The study, approved by the Zahedan University of Medical Science Multiple Institutional Review Board, was conducted with all clinical samples from MS patients who were treated at the Department of Neurology, Ali-ebn Abitaleb Hospital, Zahedan, Iran, and also, Healthy Blood Donors (HBD) who voluntarily agreed to participate in this research at the central medical laboratory of Zahedan from December 2008 through July 2010. MS patients (in southeast of Iran) were diagnosed with Magnetic Resonance Imaging (MRI) and McDonald criteria were collected.18 We analyzed 201 different samples; 78 patients and 123 people as the healthy control group. The patient group comprised 22 men (mean age, 28.8 years; age range, 17–48 years) and 56 women (mean age, 30.3 years; age range, 16–52 years). The control group of HBD comprised 34 men (mean age, 26.4 years; age range, 17–42 years) and 89 women (mean age, 26.0 years; age range, 17–50 years).19 EDSS score for all patients at the time of inclusion were below scale 5.0, except of three individuals with secondary progressive MS (SPMS) [scale 6.5] and five with relapsing remitting multiple sclerosis (RRMS) [scale 5.0]. All patients had at least one annual relapse, during two years before inclusion in the study. Serum, PBMCs and unstimulated whole saliva samples were collected by standard methods. A total of 38 CSF samples (1.5mL) were collected from MS patients (RRMS=22, SPMS=6, primary progressive multiple sclerosis – PPMS=10) after lumbar puncture (LP) in sterile containers and were centrifuged for 15min at 180g at 20oC to obtain cell-free supernatants. Serum samples from 11 patients with RRMS and six patients with SRMS (17 samples in total) were obtained during periods of disease exacerbation and the relation was tested between defined EBV reactivation periods and exacerbation rate for a mean of one year. All Specimens were stored at −70°C until the experiment was performed. Multiple specimens were available for each patient, and all of them were tested. When possible, clinical materials were tested more than once.

EBV DNA extraction was performed for 100μL of samples using RIBO-prep nucleic acid extraction kit (Interlabservice, Moscow, Russia) according to the manufacturer's protocol. Real-time PCR was performed using the AmpliSens EBV-screen-FRT kit (Inter lab service) according to the manufacturer's protocol. This real-time PCR assay showed to be sensitive, specific, and reproducible. The assay has an internal control, which allows inefficient extraction or PCR inhibition to be detected. Real-time amplification was carried out using 10μL DNA eluate combined with 10μL PCR-mix-1-FL and 5μL PCR-mix-2-FL using Rotor-Gene 3000 Instrument (Corbett Research, Sydney, Australia) with the following cycling parameters: pre-denaturation at 95°C for 15min, 95°C for 5s, 60°C for 20s and 72°C for 15s for 45 cycles. Data acquisition was performed in both JOE/HEX/Yellow channel for EBV DNA and in the FAM/Green channel for β-Globin gene DNA during the annealing (60°C) stage. For quantification of EBV DNA two standard positive samples of KSG1 (104 copies per reaction mixture) and KSG2 (102 copies per reaction mixture) were included in the run (Interlabservice). Calculations of Ct, preparation of standard curve and quantification of DNA in each sample were performed by Rotor-Gene Operating Software, version 1.8 (Corbett Research).

Concentrations of serum EBV IgG VCA and IgM VCA were measured based on EIA method in an automated instrument, according to the manufacturer's instructions (Biotrin, The Rise, Mount Merrion, Co., Dublin, Ireland).

In this study, we considered reactive EBV infection, when IgG and IgM were positive by immunoassay, and/or two or more consecutive positive qPCR, and/or load EBV≥200 copies in serum, or ≥150 copies in both saliva and PBMNCs.

The Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA), Version 16 was used for statistical analysis. χ2 analysis was applied to analyze categorical variables, t tests for continuous variables and Mann–Whitney U tests for non-parametric variables. The nominal variable groups were compared by Pearson's correlation coefficient. All p-values are two-tailed and significant at p<0.05 or p<0.01 depending on the statistical method. Relative risk was calculated using the Word Processing, Database, and Statistic Program for Public Health Epi Info 6, Version 6.04B [Centers for Disease Control and Prevention (CDC), USA, World Health Organization, Geneva, Switzerland].

The study conformed to the Helsinki declaration and was reviewed and approved by the local research committee; written informed consent was obtained from all subjects.

Recent studies have demonstrated that at least 91.02% of MS patients are positive for EBV- specific IgG (IgG+) antibodies in contrast with 82.11% of healthy controls (Table 1). 100% of SPMS patients were IgG+ in their serum samples compared to 93.47% of the RRMS, and 80.95% of PPMS samples (Table 2). The detection of anti-EBV IgM from healthy volunteer donors and MS patients, independent of EBV-DNA detection in PBMCs, is indicative only of a new infection and found in 3.25% of controls but in none of the patients (p<0.05). Moreover, among MS subtypes, only SPMS (36.36%) and RRMS (15.21%) patients showed anti-EBV-IgM as a sign of reactivation in their serum in contrast with PPMS (p<0.001). On the other hand, patients had higher concentration of both IgM and IgG compared to controls (Table 1).

EBV DNA load in serum samples did not differed between MS patients and controls (Table 1). In the saliva samples, 50% of patients were EBV+ compared to 38.21% of the controls (Table 1). Viral DNA was found in all saliva samples that previously were positive for viral DNA in their PBMCs both in patients and controls. Saliva showed much higher prevalence of viral sequence than serum samples in controls (p=0.001). In the PBMCs samples, 67.94% of patients were EBV+ in contrast to 41.46% of the controls (Table 1). EBV DNA was detected only in seven CSF samples of RRMS (31.81%) and four CSF samples of SPMS (66.66%) obtained during an exacerbation but were not found in CSF of patients with remission or patients with PPMS (Table 2). As shown in Table 2, 80.43% of patients with RRMS, 90.9% of patients with SRMS and 28.57% patients with PPMS had EBV sequences (EBV+) in PBMCs. Six patients with RRMS (13.046%) and four patients with SRMS (36.36%) showed further positivity in all specimens (Table 2). Furthermore, among patients’ samples, 11 (14.1%) individuals showed positive results in all specimens in contrast to none of the controls (Table 2).

Systemic EBV infections were found in 68.75% of patients and in only 4.54% of controls (p<0.001). As a measure of reactivation, combined qPCR results and IgG serology showed that 16.66% of the patients had reactive EBV infections compared to 3.25% of controls. Reactive viral infection in these patients was confirmed by the detection of specific anti-EBV IgM antibodies in their serum. Viral DNA in serum and specific IgM antibodies in plasma were not detected in 82.11% of healthy controls (101/123) and 48.71% of patients (38/78). A strong association was found between EBV reactivation and MS attacks when MS primary stages (4.54%) were compared to other stages (p<0.001). Neither viral DNA in serum, nor the presence of IgM specific antibodies, or elevated titers of IgG antibodies to EBV were found in 8.69% of RRMS (4/46), 18.18% of SPMM (2/11) and 42.85% of PPMS (9/21), confirming that in these patients EBV infection remained latent. Episodes of defined EBV reactivation were observed in a subgroup (11 patients with RRMS and six patients with SRMS), and these episodes were associated with increased relative risk (RR) for disease exacerbation. In these subgroup of patients, the annual number of reactivation was 3.1 in the group of 11 patients who had one or more relapses, compared to 1.12 in the group of six patients who did not experience a relapse (p<0.05). In a 4-week period beginning two weeks before reactivation and ending two weeks after reactivation, the RR of relapse was 3.5 (p<0.05) compared to all other periods. Furthermore, all patients with disease exacerbation showed reactivated infection and EBV-DNA in their CSF samples. Prevalence of EBV-DNA and anti-EBV antibodies was demonstrated by a comprehensive analysis performed among males and females in both control and patient groups (Figs. 1 and 2). In all cases, female patients showed higher positivity (Fig. 1) and systemic EBV infection was more prevalent among females compared to males (p<0.001). Female patients with SPMS showed higher prevalence of EBV-DNA and anti-EBV antibodies compared to both males and other MS subtypes (Fig. 2). Increased EBV-DNA concentrations tended to be associated with EBV systemic infection, but associations with additional components such as MS subtypes and gender were even stronger.

Fig. 1.

Prevalence of EBV-DNA and anti-EBV antibodies among male and female in healthy controls and MS patients.

(0.65MB).

Fig. 2.

Prevalence of EBV-DNA and anti-EBV antibodies among male and female in different subtypes of MS.

(0.74MB).

Significant difference and positive correlation with concentration of EBV-DNA in saliva and EBV-DNA in serum were found in both groups (p<0.005), but a significant inverse correlation with EBV-IgG and IgM response was found only in the patient group (Tables 3 and 4). No correlation was found between detection of EBV-DNA in CSF and detection of EBV-DNA in other samples, or with EBV-IgG and IgM concentrations (Table 3). Serologically, immune status showed poor correlation with IgG concentration and detection of EBV-DNA in serum for both groups (p<0.005). There were no statistically significant correlations between detection of EBV-DNA in serum and EBV-DNA in PBMCs in both groups (Tables 3 and 4). There was direct correlation between EBV-IgG concentration and detection of EBV-DNA in PBMCs in both groups (Tables 3 and 4). Again, a positive correlation was observed between EBV-DNA in saliva and increased EBV-IgG concentration only among patients (Table 3). We found a positive correlation between the detectability of EBV-DNA in CFS from patients and exacerbation, as well as a decrease in EBV IgG/IgM ratio.