Epstein-Barr Virus (EBV) Serology

Epstein-Barr virus (human herpesvirus 4) infects B lymphocytes and epithelial cells and persists for life. Serology measures antibodies to viral capsid antigen (VCA IgM and IgG), early antigen (EA-D IgG), Epstein-Barr nuclear antigen (EBNA-1 IgG) and, in some laboratories, VCA IgG avidity. The pattern of these antibodies stages the infection.

The heterophile antibody test (Monospot, Paul-Bunnell) detects non-specific IgM agglutinating animal red cells and supports a diagnosis of infectious mononucleosis in adolescents and adults. EBV DNA by quantitative PCR in blood is used for transplant, immunodeficiency and nasopharyngeal carcinoma.

Over 90% of adults worldwide have been infected. In low income settings infection occurs in early childhood and is mostly silent. In high income countries about half of infections occur in adolescence and young adulthood, with infectious mononucleosis in 30 to 50% of those infected at that age.

EBV is linked to Burkitt lymphoma (endemic in equatorial Africa and Papua New Guinea), Hodgkin lymphoma, nasopharyngeal carcinoma, post-transplant lymphoproliferative disorder, X-linked lymphoproliferative disease, haemophagocytic lymphohistiocytosis and multiple sclerosis risk.

Indications. Fever, pharyngitis, lymphadenopathy and lymphocytosis with atypical lymphocytes (infectious mononucleosis) and a negative heterophile test. Prolonged fever or hepatitis of unknown cause. Pre-transplant donor and recipient serostatus. Suspected post-transplant lymphoproliferative disorder or chronic active infection. Immunodeficiency with lymphoproliferation.

Nasopharyngeal carcinoma screening in high risk groups. Haemophagocytic syndrome. Not for chronic fatigue without other findings.

Specimen. Serum 1 mL, no fasting. Stable for days at 2 to 8 °C. EDTA whole blood or plasma 2 to 3 mL for EBV DNA PCR, processed within 24 hours.

Reference values (assay dependent). VCA IgM, VCA IgG, EA-D IgG and EBNA-1 IgG reported as negative (index under 0.8), equivocal (0.8 to 1.1) or positive (over 1.1), or as titres (VCA IgG under 1 in 10 negative). Heterophile test negative. EBV DNA not detected (quantitative results in copies/mL or IU/mL, with assay specific thresholds).

Interpretation.

• VCA IgM appears at symptom onset and falls over 4 to 8 weeks (sometimes 3 to 6 months). VCA IgG peaks at 2 to 4 weeks and persists for life. EBNA-1 IgG appears 3 to 6 months after infection and persists.
• VCA IgM positive with EBNA-1 IgG negative confirms primary infection. Sensitivity of VCA IgM is 90 to 100% and specificity 90 to 95%, but false positives occur.
• Heterophile antibody is positive in 85% of adolescents and adults with infectious mononucleosis after 2 weeks, but only 50% in the first week and 25 to 50% in children under 5 years.
• Low VCA IgG avidity indicates infection within the last 2 to 3 months.
• Raised EA-D IgG occurs in acute infection, reactivation, nasopharyngeal carcinoma (IgA to VCA and EA is used in screening) and Burkitt lymphoma.
• Quantitative EBV DNA in transplant recipients: a rising load (over the laboratory threshold, often over 1000 to 10 000 copies/mL) precedes post-transplant lymphoproliferative disorder and calls for reduced immunosuppression and imaging.
• Chronic active EBV infection: very high antibody titres (VCA IgG over 1 in 640, EA over 1 in 160) with persistent EBV DNA and compatible symptoms.

Critical values and findings needing immediate action.

• Infectious mononucleosis with stridor, tonsillar obstruction or difficulty swallowing: airway risk, steroids and admission.
• Left upper quadrant pain or shoulder tip pain after mononucleosis: splenic rupture, urgent imaging and surgical review. Contact sports are avoided for 4 to 8 weeks.
• Fever, pancytopenia, hepatosplenomegaly, very high ferritin and triglycerides: haemophagocytic lymphohistiocytosis, urgent haematology.
• Rising EBV DNA with fever, nodes or mass in a transplant recipient: post-transplant lymphoproliferative disorder, urgent biopsy and reduction of immunosuppression.
• Neurological signs (meningoencephalitis, Guillain-Barre syndrome, cerebellitis) with acute EBV serology: admission and CSF PCR.

Interferences and pitfalls.

• VCA IgM can be falsely positive in cytomegalovirus, toxoplasma, HIV seroconversion, autoimmune disease and rheumatoid factor, and can persist or reappear on reactivation. Interpretation uses the whole pattern.
• Heterophile tests give false positives in lymphoma, hepatitis, SLE and some drug reactions, and false negatives in young children and early in illness. A negative heterophile test in a child or early illness should not exclude EBV.
• EBNA-1 may be negative in patients with defective cellular immunity, in the first 3 to 6 months and in HIV, and a negative EBNA with positive VCA IgG does not always mean recent infection.
• Immunoglobulin or blood product therapy can produce passive antibody for months.
• EBV DNA in blood is detectable in healthy carriers at low levels, and results from different assays are not interchangeable. Whole blood and plasma give different values.
• Serology does not distinguish reactivation from latent carriage, and clinical context is needed.
• Differential diagnosis of mononucleosis syndrome includes cytomegalovirus, toxoplasmosis, acute HIV, streptococcal pharyngitis, hepatitis viruses, rubella, adenovirus and drug reactions (DRESS).
• Ampicillin or amoxicillin given to a patient with mononucleosis causes a rash that is not true allergy.

Special groups.

• Children: primary infection is mostly mild or asymptomatic and heterophile tests are often negative.
• Pregnancy: primary infection is usually mild, with no clear congenital syndrome, and screening is not routine.
• Older adults: primary infection is rare, and presents with prolonged fever and hepatitis, with fewer lymphoid signs.
• Immunocompromised patients (HIV, transplant, inherited immunodeficiency): antibody tests are unreliable, and EBV DNA load is the main tool.
• Regions: endemic Burkitt lymphoma follows early infection with malaria co-infection. Nasopharyngeal carcinoma is common in Southern China, Southeast Asia and North Africa, and EBV DNA and IgA antibodies are used for screening there.

Follow up tests. Full blood count with film (atypical lymphocytes), liver function tests, heterophile test, cytomegalovirus, toxoplasma and HIV serology, throat swab for group A streptococcus, quantitative EBV DNA, abdominal ultrasound for splenomegaly, lactate dehydrogenase, ferritin and triglycerides if haemophagocytosis is suspected, lymph node biopsy with EBER in situ hybridisation, and ENT endoscopy for nasopharyngeal carcinoma.