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Chronic mononucleosis: Pitfalls in the Laboratory Diagnosis
Current Topics Chronic Mononucleosis: P.iffalls in the Laboratory Diagnosis TOBY L. MERLIN, MD Since its discovery in 1964 in cultured cell lines of Burkitt's lymphoma, Epstein-Barr virus (EBV) has been implicated as a causal agent in endemic (African) Burkitt's lymphoma, nasopharyngeal carcinoma, and infectious mononucleosis. Its role in endemic Burkitt's lymphoma and nasopharyngeal carcinoma is complex; the virus probably serves as a cocarcinogen, acting in concert with chenfical carcinogens or immune-modulators. Epstein-Barr virus causes infectious mononucleosis. According to a long-standing notion, EBV might cause another relatively common nonneoplastic illness that resembles infectious mononucleosis with respect to symptoms but that, unlike infectious mononucleosis, has a prolonged clinical course of months to years. 1-7 The protracted illness is usually preceded by typical infectious mononucleosis, but the fatigue, headaches, myalgia, l y n t p h a d e n o p a t h y , and lowgrade fevers persist for months to years after the onset of illness. This protracted illness has been called chronic mononucleosis, chronic mononucleosis synd r o m e , relapsing infections mononucleosis, and chronic active Epstein-Barr viral infection. 1-7 For purposes of simplicity, it is referred to here as chronic mononucleosis. Until recently, only anecdotal collections or isolated case reports suggested the existence of chronic mononucleosis, with EBV as its cause. 1-4 Within the past year, however, a number of studies have used EBV-specific serologic tests in relatively large groups of patients in an attempt to provide evidence for the existence o f an EBV-caused chronic mononucleosis. 5-7 None of these studies has proved the existence of the illness, and none has proved the utility of the EBV-specific serologic tests in its diagnosis. Yet because chronic mononucleosis is characterized by chronic fatigue, a common complaint in our cuhure, these studies have provoked considerable lay and medical interest. They have also provoked the sometimes aggressive commercial marketing of EBV-specific serologic tests for the diagnosis of the purported disease. This article reviews EBV infections and EBVspi~cific serologic tests as they relate to chronic m o n o R e c e i v e d from the Department of Patholog)', University of New Mexico School of Medicine and Albuquerque Veterans Administration ttospital. Address correspondence and reprint requests to Dr. Merlin: Albuquerque Veterans Administration Hospital, 2100 Ridgecrest Drive SE, Albuquerque, NM 87108.
nucleosis, with particular emphasis on the pitfalls enc o u n t e r e d in attempts to diagnose the disease by EBV-specific serologic testing. OVERVIEW OF EBV INFECTIONS
Epstein-Barr viral infection, like death and taxes, is virtually inevitable among humans. The virus can first be acquired after the disappearance of protective congenitally acquired maternal antibodies, at the age of 2 to 7 months. All persons in developing nations and economically less advantaged persons in developed nations acquire EBV in childhood. 8 Most are infected by the age of 3 years, and virttmlly all are infected by adolescence. Among the econontically advantaged, however, the acquisition of EBV often does not occur until adolescence or early aduhhood. Approximately 50 per cent of persons entering college (in general, the more economically advantaged) are uninfectedP Prior to adolescence or young adulthood the acquisition of EBV usually goes unnoticed. Most acute infections in childhood are asymptomatic, or the symptoms are so mild and nonspecific that they cannot be distinguished from the frequent, mild, brief illnesses of childhood, l~ Acquisition of the virus after childhood, however, causes infectious mononucleosis in 30 to 45 per cent o f those infected. 12A3 T h e fact that the acquisition o f EBV causes infectious mononucleosis in adolescents and young aduhs but does not cause overt symptoms in children has not been explained. CHRONICITY OF EBV INFECTIONS
Infection with EBV is not only universal, but also invariably chronic. Epstein-Barr virns, a doublestranded DNA virus, integrates its genome into that of h u m a n B lymphocytes. During acute infectious mononucleosis, 0.001 to 0.01 per cent of circulating B lymphocytes are infected by the virus. In the ensuing 12 to 16 weeks, the number of infected circulating B lymphocytes diminishes to 0.00001 per cent, a level that apparently persists indefinitely.14 Epstein-Barr virus immortalizes B lymphocytes in vitro, allowing continuous replication and subculturing of the infected cells when they are provided with nutrients. Whether an in vivo equivalent of this immortalization process is responsible for the persis-
CHRONICMONONUCLEOSIS[Merlin] tence o f EBV-infected B lyinphocytes in vivo or whether the virus passes from infected to uninfected B lymphocytes in vivo is difficult to assess. A person who has acquired EBV sheds virus episodically in oropharyngeal secretions. Seventy to 90 per cent o f persons with infectious mononucleosis shed virus, and this shedding persists for eight to 24 or more weeks after the resolution of symptomatic disease. Once the initial shedding associated with the acute acquisition of the virus subsides, episodic resurgence of shedding, tmassociated with symptoms, occurs. Twenty to 30 per cent of EBV carriers shed virus at any one time. This episodic shedding of virus, albeit in miniscule quantities, is considered the reservoir for infection of the uninfected. The rate of oropharyngeal viral shedding among those not acutely infected is higher in the immunosuppressed (50 per cent) than among the immunocompetent (20 per cent).15 T h e rate is similarly higher among infected persons in third-world nations, possibly due to the immunosuppression caused by malnutrition or disease. 16 INFECTIOUS MONONUCLEOSIS
Classic infectious mononucleosis caused by EBV is characterized clinically by a triad consisting of 1) fatigue, fever, pharyngitis, lymphadenopathy, and, often, splenomegaly and mild hepatitis; 2) lympbocytosis, with a peripheral leukocyte count revealing more than 50 per cent lymphocytes, I0 to 20 per cent of which are atypical; and 3) a positive differential (Paul-Bunnell) heterophil antibody test or EBV-specific serologic results indicative of acute disease. 17 Most cases occur in persons 10 to 25 years of age. Not all cases are classic in presentation, and unusual forms of infectious mononucleosis are more frequent outside tile usual age range, l~ In approximately 10 per cent o f North Americans in whom infectious mononucleosis develops, tile differential (Paul-Bunnell) heterophil antibody test is not positive, is Most signs and symptoms of infectious mononucleosis resolve within one month. Tile usual pattern is disappearance of pharyngitis by 14 days; disappearance of fever by 21 days; and disappearance of fatigue, lymphadenopathy, and splenomegaly by 21 to 28 days. 13 T h e fatigue of infectious mononucleosis occasionally persists for months. Iz CHRONIC MONONUCLEOSIS: HISTORY
Reports of protracted or recurrent symptoms of infectious lnononucleosis first appeared at least 40 to 50 ")'ears ago, antedating currently accepted criteria for the diagnosis o f infectious mononucleosis and making comparisons of the earlier reports with current studies difficult. Sizable groups of patients with protracted symptoms after infectious mononucleosis were described in the 19,t0s and 1950s. Ill 1948 Issacs Is described the persistence of symptoms and
signs (e.g., tatigue, depression, myalgia, lymphocytosis, low serum glucose level, low blood pressure, low urinary specific gravity) in 53 of 206 patients after infectious mononucleosis. In 1950 KaufmanlV described the recurrence of symptoms and signs (e.g., fatigue, pharyngitis, lymphadenopathy, lymphocytosis with atypical lymphocytes) in 12 of 125 patients after infectious mononucleosis. In these relatively large series, the criteria used for the diagnosis of the initial and persistent illness were vague, poorly documented, and not consistent with more recently defined criteria. Interestingly, with the development of more rigid criteria for the diagnosis o f infectious mononucleosis, reports of such sizable groups of patients with chronic mononucleosis have disappeared from the medical literature. The most recent studies of protracted or recurrent symptoms after infectious mononucleosis involved batteries of EBV-specific serologic tests. 5-7 Batteries of multiple serologic tests for EBV infections are complex, and their use, until recently, was confined to research studies. The various serologic methods were extensively reviewed elsewhere. 9,2~ A brief discussion o f the serologic tests is presented here because these tests are crucial to recent attempts to demonstrate chronic mononucleosis.
EPSTEIN-BARRVIRUS SERODIAGNOSIS
Differential [PauI-Bunnell] Heterophil Antibody Test Tile differential (Paul-Bunnell) heterophil antibody test is the most familiar, most readily available serologic test for the diagnosis of EBV infection. A significant tiler o f this heterophil antibody (i.e., a positive test) is present at some point during symptomatic infectious mononucleosis in approximately 90 per cent (range, 81 to 97 per cent, depending on the test method) of non-Orientals with tile illness. 22 The heterophil antibody is not usually produced (i.e., the test is negative) with the asymptomatic acquisition of EBV by persons outside the age range usually associated with infectious mononucleosis. Thns, the acquisition of EBV that is not associated with infectious mononucleosis cannot usually be detected by a test for the Paul-Bunnell heterophii antibody. 2~ The specificity of the Paul-Bunnell heterophil antibody test ranges from 93 to 98 per cent. z2 The Paul-Bunnell heterophil antibgdy test is not an EBV-specific serologic test. The Paul-Bunnell heterophil antibody does not react with an antigen of EBV per se. In humans, it probably reacts with neoantigen(s) induced or augmented on some cells, possibly B lymphocytes, by EBV infection. These induced neoantigens are fortuitously similar or identical to common antigens on horse, sheep, or bovine erythrocytes. It is, thus, a "heterophil" antibody because it is formed in one species (i.e., humans) and cross reacts with antigens present in another species
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phoid cells in culture tile early antigens appear in detectable quantities b e f o r e viral n u c l e o c a p s i d - giving rise to the name early antigen. The early antigens are probably viral enzymes. Anti-EA is usually produced acutely with the acquisition of the virus, beginning concomitant with or shortly after the prodnction of anti-VCA. It usually persists for only eight to 12 weeks, similar to IgM anti-VCA.20, 21 Although anti-EA is usually associated with the recent acquisition of EBV, the association is far from absolute. 25 It has been reported that 4 to 20 per cent of persons with past, but not recent, EBV infections are positive for anti-EA. 24,~5After the acute acqnisition of EBV, the anti-EA diminishes or disappears in most persons but then reappears with age. 25 A number of conditions have been identified in which anti-EA is elevated, sometimes to very high titers, without recent EBV acquisition. These conditions include pregnancy, immunodeficiency states, H o d g k i n ' s disease, n o n - H o d g k i n s ' s l y m p h o m a , Burkitt's l y m p h o m a , n a s o p h a r y n g e a l carcinoma, leukemia, AIDS, and the AIDS-related complex.9.20.21,27,31-33 The demonstration of anti-EA is accomplished by immunofluorescence, with nonproducer EBV-infected cell lines that have been superinfected with EBV used as substrates. Patterns of i m m u n o f l u o rescence staining of the substrate cells have been reported as diffuse, or EA-D, and restricted, or EAR.20,21 Anti-EA-D is a pattern of nuclear and cytoplasmic staining, while the anti-EA-R pattern is localized cytoplasmic, perinuclear staining. Childhood EBV acquisition is usually associated with anti-EAR. 21 Adolescent and adult acquisition of EBV is usually associated with anti-EA-D. Antibody to EBV nuclear antigen. In virtually all immunocompetent persons antibody to EBV nuclear antigen (anti-EBNA) develops, usually two to three months and, rarely one month after viral acquisition. 2L25 T h e presence of anti-EBNA indicates that EBV acquisition is not recent, but o c c u r r e d one month to years in the past. Like IgG anti-VCA, the anti-EBNA persists indefinitely once acquired. Nuclear antigen is a polypeptide or group of polypeptides encoded by the EBV DNA. 34 It is expressed in all cells infected with EBV but is present in such small quantities in EBV-infected cells that it cannot be demonstrated by routine indirect immunofluorescence. An amplification technique, anticomplement indirect immunofluorescence, is necessary to allow demonstration of anti-EBNA. Fixed smears of nonproductively EBV-infected B-cell lymphoblastoid cell lines are used as substrates. Failure to mount the usual anti-EBNA response is seen in some immtmodeficiency states, eg., the Xlinked lymphoproliferative syndrome. 35 In addition, unusually low titers o f anti-EBNA, in the presence of high titers of IgG anti-VCA, have been reported in patients with rheumatoid arthritis, 29 and high titers
(i.e., horse, sheep, or cow). Suspensions of horse, sheep or bovine erythrocytes are used to absorb or test for the presence of the antibody. The Paul-Bunnell antibody is usually an IgM antibody. It is said to persist only eight to 12 weeks in infectious mononucleosis, but this finding is highly de per~dent on the test system employed. Horse erythrocytes detect the heterophil antibody much longer tlmn either sheep or bovine erythrocytes, and the heterophil antibody will thus appear to persist longer x~hen tested by this method. 2'~
EBV-specific Serologic Tests Serologic tests specific for EBV detect antibodies to antigens that are apparently encoded by the EBV genome. The usual method for demonstrating antibodies to these antigens is indirect immunofluorescence, although other methods (i.e., enzyme immunoassays) have been explored. Antibody to EBV viral capsid antigen. All EBV infections in immunocompetent persons result in the production of antibody to EBV viral nucleocapsid antigen (anti-VCA). 21 Immunofluorescence test systems for anti-VCA use fixed smears of B-cell lymphoblastold cell lines productively infected (i.e., producing virus) with EBV as a viral capsid antigen-containing substrate. 21 IgM, IgA, and IgG anti-VCAs are distinguished by c o m m o n methods of immunoglobulin fractionation and heavy-chain-specific fluoresceinated conjugates. Serum levels o f anti-VCA peak at three to four weeks following EBV infection. IgM anti-VCA levels decline rapidly, and IgM anti-VCA is usually undetectable at 12 weeks. 23 IgG anti-VCA levels decline somewhat after peaking but persist indefinitely. The presence of IgG anti-VCA is the most sensitive indicator available for EBV infection, recent or remote, because of this indefinite persistence3 l High levels of IgG anti-VCA are seen in acute infections but are not specific for acute infections3 4,25 Similarly high levels are seen in remote EBV infection and in a variety o f other conditions: primary and secondary immunodeficiency states, Hodgkin's disease, leukemia, sarcoidosis, l y m p h o m a , nonlymphomatous malignancies, systemic lupus erythematosus, chronic renal diseases, endemic Burkitt's lymphoma, nasopharyngeal carcinoma, rheumatoid arthritis, the acquired immunodeficiency syndrome (AIDS), the AIDS-related complex, and multiple sclerosis.9,21,26-29 Although t h e r e is some d i s a g r e e m e n t as to whether IgM anti-VCA appears only with acute infections3 4 several studies have found it a reliable marker of recent viral acquisition.25, 3~ Antibody to EBV early antigen. Seventy to 95 per cent of EBV infections (symptomatic and asymptomatic) in the immunocompetent produce antibody to EBV early antigens (anti-EA) as an initial, usually transient, response to viral acquisition. 2L25 In lyre-
4
CHRONIC MONONUCLEOSIS[Merlin]
of anti-EBNA with high titers of IgG anti-VCA have been reported in patients with muhiple sclerosis. 28
the absence of other identifiable causes of the symptoms; and 4) geometric and arithmetic mean titers of IgG anti-VCA higher than those of the control group. Three of the healthy control subjects had detectable anti-EA. In 1985 Straus et al. 7 published their findings for 23 of 31 patients followed up for four years whom they believed to have persistent symptomatic EBV disease. The initial 31 patients had been referred because of chronic fatiguing illness occurring after infectious mononucleosis or an infectious mononucleosis-like illness. The 23 patients were characterized by 1) chronic (longer than one year) fatiguing illness; 2) abnormality(ies) of EBV-specific serologic test results; and 3) the absence of other identifiable chronic infectious or immune-impairing illnesses. The EBVserologic abnormalities (sometimes detectable only intermittently) were as follows: anti-EA of 1:10 or higher in 19 of the 23 patients (versus six of 23 control subjects); IgG anti-VCA of 1:320 or higher in 19 of the 23 patients (versus three of the control subjects); anti-EBNA of 1:5 or less in seven of the 23 (versus no control subjects); IgM anti-VCA of 1:10 or higher in five of the 23 (versus no control subjects). Straus et al. 7 investigated other parameters of EBV infection or imntune flmction in these patients. T h e y quantified EBV-infected B lymphocytes and found their numbers no different from those in the control subjects. T h e y found slight immunoglobulin deficiencies in four of the 23 patients. Evidence of increased circulating i n m l u n e complexes and increased T-cell-mediated suppress!on was also found in some of the patients. In sumnmry, the recent studies revealed groups of patients with the following clmracteristics: 1) acquired, but persistent or relapsing, symptomatic illness characterized primarily by fatigue; 2) no demonstrable other disease that would have caused the symptoms; and 3) values for EBV-specific serologic tests that were, at least intermittently, different from those for healthy control subjects. According to the authors of these recent studies, the finding of abnormal EBV-specific serologic profiles (i.e., values different from those of their control subjects) suggests that EBV may be the cause of the symptoms.
EBV-specific Serologic Tests and Chronic Mononucleosis Ahhough the existence of a prolonged symptomatic infections mononucleosis-like illness caused by EBV has been suspected, it has remained poorly defi0ed and unsubstantiated because of 1) the nonspecificity o f the s y m p t o m a t o l o g y (e.g., malaise, fever, lymphadenopathy); 2) the absence of objective clinical signs; and 3) the absence of other objective criteria (e.g., laboratory studies) for the diagnosis or definition of the disease. T h e development of EBVspecific serologic tests has offered the possibility of objective criteria for defining chronic mononucleosis. Evidence that EBV-specific serologic tests might, in fact, be a tool for the investigation o f chronic mononucleosis was derived first fi'om a retrospective study in 1975 of persons with unusual patterns of EBV-specific serologic resuhs (anti-EA-R, rather than the usual anti-EA-D) after infectious mononucleosis. 1 The investigators found that three of the 14 patients with such unusual patterns complained of recurrent symptoms. In 1982 Tobi et al. 4 described the cases of seven patients who had 1) persistent (longer than one year) symptoms (e.g., malaise, fever, weight loss, myalgia, abdominal pain, anxiety); 2). persistent (longer than one year) IgM anti-VCA; and 3) no apparent manifestations of serious diseases. Only one patient had had a preceding bout of classic infectious mononucleosis. During the past year, the resuhs of three studies that attempted to use EBV-specific serologic tests to define chronic mononucleosis objectively were published. 5-7 These studies attracted considerable medical and lay interest. In 1984 Dubois et al. 5 presented data for 14 patients who had what these investigators referred to as chronic mononucleosis syndrome. 5 These 14 patients were characterized by 1) persistent (longer than six months) symptoms (e.g., fatigue, fever, myalgia depression, pharyngitis, lymphadenopathy), particularly disabling fatigue; 2) abnormal EBV serologic profiles, including, at least occasionally, lgG antiVCA of 1:160 or higher (values for control subjects, 1:40 to 1:320) or anti-EA of 1:5 or higher (values for all control subjects, less titan 1:5); and 3) the absence of malignancy, autoimmunity, or profound immunodeficiency. In 1985 Jones et al. 6 presented data for 39 of 44 patients whom they believed to have active EpsteinBarr viral infection on the basis of their EBV-specific serologic test resuhs. These 39 patients were characterized by 1) persistent or relapsing (longer than one year) symptoms (e.g., fatigue, headache, parethesia, depression, pharyngitis, fever, lymphadenopathy); 2) detectable anti-EA, at least intermittently; 3)
PROBLEMS WITH RECENT STUDIES OF CHRONIC MONONUCLEOSIS
T h e evidence that EBV causes chronic mononucleosis is weak. T h e recent studies have not provided serologic data showing clear-cut differences between patients suspected o f having chronic mononucleosis and persons not believed to have tlie disease. T h e patients suspected of having chronic nlononncleosis are characterized in these recent studies primarily by elevations of anti-EA and IgG anti-VCA levels. Yet such elevations of anti-EA and IgG anti-VCA levels are seen in many healtl W persons w
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remotely infected with EBV, as well as in persons remotely infected with EBV who have other diseases, as discussed earlier. T h e elevations of anti-EA and IgG anti-VCA levels characterizing the patients in these studies, although sometimes different from those of the control subjects, are 1) sometimes the same as control values; 2) when outside the range of control values for a research group, often still within the range of values found in healthy populations; 3) within the range of values seen for EBV-specific serologic tests in a wide variety of diseases. The recent studies simply show that elevations of anti-EA and IgG anti-VCA levels are more prevalent in tile patients with suspected chronic mononucleosis than in the control subjects. The elevations of anti-EA and IgG anti-VCA levels are not abnormalities of EBV serologic tests indicative of EBV-induced disease (i.e., found in patients with EBV-caused illness but not ill healthy persons). The presence of IgM anti-VCA after the several months immediately following acute EBV infections is distinctly unusual. 25,30 Persistent elevations of IgM anti-VCA levels unlike elevations of IgG anti-VCA and anti-EA, do not occur in heahhy persons or patients with non-EBV diseases. Thus, the convincing demonstration of persistent elevations of IgM antiVCA in patients with suspected chronic mononucleosis would support the hypothesis that EBV causes the suspected disease. Elevations of IgM anti-VCA were reported in some patients in the most recent studies: three of the 39 patients of Jones et al., G five of the 23 patients of Straus et al. 7 and none of the 14 patients of Dubois et al. 5 Yet some problems are associated with tile demonstration of IgM anti-VCA in these studies: 1) IgM anti-VCA was demonstrated in only eight of 76 patients (approximately 10 per cent) purported to have the disease in these stndies. 5-7 2) Some of these eight patients had r h e u m a t o i d factor, which may have caused apparent elevations of IgM anti-VCA that were, in fact, false-positive results. 7 3) IgM anti-VCA was demonstrated only once or intei'mittently ill each of the eight patients, 6,7 although multiple specimens from each o f the total g r o u p o f 76 patients were tested; thus, although IgM anti-VCA was found in approximately 10 per cent of tile patients, it was evidently found in a much smaller percentage (probably 1 to 2 per cent) of all specimens tested from all patients purported to have chronic mononucleosis. The demonstration of IgM anti-VCA in such a small percentage of specimens from the 76 patients certainly is not convincing evidence that these 76 patients had EBV-caused disease. In 1982 Tobi et al. 4 demonstrated tile persistence o f IgM anti-VCA for 15 to 29 months in seven patients. This observation is clearly different from tile rare, intermittent IgM anti-VCA seen in the more recent studies. The inability to duplicate such persistence of IgM anti-VCA in the more recent studies of large groups o f patients with snspected chronic mononucleosis has not been explained.
Jones et al. 6 and Straus et al. 7 reported failure to mount the usnal anti-EBNA response in eight of 31 and five of 23 patients, respectively. Again, however, for most patients ill both studies, anti-EBNA levels were normal. In summary there is little convincing serologic evidence from recent studies that the patients' symptoms were caused by EBV. As a group, the patients suspected of having chronic mononucleosis do have anti-EA and IgG anti-VCA levels that are higher than those observed ill groups of healthy persons. However, the individual serologic values cannot be used to distinguish patients from healthy persons, as many healthy persons have similarly elevated anti-EA and IgG anti-VCA levels. Elevations of the levels of antiEA and IgG anti-VCA are also seen ill a variety of abnormal and physiologic states. Very few values for IgM anti-VCA are elevated in the patients, and at least some of these values may be erroneous. Abnormalities of anti-EBNA are reported ill only a small fraction of the patients. T h e relation of EBV to the symptoms of patients with chronic fatiguing illness remains obscure, despite the serologic studies. The EBV serologic abnormalities found in these patients could bave been caused by a variety of illnesses. Such abnormalities are also often found in healthy persons. Pitfalls in Aflempts to Use EBV-specific Serologic Tests for the Diagnosis of Chronic Mononucleosis
Some hazards are involved ill the nse of EBVspecific serologic tests for tile diagnosis of chronic mononucleosis in patients with chronic fatiguing illnesses. The presence of the disease is unproved and abnormalities of EBV-specific serologic tests are not specific for tile suspected disease. As discussed earlier, abnormalities of EBV-specific serologic tests are seen in a host of pathologic and physiologic states. All of the recent studies discussed earlier of patients suspected of having chronic mononucleosis methodically excluded patients with other diseases before testing for abnormalities of EBV-specific serologic tests. It is clear that the EBV-specific serologic tests cannot be used to rule out other, non-EBV diseases, because abnormalities of EBV-specific sero- ' logic tests occur in so many diseases not caused by EBV. The exclusion of patients with other diseases in the recent studies has been by multiple, extensive medical evaluations. Yet the EBV-specific serologic tests used in these research studies are being marketed to the lnedical comntunity as diagnostic tests for the evaluation of patients with persistent or relapsing syndromes of fatigue and depression. T h e practical value of EBV serologic tests used in this fashion is clearly limited, as a positive EBV serologic result (i.e., similar to those seen in some patients ill recent studies) neither establishes tile existence of chronic mononucleosis nor excludes the possibility of other diseases. The natnre of the marketing of these serologic 6
CHRONIC MONONUCLEOSIS[Merlin]
tests is i n d i c a t e d in the f o l l o w i n g quotes f r o m a mailing to physicians from a major national reference i m m u n o l o g y laboratory36:
TABLE I. Conditions Causing Aberrations of Epstein-Barr Virus-Specific Serologic Tests Similar to Those in Suspected Chronic Mononucleosis
. . . . is proud to announce its CHRONIC ACTIVE EBV DISEASE PANEL for the evaluation of children and adults with persistent or relapsing syndromes of fatigue, deprlession and dyslogia, sometimes with low grade fever, adenopathy and weight loss. This ill-defined, flu-like syndrome is well known and widely recognized by primary care physicians; it typifies many perplexing patients Whose complaints of disability are characteristically and inexplicably unaccompanied by "objective"findings on physical and laboratory examination (including their normal sedimentation rates). Now, however, two groups of investigators report data suggesting that chronic active or recrudescent E-B virus infections are common in such patients. It is hard to overemphasize the importance of these findings. Simply based on the description of the syndrome alone and its laboratory counterpart one can expect e n o r m o u s b e n e f i t s to patients who can finally be told of a possible organic basis for their illnesses which for so many patients would otherwise be interpreted as "neurotic" with resultant feelings of self doubt.
Primary immunodeficiencystates (e.g., ataxia telangiectasia) Secondary immtmodeficiency states (e.g., steroid therapy, antithymocyte globulin therapy) Hodgkin's disease Non-Hodgkin's lymphoma Leukenfias Systemic lupus erythematosus Endemic Burkitt's lymphoma Nasopharyngeal carcinoma AIDS AIDS-related complex Rheumatoid arthritis Multiple sclerosis Chronic renal failure Renal transplantation Nonlymphomatous malignant tumors Cancer chemotherapy
of chronic mononucleosis (table 1). Use o f the EBVspecific serologic tests in the medical evaluation of chronic fatiguing illnesses should be, at least, tempered by cautious awareness o f the range of diseases other than chronic mononucleosis that can cause abnormal results a n d lead to extensive medical evaluations. For a laboratory test to be reasonably clinically useful, a positive (or negative) test result must be a reasonable indicator that a patient has (or does not have) the disease in question. This is clearly not the case with regard to EBV-specific serologic tests and chronic mononucleosis. These tests clearly do not indicate a specific disease, nor do they exclude a range o f other diseases.
T r u e , these tests do, to some extent, suggest an organic basis for the illness, but only in the most vague, and clinically most useless, s e n s e - - t h e percentage o f ill persons who have abnormalities o f these test results is greater than that o f healthy persons. T h e tests do not, however, establish a diagnosis of chronic mononucleosis or chronic active EBV infection in the patient with a chronic fatiguing illness. T h e y suggest, vaguely, that such a disease might be present. T h e y clearly do not exclude the possibility o f other diseases, which is their great liability. Abnormalities of these test results, like those mentioned in the recent studies o f suspected chronic m o n o n u cleosis, are not specific for any disease.9,20.21,27-29,31-33 Positive EBV serologic test results might indicate that a patient has chronic mononucleosis, Hodgkin's disease, n o n - H o d g k i n s ' s l y m p h o m a , systemic lupus e r y t h e m a t o s u s , AIDS, multiple sclerosis, n o n l y m p h o m a t o u s t u m o r s , or a variety o f o t h e r diseases. This is not an unrealistic list. All o f these diseases may be manifested as chronic fatigue, even chronic fatigue after infectious mononucleosis; all are associated with abnormalities o f EBV-specific serologic test results, similar to those seen in suspected chronic m o n o n u cleosis. O f the 31 patients referred to Straus et al. 7 at the N I H for the evaluation o f "histories of chronic illness with fatigue after infectious mononucleosis (or with features o f that disease)," five were excluded because they were f o u n d to have not chronic mononucleosis but rather SjOgren's syndrome, systemic lupus el)'thematosus, lymphoma, Hodgkin's disease, or multiple sclerosis. A variety o f diseases, some o f them grave, can have both clinical presentations and abnormalities of EBV-specific serologic test results that would be indistinguishable from those o f the p u r p o r t e d disease
CONCLUSIONS
Epstein-Barr virus causes chronic infections in all persons that it infects. This chronicity entails a complex a n d poorly u n d e r s t o o d i m m u n o l o g i c relation with the host. T h e various EBV-specific serologic tests that have been developed show somewhat characteristic patterns o f changes with the usual acute infections, probably reflecting the complex reactions of the host to the productive a n d latent viral infections. Attempts have been m a d e to apply these EBV-specific serologic tests to persons with protracted symptoms resembling those o f infectious mononucleosis in o r d e r to show that the symptoms are d u e to active EBV infection. These attempts have shown abnormalities o f EBV-specific serologic test resnlts in groups o f patients with such symptoms, but have not shown distinct abnormalities or proved tile existence o f active disease in the patients. T h e EBV-specific serologic tests have not been shown to be useful diagnostic tools in the evaluation o f patients with chronic fatiguing illness outside an experimental context. Because of their nonspecificity, these tests have the potential to mislead physicians to overlook other signif7
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icant diseases in patients with chronic vague symptoms.
REFERENCES 1. IIor.witz CA, ltenle W, Itenle G, et al: Clinical evaluation of patients with infectious mononucleosis and development of antibodies to the R component of the Epstein-Barr virus induced early antigen. Am J Med 58:330, 1975 2.. Chang RS, Maddock R: Recurrence of infections mononucleosis. Lancet 1:701, 1980 3. Askinazi C, Sessions C, Brusch Jl.: Positive differential heterophile antibody test: persistence in a symptomatic patient. JAMA 236:!492, 1976 4. Tobi M, Morag A, Ravid Z, et al: Prolonged atypical illness associated with serologic evidence of persistent Epstein-Barr virus infection. Lancet 1:61, 1982 5. Dubois RE, Selley JK, Brus I, et al: Chronic mononucleosis syndrome. South Med J 77:1376, 198-t 6. Jones JF, Ray G, Minnich LL, et al: Evidence for active Epstein-Barr virus infection in patients with persistent, unexplained illnesses: elevated anti-early antigen antibodies. Ann Intern Mcd 102:1, 1985 7. Strauss SE, Tosato G, Armstrong G, et al: Persisting illness and fatigue in adults with evidence of Epstein-Barr virus infection. Ann Intern Med 1:7, 1985 8. de-Th6 G: Epidemiology of Epstein-Barr virt:s and associated diseases in man. Roizman B (ed): In The llerpes Viruses, vol 1. New York, Plenum, 1982, pp 25-103 9. Henle W, Hcnle G: Seroepidemiology of the virus. In Epstein MA, Achong EG (eds): The Epstein-Barr Virus. New York, Springer-Verlag, 1979, pp 62-78 10. Fleisher G, Henle W, Henle G: l'rimary infection with EpsteinBarr virus in infants in the United States: Clinical and serologic observations. J Infect Dis 139:553, 1979 1 I. Biggar RJ, Ilenle W, Fleisher G, et al: Primary Epstein-Barr virus infections in African infants. II. Clinical and serologic observations during seroconversion, lnt J Cancer 22:239, 1978 12. Andiman WA: The Epstein-Barr virt,s and Epstein-Barr virus infections in childhood. J Pediatr 95:171, 1979 13. Rapp CE, HewetsonJF: Infections mononucleosis and the Epstein-Barr virus. A m J Dis Child 132:78, 1978 14. Rocchi C, De Feliu A, Ragona G: Quantitative evaluation of Epstein-Barr virus infected mononuclear peripheral blood leukocytes in infectious mononucleosis. N Engl J Med 296:132, 1977 15. Strauch B, Andrews L, Miller G, et al: Oropharyngeal excretion of Epstein-Barr virus by renal transplant recipients and other patients treated with immunosuppression drugs. Lancet 1:234, 197,t 16. Epstein MA, Achong BG: Introduction: discover)' and general biology of the virus. In Epstein MA, Achong BG (eds): The Epstein-Barr Virus. New York, Springer-Verlag, 1970 17. Evans AS: Infectious mononucleosis and related syndrontes. A m J Med Sci 276:325, 1978 18. lssacs R: Chronic infectious mononucleosis. Blood 3:858, 1948
19. Kaufman RE: Recurrence in infectious mononucleosis. Am Prac 1:673, 1950 20. Henle W, tlenle GE, llorwitz CA: Epstein-Ban virus specific diagnostic tests in infectious mononucleosis. Ilum Pathol 5:551, 1974 21. l-tenle W, Ilenle G: hnmunology of Epstein-Barr Virus. In Roizman B (ed): The tlerpes Viruses, vol 1. New York, Plenum, 1982, pp 209-252 22. Evans AS, Niederman JC, Cenabre I.C, et al: A prospective evahmtion of heterophile and Epstein-Barr virus specific IgM antibody tests in clinical and subclinical infectious ntononucleosis: specificity attd sensitivity of the tests and persistence of antibody. J Infect Dis 132:546, 1975 23. Schmitz H, Scherer M: lgM antibodies to Epstein-Barr virus in infections mononucleosis. Arch Gesam I'ir~tsforsch 37:332, 1972 24. Sumaya CU: Endogenous reactivation of Epstein-Barr virus infections. J Infect Dis 135:374, 1977 25. Lamy ME, Faurt AM, Cornur C, et al: Stt,dy of Epstein-Barr Virus (EBV) antibodies IgG and IgM anti-VCA, IgG antiEA; anti lg anti-EBNA obtained with an original microtiter technique. Acta Clin Belg 37:281, 1982 26. Wagh-Mathur V, Eulow RW, Splgland I, et al: l.ongitudinal study of persistent generalized l)'mphadenopathy in homosexual men: relation to acquired immunodeficiency syndrome. Lancet 1: 1033, 198-t 27. Chang RS, Thompson H, l'omerantz S: Epstein-Barr virus infection in homosexual nten with chronic persistent generalized l)mplmdenopathy. J Infect Dis 151:459, 1985 28. Larsen PD, Bloomer LC, Bray PF: Epstein-Barr nuclear and viral capsid antigen antibody titers in multiple sclerosis. Neurology 35:435, 1985 29. Cohen Jll, Vischer TL, Carquin J, et al: A subset of rheumatoid arthritic patients with a pattern of Epstein-Barr virus antibodies similar to that in primary and secondary immunodeficiency diseases. Arthritis Rheum 28:339, 1985 30. Nikoskelainen J, Hanninen P: Epstein-Barr virus (EBV) specific lgM antibodies in diagnosis of recent infection. Scand J Clin Lab Invest 30(suppl 130):32, 1973 31. Fleisher G, Bolognese R: Persistent Epstein-Barr virus infection and pregnancy. J Infect Dis 147:982, 1983 32. VirelizierJ, Lenair G, Griscelli C: Persistent Epstein-Barr virus infection in a clfild with hypergammaglobulinemia and immunoblastic proliferation associated with a selective defect in immt,ne interferon secretion. Lancet 2:231, 1978 33. Henle W, tlenle G: Epstein-Barr virus-specific serology in imntunologically compromised individuals. Cancer Res 41:4222, 1981 34. Fischer DK, Robert MF, Shedd D, et al: hldentification of Epstein-Barr nuclear antigen polypeptide in mouse and monkey cells after gene transfer with a cloned 2.9 kilobasepair subfragment of the genome. Proc Natl Acad Sci 81:43, 198-t 35. Sakamoto K, Freed llJ, Purtilo DT: Antibody to Epstein-Barr virus in families with the X-linked lymphoproliferative s)'ndrome. J lmmunol 125:921, 1980 36. Baker CW: Persisting illness and fatigue: chronic active E-B virus disease (mass mailing). Los Angeles, Specificity Laboratories, 1985
nucleosis, with particular emphasis on the pitfalls enc o u n t e r e d in attempts to diagnose the disease by EBV-specific serologic testing. OVERVIEW OF EBV INFECTIONS
Epstein-Barr viral infection, like death and taxes, is virtually inevitable among humans. The virus can first be acquired after the disappearance of protective congenitally acquired maternal antibodies, at the age of 2 to 7 months. All persons in developing nations and economically less advantaged persons in developed nations acquire EBV in childhood. 8 Most are infected by the age of 3 years, and virttmlly all are infected by adolescence. Among the econontically advantaged, however, the acquisition of EBV often does not occur until adolescence or early aduhhood. Approximately 50 per cent of persons entering college (in general, the more economically advantaged) are uninfectedP Prior to adolescence or young adulthood the acquisition of EBV usually goes unnoticed. Most acute infections in childhood are asymptomatic, or the symptoms are so mild and nonspecific that they cannot be distinguished from the frequent, mild, brief illnesses of childhood, l~ Acquisition of the virus after childhood, however, causes infectious mononucleosis in 30 to 45 per cent o f those infected. 12A3 T h e fact that the acquisition o f EBV causes infectious mononucleosis in adolescents and young aduhs but does not cause overt symptoms in children has not been explained. CHRONICITY OF EBV INFECTIONS
Infection with EBV is not only universal, but also invariably chronic. Epstein-Barr virns, a doublestranded DNA virus, integrates its genome into that of h u m a n B lymphocytes. During acute infectious mononucleosis, 0.001 to 0.01 per cent of circulating B lymphocytes are infected by the virus. In the ensuing 12 to 16 weeks, the number of infected circulating B lymphocytes diminishes to 0.00001 per cent, a level that apparently persists indefinitely.14 Epstein-Barr virus immortalizes B lymphocytes in vitro, allowing continuous replication and subculturing of the infected cells when they are provided with nutrients. Whether an in vivo equivalent of this immortalization process is responsible for the persis-
CHRONICMONONUCLEOSIS[Merlin] tence o f EBV-infected B lyinphocytes in vivo or whether the virus passes from infected to uninfected B lymphocytes in vivo is difficult to assess. A person who has acquired EBV sheds virus episodically in oropharyngeal secretions. Seventy to 90 per cent o f persons with infectious mononucleosis shed virus, and this shedding persists for eight to 24 or more weeks after the resolution of symptomatic disease. Once the initial shedding associated with the acute acquisition of the virus subsides, episodic resurgence of shedding, tmassociated with symptoms, occurs. Twenty to 30 per cent of EBV carriers shed virus at any one time. This episodic shedding of virus, albeit in miniscule quantities, is considered the reservoir for infection of the uninfected. The rate of oropharyngeal viral shedding among those not acutely infected is higher in the immunosuppressed (50 per cent) than among the immunocompetent (20 per cent).15 T h e rate is similarly higher among infected persons in third-world nations, possibly due to the immunosuppression caused by malnutrition or disease. 16 INFECTIOUS MONONUCLEOSIS
Classic infectious mononucleosis caused by EBV is characterized clinically by a triad consisting of 1) fatigue, fever, pharyngitis, lymphadenopathy, and, often, splenomegaly and mild hepatitis; 2) lympbocytosis, with a peripheral leukocyte count revealing more than 50 per cent lymphocytes, I0 to 20 per cent of which are atypical; and 3) a positive differential (Paul-Bunnell) heterophil antibody test or EBV-specific serologic results indicative of acute disease. 17 Most cases occur in persons 10 to 25 years of age. Not all cases are classic in presentation, and unusual forms of infectious mononucleosis are more frequent outside tile usual age range, l~ In approximately 10 per cent o f North Americans in whom infectious mononucleosis develops, tile differential (Paul-Bunnell) heterophil antibody test is not positive, is Most signs and symptoms of infectious mononucleosis resolve within one month. Tile usual pattern is disappearance of pharyngitis by 14 days; disappearance of fever by 21 days; and disappearance of fatigue, lymphadenopathy, and splenomegaly by 21 to 28 days. 13 T h e fatigue of infectious mononucleosis occasionally persists for months. Iz CHRONIC MONONUCLEOSIS: HISTORY
Reports of protracted or recurrent symptoms of infectious lnononucleosis first appeared at least 40 to 50 ")'ears ago, antedating currently accepted criteria for the diagnosis o f infectious mononucleosis and making comparisons of the earlier reports with current studies difficult. Sizable groups of patients with protracted symptoms after infectious mononucleosis were described in the 19,t0s and 1950s. Ill 1948 Issacs Is described the persistence of symptoms and
signs (e.g., tatigue, depression, myalgia, lymphocytosis, low serum glucose level, low blood pressure, low urinary specific gravity) in 53 of 206 patients after infectious mononucleosis. In 1950 KaufmanlV described the recurrence of symptoms and signs (e.g., fatigue, pharyngitis, lymphadenopathy, lymphocytosis with atypical lymphocytes) in 12 of 125 patients after infectious mononucleosis. In these relatively large series, the criteria used for the diagnosis of the initial and persistent illness were vague, poorly documented, and not consistent with more recently defined criteria. Interestingly, with the development of more rigid criteria for the diagnosis o f infectious mononucleosis, reports of such sizable groups of patients with chronic mononucleosis have disappeared from the medical literature. The most recent studies of protracted or recurrent symptoms after infectious mononucleosis involved batteries of EBV-specific serologic tests. 5-7 Batteries of multiple serologic tests for EBV infections are complex, and their use, until recently, was confined to research studies. The various serologic methods were extensively reviewed elsewhere. 9,2~ A brief discussion o f the serologic tests is presented here because these tests are crucial to recent attempts to demonstrate chronic mononucleosis.
EPSTEIN-BARRVIRUS SERODIAGNOSIS
Differential [PauI-Bunnell] Heterophil Antibody Test Tile differential (Paul-Bunnell) heterophil antibody test is the most familiar, most readily available serologic test for the diagnosis of EBV infection. A significant tiler o f this heterophil antibody (i.e., a positive test) is present at some point during symptomatic infectious mononucleosis in approximately 90 per cent (range, 81 to 97 per cent, depending on the test method) of non-Orientals with tile illness. 22 The heterophil antibody is not usually produced (i.e., the test is negative) with the asymptomatic acquisition of EBV by persons outside the age range usually associated with infectious mononucleosis. Thns, the acquisition of EBV that is not associated with infectious mononucleosis cannot usually be detected by a test for the Paul-Bunnell heterophii antibody. 2~ The specificity of the Paul-Bunnell heterophil antibody test ranges from 93 to 98 per cent. z2 The Paul-Bunnell heterophil antibgdy test is not an EBV-specific serologic test. The Paul-Bunnell heterophil antibody does not react with an antigen of EBV per se. In humans, it probably reacts with neoantigen(s) induced or augmented on some cells, possibly B lymphocytes, by EBV infection. These induced neoantigens are fortuitously similar or identical to common antigens on horse, sheep, or bovine erythrocytes. It is, thus, a "heterophil" antibody because it is formed in one species (i.e., humans) and cross reacts with antigens present in another species
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phoid cells in culture tile early antigens appear in detectable quantities b e f o r e viral n u c l e o c a p s i d - giving rise to the name early antigen. The early antigens are probably viral enzymes. Anti-EA is usually produced acutely with the acquisition of the virus, beginning concomitant with or shortly after the prodnction of anti-VCA. It usually persists for only eight to 12 weeks, similar to IgM anti-VCA.20, 21 Although anti-EA is usually associated with the recent acquisition of EBV, the association is far from absolute. 25 It has been reported that 4 to 20 per cent of persons with past, but not recent, EBV infections are positive for anti-EA. 24,~5After the acute acqnisition of EBV, the anti-EA diminishes or disappears in most persons but then reappears with age. 25 A number of conditions have been identified in which anti-EA is elevated, sometimes to very high titers, without recent EBV acquisition. These conditions include pregnancy, immunodeficiency states, H o d g k i n ' s disease, n o n - H o d g k i n s ' s l y m p h o m a , Burkitt's l y m p h o m a , n a s o p h a r y n g e a l carcinoma, leukemia, AIDS, and the AIDS-related complex.9.20.21,27,31-33 The demonstration of anti-EA is accomplished by immunofluorescence, with nonproducer EBV-infected cell lines that have been superinfected with EBV used as substrates. Patterns of i m m u n o f l u o rescence staining of the substrate cells have been reported as diffuse, or EA-D, and restricted, or EAR.20,21 Anti-EA-D is a pattern of nuclear and cytoplasmic staining, while the anti-EA-R pattern is localized cytoplasmic, perinuclear staining. Childhood EBV acquisition is usually associated with anti-EAR. 21 Adolescent and adult acquisition of EBV is usually associated with anti-EA-D. Antibody to EBV nuclear antigen. In virtually all immunocompetent persons antibody to EBV nuclear antigen (anti-EBNA) develops, usually two to three months and, rarely one month after viral acquisition. 2L25 T h e presence of anti-EBNA indicates that EBV acquisition is not recent, but o c c u r r e d one month to years in the past. Like IgG anti-VCA, the anti-EBNA persists indefinitely once acquired. Nuclear antigen is a polypeptide or group of polypeptides encoded by the EBV DNA. 34 It is expressed in all cells infected with EBV but is present in such small quantities in EBV-infected cells that it cannot be demonstrated by routine indirect immunofluorescence. An amplification technique, anticomplement indirect immunofluorescence, is necessary to allow demonstration of anti-EBNA. Fixed smears of nonproductively EBV-infected B-cell lymphoblastoid cell lines are used as substrates. Failure to mount the usual anti-EBNA response is seen in some immtmodeficiency states, eg., the Xlinked lymphoproliferative syndrome. 35 In addition, unusually low titers o f anti-EBNA, in the presence of high titers of IgG anti-VCA, have been reported in patients with rheumatoid arthritis, 29 and high titers
(i.e., horse, sheep, or cow). Suspensions of horse, sheep or bovine erythrocytes are used to absorb or test for the presence of the antibody. The Paul-Bunnell antibody is usually an IgM antibody. It is said to persist only eight to 12 weeks in infectious mononucleosis, but this finding is highly de per~dent on the test system employed. Horse erythrocytes detect the heterophil antibody much longer tlmn either sheep or bovine erythrocytes, and the heterophil antibody will thus appear to persist longer x~hen tested by this method. 2'~
EBV-specific Serologic Tests Serologic tests specific for EBV detect antibodies to antigens that are apparently encoded by the EBV genome. The usual method for demonstrating antibodies to these antigens is indirect immunofluorescence, although other methods (i.e., enzyme immunoassays) have been explored. Antibody to EBV viral capsid antigen. All EBV infections in immunocompetent persons result in the production of antibody to EBV viral nucleocapsid antigen (anti-VCA). 21 Immunofluorescence test systems for anti-VCA use fixed smears of B-cell lymphoblastold cell lines productively infected (i.e., producing virus) with EBV as a viral capsid antigen-containing substrate. 21 IgM, IgA, and IgG anti-VCAs are distinguished by c o m m o n methods of immunoglobulin fractionation and heavy-chain-specific fluoresceinated conjugates. Serum levels o f anti-VCA peak at three to four weeks following EBV infection. IgM anti-VCA levels decline rapidly, and IgM anti-VCA is usually undetectable at 12 weeks. 23 IgG anti-VCA levels decline somewhat after peaking but persist indefinitely. The presence of IgG anti-VCA is the most sensitive indicator available for EBV infection, recent or remote, because of this indefinite persistence3 l High levels of IgG anti-VCA are seen in acute infections but are not specific for acute infections3 4,25 Similarly high levels are seen in remote EBV infection and in a variety o f other conditions: primary and secondary immunodeficiency states, Hodgkin's disease, leukemia, sarcoidosis, l y m p h o m a , nonlymphomatous malignancies, systemic lupus erythematosus, chronic renal diseases, endemic Burkitt's lymphoma, nasopharyngeal carcinoma, rheumatoid arthritis, the acquired immunodeficiency syndrome (AIDS), the AIDS-related complex, and multiple sclerosis.9,21,26-29 Although t h e r e is some d i s a g r e e m e n t as to whether IgM anti-VCA appears only with acute infections3 4 several studies have found it a reliable marker of recent viral acquisition.25, 3~ Antibody to EBV early antigen. Seventy to 95 per cent of EBV infections (symptomatic and asymptomatic) in the immunocompetent produce antibody to EBV early antigens (anti-EA) as an initial, usually transient, response to viral acquisition. 2L25 In lyre-
4
CHRONIC MONONUCLEOSIS[Merlin]
of anti-EBNA with high titers of IgG anti-VCA have been reported in patients with muhiple sclerosis. 28
the absence of other identifiable causes of the symptoms; and 4) geometric and arithmetic mean titers of IgG anti-VCA higher than those of the control group. Three of the healthy control subjects had detectable anti-EA. In 1985 Straus et al. 7 published their findings for 23 of 31 patients followed up for four years whom they believed to have persistent symptomatic EBV disease. The initial 31 patients had been referred because of chronic fatiguing illness occurring after infectious mononucleosis or an infectious mononucleosis-like illness. The 23 patients were characterized by 1) chronic (longer than one year) fatiguing illness; 2) abnormality(ies) of EBV-specific serologic test results; and 3) the absence of other identifiable chronic infectious or immune-impairing illnesses. The EBVserologic abnormalities (sometimes detectable only intermittently) were as follows: anti-EA of 1:10 or higher in 19 of the 23 patients (versus six of 23 control subjects); IgG anti-VCA of 1:320 or higher in 19 of the 23 patients (versus three of the control subjects); anti-EBNA of 1:5 or less in seven of the 23 (versus no control subjects); IgM anti-VCA of 1:10 or higher in five of the 23 (versus no control subjects). Straus et al. 7 investigated other parameters of EBV infection or imntune flmction in these patients. T h e y quantified EBV-infected B lymphocytes and found their numbers no different from those in the control subjects. T h e y found slight immunoglobulin deficiencies in four of the 23 patients. Evidence of increased circulating i n m l u n e complexes and increased T-cell-mediated suppress!on was also found in some of the patients. In sumnmry, the recent studies revealed groups of patients with the following clmracteristics: 1) acquired, but persistent or relapsing, symptomatic illness characterized primarily by fatigue; 2) no demonstrable other disease that would have caused the symptoms; and 3) values for EBV-specific serologic tests that were, at least intermittently, different from those for healthy control subjects. According to the authors of these recent studies, the finding of abnormal EBV-specific serologic profiles (i.e., values different from those of their control subjects) suggests that EBV may be the cause of the symptoms.
EBV-specific Serologic Tests and Chronic Mononucleosis Ahhough the existence of a prolonged symptomatic infections mononucleosis-like illness caused by EBV has been suspected, it has remained poorly defi0ed and unsubstantiated because of 1) the nonspecificity o f the s y m p t o m a t o l o g y (e.g., malaise, fever, lymphadenopathy); 2) the absence of objective clinical signs; and 3) the absence of other objective criteria (e.g., laboratory studies) for the diagnosis or definition of the disease. T h e development of EBVspecific serologic tests has offered the possibility of objective criteria for defining chronic mononucleosis. Evidence that EBV-specific serologic tests might, in fact, be a tool for the investigation o f chronic mononucleosis was derived first fi'om a retrospective study in 1975 of persons with unusual patterns of EBV-specific serologic resuhs (anti-EA-R, rather than the usual anti-EA-D) after infectious mononucleosis. 1 The investigators found that three of the 14 patients with such unusual patterns complained of recurrent symptoms. In 1982 Tobi et al. 4 described the cases of seven patients who had 1) persistent (longer than one year) symptoms (e.g., malaise, fever, weight loss, myalgia, abdominal pain, anxiety); 2). persistent (longer than one year) IgM anti-VCA; and 3) no apparent manifestations of serious diseases. Only one patient had had a preceding bout of classic infectious mononucleosis. During the past year, the resuhs of three studies that attempted to use EBV-specific serologic tests to define chronic mononucleosis objectively were published. 5-7 These studies attracted considerable medical and lay interest. In 1984 Dubois et al. 5 presented data for 14 patients who had what these investigators referred to as chronic mononucleosis syndrome. 5 These 14 patients were characterized by 1) persistent (longer than six months) symptoms (e.g., fatigue, fever, myalgia depression, pharyngitis, lymphadenopathy), particularly disabling fatigue; 2) abnormal EBV serologic profiles, including, at least occasionally, lgG antiVCA of 1:160 or higher (values for control subjects, 1:40 to 1:320) or anti-EA of 1:5 or higher (values for all control subjects, less titan 1:5); and 3) the absence of malignancy, autoimmunity, or profound immunodeficiency. In 1985 Jones et al. 6 presented data for 39 of 44 patients whom they believed to have active EpsteinBarr viral infection on the basis of their EBV-specific serologic test resuhs. These 39 patients were characterized by 1) persistent or relapsing (longer than one year) symptoms (e.g., fatigue, headache, parethesia, depression, pharyngitis, fever, lymphadenopathy); 2) detectable anti-EA, at least intermittently; 3)
PROBLEMS WITH RECENT STUDIES OF CHRONIC MONONUCLEOSIS
T h e evidence that EBV causes chronic mononucleosis is weak. T h e recent studies have not provided serologic data showing clear-cut differences between patients suspected o f having chronic mononucleosis and persons not believed to have tlie disease. T h e patients suspected of having chronic nlononncleosis are characterized in these recent studies primarily by elevations of anti-EA and IgG anti-VCA levels. Yet such elevations of anti-EA and IgG anti-VCA levels are seen in many healtl W persons w
HUMANPATHOLOGY Volume17,No. '1 [January '1986)
remotely infected with EBV, as well as in persons remotely infected with EBV who have other diseases, as discussed earlier. T h e elevations of anti-EA and IgG anti-VCA levels characterizing the patients in these studies, although sometimes different from those of the control subjects, are 1) sometimes the same as control values; 2) when outside the range of control values for a research group, often still within the range of values found in healthy populations; 3) within the range of values seen for EBV-specific serologic tests in a wide variety of diseases. The recent studies simply show that elevations of anti-EA and IgG anti-VCA levels are more prevalent in tile patients with suspected chronic mononucleosis than in the control subjects. The elevations of anti-EA and IgG anti-VCA levels are not abnormalities of EBV serologic tests indicative of EBV-induced disease (i.e., found in patients with EBV-caused illness but not ill healthy persons). The presence of IgM anti-VCA after the several months immediately following acute EBV infections is distinctly unusual. 25,30 Persistent elevations of IgM anti-VCA levels unlike elevations of IgG anti-VCA and anti-EA, do not occur in heahhy persons or patients with non-EBV diseases. Thus, the convincing demonstration of persistent elevations of IgM antiVCA in patients with suspected chronic mononucleosis would support the hypothesis that EBV causes the suspected disease. Elevations of IgM anti-VCA were reported in some patients in the most recent studies: three of the 39 patients of Jones et al., G five of the 23 patients of Straus et al. 7 and none of the 14 patients of Dubois et al. 5 Yet some problems are associated with tile demonstration of IgM anti-VCA in these studies: 1) IgM anti-VCA was demonstrated in only eight of 76 patients (approximately 10 per cent) purported to have the disease in these stndies. 5-7 2) Some of these eight patients had r h e u m a t o i d factor, which may have caused apparent elevations of IgM anti-VCA that were, in fact, false-positive results. 7 3) IgM anti-VCA was demonstrated only once or intei'mittently ill each of the eight patients, 6,7 although multiple specimens from each o f the total g r o u p o f 76 patients were tested; thus, although IgM anti-VCA was found in approximately 10 per cent of tile patients, it was evidently found in a much smaller percentage (probably 1 to 2 per cent) of all specimens tested from all patients purported to have chronic mononucleosis. The demonstration of IgM anti-VCA in such a small percentage of specimens from the 76 patients certainly is not convincing evidence that these 76 patients had EBV-caused disease. In 1982 Tobi et al. 4 demonstrated tile persistence o f IgM anti-VCA for 15 to 29 months in seven patients. This observation is clearly different from tile rare, intermittent IgM anti-VCA seen in the more recent studies. The inability to duplicate such persistence of IgM anti-VCA in the more recent studies of large groups o f patients with snspected chronic mononucleosis has not been explained.
Jones et al. 6 and Straus et al. 7 reported failure to mount the usnal anti-EBNA response in eight of 31 and five of 23 patients, respectively. Again, however, for most patients ill both studies, anti-EBNA levels were normal. In summary there is little convincing serologic evidence from recent studies that the patients' symptoms were caused by EBV. As a group, the patients suspected of having chronic mononucleosis do have anti-EA and IgG anti-VCA levels that are higher than those observed ill groups of healthy persons. However, the individual serologic values cannot be used to distinguish patients from healthy persons, as many healthy persons have similarly elevated anti-EA and IgG anti-VCA levels. Elevations of the levels of antiEA and IgG anti-VCA are also seen ill a variety of abnormal and physiologic states. Very few values for IgM anti-VCA are elevated in the patients, and at least some of these values may be erroneous. Abnormalities of anti-EBNA are reported ill only a small fraction of the patients. T h e relation of EBV to the symptoms of patients with chronic fatiguing illness remains obscure, despite the serologic studies. The EBV serologic abnormalities found in these patients could bave been caused by a variety of illnesses. Such abnormalities are also often found in healthy persons. Pitfalls in Aflempts to Use EBV-specific Serologic Tests for the Diagnosis of Chronic Mononucleosis
Some hazards are involved ill the nse of EBVspecific serologic tests for tile diagnosis of chronic mononucleosis in patients with chronic fatiguing illnesses. The presence of the disease is unproved and abnormalities of EBV-specific serologic tests are not specific for tile suspected disease. As discussed earlier, abnormalities of EBV-specific serologic tests are seen in a host of pathologic and physiologic states. All of the recent studies discussed earlier of patients suspected of having chronic mononucleosis methodically excluded patients with other diseases before testing for abnormalities of EBV-specific serologic tests. It is clear that the EBV-specific serologic tests cannot be used to rule out other, non-EBV diseases, because abnormalities of EBV-specific sero- ' logic tests occur in so many diseases not caused by EBV. The exclusion of patients with other diseases in the recent studies has been by multiple, extensive medical evaluations. Yet the EBV-specific serologic tests used in these research studies are being marketed to the lnedical comntunity as diagnostic tests for the evaluation of patients with persistent or relapsing syndromes of fatigue and depression. T h e practical value of EBV serologic tests used in this fashion is clearly limited, as a positive EBV serologic result (i.e., similar to those seen in some patients ill recent studies) neither establishes tile existence of chronic mononucleosis nor excludes the possibility of other diseases. The natnre of the marketing of these serologic 6
CHRONIC MONONUCLEOSIS[Merlin]
tests is i n d i c a t e d in the f o l l o w i n g quotes f r o m a mailing to physicians from a major national reference i m m u n o l o g y laboratory36:
TABLE I. Conditions Causing Aberrations of Epstein-Barr Virus-Specific Serologic Tests Similar to Those in Suspected Chronic Mononucleosis
. . . . is proud to announce its CHRONIC ACTIVE EBV DISEASE PANEL for the evaluation of children and adults with persistent or relapsing syndromes of fatigue, deprlession and dyslogia, sometimes with low grade fever, adenopathy and weight loss. This ill-defined, flu-like syndrome is well known and widely recognized by primary care physicians; it typifies many perplexing patients Whose complaints of disability are characteristically and inexplicably unaccompanied by "objective"findings on physical and laboratory examination (including their normal sedimentation rates). Now, however, two groups of investigators report data suggesting that chronic active or recrudescent E-B virus infections are common in such patients. It is hard to overemphasize the importance of these findings. Simply based on the description of the syndrome alone and its laboratory counterpart one can expect e n o r m o u s b e n e f i t s to patients who can finally be told of a possible organic basis for their illnesses which for so many patients would otherwise be interpreted as "neurotic" with resultant feelings of self doubt.
Primary immunodeficiencystates (e.g., ataxia telangiectasia) Secondary immtmodeficiency states (e.g., steroid therapy, antithymocyte globulin therapy) Hodgkin's disease Non-Hodgkin's lymphoma Leukenfias Systemic lupus erythematosus Endemic Burkitt's lymphoma Nasopharyngeal carcinoma AIDS AIDS-related complex Rheumatoid arthritis Multiple sclerosis Chronic renal failure Renal transplantation Nonlymphomatous malignant tumors Cancer chemotherapy
of chronic mononucleosis (table 1). Use o f the EBVspecific serologic tests in the medical evaluation of chronic fatiguing illnesses should be, at least, tempered by cautious awareness o f the range of diseases other than chronic mononucleosis that can cause abnormal results a n d lead to extensive medical evaluations. For a laboratory test to be reasonably clinically useful, a positive (or negative) test result must be a reasonable indicator that a patient has (or does not have) the disease in question. This is clearly not the case with regard to EBV-specific serologic tests and chronic mononucleosis. These tests clearly do not indicate a specific disease, nor do they exclude a range o f other diseases.
T r u e , these tests do, to some extent, suggest an organic basis for the illness, but only in the most vague, and clinically most useless, s e n s e - - t h e percentage o f ill persons who have abnormalities o f these test results is greater than that o f healthy persons. T h e tests do not, however, establish a diagnosis of chronic mononucleosis or chronic active EBV infection in the patient with a chronic fatiguing illness. T h e y suggest, vaguely, that such a disease might be present. T h e y clearly do not exclude the possibility o f other diseases, which is their great liability. Abnormalities of these test results, like those mentioned in the recent studies o f suspected chronic m o n o n u cleosis, are not specific for any disease.9,20.21,27-29,31-33 Positive EBV serologic test results might indicate that a patient has chronic mononucleosis, Hodgkin's disease, n o n - H o d g k i n s ' s l y m p h o m a , systemic lupus e r y t h e m a t o s u s , AIDS, multiple sclerosis, n o n l y m p h o m a t o u s t u m o r s , or a variety o f o t h e r diseases. This is not an unrealistic list. All o f these diseases may be manifested as chronic fatigue, even chronic fatigue after infectious mononucleosis; all are associated with abnormalities o f EBV-specific serologic test results, similar to those seen in suspected chronic m o n o n u cleosis. O f the 31 patients referred to Straus et al. 7 at the N I H for the evaluation o f "histories of chronic illness with fatigue after infectious mononucleosis (or with features o f that disease)," five were excluded because they were f o u n d to have not chronic mononucleosis but rather SjOgren's syndrome, systemic lupus el)'thematosus, lymphoma, Hodgkin's disease, or multiple sclerosis. A variety o f diseases, some o f them grave, can have both clinical presentations and abnormalities of EBV-specific serologic test results that would be indistinguishable from those o f the p u r p o r t e d disease
CONCLUSIONS
Epstein-Barr virus causes chronic infections in all persons that it infects. This chronicity entails a complex a n d poorly u n d e r s t o o d i m m u n o l o g i c relation with the host. T h e various EBV-specific serologic tests that have been developed show somewhat characteristic patterns o f changes with the usual acute infections, probably reflecting the complex reactions of the host to the productive a n d latent viral infections. Attempts have been m a d e to apply these EBV-specific serologic tests to persons with protracted symptoms resembling those o f infectious mononucleosis in o r d e r to show that the symptoms are d u e to active EBV infection. These attempts have shown abnormalities o f EBV-specific serologic test resnlts in groups o f patients with such symptoms, but have not shown distinct abnormalities or proved tile existence o f active disease in the patients. T h e EBV-specific serologic tests have not been shown to be useful diagnostic tools in the evaluation o f patients with chronic fatiguing illness outside an experimental context. Because of their nonspecificity, these tests have the potential to mislead physicians to overlook other signif7
HUMAN PATHOLOGY
Volume 1L No. 1 (January 1986)
icant diseases in patients with chronic vague symptoms.
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