SIR, Although recent studies have demonstrated a clear association of Epstein-Barr virus (EBV) infection with multiple sclerosis (Zaadstra et al., 2008; Lunemann and Munz, 2009; Salvetti et al., 2009), there has been much debate if and where the virus acts in the pathogenic cascade of multiple sclerosis and whether the virus needs to gain entry to the central nervous system (CNS). A recent study reported in Brain by Willis et al.(2009) showed that there is little evidence for the presence of EBV in the central nervous system of people with multiple sclerosis. These findings contrast greatly with the studies by Serafini et al.(2007) that have described abundant EBV positive cells in multiple sclerosis, and the presence of ectopic B cell follicles enriched with EBV infected cells in some patients. Willis and co-workers used a variety of validated methods to determine the presence of EBV in the CNS of patients with B cell infiltrates within the meninges and parenchyma. The paper described two crucial observations. Unlike Serafini et al.(2007), Willis et al. did not find the presence of EBV to be a characteristic feature of multiple sclerosis (aptly the title of the paper). Second, ectopic follicles, suggested by Serafini et al. to harbour EBV infected B cells, were not observed, despite scrutiny of meningeal tissues where these follicles should have been present.

Clearly, the issue of whether EBV is indeed present in the CNS is crucial not only to determine the impact of the virus on the disease in the CNS, but also for diagnostic pathology in general. The studies reveal the vagaries of pathological detection methods for infectious agents such as EBV. Care in the practice and interpretation of such methodologies are of course key for correct diagnosis and proving pathogenic involvement and ultimately, to ensure correct treatment of patients with multiple sclerosis. In our laboratory (VUMC), an in situ hybridization technique is widely used to detect EBV encoded RNA in various pathological specimens (Middeldorp et al., 2003) and our degree of positive samples does not differ significantly from that reported in literature. EBV encoded RNA is generally localized to the nucleus. This is the case for all EBV-associated malignancies, including Hodgkin’s disease, Burkitt’s lymphoma and brain lymphomas. EBV encoded RNA detected in the cytoplasm of cells in interphase, or in plasma cells or eosinophils are considered false positives, due to the application of over-sensitive methods (Gulley and Tang, 2008).