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Infectious mononucleosis
Clinical Microbiology Newsletter February 1, 1984
Vol. 6, No. 3
Infectious Mononucleosis Laurence R. McCarthy,Ph.D. Hynson, Westcott & Dunning
Division of Becton Dickinson & Company Balthnore, Maryland 21201 Infectious mononucleosis is an acute infection characterized by posterior cervical lymphadenopathy, pharyngitis, splenomegaly, and the presence of abnormal lymphocytes in the peripheral blood. The disease primarily affects those under 22 years of age. Emil Pfeiffer described the disease as drusenfieber, glandular fever, and is generally credited as being the first to describe infectious mononucleosis (4, 14). Pfeiffer's original article identified two forms of the disease: a shortduration febrile illness with assbciated lymphadenopathy, and a second, more severe and prolonged illness characterized by fever, pharyngitis, lymphadenopathy, splenomegaly, and fatigue: In 1920 Sprunt and Evans associated the disease with the presence of abnormal lymphocytes and were the first to describe the disease as infectious mononucleosis (5, 20). The symptoms of infectious mononucleosis are varied. The prodromal phase typically lasts four to six days and is followed by the acute clinical disease with associated fever, which may last from four to six days to two or three weeks. Approximately 50% of patients with the disease may exhibit splenomegaly. Care during the physical exam is indicated as palpation of the spleen may lead to splenic rupture. (The spleen is delicate and
fragile during the disease.) A variety of symptoms may be observed. Table 1 lists several symptoms associated with the disease and their approximate frequency (15). Lymphadenopathy may not be limited to cervical lymph nodes and may either be more generalized or localized in other anatomic sites. Occasionally patients may display a mild skin rash or one that is extensive and maculopapular in nature. Approximately 80% of patients present with pharyngitis and/or lymphadenopathy whereas the remaining 20% display fever without either pharyngitis or lymphadenopathy (19). Those with pharyngitis often present with exudative pharyngitis, therefore the diagnosis of streptococcal pharyngitis must be excluded. Younger patients typically have a mild and short-duration illness, whereas older patients display more prolonged illness.
when a technician, working in the Henle's laboratory, developed the disease. The technician, who lacked EBV antibodies before developing infectious mononucleosis, displayed a rise in anti-EBV antibody. White blood cells obtained from the patient grew in continuous culture and displayed EBV associated antigens. Subsequent large-scale testing documented the association of infectious mononucleosis with EBV. Patients who developed the disease displayed increases in anti-EB.V antibody, and EBV was cultured from lymphocytes obtained from several such patients (4). In addition to infectious mononucleosis caused by EBV, some patients, have similar disease symptoms without evidence of EBV infection. These "monolike" diseases are caused by a variety of etiologic agents, which include cytomegalovirus, To_roplasma gondii, and rubella (5, 10). It is be-
A g e n t s o f Disease
Infectious mononucleosis is caused by the Epstein-Barr virus (EBV). A causal role for this virus was established in 1968 by Werner and Gertrude Henle, together with Wemer Diehl (8). In 1958, Burkitt described a tumor that occurred in African childlZen now termed Burkitt's lymphoma. In 1964, Epstein and Barr reported the observation of herpes-like viral particles in cultured Burkitt tumor cells. In 1966, the Henles described an indirect immunofluorescence test antibody to EBV. Their discovery of EBV as the agent of infectious mononucleosis occurred
In This Issue
Infectious Mononucleosis . . . . . . . .
17
Diagnostic tests for Epsteh~-Barr virus Latex Agglutination Testing . . . . . 20
The hnportance of environmental effects on results Letters to the Editors . . . . . . . . . . . . NCCLS News . . . . . . . . . . . . . . . . . . . . .
21
23
0196-4399/84/S0.u0 + 02.00
Table 1 Symptoms Observed in Patients with Infectious Mononucleosis (15) Symptoms
Percent of Patients
Fatigue and malaise Pharyngitis Anorexia Headache Chills Cough Myalgia Chest pain Arthralgic Photophobie
90-100 80-85 50-80 40-70 40-60 30-50 12-30 5-20 5 - I0 5-10
munosuppressed patients after they have received blood transfusions (1, 2, 16).
Diagnostic Tests Whereas disease diagnosis may be accomplished by the in vitro cultivation of EBV from patients' mononuclear leukocytes during the acute phase of disease (17), the most widely used tests are antibody detection tests.
Heterophile Antibodies
lieved that these agents account for less than 2% of cases ~dlagnosed as infectious mononucleosis. Although it is recognized that these other agents may cause a disease similar to that caused by EBV, this review deals exclusively with diagnosis of EBV disease. Epidemiology The incidence of infectious mononucleosis in the United States has been found to approximate 50 cases per 100,000 population. The disease is observed to occur most frequently in older children and young adults. Highest incidence of disease has been reported to occur in patients 15 to 24 years of age and highest frequency has been reported to occur in college-age students and in military recruits. Typical spread is via the upper respiratory tract secretions, wherein the virus is shed intermittently or continuously during the course of the clinical disease and up to three months after acute illness (12). Occasionally, the disease has also been observed to occur in im-
Serologic diagnosis of infectious mononucleosis was first accomplished by Paul and Bunnell in 1932 (13). Their finding that heterophile antibody titers were of value in the diagnosis of infectious mononucleosis followed an initial report that heterophile anti-sheep red blood cell antibodies were associated with serum sickness. While determining if heterophile antibodies were associated with rheumatic fever, Dr. Paul serendipitously discovered the presence of an elevated heterophile antibody titer in a medical student who did not have rheumatic fever but rather had infectious mononucleosis (4). Subsequent studies on an expanded number of patients revealed the association of heterophile antibodies with infectious mononucleosis. Subsequently, the now-recognized Davidsohn differential test was developed by Davidsohn in 1937 (3). In the Davidsohn test, serum specimens that display heterophile antibodies are individually treated with beef erythrocytes and guinea pig kidney cells. Heterophile antibodies may be observed in a variety of diseases, and are not specific to infectious mononucleosis. They may be naturally occurring
Table 2 Davidsohn Differential Test: Differentiation of Heterophile Antibodies Resulting from Infectious Mononucleosis, Serum Sickness, and Normal Forssman Antibodies
18
Disease
Observation after Absorption with Guinea Pig Kidney
Beef RBC
Infectious mononucleosis Serum sickness
Antibody remains Antibody absorbed
Antibody absorbed Antibody absorbed
No disease Forssman antibodies
Antibody absorbed
Antibody remains
0196-4399,84/$0.00+ 02.00
Forssman antibodies, be formed as a part of serum sickness, or be formed during infectious mononucleosis. The Davidsohn differential test results shown in Table 2 clearly differentiate between the three possible sources of heterophile antibody. Additional tests for infectious mononucleosis have included testing for ox cell hemolysins (4) and a variety of commercial kits that permit the rapid diagnosis of the disease by mixing drops of serum on a slide with either sheep or horse red blood cells (with or without guinea pig and beef cell absorption). Another slide test relies upon the finding that papain-treated sheep cells loose binding sites to which antibodies from infectious mononucleosis patients bind. Papaintreated cells, however, will be agglutinated normal Forssman antibody. Agglutination of untreated cells and failure to agglutinate papain-treated cells allow diagnosis of the disease. Patients with Forssman antibody will agglutinate both types of cells. Because of the different principles employed in these rapid slide tests, it is imperative that the manufacturer's instructions be followed with care. E p s t e i n - B a r r Virus
Specific Antibodies Not all patients with infectious mononucleosis develop heterophile antibodies. At least 10% of adolescents and adults with the disease fail to produce such antibodies (11, 15) as do a greater percentage of children with the disease (18). Thus, determination of viral-specific antibodies may offer an advantage in diagnosis. Several EBV antigen preparations have been employed (9). This discussion will be limited to indirect immunofluorescent tests that appear to have proven useful in diagnosis. Viral capsid antigens (VCA) are produced by tissue culture cell lines, which produce whole viruses after infection and thus are termed producer cell lines. Acetone-fixed preparations of these cells are used for anti-VCA testing. IgG and IgM-specific antiVCA tests are performed using this antigen (9). Early antigens (EA) have also been
ClinicalMicrobiologyNewsletter
employed for serodiagnosis. Early antigens are produced by infecting culture cells (e.g., Raji cells), which do not normally support the replication of the virus. Only early antigens are produced rather than progressing to the development of VCA and complete virus particles. Acetone fixed cells are used as antigen. Two patterns of fluorescence have been observed with EA. One pattern is the diffuse pattern where diffuse staining of the nucleus and cytoplasm is observed. The second pattern is the restricted pattern where staining is limited to cytoplasmic aggregates of antigen. The third EBV test involves the detection of anti-EBV antibody that is directed against nuclear antigen (EBNA). The antigen is prepared from infected nonproducing cell lines, but is also present in infected producer cell lines. The test is not the typical indirect immunofluorescent test, rather it is an anti-complement immunofluorescent test. In this test, the patient's serum is first mixed with human complement and the resulting mixture is'used to cover a cell preparation containing EBNA. The presence of complement enhances subsequent staining. After incubation and washing, the slide is then stained with anti-131C globulins that are conjugated to fluorescein (9). Anti-EBNA antibody only reacts with cells that contain EBV genome. The value of each of these EBV tests varies somewhat between authors (7, 9, 21). Each test is listed in Table 3 together with its value in the diagnosis of the disease. Though EBV serologic testing is valuable, it is more expensive than heterophile tests. There are reports that recommend that EBV serology be reserved for diagnosis of the disease in patients who fail to display heterophile antibodies (7). IgG anti-VCA antibodies are not diagnostic by themselves unless a fourfold increase in antibody is observed. Because lgG anti-VCA antibody remains elevated long after infection, a single elevated titer may represent past infection instead of current infection. The significance of IgG-VCA antibodies may be assessed by testing for anti-EBNA antibodies. EBNA anti-
© 1984by ElsevierSciencePublishingCo.. Inc.
Table 3
EBV Specific Tests for the Diagnosis of Infectious Mononucleosis EBV Test
Comment
IgM anti-VCA
Antibody formed early in course of disease, disappears in convalescent. Positive test indicates infection.
IgG anti-VCA
Antibody formed during acute illness; antibody remains elevated for prolonged duration. Fourfold increase in titer diagnostic. Formation of IgG may be delayed, and not present in early stage of acute infection.
Anti-EA
Antibodies showing diffuse pattern appear in 70-80% of patients during acute disease. Rarely observed in patients without EBV disease. Restricted pattern not usually seen in infectious mononucleosis.
Anti-EBNA
Antibody formed during convalescence. Presence of antibody indicates past disease.
Virus culture throat
Positive during acute disease; intermittent/continuous shedding up to three months after disease,
Leukocyte culture
Positive during acute disease. Cultures may be positive during convalescence.
bodies are observed only during convalescence and persist for some time. Thus, elevated IgG-VCA antibodies together with anti-EBNA antibodies suggest past infection, whereas the presence of IgG-VCA in the absence of anti-EBNA antibodies suggests active disease. Anti-EA antibodies, especially those which yield the diffuse pattern of staining, are typically seen during acute infection. Seventy to 80% of patients with the disease demonstrate anti-EA antibodies (9, 21). The IgManti-VCA test is specific for diagnosis, although IgM antibodies may not be synthesized by all infected patients (6). In a recent evaluation of IgG-VCA, IgM-VCA, EA, EBNA, and heterophile antibody tests, Fleisher and coworkers (7) reported that the best combination of tests for diagnosis of the disease was to test all patients with a heterophile antibody slide test and test only those sera that were slide test negative with EBV-specific antibody tests. They recommend that if the initial IgG-VCA test fails to reveal antiEBV antibodies then sera should be tested for IgM VCA antibodies. If both tests are negative, a second serum obtained two weeks later should be ex-
amined again for IgG and IgG antibody. The diagnosis of infectious mononucleosis remains complex with no single test being entirely appropriate for adequate and cost-effective diagnosis. Laboratorians charged with offering tests to diagnose infectious mononucleosis need to be aware of the strengths and weaknesses of each. References
I. Blacklow, N. R. el al. 1971. Mononucleosis with heterophil antibodies and EB virus infection: Acquisition by an elderly patient in hospital. Am. J. Med. 51:549-552. 2. Cheeseman, S. H. et al. 1980. Epstein-Burr virus infection in renal transplant recipients. Ann. Intern. Med. 93:39-42. 3. Davidsohn, I. 1937. Serologic diagnosis of infectious mononucleosis. J. Am. Med. Assoc. 108:287-294. 4. Evans, A. S. 1974. The history of infectious mononucleosis. Am. J. Med. Sci; 267:189-195. 5. Evans, A. S. 1972.'infectious mononucleosis and other mono-like syndromes. N. Engl. J. Med. 286:836838. 6. Evans, A. S. el al. 1975. A prospective evaluation of heterophile and Epstein-Burr virus-specific IgM antibody tests in clinical and subclinical infec-
01964399;84/$0.00 + 02.00
19
11. Middleton, P. J. 1974. Epstein-Barr virus infections. Can. Med. Assoc. J. 110:9-10. 12. Niederman, J. C. et al. 1976. Infectious mononucleosis: Epstein-Barr virus shedding in saliva and the oropharynx. New Engl. J. Med. 294:1355-1359. 13. Paul, J. R., and W. W. Bunnell. 1932. The presence of heterophile antibodies in infectious mononucleosis. Am. J. Med. Sci. 183:91-104. 14. Pfeiffer, E. 1889. Drusenfieber. Jahrb f Kinderheilk. 29:257-264. 15. Rapp, C. E., and J. F. Hewetson. 1978. Infectious mononucleosis and the Epstein-Bart" virus. Am. J. Dis. Child. 132:78-86. 16. Ritchey, A. K. et al. 1980. Mononucleosis syndrome following granulocyte transfusion in patients with leukemia. J. Ped. 97:267-269.
17. Rocchi, G. et al. 1977. Quantitative evaluation of Epstein-Barr virus infected mononuclear peripheral blood leukocytes in infectious mononucleosis. New Eng. J. Med. 296:132134. 18. Shapiro, S. A., and J. M. Gerson. 1978. The values of Epstein-Barr virus titers as a diagnostic shortcut in infectious mononucleosis: two case reports. Clin. Ped. 17:797-798. 19. Shurin, S. 1979. Infectious mononucleosis. Ped. Clin. N. Amer. 26:315326. 20. Sprunt, T. P., and F. A. Evans. 1920. Mononucleosis leukocytosis in reaction to acute infections (infectious mononucleosis). Johns Hopkins Hosp. Bull. 31:409-417. 21. Sumaya, C. V. 1977. Primary Epstein-Barr virus infections in children. Pediatrics. 59:16-20.
Latex Agglutination Testing: The Importance of the Microenvironment
esting and important phenomena which should be taken into account by the microbiologist when comparing direct late~ agglutination tests to each other and to other testing methods.
Kenneth D. Noonan, Ph.D.
A s s a y Sensitivity The sensitivity of a latex-based test is determined by the avidity of the antibody used, the amount of antibody conjugated to the solid phase (latex), the length of time the patient specimen and the latex reagent are in contact, and the microenvironment in which the latex reagent is placed. The latter determinant of sensitivity is probably the most interesting and least understood of the four factors relating to reagent sensitivity. Furthermore, environmental effects on latex reagent sensitivity are even more important when monoclonal antibodies (which recognize a very specific epitope of an antigen) are used in immunodiagnostics. All immunodiagnostics are developed for use with specific body fluids. In the case of meningitis testing, the specimens of interest are predominantly cerebrospina'l fluid (CSF), urine, and serum. Each of these specimens has its own microenvironment. The challenge to the manufacturer is to develop reagents and methods that will
optimize their sensitivity, specificity, and stability in the appropriate patient specimens. Sometimes certain types of specimens must be processed in a specific manner to insure that pH, salt and/or protein concentrations are optimized. The reason that the microenvironment must be so closely controlled relates to the fact that adverse alteration in pH, salt, and/or protein concentration changes either the three-dimensional structure of the latex-bound antibody or the antigen to be detected such that the ability of the diagnostic test is severely compromised. Latex reagents employing monoclonal antibodies are even more likely to respond to changes in the microenvironment than are reagents based on polyspecific antisera because all of the.molecules on a monoclonal antibody-coated latex reagent are identical. Any change in the microenvironment which affects one antibody molecule will affect all antibody molecules identically. Under inappropriate environmental conditions, a very effective diagnostic monoclonal antibody-based latex reagent can be changed into a less sensitive or less stable reagent tlian the one claimed by the manufacturer. All manufacturers develop test kits which operate most effectively with
7.
8.
9.
10.
tious mononucleosis: specificity and sensitivity of the tests and persistence of antibody. J. Inf. Dis. 132:546-554. Fleisher, G. R., M. Collins, and S. Fager. 1983. Limitations of available tests for diagnosis of infectious mononucleosis. J. Clin. Microbiol. 17:619624. Henle, G., W. llenle, and V. Diehl. 1968. Relation of Burkitt's tumor-associated herpes-type virus to infectious mononucleosis. Proc. Nat. Acad. Sei. 59:94-101. ltenle, W., G. E. Henle, and C. A. Horwitz. 1974. Epstein-Barr virus specific diagnostic tests in infectious mononucleosis. Hum. Path. 5:551565. llorwitz, C. A. et al. 1977. Heterophil-negative infectious mononucleosis and mononucleosis-like illnesses. Am. J. Med. 63:947-957'."
Editorial
Director of Research & Development Hyt,son, IVestcott & Dunning Division of Becton Dickhzson & Company Balthnore, Maryland 21201 A number of new immunodiagnostic assays for the direct detection of antigens in patient specimens have been introduced into the clinical microbiology laboratory over the last two years. This has been particularly true in the area of meningitis testing, where at least three manufacturers have introduced latex agglutination tests for detecting antigens from Hemophilus in-
fluenzae B, Streptococus pneumoniae, Neisseria meningitidis A, B, C, Y, and group B streptococcus. All of these tests have been developed with the intention of maximizing the sensitivity, specificity, and stability of the reagents so that the clinical microbiologist has an effective alternative to counterimmunoelectrophoresis (CIE). In developing these assay systems, we have encountered a number of inter1
20
0196-4399/84/$0.00 + 02.00
Clinical Microbiology Newsletter
Vol. 6, No. 3
Infectious Mononucleosis Laurence R. McCarthy,Ph.D. Hynson, Westcott & Dunning
Division of Becton Dickinson & Company Balthnore, Maryland 21201 Infectious mononucleosis is an acute infection characterized by posterior cervical lymphadenopathy, pharyngitis, splenomegaly, and the presence of abnormal lymphocytes in the peripheral blood. The disease primarily affects those under 22 years of age. Emil Pfeiffer described the disease as drusenfieber, glandular fever, and is generally credited as being the first to describe infectious mononucleosis (4, 14). Pfeiffer's original article identified two forms of the disease: a shortduration febrile illness with assbciated lymphadenopathy, and a second, more severe and prolonged illness characterized by fever, pharyngitis, lymphadenopathy, splenomegaly, and fatigue: In 1920 Sprunt and Evans associated the disease with the presence of abnormal lymphocytes and were the first to describe the disease as infectious mononucleosis (5, 20). The symptoms of infectious mononucleosis are varied. The prodromal phase typically lasts four to six days and is followed by the acute clinical disease with associated fever, which may last from four to six days to two or three weeks. Approximately 50% of patients with the disease may exhibit splenomegaly. Care during the physical exam is indicated as palpation of the spleen may lead to splenic rupture. (The spleen is delicate and
fragile during the disease.) A variety of symptoms may be observed. Table 1 lists several symptoms associated with the disease and their approximate frequency (15). Lymphadenopathy may not be limited to cervical lymph nodes and may either be more generalized or localized in other anatomic sites. Occasionally patients may display a mild skin rash or one that is extensive and maculopapular in nature. Approximately 80% of patients present with pharyngitis and/or lymphadenopathy whereas the remaining 20% display fever without either pharyngitis or lymphadenopathy (19). Those with pharyngitis often present with exudative pharyngitis, therefore the diagnosis of streptococcal pharyngitis must be excluded. Younger patients typically have a mild and short-duration illness, whereas older patients display more prolonged illness.
when a technician, working in the Henle's laboratory, developed the disease. The technician, who lacked EBV antibodies before developing infectious mononucleosis, displayed a rise in anti-EBV antibody. White blood cells obtained from the patient grew in continuous culture and displayed EBV associated antigens. Subsequent large-scale testing documented the association of infectious mononucleosis with EBV. Patients who developed the disease displayed increases in anti-EB.V antibody, and EBV was cultured from lymphocytes obtained from several such patients (4). In addition to infectious mononucleosis caused by EBV, some patients, have similar disease symptoms without evidence of EBV infection. These "monolike" diseases are caused by a variety of etiologic agents, which include cytomegalovirus, To_roplasma gondii, and rubella (5, 10). It is be-
A g e n t s o f Disease
Infectious mononucleosis is caused by the Epstein-Barr virus (EBV). A causal role for this virus was established in 1968 by Werner and Gertrude Henle, together with Wemer Diehl (8). In 1958, Burkitt described a tumor that occurred in African childlZen now termed Burkitt's lymphoma. In 1964, Epstein and Barr reported the observation of herpes-like viral particles in cultured Burkitt tumor cells. In 1966, the Henles described an indirect immunofluorescence test antibody to EBV. Their discovery of EBV as the agent of infectious mononucleosis occurred
In This Issue
Infectious Mononucleosis . . . . . . . .
17
Diagnostic tests for Epsteh~-Barr virus Latex Agglutination Testing . . . . . 20
The hnportance of environmental effects on results Letters to the Editors . . . . . . . . . . . . NCCLS News . . . . . . . . . . . . . . . . . . . . .
21
23
0196-4399/84/S0.u0 + 02.00
Table 1 Symptoms Observed in Patients with Infectious Mononucleosis (15) Symptoms
Percent of Patients
Fatigue and malaise Pharyngitis Anorexia Headache Chills Cough Myalgia Chest pain Arthralgic Photophobie
90-100 80-85 50-80 40-70 40-60 30-50 12-30 5-20 5 - I0 5-10
munosuppressed patients after they have received blood transfusions (1, 2, 16).
Diagnostic Tests Whereas disease diagnosis may be accomplished by the in vitro cultivation of EBV from patients' mononuclear leukocytes during the acute phase of disease (17), the most widely used tests are antibody detection tests.
Heterophile Antibodies
lieved that these agents account for less than 2% of cases ~dlagnosed as infectious mononucleosis. Although it is recognized that these other agents may cause a disease similar to that caused by EBV, this review deals exclusively with diagnosis of EBV disease. Epidemiology The incidence of infectious mononucleosis in the United States has been found to approximate 50 cases per 100,000 population. The disease is observed to occur most frequently in older children and young adults. Highest incidence of disease has been reported to occur in patients 15 to 24 years of age and highest frequency has been reported to occur in college-age students and in military recruits. Typical spread is via the upper respiratory tract secretions, wherein the virus is shed intermittently or continuously during the course of the clinical disease and up to three months after acute illness (12). Occasionally, the disease has also been observed to occur in im-
Serologic diagnosis of infectious mononucleosis was first accomplished by Paul and Bunnell in 1932 (13). Their finding that heterophile antibody titers were of value in the diagnosis of infectious mononucleosis followed an initial report that heterophile anti-sheep red blood cell antibodies were associated with serum sickness. While determining if heterophile antibodies were associated with rheumatic fever, Dr. Paul serendipitously discovered the presence of an elevated heterophile antibody titer in a medical student who did not have rheumatic fever but rather had infectious mononucleosis (4). Subsequent studies on an expanded number of patients revealed the association of heterophile antibodies with infectious mononucleosis. Subsequently, the now-recognized Davidsohn differential test was developed by Davidsohn in 1937 (3). In the Davidsohn test, serum specimens that display heterophile antibodies are individually treated with beef erythrocytes and guinea pig kidney cells. Heterophile antibodies may be observed in a variety of diseases, and are not specific to infectious mononucleosis. They may be naturally occurring
Table 2 Davidsohn Differential Test: Differentiation of Heterophile Antibodies Resulting from Infectious Mononucleosis, Serum Sickness, and Normal Forssman Antibodies
18
Disease
Observation after Absorption with Guinea Pig Kidney
Beef RBC
Infectious mononucleosis Serum sickness
Antibody remains Antibody absorbed
Antibody absorbed Antibody absorbed
No disease Forssman antibodies
Antibody absorbed
Antibody remains
0196-4399,84/$0.00+ 02.00
Forssman antibodies, be formed as a part of serum sickness, or be formed during infectious mononucleosis. The Davidsohn differential test results shown in Table 2 clearly differentiate between the three possible sources of heterophile antibody. Additional tests for infectious mononucleosis have included testing for ox cell hemolysins (4) and a variety of commercial kits that permit the rapid diagnosis of the disease by mixing drops of serum on a slide with either sheep or horse red blood cells (with or without guinea pig and beef cell absorption). Another slide test relies upon the finding that papain-treated sheep cells loose binding sites to which antibodies from infectious mononucleosis patients bind. Papaintreated cells, however, will be agglutinated normal Forssman antibody. Agglutination of untreated cells and failure to agglutinate papain-treated cells allow diagnosis of the disease. Patients with Forssman antibody will agglutinate both types of cells. Because of the different principles employed in these rapid slide tests, it is imperative that the manufacturer's instructions be followed with care. E p s t e i n - B a r r Virus
Specific Antibodies Not all patients with infectious mononucleosis develop heterophile antibodies. At least 10% of adolescents and adults with the disease fail to produce such antibodies (11, 15) as do a greater percentage of children with the disease (18). Thus, determination of viral-specific antibodies may offer an advantage in diagnosis. Several EBV antigen preparations have been employed (9). This discussion will be limited to indirect immunofluorescent tests that appear to have proven useful in diagnosis. Viral capsid antigens (VCA) are produced by tissue culture cell lines, which produce whole viruses after infection and thus are termed producer cell lines. Acetone-fixed preparations of these cells are used for anti-VCA testing. IgG and IgM-specific antiVCA tests are performed using this antigen (9). Early antigens (EA) have also been
ClinicalMicrobiologyNewsletter
employed for serodiagnosis. Early antigens are produced by infecting culture cells (e.g., Raji cells), which do not normally support the replication of the virus. Only early antigens are produced rather than progressing to the development of VCA and complete virus particles. Acetone fixed cells are used as antigen. Two patterns of fluorescence have been observed with EA. One pattern is the diffuse pattern where diffuse staining of the nucleus and cytoplasm is observed. The second pattern is the restricted pattern where staining is limited to cytoplasmic aggregates of antigen. The third EBV test involves the detection of anti-EBV antibody that is directed against nuclear antigen (EBNA). The antigen is prepared from infected nonproducing cell lines, but is also present in infected producer cell lines. The test is not the typical indirect immunofluorescent test, rather it is an anti-complement immunofluorescent test. In this test, the patient's serum is first mixed with human complement and the resulting mixture is'used to cover a cell preparation containing EBNA. The presence of complement enhances subsequent staining. After incubation and washing, the slide is then stained with anti-131C globulins that are conjugated to fluorescein (9). Anti-EBNA antibody only reacts with cells that contain EBV genome. The value of each of these EBV tests varies somewhat between authors (7, 9, 21). Each test is listed in Table 3 together with its value in the diagnosis of the disease. Though EBV serologic testing is valuable, it is more expensive than heterophile tests. There are reports that recommend that EBV serology be reserved for diagnosis of the disease in patients who fail to display heterophile antibodies (7). IgG anti-VCA antibodies are not diagnostic by themselves unless a fourfold increase in antibody is observed. Because lgG anti-VCA antibody remains elevated long after infection, a single elevated titer may represent past infection instead of current infection. The significance of IgG-VCA antibodies may be assessed by testing for anti-EBNA antibodies. EBNA anti-
© 1984by ElsevierSciencePublishingCo.. Inc.
Table 3
EBV Specific Tests for the Diagnosis of Infectious Mononucleosis EBV Test
Comment
IgM anti-VCA
Antibody formed early in course of disease, disappears in convalescent. Positive test indicates infection.
IgG anti-VCA
Antibody formed during acute illness; antibody remains elevated for prolonged duration. Fourfold increase in titer diagnostic. Formation of IgG may be delayed, and not present in early stage of acute infection.
Anti-EA
Antibodies showing diffuse pattern appear in 70-80% of patients during acute disease. Rarely observed in patients without EBV disease. Restricted pattern not usually seen in infectious mononucleosis.
Anti-EBNA
Antibody formed during convalescence. Presence of antibody indicates past disease.
Virus culture throat
Positive during acute disease; intermittent/continuous shedding up to three months after disease,
Leukocyte culture
Positive during acute disease. Cultures may be positive during convalescence.
bodies are observed only during convalescence and persist for some time. Thus, elevated IgG-VCA antibodies together with anti-EBNA antibodies suggest past infection, whereas the presence of IgG-VCA in the absence of anti-EBNA antibodies suggests active disease. Anti-EA antibodies, especially those which yield the diffuse pattern of staining, are typically seen during acute infection. Seventy to 80% of patients with the disease demonstrate anti-EA antibodies (9, 21). The IgManti-VCA test is specific for diagnosis, although IgM antibodies may not be synthesized by all infected patients (6). In a recent evaluation of IgG-VCA, IgM-VCA, EA, EBNA, and heterophile antibody tests, Fleisher and coworkers (7) reported that the best combination of tests for diagnosis of the disease was to test all patients with a heterophile antibody slide test and test only those sera that were slide test negative with EBV-specific antibody tests. They recommend that if the initial IgG-VCA test fails to reveal antiEBV antibodies then sera should be tested for IgM VCA antibodies. If both tests are negative, a second serum obtained two weeks later should be ex-
amined again for IgG and IgG antibody. The diagnosis of infectious mononucleosis remains complex with no single test being entirely appropriate for adequate and cost-effective diagnosis. Laboratorians charged with offering tests to diagnose infectious mononucleosis need to be aware of the strengths and weaknesses of each. References
I. Blacklow, N. R. el al. 1971. Mononucleosis with heterophil antibodies and EB virus infection: Acquisition by an elderly patient in hospital. Am. J. Med. 51:549-552. 2. Cheeseman, S. H. et al. 1980. Epstein-Burr virus infection in renal transplant recipients. Ann. Intern. Med. 93:39-42. 3. Davidsohn, I. 1937. Serologic diagnosis of infectious mononucleosis. J. Am. Med. Assoc. 108:287-294. 4. Evans, A. S. 1974. The history of infectious mononucleosis. Am. J. Med. Sci; 267:189-195. 5. Evans, A. S. 1972.'infectious mononucleosis and other mono-like syndromes. N. Engl. J. Med. 286:836838. 6. Evans, A. S. el al. 1975. A prospective evaluation of heterophile and Epstein-Burr virus-specific IgM antibody tests in clinical and subclinical infec-
01964399;84/$0.00 + 02.00
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11. Middleton, P. J. 1974. Epstein-Barr virus infections. Can. Med. Assoc. J. 110:9-10. 12. Niederman, J. C. et al. 1976. Infectious mononucleosis: Epstein-Barr virus shedding in saliva and the oropharynx. New Engl. J. Med. 294:1355-1359. 13. Paul, J. R., and W. W. Bunnell. 1932. The presence of heterophile antibodies in infectious mononucleosis. Am. J. Med. Sci. 183:91-104. 14. Pfeiffer, E. 1889. Drusenfieber. Jahrb f Kinderheilk. 29:257-264. 15. Rapp, C. E., and J. F. Hewetson. 1978. Infectious mononucleosis and the Epstein-Bart" virus. Am. J. Dis. Child. 132:78-86. 16. Ritchey, A. K. et al. 1980. Mononucleosis syndrome following granulocyte transfusion in patients with leukemia. J. Ped. 97:267-269.
17. Rocchi, G. et al. 1977. Quantitative evaluation of Epstein-Barr virus infected mononuclear peripheral blood leukocytes in infectious mononucleosis. New Eng. J. Med. 296:132134. 18. Shapiro, S. A., and J. M. Gerson. 1978. The values of Epstein-Barr virus titers as a diagnostic shortcut in infectious mononucleosis: two case reports. Clin. Ped. 17:797-798. 19. Shurin, S. 1979. Infectious mononucleosis. Ped. Clin. N. Amer. 26:315326. 20. Sprunt, T. P., and F. A. Evans. 1920. Mononucleosis leukocytosis in reaction to acute infections (infectious mononucleosis). Johns Hopkins Hosp. Bull. 31:409-417. 21. Sumaya, C. V. 1977. Primary Epstein-Barr virus infections in children. Pediatrics. 59:16-20.
Latex Agglutination Testing: The Importance of the Microenvironment
esting and important phenomena which should be taken into account by the microbiologist when comparing direct late~ agglutination tests to each other and to other testing methods.
Kenneth D. Noonan, Ph.D.
A s s a y Sensitivity The sensitivity of a latex-based test is determined by the avidity of the antibody used, the amount of antibody conjugated to the solid phase (latex), the length of time the patient specimen and the latex reagent are in contact, and the microenvironment in which the latex reagent is placed. The latter determinant of sensitivity is probably the most interesting and least understood of the four factors relating to reagent sensitivity. Furthermore, environmental effects on latex reagent sensitivity are even more important when monoclonal antibodies (which recognize a very specific epitope of an antigen) are used in immunodiagnostics. All immunodiagnostics are developed for use with specific body fluids. In the case of meningitis testing, the specimens of interest are predominantly cerebrospina'l fluid (CSF), urine, and serum. Each of these specimens has its own microenvironment. The challenge to the manufacturer is to develop reagents and methods that will
optimize their sensitivity, specificity, and stability in the appropriate patient specimens. Sometimes certain types of specimens must be processed in a specific manner to insure that pH, salt and/or protein concentrations are optimized. The reason that the microenvironment must be so closely controlled relates to the fact that adverse alteration in pH, salt, and/or protein concentration changes either the three-dimensional structure of the latex-bound antibody or the antigen to be detected such that the ability of the diagnostic test is severely compromised. Latex reagents employing monoclonal antibodies are even more likely to respond to changes in the microenvironment than are reagents based on polyspecific antisera because all of the.molecules on a monoclonal antibody-coated latex reagent are identical. Any change in the microenvironment which affects one antibody molecule will affect all antibody molecules identically. Under inappropriate environmental conditions, a very effective diagnostic monoclonal antibody-based latex reagent can be changed into a less sensitive or less stable reagent tlian the one claimed by the manufacturer. All manufacturers develop test kits which operate most effectively with
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tious mononucleosis: specificity and sensitivity of the tests and persistence of antibody. J. Inf. Dis. 132:546-554. Fleisher, G. R., M. Collins, and S. Fager. 1983. Limitations of available tests for diagnosis of infectious mononucleosis. J. Clin. Microbiol. 17:619624. Henle, G., W. llenle, and V. Diehl. 1968. Relation of Burkitt's tumor-associated herpes-type virus to infectious mononucleosis. Proc. Nat. Acad. Sei. 59:94-101. ltenle, W., G. E. Henle, and C. A. Horwitz. 1974. Epstein-Barr virus specific diagnostic tests in infectious mononucleosis. Hum. Path. 5:551565. llorwitz, C. A. et al. 1977. Heterophil-negative infectious mononucleosis and mononucleosis-like illnesses. Am. J. Med. 63:947-957'."
Editorial
Director of Research & Development Hyt,son, IVestcott & Dunning Division of Becton Dickhzson & Company Balthnore, Maryland 21201 A number of new immunodiagnostic assays for the direct detection of antigens in patient specimens have been introduced into the clinical microbiology laboratory over the last two years. This has been particularly true in the area of meningitis testing, where at least three manufacturers have introduced latex agglutination tests for detecting antigens from Hemophilus in-
fluenzae B, Streptococus pneumoniae, Neisseria meningitidis A, B, C, Y, and group B streptococcus. All of these tests have been developed with the intention of maximizing the sensitivity, specificity, and stability of the reagents so that the clinical microbiologist has an effective alternative to counterimmunoelectrophoresis (CIE). In developing these assay systems, we have encountered a number of inter1
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Clinical Microbiology Newsletter