What tests are used to diagnose Epstein-Barr virus (EBV) infection?

How to Test for EBV

Begin with a heterophile antibody test (Monospot) combined with a complete blood count with differential, but if the heterophile test is negative and clinical suspicion remains high, proceed immediately to EBV-specific antibody testing including VCA IgM, VCA IgG, and EBNA antibodies. 1, 2

Initial Testing Strategy

First-Line Testing

• Order a rapid heterophile antibody test (Monospot or Paul-Bunnell test) as the initial screening test in adolescents and adults with suspected infectious mononucleosis 1, 2
• Obtain a complete blood count with differential to look for elevated white blood cell count with increased atypical lymphocytes, which is a hallmark of EBV-associated mononucleosis 1
• The heterophile antibody becomes detectable between days 6-10 after symptom onset and peaks during weeks 2-3 of illness 2

Critical Limitations of Heterophile Testing

• Do not rely solely on heterophile testing in children under 10 years—proceed directly to EBV-specific antibodies, as false-negative rates are significantly higher in this age group 1, 2
• Approximately 10% of patients will have false-negative heterophile tests even with acute EBV infection 2, 3
• False-positive results can occur with leukemia, pancreatic carcinoma, viral hepatitis, and CMV infection 2

EBV-Specific Antibody Panel

When to Order

• Order EBV-specific antibodies immediately when the heterophile test is negative but clinical suspicion remains high 1, 2
• The Infectious Diseases Society of America explicitly states that heterophile tests are suboptimal and recommends EBV-specific antibody testing when these are negative 2

Essential Components of the Panel

• VCA IgM: Indicates acute or recent infection 1, 2, 4
• VCA IgG: Develops rapidly in acute infection and persists for life 1, 2, 4
• EBNA antibodies: Critical for timing the infection; develops 1-2 months after primary infection and persists for life 1, 2, 4

Interpreting EBV Antibody Results

Primary Acute Infection

• VCA IgM positive + EBNA antibodies absent = recent primary EBV infection (less than 6 weeks) 1, 2, 4
• VCA IgG may or may not be present initially 4

Past Infection

• EBNA antibodies present + VCA IgM absent = past infection (more than 6 weeks ago), making EBV unlikely as the cause of current symptoms 1, 2, 4
• Over 90% of normal adults have IgG antibodies to VCA and EBNA from past infection 2

Equivocal Patterns

• If VCA IgG is present without VCA IgM or EBNA antibodies, determine IgG avidity to distinguish acute from past infection 4
• If all three parameters (VCA IgM, VCA IgG, and EBNA) are positive simultaneously, this may indicate recent infection or reactivation 4

Testing in 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 test (NAAT) in peripheral blood rather than relying solely on serology 1, 2
• EBV DNA levels > 10^2.5 copies/mg DNA in peripheral blood mononuclear cells indicate active infection 5, 1
• Post-transplant patients require EBV DNA surveillance due to high risk of lymphoproliferative disease 2

Children Under 10 Years

• Proceed directly to EBV-specific antibody testing (VCA IgM, VCA IgG, EBNA) rather than heterophile testing due to higher false-negative rates 1, 2

CNS Involvement

• For suspected EBV-associated encephalitis, perform both CSF PCR and serology (VCA IgM/IgG and EBNA) 1
• Collect cerebrospinal fluid in a sterile tube and transport at room temperature within 2 hours 1

Chronic Active EBV Infection (CAEBV)

Diagnostic Criteria

• CAEBV requires: (1) persistent or recurrent infectious mononucleosis-like symptoms, (2) unusual antibody patterns with raised anti-VCA and anti-EA, and (3) chronic illness that cannot be explained by other known disease processes 5
• Markedly elevated VCA IgG titers (≥1:640) combined with elevated EA IgG (≥1:160) suggest CAEBV 5, 1
• Positive IgA antibodies to VCA and/or EA are often demonstrated in CAEBV 5, 1

Recommended Specific Laboratory Tests for CAEBV

• Quantitative PCR showing > 10^2.5 copies/mg DNA in peripheral blood mononuclear cells 5
• In situ hybridization for EBV-encoded RNAs (EBERs) in affected tissues 5
• Double staining to clarify target cells of EBV infection (B, T, NK cells or monocytes/macrophages) 5

Specimen Collection

Timing and Type

• Collect serum specimens as soon as possible after symptom onset for serologic testing 1
• For EBV DNA detection, use whole blood, peripheral blood lymphocytes, or plasma in EDTA tube at room temperature, transported within 2 hours 1

Common Pitfalls to Avoid

• Do not order EBV testing from throat swabs—EBV can persist 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; these develop 1-2 months post-infection and indicate past exposure 1, 2
• Remember that 5-10% of infected patients, particularly immunocompromised individuals, may not develop EBNA antibodies despite infection 1, 2
• Consider alternative diagnoses including CMV, adenovirus, HIV, and Toxoplasma gondii when evaluating mononucleosis-like illness 2