Epstein-Barr Virus - EBV

Epstein-Barr virus (EBV), a pervasive virus that infects over 90% of the world’s population by adulthood, causes infectious mononucleosis (IM) in immunocompetent individuals and lymphoproliferative disease in immunocompromised patients.     Most people are infected in childhood, at which time the infection is most often subclinical, but the virus remains as a permanent, latent infection and can reactivate. Early and accurate diagnosis of EBV IM is important because it allows clinicians to create a targeted treatment plan and avoid the inappropriate use of antibiotics.

Several different tests and methods are available to diagnose EBV infection and the resulting diseases. Rapid heterophile antibody testing (also referred to as Monospot) is often used as a first-line test in the diagnosis of EBV IM because it is inexpensive and has a fast turnaround. However, false-negative results can occur in young children and early in the course of the illness, necessitating follow-up testing by EBV-specific serology. EBV-specific serology may also be used in the place of heterophile antibody testing as an initial test.  Molecular evaluation by polymerase chain reaction (PCR) and histologic examination with in situ hybridization (ISH) are used for the diagnosis and monitoring of EBV-related lymphoproliferative diseases and cancers.

Quick Answers for Clinicians

When should Epstein-Barr virus testing be considered?

Epstein-Barr virus (EBV)-induced infectious mononucleosis (IM) might be suspected when a child, adolescent, or young adult (generally between the ages of 5 and 25 years, especially between 16 and 20 years) presents with sore throat, fever, and malaise in combination with lymphadenopathy and pharyngitis on physical examination. Palatal petechiae, splenomegaly, and posterior cervical adenopathy are highly suggestive of IM. 

Which tests are available to diagnose Epstein-Barr virus infection? What are the advantages and disadvantages of those tests?

Heterophile antibody testing (also known as Monospot) is often used as a first-line test in the diagnosis of Epstein-Barr virus (EBV) infectious mononucleosis (IM). Although the CDC does not recommend the Monospot test for general use, the heterophile antibody test is often used as a first-line test because it is fast and inexpensive.  In general, the heterophile antibody test should not be used in children younger than 5 years, most of whom do not produce the heterophile antibody.  Negative heterophile antibody test results (in patients of any age) must be followed with EBV-specific serology.  

Serologic assays for EBV-specific antibodies are necessary to diagnose asymptomatic EBV infection and can confirm a diagnosis in symptomatic patients. EBV-specific serology may also be used to diagnose EBV IM in place of a heterophile antibody test. If results are negative for EBV, serologies for other virus-specific antigens (eg, cytomegalovirus, Toxoplasma gondii, human herpesvirus 6, and HIV) may be considered. 

Molecular evaluation by polymerase chain reaction (PCR) is often preferred over serology in cases of reactivation, but serology may be more sensitive and specific in cases of acute EBV infection because a positive PCR result with a lower copy number may not distinguish between clinically insignificant latently infected cells and primary infection. 

Indications for Testing

EBV testing is used to:

  • Diagnose either symptomatic or asymptomatic EBV infection, estimate timing of infection, and, in some cases, aid in the diagnosis of EBV-related cancers
  • Diagnose or determine risk for EBV-associated posttransplant lymphoproliferative disorders (PTLDs)
  • Evaluate for or monitor EBV DNAemia (positive findings of EBV DNA) in immunosuppressed patients to aid in therapy decisions

Laboratory Testing

Diagnosis

Serologic Assays

Heterophile Antibody Test

The heterophile antibody test (Monospot) is a standard diagnostic test for EBV IM in patients with symptoms of IM.   Although the CDC does not recommend using the test for initial EBV IM evaluation, it is often used and is recommended by some groups, including the Infectious Diseases Society of America (IDSA), because it is rapid and inexpensive.     There are several limitations of heterophile antibody testing. The test is not useful for children younger than 5 years because many do not produce the heterophile antibody. The test also offers low sensitivity and low negative predictive value in older children and adolescents.  Additionally, other conditions can cause positive heterophile antibody results.   

Negative results should be followed by serologic assays to identify specific EBV antigens and to confirm the presence of EBV infection.    Positive results can be followed by EBV antibody testing to determine the stage of infection.  

EBV-Specific Antibody Tests

EBV-specific antibody testing for immunoglobulin G (IgG)- and IgM-class antibodies to viral capsid antigen (VCA) and Epstein-Barr nuclear antigen (EBNA) may be used to follow up on negative or positive heterophile antibody test results. These tests may also be used in place of heterophile antibody testing. Although EBV-specific antibody tests are more sensitive than heterophile antibody tests, they are labor intensive to perform and have longer turnaround times. 

Given that antibodies often appear later in the course of illness, repeat testing in 10-14 days may be helpful if results are equivocal. The presence or absence of certain antibodies, as detailed below, can indicate the stage of infection.

Interpretation of EBV-Specific Serologic Results
Infection VCA IgM VCA IgG EA EBNA
No previous Negative Negative Negative Negative
Acute/primary Positive Positive Positive/negative Negative
Recent Positive/negative Positive Positive/negative Positive/negative
Past Negative Positive Negative Positive
Reactivationa Positive/negative Positive Positive Positive
aAntibody to early antigen in the presence of a positive EBNA test does not automatically indicate that a patient's current medical condition is caused by EBV reactivation. Healthy individuals with no symptoms can have antibodies to early antigen for years after initial EBV infection. Reactivation can occur subclinically.

EA, early antigen

Sources: CDC , Morrison 

Serologic testing may also be useful in the evaluation for certain EBV-associated malignancies, but its application is limited.  For example, elevated titers of IgA directed toward EBV early antigens and VCAs and IgG directed against the EBV BRLF1 gene product Rta are indicative of nasopharyngeal carcinoma.  But because serology is generally not beneficial for diagnosing EBV-related lymphoproliferative diseases and cancers, molecular testing is preferable in the diagnosis and management of these disorders. 

Polymerase Chain Reaction

PCR, which can offer high sensitivity and specificity, is used to detect and/or quantify EBV DNA. It may be preferred over serology in cases of reactivation, but serology may be more sensitive and specific in cases of acute EBV infection because a positive PCR result with a lower copy number may not distinguish between clinically insignificant latently infected cells and primary infection.  Peripheral blood EBV viral load by PCR is part of the diagnostic workup for EBV-associated lymphoproliferative diseases and EBV-associated PTLD, is used to assess risk for developing PTLD, and is recommended for monitoring EBV DNAemia in patients who have undergone hematopoietic stem cell transplantation (HSCT).  

In Situ Hybridization

Along with physical examination and imaging studies, diagnosis of EBV-associated lymphoproliferative diseases requires histologic examination of biopsy tissue by ISH for EBV-encoded RNA transcripts.   In patients with lymphoproliferative lesions of the head and neck, ISH for EBV-encoded RNA transcripts should be routinely performed because it is the most sensitive available test to detect EBV in this setting. 

Monitoring

In patients who have undergone HSCT, EBV viral load monitoring should begin no later than 4 weeks after HSCT, should continue for at least 4 months, and should be performed at least once per week in high-risk patients; longer or more frequent monitoring may be indicated in some in patients. 

ARUP Lab Tests

Heterophile Antibody Tests
Antibody Panels for EBV Infection

Evaluate for primary EBV IM after a suspected false-negative heterophile antibody (Monospot) test 

Components: EBV antibody to VCA, IgG and IgM; EBV antibody to nuclear antigen, IgG; and EBV antibody to early D antigen (EA-D), IgG

Determine past or present EBV infection as well as susceptibility to future EBV infection

Use in conjunction with EBV nuclear antigen to diagnose primary EBV IM 

Components: EBV antibody to VCA, IgG and IgM

Individual Antibody Tests for EBV Infection

Use in conjunction with other serologic tests to diagnose primary EBV IM

Document past EBV exposure

Use in conjunction with other serologic tests to diagnose primary EBV IM 

Use in conjunction with other serologic tests to diagnose acute EBV infection and EBV reactivation

Antibody Testing for EBV- Associated Nasopharyngeal Carcinoma

Detect nasopharyngeal carcinoma and aid in prognosis

Do not use for diagnosis of EBV IM

Polymerase Chain Reaction

Detect EBV DNA in individuals suspected of having EBV-related disease

Do not use for diagnosis of IM

Quantify EBV viral load as an aid in monitoring EBV-related disease

In Situ Hybridization

Detect EBV in tissue

Medical Experts

Contributor
Contributor

Slev

Patricia R. Slev, PhD
Associate Professor of Clinical Pathology, University of Utah
Section Chief, Immunology; Medical Director, Immunology Core Laboratory, ARUP Laboratories

References

Resources from the ARUP Institute for Clinical and Experimental Pathology®