Recommendations for Testing EBV Exposure
Primary Testing Approach
For suspected acute EBV infection (infectious mononucleosis), begin with a heterophile antibody test (Monospot), and if negative but clinical suspicion remains high, proceed immediately to EBV-specific antibody testing including VCA IgM, VCA IgG, and EBNA antibodies. 1
Initial Testing Strategy
Heterophile antibody testing is the recommended first-line test for suspected infectious mononucleosis in immunocompetent patients, collected as serum in a clot tube at room temperature and transported within 2 hours 1
Important caveat: Heterophile tests have approximately 10% false-negative rates overall, with significantly higher false-negative rates in children under 10 years of age 2, 3
When heterophile testing is negative but clinical suspicion persists, EBV-specific antibody panels must be ordered rather than stopping the diagnostic workup 2
EBV-Specific Serologic Testing
The definitive serologic panel includes three key antibodies: IgG and IgM to viral capsid antigen (VCA), and antibodies to Epstein-Barr nuclear antigen (EBNA) 1, 2
Interpretation of Serologic Patterns
Recent primary infection (< 6 weeks): VCA IgM positive (with or without VCA IgG) AND EBNA antibodies absent 1, 2
Past infection (> 6 weeks): EBNA antibodies present, which develop 1-2 months after primary infection and persist for life, making EBV unlikely as the cause of current acute symptoms 1, 2
Critical timing note: Over 90% of normal adults have IgG antibodies to both VCA and EBNA from past exposure, though 5-10% of infected patients never develop EBNA antibodies 1
Problematic Serologic Patterns Requiring Additional Testing
When all three antibodies (VCA IgM, VCA IgG, and EBNA) are simultaneously positive, this creates diagnostic ambiguity as it can represent either late primary infection or reactivation 4, 5:
IgG avidity testing should be performed to distinguish these scenarios: low avidity indicates recent primary infection (42% of such cases), while high avidity indicates reactivation or past infection (49% of cases) 6, 5
Heterophile antibodies are highly discriminatory in this setting: 94% of patients with true primary infection have positive heterophile antibodies, while only 5% of reactivation cases do 5
EBV PCR can provide additional diagnostic clarity, detecting EBV DNA in serum in primary infections (particularly when heterophile antibodies are absent) but in only 3% of reactivation cases 7
Testing in Special Populations
Pediatric Patients
Children under 10 years require EBV-specific antibody testing as the primary approach due to unreliable heterophile antibody tests in this age group 2, 3
Serum specimens should be collected as soon as possible after symptom onset 2
Immunocompromised Patients
For transplant recipients, HIV-infected individuals, and those with congenital immunodeficiencies, quantitative EBV viral load testing by nucleic acid amplification test (NAAT) is the recommended approach rather than relying solely on serology 1, 2
Specimen requirements: Whole blood, peripheral blood lymphocytes, or plasma in EDTA tubes at room temperature, transported within 2 hours 1
Threshold for concern: EBV DNA levels > 10^2.5 copies/mg DNA in peripheral blood mononuclear cells indicate active infection 1, 3
Clinical significance: Rising EBV viral loads may precede development of EBV-associated lymphoproliferative disease, and levels typically decrease with effective therapy 1
Tissue diagnosis requirement: Definitive diagnosis of EBV-associated lymphoproliferative disease requires demonstration of EBV DNA, RNA, or protein in biopsy tissue 1
CNS Involvement
For suspected EBV-associated encephalitis, both CSF PCR and serology (VCA IgM/IgG and EBNA) should be performed, as PCR alone can yield false-negative and false-positive results 1
CSF specimen: Collect in sterile tube at room temperature, transport within 2 hours 1, 2
Important limitation: EBV DNA in CSF can be nonspecific, appearing with other CNS pathologies including toxoplasmosis and pyogenic abscesses 1
Common Pitfalls to Avoid
Do not stop at a negative heterophile test when clinical suspicion is high—proceed to EBV-specific antibodies 2
Do not interpret isolated VCA IgG positivity without additional testing, as this can represent acute infection, past infection, or indeterminate status 4
Do not rely on early antigen (EA) antibodies for diagnosing reactivation, as their clinical utility is questionable with only 3% correlation with detectable EBV DNA 7
Do not assume EBNA antibodies will always develop—5-10% of infected patients remain EBNA-negative despite true infection 1, 3
In immunocompromised patients, do not interpret positive PCR results without considering that approximately 5-10% may not mount EBNA antibody responses 3