV EBNA-1 in human serum and plasma, using chemiluminescent microparticle immunoassay technology. The VCA IgM assay includes a predilution step to minimize rheumatoid factor interference. To capture EBV specific IgG or IgM antibodies in a patient sample, VCA or EBNA-1 antigen coated paramagnetic microparticles are added for the first incubation. After washing, anti-human IgG: or anti-human IgM: acridinium labeled conjugate is added to create a reaction mixture in the second step. This is followed by another wash cycle and adding of pre-trigger and trigger solutions to initiate a chemiluminescent reaction the result of which is measured as relative light units (RLUs). The presence or absence of EBV VCA IgM, VCA IgG, or EBNA-1 IgG antibodies in the sample is determined by comparing the chemiluminescent signal in the reaction to the cutoff signal determined from an active calibration. Assay results are presented as a ratio of the specimen signal (in RLUs) to the cutoff RLU value (S/CO). For the EBV VCA IgM and EBV EBNA-1 IgG assays, S/CO values of ≥1.00 are considered reactive, S/CO values from 0.50 to b1.00 are considered grayzone, and S/CO values b0.50 are considered nonreactive. For the EBV VCA IgG assay, S/CO values of ≥1.00 S/CO are considered reactive, S/CO values from 0.75 to b1.00 are considered grayzone, and S/CO values b0.75 are considered nonreactive. 2.2. DiaSorin LIAISON ® EBV VCA IgM, EBV VCA IgG, and EBV EBNA IgG assays For the evaluation of relative sensitivity, relative specificity, and infection stage determination, the ARCHITECT ® EBV VCA IgM, EBV VCA IgG, and EBV EBNA-1 IgG results were compared to those obtained by the respective LIAISON® EBV IgM, VCA IgG, and EBNA IgG (DiaSorin, Saluggia, Italy) assays. The interpretation of the LIAISON ® EBV assay results was performed according to their respective package insert instructions. 2.3. Specimens 2.3.1. Specimens for sensitivity, specificity, and infection stage determination Specificity and sensitivity were assessed on a total of 1113 specimens, consisting of 995 random diagnostic specimens and 118 presumed acute infection specimens. The presumed acute infection specimens had been preselected based on a positive heterophilic

311

antibody (Monospot) result, which provides a patient population with a high probability of acute EBV infections without favouring any of the 2 methods under evaluation. Of the 1113 specimens, 200 were fresh, and the remaining specimens were collected prior to study start and stored frozen. Frozen specimens were thawed once for testing. Based on the high seroprevalence for EBV antibodies, it was expected that the vast majority of diagnostic samples would be EBNA1 IgG and VCA IgG positive. In order to be able to evaluate specificity on a sufficient number of specimens for these markers, 250 selected seronegative specimens were analysed separately. These samples were chosen based on a negative result by Enzygnost anti-EBV IgG (Siemens Healthcare Diagnostic Products, Marburg, Germany) and anti-EBV EBNA IgG (BIO-RAD Medical Diagnostics GmbH, Dreieich, Germany) and sourced from a routine clinical laboratory in Germany. The preselected seronegative specimens were collected prior to study start and stored frozen. They were thawed once for testing. 2.4. Resolution testing Any specimen with discrepant results between the ARCHITECT ® EBV assay and the corresponding LIAISON ® EBV assay or specimens within the grayzone (equivocal results) underwent supplemental testing. 2.4.1. EBNA-1 IgG Specimens with discordant results between ARCHITECT ® EBV EBNA-1 IgG and LIAISON® EBNA IgG were tested with Euroimmun EBV-Profile 2 Euroline IgG (EUROIMMUN, Lübeck, Germany) and Mikrogen recomLine EBV IgG (MIKROGEN GmbH, Neuried, Germany) immunoblots. Only the EBNA-1 band in each blot was considered for EBNA-1 IgG reactivity. When the 2 immunoblots gave discordant results, the bioMérieux VIDAS ® EBV EBNA IgG (bioMérieux, Marcy l'Etoile, France) was performed, and its result was used to determine the final interpretation, based on agreement between 2 out of the 3 assays. Any specimen with a discordant EBNA-1 IgG result but at the same time concordantly negative for VCA IgG was considered as false reactive for EBNA-1 IgG and not tested further. Specimens with discordant results for EBNA-1 IgG but concordantly reactive for VCA IgM and VCA IgG were tested for VCA IgG avidity by immunofluorescence to determine whether the specimen was likely to be from an acute or past infection. Testing by in-house immunofluorescence tests (IFT) was performed at Virology Department of the Institute for Medical Microbiology and Hygiene, University of Freiburg, Germany. 2.4.2. EBV VCA IgG Specimens discordant between ARCHITECT ® EBV VCA IgG and LIAISON ® VCA IgG were resolved by IFT EBV VCA IgG, since this is considered to be the most widely accepted confirmation assay for EBV VCA IgG. Additionally, specimens were evaluated using Euroimmun EBV-Profile 2 Euroline IgG and Mikrogen recomLine EBV IgG Immunoblots. Any specimen with a discrepant VCA IgG result but concordantly VCA IgM non-reactive and EBNA-1 IgG reactive was assumed to be VCA IgG reactive, and no further confirmation testing was done. 2.4.3. EBV VCA IgM Specimens with discordant results between ARCHITECT ® EBV VCA IgM and LIAISON® EBV IgM were resolved using either a) IFT VCA IgM and IFT VCA IgG avidity when they were concordantly nonreactive for EBNA-1 IgG indicating an acute primary EBV infection or b) by Euroimmun EBV-Profile 2 Euroline IgM, Mikrogen recomLine EBV IgM immunoblots, and IFT EBV-VCA-IgM when EBNA-1 IgG was concordantly reactive indicating a past infection. These testing strategies were chosen to differentiate between acute phase specimens with low-avidity VCA IgG when IgM reactivity is likely to be present and those of later infection stages when VCA IgM reactivity should not be

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detected. Specimens with proven VCA IgM reactivity in past infection were therefore excluded from calculation of sensitivity and specificity.

2.5. Data analysis In a first step, test results of each individual ARCHITECT ® EBV assay were compared to those generated by the corresponding LIAISON ® EBV assay to calculate sensitivity and specificity. Specimens giving discordant results between both assays were resolved as outlined above to reach a final interpretation. Specimens with grayzone results in any assay were excluded from calculation of sensitivity and specificity. Secondly, the interpretation of the combined assay results for all 3 markers of one system was used to determine the stage of EBV infection for each clinical specimen as indicated by the manufacturers. The results of both panels were compared to the “true” status of the specimens as obtained after resolution testing of any discordant or grayzone specimen. Additionally, the data set was retrospectively analyzed as if specimens had been initially tested solely by ARCHITECT® and LIAISON ® EBNA-1 IgG; followed by parallel testing of ARCHITECT ® and LIAISON® VCA IgM/IgG for all EBNA-1 IgG nonreactive or grayzone/equivocal specimens (see Fig. 1) to determine its impact on accuracy of results and number of tests needed for a final interpretation. The following stages of EBV infections were distinguished per final interpretation based on the results generated for all 3 markers: seronegative, early phase acute primary infection, acute primary infection, transient infection, past infection, isolated VCA IgG, and isolated EBNA-1 IgG reactivity. The term “transient” infection is used for specimens showing triple reactivity for VCA IgM, VCA IgG, and EBNA-1 IgG antibodies according to Gärtner et al. (2003), suggesting that all 3 analytes can be found in patients with primary EBV infections when VCA IgM persists while EBNA-1 IgG is already produced or during EBV reactivation, when VCA IgM levels are increased and EBNA-1 IgG is not yet lost. In these cases, if clinically relevant, further evaluation such as avidity testing of VCA IgG or immunoblot analysis can possibly provide more insight into the stage of the EBV infection. Specimens classified as early phase acute primary infection, transient infection, isolated VCA IgG, and isolated EBNA-1 IgG were regarded as “unresolved” because they require either additional testing of the same or a second sample. Grayzone results were included for infection stage determination and interpreted as follows: for ARCHITECT ®, a sample with a grayzone test result in the VCA IgM or VCA IgG assay is always considered reactive, while a grayzone EBNA-1 IgG result is considered as non-

reactive. There is one exception: a grayzone VCA IgM result in combination with reactivity in both VCA IgG and EBNA-1 IgG assays is considered as non-reactive, to be interpreted as past infection rather than transient phase. The interpretation of the LIAISON ® EBV assay panel was determined as indicated in the package insert: VCA IgM and EBNA IgG equivocal results in the LIAISON® EBV assays are interpreted either as reactive or nonreactive for the purpose of infection stage determination, depending on the result of the other 2 markers tested. 3. Results 3.1. Specificity and sensitivity of ARCHITECT ® and LIAISON ® individual EBV assays Specificity and sensitivity were assessed on a total of 1113 diagnostic specimens. Specificity was also separately assessed on 250 preselected EBV seronegative specimens. 3.1.1. EBV VCA IgM Of 1113 specimens tested, 154 specimens were classified as VCA IgM positive, and 841 specimens, as VCA IgM negative, while 118 specimens were excluded from the sensitivity and specificity calculation. Twelve of these showed inconclusive results after confirmation, and 23 specimens were positive for all 3 markers (Table 1), the remainder being excluded due to grayzone/equivocal results. Grayzone rates were 4.9% (n = 54) and 6.4% (n = 71) for the ARCHITECT® and LIAISON ® EBV IgM assays, respectively (Table 1). Sensitivity was 100% for both ARCHITECT ® and LIAISON® EBV IgM assays. In the non-selected diagnostic population, ARCHITECT ® EBV VCA IgM generated 1 false-positive result, indicating a specificity of 99.9%. In comparison, 9 false-positive results were obtained for the LIAISON ® EBV IgM assay, resulting in a specificity of 98.9%. Specificity in the group of preselected seronegative samples was 100.0% for ARCHITECT® and 99.5% for LIAISON® VCA IgM (Table 2). 3.1.2. EBV VCA IgG Based on the results after resolution testing, 986 specimens were classified as VCA IgG positive and 109 specimens as VCA IgG negative. Three specimens gave inconclusive confirmation results, and 15 specimens (1.3 %) had a grayzone result in the ARCHITECT ® EBV VCA IgG assay and were excluded from calculation of sensitivity and specificity. The LIAISON ® VCA IgG assay does not include a grayzone interpretation (Table 1).

EBNA-1 IgG

Reactive

Past Infection

Non-Reactive

VCA IgG & IgM - IgM

Both Reactive

Single Reactive VCA IgG

Acute Infection

Unresolved: Additional Testing or Follow Up Sample (Avidity, Blot, PCR)

Both Non-Reactive

VCA IgM

Suspected Primary Infection (follow-up)

Fig. 1. EBNA-based testing algorithm.

Seronegative

E. Sickinger et al. / Diagnostic Microbiology and Infectious Disease 79 (2014) 310–316 Table 1 Specimens (n = 1113) used to evaluate specificity and sensitivity of ARCHITECT® and LIAISON® individual EBV assays. Marker/ instrument

Grayzone, no. (%)

Positive/Discordant reactive in past infection

Confirmation inconclusive

Specimens excluded (n)

VCA IgM ARCHITECT® LIAISON®

54 (4.9) 71 (6.4)

23a

12

118

VCA IgG ARCHITECT® LIAISON®

15 (1.3) N/A

N/A

3

18

EBNA-1 IgG ARCHITECT® LIAISON®

8 (0.7) 57 (5.1)

12b

4

68

a b

Positive in past infection. Discordant reactive in past infection.

Table 2 Sensitivity and specificity of ARCHITECT® and LIAISON® individual EBV assays. ARCHITECT®

VCA IgM Sensitivity (acute infections) Overall specificity (past or no infections) Specificity preselected seronegatives VCA IgG Overall sensitivity Overall specificity Specificity preselected seronegatives EBNA-1 IgG Sensitivity (past infections) Overall specificity (acute and no infections) Specificity preselected seronegatives

% (n)

3.1.3. EBV EBNA-1 IgG Of 1113 specimens tested, 764 specimens were classified as EBNA1 IgG positive, and 281 specimens were EBNA-1 IgG negative. Sixtyeight specimens were excluded from the sensitivity and specificity calculation. Of these, 4 showed inconclusive results after confirmation, and 12 were discordant reactive in past infection, the remainder were grayzone/equivocal reactive: 8 (0.7%) in ARCHITECT ® EBV EBNA-1 IgG in comparison to 57 (5.1%) for LIAISON ® EBNA (Table 1). Sensitivity was 100.00% for ARCHITECT® EBNA-1 IgG and 99.2% for the LIAISON ® EBNA-1 IgG assay (Table 2). Both assays had 1 falsereactive result and a calculated specificity of 99.6% in the random diagnostic population. When using the well-defined preselected negative samples to assess specificity, ARCHITECT ® EBNA-1 IgG showed no false-positive results compared to one for LIAISON ® EBNA IgG. 3.2. EBV panel performance

The ARCHITECT® EBV VCA IgG assay had 17 false-negative results compared to 28 for the LIAISON® EBV IgG assay, resulting in sensitivities of 98.3% and 97.2% for ARCHITECT® and LIAISON®, respectively (Table 2). A further analysis of those samples missed by either assay revealed that all 17 specimens missed by ARCHITECT® EBV VCA IgG were in the early phase of an acute infection indicated by VCA IgM reactivity. For 14 samples, the Monospot result was positive; for the remaining 3 samples, the Monospot result is not known. In contrast, all samples classified as past infections were detected by the ARCHITECT VCA IgG assay. LIAISON® missed VCA IgG in 9 samples in the acute infection phase and in 17 specimens in the phase of past EBV infection and 1 sample classified as VCA IgG reactive only. One specimen was excluded from this analysis due to inconclusive results upon resolution testing. In the group of preselected seronegative specimens, ARCHITECT ® EBV VCA IgG had 3 false-positive results and a calculated specificity of 98.8% versus 9 false-positive results for LIAISON ® EBV VCA IgG and 96.3% specificity (Table 2). When looking at specificity in the routine diagnostic population, specificity was 95.4% (104/109) for ARCHITECT ® EBV VCA IgG and 99.1% (108/109) for LIAISON ® VCA IgG after resolution of discordant results by VCA IgG immunofluorescence. Two out of 5 samples with a false-positive result in the ARCHITECT ® EBV VCA IgG assay showed VCA IgG reactive bands in 1 or 2 immunoblots. The 1 false-positive specimen in the LIAISON® EBV VCA IgG assay was positive in the Mikrogen recomLine EBV IgG blot.

Marker/population

313

LIAISON® FN or FP

% (n)

FN or FP

100.0 (154/154) 99.9 (840/841)

0 1

100.0 (154/154) 98.9 (832/841)

0 9

100.0 (209/209)

0

99.5 (208/209)

1

98.3 (969/986) 95.4 (104/109) 98.8 (239/242)

17 5 3

97.2 (958/986) 99.1 (108/109) 96.3 (233/242)

28 1 9

100.0 (764/764) 99.6 (280/281)

0 1

99.2 (758/764) 99.6 (280/281)

6 1

100.0 (238/238)

0

99.6 (237/238)

1

Results generated by all 3 assays of the respective ARCHITECT ® or LIAISON ® EBV panels and their combined interpretation to determine the stage of an EBV infection were compared to those obtained after resolution of discordant results. Based on the final interpretation after resolution, samples were categorized as follows: 92 were EBV seronegative, 153 showed a pattern of acute infection (combining 2 categories of early acute and acute primary infections), and 790 were from past infection. Moreover, 60 samples (5.5 %) had an unclear pattern (Tables 3 and 4). The ARCHITECT® EBV panel gave a correct result for 85 of 92 specimens defined as seronegative, resulting in a specificity of 92.4%, whereas the LIAISON® EBV panel correctly classified 86 specimens as seronegative, with a calculated specificity of 93.5% (Table 3). Out of 153 samples with a confirmed pattern of acute infection (taking together samples with isolated VCA IgM or combined VCA IgM and IgG reactivity in the absence of EBNA-1 IgG antibodies) 149 and 152, respectively, were correctly determined by the ARCHITECT® and LIAISON® EBV panel giving a calculated sensitivity of 97.4% and 99.3%. In the group of past infection specimens, 773 specimens were correctly identified by ARCHITECT ®, and 745, by LIAISON®, yielding a sensitivity of 97.8% and 94.3%, respectively (Table 3). In this group, 17 specimens interpreted differently by ARCHITECT® were either transient, with 3-marker reactivity, or isolated VCA IgG reactive. In contrast, amongst the 45 specimens that were misclassified by LIAISON®, 6 were apparently seronegative, and another 2 gave a pattern of an acute infection. The overall agreement of the individual EBV panels versus the “true” infection stage based on the results after resolution testing was 95.6% for the ARCHITECT® EBV panel versus 93.4% for LIAISON ® EBV panel when considering parallel testing with all 3 parameters. The data were further analysed as if all specimens had been tested for EBNA-1 IgG antibodies in a first step and only for those that were not EBNA-1 IgG reactive, test results for VCA IgG and IgM were taken into account. According to this approach, ARCHITECT ® EBNA-1 IgG categorised 808 specimens as past infection due to a positive EBNA-1 IgG test, compared to 759 specimens by the LIAISON ® EBNA IgG assay (note: due to exclusion of 18 inconclusive specimens per final interpretation, the results for only 805 are shown in Table 4). Consequently, 305 samples would have needed further testing for VCA IgG and VCA IgM on ARCHITECT ® resulting in a total of 1723 tests (1113 and 2 × 305), an average of 1.55 tests per sample. For LIAISON®, the same calculation gave a total number of 1821 tests and an average of 1.64 tests per sample. The classification of patients as having past EBV infection based on initial EBNA testing was accurate for all but 1 case for each assays system: one acute primary infection specimen was classified as past infection instead of transient infection by the ARCHITECT® EBNA-1 IgG assay as well as a different sample by the LIAISON® EBNA IgG assay (Table 4). Based on the results of initial EBNA-1 IgG testing, the agreement versus the “true” infection stage was 94.0% for the ARCHITECT ® EBV panel and 93.9% for the LIAISON® EBV panel.

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Table 3 Parallel testing with the ARCHITECT® compared to the LIAISON® EBV Panel. Serological profile

Staging per final interpretation

VCA IgM/VCA IgG/EBNA-1 IgG

Seronegative Early phase acute Acute primary Transient Past Isolated Isolated Total primary infection infection infection infection VCA IgG EBNA-1 IgG

ARCHITECT® EBV Panel −/−/− +/−/− +/+/− +/+/+ −/+/+ −/+/− −/−/+

Seronegative 85 Early phase acute primary infection 2 Acute primary infection 0 Transient infection 0 Past infection 0 Isolated VCA IgG 5 Isolated EBNA-1 IgG 0 Total 92 Agreement to final interpretation 95.6% LIAISON® EBV Panel −/−/− Seronegative 86 +/−/− Early phase acute primary infection 5 +/+/− Acute primary infection 0 +/+/+ Transient infection 0 −/+/+ Past infection 0 −/+/− Isolated VCA IgG 1 −/−/+ Isolated EBNA-1 IgG 0 Total 92 Agreement to final interpretation 93.4% a

2 9 0 0 0 0 0 11

1 15 125 1 0 0 0 142

0 0 1 22 1 0 0 24

0 0 0 6 773 11 0 790

0 0 3 0 0 31 0 34

0 0 0 0 0 0 2 2

88 26 129 29 774 47 2 1095a

0 11 0 0 0 0 0 11

0 10 131 1 0 0 0 142

0 0 1 19 4 0 0 24

6 0 2 13 745 11 13 790

1 0 4 0 0 29 0 34

0 0 0 0 0 0 2 2

93 26 138 33 749 41 15 1095a

18/1113 specimens were excluded due to inconclusive results.

serologic profiles due to isolated VCA IgM, VCA IgG, or EBNA-1 IgG reactivity or positivity for all 3 parameters are not uncommon and are observed in up to 10% of cases (De Paschale et al., 2009; Garcia et al., 2008; Klutts et al., 2004; Klutts et al., 2009). In the present study, 7.3% of samples were unresolved after initial EBV testing with VCA IgG, VCA IgM, and EBNA-1 IgG. Increasing the number of markers for routine serological status determination, e.g., by adding EA-D IgG (IgG antibodies to EBV early antigen-diffuse) generally does not improve the accuracy of the diagnosis (Klutts et al., 2009). Since each individual assay contributes to the performance of the total ARCHITECT® or LIAISON ® EBV panel, specificity and sensitivity of the VCA IgM, VCA IgG, and EBNA-1 IgG ARCHITECT ® and LIAISON® assays were determined separately. Both VCA IgM assays had a sensitivity of 100% for detection of specimens from an acute phase EBV infection. Slightly better specificity was observed for the ARCHITECT® EBV VCA IgM assay with only 1 false-positive result compared to 9 false positives for the LIAISON ® VCA IgM assay, resulting in calculated specificities of 99.9% and 98.9%, respectively. If grayzone results were interpreted as false

4. Discussion The samples investigated in this study are representative of those obtained in daily routine testing in a primary care lab. They are typically sent in by general practitioners with a request for EBV IgM testing. When excluding the preselected acute infection specimens (heterophilic antibody positive), the vast majority of those specimens, i.e., 80.9%, were actually determined to be past infections and only 2.4% turned out to be acute infections. The rate of seronegative specimens was 9.4%. These numbers are in accordance with previously published findings (Hess, 2004; Kreuzer et al., 2013). They underline that in routine clinical testing, more than 70% of the serum samples originate from patients with past EBV infection because of the high seroprevalence of close to 95% in adults (Hess, 2004). VCA IgG, VCA IgM, and EBNA-1 IgG are thought to be the most relevant EBV markers because they generally allow differentiation of acute EBV infection from past or no infection. They are suitable to diagnose EBV-associated IM in immune competent individuals (Garcia et al., 2008; Hess, 2004; Ooka et al., 1991). However, unresolved

Table 4 EBNA-based testing with the ARCHITECT® compared to the LIAISON® EBV Panel. Serological profile

Staging per final interpretation

VCA IgM/VCA IgG/EBNA-1 IgG

Seronegative Early phase acute Acute primary Transient Past Isolated Isolated Total primary infection infection infection infection VCA IgG EBNA-1 IgG

ARCHITECT® EBV Panel −/−/− +/−/− +/+/− −/+/+ −/+/−

Seronegative 85 Early phase acute primary infection 2 Acute primary infection 0 Past infection 0 Isolated VCA IgG 5 Total 92 Agreement to final interpretation 94.0% LIAISON® EBV Panel −/−/− Seronegative 86 +/−/− Early phase acute primary infection 5 +/+/− Acute primary infection 0 −/+/+ Past infection 0 −/+/− Isolated VCA IgG 1 Total 92 Agreement to final interpretation 93.9%

a

18/1113 specimens were excluded due to inconclusive results.

2 9 0 0 0 11

1 15 125 1 0 142

0 0 1 23 0 24

0 0 0 779 11 790

0 0 3 0 31 34

0 0 0 2 0 2

88 26 129 805 47 1095a

0 11 0 0 0 11

0 10 131 1 0 142

0 0 1 23 0 24

6 0 2 771 11 790

1 0 4 0 29 34

0 0 0 2 0 2

93 26 138 797 41 1095a

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positives, the specificity of the ARCHITECT ® VCA IgM assay decreased from 99.9% in the seronegative population to 94.4%, compared to a decrease from 98.9% to 91.7% for the LIAISON® VCA IgM assay. The ARCHITECT ® EBV VCA IgG assay showed better sensitivity: 17 false-negative results were obtained in comparison to 28 false negatives for the corresponding LIAISON ® EBV VCA IgG assay. This seems to agree with the slightly lower specificity of the ARCHITECT® EBV VCA IgG assay compared to LIAISON ® EBV VCA IgG assay (95.4% versus 99.1%) observed in the non-selected diagnostic population. However, due to the high seroprevalence rate, the total number of VCA IgG negative samples was relatively low (n = 109). Interestingly, the opposite was seen in the larger group of preselected seronegative samples: the ARCHITECT ® EBV VCA IgG assay had a similar or even better specificity compared to the LIAISON ® EBV VCA IgG assay (98.8% versus 96.3%, respectively). Further analysis of the false-reactive specimens in immunoblot, although not part of the confirmation strategy, revealed VCA IgG–positive results for at least 2 of the 5 false positives by 1 or 2 blots. It may be questioned whether these specimens are truly false positive or potentially isolated VCA IgG being missed by Immunofluorescence assay (IFA) when used as the only confirmatory assay. Basically, Enzyme immunoassay (EIA) is more sensitive than IFA, but IFA is more specific than EIA because nonspecific reactions, such as anticellular reactivity, are detected unambiguously (Bauer, 2001; Gärtner et al., 2003). Due to the fact that specimens with grayzone results were excluded from analysis of sensitivity and specificity, similar data were obtained for both EBNA-1 IgG assays. The difference in grayzone results between the 2 systems was striking: 57 (5.1%) by LIAISON ® EBNA IgG assay versus only 8 (0.7%) for the ARCHITECT ® EBNA-1 IgG assay. This relatively high number has been reported before (de Ory et al., 2011). If grayzone results are interpreted as non-reactive in the LIAISON ® EBNA IgG assay, the number of false-negative samples increases from 6 to 46, resulting in a sensitivity of 94.3%, whereas for the ARCHITECT® EBNA-1 IgG assay, sensitivity decreases slightly from 100% to 99.6%. If on the other hand grayzone specimens are counted as reactive, the overall specificity decreases from 99.6% to 98.6% for the ARCHITECT® EBNA-1 IgG assay and from 99.6% to 96.2% for the LIAISON ® EBNA IgG assay. This is even more pronounced in the group of preselected seronegative samples: ARCHITECT ® EBNA-1 IgG remains at 100% specificity, while LIAISON® EBNA IgG has 13 false positives and specificity drops to 94.8%. Sensitivity and specificity performance characteristics of immunoassays greatly depend on the antigens selected and their preparation. It has been suggested that antibodies against the p18 VCA antigen are produced late during primary infection (Bauer, 2001), and sensitivity of VCA IgM detection might be improved by combined use of p18 and gp 125 (Berth and Bosmans, 2010). Choice and nature of antigens, i.e,. native, recombinant, or synthetic, indeed lead to considerable variations in sensitivity and specificity for different assays (Bruu et al., 2000; de Ory et al., 2011; Gärtner et al., 2003; Hess, 2004; Klutts et al., 2004). However, those differences cannot always solely be attributed to the antigens used, as even assays using the very same antigen, i.e., p18, showed differences in sensitivities of more than 20% as observed for the DiaSorin assays (Chemiluminescent immunoassay-Liaison (CLIA-L) and enzyme-linked immunosorbent assay) (de Ory et al., 2011). Other factors, such as choice of dilution buffer, assay format, and detection system, may have a significant influence as well. In addition, the interpretation of results varies for different test systems according to each manufacturer's package insert. Overall, acceptable results for both sensitivity and specificity were obtained for all assays evaluated in our study. However, a large number of specimens were excluded from the analysis due to grayzone results or inconclusive confirmation results. This was most prominent for the VCA IgM assays reflecting the dilemma of EBV serology in general. In the absence of a proper reference test and the necessity of high sensitivity for detection of early acute infections, VCA IgM assays require a large

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grayzone. When used in parallel to VCA IgG and EBNA-1 IgG assays, a large number of grayzone results is, therefore, inevitable. To overcome this inherent general lack of clinically sufficient performance of any EBV IgM test and to avoid overuse of laboratory tests, testing algorithms may be a valuable and economic approach. In this study, we compared 2 strategies, i.e., initial testing with all 3 EBV assays in parallel versus initial testing using solely the EBNA-1 IgG assay (EBNA testing) followed by VCA IgG and IgM for EBNA-1 IgG nonreactive or grayzone/equivocal samples only. The ability to generate the correct serological result and the total number of tests needed was calculated for each system separately. Applying EBNA-1 IgG testing initially, higher average tests per sample of 1.64 (1821/ 1113) are needed when using the LIAISON® EBV panel compared to only 1.55 tests per sample (1723/1113) using the ARCHITECT ® EBV panel. The ARCHITECT® EBV EBNA-1 IgG assay interpreted 808/1113 specimens as past infection on first approach requiring no further testing compared to 759 by the LIAISON ® EBV EBNA IgG assay. This is mainly due to the large number of grayzone results in the LIAISON ® EBNA-IgG test. Thirty-eight of 57 specimens initially grayzone in the LIAISON ® EBNA IgG assay could only be interpreted as past infection on the basis of additional VCA IgG testing results. Notably, using this algorithm, only 1 sample in each system was incorrectly interpreted compared to the simultaneous use of all 3 tests (VCA IgM, VCA IgG, EBNA-1 IgG). One sample was judged per final status as acute infection but classified as past infection based on ARCHITECT® EBNA-1 IgG testing alone. Resolution testing for this sample showed a grayzone VCA IgG avidity result suggesting that the infection was not from the early acute phase. For LIAISON ® EBNA IgG, a different specimen was found to be EBNA-1 IgG positive and, therefore, treated as past infection but actually had low VCA IgG avidity indicating an acute infection specimen. When parallel testing with all 3 assays was done, larger differences in sensitivity and specificity between both panels were observed. Only 745 of 790 past infection specimens were correctly identified by the LIAISON ® EBV panel, 13 were classified as transient, and another 13, as isolated EBNA-1 IgG, due to a lower VCA IgG sensitivity as outlined above. Using the ARCHITECT® EBV panel, 6 specimens were classified as transient infection, highlighting the benefit of EBNA-1 IgG testing, as long as a specific and sensitive test is used. The agreement with infection stage determination based on interpretation after resolution testing was a little lower when using an EBNA-1 IgG based testing for ARCHITECT®, e.g., 94.0% versus 95.6% when all markers are evaluated in parallel, because all specimens showing 3 markers indicative of a transient phase are tagged as past infection based on the positive EBNA-1 IgG result and, as a result, do not require further testing. This is acceptable as a simplification and more efficient way of diagnosis as long as the EBNA-1 IgG assay does not lead to the wrong classifications of seronegative samples or those in an acute phase of EBV infection. Vice versa, the agreement with infection stage determination based on interpretation after resolution testing was slightly better for LIAISON ® when using initial EBNA IgG testing versus when performing parallel testing of all 3 markers, masking the lack of VCA IgG reactivity in 13 samples from past infection. For interest, we also calculated the average number of tests needed when all specimens are tested for VCA IgG and IgM initially, followed by EBNA-1 IgG for all VCA IgG reactive samples, a strategy that is not uncommon in labs since physicians usually suspect IM in the majority of samples sent for analysis. In this case, 2.9 tests per sample are required to come to the same conclusions, almost as many as using the parallel testing of all 3 markers. This study has some limitations. In the absence of a “gold standard” reference method (although IFT is sometimes perceived as such) and missing clinical data for the evaluated specimens, sensitivity and specificity of the 3 ARCHITECT® EBV assays were established in comparison to a generally accepted method, i.e., the LIAISON® EBV panel. The LIAISON® EBV panel was chosen as it is the most widely used

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automated EBV assay panel in serologic testing. During evaluation of sensitivity and specificity, confirmatory testing was only performed for samples with discordant results between the 2 methods. This approach is acceptable as the reliability of the comparator assay has been clearly established (de Ory et al., 2011; Gärtner et al., 2003). Nevertheless, misclassifications based on the reference method employed cannot be excluded totally. This may have an impact on the accuracy of the reported sensitivities and specificities. In summary, using an algorithm based on initial EBNA-1 IgG testing for the ARCHITECT® EBV panel leads to determination of correct immune status and stage of infection during initial testing in approximately 81% of specimens received during daily routine testing in a primary care lab. It reduces the number of tests needed per specimen by nearly 50% compared to parallel testing by VCA IgM and VCA IgG or by testing of all 3 markers in parallel. The high specificity of the ARCHITECT® EBNA-1 IgG assay combined with a good sensitivity of the VCA IgM assay for detection of early acute EBV infections makes this system suitable for accurate and cost-efficient EBV serodiagnosis in a routine clinical laboratory. However, in cases where the clinical picture is more complex, testing all 3 EBV markers in parallel may be considered a necessary option. Acknowledgments The authors thank Dr Huzly for many invaluable discussions on the topic of EBV serology, on confirmatory algorithms, and finally for performing IFT at Institut für Medizinische Mikrobiologie und Hygiene, Abteilung Virologie, Universitätsklinikum Freiburg, Germany. References Balfour Jr HH, Odumade OA, Schmeling DO, Mullan BD, Ed JA, Knight JA, et al. Behavioral, virologic, and immunologic factors associated with acquisition and severity of primary Epstein-Barr virus infection in university students. J Infect Dis 2013;207:80–8. Bauer G. Simplicity through complexity: immunoblot with recombinant antigens as the new gold standard in Epstein-Barr virus serology. Clin Lab 2001;47:223–30. Berth M, Bosmans E. Comparison of three automated immunoassay methods for the determination of Epstein-Barr virus-specific immunoglobulin M. Clin Vaccine Immunol 2010;17:559–63. Bruu AL, Hjetland R, Holter E, Mortensen L, Natas O, Petterson W, et al. Evaluation of 12 commercial tests for detection of Epstein-Barr virus-specific and heterophile antibodies. Clin Diagn Lab Immunol 2000;7:451–6. Crawford DH, Swerdlow AJ, Higgins C, McAulay K, Harrison N, Williams H, et al. Sexual history and Epstein-Barr virus infection. J Infect Dis 2002;186:731–6. de Ory F, Guisasola ME, Sanz JC, Garcia-Bermejo I. Evaluation of four commercial systems for the diagnosis of Epstein-Barr virus primary infections. Clin Vaccine Immunol 2011;18:444–8.

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