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INFECTIOUS MONONUCLEOSIS. CONTENTS. INTRODUCTION
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CONTENTS • INTRODUCTION • EPSTEIN-BARR VIRUS Viral Capsid Antigen Early Antigen Nuclear Antigen • SIGNS AND SYMPTOMS • CLINICAL MANIFESTATIONS • DAVIDSON DIFFERENTIAL TEST • MONO-PLUS TEST Sample Requirements Principle Procedure Interpretation of Results False Positives False Negatives • CONCLUSION • REFERENCES
Introduction • Epstein-Barr virus was first discovered in 1964 as the cause of infectious mononucleosis. • The mode of transmission is not known, but may be facilitated by saliva exchange. • This disorder is usually an acute, benign, and self-limiting lymphoproliferative condition. • The virus is shed in the throat during the illness and for up to a year after infection. • After the initial infection, the virus tends to become dormant for a prolonged period and can later reactivate and be shed from the throat again.
Introduction • The virus is spread by person-to-person contact, via saliva. In rare instances, the virus has been transmitted by blood transfusion or transplacentally. • In underdeveloped countries, people are exposed in early childhood where they are less likely to develop noticeable symptoms. • In developed countries such as the United States, the age of first exposure may be delayed to older childhood and young adult age when symptoms are more likely to result.
Introduction • Infectious Mono is recognized more often in high school and college students. • The disease usually runs its course in two to four weeks, although cases may be as brief as a week or last six to eight weeks. • After recovery, weakness may continue for several months.
Epstein-Barr Virus • Epstein-Barr virus (EBV) is a human herpes DNA virus. • It is estimated that 95 percent of the world population is exposed to the virus. • In Infectious Mono the virus affects B-lymphocytes. • There are two techniques used to identify EBV; immunofluorescence and complement fixation.
Epstein-Barr Viral Infection • It is a systematic immune complex disease of soluble and tissue-fixed antigen involvement characterized by fever, fatigue, chills, headache, myalgia, skin rash, splenomegaly and cervical adenopathy. • EBV infected B-lymphocytes express a variety of “new” antigens encoded by the virus. Infection with EBV results in expression of: 1. Viral Capsid Antigen (VCA) 2. Early Antigen (EA) 3. Nuclear Antigen (NA) Each antigen expression has corresponding antibody responses.
Epstein-Barr Virus (VCA) • Viral capsid antigen (VCA) is produced by infected B cells and can be found in the cytoplasm. • Anti-VCA IgM is usually detectable early in the course of infection, 4 to 7 days after onset of signs and symptoms, but it is low in concentration and disappears within 2 to 4 months.
Epstein-Barr Virus (EA) • Early antigen (EA) is a complex of two components, early antigen-diffuse (EA-D), which is found in both the nucleus and cytoplasm of the B cells, and early antigen-restricted (EA-R), which is 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 IM. 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. 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 Virus (EBNA) • Epstein-Barr nuclear antigen (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, EBV-NA does not become available for antibody stimulation until after the incubation period of Infectious Mono, when activated T lymphocytes destroy the EBV genome-carrying B cells. As a result, antibodies to NA are absent or barely detectable during acute IM. • Anti-EBNA IgG does not appear until a patient has entered the convalescent period. EBV-NA antibodies are almost always present in sera containing IgG antibodies to VCA of EBV unless the patient is in the early acute phase of IM. Patients with severe immunologic defects or immunosuppressive disease may not have EBV-NA antibodies, even if antibodies to VCA are present.
Epstein-Barr Virus (EBNA) • Under normal conditions, antibody titers to NA gradually increase through convalescence and reach a plateau between 3 and 12 months postinfection. The antibody titer remains at a moderate, measurable level indefinitely because of the persistent viral carrier state established following primary EBV infection. • Test results of antibodies to EBV-NA should be evaluated in relationship to patient symptoms, clinical history, and antibody response patterns to EBV-VCA and EA to establish a diagnosis.
Signs and Symptoms • Mononucleosis is characterized by fever, sore throat, fatigue, malaise, and loss of appetite. • Patients generally have swelling of the lymph nodes in the neck and often have an enlarged spleen. • No treatment, other than rest, is needed in the vast majority of cases and there is no vaccine available to prevent IM.
Signs and Symptoms • In children and infants the time of onset is usually vague and the duration of prodromal symptoms is difficult to determine. • Anorexia, sometimes accompanied by nausea and vomiting, is a common and non-specific early symptom of this infection. • The most important and most characteristic symptom of IM is a sore throat. This usually develops a few days after the onset of the illness, increases in severity during the first week, and then rapidly subsides during the next five to seven days. • In many young adults sore throat is the first indication of sickness and in some it is the only major symptom throughout the entire illness.
Signs and Symptoms • Although anorexia may persist for as long as there is fever, its intensity and duration are more directly related to the severity of sore throat and dysphagia. • Gross tonsillar and pharyngeal edema may cause virtually complete pharyngeal obstruction with harsh-sounding breathing and complete inability to swallow either food or fluids. • In some patients the soreness of the throat is so severe that swallowing even a few sips of water is extremely painful.
Signs and Symptoms • The headaches of early IM are often retro-orbital in location but have no characteristic features. • They may be moderately severe for one or two days but usually they are mild and rarely last for more than three or four days. • Ocular symptoms may be in the form of photophobia, ocular muscle aching or the awareness of puffiness.
Signs and Symptoms • Lymphadenopathy, disease of the lymph nodes, is sometimes accidentally discovered or detected during self-examination following the development of systemic symptoms. • In about 3 percent of all cases of IM, the gross cervical lymphadenopathy imparts a “bull neck” appearance. • Enlargement of lymph nodes usually begins two or three days after the onset of the first symptoms and, by the end of the week, palpable lymphadenopathy is present in 70-80 percent of all patients.
Signs and Symptoms • Jaundice is a moderately important symptom of infectious mono as 8-10 percent of patients eventually become visibly jaundiced. • In most instances, however, it is not noticed since it consists of only a transient icteric tint to the sclerae and mucous membranes, lasting for a few days.
Clinical Manifestations • Examination of the blood usually shows an increase in the white blood cells, due to the appearance of many atypical lymphocytes in the blood. • Blood serum in IM often contains an antibody known as heterophil antibody that agglutinates, or clumps, the red blood cells of sheep. • Heterophil antibodies are antibodies that are stimulated by one antigen and react with an entirely unrelated surface antigen present on cells from different mammalian species.
Clinical Manifestations • Heterophil antibody titers rise during the first two or three weeks with half or more developing a significant titer during the first week of illness. • The level of antibody gradually declines and usually disappears in eight to twelve weeks following the onset. • Elevated titers sometimes linger for four to six months up to a year or more. • Heterophil antibody most commonly used in the serological diagnosis of IM is an IgM antibody which agglutinates sheep red blood cells.
Clinical Manifestations • The original Paul-Bunnell test was a simple titration of sheep cell agglutinins but this procedure was subsequently modified in order to distinguish between sheep cell agglutinins formed in IM and the Forssman-type antibodies found in normal serum, serum sickness and in certain other conditions. • Tissues rich in Forssman antigen (guinea pig kidney) absorb Forssman antibodies but do not affect the heterophil antibodies in IM. • Heterophil antibodies are absorbed by beef cells, • Forssman hapten is a glycolipid usually associated with a protein, the determinant being largely carbohydrate and therefore heat stable.
Davidsohn Differential • The principle behind the Paul-Bunnell-Davidsohn test is that the two types of sheep agglutinins are distinguished by titrating them before and after absorption with guinea pig kidney and ox cells. • Patients serum containing antibodies due to IM is added to guinea pig kidney cells. These antibodies are not absorbed by the kidney cells. These antibodies then react with Beef (Ox) red blood cells which causes agglutination and is a positive test for IM. • Patients serum containing Forssman antibodies are added to guinea pig kidney cells. Antibodies are absorbed by the kidney cells. These antibodies are then allowed to react with Beef red blood cells which does not cause agglutination. This is a positive test for Forssman antigens.
Davidsohn Differential* To be considered absorbed there must be greater than a three tube difference between the presumptive titer and the differential titer.
Advantages When properly performed, this test is specific for Infectious Mononucleosis and false-positive results are rare. Disadvantages Davidsohn Differential test is very time consuming and burdensome. Davidsohn Differential
Mono-Plus Sample Requirements • Red top tube of blood (Serum) • Green top tube of blood (Plasma) • Purple top tube of blood (Plasma) • CPDA-1 (Plasma) • Capillary blood from fingertip (Whole Blood)
Mono-PlusPrinciple • Qualitative detection of IM heterophil antibodies in human serum, plasma and whole blood using direct solid-phase immunoassay technology. • A band of bovine (Ox) erythrocyte extracts are impregnated in the test membrane. • If IM-specific heterophil antibody is present in the sample, it will be captured by the bovine erythrocyte extracts.
Mono-PlusPrinciple • The Developer Solution is then added to the sample well. • The solution mobilizes the dye conjugated to the anti-human IgM antibodies. • The antigen band can be seen in the Test Window (T) only when the antibody-dye conjugate binds to the IM-specific heterophil antibody which has been bound to the bovine erythrocyte extract.
Mono-PlusPrinciple • The antibody-dye conjugate will bind to another band located in the Control Window (C) to generate a colored band regardless of the presence of IM heterophil antibodies in the sample. • The presence of two colored bands or lines, one in the Test Window (T) and one in the Control Window (C), indicates a positive test. • The presence of a colored band in the Control Window (C) only indicates a negative result.
Mono-PlusProcedure • Step 1 • Pipette 10 uL of serum or plasma in the upper well.
Mono-PlusProcedure • Step 2 • Add 2-3 drops of Developer Solution to the lower end of the sample well.
Mono-PlusProcedure • Step 3 • Read test results in 8 minutes. • Strong positive may appear in less than 3 minutes. • Must wait 8 minutes to report negative result. • Results are stable 15 minutes after Developer is added.
Positive Result A pink-purple horizontal bar in the Test Window (T) and the Control (C). Negative Result A pink-purple horizontal bar in the Control Window (C). No horizontal bar in the Test Window (T). Mono-PlusInterpretation of Results
Mono-PlusInterpretation of Results • Invalid Result • If no bar appears in the Control Window (C) the test is invalid. • A distinct horizontal bar should always appear in the Control Window (C).
Mono-PlusFalse Positives • IM heterophil has been associated with disease states such as: Burkitt’s Lymphoma, viral hepatitis, adenovirus, leukemia, cytomegalovirus, rheumatoid arthritis and Toxoplasma gondii. EBV-specific lab diagnosis may be used for persons with these illnesses. • Sera of patients with IM react not only with beef erythrocytes but also other bovine antigens. False positives have occurred with bovine heart extract (cardiolipin).
Mono-PlusFalse-Negatives • Although most patients will have detectable heterophile levels within three weeks of infection, occasionally a patient with strong clinical signs of IM may take as long as three months to develop a detectable level. This can be resolved by taking additional specimens every few days and retesting. • Some segments of the population who contract IM do not produce measurable levels of heterophil antibody. Approximately 50% of children under 4 years of age who have IM may test as IM heterophil negative. EBV-specific laboratory diagnosis may be helpful in these cases.
Conclusion • In addition to clinical signs and symptoms, laboratory testing is necessary to establish or confirm the diagnosis of IM. This can provide important information for both the diagnosis and management of EBV-associated disease. • If the classic signs and symptoms of IM are absent, a diagnosis of IM is more difficult to make. A definite diagnosis of IM can be established by serologic antibody testing. The antibodies present in IM are heterophil and EBV antibodies. • EBV is widely disseminated. It is estimated that 95% of world’s population is exposed to the virus, which makes it the most ubiquitous virus known to man. • EBV is only a minor problem for immunocompetent persons, but it can become a major one for immunologically compromised patients • After primary exposure a person is considered to be immune and generally no longer susceptible to overt reinfection.
References • Mono-Plus; Wampole Laboratories, Dist.; Cranbury Laboratories; 1999. • Infectious Mononucleosis; Robert J. Hoagland; Grune and Stratton Inc.; New York and London; 1967. • Immunology and Serology in Laboratory Medicine; Mary Louise Turgeon; The C.V. Mosby Company; St Louis; 1990. • Infectious Mononucleosis; Sidney Leibowitz, M.D.; Grune and Stratton; New York; 1953.
