Laboratory-based investigations of IM and Epstein-Barr virus.

Infectious mononucleosis (IM) also known as mono or glandular fever, is a clinical syndrome caused by Epstein-Barr virus (EBV), the fourth human herpes virus. EBV is a member of the herpes-virus family--a gamma herpes virus, in fact--and infects over 95% of the world's population, mostly with no adverse effects. (1) The timing of initial infection is crucial in dictating the symptoms experienced by the host. IM is the typical illness experienced by adolescents newly infected with EB whereas, in contrast, infants nearly always have asymptomatic infection. Infections are lifelong and do not usually carry any risk of future disease. But some features of the acute infection have significant clinical importance. Under certain circumstances, most commonly immunosuppression, EBV infection has been implicated in several types of human malignancy--Burkitt's lymphoma being perhaps the best recognized, but also nasopharyngeal carcinoma and others. The laboratory can play a crucial role in diagnosing and managing EBV infection.

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EBV was first detected by Epstein, Barr, and Achong in a Burkitt's lymphoma cell line in 1964, (2) and holds the dubious honor of being the first human tumor virus described. Four years later, Henle reported an association between EBV and infectious mononucleosis, (3) an observation that was later confirmed in a study of students at Yale University. (4)

EBV shares the classic structural features of the other herpes viruses: it is enveloped, contains a nucleoid and capsid, and has a linear double-stranded DNA genome, which is contained within the nucleoid structure. EBV can enter into a latent phase of replication, during which the genome persists as a circular episome in the cell nucleus, with only minimal activity of select viral genes. These genes ensure that the episome is replicated and transmitted to daughter cells as if it were a normal host chromosome, as well as being involved in reactivation and tumorigenesis.

The cell types naturally infected with EBV are the B lymphocyte, responsible for antibody production, and epithelial cells. The typical portal of entry is the mouth, with initial replication occurring in the nasopharynx, and EBV is secreted in saliva from infected salivary glands (which is why IM is also sometimes called the kissing disease). (5) Systemic spread results in seeding of organs such as lymph nodes, spleen, and liver; and abnormal liver enzyme levels are a common finding. The mononucleosis is not, in fact, from stimulation of B cells by viral infection (although EBV will transform cell lines in vitro), but is from a large, effective, CD8 cytotoxic T-cell response against the EBV-infected circulating B cells. (6) Once latent infection is established in epithelial cells and B lymphocytes, they become immortalized with occasional reactivation of lytic viral replication. After the initial infection, EBV may be continually secreted in saliva for up to 18 months, but even once the infection is brought under immune control, a large percentage of healthy asymptomatic people infected with EBV will shed the virus in saliva at any one time.

Populations affected; syndromes mimicked

Most of the clinical effects of EBV infection are found in the adolescent and adult populations, even though worldwide most infections occur in infants. This is due to the fact that in the developed world, the epidemiology is very different from that in developing nations. Additionally, dietary difference and exposures to other infectious agents modify the effects of EBV considerably.

In the United States, approximately 50% of the population is infected by five years of age, (7) although exact numbers are difficult to come by as EBV is not a reportable infection. Ninety percent of adults are infected by 25 years of age (in contrast to developing countries where 90% of people are infected by two years of age). In lower socio-economic groups, EBV infection tends to occur at younger ages and with a consequently lower rate of IM. Studies in the 1970s showed a thirtyfold higher risk of IM in the Caucasian population vs. African-Americans, but this was due to social differences and not any real racial predilection. (8) Approximately half of adolescents and adults newly infected with EBV will become symptomatic with IM. (9) The male to female ratio of cases is 1:1 although the age of IM is approximately two years younger in females in the United States.

EBV infection can mimic a number of clinical syndromes. Acute infection with EBV is easily confused with that of a streptococcal pharyngitis (which, in fact, may follow IM in a quarter of cases). Sore throat, fever, enlarged red tonsils with exudates, and easily palpable lymph nodes in the neck are all common features. Some differences do exist (for example, the lymph nodes are usually tender with streptococcal pharyngitis), but it can be important to make the distinction. Inadvertently treating IM with ampicillin (a typical antibiotic use in treating strep throat) results in the appearance of a maculopapular rash. In addition, the lymphadenopathy may raise cause for concern about the possibility of leukemia or lymphoma, especially when it is associated with lethargy and other non-specific symptoms.

Acute IM, however, is important to recognize. The seeding of EBV in the spleen can predispose it to rupture; (10) and although this is rare, it is an important cause of mortality from IM. Athletics and contact sports should be avoided until a few weeks after symptoms resolve from the acute infection. Other symptoms that are typical of IM include abdominal pain, jaundice, and central nervous system (CNS) signs that may mimic meningitis or encephalitis.

There is a well-recognized post-viral syndrome associated with EBV infection, predominantly consisting of significant tiredness which may be relapsing and remitting. EBV, therefore, may be in the minds of clinicians for extended periods after the initial acute infection has passed. In this setting, the potential other diagnoses include systemic inflammatory diseases, such as systemic lupus erythematosus, psychological illness such as depression, and less easily defined entities such as chronic fatigue syndrome, which for a while was thought to be caused by EBV. With any kind of chronic disease, co-morbidity is common; most specifically, depression is well recognized as a result of persistent tiredness and not just a cause. Often, finding a specific cause for the initial symptoms can be very helpful in the minds of clinician and patient alike.

Classic lab findings

The classic laboratory findings of acute EBV infection are a lymphocytosis with a high (around 20%) percentage of reactive or atypical lymphocytes (see Table 1). Although frequently seen in cases of IM, these findings are also seen in other viral infections. The acute infection may be diagnosed more specifically using serologic testing. The Paul-Bunnell or Monospot test detects heterophile antibodies, which bind to red blood cells of other species (sheep or horse, respectively). The accuracy of the test can vary, but it is generally fairly sensitive and specific--up to 84% and 100%, respectively. (11) The reaction can persist for several months after the acute infection. False-positive results are uncommon but do occur--they may be due, in some cases, to true positive reactions after asymptomatic infection. False-negative results, however, are seen more frequently, especially in younger patients. Under the age of 14, the test is decreasingly sensitive; and in young children, it may not be helpful at all. (12)

Table 1. Typical laboratory findings in acute infectious mononucleosis.

     Laboratory findings               Complications

Atypical lymphocytosis           Sensitive but not specific

Positive heterophile antibody    Not accurate in young children
(Monospot) test

High levels of anti-VCA IgM      May cross-react with rheumatoid
                                 factor

Raised liver enzymes (AST, ALT)  Sensitive but not specific

Specific anti-EBV antibodies are used less frequently for diagnosis of acute IM because the tests are more time-consuming. Anti-VCA IgM is a good indicator of acute infection, but there are cross-reactions with rheumatoid factor. To avoid this, serum specimens should be mixed with staphylococcal protein A to adsorb the rheumatoid factor--an unnecessary complication with the existence of the heterophile antibody test.

Anti-EBNA2 antibodies rise early in infection and drop in convalescence. In contrast, anti-EBNA1 antibodies rise in convalescence. In the case of chronic IM, which is a diagnosis based upon at least 12 months of symptoms after the acute illness, the antibody profile is reversed, with higher anti-EBNA2 antibody titers compared to anti-EBNA1. Anti-VCA and anti-EA antibodies are frequently observed, suggesting ongoing viral replication.

Tumors and more severe disease

There are several conditions associated with EBV infection that are forms of abnormal cell replication (see Table 3). In areas where malaria is endemic, co-infection with Plasmodium falciparum is associated with the development of Burkitt's lymphoma. (13) This malignant lymphoid tumor (commonly facial) is, in some areas, the most common childhood cancer. The actual transition from B-cell hyperproliferation (driven by EBV and P falciparum) to malignancy is multistep, including a characteristic translocation of the c-myc proto-oncogene on chromosome 8 to the immunoglobulin promoter regions on chromosomes 14, 2, or 22 (the heavy, kappa light, and lamba light chains, respectively) with the majority being eight to 14 translocations. (14) The proximity of the altered c-myc gene to the immunoglobulin gene promoters in hyperactive B cells is an intuitive mechanism for the disease. In addition, the relatively simple genetics may explain why Burkitt's lymphoma is so responsive to chemotherapy (over 90% cure rate). In Burkitt's lymphoma, EBV is typically found in a latent form in the B cells, with its episome expressing a pattern of gene transcripts which are composed of two small RNA molecules (the EBV encoded RNAs--EBERs) and the EB nuclear antigen 1 (EBNA1), so-called type I latency. (15)

Table 3. EBV-associated malignancies, their predisposing factors, and
preferred diagnostic methods.

    Malignancy       Predisposing factors        Diagnostic methods

Burkitt's lymphoma   Plasmodium falciparum     Histology, (EBV genome
                     co-infection, AIDS        or antigen detection
                                               technically difficult)

Nasopharyngeal       Chinese race, diets rich  Histology, EBV genome,
carcinoma            in nitrosamines, HLA-A2   EBNA1 antigen in tumor
                                               cells. Antibody levels
                                               (IgA to VCA, EA, EBNA1)
                                               may help in monitoring

Hodgkin's lymphoma   Uncertain                 Histology, (EBV PCR not
                                               helpful but of academic
                                               interest)

Non-Hodgkin's        AIDS                      Histology
lymphoma

Primary CNS          AIDS                      Histology
lymphoma

Post-transplant      Immunosuppression         Histology, EBV genome,
lymphoproliferative                            high VCA, and EA
disorder                                       antibody titers

Oral hairy           AIDS                      Detection of EBV
leukoplakia                                    genomes in tissue

X-linked             SAP gene mutation         Detection of EBV genome
lymphoproliferative                            in tissue. High levels
disease (Duncan's                              of anti-VCA and anti-EA
disease)                                       IgM and relatively low
                                               anti-EBNA antibodies.
                                               Poor anti-EBV T cell
                                               activity

Burkitt's lymphoma is usually best diagnosed based upon tissue histology, as the detection of EBV-specific antigen or genome in the cells is more technically difficult. Histology shows a monoclonal, poorly differentiated lymphocytic tumor of B-cell origin. EBV antibody titers in children are often very high, may decline in response to therapy, and can be a useful monitoring tool.

EBV is also associated with undifferentiated nasopharyngeal carcinoma (NPC), most notably in Southern China. (16) This aggressive tumor is difficult to treat with very poor survival rates. In this situation, EBV is in type II latency, expressing additional transcripts, including three latent membrane proteins (LMP1, LMP2A, LM2B) with the two EBERs and EBNA1. (15) Multiple copies of the viral genome are usually present in the tumor cells. It appears as if dietary factors play a role in the development of the disease: salted fish and nitrosamines have been implicated. There is also, however, a clear genetic role, with linkage to HLA-A2 haplotypes being a risk factor. First-generation immigrants from China retain a high risk for NPC. Caucasians are at a lower risk for NPC, with other races falling in between.

NPC has similar technical difficulties as BL, and diagnosis is usually based on histology, despite the ability to frequently detect EBV genome and EBNA1 in tumor cells. High levels of serum IgA to VCA, EA, and EBNA1 can be used to monitor the response to treatment (see Table 2).

Table 2. Antibody profiles of various EBV-associated conditions.

             Condition                       Antibody profile

Acute infectious mononucleosis       Raised anti-VCA and high anti-EBNA2
                                     initially, later high levels of
                                     anti-EBNA1 and low anti-EBNA2

Chronic infectious mononucleosis     Low levels of anti-EBNA1 and high
                                     anti-EBNA2. Persistent anti-VCA and
                                     anti-EA antibodies

X-linked lymphoproliferative         High anti-VCA and anti-EA, low
syndrome                             anti-EBNA antibodies (may be seen
                                     in female carriers as well as
                                     affected males)

Post-transplant lymphoproliferative  High anti-VCA and anti-EA
disorder                             antibodies. Seroconversion seen in
                                     pre-transplant seronegative
                                     patients

Nasopharyngeal carcinoma             Antibody levels (IgA to VCA, EA,
                                     EBNA1) may be raised and may help
                                     in monitoring

In healthy individuals, the third form of latent infection occurs, type III latency, in which six EBNAs (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNALP) are produced, along with the EBERs and the LMPs. (15) The functional roles of the genes are in promoting immortalization of the host cell, stability and persistence of the viral genome, and in allowing the virus to reactivate from its latent state.

Another tumor type associated with EBV is Hodgkin's disease, and EBV can be detected by PCR in some specimens within the characteristic Reed-Sternberg cells. (17)

Other populations at risk

Immunocompromised individuals can suffer from more severe disease. A rare entity called x-linked lymphoproliferative syndrome (XLPS), also known as Duncan's disease or Duncan's syndrome, results in an inability to effectively fight off EBV infection. (18) The gene responsible, SAP (signaling lymphocyte activation molecule [SLAM]-associated protein), is found on the X chromosome. EBV infection either rapidly leads to death from overwhelming viral replication and hemophagocytosis (accounting for approximately half of all deaths from IM) or a chronic B-cell lymphoproliferative disease with tumors typically occurring in the CNS or gastro-intestinal, or GI, tract. Males are always affected with poor T-cell responses to EBV and skewed antibody responses (far fewer antibodies to the EBNA proteins but higher responses to the capsid and early antigen proteins), but female carriers may demonstrate some defects in their immune responses to EBV, usually only in their antibody profiles (see Table 2).

XLPS is diagnosed similarly to IM, with high levels of anti-VCA and anti-EA IgM and relatively low anti-EBNA antibodies. There may be abnormal natural killed, or NK, cell activity and low anti-EBV cytotoxic T-cell activity. Unfortunately, many diagnoses of XLPS are made post-mortem based upon the detection of EBV-containing lymphocytic infiltrates. Females in affected families may show the typical skewed antibody profile in the absence of clinical disease--in such cases, genetic counseling is warranted, as half of all males born from that woman will be affected and half of all females will be carriers.

Transplant recipients are at risk of post-transplant lymphoproliferative disorder (PTLD), which is directly associated with the degree of immunosuppression involved. (19) EBV-infected B cells (and sometimes reactive T cells) form sheets of atypical lymphoid tissue similar to non-Hodgkin's lymphoma. It appears to be due to acute infection with EBV after transplantation, rather than a reactivation of latent infection. Lymphoma, though, is recognized as a common complication of chronic immune suppression and is associated with both acute and chronic EBV infection--EBV genome is found in the tissues, and there are high titers of antibodies to the capsid (VCA) and early antigen (EA) proteins suggestive of ongoing viral replication. Interestingly, though, the tumors are usually a monoclonal rather than a polyclonal response to EBV infection. Similar to the situation with Burkitt's lymphoma, additional genetic mutations are required to result in a malignancy. PTLD lesions contain EBNA-positive cells but also display other viral antigens more typically seen in lytic infection. Because of their immune suppression, people with PTLD do not usually show clinical signs of the acute infection that typically precedes the condition. It may be possible, however, to demonstrate seroconversion from serum specimens taken prior to transplantation (some centers will test EBV antibody levels, among other viruses, for precisely this purpose) (see Table 2).

People with AIDS are also at risk of complications from EBV--unsurprisingly, they tend to involve neoplastic disease (reviewed in Reference 20). The association is complicated by the fact that HIV infection itself predisposes people to lymphomas (B-cell hyperactivation is a consistent finding in HIV infection; but unlike EBV, the mechanism is secondary to the loss of CD4+ helper T-cell regulation). Burkitt's lymphoma occurs at an increased frequency as well as lymphoma of the brain, although only half of the AIDS-associated Burkitt's lymphoma contains EBV genomes. One neoplastic process that has been recently recognized to be linked to EBV is oral hairy leukoplakia--these lesions on the tongue of some HIV-positive people have been shown to contain the EBV genome and expressed EBV proteins. (21), (22) EBV has also been associated with a pneumonitis, similar to cytomegalovirus, or CMV, another herpes virus that causes opportunistic infections in people with AIDS. Detection of EBV in these settings can be important, as some studies have shown that acyclovir can be beneficial, (23) even though it has little to no effect on IM. (24), (25)

Despite the association between EBV and human disease, as a pathogen, it is probably a good example of how one might coexist benignly with its host. EBV infection is typically asymptomatic and lifelong. The diseases it does cause do not affect host survival enough to be detrimental to its own chances of spread. The laboratory can play an important role in diagnosing and managing EBV-associated illness, but understanding the applications and limitations of the techniques is important for diagnosticians and clinicians alike.

References

(1.) Kangro HO, Osman HK, Lau YL, Heath RB, Yeung CY, Ng MH. 1994. Seroprevalence of antibodies to human herpesviruses in England and Hong Kong. J Med Virol. 43:91-96.

(2.) Epstein MA. Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet. 1964;1:702.

(3.) Henle G, Henle W, Diehl V: Relation of Burkitt's tumor-associated herpes-type virus to infectious mononucleosis. Proc Natl Acad Sci USA. 1968;59(1):94-101.

(4.) Sawyer RN, Evans AS, Niederman JC, et al: Prospective studies of a group of Yale University freshmen. I. Occurrence of infectious mononucleosis. J Infect Dis. 1971;123(3):263-270.

(5.) Miller CS, Avdiushko SA, Kryscio RJ, Danaher RJ, Jacob RJ. Effect of prophylactic valacyclovir on the presence of human herpesvirus DNA in saliva of healthy individuals after dental treatment. J Clin Microbiol. 2005;43(5):2173-2180.

(6.) Tomkinson BE, Wagner DK, Nelson DL, et al. Activated lymphocytes during acute Epstein-Barr virus infection. J Immunol. 1987;139(11):3802-3807.

(7.) Schooley RT. Epstein-Barr virus (infectious mononucleosis). In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 4th ed. New York: Churchill Livingstone; 1995:1364-1377.

(8.) Auwaerter PG. Infectious mononucleosis in middle age. JAMA. 1999;281(5):454-459.

(9.) Svedmyr E, Ernberg I, Seeley J, Weiland O, Masucci G. Virologic, immunologic, and clinical observations on a patient during the incubation, acute, and convalescent phases of infectious mononucleosis. Clin Immunol Immunopathol. 1984;30:437.

(10.) Aldrete JS. Spontaneous rupture of the spleen in patients with infectious mononucleosis. Mayo Clin Proc. 1992; 67(9):910-912.

(11.) Linderholm M, Boman J, Juto P, et al. Comparative evaluation of nine kits for rapid diagnosis of infectious mononucleosis and Epstein-Barr virus-specific serology. J Clin Microbiol. 1994;32(1):259-261.

(12.) Sumaya CV, Ench Y. Epstein-Barr virus infectious mononucleosis in children: part II. Heterophil antibody and viral-specific responses. Pediatrics. 1985;75(6):1011-1019.

(13.) Ferry J. Burkitt's Lymphoma: Clinicopathologic Features and Differential Diagnosis. The Oncologist. 2006;11(4):375-383.

(14.) Bernheim B, Bernheim L. Cytogenetic studies on African Burkitt's lymphoma cell lines: t(8;14), t(2;8) and t(8;22) translocations. Cancer Genet Cytogenet. 1981;3(4):307-315.

(15.) Kieff E, Rickinson AB. Epstein-Barr virus and its replication. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, and Straus SE, eds. Fields virology. 4th ed. Philadelphia, PA: Lippincott-Williams & Wilkins. 2001:2511-2573.

(16.) Liebowitz D. Nasopharyngeal carcinoma: the Epstein-Barr virus association. Semin Oncol. 1994;21(3):376-381.

(17.) Roth J, Daus H, Gause A, Trumper L, Pfreundschuh M. Detection of Epstein-Barr virus DNA in Hodgkin-and Reed-Sternberg-cells by single cell PCR. Leuk Lymphoma. 1994;13:137.

(18.) Dupre L, Andolfi G, Tangye SG, Clementi R, Locatelli F, Arico M, et al. SAP controls the cytolytic activity of CD8+ T cells against EBV-infected cells. Blood. 2005;105(11):4383-4389.

(19.) Smets F, Latinne D, Bazin H, Reding R, Otte J-B, Buts J-P, Sokal EM. Ratio between Epstein-Barr viral load and anti-Epstein-Barr virus specific T-cell response as a predictive marker of posttransplant lymphoproliferative disease. Transplantation. 2002;73(10):1603-1610.

(20.) Navarro WH, Kaplan LD. AIDS-related lymphoproliferative disease. Blood. 2006;107(1):13-20.

(21.) Webster-Cyriaque J, Middeldorp J, Raab-Traub N. Hairy leukoplakia: an unusual combination of transforming and permissive Epstein-Barr virus infections. J Virol. 2000;74(16):7610-7618.

(22.) Greenspan JS, Greenspan D, Lennette ET, et al. Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion. N Engl J Med. 1985;313(25):1564-1571.

(23.) Resnick L, Herbst JS, Ablashi DV, et al. Regression of oral hairy leukoplakia after orally administered acyclovir therapy. JAMA. 1988;259(3):384-388.

(24.) Andersson J, Britton S, Ernberg I, et al. Effect of acyclovir on infectious mononucleosis: a double-blind, placebo-controlled study. J Infect Dis. 1986;153(2):283-290.

(25.) Tynell E, Aurelius E, Brandell A, et al. Acyclovir and prednisolone treatment of acute infectious mononucleosis: a multicenter, double-blind, placebo-controlled study. J Infect Dis. 1996;174(2):324-331.

RELATED ARTICLE: CONTINUING EDUCATION

To earn CEUs, see test on page 16.

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Describe the physical structure of the Epstein-Barr virus (EBV) and explain how the virus is able to replicate and infect the host's cells.

2. Differentiate an acute infection with EBV from a chronic infection with EBV based on clinical laboratory data as well as clinical presentation.

3. Cite relevant statistics associated with EBV infection.

4. Compare and contrast the various clinical conditions and associated malignancies associated with EBV infection.

5. Correlate laboratory testing and methodologies with the appropriate clinical conditions caused by EBV.

6. Explain how screening tests can be beneficial with respect to EBV illnesses.

By Nick Bennett, MA(Cantab), MB/BChir, PhD

Nick Bennett, MA(Cantab), MB/BChir, PhD, is a resident physician in the Department of Pediatrics at SUNY Upstate Medical University, Syracuse, NY. His background is in the viral packaging mechanisms of HIV. Currently, his interests range from molecular virology, through software-enhanced database analysis, to the promotion of effective communication skills and humanism in medicine.