Transcript INFECTIOUS MONONUCLEOSIS

INFECTIOUS
MONONUCLEOSIS
CONTENTS
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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
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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
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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
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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 Viral Infection
Epstein-Barr Virus
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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
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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)
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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)
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Early antigen (EA) is a complex of two components, early antigendiffuse (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)
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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)
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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 of
Infectious Mononucleosis
Signs and Symptoms
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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
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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
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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
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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
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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
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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 of
Infectious Mono
Clinical Manifestations
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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
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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
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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
SLIDE TEST
Davidsohn Differential
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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.
Heterophil Antibody
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Kidney Extract
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Beef Erythrocyte
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Infectious Mono
Not Absorbed
Absorbed
Forssman
Absorbed
Not Absorbed
Serum Sickness
Absorbed
Absorbed
Davidsohn Differential
Advantages
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When properly performed, this
test is specific for Infectious
Mononucleosis and falsepositive results are rare.
Disadvantages
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Davidsohn Differential test is
very time consuming and
burdensome.
MONO-PLUS TEST
Mono-Plus
Sample Requirements
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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-Plus
Principle
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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-Plus
Principle
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The Developer Solution is then added to the sample well.
The solution mobilizes the dye conjugated to the antihuman 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-Plus
Principle
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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-Plus
Procedure
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Step 1
Pipette 10 uL of serum or plasma in the upper
well.
Mono-Plus
Procedure
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Step 2
Add 2-3 drops of Developer
Solution to the lower end of the
sample well.
Mono-Plus
Procedure
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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.
Mono-Plus
Interpretation of Results
Positive Result
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A pink-purple horizontal bar in
the Test Window (T) and the
Control (C).
Negative Result
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A pink-purple horizontal bar in
the Control Window (C).
No horizontal bar in the Test
Window (T).
Mono-Plus
Interpretation of Results
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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-Plus
False Positives
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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-Plus
False-Negatives
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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
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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
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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.