Pediatric Mononucleosis and Epstein-Barr Virus Infection Workup

Updated: Dec 05, 2018
  • Author: Nicholas John Bennett, MBBCh, PhD, MA(Cantab), FAAP; Chief Editor: Russell W Steele, MD  more...
  • Print

Laboratory Studies

Classic criteria

The 3 classic criteria for laboratory confirmation of acute infectious mononucleosis include (1) lymphocytosis, (2) the presence of at least 10% atypical lymphocytes on peripheral smear, and (3) a positive serologic test result for Epstein-Barr virus (EBV).

CBC count

Leukocytosis with a WBC count of 10,000-20,000 cells/mL (10-20 X 109/L) is found in 40-70% of patients with acute infectious mononucleosis. By the second week of illness, approximately 10% of patients have a WBC count greater than 25,000 cells/mL.

Approximately 80-90% of patients have lymphocytosis with more than 50% lymphocytes. Lymphocytosis is most severe during the second and third weeks of illness and lasts for 2-6 weeks. Usually, 20-40% of the lymphocytes are atypical, although not all patients have more than 10% atypical lymphocytes.

The atypical lymphocytes of 3 Downey types are larger, have a lower nuclear-to-cytoplasmic ratio, and have a nucleus that is less dense than that of normal lymphocytes. Most of these atypical lymphocytes are polyclonal-activated CD8 cytotoxic-suppressor T lymphocytes, although CD4 helper T cells and CD11 natural killer cells are also present.

Mild thrombocytopenia occurs in 25-50% of patients.

Liver function tests

Most (ie, 80-100%) patients with acute infectious mononucleosis have elevated liver function test results.

Alkaline phosphatase, aspartate aminotransferase (AST), and bilirubin levels peak 5-14 days after onset, and gamma-glutamyltransferase (GGT) levels peak at 1-3 weeks after onset.

Occasionally, GGT levels remain mildly elevated for as long as 12 months, but most liver function test results are normal within 3 months.

Lactic acid dehydrogenase (LDH) levels are increased in approximately 95% of patients.

Serum ferritin can also be increased.

Heterophile antibody test

Epstein-Barr virus infection stimulates polyclonal secretion of antibodies by infected B cells, including transient production of heterophile antibodies. These are antibodies that agglutinate cells from other species and are not directed against Epstein-Barr virus. The Paul-Bunnell test for heterophile antibodies is based on the fact that serum obtained from patients with acute mononucleosis contains antibodies that agglutinate sheep RBCs in a tube dilution assay, whereas such antibodies are absent or nearly absent in the serum of healthy persons.

Differential absorption includes the following:

Antibodies other than those produced during acute infectious mononucleosis can agglutinate sheep RBCs. Such antibodies include those formed in serum sickness and during drug reactions and naturally occurring antibodies to the Forssman antigen.

Differential absorption permits identification of the antibody type. Bovine RBCs absorb infectious mononucleosis heterophile antibodies from serum but do not absorb Forssman antibodies. Guinea pig kidney cells absorb Forssman antibodies, leaving the infectious mononucleosis heterophile antibodies. Antibodies formed in serum sickness are absorbed by both guinea pig kidney cells and bovine RBCs. Thus, in terms of absorbing infectious mononucleosis heterophile antibodies, clinicians use the saying, "cow can, pig can't."

Serum from a patient with infectious mononucleosis agglutinates sheep RBCs after absorption with guinea pig cells, but no agglutination occurs after absorption with bovine RBCs.

Heterophile antibody titers include the following:

  • The titer of Paul-Bunnell—heterophile antibody is determined with tube dilution.

  • Depending on the dilution system, a titer of 1:40 or 1:28 after absorption with guinea pig cells is considered positive for acute infectious mononucleosis.

Titer level does not correlate with severity of clinical illness.

  • Heterophile antibodies are measurable in approximately 50% of patients in the first week of illness, and 60-90% of patients have test results that are positive for heterophile antibodies in the second or third weeks. The titer begins to decline during the fourth or fifth week and is often less than 1:40 within 2-3 months after symptom onset.

  • As many as 20% of patients have positive titer results 1-2 years after acquisition. Also, because horse RBC agglutinins are more sensitive than sheep RBCs, 75% of patients have positive horse RBC agglutinin findings at 1 year.

  • Only 10-30% of children younger than 2 years and 50-75% of children aged 2-4 years develop heterophile antibodies with primary Epstein-Barr virus infection.

Monospot includes the following:

  • Rapid slide agglutination tests, including Monospot assays, have been developed to measure acute infectious mononucleosis heterophile antibodies in a rapid qualitative fashion. Slide tests use either horse RBCs or bovine RBCs. Horse RBCs are more sensitive than sheep RBCs or bovine RBCs and can be treated with formalin to extend the shelf life of the test. Bovine RBCs are specific for acute infectious mononucleosis heterophile antibodies and, thus, do not require differential absorption.

  • All commercial kits for rapid diagnosis of acute infectious mononucleosis heterophile antibodies have low sensitivity (63-84%), with a negative predictive value of more than 10%.

  • Spot tests rarely yield false-positive results in patients with lymphoma or hepatitis.

Epstein-Barr virus serology

Infection with Epstein-Barr virus is characterized by development of the specific antibodies to antigenic components of the virus. These antigens appear at different stages of infection and differ in lytic versus latent infection.

Antibodies to Epstein-Barr virus antigens measured for clinical purposes include antibodies to viral capsid antigen (VCA), early antigens (EAs), and EBNA. EAs are expressed early in the lytic cycle, whereas VCA and membrane antigens are structural viral proteins expressed late in the lytic cycle. EBNA is expressed in latently infected cells.

Antibodies to membrane antigens are not usually measured, but their presence correlates with viral-neutralizing activity.

Antibodies to these proteins are measured with enzyme immunoassays, indirect immunofluorescence assays, and immunoblot assays.

EAs are expressed in cells early in the lytic cycle. These antigens are nonstructural Epstein-Barr virus proteins, which are classified into 2 groups based on cell distribution and stability with methanol treatment.

The restricted component of early antigens (EA/R) is found in the cytoplasm of infected cells and is methanol sensitive. Antibody to EA/R is usually measurable in children younger than 4 years with primary Epstein-Barr virus infection or in patients with nonsymptomatic infection.

Approximately 80% of patients with infectious mononucleosis have antibodies to the diffuse-staining component of EA (EA/D).

EA/D antibody levels are elevated in patients with nasopharyngeal carcinoma, and the levels of antibodies to EA/R are high in individuals with Epstein-Barr virus–associated Burkitt lymphoma. Patients who are immunocompromised and have persistent or reactivated Epstein-Barr virus infections often have high levels of antibodies to EA/D or EA/R.

In early primary Epstein-Barr virus infection, oropharyngeal epithelial cells are lytically infected, and the above antigens are expressed. Antibodies are measurable at the onset of clinical symptoms or even slightly before.

Although not always measurable, EA antibody levels increase upon symptom onset. EA/D is more common, although EA/R is present more often in patients with asymptomatic infection or in children younger than 4 years. The levels of antibodies to EA rise for 3-4 weeks, then usually quickly decline to undetectable levels by 3-4 months, although low levels may be intermittently detected for years. However, in patients with a more prolonged symptomatic illness, EA/D may become unmeasurable, and EA/R results may become positive.

VCA-IgM levels are usually measurable at symptom onset, peak at 2-3 weeks, and then decline and become unmeasurable within 3-4 months. VCA-IgG levels rise shortly after symptom onset, peak at 2-3 months, then drop slightly but persist for life. Antibodies to EBNA appear during convalescence and remain present for life.

Primary acute Epstein-Barr virus infection is associated with VCA-IgM, VCA-IgG, and absent EBNA antibodies.

The antibody pattern in recent infection (3-12 mo) includes positive findings for VCA-IgG and EBNA antibodies, negative VCA-IgM antibodies, and, usually, positive EA antibodies.

After 12 months, the pattern is the same as in recent infection, except EA antibodies are not present.

EBV viral polymerase chain reaction (PCR)

Viral PCR can be performed on whole blood and can be either qualitative or quantitative. Quantitative PCR can be useful in investigating EBV-associated disease in the context of immunosuppressed or immunodeficient patients. The symptoms of severe EBV-associated disease may mimic those of malignancy, graft-versus-host disease, or organ rejection, and measuring the viral load can help with deciding whether to increase or decrease immunosuppressive medications or perform further invasive testing.

Quantitative PCR can be used to measure Epstein-Barr virus DNA in plasma during acute infectious mononucleosis. Levels decline during convalescence and are rarely measurable in latently infected individuals. However, Epstein-Barr virus DNA in serum may be detectable with PCR with reactivation of infection, such as in patients with PTLD.

An Epstein-Barr early region (EBER) probe can be used to identify the Epstein-Barr virus messenger RNA in the nuclei of Epstein-Barr virus–infected lymphoid cells by in situ hybridization.

Studies have suggested that quantitative PCR in bronchoalveolar lavage fluid (BALF) for Epstein-Barr virus may be predictive of PTLD in lung-transplant recipients and is superior to plasma levels. [17]

In some patients who have an immune deficiency, the serologic response to EBV may be blunted or absent; the only way to detect EBV in that setting is with the use of PCR.

Longitudinal tracking of the quantitative viral load can help with judging the effectiveness of antiviral medications or the immune response to the infection.


Viral inclusions may be visible. Certain EBV-associated tumors have characteristic histology (eg, Reed Sternberg Cells). In situ testing with DNA probes or immunohistochemistry for viral proteins can be done to further demonstrate infection.


Imaging Studies

Acute infectious mononucleosis

No specific imaging studies are indicated in diagnosing acute infectious mononucleosis.

Chest radiography reveals mediastinal adenopathy in fewer than 1% of patients. Mediastinal lymph node enlargement should prompt consideration of other diagnoses.

Abdominal CT scanning is the preferred imaging modality to assess for splenic rupture but can be performed only in patients who are hemodynamically stable. Ultrasonography or radionuclide scanning of the spleen may also assist in ascertaining the diagnosis.

Lateral neck films are occasionally helpful to document tonsillar hypertrophy and exclude epiglottis or retropharyngeal abscess in a patient with upper airway obstruction or stridor.

Posttransplant lymphoproliferative disorder (PTLD)

In bone marrow or solid organ transplant patients with PTLD, chest radiography may reveal nodular lesions. Chest CT scanning with contrast may reveal the characteristic peripheral nodules, and abdominal CT scanning with contrast can define the extent of intra-abdominal lesions.


Histologic Findings

Acute mononucleosis

See the list below:

  • Serum: Epstein-Barr virus infection is characterized by the presence of atypical lymphocytes in the peripheral blood. The cells are activated CD8 T cells, which are not infected but are mobilized to destroy the infected B cells.

  • Lymph nodes: During acute mononucleosis, lymph nodes are enlarged, with enlarged germinal centers and lymphoid follicles. Perifollicular areas of the tonsils contain many infected B lymphocytes, which express Epstein-Barr virus–specific antigens, including LMP1, EBNA1, and EBNA2.

  • Spleen: The spleen is larger, with lymphocytic infiltration of the capsule and trabeculae. Pleomorphic blast cells are present in the hyperplastic red pulp. Vascular congestion is coupled with focal and subcapsular hemorrhages.

  • Liver: Histologic changes in the liver are usually minimal, with mild swelling in hepatic sites and bile ducts and lymphocytic portal infiltration.

  • CNS: In fatal infectious mononucleosis, degenerative changes are observed in the neurons of the CNS. Neuronal degeneration, perivascular cuffing, and astrocytic hyperplasia may be present.


This is characterized by homogeneous lymphocytic proliferation with an immunoblastic component. Lesions may efface lymphoid organ architecture or develop ectopically in nonlymphoid organs. The Epstein-Barr virus–infected cells in patients with PTLD express EBER.


Demonstration of hemophagocytosis in a bone marrow aspirate is diagnostic for HLH, although nonspecific findings such as dyserythropoiesis are common.