Epstein-Barr Virus (EBV)
The Epstein-Barr virus is a member of the herpesvirus group and is reported to infect 80% or more of the U.S. population. It is thought to be spread from person to person, most likely through saliva, with the majority of infections occurring in childhood, adolescents, and young adults. The EBV infects B-lymphocytes. In common with the other herpesviruses, once infection (with or without symptoms) takes place the virus eventually becomes dormant but can be reactivated later into clinical disease. Reactivation is said to occur in 15%-20% of healthy persons and in up to 85% in some groups of immunosuppressed patients. Epstein-Barr virus infection in young children is usually asymptomatic. Primary infection by EBV in older children, adolescents, or young adults produces the infectious mononucleosis syndrome in up to 50% of cases. The EBV is also strongly associated with Burkitt’s lymphoma in Africa and nasopharyngeal carcinoma in southern China.
Infectious mononucleosis (infectious mono; IM)
Infectious mononucleosis (IM) patients are most often adolescents and young adults, but a significant number are older children and middle-aged or even older adults. When IM is part of a primary infection, the incubation period is 3-7 weeks (range, 2-8 weeks). The acute phase of illness in those patients who are symptomatic lasts about 2-3 weeks (range, 0-7 weeks). Convalescence takes about 4-8 weeks. The most common features of the acute illness are fever, pharyngitis, and adenopathy, with lymph node enlargement occurring in 80%-90% of patients. The posterior cervical nodes are the ones most commonly enlarged. Soft palate petechiae are found in 10%-30% of cases. Jaundice, usually mild, is found in about 5%-10% (range, 4%-45%) of patients in large series. The spleen is mildly enlarged in about 50% of patients (range, 40%-75%) and hepatomegaly is present in about 10% (range, 6%-25%).
Laboratory findings. Patients usually have normal hemoglobin values. Mild thrombocytopenia is reported in 25%-50% of patients (range, 15%-50%). Leukocytosis between 10,000 and 20,000/ mm3 (10 Ч 109-20 Ч 109/L) occurs in 50%-60% of patients (range, 40%-70%) by the second week of illness. About 10% (range, 5%-15%) of patients develop a leukocytosis over 25,000/mm3 (25 Ч 109/L). However, during the first week there may be leucopenia. About 85%-90% (range, 80%-100%) of patients with IM have laboratory evidence of hepatic involvement (Table 17-2). Peak values are reported to occur 5-14 days after onset of illness for aspartate aminotransferase (AST), bilirubin, and alkaline phosphatase (ALP); and between 7 and 21 days for gamma-glutamyltransferase (GGT). The AST and ALP levels return to normal in nearly all patients by 90 days, but occasionally there may be some degree of GGT elevation persisting between 3-12 months. Total LDH is elevated in about 95% of patients. LDH isoenzyme fractionation by electrophoresis can show three patterns: elevation of all five fractions; elevation of LDH 3, 4, and 5; or elevation of LDH-5 only.
Table 17-2 Liver function tests in EBV-induced infectious mononucleosis
Peripheral blood smear. The first of three classic findings is a lymphocytosis, with lymphocytes making up more than 50% of the total white blood cells (WBCs). Lymphocytosis is said to be present in 80%-90% of patients (range, 62%-100%), peaks during the second or third week, and lasts for an additional 2-6 weeks. The second classic criterion is the presence of a “significant number” of atypical lymphocytes on Wright-stained peripheral blood smear. There is disagreement as to whether greater than 10% or greater than 20% must be atypical. These atypical lymphocytes are of three main types (Downey types). Type I has vacuolated or foamy blue cytoplasm and a rounded nucleus. Type II has an elongated flattened nucleus and large amounts of pale cytoplasm with sharply defined borders and often some “washed-out” blue cytoplasm coloring at the outer edge of the cytoplasm. Type III has an irregularly shaped nucleus or one that may be immature and even may have a nucleolus and resemble a blast. All three types are larger than normal mature lymphocytes, and their nuclei are somewhat less dense. Most of the atypical lymphocytes are activated T-lymphocytes of the CD-8 cytotoxic-suppressor type. Some of the Downey III lymphocytes may be EBV-transformed B lymphocytes, but this is controversial. These atypical lymphocytes are not specific for IM, and may be found in small to moderate numbers in a variety of diseases, especially cytomegalovirus and hepatitis virus acute infections. In addition, an appearance similar to that of the type II variety may be created artificially by crushing and flattening normal lymphocytes near the thin edge of the blood smear. IM cells are sometimes confused with those of acute leukemia or disseminated lymphoma, although in the majority of cases there is no problem.
Although most reports state or imply that nearly all patients with IM satisfy the criteria for lymphocytosis and percent atypical lymphocytes, one study found only 55% of patients had a lymphocytosis and only 45% had more than 10% atypical lymphocytes on peripheral smear when the patients were first seen. Two studies found that only about 40% of patients with IM satisfied both criteria.
Serologic tests. The third criterion is a positive serologic test for IM either based on heterophil antibodies or specific anti-EBV antibodies. The classic procedure is the heterophil agglutination tube test (Paul-Bunnell test). Rapid heterophil antibody slide agglutination tests have also been devised. Slide tests now are the usual procedure done in most laboratories. However, since the basic principles, interpretation, and drawbacks of the slide tests are the same as those of the older Paul-Bunnell tube test, there are some advantages in discussing the Paul-Bunnell procedure in detail.
Serologic tests based on heterophil antibodies.
Paul-Bunnell antibody is an IgM-type antibody of uncertain origin that is not specific for EBV infection but is seldom found in other disorders (there are other heterophil antibodies that are not associated with EBV infection). Paul-Bunnell antibodies begin to appear in the first week of clinical illness (about 50% of patients detectable; range, 38%-70%), reaching a peak in the second week (60%-78% of patients positive) or third (sometimes the fourth) week (85%-90% positive; range, 75%-100%), then begin to decline in titer during the fourth or fifth week, most often becoming undetectable 8-12 weeks after beginning of clinical illness. However in some cases some elevation is present as long as 1-2 years (up to 20% of patients). In children less than 2 years old, only 10%-30% develop heterophil antibodies; about 50%-75% of those 2-4 years old develop heterophil antibodies. One report states that these antibodies are rarely elevated in Japanese patients of any age. Once elevated and returned to undetectable level, heterophil antibodies usually will not reelevate in reactivated IM, although there are some reports of mild heterophil responses to other viruses.
The original Paul-Bunnell test was based on the discovery that the heterophil antibody produced in IM would agglutinate sheep red blood cells (RBCs). In normal persons the sheep cell agglutination titer is less than 1:112 and most often is almost or completely negative. The Paul-Bunnell test is also known as the “presumptive test” because later it was found that certain antibodies different from those of IM would also attack sheep RBCs. Examples are the antibodies produced to the Forssman antigen found naturally in humans and certain other animals and the antibody produced in “serum sickness” due to certain drug reactions. To solve this problem the differential absorption test (Davidsohn differential test) was developed. Guinea pig kidney is a good source of Forssman antigen. Therefore, if serum containing Forssman antibody is allowed to come in contact with guinea pig kidney material, the Forssman antibody will react with the kidney antigen and be removed from the serum when the serum is taken off. The serum will then show either a very low or a negative titer, whereas before it was strongly positive. The IM heterophil antibody is not significantly absorbed by guinea pig kidney but is nearly completely absorbed by bovine (beef) RBCs, which do not significantly affect the Forssman antibody. The antibody produced in serum sickness will absorb both with beef RBCs and guinea pig kidney.
The level of Paul-Bunnell titer does not correlate well with the clinical course of IM. Titer is useful only in making a diagnosis and should not be relied on to follow the clinical course of the disease or to assess results of therapy.
In suspected IM, the presumptive test is performed first; if necessary, it can be followed by a differential absorption procedure.
“Spot” tests were eventually devised in which the Paul-Bunnell and differential absorption tests are converted to a rapid slide agglutination procedure without titration. Most of the slide tests use either horse RBCs, which are more sensitive than sheep RBCs, or bovine RBCs, which have sensitivity intermediate between sheep and horse RBC but which are specific for IM heterophil antibody and therefore do not need differential absorption. Slide test horse cells can also be treated with formalin or other preservatives that extend the shelf life of the RBC but diminish test sensitivity by a small to moderate degree.
Heterophil-negative infectious mononucleosis.
This term refers to conditions that resemble IM clinically and show a similar Wright-stained peripheral blood smear picture, but without demonstrable elevation of Paul-Bunnell heterophil antibody (see the box) About 65% (range, 33%-79%) are CMV infection, about 25% (15%-63%) are heterophil-negative EBV infections, about 1%-2% are toxoplasmosis, and the remainder are other conditions or of unknown etiology.
Diagnosis of infectious mononucleosis. When all three criteria for IM are satisfied, there is no problem in differential diagnosis. When the results of Paul-Bunnell test or differential absorption test are positive, most authors believe that the diagnosis can be made, although there are reports that viral infections occurring after IM can cause anamnestic false positive heterophil reelevations. When the clinical picture is suggestive of IM but results of the Paul-Bunnell test, differential absorption procedure, or the spot test are negative, at least one follow-up specimen should be obtained in 14 days, since about 20%-30% of IM patients have negative heterophil test results when first seen versus 10%-15% negative at 3 weeks after onset of clinical symptoms (although the usual time of antibody appearance is 7-10 days, it may take as long as 21 days and, uncommonly, up to 30 days). About 10% (range, 2%-20%) of patients over age 5 years and 25%-50% or more under age 5 years never produce detectable heterophil antibody. Another potential problem is that several evaluations of different heterophil kits found substantial variation in sensitivity between some of the kits. If the clinical picture is typical and the blood picture is very characteristic (regarding both number and type of lymphocytes), many believe that the diagnosis of IM can be considered probable but not established. This may be influenced by the expense and time lapse needed for specific EBV serologic tests or investigation of CMV and the various other possible infectious etiologies.
Some “Heterophil-Negative” Mononucleosis Syndrome Etiologies
Viruses
EBV heterophil-negative infections
Cytomegalovirus
Hepatitis viruses
HIV-1 seroconversion syndrome
Other (rubella, herpes simplex, herpesvirus 6, mumps, adenovirus)
Bacteria
Listeria, tularemia, brucellosis, cat scratch disease, Lyme disease, syphilis, rickettsial diseases
Parasites
Toxoplasmosis, malaria
Medications
Dilantin, azulfidine, dapsone, “serum sickness” drug reactions
Other
Collagen diseases (especially SLE, primary or drug-induced)
Lymphoma
Postvaccination syndrome
Subacute bacterial endocarditis (SBE)
In summary, the three classic criteria for the diagnosis of IM are the following:
1. Lymphocytes comprising more than 50% of total WBC count.
2. Atypical lymphocytes comprising more than 10% (liberal) or 20% (conservative) of the total lymphocytes.
3. Significantly elevated Paul-Bunnell test and/or differential absorption test result. A positive slide agglutination test result satisfies this criterion.
Serologic tests based on specific antibodies against EBV. The other type of serologic test for IM detects patient antibodies against various components of the EBV (Table 17-3). Tests are available to detect either viral capsid antigen-IgM or IgG (VCA-IgM or IgG) antibodies. VCA-IgM antibody is usually detectable less than one week after onset of clinical illness and becomes nondetectable in the late convalescent stage. Therefore, when present it suggests acute or convalescent EBV infection. Rheumatoid factor (RF) may produce false positive results, but most current kits incorporate some method to prevent RF interference. VCA-IgG is usually detectable very soon after VCA-IgM, but remains elevated for life after some initial decline from peak titer. Therefore, when present it could mean either acute, convalescent, or old infection. Tests are available for Epstein-Barr nuclear antigen (EBNA) IgM or IgG antibody, located in nuclei of infected lymphocytes. Most kits currently available test for IgG antibody (EBNA-IgG or simply EBNA). EBNA-IgM has a time sequence similar to that of VCA-IgM. The more commonly used EBNA-IgG test begins to rise in late acute stage (10%-34% positive) but most often after 2-3 weeks of the convalescent stage. It rises to a peak after the end of the convalescent stage (90% or more positive), then persists for life. Elevated EBNA/EBNA-IgG is suggestive of nonacute infection when positive at lower titers and older or remote infection at high or moderately high titer. A third type of EBV test is detection of EBV early antigen (EA), located in cytoplasm of infected cells. There are two subtypes; in one the antigen is spread throughout the cytoplasm (“diffuse”; EA-D) and in the other, the antigen is present only in one area (“restricted”; EA-R). The EA-D antibody begins to rise in the first week of clinical illness, a short time after the heterophil antibody, then peaks and disappears in the late convalescent stage about the same time as the heterophil antibody. About 85% (range, 80%-90%) of patients with IM produce detectable EA-D antibody, which usually means EBV acute or convalescent stage infection, similar to VCA-IgM or heterophil antibody. However, EA-D may rise again to some extent in reactivated EBV disease, whereas VCA-IgM does not (whether heterophil antibody ever rises is controversial, especially since it may persist for up to a year or even more in some patients). EA-D is typically elevated in EBV-associated nasopharyngeal carcinoma. EA-R is found in about 5%-15% of patients with clinical IM. It is more frequent (10%-20%) in children less than 2 years old with acute EBV infection and is typically elevated in patients with EBV-related Burkitt’s lymphoma. Expected results from the various serologic tests in different stages of EBV infection are summarized in Table 17-3 and Fig. 17-9.
Table 17-3 Antibody tests in EBV infection
Fig. 17-9 Tests in EBV infection.
Specific serologic tests for EBV are relatively expensive compared to heterophil antibody tests and are available predominantly in university centers and large reference laboratories. Such tests are not needed to diagnose IM in the great majority of cases. The EBV tests are useful in heterophil-negative patients, in problem cases, in patients with atypical clinical symptoms when serologic confirmation of heterophil results is desirable, and for epidemiologic investigations. If the initial heterophil test is nonreactive or equivocal, it is desirable to freeze the remainder of the serum in case specific EBV tests are needed later.
Summary of Epstein-Barr Antibody Test Interpretation
Never infected (susceptible) = VCA-IgM and IgG both negative.
Presumptive primary infection = Clinical symptoms, heterophil positive.
Primary infection: VCA-IgM positive (EBNA-IgG negative; heterophil positive or negative)
Reactivated infection: VCA-IgG positive; EBNA-IgG positive; EA-D positive (heterophil negative, VCA-IgM negative)
Old previous infection: VCA-IgG positive; EBNA-IgG positive; EA-D negative (VCA-IgM negative, heterophil negative).