Epstein-Barr Virus (EBV) Infectious Mononucleosis (Mono) Workup

Updated: Jul 11, 2017
  • Author: Burke A Cunha, MD; Chief Editor: Michael Stuart Bronze, MD  more...
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Laboratory Studies

Epstein-Barr virus (EBV) infection induces specific antibodies to EBV and various unrelated non-EBV heterophile antibodies. These heterophile antibodies react to antigens from animal RBCs.

  • Sheep RBCs agglutinate in the presence of heterophile antibodies and are the basis for the Paul-Bunnell test.
  • Agglutination of horse RBCs on exposure to heterophile antibodies is the basis of the Monospot test.

Heterophile test antibodies are sensitive and specific for EBV heterophile antibodies, they are present in peak levels 2-6 weeks after primary EBV infection, and they may remain positive in low levels for up to a year.

The latex agglutination assay, which is the basis of the Monospot test using horse RBCs, is highly specific. Sensitivity is 85%, and specificity is 100%.

The heterophile antibody test (eg, the Monospot test) results may be negative early in the course of EBV infectious mononucleosis. Positivity increases during the first 6 weeks of the illness. Patients who remain heterophile negative after 6 weeks with a mononucleosis illness should be considered as having heterophile-negative infectious mononucleosis.

  • Patients with heterophile infectious mononucleosis should be tested for EBV-specific antibodies before definitively diagnosing heterophile-negative infectious mononucleosis.
  • Patients with heterophile- or Monospot-negative infectious mononucleosis should be tested serologically as are patients who present with a mononucleosislike illness who are negative for heterophile antibodies. The heterophile test is less useful in children younger than 2 years, in whom the results are frequently negative.
  • Although virtual 100% specificity exists with the Monospot test, rarely, other disorders have been reported that may produce a false-positive Monospot test result. These causes of false-positive Monospot test results include toxoplasmosis, rubella, lymphoma, and certain malignancies, particularly leukemias and/or lymphomas.

Testing for EBV-specific antibodies is as follows:

  • EBV induces a serological response to the various parts of the Epstein-Barr viral particle. IgM and IgG antibodies directed against the VCA of EBV are useful in confirming the diagnosis of EBV and in differentiating acute and/or recent infection from previous infection. EBV IgM VCA titers decrease in most patients after 3-6 months but may persist in low titer for up to 1 year. EBV IgG VCA antibodies rise later than the IgM VCA antibodies but remain elevated with variable titers for life.
  • False-positive VCA antibody titer results may occur on the basis of cross-reactivity with other herpes viruses, eg, CMV, or with unrelated organisms, eg, Toxoplasma gondii.

Other antigens indicating EBV infection are less useful diagnostically and include early antigen (EA), which is present early in EBV infectious mononucleosis. EBV nuclear antigen (EBNA) appears after 1-2 months and persists throughout life. The presence of elevated EBNA titers has the same significance as elevated IgG VCA titers. The presence of these antibodies suggests previous exposure to the antigen (past infection) and excludes EBV infection acquired in the previous year.

As with heterophile antibody responses, specific EBV antibodies may not be present in children younger than 2 years.

Nonspecific tests are as follows:

  • Patients with infectious mononucleosis in the differential diagnoses should have a CBC count with differential and an evaluation of the erythrocyte sedimentation rate (ESR). The CBC count is more useful in ruling out other diagnoses that may mimic infectious mononucleosis than in providing any specific diagnostic information. Because leukocytosis is the rule in infectious mononucleosis, the presence of a normal or decreased WBC count should suggest an alternative diagnosis. Lymphocytosis accompanies infectious mononucleosis, increases during the first few weeks of illness, and then gradually returns to normal. The appearance, peak, and disappearance of atypical lymphocytes follow the same time course as lymphocytosis. Patients with fever, pharyngitis, and lymphadenopathy are likely to have EBV infectious mononucleosis if the relative atypical lymphocyte count is equal to or greater than 20%.
  • Atypical lymphocytes should be differentiated from abnormal lymphocytes. Abnormal lymphocytes are associated with lymphoreticular malignancies, whereas atypical lymphocytes are associated with various viral and noninfectious diseases, as well as drug reactions. Atypical lymphocytes are each different in their morphology as observed on the peripheral smear, whereas abnormal lymphocytes are monotonous in their sameness, which readily permits differentiation on the peripheral smear.
  • Because anemia is so rare with EBV infectious mononucleosis, patients with anemia should undergo workup for another cause of their anemia.
  • Thrombocytopenia not uncommonly accompanies EBV infectious mononucleosis, but it may be present in various other viral illnesses, including in patients with heterophile-negative infectious mononucleosis.
  • An ESR is most useful in differentiating group A streptococcal pharyngitis from EBV infectious mononucleosis. The sedimentation rate is elevated in most patients with EBV infectious mononucleosis, but it is not elevated in group A streptococcal pharyngitis. However, an elevated ESR does not differentiate EBV from the other heterophile-negative causes of infectious mononucleosis, nor does it differentiate infectious mononucleosis from malignancies.
  • Because the liver is uniformly involved in EBV infectious mononucleosis, mild elevation of the serum transaminases is a constant finding in early EBV infectious mononucleosis. Mild increases in the serum transaminases are also a feature of the infectious agents responsible for heterophile-negative infectious mononucleosis. High elevation of the serum transaminases should suggest viral hepatitis. The serum alkaline phosphatase and gamma-glutamyl transpeptidase (GGTP) levels are not usually elevated in individuals with EBV infectious mononucleosis.

Specific tests are as follows:

  • Heterophile antibody tests
    • Patients with infectious mononucleosis should first be tested with a heterophile antibody test. The most commonly used is the latex agglutination assay using horse RBCs, and it is marketed as the Monospot test. Enzyme-linked immunosorbent assay (ELISA) rapid diagnostic tests are also available, which are based on the detection of heterophile antibodies. Physicians should remember that heterophile antibody responses require 1-2 weeks to become positive. In a group of patients with EBV mononucleosis, the number of patients becoming positive increases to a maximum 6 weeks after the onset of the illness.
    • If results are initially negative, a Monospot test should be ordered weekly for 6 weeks in patients with suspected EBV infectious mononucleosis. If the Monospot test remains persistently negative after 6 weeks of weekly serial testing, then a specific EBV serological test should be ordered. Before patients with an infectious mononucleosis–like syndrome are labeled as having heterophile-negative infectious mononucleosis, specific EBV serological tests should be obtained, and the results should be negative (see below).
    • Major antibodies - Heterophile (Paul-Bunnell), EBV antigens, cold agglutinins (anti-1), smooth muscle antibodies (SMA)
    • Minor antibodies - Rheumatoid factor (RF), antinuclear antibodies (ANA), antimitochondrial antibodies, antireticulin antibodies, antimicrosomal antibodies, anti–intermediate filaments (IMF), lymphocytotoxin, Wasserman reagin
    • The Monospot test has high sensitivity and specificity, eg, 85% and nearly 100%, respectively. Rarely, Monospot test results may be falsely positive, particularly in patients with CMV or rubella but also in patients with SLE and rheumatoid arthritis. Potential false-positive reactions may occur in those with HIV infection or herpes simplex virus (HSV). If a false-positive Monospot test result is suspected, then specific testing using an EBV-based antibodies serological test is indicated. A false-negative Monospot test result may occur if testing is performed too early in the course of the illness or in very young children (< 2 y) and occasionally in elderly patients.
  • Specific EBV antibody tests
    • Specific EBV antibody testing is more time-consuming and expensive than the Monospot test. EBV serological tests should be obtained in patients with a mononucleosislike illness and a negative finding on the Monospot test. As with the heterophile test, the EBV antibody response may be falsely negative early in the course of the infection. False negativity may also occur in young children (< 2 y).
    • The antibody response to specific EBV serological testing consists of measuring the antibody response to surface and core EBV viral proteins. For clinical purposes, the most useful EBV-specific antibodies are the VCAs and the EBNA. Both VCA and EBNA antibodies are usually reported as IgM or IgG antibodies. Acute infection is diagnosed in patients who have an increased EBV IgM VCA titer. Later in the course of infection, the increase in IgM VCA antibodies may be accompanied by an increase in IgG VCA antibodies and an increase in IgG EBNA antibodies. Many laboratories report EBNA titers only, which usually measure the IgG EBNA.
    • Increased IgG VCA and/or increased IgG EBNA titers indicate past exposure to EBV, which may have been subclinical or clinical. Increased IgG VCA titers are not synonymous with chronic infectious mononucleosis, and these titers are not diagnostic of CFS. Following acute infection, the increase in IgM titers peaks after 4-8 weeks and usually remain positive for as long as 1 year. The Monospot heterophile antibodies follow the same time course as the IgM VCA titers.
    • Rarely, cross-reactivity occurs between VCA antibodies to EBV and those to CMV or toxoplasmosis. False-positive cross-reactivity to specific EBV antibodies is extremely rare. Such patients have high elevations of IgM CMV or toxoplasmosis titers, which helps to differentiate between the primary infectious agent and the serological cross-reactivity resulting in a false-positive test result.
    • Patients with heterophile-negative infectious mononucleosis, eg, those with persistently negative Monospot test results for 6 weeks and those with a negative EBV-specific test result, should be tested serologically for the infectious agents that cause heterophile-negative infectious mononucleosis (eg, HIV, HHV-6, toxoplasmosis, CMV, rubella, anicteric viral hepatitis).

Table 2. EBV Serologic Responses in EBV-Associated Diseases (Open Table in a new window)

EBV Diseases EBV Antibody Responses
Anti-VCA Anti-EA



IgM IgG Diffuse EA Restricted EA Anti-EBNA
Acute EBV mononucleosis + + + + - -
Past EBV infection - - + - - +
Chronic active EBV infection - - +++ + + +
Burkitt lymphoma - - +++ +/- + +
Nasopharyngeal carcinoma - - +++ + +/- +


Other tests are as follows:

  • Patients with suspected infectious mononucleosis should not have their throats cultured for group A streptococci because the carriage rate is approximately 30% in these patients. The mere recovery of group A streptococci from the oropharynx does not signify the cause of the patient's pharyngitis; it does not differentiate colonization from infection. In such patients, a Gram stain of the oropharynx is used to differentiate patients who have pharyngitis with positive cultures for group A streptococci from those colonized with group A streptococci.
  • Patients with EBV infectious mononucleosis or other causes of viral pharyngitis and group A streptococcal colonization have little or no white cell response on the Gram stain of the pharynx. Patients with group A streptococcal pharyngitis also have a positive finding on throat culture, but, in contrast to the patients with colonization, they show an intense polymorphonuclear cellular response with cellular debris and fibrous fragments indicating acute infection. The rapid streptococcal test cannot be used to differentiate colonization from infection any more than throat cultures.
  • Patients with presumed CNS involvement with EBV infectious mononucleosis should undergo serological tests for other causes of viral encephalitis appropriate to the patient's exposure history.

Imaging Studies

Patients with presumed CNS involvement with EBV infectious mononucleosis should undergo a CT scan and/or MRI to rule out other causes of encephalitis.


Other Tests

Patients with presumed CNS involvement with EBV infectious mononucleosis should also undergo an EEG to rule out other causes of encephalitis.



Rarely, if ever, is a bone marrow biopsy or lymph node biopsy needed in patients with EBV infectious mononucleosis. In the diagnosis of EBV infectious mononucleosis, the assessment of lymph node enlargement can be made confidently based on specific EBV antibody testing, and surgery is almost never necessary.

Patients with presumed CNS involvement with EBV infectious mononucleosis should also undergo a lumbar puncture to rule out other causes of encephalitis.


Histologic Findings

Oropharyngeal epithelium demonstrates an intense lymphoproliferative response in the cells of the oropharynx. The lymph node and spleen show lymphocytic infiltration primarily in the periphery of a lymph node.