Multiple sclerosis and Epstein-Barr virus

1Montreal Neurological Institute, McGill University; 2Department of Epidemiology and Biostatistics, McGill University, and Centre for Clinical Epidemiology and Community Studies, Sir Mortimer B Davis Jewish General Hospital, Montreal, Quebec Correspondence and reprints: Dr Christina Wolfson, Director, Centre for Clinical Epidemiology and Community Studies, Sir Mortimer B Davis Jewish General Hospital, Room A127, 3755 Chemin de la Cote Ste Catherine, Montreal, Quebec H3T 1E2. Telephone 514-340-7563, fax 514-340-7564, e-mail tinaw@epid.jgh.mcgill.ca Received for publication June 5, 2000. Accepted January 24, 2001 RA Marrie, C Wolfson. Multiple sclerosis and Epstein-Barr virus. Can J Infect Dis 2002;13(2):111-118.

M ultiple sclerosis (MS) is an inflammatory, demyelinat- ing disease of the central nervous system (CNS) of unknown etiology.Recently, a multifactorial etiology was proposed, in which multiple environmental factors act together in a genetically susceptible individual to cause the disease (1).Geographic and temporal variation in incidence and prevalence, as well as an apparently age-depend-ent change in disease risk with migration, support an etiological role for environmental factors (1).
Viral infection is touted as a putative etiological factor in MS.Animal models of virally mediated CNS demyelination exist, although the mechanisms are unknown (2).Viruses of the herpesvirus family are of interest because of their neurotropism, ubiquitous nature and tendency to pro- Acute neurological complications occur in 5.5% to 18% of those with IM, and include encephalitis, transverse myelitis and postinfectious encephalomyelitis (5,6).These may occur in the absence of typical manifestations of IM (7).This finding prompted unsuccessful efforts to identify EBV in the brains of MS patients (8).This lack of success may be due to insensitivity of detection methods, the fact that EBV does not have an etiological role, or that EBV merely initiates or perpetuates the disease process.In the present article, we evaluate the epidemiological evidence that EBV infection increases the subsequent risk of MS.

METHODS
Searches of the medical literature (1965 to 1999) were conducted with subject headings 'demyelination' or 'multiple sclerosis' AND 'Epstein-Barr virus' or 'infectious mononucleosis' or 'case-control' or 'cohort' or 'seroepidemiol*' or 'risk' using MEDLINE and the Cochrane Library to identify observational studies.Bibliographies of all articles retrieved were searched for additional articles not indexed in either database.Case reports, case series and reviews were excluded.Twenty-five observational studies evaluating EBV infection (or more specifically IM) and MS etiology were identified.
Studies were categorized by methodology into three groups -seroepidemiological, case-control and historical cohort studies.Ratings of methodological rigour were assigned using criteria derived from published guidelines for the epidemiological study of MS (Appendix 1) (9,10).In each category, the criteria for a rating of 'A' were those considered to characterize an ideal study.The lowest rating assigned usually indicated a study lacking all of the ideal characteristics needed for an 'A' rating.Ratings between the highest and lowest ratings identified studies with some but not all of these ideal characteristics.Where necessary for complete evaluation of a study, prior publications referenced were retrieved.
The prevalence of antibodies in cerebral spinal fluid was measured by Bray et al (12) ('C' study) and Enbom et al (15) ('B' study).Enbom et al (15) found increased antibody prevalence and titres in cases.Another 'B' study by Sumaya et al (24) did not find increased titres among cases.The 'C' Some studies included patients without MS as cases or failed to report diagnostic criteria.The studies frequently failed to blind laboratory procedures or describe their sources of cases and controls.
The 'A' study by the Italian Multiple Sclerosis Study Group found no difference in the reported frequency of IM among cases and controls (29).
One of the 'B' studies found an increased risk of MS in subjects with a history of IM (35).The odds ratio (OR) was 5.5 (95% CI 1.5 to 19.7).The OR, when IM occurred after the age of 17 years, was 6.0 (95% CI 1.4 to 25.4).In this study, only 11 subjects of 225 cases and 900 controls had IM.While definitions of cases and controls were clearly described, the diagnostic criteria used were not previously established standard criteria such as those of Poser et al (36).The criteria used were created for the purposes of that study because of the limitations imposed by the information available in the database being used.None of the other 'B' studies detected a difference in the reported frequency of IM among cases and controls (27,28,33).Their sample sizes ranged from 81 to 225.Neither of the other 'B' studies assessing age at occurrence of IM found a significant difference between groups (27,33).
Souberbielle et al (34) ('C' study) also found no difference in the reported frequency of IM.Martyn et al (31) ('C' study) enrolled cases with acutely relapsing MS, optic neuritis or other isolated demyelinating lesions.Past exposure was assessed using an interview and serology.In seropositive individuals reporting a history of IM, the OR was 2.9 (95% CI 1.1 to 7.2).The OR, when IM occurred before the age of 17 years, was 7.9 (95% CI 1.7 to 37.9).
There were two 'D' studies (30,32).Operskalski et al (32) found that cases were much more likely than controls to report a history of IM (OR 17.0, 95% CI 2.0 to 81.8).This study used prevalent cases without stating diagnostic criteria, and a study collaborator interviewed all subjects.Lenman and Peters (30) interviewed 50 consecutive inpatients and outpatients with MS, and 50 matched controls from the same ward.Diagnostic criteria were not described.There was no difference in the reported frequency of IM.
Statistically significant differences emerged only in casecontrol studies with weaker methodologies and in one 'B' study.The diagnostic criteria were adequate in most studies,  but prevalent cases were enrolled, raising the problem of selective survivor bias.Lack of blinding in some studies and the uncertain accuracy of recalled, self-reported diagnoses of IM are important sources of measurement bias.

Historical cohort studies
Two historical cohort studies were identified, both using adequate diagnostic criteria (37,38).Both were rated as 'A' (Table 5).Lindberg et al (38) assembled a cohort of 494 individuals with heterophil antibody (HA)-positive IM from a hospital registry, and linked it to subsequent hospital records and an MS register.On comparison of observed and expected MS rates, the estimated relative risk in IM patients was 3.7 (no CI given).

DISCUSSION
There is geographic and temporal variation in MS incidence rates, and there is an apparent change in disease risk associated with migration between areas with differing risks of MS, depending on the age at migration (39).Migration studies have been interpreted to indicate the importance of exposure to an environmental factor in early life between the ages of 10 and 15 years (39).This has led to hypotheses that infections are etiological factors in MS and that the timing of these infections is important.
It has been argued that if MS is secondary to persistent viral infection or an immune reaction to a resident virus, it should be possible to identify the virus in the central nervous system at some phase of the disease, as has been the case in other chronic, progressive viral diseases (40).Others have tempered this argument, suggesting that in chronic infections, virus gene expression may be restricted, and traditional techniques may not be sensitive enough to detect viral material (41).Hilton et al (42) performed in situ hybridization with EBV-specific RNA probes on 21 plaques from 10 postmortem MS cases but were unable to detect a signal.Direct evidence of EBV infection of the CNS has not been demonstrated to date.However, in chronic infections, virus gene expression may be restricted, and traditional techniques may be insensitive for the detection of viral material (41).
Acute neurological complications are associated with IM (5,6).A single case series, consisting of five patients with primary EBV infection complicated by neurological involvement, described the development of classical MS in four patients and diffuse demyelinating disorder in the fifth (43).This argues for a role of EBV in the development of MS.
To date, evidence for an etiological role of EBV in MS exists largely in the epidemiological literature, which has examined only the evidence of systemic, not CNS, EBV infection.The etiological role of EBV in MS was reviewed in the present paper using seroepidemiological case-control and cohort studies.The strongest epidemiological evidence was derived from a randomized controlled trial (experimental study).This sort of evidence is obviously not available or appropriate in the study of risk factors for MS.Observational studies are the only feasible way of studying most questions concerning risk.Cohort studies provide the strongest level of evidence available.These studies have the advantage of establishing exposure without the bias of already knowing the disease outcome, and can assess the relationship between exposure and many diseases.Unfortunately, these studies require large numbers of subjects and prolonged follow-up, and are expensive.
Case-control studies provide the next level of evidence.These studies are easier to conduct than cohort studies, and are particularly useful for the study of rare diseases but are susceptible to bias.These studies are strongest if incident cases are used, thus avoiding the risk of selective survivor biases.
Seroepidemiological studies document evidence of prior infection but cannot establish when an infection occurred, its severity or even whether it was clinically symptomatic or asymptomatic.These studies are more appropriate for generating, rather than testing, etiological hypotheses.
There was a large variation in the quality and type of evidence available, and this impeded the synthesis of the data.Differences in the reporting of data and the amount of raw data available in the studies precluded statistical combination of the data.Whether the occurrence of IM as a manifestation of EBV infection or the occurrence of any EBV infection has differential importance is unclear, and all studies did not address the same question in this respect.
While both well designed ('A') historical cohort studies and one case-control study ('B') found increased risks of MS in subjects with HA-positive IM, results from other studies were less convincing.In historical cohort studies, the subjects were identified only if they had a test confirming exposure status, while in case-control studies, they were identified by disease status.This may explain the differences in their results.Overall, the evidence is insufficient to accept or reject the hypothesis that EBV increases subsequent risk of MS.A recent review concluded that there was evidence to support a role of EBV in the etiology of MS.Ascherio and Munch (44) evaluated studies comparing EBV serology in MS patients with controls, but did not examine the characteristics of the studies in detail.They concluded that the summary OR of MS comparing EBVseropositive individuals with EBV-seronegative individuals was 13.5 and that the strength of the association supported a role of EBV in MS.Several studies have demonstrated higher seroprevalence and higher antibody titres to other viruses, including measles, mumps and rubella (11,20,21).Elevated viral antibody titres have also been demonstrated in other disorders.One study, in particular, conducted by Shirodaria et al (21), demonstrated an increased rate of seropositivity, as well as elevated titres in MS and rheumatoid arthritis patients, compared with controls.There were no differences between the MS and rheumatoid arthritis patients.Serological data alone would thus seem insufficient to support a role of EBV in MS.
Given the establishment of latent infections, the association of neurological complications with IM and the above results, a potential etiological role for this virus remains attractive, although difficult to study.EBV may be one of many factors capable of causing MS in a genetically susceptible individual and may be neither a necessary nor a sufficient cause (1,45).Exposure is likely to be highly prevalent among subjects with and without MS, requiring large sample sizes to identify an effect.MS is relatively rare, probably with a long latent period between exposure and symptom onset, making it difficult to verify the temporal relationship between exposure and disease onset.It would be of interest to determine whether EBV infection alone or its clinical manifestation as IM is of more importance in MS risk.Does EBV interact with other exposures or genetic factors to cause MS?Further study, ideally using large samples of incident cases with blinded, trained interviewers using confirmatory sources for recalled data, is justified to explore these and other questions.

TABLE 1 Seroepidemiological studies -Characteristics
(4)S Clinically definite multiple sclerosis; CPMS Clinically probable multiple sclerosis; CPoMSClinically possible multiple sclerosis; LSDMS Laboratorysupported definite multiple sclerosis; LSPMS Laboratory-supported probable multiple sclerosis; MS Multiple Sclerosis; NR Not reported duce latent, recurrent infections(3).Epstein-Barr virus (EBV), in particular, is hypothesized to play an etiological role in this disorder.Acute infection leads to lifelong, latent infection of B lymphocytes(4).By adulthood, 90% of the population has been infected with EBV, as demonstrated by antibodies to this virus.Infection is often mild or not apparent in young children, while infection later in life may present as infectious mononucleosis (IM).

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TABLE 4 Case-control studies -Methods and results
*Blinded to subject status and/or study hypotheses; † Statistically significant results.IM Infectious mononucleosis; NR Not reported

TABLE 5
Haahr et al (37)used a Danish registry comprising all patients with positive HA tests between1968 and 1978 (except 1975), and all patients with negative HA tests in 1978 and 1968 to 1970.On comparison of observed and expected MS rates, the estimated relative risk among the HA-positive group was 2.8 (no CI given).The advantages of using registries include elimination of recall bias and reduction of selection bias.

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