In an adolescent or young adult with fever, sore throat, fatigue, and cervical lymphadenopathy and a negative heterophile (Monospot) test, what is the next step in the diagnostic work‑up?

Next Steps After Negative Monospot Test

When an adolescent or young adult presents with fever, sore throat, fatigue, and cervical lymphadenopathy but has a negative heterophile (Monospot) test, immediately order EBV-specific antibody testing including VCA IgM, VCA IgG, and EBNA antibodies, along with CMV IgM and IgG serology. 1, 2

Primary Diagnostic Approach

Order the following tests immediately:

• EBV-specific antibody panel: VCA IgM, VCA IgG, and EBNA antibodies 3, 1, 2
• CMV serology: CMV IgM and IgG antibodies 3, 2
• Complete blood count with differential if not already done, looking for >40% lymphocytes and >10% atypical lymphocytes 4

The heterophile antibody test has a false-negative rate of approximately 10% in adolescents and young adults, and is particularly unreliable during the first week of illness when it may not yet be positive 2, 5, 4. Rather than waiting 7-10 days to repeat the Monospot, proceed directly to definitive EBV-specific testing 1, 2.

Interpreting EBV-Specific Antibody Results

Primary acute EBV infection is confirmed when:

• VCA IgM is positive AND
• EBNA antibodies are absent 1, 2

Past EBV infection (not causing current symptoms) is indicated when:

• EBNA antibodies are present (these develop 1-2 months after primary infection and persist for life) 1, 2

Note that 5-10% of patients with confirmed EBV infection fail to develop EBNA antibodies, so their absence alone doesn't rule out past infection if VCA IgG is present 3, 2.

Why Include CMV Testing

CMV is a common cause of heterophile-negative mononucleosis-like illness and should be tested simultaneously with EBV 2, 6. For immunocompetent patients, CMV IgM and IgG antibody testing is the recommended first-line approach 3, 2. Be aware that false-positive CMV IgM results can occur in patients with EBV infection, which is why testing both simultaneously helps avoid diagnostic confusion 2.

Additional Differential Diagnoses to Consider

If both EBV and CMV testing are negative, consider:

• HIV (acute retroviral syndrome) - particularly important given similar presentation and need for early diagnosis 6
• Toxoplasma gondii 6
• Adenovirus 6
• Human herpesvirus 6 (HHV-6) 6
• Streptococcus pyogenes (though typically doesn't cause the full mononucleosis syndrome) 6

Common Pitfalls to Avoid

Do not rely solely on repeating the heterophile test. While the Infectious Diseases Society of America notes that heterophile antibodies become more likely to be positive 7-10 days after symptom onset, proceeding directly to EBV-specific antibody testing is more definitive and avoids diagnostic delay 1, 2.

Do not order throat swabs for EBV detection. EBV can persist in throat secretions for weeks to months after infection and does not confirm acute infection 1, 2.

Check liver enzymes if not already done. Elevated transaminases increase clinical suspicion for infectious mononucleosis even with a negative heterophile test, and markedly elevated transaminases (>10x normal) suggest EBV-associated acute hepatitis 1, 4.

Special Population Considerations

For immunocompromised patients (transplant recipients, HIV-infected individuals, those on immunosuppression), order quantitative EBV and CMV viral load testing by nucleic acid amplification (NAAT) rather than relying solely on serology, as these patients are at high risk for EBV-associated lymphoproliferative disease 3, 1, 2, 7.