What testing is used to diagnose mononucleosis?

Diagnostic Testing for Infectious Mononucleosis

The primary diagnostic approach for infectious mononucleosis should begin with a heterophil antibody test (Monospot), followed by EBV-specific antibody testing when the Monospot is negative but clinical suspicion remains high. 1

Initial Diagnostic Testing

First-Line Testing

• Complete Blood Count (CBC) with differential

  • Look for lymphocytosis with >10% atypical lymphocytes
  • This finding has high specificity (99%) but limited sensitivity (39%) 2

• Heterophil antibody test (Monospot)

  • Rapid and widely available screening test
  • Sensitivity: 86%, Specificity: 99% 2
  • Usually becomes detectable between 6-10 days after symptom onset 1
  • False negatives occur in approximately 10% of cases, especially in:
    • Children younger than 10 years
    • Early in the disease course (first week)
    • Some adults with true EBV infection 1, 3

Second-Line Testing (When Monospot is Negative)

When clinical suspicion remains high but the heterophil test is negative, EBV-specific serologic testing is recommended 1, 3:

• EBV-specific antibody panel:
  • IgM antibodies to viral capsid antigen (VCA-IgM): Present in acute infection
  • IgG antibodies to viral capsid antigen (VCA-IgG): Appears in acute phase and persists indefinitely
  • Antibodies to early antigen (EA): Present in acute infection
  • Antibodies to Epstein-Barr nuclear antigen (EBNA): Typically absent in acute infection, appears weeks to months later and persists indefinitely

Interpretation of EBV Serology

Serologic Pattern	Interpretation
VCA-IgM (+), VCA-IgG (+), EBNA (-)	Acute EBV infection
VCA-IgM (-), VCA-IgG (+), EBNA (+)	Past EBV infection
VCA-IgM (-), VCA-IgG (-), EBNA (-)	No prior EBV infection

Important Clinical Considerations

• In patients with negative heterophil test but strong clinical suspicion, consider:

  • Testing may be falsely negative early in the disease course
  • Repeat testing in 7-10 days if symptoms persist 1, 3
  • Proceed directly to EBV-specific antibody testing in children under 10 years 1

• If both heterophil test and EBV serology are negative but clinical presentation suggests mononucleosis, consider:

  • CMV infection (causes mononucleosis-like syndrome)
  • HIV acute infection
  • Toxoplasma gondii infection
  • Adenovirus infection 1

Pitfalls to Avoid

• Do not rely solely on clinical features for diagnosis, as the classic triad of fever, pharyngitis, and lymphadenopathy can be caused by other infections
• Do not dismiss the diagnosis based on a single negative heterophil test, especially in children or early in the disease course
• Avoid unnecessary antibiotic treatment for presumed streptococcal pharyngitis without appropriate testing, as 30% of EBV infections can have a false-positive rapid strep test 4
• Be aware that false-positive heterophil tests can occur in patients with leukemia, pancreatic carcinoma, viral hepatitis, and CMV infection 1

Additional Testing in Complicated Cases

• Liver function tests: Consider when hepatomegaly or jaundice is present
• Abdominal ultrasound: When splenomegaly is suspected but not clinically apparent
• Additional testing should be pursued when complications are suspected (neurological, hematological, etc.) 4