Approach Considerations
There is no definite evidence-based consensus or guidelines for criteria to diagnose Epstein-Barr virus (EBV) infection. Patients between 10 and 30 years of age with sore throat, fever, and significant anterior cervical adenopathy, fatigue, posterior cervical adenopathy, inguinal adenopathy, palatal petechiae, eyelid edema, and splenomegaly are at high risk for infectious mononucleosis.
A white blood cell (WBC) count and heterophile antibody test should be done, along with a rapid test for streptococcal pharyngitis. If the patient has more than 20% atypical lymphocytes or more than 50% lymphocytes, with at least 10% atypical lymphocytes, infectious mononucleosis is quite likely, and further confirmation of the diagnosis is not needed. A positive result of a heterophile antibody test also is strong evidence in favor of a diagnosis of infectious mononucleosis. [48]
A negative result of an antibody test, particularly during the first week of illness, may indicate that the patient does not have infectious mononucleosis or it could be an infectious mononucleosis-like illness. A second heterophile antibody test can be ordered after 1-2 weeks in such cases. [46] If confirmatory testing is required, viral capsid antigen (VCA)-IgM antibody testing should be done, and a negative result strongly rules out infectious mononucleosis.
Laboratory Studies
Classic criteria
Hoagland’s 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 EBV with typical clinical features of EBV infection
Complete blood cell 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.
Lymphocytosis is most severe during the second and third week 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 are mature T cells, which are antigenically stimulated. They are larger with eccentrically placed indented and folded nucleus and have a lower nuclear-to-cytoplasm ratio (Downey cells). [49] 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. Only rarely purpura is seen.
Liver function tests
Most (ie, 80-100%) of patients with acute infectious mononucleosis have elevated liver enzymes, even though jaundice is not usually present.
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
Heterophile antibody tests are rapid, inexpensive, and specific tests that can be performed from the onset of symptoms of infectious mononucleosis.
Epstein-Barr virus infection stimulates polyclonal secretion of antibodies by infected B cells. Transient production of heterophile antibodies is seen in EBV infection and is known as Paul-Bunnell antibodies. [50] 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, or more sensitivity, horse red blood cells (RBCs) but not guinea pig kidney cells in a tube dilution assay. Similar response is seen in serum sickness and rheumatic diseases but with different adsorption property. Thus, in terms of adsorbing infectious mononucleosis heterophile antibodies, clinicians use the saying, "Cow can, pig can't." Titers above 1:28 or higher than 1:40 are considered positive, depending on the dilution system used..
In this latex agglutination assay, serum from a patient with infectious mononucleosis agglutinates sheep RBCs after adsorption with guinea pig cells, but no agglutination occurs after adsorption with bovine RBCs.
Titer level does not correlate with the severity of clinical illness. Even though the tests have low sensitivity and low negative predictive value in young children under the age of 4, in adolescents, heterophile antibody tests have high specificity and sensitivity in the diagnosis of primary acute EBV infection.
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 onset of symptoms.
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 test
The Monospot test is a rapid slide agglutination test that was 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 adsorption. [50]
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
In young children, virus-specific serology appears to have better sensitivity, but there is cross-reaction with other herpesvirus infections, a longer turnaround time, and it is more expensive to perform.
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 EB nuclear antigen (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. [51]
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 earlier.
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. [1]
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. [52]
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.
Pathology/histology
Viral inclusions may be visible. Certain EBV-associated tumors, such as Hodgkin's lymphoma, have characteristic histology (eg, Reed Sternberg cells). In situ testing with DNA probes or immunohistochemistry for viral proteins can be done further to 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.
Clinical examination is less reliable to assess splenomegaly, and an ultrasound scan is confirmatory to measure splenic size. Almost always splenic rupture occurs in the first 3 weeks. If splenomegaly is detected, contact sports must be avoided at least for 6 weeks and follow-up ultrasound scans are required.
Abdominal CT scanning is the preferred imaging modality to assess for splenic rupture if suspected.
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, which can be present in EBV-infected young adults.
Post-transplant lympho-proliferative 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 infectious mononucleosis
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.
PTLD
PTLD 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.
HLH
Demonstration of hemophagocytosis in a bone marrow aspirate is diagnostic for hemophagocytic lymphohistiocytosis (HLH), although nonspecific findings such as dyserythropoiesis are common.
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Infectious mononucleosis. Antibody response to Epstein-Barr virus. Adapted with permission from Johnson DH, Cunha BA. Epstein-Barr virus serology. Infect Dis Pract. 1995;19:26-27.
