In an adult or adolescent with suspected infectious mononucleosis, how useful is EBNA (Epstein‑Barr nuclear antigen) serology for diagnosing a new EBV infection?

EBNA Testing for Diagnosing Acute EBV Infection

EBNA (Epstein-Barr nuclear antigen) antibody testing is highly useful for diagnosing new EBV infection because the absence of EBNA antibodies combined with positive VCA IgM confirms acute primary infection, while the presence of EBNA antibodies effectively rules out recent infection. 1, 2

Diagnostic Role of EBNA in Acute EBV Infection

The critical diagnostic principle is that EBNA antibodies develop 1-2 months after primary infection and persist for life, making their absence a key marker of recent infection. 1, 2, 3

Primary Acute Infection Pattern

• Primary acute EBV infection is diagnosed when VCA IgM is positive AND EBNA antibodies are absent 4, 1, 2
• This serological pattern indicates infection occurring within the past 6 weeks 2, 3
• The combination of negative EBNA with low-avidity VCA IgG has 100% sensitivity and specificity for primary infection 5

Past Infection Pattern

• The presence of EBNA antibodies indicates infection occurring more than 6 weeks prior, making EBV unlikely as the cause of current acute symptoms 2, 3
• Over 90% of normal adults have both VCA IgG and EBNA antibodies from past infection 1, 2
• This pattern effectively excludes acute infectious mononucleosis as the diagnosis 3

Recommended Testing Algorithm

The Infectious Diseases Society of America provides clear guidance on when and how to use EBNA testing 4, 1:

Initial Approach

• Begin with heterophile antibody test (Monospot) and complete blood count with differential 1, 2
• The heterophile test becomes detectable between days 6-10 after symptom onset 1, 2

When Heterophile is Negative

• If heterophile test is negative but clinical suspicion remains high, proceed directly to EBV-specific antibody testing with VCA IgM, VCA IgG, AND EBNA antibodies 1, 2
• Paul-Bunnell and Monospot tests are explicitly considered suboptimal for diagnosis 4, 1
• This is particularly important because false-negative heterophile results occur in approximately 10% of patients 1

Interpretation Framework

• VCA IgM positive + EBNA absent = recent primary infection (less than 6 weeks) 2, 3
• EBNA present + VCA IgM absent = past infection (more than 6 weeks ago) 2, 3
• VCA IgM positive + EBNA present = likely reactivation or false-positive IgM, not acute primary infection 6, 7

Critical Limitations and Pitfalls

False-Positive VCA IgM

• VCA IgM can be falsely positive during other viral infections, particularly CMV (60.7% cross-reactivity rate) 7
• VCA IgM can also indicate reactivation rather than primary infection when EBNA antibodies are present 8, 6
• In patients with VCA IgM, VCA IgG, and EBNA all positive, only 42% actually had late primary infection—the majority had reactivation 6

EBNA Antibody Absence in Some Patients

• Approximately 5-10% of infected patients fail to develop EBNA antibodies, which should be considered when interpreting results 1, 3
• This is more common in immunocompromised individuals 2, 3

Age-Related Considerations

• Do not rely solely on heterophile testing in children under 10 years—proceed directly to EBV-specific antibodies including EBNA 1, 2
• False-negative heterophile results are especially common in this age group 1, 3

Special Populations

Immunocompromised Patients

• For transplant recipients, HIV-infected individuals, and those with congenital immunodeficiencies, order quantitative EBV viral load testing by nucleic acid amplification (NAAT) rather than relying solely on serology 2, 3
• EBV DNA levels >10^2.5 copies/mg DNA in peripheral blood mononuclear cells indicate active infection 2, 3
• Post-transplant patients require EBV DNA surveillance due to high risk of lymphoproliferative disease 4, 1

Chronic Active EBV Infection (CAEBV)

• CAEBV shows unusual antibody patterns with markedly elevated VCA IgG (≥1:640) and EA IgG (≥1:160) 4, 2
• Various EBNA titers are reported in CAEBV, ranging from nondetectable to increased levels 4
• Positive IgA antibodies to VCA and/or EA are often demonstrated in CAEBV 4, 2

Cost-Effective Testing Strategy

• A sequential testing algorithm starting with EBNA IgG analysis provides substantial cost savings (23%) compared to testing all markers simultaneously 7
• If EBNA is positive, acute infection is excluded and further testing is unnecessary 7
• If EBNA is negative, proceed with VCA IgM and VCA IgG to confirm acute infection 7

Common Diagnostic Errors to Avoid

• Do not order EBV testing from throat swabs—EBV persists in throat secretions for weeks to months after infection and does not confirm acute infection 1, 2
• Do not interpret the presence of EBNA antibodies as indicating acute infection 2, 3
• Do not assume VCA IgM alone confirms primary infection without checking EBNA status 6, 7
• Always consider CMV testing concomitantly, as CMV can cause false-positive VCA IgM results and presents with similar clinical features 7