Infectious Mononucleosis

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Infectious Mononucleosis

Learning Objectives

At the conclusion of this chapter, the reader should be able to:

• Describe the etiology, epidemiology, and signs and symptoms of infectious mononucleosis.

• Explain the immunologic manifestations of infectious mononucleosis, including heterophile antibodies.

• Discuss the elements of Epstein-Barr virus (EBV) serology and the diagnostic clinical applications of the presence of each component.

• Analyze and apply laboratory data to a case study.

• Correctly answer case study related multiple choice questions.

• Be prepared to participate in a discussion of critical thinking questions.

• Compare the serologic procedures and clinical applications of the Paul-Bunnell, Davidsohn differential, and rapid agglutination techniques.

• Correctly answer end of chapter review questions.

Key Terms

Etiology

The Epstein-Barr virus (EBV), a human herpesvirus, was discovered in 1964 by Dr. M. Anthony Epstein and his colleague, Yvonne Barr. Subsequently, Drs. Werner and Gertrude Henle screened human serum samples for antibodies to viral capsid antigens of EBV and established the relationship of EBV to several cancers (e.g., Burkitt’s lymphoma). EBV became the most intensively studied human cancer virus. The entire genome of one EBV strain was completely sequenced in 1984. The virus parasitizes every cell system—signal transduction, cell cycle control, regulation of gene expression, posttranscriptional RNA processing, protein modification and stability, and DNA replication.

Infectious mononucleosis, caused by EBV, is usually an acute, benign, and self-limiting lymphoproliferative condition. EBV is also the cause of Burkitt’s lymphoma (a malignant tumor of the lymphoid tissue occurring mainly in African children), nasopharyngeal carcinoma, and neoplasms of the thymus, parotid gland, and supraglottic larynx. EBV is an important factor in the development of nasopharyngeal carcinoma, an epithelial cancer. Although nasopharyngeal carcinoma is rare in North American and European whites, it is one of the most common cancers in southern China and parts of Southeast Asia. Genetics and environmental factors appear to contribute to the elevated risk of nasopharyngeal carcinoma among the Chinese.

EBV infections can result in complications involving the cardiac, ocular, respiratory, hematologic, digestive, renal, and neurologic systems. EBV-associated neurologic syndromes include Bell’s palsy, Guillain-Barré syndrome, meningoencephalitis, Reye’s syndrome, myelitis, cranial nerve neuritis, and psychotic disorders. Respiratory paralysis caused by bulbar involvement can be fatal.

Epidemiology

EBV is widely disseminated. It is estimated that 95% of the world’s population is exposed to the virus, which makes EBV the most ubiquitous virus known to humans. EBV is a human herpes DNA virus that infects B lymphocytes. The variant lymphocytes produced in response to and seen in microscopic examination of the peripheral blood have T cell characteristics. The mononucleosis is not from stimulation of B cells by viral infection (EBV will transform cell lines in vitro) but is from a large, effective, CD8 cytotoxic T cell (Tc) response against the EBV-infected circulating B lymphocytes. One of the habitats of the persisting viral genome in hosts with a latent infection is the B lymphocytes of the lymphoreticular system and epithelial cells of the oropharynx.

Although transmitted primarily by close contact with infectious oral pharyngeal secretions, EBV is reportedly transmitted by blood transfusion and transplacental routes. Under normal conditions, EBV transmission through transfusion or transplacental exposure is unlikely. In addition, EBV-associated posttransplantation lymphoproliferative disorder (PTLD) develops in 1% to 10% of organ transplant recipients.

The frequency of seronegative patients is almost 100% in early infancy but declines with increasing age, more or less rapidly, depending on socioeconomic conditions, to less than 10% in young adults. After primary exposure, a person is considered to be immune and generally no longer susceptible to overt reinfection. In Western societies, primary exposure to EBV occurs in two waves. Approximately 50% of the population is exposed to the virus before age 5 years; a second wave of seroconversion occurs during late adolescence (age 15 to 24 years). Approximately 90% of adult patients demonstrate antibodies to the virus.

Individuals at risk include those who lack antibodies to the virus. EBV is only a minor problem for immunocompetent persons, but can become a major concern for immunocompromised patients. Blood transfusion from an immune donor to a nonimmune recipient may produce a primary infection in the recipient known as infectious mononucleosis postperfusion syndrome. Infectious mononucleosis or an infectious mononucleosis–like illness after blood transfusion often may result from a concomitant cytomegalovirus (CMV) infection rather than EBV. In addition, the association with EBV appears to be a specific finding in malignant lymphoma developing after severe immunosuppression, such as that induced by cyclosporine therapy.

A low percentage of patients experience symptomatic reactivation. Reactivation of latent EBV infection has been implicated in a persistent illness referred to as EBV-associated fatigue syndrome, but this phenomenon is not universally accepted.

Clinically apparent infectious mononucleosis has an estimated frequency of 45/100,000 in adolescents. In immunosuppressed patients, the incidence of EBV infection ranges from 35% to 47%. As with other herpesviruses, there is a carrier state after primary infection.

Signs and Symptoms

Although EBV infects more than 95% of the world’s population, most individuals experience no adverse effects. Infants typically have asymptomatic infection. The timing of initial infection is a key indicator of the ensuing symptoms. Infectious mononucleosis is the typical illness experienced by adolescents newly infected with EBV.

Most individuals experience seroconversion without any significant clinical signs or symptoms of disease. Immunocompetent persons maintain EBV as a chronic latent infection. In children younger than 5 years, infection is asymptomatic or frequently characterized by mild, poorly defined signs and symptoms. Although anyone can suffer from this viral disorder, it is typically manifested in young adults.

The incubation period of infectious mononucleosis is from 10 to 50 days; once fully developed, it lasts for 1 to 4 weeks. Clinical manifestations include extreme fatigue, malaise, sore throat, fever, and cervical lymphadenopathy. Splenomegaly occurs in about 50% of cases. Jaundice is infrequent, although the most common complication is hepatitis. A smaller percentage of patients develop hepatomegaly or splenomegaly and hepatomegaly. Because abnormal liver function is more marked with EBV-induced than CMV-associated infectious mononucleosis, EBV must be considered in the differential diagnosis of hepatitis.

A significant number of patients with infectious mononucleosis do not manifest classic signs and symptoms.

Laboratory Diagnostic Evaluation

In addition to clinical signs and symptoms, laboratory testing is necessary to establish or confirm the diagnosis of infectious mononucleosis (Table 22-1).

Table 22-1

Classic Laboratory Findings in Acute Infectious Mononucleosis

Assay Result
Heterophile antibody test Positive
Anti-VCA IgM Elevated titer
Liver enzymes Elevated
Leukocyte differential Increased number of variant (atypical) lymphocytes

VCA, Viral capsid antigen; IgM, immunoglobulin M.

Hematologic studies reveal a leukocyte count ranging from 10 to 20 × 109/L in about two thirds of patients; about 10% of the patients demonstrate leukopenia. A differential leukocyte count may initially disclose a neutrophilia, although mononuclear cells usually predominate as the disorder develops. Typical relative lymphocyte counts range from 60% to 90%, with 5% to 30% variant lymphocytes. These variant lymphocytes exhibit diverse morphologic features and persist for 1 to 2 months and as long as 4 to 6 months (Fig. 22-1).

image
Figure 22-1 Variant lymphocytes seen in Epstein-Barr virus infection (mononucleosis). (From Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St Louis, 2013, Saunders.)

If the classic signs and symptoms are absent, a diagnosis of infectious mononucleosis is more difficult to make. A definitive diagnosis can be established by serologic antibody testing. The antibodies present in infectious mononucleosis are heterophile and EBV antibodies.

Immunologic Manifestations

Heterophile Antibodies

Heterophile antibodies are composed of a broad class of antibodies. These antibodies are stimulated by one antigen and react with an entirely unrelated surface antigen present on cells from different mammalian species. Heterophile antibodies may be present in normal individuals in low concentrations (titers), but a titer of 1:56 or greater is clinically significant in patients with suspected infectious mononucleosis.

The immunoglobulin M (IgM) type of heterophile antibody usually appears during the acute phase of infectious mononucleosis, but the antigen that stimulates its production remains unknown. IgM heterophile antibody is characterized by the following features:

Paul and Bunnell first associated infectious mononucleosis with sheep cell agglutination and developed a test for the infectious mononucleosis heterophile. Davidsohn modified the original Paul-Bunnell test, introducing a differential adsorption aspect to remove the cross-reacting Forssman and serum sickness heterophile antibodies. Rapid agglutination slide tests are now available.

Epstein-Barr Virus Serology

Within the adult population, 10% to 20% of individuals with acute infectious mononucleosis do not produce infectious mononucleosis heterophile antibody. The pediatric population is of particular concern because more than 50% of children younger than 4 years with infectious mononucleosis are heterophile negative. In diagnostically inconclusive cases of infectious mononucleosis, a more definitive assessment of immune status may be obtained through an EBV serologic panel. Candidates for EBV serology include those who do not exhibit classic symptoms of infectious mononucleosis, who are heterophile negative, or who are immunosuppressed.

Epstein-Barr–infected B lymphocytes express a variety of new antigens encoded by the virus. Infection with EBV results in the expression of viral capsid antigen (VCA), early antigen (EA), and nuclear antigen (NA), with corresponding antibody responses. Assays for IgM and IgG antibodies to these EBV antigens are available. EBV-specific serologic studies are beneficial in defining immune status, and their time of appearance may indicate the stage of disease (Fig. 22-2; Table 22-2). This can provide important information for the diagnosis and management of EBV-associated disease. Patients with nasopharyngeal carcinoma have elevated titers of IgA antibodies to EBV replicative antigens, including VCA. These antibodies, which frequently precede the appearance of the tumor, serve as a prognostic indicator of remission and relapse.

Table 22-2

Characteristic Antibody Formation in Infectious Mononucleosis

Parameter VCA IgM VCA IgG EA-D EA-R EBNA IgG Heterophile
No previous exposure − − − − − −
Recent (acute) infection + + ± − − +
Past infection (convalescent) period − + − − + −
Reactivation of latent infection ± + ± ± + ±

image

VCA, Viral capsid antigen; EA-D, early antigen–diffuse; EA-R, early antigen–restricted; EBNA, Epstein-Barr nuclear antigen; IgG, immunoglobulin G.

image
Figure 22-2 Epstein-Barr virus (EBV) antibody response during the course of infectious mononucleosis. EA, Early antigen; VCA, viral capsid antigen; EBNA, Epstein-Barr nuclear antigen; CF, complement fixation test. (Redrawn from Krugman S, et al: Infectious diseases of children, ed 9, St Louis, 1992, Mosby.)

Early Antigen (EA)

EA is a complex of two components, early antigen–diffuse (EA-D), which is found in the nucleus and cytoplasm of the B cells, and early antigen–restricted (EA-R), usually found as a mass only in the cytoplasm.

Anti–EA-D of the IgG type is highly indicative of acute infection, but it is not detectable in 10% to 20% of patients with infectious mononucleosis. EA-D disappears in about 3 months; however, a rise in titer is demonstrated during reactivation of a latent EBV infection.

Anti–EA-R IgG is not usually found in young adults during the acute phase but may be seen in the serum of very young children during the acute phase. Anti–EA-R IgG appears transiently in the later, convalescent phase. In general, anti–EA-D and anti–EA-R IgG are not consistent indicators of the disease stage.

Epstein-Barr Nuclear Antigen (EBNA)

EBNA is found in the nucleus of all EBV-infected cells. Although the synthesis of NA precedes EA synthesis during the infection of B cells, EBNA does not become available for antibody stimulation until after the incubation period of infectious mononucleosis, when activated T lymphocytes destroy the EBV genome–carrying B cells. As a result, antibodies to NA are absent or barely detectable during acute infectious mononucleosis.

Anti-EBNA IgG does not appear until a patient has entered the convalescent period. EBNA antibodies are almost always present in sera containing IgG antibodies to VCA of EBV unless the patient is in the early acute phase of infectious mononucleosis. Patients with severe immunologic defects or immunosuppressive disease may not have EBNA antibodies, even if antibodies to VCA are present.

Under normal conditions, antibody titers to NA gradually increase through convalescence and reach a plateau 3 to 12 months after infection. The antibody titer remains at a moderate, measurable level indefinitely because of the persistent viral carrier state established after primary EBV infection. Most healthy individuals with previous exposure to EBV have antibody titers to EBNA that range from 1:10 to 1:160. In EBV-associated malignancies, the levels of EBNA antibody are usually high in patients with nasopharyngeal carcinoma and can range from barely detectable to very high levels in patients with Burkitt’s lymphoma.

Test results of antibodies to EBNA should be evaluated in relation to patient symptoms, clinical history, and antibody response patterns to VCA and EA to establish a diagnosis (Table 22-3). The antibody profile can be especially useful. For example, a patient with an infectious mononucleosis–like illness caused by reactivation of a persistent EBV infection resulting from an immunosuppressive malignancy or nonmalignant disease can demonstrate high titers of IgM and IgG VCA antibodies. If the antibody to EBNA is also elevated, however, a diagnosis of primary EBV infection can be excluded.

Table 22-3

Characteristic Diagnostic Profile of Epstein-Barr Virus

Stage Description
Susceptibility If the patient is seronegative (lacks antibody to VCA)
Primary infection Antibody (IgM) to VCA is present; EBNA is absent.
High or rising titer of antibody (IgG) to VCA and no evidence of antibody to EBNA after at least 4 wk of symptoms
Reactivation If antibody to EBNA and increased antibodies to EA are present, patient may be experiencing reactivation.
Past infection Antibodies to VCA and EBNA are present.

VCA, Viral capsid antigen; EBNA, Epstein-Barr nuclear antigen; EA, early antigen.

Additional Testing

Immunofluorescence is a common methology in EBV serology testing. Antigen substrate slides containing EBV-infected B cells are incubated with the patient’s serum. The presence of specific antibody is detected by the addition of fluorescein-conjugated antihuman IgG or IgM. The disadvantages of this type of testing are that it is time-consuming, difficult to interpret, and prone to interference from other serum components (e.g., rheumatoid factor).

The enzyme-linked immunosorbent assay (ELISA) may be used to detect antibodies to EBNA. This ELISA uses a synthetic peptide antigen to determine the relative amounts of IgM and IgG antibodies in patient serum or plasma. Its sensitivity is reportedly 98.9%, with a specificity of 99.0%.

CASE STUDY

image Paul-Bunnell Screening Test

Principle

The classic Paul-Bunnell test is a hemagglutination test designed to detect heterophile antibodies in patient serum when mixed with antigen-bearing sheep erythrocytes. Dilutions of inactivated patient serum are mixed with sheep erythrocytes, incubated, centrifuged, and macroscopically examined for agglutination. Positive reactions are preliminary.

See image for the procedural protocol on the Evolve website.

image Davidsohn Differential Test

Principle

This classic procedure distinguishes between the heterophile antibodies that agglutinate the antigen-bearing erythrocytes of sheep. The differential nature of the test is predicated on the fact that sheep and beef (ox) erythrocytes bear some common antigens not present on the kidney cells of the guinea pig. Exposure of patient serum to guinea pig cells, which are rich in Forssman antigen, and to beef erythrocytes, which are poor in Forssman antigen, produces differential absorption. Any absorbed antibodies are removed by centrifugation and the supernatant fluid is tested with sheep erythrocytes. This classical test differentiates the heterophile types of antibody associated with infectious mononucleosis or serum sickness (Table 22-4).

Table 22-4

Agglutinins for Sheep Erythrocytes in Human Serum

Type of Serum Absorbed by Guinea Pig Kidney Absorbed by Beef Erythrocytes
Normal Positive (+) Negative (−)
Infectious mononucleosis Negative (−) Positive (+)
Serum sickness Positive (+) Positive (+)

The Davidsohn differential procedure is performed only if the preliminary Paul-Bunnell test is positive in a titer of 1:56 or greater. Serum sickness occurs as the result of sensitization to animal serum, usually horse serum.

See image for the procedural protocol on the Evolve website.

image MonoSlide Test

Principle

The BBLMonoSlide procedure (BD BBLMonoSlide, Becton, Dickinson, Franklin Lakes, NJ) is based on the agglutination of horse erythrocytes by heterophile antibody present in infectious mononucleosis. Because horse red blood cells (RBCs) exhibit antigens directed against both Forssman and infectious mononucleosis antibodies, a differential absorption of the patient’s serum is necessary to distinguish the specific heterophile antibody from those of the Forssman type.

The basic principle of the absorption steps in this procedure is comparable to that originally described by Davidsohn in the sheep agglutinin test. Serum or plasma is absorbed with both guinea pig kidney and beef erythrocyte stroma. Guinea pig kidney contains only the Forssman antigen, and beef erythrocytes contain only the antigen associated with infectious mononucleosis. Guinea pig kidney will absorb only heterophile antibodies of the Forssman type and beef erythrocytes will absorb only the heterophile antibody of infectious mononucleosis. Agglutination of horse RBCs by the absorbed patient specimen indicates a positive reaction for heterophile antibody.

The BBLMonoSlide Test uses a disposable card, guinea pig kidney antigen for absorption, and specially treated horse erythrocytes (color-enhanced) to increase specificity and sensitivity and to enhance readability. No special equipment is required to read the BBLMonoSlide Test results.

Chapter Highlights

• Epstein-Barr virus (EBV), a DNA virus, is the cause of infectious mononucleosis.

• An estimated 95% of the world’s population is exposed to EBV, making it the most ubiquitous virus known. The virus infects B lymphocytes. Although transmitted primarily by infectious oral-pharyngeal secretions, EBV may also be transmitted by blood transfusion and transplacentally.

• The frequency of seronegative patients is almost 100% in early infancy but declines with increasing age to less than 10% in young adults. After primary exposure, a person is considered immune and generally no longer susceptible to overt reinfection.

• In Western societies, primary exposure to EBV occurs in two waves among children and adolescents. EBV is only a minor problem for immunocompetent persons but can become a major concern for immunocompromised individuals.

• The antibodies present in infectious mononucleosis are heterophile and EBV antibodies.

• EBV-infected B lymphocytes express a variety of new antigens encoded by the virus. Infection with EBV results in the expression of viral capsid antigen VCA, early antigen EA, and nuclear antigen NA, with corresponding antibody responses. Assays for IgM and IgG antibodies to these EBV antigens are available.

• EBV-specific serologic studies are beneficial for defining immune status. The time of antibody appearance may indicate the stage of the disease.

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