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Primary and Recurrent Herpes Simplex Virus Type 2-Induced Meningitis Tomas Bergstrom, Anders Vahlne, Kjell Alestig, Stig Jeansson, Marianne Forsgren, and Erik Lycke

~rom

the Departments of Clinical Virology and Infectious Diseases, University of G6teborg; and the Department of Virology, Central Microbiological Laboratory, Stockholm County Council, Sweden

Although herpes simplex virus type 2 (HSV-2) can cause futal encephalitis in neonates or immunocompromised patients, severe central nervous system (CNS) infections of this virus postnatally are rare [1]. However, when reaching the CNS, HSV2 might induce a nonfutal infection of the meninges [2]. In contrast to HSV-2, HSV type 1 (HSV-l) is the most common etiologic agent in sporadic necrotizing encephalitis [1]. In patients with primary genital infection, symptoms such as stiff neck, headache, and photophobia are common, suggesting a probable involvement of the meninges [3]. However, because lumbar puncture is infrequently used in venereologic or gynecologic diagnosis, meningitis is seldom confirmed. On the other hand, in patients with serous meningitis who are admitted to other wards, history and symptoms of genital HSV-2 infection may be overlooked. Consequently, few patients with confirmed HSV-2-induced meningitis have been described [2, 4-11]. Complications such as persistent myalgia [4], ascending myelitis [2], urinary retention [2, 10, 11], and sensory polyneuropathy [11] have been noted, but the longterm outcome of these cases has not been properly studied. HSV-2 has also been suggested as the causative agent in recurrent meningitis [4, 5, 7]. However, patients with HSVinduced recurrent meningitis pose diagnostic problems. Except for one report of HSV-l isolated from the cerebrospinal fluid (CSF) [12], HSV seems not to have been isolated from the CNS of patients with recurrent meningitis. HSV-2-induced recurrent blisters in the pelvic area have been reported as preceding meningitis in a few cases [4, 7]. Genital recrudesReceived 18 September 1989; revised 16 January 1990. Financial support: Swedish Medical Research Council, Goteborg Medical Society, and Medical Faculty, University of Goteborg. Reprints and correspondence: Dr. Tomas Bergstrom, Department of Clinical Virology, University of Goteborg, Guldhedsgatan 10 B, 41346 GOteborg, Sweden. The Joumal of lDfectious Diseases 1990;162:322-330 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6202-0005$01.00

cence may escape the clinician's notice by a hidden localization (e.g., in the cervix), or the lesion may be healed at onset of recurrent meningitis. We present here the clinical course and laboratory findings of 27 patients followed from their first episode of confirmed HSV-2-induced meningitis through episodes of recurrent meningitis and of 7 additional patients with recurrent meningitis possibly due to HSV':2. Because patients with reactivated HSV seldom show signs of an ongoing infection by conventional serologic assays, a possible method for assessing recurrent HSV infection of the CNS would be to measure the patients'intrathecal antibody responses. Immunoblotting analyses of intrathecal IgG antibodies to individual HSV-2 proteins were performed in patients from whom paired serum and CSF samples were available and were compared to virus isolation and antigen detection.

Materials and Methods Patients. Patients (27: 9 men, 18 women; mean age, 26.4 ± 7.2 years) with HSV-2-induced meningitis were selected on the basis ofthe following diagnostic criteria: isolation of HSV-2 from the CSF during the first episode of meningitis (21 patients) or seroconversion against HSV-2 concurrent with the first episode of meningitis (6 patients). Seven patients with recurrent meningitis possibly due to HSV-2 were selected on the basis of HSV-2 isolation from peripheral lesions concurrent with episodes of meningitis (5 patients) or findings ofIgG antibodies against HSV-2 by ELISA in CSF samples collected during episodes of meningitis (2 patients). Three patients fulfilled both criteria. Virologic diagnoses of all patients were performed in either the Department of Clinical Virology, University of GOteborg or the Central Microbiological Laboratory, Stockholm County Council, during 1978-1987. Clinical data were collected from patient records, where host vulnerability factors had been noted according to a structured questionnaire. Controls. CSF samples were collected from 12 patients under-

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In 27 patients with a first episode of herpes simplex virus type 2 (HSV-2)-induced meningitis, confirmed by virus isolation from the cerebrospinal fluid (CSF) or seroconversion to HS\'-2, initial neurologic complications were found in 10 (37%) but subsided before 6 months in all patients. Long-term complications were recurrent meningitis in 5 (19%) and periodic headache related to genital HSV recrudescences in 4 (15%). Seven additional patients had possible HS\'2-induced recurrent meningitis. In contrast to the first episode of meningitis, virus isolation, HSV antigen detection, and IgG analyses in consecutive serum samples were of no diagnostic value in episodes of HS\'-2-induced recurrent meningitis. Instead, immunoblotting was used to assay intrathecally produced IgG antibodies to the HS\'-2 type-specific protein gG-2 in recurrent meningitis, when CSF was collected at a minimum of 3 days after onset.

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Table 1. Comparisons of diagnostic methods used with 27 patients with first episode of confirmed herpes simplex virus type 2 (HSY-2)-induced meningitis and in their 10 recurrences.

Seroconversion or rise in titer to gG-2 Isolation of HSV-2 in CSF HSV antigen detection in CSF Intrathecal IgG antibodies to gG-2 (immunoblot) NOTE.

First episodes (no. positivel no. tested)

Recurrences (no. positivel no. tested)

17/17 21/27 5/7

OlIO OlIO OlIO

3/7

3/6

CSF = cerebrospinal fluid.

To assess intrathecal IgG antibody production, paired CSF and serum samples were diluted to an IgG concentration of 10 mg/l and allowed to react for identical time periods during all steps of the procedure. On strips incubated with CSF, stronger reactions against HSY-2-specific proteins than seen on strips incubated with serum samples from the same patient were judged as evidence of intrathecally produced IgG antibodies to HSY-2. The designations of the different HSY-2-infected cell polypeptides used were those described by Powell and Courtney [22]. Polypeptides were identified on strips by use of mouse MAbs or polyclonal rabbit antibodies obtained after immunizing animals with purified HSY-2 antigens. Radioimmunoprecipitation (RIP) was performed by allowing 100 #-tl ofpatient serum, CSF samples, or anti-gG-2 monospecific rabbit serum (obtained after immunizing a rabbit with monoclonal-immunosorbent-purified gG-2) to react with [3H]glucosamine-Iabeled HSY-2 antigen under gentle agitation for 1 h at 4°C as described by Olofsson et al. [16]. A 10% (wt/wt) suspension (200 #-tl) of Staphylococcus aureus (strain Cowan 1) was added, and the mixture was subjected to gentle agitation for 1 h at 4°C. After washing and centrifuging, the pellets were solubilized and subjected to PAGE as described for immunoblotting. The gels were treated with amplifier (Amersham, Solna, Sweden), dried, and subjected to autoradiography on Hyper-MP film (Amersham). [l4C]methylated proteins (14,300-200,000, Amersham) were used as molecular weight standards.

Results Patients with Confirmed HSJL2-Induced Meningitis HSV isolation and antigen detection. HSV was isolated from CSF during the first episode of meningitis in 21 patients, and all isolates were typed as HSV-2. For the 6 additional patients in whom the diagnosis was based on seroconversion against HSV-2, attempts to isolate virus from CSF were negative. HSV antigen detection was positive in CSF of 5 patients (2 of whom were HSV isolation-negative) and negative in 2 patients (also HSV isolation-negative) with the first episode of meningitis (table 1). In patients with the first episode of meningitis, HSV-2 virus isolation was attempted from peripherallesions in 10 patients and was successful in 7, 4 of whom were included on the basis of seroconversion. In the other

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going spinal anesthesia for orthopedic operations. None showed signs or symptoms of infection. Serum and CSF samples were drawn from 20 patients with serous meningitis (10 patients from whom enterovirus and one from whom parotitis virus was isolated from CSF, 1 with serologic evidence of Epstein-Barr virus infection, and 8 with serous meningitis of presumed viral etiology but with negative virus isolation from CSF and negative serology for HSY, enterovirus, and parotitis virus) and from 6 patients with meningitis and serologic signs of infection with Borrelia burgdoiferi. Serum and CSF samples were also collected from 11 patients with suspected noninfectious neurologic disorders, selected for presence of HSY-2 serum antibodies (see below) but lacking intrathecally produced HSY antibodies as diagnosed by ELISA. CSF studies. Cells were counted in a Fuchs-Rosenthal chamber. Cytologic preparations were done in a cytocentrifuge, and the cells were stained with May-Griinewald-Giemsa solution. The protein concentration of the CSF was determined as described by Lowry et al. [13]. IgG WetS quantified by electroimmunodiffusion in agarose gel [14]. HSV isolation and antigen detection. CSF samples and secretions from peripheral blisters were inoculated onto cell cultures of green monkey kidney (GMK-AHl) and Yero cells, which were then incubated at 37°C and read daily for 14 days. HSY isolates were typed using monoclonal antibodies [15, 16]. Detection ofHSY antigen in CSF samples was attempted by using a newly developed cyclical enzyme-amplified detection system based 011 monoclonal antibodies (MAbs) (Wellcozyme HSY; Wellcome, Stockholm) of reported high sensitivity and specificity [17, 18]. The CSF samples were incubated for 24 h at 37°C; otherwise the protocol of the manufacturer was followed exactly. Serology. HSY-specific IgG antibodies in serum and CSF samples were determined by ELISA as described [19, 20]. The HSY-l antigen was a type-common membrane-associated antigen, that is, reactive with antibodies to both HSY-l and HSY-2. The HSY-2 antigen, purified on a Helix pomatia lectin-coupled sepharose column that binds preferentially to clustered O-linked oligosaccharides, consisted of a single glycoprotein that was identified as glycoprotein G (gG-2) [16] and reacted selectively to HSY-2 [20]. Morbilli antibodies were determined by ELISA using antigen produced by infection of GMK-AHI cells. Antibodies to B. burgdoiferi were analyzed by immunofluorescence. Further characterization of the HSY-2 IgG response was done by immunoblotting. HSY-2 antigen was prepared by infecting confluent monolayers of HEp-2 cells with HSY-2 (strain B4327UR; S. Jeansson, GOteborg) at a multiplicity of infection of 10 plaque-forming units/cell. Cells were harvested after a 24-h incubation, washed, and pelleted in an Eppendorf centrifuge. Pellets were solubilized in 0.5 ml of sample buffer (0.07 MTris-CI, pH 6.8, 10% glycerol, 2% sodium dodegl sulfate, 5 % mercaptoethanol, and bromphenol blue dye) and ultrasonicated. Antigens were separated by polyacrylamide gel electrophoresis (PAGE) using 9.25 % polyacrylamide gels linked with N, N'-diallyltartardiamide and transferred to nitrocellulose membranes as described by Towbin et al. [21]. Strips were washed in Tris-buffered saline with 0.3% Tween and incubated with serum and CSF samples overnight after blocking with 3 % bovine serum albumin and 4 % fetal calf serum. Peroxidase-labeled rabbit anti-human IgG (DAKO, immunoglobulin a/s, Copenhagen, Denmark) was used as conjugate and orthodianisidine as substrate. Sera were used in a dilution of 1:100 when consecutive samples were studied.

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Table 2. Findings in cerebrospinal fluid (CSF) from 27 patients with confirmed herpes simplex virus type 2-induced meningitis during the first episode and during recurrences. First episode (n = 27) No. of leukocytes/mm3 No. of mononuclear cells/mm3 Protein (gil) Glucose (mmol/l)

431 367 1.6 3.0

± ± ± ±

316 (4-1100) 293 (4-1070) 1.1 (0.4-3.0) 0.7 (1.5-4.5)

Recurrences (n = 7) 280 263 1.0 3.5

± ± ± ±

170 (77-546) 166 (72-528) 0.3 (0.7-1.5) 0.9 (2.3-4.8)

Non-HSV serous meningitis (n = 20) 247 164 0.8 3.1

± 217 (28-696) ± 119 (14-384) ± 0.4 (0.3-1.5)

± 0.6 (2.4-4.2)

NOTE. Values are mean ± SD (range). No patient had an acellular CSF.

(mean ± SO) and 19.7 ± 8.3 days to seroconversion (mean ± SO). eSF antibodies (dilution 1:10) to the HSY-2 type-selective gO-2 antigen were detected by ELISA in one of nine patients examined during the first episode of meningitis and in all 10 recurrences in five patients. Antibodies to morbilli virus were not detected in any sample despite high titers (1600-3200) in corresponding serum samples. CSF antibodies to gO-2 were not detected by ELISA in any ofthe controls with normal eSF, serous meningitis of other origin, or other neurologic diseases. CSF findings. The cytological findings and protein and glucose concentrations in eSF collected at the first episode of meningitis (27 specimens) and at seven recurrences from follOW-tip of five patients are shown in table 2. Mean numbers of cells and protein concentrations were elevated, and this finding was more pronounced in CSF collected at primary episodes ofmeningitis compared with samples collected at recurrences. Mononuclear cells were predominant in all samples tested. Mean eSF values ± SO for the first episodes of meningitis of the six patients for whom diagnosis was based on seroconversion were 324 ± 342 cells/mm3 ; 283 ± 299 mononuclear cells/mm3 ; 1.1 ± 0.6 g of protein/I; and 2.8 ± 0.3 mmol of glucose/I. Clinical course and complications. Oenitallesions were noted in 23 patients and preceded meningitis by 7.5 ± 5.9 days (mean ± SO; range, 3-27 W,iys). All patients showed symptoms consistent with meningitis, that is, severe headache and stiff neck. Headache was usually described as intense and developed during 2-3 days in most patients. Neurologic symptoms remaining when the meningitis subsided were considered as complications (table 3). A total of 26 neurologic complications were noted in 17 patients (6 men, 11 women). Ten patients experienced complications with onset during the first episode of meningitis (considered initial complications), and 4 showed more than one complication. A woman with urinary retention also had paresthesia of the left leg and foot. Another woman with urinary retention had motor weakness in one leg and alternating neuralgia in both legs. One woman who developed dysesthesia in the pelvic area, left hand, left leg, and right foot also showed weakness in the abdominal muscles. In these patients, initial complications occurred between 7 days and 4 months,

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13 patients, in whom genital lesions preceded meningitis, virus isolation was not performed. Five patients, all with diagnoses confirmed by recovery of HSY-2 from eSF, experienced recurrences of meningitis. Yirus isolation and HSY antigen detection were negative in all eSF samples collected during these 10 recurrences, but HSY-2 was isolated from peripheral genital lesions during both recurrences in one patient (who also had HSY-2 isolation-positive lesions on her left elbow during the last recurrence) and all 3 recurrences in one patient but not from HSY-typical genital lesions in one patient who had 1 recurrence. Of the two patients with no peripheral lesions, one had tonsillitis during all 3 recurrences ofmeningitis, but virus isolations from throat secretions were negative. HSV antibodies. Patients in whom serologic data indicated that the HSY-2 infection occurred without prior HSY-l infection are described as having primary HSY infection. Two of the HSY-2 eSF isolation-positive patients already had HSY serum IgO antibodies (as judged by the type-common HSY-l antigen, though they were negative in the HSY-2 ELISA) at the onset ofmeningitis (the only serum sample available) and were therefore not considered as having a primary HSY infection. Serum samples were not available from four patients. In one patient, a low HSY-l IgO titer was found at onset and did not rise significantly. HSY-2-specific serology was not done because of lack of material, so it could not be determined whether her eSF HSY-2 isolation-positive meningitis resulted from a primary or secondary HSY infection. Serologic evidence of primary HSY infection was obtained for the other 20 patients. Three patients from whom HSY-2 was isolated from the CSF were also considered as having primary HSY infection, as the only serum samples available for serologic analysis and obtained at onset ofmeningitis were HSY-l and HSY-2 IgO-negative. Seventeen patients showed seroconversion (defined as change from negative to a titer ~log 2.3) against the type-common HSY-l and the type-specific HSY-2 antigen in ELISA, including the 6 for whom diagnosis was based on seroconversion. Mean log titers to HSY-2 after seroconversion in the 17 patients were 2.86 ± 0.41, and mean time to seroconversion was 20.6 ± 13.0 days (±SO). Corresponding results for the 6 patients for whom diagnosis was based on seroconversion only were log titer 2.75 ± 0.34

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Table 3. Complications of confirmed herpes simplex virus type 2-induced meningitis in 27 patients. No. of patients

Complication Urinary retention Dysesthesia, paresthesia Neuralgia Motor weakness, paraparesis Concentration difficulties (rv 3 month's duration) Impaired hearing Permanent sequele at 6-month follow-up Recurrent meningitis Periodic headache not diagnosed as meningitis None

4 4 3 3 2 1

o 5 4

10

6 years, and one for 9 years), 10 recurrences of meningitis followed during the observation period (3 in two patients, 2 in one patient, and 1 in two patients), with a mean interval ± SD of 24 ± 21 months (range, 4-60 months). Four patients also suffered from repeated episodes of severe headache, and four additional patients had periodic headache not diagnosed as recurrent meningitis. One patient who develoPed recurrent meningitis and one patient with periodic headache suffered from acute complications during their first episodes of meningitis. Concerning possible host vulnerability factors, the patients' histories were unremarkable except for three who had been admitted for parotitis-induced meningitis during childhood. None of the patients with a first episode of HSV-2 meningitis admitted homosexual contacts. One patient (CSF HSV-2 isolation-positive) might have had a concurrent brucellosis according to clinical and serologic findings. No findings supported the occurrence of other infections, despite diagnostic efforts aimed at parotitis virus, HSV-l, varicella-zoster virus, cytomegalovirus, human immunodeficiency virus (HIV), enterovirus, and B. burgdorferi.

Patients with Possible HSV--2-Induced Recurrent Meningitis Clinical and laboratory findings in seven patients with recurrent meningitis possibly due to HSV-2 are shown in table 4. One of them, at 26 years of age in 1950, fell ill with penile blisters, fever, headache, and general malaise. Lumbar puncture findings suggested acute meningitis, but no attempts to isolate virus were made. After being free of symptoms for 1 year, he suffered from recurrent vesicular lesions on his right gluteal region or penis as often as once every month. Meningitis followed at irregular intervals, and CSF samples repeatedly demonstrated pleocytosis and a preponderance of mononuclear cells. On several occasions, HSV-2 was recovered from his skin vesicles. He demonstrated concentration difficulties

Table 4. Clinical and laboratory findings (from last episode) in seven patients with recurrent meningitis possibly due to herpes simplex virus type 2 (HSV-2).

Patient no., gender (y) 1, F (30) 2, F (31) 3, M (35)

Type of lesion/HSV-2 isolation

4/0.5 4/4 3/4

4, M (40) 5, M (41) 6, F (41)

9/4 2/6 5/0.5

7, M (64) Mean ± SD NOTE.

No. of episodes of meningitis/ interval (y)

CSF

25/2

Genital/Sacral/+

Cells/mm3 (mononuclear cells)

Protein (gil)

Glucose (mmol/l)

10/1600 20/3200 10/400

160/180 64/70 82/96

1.0 1.1 1.6

3.2 3.6 3.0

Neuralgia Neuralgia

40/3200 ND/I600 20/800

334/334 460/460 98/98

0.7 0.8 0.5

3.3 3.4 3.0

Neuralgia

ND/6400

Complications Depression, neuralgia

Anal/-

Oral/+ Sacral/+ Genital/+ Sacral/+ Shoulder/+ Glutea1/+

CSF measurements

ELISA HSV-2 titers (CSF/serum)

16/26 173

= cerebrospinal fluid,

ELISA

= enzyme-linked immunosorbent assay, + = HSV-2

isolated, -

± 162/181 ± 159 = HSV-2 not isolated,

1.0 1.0 NO

± 0.4

= not done.

3.2 3.2

± 0.2

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but all the permanent symptoms had subsided at follow-up at 6 months. A 30-year-old woman had an isolation-positive and serologically confirmed primary HSV-2 genital infection followed by an erythema multiforme-like rash on her legs. She develoPed paresthesia in both feet that gradually involved both legs. Six days later she showed symptoms and signs ofa mild meningitis complicated by urinary retention, constipation, and radiating pain from the pelvic area to the feet. Examination of her CSF showed normal concentrations of protein and glucose and a low-grade pleocytosis with only mononuclear cells. Neurologic examination revealed a dysesthesia from toes to waist and enhanced tendon reflexes in both legs, which later changed to hyporeflexia. She was unable to walk because of weakness in both legs, where neuralgia was present. The course was suggestive of ascending myelitis. After 1 week her condition improved and her mild CSF pleocytosis vanished. At dismissal from the hospital after 14 days, genital-tract pain was the only symptom remaining; she fully recovered after 1 month. However, sensory symptoms have returned regularly at menstruations during 3 years of follow-up. In five patients (three patients followed for 3 years, one for

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and neuralgia after each episode of meningitis, but a recent neurologic examination failed to show permanent sequelae. He experienced his 26th episode of meningitis in 1988. From this group of seven patients, virus could not be isolated from 23 CSF samples drawn during episodes of meningitis. Likewise, HSV antigen was not detected in the 5 CSF samples examined. Peripheral HSV-like lesions, which made us suspect HSV as a causative agent for recurrent meningitis in these patients, preceded episodes of meningitis in all except one patient, in whom peripheral lesions were not related in time to episodes of meningitis. HSV-2 was repeatedly isolated from lesions in five patients. In one patient, HSV-2 was not isolated from genital lesions, but her husband had HSV-2 isolation-positive recurrent vesicles in the gluteal region combined with neuralgia. Another patient was a homosexual man with recurrent anal and oral HSV-like lesions that were isolation-negative. All patients had serum IgG antibodies to the type-selective HSV-2 antigen and in higher titers than to the type-common HSV-l antigen (unpublished data). HSV-2 IgG antibodies (but not morbilli antibodies) were also found in CSF during recurrent meningitis in all five patients from whom paired serumCSF samples were available for testing. Cytologic examination of CSF samples from the most recent episodes of meningitis showed pleocytosis with almost total dominance of mononuclear cells. Protein levels were

moderately elevated and glucose concentrations were normal in all patients. Although neuralgia in the areas of peripheral lesions were experienced at the onset of recurrent meningitis by three patients, no neurologic deficits were found by clinical examination. In addition to neuralgia, one patient suffered repeatedly from depression at the onset of recurrent sacral HSV-2 isolation-positive lesions. Another patient showed symptoms of a persistent neuropathy with continuous paresthesia, dysesthesia, and neuralgia in both legs, slightly aggravated during eruption of vesicles.

Patients with Confirmed or Possible HSV-2-Induced Meningitis: Immunoblotting and RIP In 17 patients tested who had confirmed HSV-2 meningitis (including the 6 for whom diagnosis was based on seroconversion to HSV-2 in ELISA), immunoblotting performed on sera collected during and after the first episode of meningitis was consistent with seroconversion against HSV-2 by ELISA (figure 1). CSF IgG antibodies were more strongly reactive to gG-2 than to glycoproteins Band D during the first episodes of meningitis caused by a primary HSV-2 infection. In three CSF samples from 1patient, maximum intensity of this reactivity was noted on 'day 9 after onset of the meningitis. When intrathecal HSV-2 antibody production was assessed on sera and CSF diluted to equal IgG concentrations of 10 mg/I, CSF from 3 patients (collected 4, 5, and 6 days after the onset of meningitis) gave a markedly stronger reaction when compared visually with the paired sera (table 1, figure 2). In 4 patients, CSF was collected within 2 days after the onset of meningitis; in 1 of these equal reactivity was seen compared with the paired serum. The remaining three CSF samples were considered negative. In recurrent meningitis, no changes in immunoblot pattern were found in sera collected at onset and after episodes of meningitis (figure 1, table 1). CSF IgG antibodies were found to all proteins that induced serum antibodies. CSF from six of nine patients tested (three with recurrences after a confirmed first-episode HSV-2-induced meningitis, and three with probable HSV-2-induced recurrent meningitis) possessed markedly stronger reactivity to gG-2 when compared visually with the paired sera (figure 2, lanes Cs and Sd)' All positive CSF samples were collected 3-5 days after the onset of symptoms of meningeal irritation. In the three remaining patients with recurrent meningitis, samples were drawn on the first day after onset, and no intrathecal anti-HSV-2 antibody production was noted. All controls with B. burgdoiferi-induced meningitis (n = 6) and serous meningitis of viral origin (n = 20) were negative for CSF-derived HSV-2 antibodies by immunoblotting, although 4 were seropositive for HSV-2 (unpublished data). Likewise, no evidence of intrathecally produced HSV-2 antibodies were seen in 11 HSV-2 high-titer-seropositive patients

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Figure 1. Immunoblotting images of IgO antibodies to HSV-2 in successive serum samples in a patient followed through the first episode of HSV-2-induced meningitis showing seroconversion from acute (a) to convalescent sera after 2 weeks (2w) and through three recurrences during which no differences were noted between acute and convalescent sera after 1 (lw) and 2 weeks (2w). At right are an HSV-I and -2-seronegative (n) and an HSV-l-seropositive, HSV-2seronegative human serum sample (1). The HSV-2 gO-2 is identified by rabbit serum obtained after immunization with immunosorbentpurified gG-2. Molecular weight markers are indicated at left.

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1

2 Presence of antibodies to gG-2 in CSF in the six patients with evidence of intrathecal gG-2 antibodies by immunoblotting was confirmed by RIP of glucosarnine-Iabeled HSY-2 antigen. Figure 4 shows autoradiographic images of immunoprecipitation with monospecific rabbit anti-gG-2 antibodies and serum and CSF from a patient with possible HSY-2-induced recurrent meningitis (patient 6, table 4).

100 _

Discussion 69 _

Figure 2. Intrathecal anti-HSV-2 IgG antibodies during the first episode and three recurrences of HSV-2-induced meningitis in a patient. Serum and CSF samples were diluted to an equal concentration of 10 mglml IgG and compared visually. The stronger reactivity noted on lane C6 (CSF from day 6 after onset of first episode of meningitis) and lane Cs (CSF from day 5 after onset of fourth episode) compared with their paired and equally diluted serum samples (Sd) was taken as evidence of intrathecal synthesis of HSV-2 antibodies. In lane S, the serum sample from the fourth episode of meningitis was used in the standard dilution of I:100 for comparison. Molecular weight markers are at left. Positions of gG-2 were identified using the rabbit serum described in figure 1 and glycoproteins B and D by monoclonal antibodies.

with neurologic disorders of suspected noninfectious origin (figure 3). All CSF samples from uninfected orthopedic patients lacked HSY-2 antibodies as determined by immunoblotting. n

Figure 3. Serum (s) and CSF (c) samples from 11 controls with various neurologic disorders of presumed noninfectious origin and with high (>800) IgG anti-HSV-2 antibody titers in serum, diluted to an equal IgG concentration of 10 mglml. Reactions on HSV-2 strips incubated with CSF samples were weaker compared with those on strips incubated with corresponding serum samples, judged as absence of intrathecal antibodies to HSV-2. The positions of rabbit serum to gG-2 (r), HSV-l seropositive, HSV-2 seronegative human serum (I), HSV-I and HSV-2 seronegative human serum (n), and gG-2 (in brackets) are shown at left.

1

se

se

se

se

se

se

se

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sc

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se

_ 100

_ 69

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The incidence of genital HSY-2 infections appears to be increasing [23, 24]. Recent seroepidemiologic findings suggest a decreasing age-specific prevalence of HSY-I infection in Western industrialized countries [25]. A substantial proportion of the young population may become sexually active without prior HSY-l infection, and the risk of acquiring HSY-2 as a primary infection might therefore be greater [26]. Both clinical data [23] and experimental studies in animals [27, 28] have shown that symptoms are more severe and complications more common in primary genital HSY-2 infection compared with nonprimary infection. In our study, 20 of 22 meningitis patients with evaluable serologic data showed evidence of primary HSY-2 infection. Also, the large number of patients with confirmed HSY-2 meningitis in this study compared with earlier reports might suggest that the frequency of this complication is increasing. Hence, attempts to isolate HSY-2 from CSF and from subsequent genital lesions and HSY-2-directed serology should be included in the routine diagnosis of serous meningitis. Complications of genital HSY-2 infection such as meningitis are seen more often in women than in men. The reason is not known, but involvement of a larger area, including urethra and cervix, has been hypothesized [25]. Clinical symptoms of meningitis have been reported in 36 % of women with primary HSY-2 infection versus 11 % of men [3]. In published reports of 16 patients with confirmed HSY-2 meningitis, 12

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R

5+

(+

5- H5V-2

200

100

Figure 4. Presence of CSF antibodies to gG-2, confinned as intrathecal by immunoblotting, achieved by radioimmunoprecipitation (RIP) of [3H]glucosamine-labeled HSV-2 antigen. Figure shows autography after RIP with 100 III of anti-gG-2 monospecific rabbit serum obtained after immunizing a rabbit with immunosorbentpurified (with a monoclonal antibody) gG-2 (lane R), serum (S+), and CSF (C+) from a patient with recurrent meningitis, and a control human serum negative for HSV-2 antibodies by ELISA and immunoblotting (S-). Labeled HSV-2 antigen and molecular weight markers are at right.

were female. In accordance, we found a male-to-female ratio of 1:2 (9 men, 18 women). Whether HSY-2 reaches the meninges by a neuronal or hematogenous route in HSY-2 meningitis had been debated. Data from animal models have shown a neuronal spread from infected genital sites through peripheral nerves into the CNS [29]. Hematogenous dissemination was suggested after isolation of HSY-2 from buffy-coat leukocytes in two patients with meningitis [2], but isolation from blood cells could not be repeated in a trial of patients with primary genital HSY infection [30]. In 85 % ofour patients, genital lesions preceded meningitis with a mean interval of rv 1 week. At onset, meningitis symptoms often developed gradually over 2-3 days. Also, focal neurologic symptoms in the lumbosacral region were common and occurred early. These clinical findings suggest a local HSY-2 replication in the genitalia followed by a neuronal spread to the meninges but do not exclude a viremic spread. Additional neurologic complications were found in 63 % of patients with confirmed HSY-2 meningitis and could be classified as initial or recurrent. Of the initial complications, urinary retention was described as the inability to urinate rather than pain from local genital lesions, suggesting involve-

ment of the sacral autonomic nervous system in addition to motor and sensory neurons. The course in all patients was benign, in agreement with an earlier report [4], and none had permanent symptoms after 6 months. Initial complications did not predispose to the recurrent ones experienced by 9 of 27 patients, because only 1 patient who suffered from periodic headache and I who developed recurrent meningitis showed acute neurologic complications during their first episodes of meningitis. Conversely, 15 of 17 patients who suffered initial neurologic complications during the first episode of meningitis did not develop recurrent meningitis or periodic headache. The recurrent complications seemed unpredictable, and there was no tendency towardS a milder or vanishing clinical course during the study period, although cytologic findings in CSF indicated a diminished inflammatory response during relapses compared with that in first episodes of meningitis. After primary infection, HSY-2 establishes a latent infection, generally in the sacral ganglia, from which virus can be recovered at autopsy [31]. Notably, 6 of 12 patients with recurrent meningitis of confirmed or possible HSY-2 etiology presented here had extragenital vesicles compared with none seen during the first episode of HSY-2 meningitis. Although these findings might be explained by autoinoculation to other areas, spread of virus to ganglia other than those first infected might also follow viral involvement and spread within the CNS. Focal neurologic complications seen in first episodes of HSY-2 meningitis suggest that such spread might be an early event. A similar pattern was shown in mice, in which neurologic complications of genital HSY-2 infection correlated with HSY-2 antigen presence and demyelination in the spinal cord [29]. The case history of the patient with symptoms of ascending myelitis is compatible with widespread dissemination of HSY-2 and demyelination in the CNS. In contrast to the first episode of HSY-2 meningitis, diagnosis of recurrent HSY-2-induced meningitis poses a methodologic problem. We could not isolate HSY-2 from 33 CSF samples collected during recurrent meningitis episodes in 5 patients with confirmed and 7 patients with possible HSY2-induced meningitis. If peripheral lesions are present, virus isolation might support the diagnosis, as in 2 patients with recurrences after HSY-2-confirmed first episode of meningitis and in 5 of our patients with possible HSY-2-induced meningitis, but peripheral HSY-2 reactivation could be a concurrent phenomenon without etiologic relevance. For reliable serologic detection of HSY-2 antibodies, crossreactivity with HSY-l must be ruled out. We used a typeselective ELISA based on the gG-2 purified by Helix pomaria-lectin chromatography [19, 20]. This assay showed seroconversion to HSY-2 in all 17 patients tested at the first episode of meningitis. In contrast, significant rises in titer in consecutive serum samples were not found in any of our patients with either confirmed or possible HSY-2-induced recurrent meningitis. In a study of patients with encephalitis caused by HSY-l in which repeated CSF samples were collected [32,

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HSV-2 Meningitis

Acknowledgment We thank Hans Ahme, Saren Elowsson,and Per Lundberg for clinical records and Mia Boethius and Annkatrin Gusdal for technical assistance.

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33], we found a rise in intrathecal titers of HSV-l IgG antibodies. However, because of the brief duration of HSV-2-induced meningitis, successive lumbar punctures are impractical and unethical. Although we detected HSV-2 IgG antibodies in eSF in all 10 recurrences ofmeningitis in patients with confirmed HSV-2 etiology of the first episode of meningitis, the sensitivity of the ELISA did not permit us to determine whether these antibodies were intrathecally produced. In patients with chronic or frequently recurring infections in the eNS, intrathecally produced IgG antibodies may be assessed by detecting a stronger antibody reactivity in eSF when immunoblotting is performed on serum and eSF samples diluted to identical concentrations of IgG as has been described for eNS infection by HIV [34, 35]. By using this method, we detected a relatively stronger intrathecal response to HSV-2 proteins such as glycoprotein G compared with serum reactivity in three patients with recurrent meningitis after the first episode of confirmed HSV-2 etiology. However, because the HSV-2 immunoblotting strips might also contain cell-produced nonHSV proteins, we confirmed the specificity of eSF anti-gG-2 antibodies by using RIP with glucosamine-labeled HSV-2 antigen, allowing only antibodies to HSV-2 glycoproteins to be visualized. This finding was repeated in all three patients analyzed who had recurrent possible HSV-2-induced meningitis but in none of the controls, suggesting that immunoblotting might be a useful diagnostic method for this disease. A prerequisite seems to be that eSF is collected on day 3 or later after onset of meningitis. Furthermore, the intrathecal response seemed more enhanced with increasing numbers ofepisodes, suggesting a memory function in the intrathecal IgG response separate from the serum IgG response. Therefore, screening for HSV-2 serum antibodies followed by HSV-2 immunoblotting analysis of serum and eSF might be diagnostic tools included in the search for causes of recurrent meningitis and possibly also of severe periodic headache.

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