What is the first‑line diagnostic test for suspected infectious mononucleosis in a teenager or young adult presenting with fever, sore throat, cervical lymphadenopathy, and fatigue?

First-Line Diagnostic Test for Infectious Mononucleosis

Begin 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 immediately to EBV-specific antibody testing with VCA IgM, VCA IgG, and EBNA antibodies. 1

Initial Testing Strategy

The most cost-effective and efficient approach combines:

• Complete blood count with differential to assess for >40% lymphocytes and >10% atypical lymphocytes 2
• Rapid heterophile antibody test (Monospot) as the first-line serologic test 1, 2

The heterophile antibody test has 87% sensitivity and 91% specificity in the appropriate clinical context 2. A positive Monospot is diagnostic and requires no further EBV-specific testing 3.

Critical Timing and Limitations

When Heterophile Testing Fails

The heterophile antibody becomes detectable between days 6-10 after symptom onset and peaks during weeks 2-3 of illness 1. False-negative results occur in approximately 10% of patients and are especially common in:

• Children younger than 10 years - proceed directly to EBV-specific antibodies in this population 1, 3
• First week of illness - the test has a 25% false-negative rate during this period 4

If the initial Monospot is negative but clinical suspicion remains high, you can repeat the test on a serum specimen obtained 7-10 days later, when heterophile antibodies are more likely to be positive 3. However, the IDSA recommends proceeding directly to EBV-specific antibody testing rather than waiting 1.

EBV-Specific Antibody Panel (Second-Line)

When heterophile testing is negative or unavailable, order the complete EBV antibody panel 1, 3:

• VCA IgM - indicates acute/recent infection 1
• VCA IgG - develops rapidly in acute infection 1
• EBNA antibodies - critical for timing the infection 1

Interpreting EBV Antibody Results

Primary acute EBV infection is diagnosed when:

• VCA IgM is positive AND
• EBNA antibodies are absent 1, 3

Past infection (not current acute disease) is indicated when:

• EBNA antibodies are present 1, 5
• EBNA develops 1-2 months after primary infection and persists for life 1

Important caveat: Approximately 5-10% of EBV-infected patients fail to develop EBNA antibodies, which should be considered when interpreting results 1, 3.

Supporting Laboratory Findings

Infectious mononucleosis is unlikely if:

• Lymphocyte count is <4,000/mm³ 4

Elevated liver enzymes increase clinical suspicion for infectious mononucleosis when the heterophile test is negative 2.

Special Population Considerations

Immunocompromised Patients

For transplant recipients, HIV-infected individuals, and those with congenital immunodeficiencies, order quantitative EBV viral load testing by nucleic acid amplification (NAAT) rather than relying solely on serology 3. These patients are at high risk for EBV-associated lymphoproliferative disease 1, 3.

Pediatric Patients

In children under 10 years, heterophile antibody tests have higher false-negative rates 3. Consider proceeding directly to EBV-specific antibody testing in this age group 1, 3.

Common Pitfalls to Avoid

• Do not rely solely on heterophile testing in children under 10 years - the false-negative rate is unacceptably high 1, 3
• 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, 3
• Do not interpret VCA IgG alone - without IgM and EBNA status, you cannot distinguish between recent and remote infection 1
• Consider alternative diagnoses including CMV, adenovirus, HIV, and Toxoplasma gondii when evaluating mononucleosis-like illness 1

False-Positive Heterophile Results

False-positive results can occur with leukemia, pancreatic carcinoma, viral hepatitis, and CMV infection 1. When the clinical picture doesn't fit, consider EBV-specific testing to confirm.