Repeatedly high levels of an Epstein-Barr virus (EBV) antibody on serial blood tests may offer a new way to distinguish multiple sclerosis (MS) from its closest inflammatory mimics — myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) and neuromyelitis optica spectrum disorder (NMOSD).

Results of the case-control study showed the odds of an MS diagnosis were significantly more likely in individuals with serially elevated Epstein-Barr nuclear antigen 1 (EBNA-1) peptide antibody levels — 300 times more likely than a diagnosis of MOGAD and 100 times more likely than NMOSD.

However, the association was not absolute. A small number of patients with neuroinflammatory diseases other than MS also had persistently elevated EBNA-1 peptide antibody levels, and some patients with MS lacked the antibodies altogether.

Still, lead author Hannes Vietzen, PhD, Center for Virology, Medical University of Vienna, Vienna, Austria, told Medscape Medical News that the findings show that the marker could serve as “a valuable adjunct to enhance diagnostic sensitivity and specificity of MS.”

The study was published online on March 9 in JAMA Neurology.

Diagnostic Challenges

Differentiating MS from MOGAD and NMOSD is challenging because of overlapping clinical and imaging features, particularly in seronegative cases. The investigators focused on EBNA-1 because EBV has been strongly linked to MS pathogenesis, and previous research suggests persistently high EBNA-1 peptide antibody levels may be largely specific to MS.

To investigate, the researchers evaluated plasma samples from 2091 patients with a confirmed neuroinflammatory disease and 1975 healthy control individuals. Samples came from centers in Austria, Germany, and the US.

A validation cohort of 183 patients included 142 with MS, 42 with MOGAD, and 17 with NMOSD. In addition, a 177-patient test cohort provided independent confirmation that serial EBNA-1 differentiates MS from MOGAD, and NMSOD.

Serial measurement proved to be key for optimizing sensitivity and specificity. Although EBV titers were more often elevated at first encounter in MS patients than in others, it was the persistence of the elevation that was required for differentiation that could be clinically useful.

High plasma titers of immunoglobulin G antibodies against a specific EBNA-1 peptide labelled 381-452 were measured with enzyme-linked immunosorbent assay at the time of initial evaluation and at three subsequent timepoints.

In the test cohort, 96.2% of those with MS vs 7.7% of those with MOGAD had elevated titers of the EBNA-1 381-452 peptide antibody, defined in a previous study as having an optical density of at least 1.7. This translated into an MS odds ratio (OR) of 303.4 (95% CI, 94.36-908.60).

Among NMOSD patients, 18.0% had elevated titers, producing an OR for MS diagnosis of 114.9 (95% CI, 94.4-908.6).

Of the 61 NMSOD patients, 12 were seronegative for the aquaporin-4 (AQP4) antibody, which was identified as diagnostic for updated NMOSD guidelines. Among these, only one patient (11%) had persistently high EBNA-peptide antibody titers, raising the OR of MS to 236.0 (95% CI, 178.6-2588.0).

In the validation cohort, the OR for MS relative to MOGAD (OR, 96.4; 95% CI, 26.6-293.0) and NMSOD (OR, 90.0; 95% CI, 19.70-319.87) were nearly 100-fold greater. 

Additional strategies to differentiate MS from alternative neuroinflammatory disorders might be particularly helpful in specific situations. In the case of AQP4-seronegative NMOSD, for example, there is a particular overlap of clinical and imaging features with MS, yet the treatments differ, Vietzen reported.

The fact that some of the treatments that favorably affect MS, such as sphingosine-1 phosphate modulators and natalizumab, pose a potential risk of triggering NMSOD relapse is a basis for seeking additional diagnostic tools.

MS EBV Seropositivity 100% Likely

Outside experts said the study findings fit into a broader body of evidence linking EBV to MS, while also raising questions about the role of the biomarker in clinical practice.

Joseph J. Sabatino Jr, MD, PhD, senior author of a 2025 review summarizing the EBV-MS relationship, said that the commonly cited estimate that EBV seroprevalence rises from about 90% in the general population to 99% among people with MS may still be an underestimate. 

“EBV seropositivity is likely 100% in patients with MS when considering assay sensitivity and increased diagnostic stringency,” Sabatino, assistant professor of neurology at the University of California San Francisco, told Medscape Medical News.

Rather than a risk factor, Sabatino said studies indicate that EBV is key to the etiology of MS because it triggers a B cell-related immune dysfunction in the central nervous system that produces autoimmune neuroinflammation.

The central role of EBV in MS risk was the basis for research evaluating elevated levels of EBNA-1 peptide antibodies as a “serological biomarker” for differential diagnosis.

It is widely recognized that EBV antibodies are less common in individuals with MOGAD, NMOSD, and other neuroinflammatory disorders, but whether that distinction is clinically meaningful remains unclear, Sabatino said.

Also commenting on the research for Medscape Medical News, Dalia Rotstein, MD, associate professor and MS specialist at the University of Toronto, Toronto, Ontario, Canada, said the case-control design and screening process were study weaknesses.

She said she would like to see the findings “replicated in a general MS cohort without reselection” in order to evaluate the potential clinical value of EBV-1 antibody titers as a discriminator.

More practically, Rotstein also expressed concern about the need for serial titer measurements to distinguish MS from other neuroinflammatory disorders.

“A need for serial measurements makes EBNA-1 titers less useful,” she said, noting that this by itself delays obtaining a measure that has only adjunctive utility.

Still, the problem of ambiguous findings and diagnostic delays in patients with symptoms of neuroinflammatory disease remains unresolved, Sabatino said. He acknowledged that persistently high EBNA-1 antibody levels have the “potential to assist with scenarios of clinical uncertainty,” but he believes it is too early to draw conclusions.

“There is a lot more research to do before that will be feasible,” Sabatino said.

Vietzen and Sabatino reported having no potential conflicts of interest. Rotstein reported financial relationships with Alexion, Amgen, Biogen, EMD Serono, Novartis, and Roche.