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

Epstein-Barr virus should be suspected when an adolescent or young adult complains of sore throat, fever, and malaise, with lymphadenopathy and pharyngitis on physical exam. The presence of lymphocytosis and increased circulating atypical lymphocytes supports the diagnosis of EBV infection; however, the diagnosis should be confirmed with a heterophile antibody test or through EBV-specific antibodies. Confirmatory testing is helpful to inform patients with infecitous mononucleosis of risks such as splenic rupture and airway obstruction. Once a reactive heterophile antibody is present, it is not necessary to test for specific antibodies to EBV. Specific antibody testing may be necessary to identify the cause of an illness in an individual who does not have a typical case of infectious mononucleosis or has other illnesses that can be cause by EBV. Timeline should be considered when ordering tests.

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 antibodies

Since heterophile antibodies typically return quicker than EBV specific antibodies, it is the test of choice in most clinical settings to determine if an individual has infectious mononucleosis. Reactive heterophile antibodies in a patient with the typical symptoms are diagnostic of EBV and further testing for specific antibodies to EBV is not necessary. Studies have shown that the Monospot test can lead to false negative and false positive results. 

One must consider that the heterophile antibody test results may be negative early in the course of EBV infectious mononucleosis. Positivity increases during the first 6 weeks of the illness. In patients with a compatible syndrome and negative heterophile antibodies, the test can be repeated if the patient is early in their clinical course. ^[29]  Another consideration is that heterophile antibody tests are often negative in infants and children younger than 4 years; thus, EBV-specific serologies are generally favored for diagnosing acute EBV infection in young children. ^[30]  Although rare, false-positive heterophile tests have been reported in those with leukemia, lymphoma, pancreatic cancer, systemic lupus erythematosus, toxoplasmosis, HIV infection, and rubella. ^[31]  

Patients who remain heterophile negative after 6 weeks with a mononucleosis illness should be considered as having heterophile-negative infectious mononucleosis.

Epstein-Barr virus-specific antibodies

These may be considered in patients with suspected infectious mononucleosis who have a negative heterophile test. Specific EBV testing should be obtained in those with prolonged illness or patients who do not fit the classical diagnostic criteria. IgM and IgG antibodies directed against viral capsid antigen has high sensitivity and specificity. ^[32]

Viral capsid antigen (VCA)  – Anti-VCA IgM and anti-VCA IgG antibodies directed against EBV viral capsid antigen are present at the onset of infection. Anti-VCA IgM appears early and usually disappears within 4 to 6 weeks. Anti-VCA IgG appears in the acute phase of the infection and peaks at 2 to 4 weeks and will remain for life. ^[32]  Although anti-VCA IgM is highly suggestive of acute EBV infection, other herpes viruses (eg, cytomegalovirus) can induce IgM antibodies to cell lines that express EBV antigens.

EBV Nuclear antigen (EBNA) – This is a protein expressed only when the virus begins to establish latency. It begins to appear 6 - 12 weeks after the onset of initial symptoms and persists throughout life. Its presence early in the disease course of an illness excludes acute EBV infection. 

Early antigen (EA)  – Anti-EA IgG appears during the acute phase and falls to undetectable levels after 3 to 6 months. ^[32]  The presence of anti-EA IgG anti-D antibodies is consistent with recent infection, but their absence does not exclude acute illness. 

EBV DNA Quantification with PCR is usually used in the management of transplant recipients who develop lymphoproliferative disorders related to EBV infection. 

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

Additional general laboratory studies

Complete blood cell count 

The complete blood cell (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 similar appearing, which readily permits differentiation on the peripheral smear.
• Leukopenia, rather than leukocytosis, is expected in patients with cytomegalovirus (CMV), rubella, HHV-6, acute HIV, and anicteric hepatitis-related infectious mononucleosis.
• Thrombocytopenia: Mild transient thrombocytopenia is not uncommon in EBV infectious mononucleosis. Severe or persistent thrombocytopenia should suggest an alternate diagnosis, eg, acute HIV or other viral infectious diseases. Thrombocytosis is not a feature of EBV infectious mononucleosis, and its presence should suggest an alternate diagnosis, eg, malignancy due to lymphoma in adults or, in children, Kawasaki disease.

Erythrocyte sedimentation rate

An erythrocyte sedimentation rate (ESR) is 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 in group A streptococcal pharyngitis. An elevated ESR does not differentiate EBV from the other heterophile-negative causes of infectious mononucleosis.

A liver function test (LFT) is abnormal in >90% of cases. Serum levels of aminotransferases and alkaline phosphatase are usually mildly elevated. The serum concentration of bilirubin is elevated in ~40% of cases. 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. Mild-to-moderate elevations of the serum transaminases that persist over months in a patient with a mononucleosislike illness should suggest CMV rather than EBV infectious mononucleosis.

Group A Streptococci co-testing

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 rapid streptococcal test cannot be used to differentiate colonization from infection either. 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, in contrast to the patients with colonization, show an intense polymorphonuclear cellular response with cellular debris and fibrous fragments indicating acute infection.

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

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.

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.