AAN 2000 Poster Presentation 2

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Active HHV-6 Viremia in Patients with Multiple Sclerosis: Correlation with Clinical Disease Relapse

Lorri J. Lobeck¹, Konstance K. Knox², , Eric F. Maas¹, Gregory Harrington, MD¹, and Donald R. Carrigan².
Wisconsin and Department of Neurology, Medical College of Wisconsin¹, 9200 W Wisconsin Ave, Milwaukee, WI 53226
Institute for Viral Pathogenesis², 10437 Innovation Drive, Milwaukee, WI 53226

Presented at the 125th Annual Meeting of the American Neurological Association
October 16, 2000
Boston, Massachusetts

Abstract

The studies described here assessed the use of a rapid culture technique to identify active human herpesvirus six (HHV-6) in the peripheral blood of patients with multiple sclerosis (MS) during disease relapse. Active HHV-6 viremia was detected in only 1% of healthy control subjects. In contrast, roughly 50% of patients with MS from two medical centers tested positive for the presence of active HHV-6 viremia. Study of paired samples from a portion of the MS patients demonstrated a significant correlation between clinical disease relapses and active HHV-6 viremia.

Introduction

HHV-6 is a cause of encephalitis in both immunologically compromised and immunologically intact patients.
The most frequently noted histopathological finding in HHV-6 encephalitis is demyelination.
In a recently published study, Knox et al. (Clin Infect Dis 2000; 31: 894-903) identified, by means of immunohistochemical staining, active HHV-6 infections in the brains of 8 of 11 (73%) of a cross section of patients with MS and only 7% (2/28) of normal and other neurologic disease controls.
In the MS patient derived CNS tissues, areas of active demyelination were positive for the HHV-6 infected cells more frequently (90%) than areas free of active demyelination (13%).
Based on these findings and the tropism of HHV-6 for blood leukocytes, the possibility was raised that the CNS lesions of MS are the result of invasion of the CNS by HHV-6 from the peripheral blood.
As an initial test of this hypothesis, blood samples from patients with relapsing-remitting MS were assessed by means of a rapid culture procedure that detects actively replicating HHV-6 in blood leukocytes.
Further, the relationship between new clinical disease relapses and the presence of such viremias was investigated.

Patients and Methods

Patients

Thirty-seven patients, with relapsing remitting MS had blood samples drawn at the time of a clinical relapse.
No patients were receiving corticosteroids when the specimens were drawn. Most patients received corticosteroid treatment after onset of their disease relapse.
A second specimen was drawn from thirty of the same patients six to ten weeks later.

Detection of Active HHV-6 Viremia

In this procedure, purified blood leukocytes from the patient are co-cultivated with human diploid fibroblasts. The fibroblasts are stained by indirect immunofluorescence with an affinity purified rabbit polyclonal antibody specific for the major immediate early protein (U89/U90) for HHV-6.
The detected infection in the patient’s leukocytes must be active since the infection has to be transferred into the target fibroblasts.

Results

Cross-Sectional Study of Active HHV-6 Viremia in Patients with MS

Results are summarized in Figure 1.
Table 1: For the Medical College of Wisconsin based patients, there were no significant differences between the HHV-6 positive and HHV-6 negative patients with respect to age, sex, disease duration, EDSS prior to relapse or severity of relapse.
There was also no significant difference in use of immunomodulator therapy.

Correlation Between Clinical Relapse and Active HHV-6 Viremia

When blood samples from patients with relapsing-remitting MS were obtained and assessed for active HHV-6 viremia at the time of clinical relapse, 50% (15/30) of the samples were positive (Figure 2). In contrast, when the same 30 patients were retested 8 to 10 weeks later (mean of 64 days), the positivity rate for active HHV-6 viremia was decreased to 17% (5/30) (p < 0.015 by two sided Fisher's exact test).
Patients were clinically evaluated at the time of the second sample; 37% (11/30) had returned to baseline, 50% (15/30) had significantly improved, 13% (4/30) had remained the same and 0% (0/30) had clinically worsened.

Click thumbnail to view larger version of figure.

Figure 1

Cross Sectional Study of the Incidence of Active HHV-6 Viremia in Symptomatic Patients with Multiple Sclerosis. The Kansas City patients are from a previously published study (Knox et al. Clin Infect Dis 2000; 31: 894-903). Normal control subjects consisted of healthy laboratory and hospital workers and well screened healthy blood donors.

Click thumbnail to view larger version of figure.

Table 1

Click thumbnail to view larger version of figure.

Figure 2

Positivity Rates for Active HHV-6 Viremia in MS Patients at the Time of a New Clinical Relapse and Same Patients Several Weeks Later.

When the time intervals between the two blood samples for each patient were analyzed, it was found that the interval was significantly shorter for those patients whose samples were congruent [i.e. negative/negative or positive/positive] than that for patients whose samples were incongruent [i.e. positive/negative or negative positive].
Congruent samples had a mean interval of 40 days (95% confidence interval of 36 to 45 days) compared to incongruent samples whose mean interval was 79 days (95% confidence interval of 49 to 110 days) ( p < 0.04 by Mann-Whitney Test).

Conclusions

Patients with relapsing-remitting MS are significantly more likely to have an active HHV-6 viremia at the time of clinical relapse than after the relapse has resolved.
Repeat testing of patients by the rapid culture technique showed that more closely spaced samples tended to test alike, while those separated by more time were more likely to be different.
Thus, HHV-6 viremia appears to be present intermittently in patients with relapsing-remitting MS.
The correlation between the presence of active HHV-6 viremia and clinical disease relapses in patients with relapsing-remitting MS observed suggests that HHV-6 may be playing a role in the disease. That role may involve a cytokine mediated immunopathologic process within the CNS or an autoimmune reaction due to antigenic similarity between viral proteins and the components of myelin.
The relatively noninvasive rapid HHV-6 culture technique should be incorporated into clinical trials assessing the role of anti-viral therapies for individuals with MS since the data obtained may provide insights as to which patients are most likely to respond to treatment.

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Active HHV-6 Viremia in Patients with Multiple Sclerosis: Correlation with Clinical Disease Relapse

Lorri J. Lobeck1, Konstance K. Knox2, , Eric F. Maas1, Gregory Harrington, MD1, and Donald R. Carrigan2. Wisconsin and Department of Neurology, Medical College of Wisconsin1, 9200 W Wisconsin Ave, Milwaukee, WI 53226 Institute for Viral Pathogenesis2, 10437 Innovation Drive, Milwaukee, WI 53226

Presented at the 125th Annual Meeting of the American Neurological Association October 16, 2000 Boston, Massachusetts

Abstract

Introduction

HHV-6 is a cause of encephalitis in both immunologically compromised and immunologically intact patients.

The most frequently noted histopathological finding in HHV-6 encephalitis is demyelination.

In a recently published study, Knox et al. (Clin Infect Dis 2000; 31: 894-903) identified, by means of immunohistochemical staining, active HHV-6 infections in the brains of 8 of 11 (73%) of a cross section of patients with MS and only 7% (2/28) of normal and other neurologic disease controls.

In the MS patient derived CNS tissues, areas of active demyelination were positive for the HHV-6 infected cells more frequently (90%) than areas free of active demyelination (13%).

Based on these findings and the tropism of HHV-6 for blood leukocytes, the possibility was raised that the CNS lesions of MS are the result of invasion of the CNS by HHV-6 from the peripheral blood.

As an initial test of this hypothesis, blood samples from patients with relapsing-remitting MS were assessed by means of a rapid culture procedure that detects actively replicating HHV-6 in blood leukocytes.

Further, the relationship between new clinical disease relapses and the presence of such viremias was investigated.

Patients and Methods

Patients

Thirty-seven patients, with relapsing remitting MS had blood samples drawn at the time of a clinical relapse.

No patients were receiving corticosteroids when the specimens were drawn. Most patients received corticosteroid treatment after onset of their disease relapse.

A second specimen was drawn from thirty of the same patients six to ten weeks later.

Detection of Active HHV-6 Viremia

In this procedure, purified blood leukocytes from the patient are co-cultivated with human diploid fibroblasts. The fibroblasts are stained by indirect immunofluorescence with an affinity purified rabbit polyclonal antibody specific for the major immediate early protein (U89/U90) for HHV-6.

The detected infection in the patient’s leukocytes must be active since the infection has to be transferred into the target fibroblasts.

Results

Cross-Sectional Study of Active HHV-6 Viremia in Patients with MS

Results are summarized in Figure 1.

Table 1: For the Medical College of Wisconsin based patients, there were no significant differences between the HHV-6 positive and HHV-6 negative patients with respect to age, sex, disease duration, EDSS prior to relapse or severity of relapse.

There was also no significant difference in use of immunomodulator therapy.

Correlation Between Clinical Relapse and Active HHV-6 Viremia

Patients were clinically evaluated at the time of the second sample; 37% (11/30) had returned to baseline, 50% (15/30) had significantly improved, 13% (4/30) had remained the same and 0% (0/30) had clinically worsened.

Table 1

Figure 2

Positivity Rates for Active HHV-6 Viremia in MS Patients at the Time of a New Clinical Relapse and Same Patients Several Weeks Later.

Congruent samples had a mean interval of 40 days (95% confidence interval of 36 to 45 days) compared to incongruent samples whose mean interval was 79 days (95% confidence interval of 49 to 110 days) ( p < 0.04 by Mann-Whitney Test).

Conclusions

Patients with relapsing-remitting MS are significantly more likely to have an active HHV-6 viremia at the time of clinical relapse than after the relapse has resolved.

Repeat testing of patients by the rapid culture technique showed that more closely spaced samples tended to test alike, while those separated by more time were more likely to be different.

Thus, HHV-6 viremia appears to be present intermittently in patients with relapsing-remitting MS.

The relatively noninvasive rapid HHV-6 culture technique should be incorporated into clinical trials assessing the role of anti-viral therapies for individuals with MS since the data obtained may provide insights as to which patients are most likely to respond to treatment.

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Presented at the 125th Annual Meeting of the American Neurological Association
October 16, 2000
Boston, Massachusetts