How is mononucleosis (mono) diagnosed?

How Mononucleosis is Tested For

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

Initial Testing Strategy

• Order a CBC with differential to look for lymphocytosis (≥50% lymphocytes) and atypical lymphocytes (≥10% of total lymphocyte count), which strongly support the diagnosis 1, 2, 3
• Perform a rapid heterophile antibody test (Monospot) as the first-line serologic test, which has 86% sensitivity and 99% specificity 1, 4
• The heterophile antibody becomes detectable between days 6-10 after symptom onset and peaks during weeks 2-3 of illness 1

Critical Limitations You Must Know

• False-negative heterophile tests occur in approximately 10% of patients and are especially common in children younger than 10 years 1, 4
• In children under 10 years, skip the heterophile test entirely and proceed directly to EBV-specific antibodies 1
• False-positive results can occur with leukemia, pancreatic carcinoma, viral hepatitis, and CMV infection 1
• Early in infection (first week), heterophile tests are commonly negative even in true cases 3

EBV-Specific Antibody Panel (When Heterophile is Negative)

Order all three tests together to properly time the infection: 1

• VCA IgM: Indicates acute/recent infection when positive 1, 5
• VCA IgG: Develops rapidly in acute infection 1, 5
• EBNA antibodies: Critical for timing—absent in acute infection, present in past infection 1, 5

Interpreting EBV Antibody Results

Primary acute EBV infection (infectious mononucleosis) is diagnosed when: 1, 5

• VCA IgM is positive
• EBNA antibodies are absent
• VCA IgG may be positive or negative

Past infection (not acute mono) is indicated when: 1, 6

• EBNA antibodies are present
• VCA IgM is negative
• VCA IgG is positive

EBNA antibodies develop 1-2 months after primary infection and persist for life, so their presence rules out acute infection 1

Common Pitfalls to Avoid

• Do not rely solely on heterophile testing in children under 10 years—proceed directly to EBV-specific antibodies 1
• 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
• Do not assume a positive VCA IgG alone means acute infection—it indicates past infection unless accompanied by positive IgM and negative EBNA 7, 6
• Consider alternative diagnoses including CMV, adenovirus, HIV, and Toxoplasma gondii when evaluating mononucleosis-like illness 1, 3

Special Populations

• Immunocompromised patients: Use quantitative EBV viral load testing by nucleic acid amplification for suspected EBV-associated lymphoproliferative disease 1
• Post-transplant patients: Require EBV DNA surveillance due to high risk of lymphoproliferative disease 1