Would you order Cytomegalovirus (CMV) and Epstein-Barr Virus (EBV) tests on a patient with Fever of Unknown Origin (FUO) and a negative Monospot (Mononucleosis spot test)?

Should You Order CMV and EBV Testing in FUO with Negative Monospot?

Yes, you should order EBV-specific antibody testing (VCA IgM, VCA IgG, and EBNA) when clinical suspicion for infectious mononucleosis remains high despite a negative Monospot test, and CMV testing should be included in the workup of FUO as it represents a common cause of mononucleosis-like syndromes. 1, 2, 3

Why the Monospot is Insufficient

The heterophile antibody test (Monospot) has significant limitations that make it unreliable as a standalone test:

• False-negative rates approach 10% in adults and are substantially higher in children under 10 years of age 2, 3
• The Infectious Diseases Society of America explicitly states that Paul-Bunnell and Monospot tests are suboptimal for diagnosis and recommends proceeding directly to EBV-specific antibody testing when these are negative 3
• The heterophile antibody typically becomes detectable only between days 6-10 after symptom onset, meaning early testing may miss acute infection 3

The Correct EBV Testing Approach

When the Monospot is negative but clinical suspicion remains:

• Order a complete EBV-specific antibody panel including VCA IgM, VCA IgG, and EBNA antibodies 1, 2, 3
• VCA IgM positive with EBNA negative indicates recent primary EBV infection 2, 3
• EBNA antibodies present indicate past infection (develops 1-2 months after primary infection) and make EBV unlikely as the cause of current symptoms 2, 3
• Approximately 5-10% of EBV-infected patients fail to develop EBNA antibodies, which should be considered when interpreting results 1, 2

Why CMV Testing is Essential

CMV is a critical consideration in FUO workup:

• CMV infectious mononucleosis is a well-documented cause of FUO and presents with a clinical picture nearly identical to EBV mononucleosis 4
• Mononucleosis-like syndromes (including both EBV and CMV) account for 0.8% of fever cases in returning travelers, representing a significant diagnostic category 1
• False-positive CMV IgM results can occur in patients infected with EBV, highlighting the importance of testing both simultaneously to avoid diagnostic confusion 1
• For immunocompetent patients with suspected acute CMV infection, CMV-specific IgM and IgG antibody testing is recommended as the first-line diagnostic test 1

The FUO Diagnostic Context

In the broader FUO workup:

• Microbiologic serology has proven screening value in FUO when potentially diagnostic clues are present 5
• The focused diagnostic approach based on clinical syndrome is more effective than comprehensive nonfocused testing 6, 7
• Both EBV and CMV fall into the infectious category of FUO causes and should be considered when patients present with lymphadenopathy, splenomegaly, or atypical lymphocytosis 4, 7

Practical Testing Algorithm

Order the following tests together:

1. EBV-specific antibodies: VCA IgM, VCA IgG, and EBNA 2, 3
2. CMV-specific antibodies: CMV IgM and CMV IgG 1
3. Specimen: Serum in clot tube, room temperature, transport within 2 hours 1, 2

Critical Pitfalls to Avoid

• Do not rely solely on the Monospot in any age group, but especially in children under 10 years 2, 3
• 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 2, 3
• Do not interpret VCA IgG alone without knowing IgM and EBNA status, as over 90% of normal adults have IgG antibodies from past infection 1, 3
• Consider alternative diagnoses including HIV, Toxoplasma gondii, and adenovirus when evaluating mononucleosis-like illness 3

Special Considerations for Immunocompromised Patients

If your patient is immunocompromised (transplant recipient, HIV-infected, congenital immunodeficiency):

• Order quantitative EBV and CMV viral load testing by nucleic acid amplification (NAAT) rather than relying solely on serology 1, 2, 3
• Use whole blood, peripheral blood lymphocytes, or plasma in EDTA tube 1, 2
• These patients are at high risk for EBV-associated lymphoproliferative disease, which requires viral load monitoring 1, 2